metabolism of mono trans arachidonic acids by …epubs.surrey.ac.uk/856646/1/27731958.pdf · 2020....
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METABOLISM OF MONO TRANS ARACHIDONIC ACIDS BY
PLATELETS, HEPATIC MICROSOMES AND
POLYMORPHONUCLEAR LEUKOCYTES
Uzzal Roy, MD
New York Medical College
New York, USA
A dissertation presented for the award of Doctor of Philosophy to the
School of Biomedical and Molecular Sciences University of Surrey
Guildford, UK
New York, USA
September 2005
ProQuest Number: 27731958
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SUMMARY
Arachidonic acid (AA) is metabolized by cyclooxygenase (COX), lipoxygenase
(LOX) and cytochrome P450 (CYP) in human cells to metabolites with various
physiological and pathological functions. There are two forms of AA - cis and tram.
The purpose of these studies was to identify, characterize and compare patterns of
trans-AA and AA metabolism by human platelets, hepatic microsomes and
polymorphonuclear leukocytes (PMNs), and observe the functional aspects of trans-
AA on these cells. To identify and characterize the metabolites formed from trans-AA
metabolism different pharmacological probes, HPLC and mass spectrometry were
used. Aggregometry was used to evaluate the effects of trans-AA on platelets.
In platelets incubated with \4-trans-PJ±, the COX pathway gave rise to all-ds-15-
HETE, \A-trans-\ 1-HETE and 14-£rarcs-5-HETE; and the LOX pathway gave rise to
14-fnms-12-HETE and a unique thromboxane like molecule, the structure of which
was characterized. AA-induced aggregation was inhibited by \A-trans-AA with an
IC50 of 7.2 pM and two unique characteristics of trans-AA were observed: (1) 14-
trans-AA was not metabolized extensively by the human platelets like AA and (2) co
incubation with AA stimulated basal metabolism of 14-trans-AA. 14-trans-AA is
likely to interact with platelets both via metabolism and via incorporation into the
platelet membrane.
Profiling of 5-trans-AA metabolites generated by hepatic microsomes revealed that
all four bonds participated in epoxidation and generated various epoxides, diols and
hydroxyeicosatetraenoic acids (HETEs). Unlike its ds-isomer, 5,6-trans-EET was
significantly more stable and resisted microsomal hydrolysis and glutathione
conjugation. The microsomal metabolic profile of 5-trans-AA was different from that
of AA and suggested that trans-AA are trans fatty acids that appear to be
metabolically similar to xenobiotics. Experiments with PMNs suggested that 5-trans-
AA was metabolized to different trans-BETEs and that these modulate cell
proliferation and viability.
II
INDEX
Page
I SUMMARY........................................................................................................ n
II INDEX .................... HI
III LIST OF FIGURES.................................................................................... VIII
IV LIST OF TABLES ............ XI
V PUBLICATIONS................................................ XII
VI LIST OF ABBREVIATIONS......................... XIII
VII ACKNOWLEDGEMENTS......................................................................... XIV
1 INTRODUCTION ......... 1
1.1 General - Arachidonic acid cascade...........................................................1
1.2 Arachidonic acid ..................... 3
1.3 M etabolism of A A ................... 5
1.3.1 Physical properties and their relevance to AA distribution 5
1.3.2 Cellular concentration of A A ........................ 6
1 .3.3 Pathways of AA release........................... 6
1.3.3.1 Cytosolic AA and phospholipase A2 ................ 7
1.3.3.2 Regulation of cPLA2 activity.............................. 7
1.3.3.3 Phospholipase C/diacylglycerol lipase................................ 8
1.3.4 Oxygenation of fatty acids..................... 8
1.3.5 Cyclooxygenase pathway..........................................................................10
1.3.6 Lipoxygenase pathway...................................... 12
1.3.7 Cytochrome P450 pathway................................................... ................... 13
1.4 Biological activities of AA m etabolites....................................................14
1.5 Trans fatty ac id s .............................................................................. 16
1.5.1 NOz-dependent cis-trans isomerization of A A .......................................18
III
1.5.2 Origin of trans-AKs - Summary................... ....................................... 21
1.5.3 Origin of reactive nitrogen species............................................ ........... 21
1.5.4 Biological role of fatty acids ............... 24
1.6 Inhibition of AA metabolism................ 25
1.7 Platelets and arachidonic acid.............................. 27
1.7.1 Platelet adhesion............................ 30
1.7.2 Platelet activation and procoagulant activity.........................................31
1.7.3 Platelet aggregation .........................................................................33
1.7.4 Platelet and the development of atherosclerosis.................................... 34
1.7.5 Platelets and arterial thrombosis... ..................................................35
1.7.6 Platelet inhibition...................................................................................... 35
1.8 Hepatic microsomes and arachidonic acid .......................................... 37
1.8.1 Cytochrome P450 ........................ 37
1.8.1.1 Terminal and sub-terminal hydroxylation of AA........................ 38
1.8.1.2 Epoxidation of AA....................................................................................... 39
1.8.1.3 Bisallylic hydroxylation of AA ................................................................... 41
1.8.1.4 Hydroxylation with double bond migration of AA.......................................42
1.8.2 Relation of cytochrome P450 with COX and LOX pathway................43
1.9 Polymorphonuclear leukocytes and arachidonic acid................... 44
1 .9.1 Role o f AA on apoptosis ..................... 45
1.10 Aims of the study........................................................................................ 46
2 METHODS............................... 48
2.1 General and analytical methods................................................ 48
2.1.1 Synthesis and purification of trans-AAs.................................................. 48
2.1.2 HPLC analyses................................................................................ 49
2.1.3 UV spectroscopy.......................... ...50
rv
2.1.4 Mass spectrometry................. ................................................................. 51
2.1.4.1 Preparation of derivatives for GC/MC......................................................... 51
2.1.4.2 Gas chromatography/mass spectrometry.............. 52
2.1.4.3 Liquid chromatography/mass spectrometry................ 53
2.1.5 Trans-AA metabolism by LOX ............................................... 56
2.2 Methods for studies with platelets ............................................56
2.2.1 Platelet preparation....................................................................................56
2.2.2 Methods for observation of metabolism of trans-AA by platelets 57
2.2.3 Methods for observation of effects of trans-AA on p la te le t............... 57
2.2.3.1 Thromboxane Bz assay ...........................................................................59
2.2.3.2 Membrane incorporation study.................... 60
2.3 Methods for studies with hepatic microsomes ........................... 60
2.3.1 Generation, isolation, purification and analysis of microsomal1 A-trans-AA metabolites.......................................................... 60
2.3.2 Isomerization of HETEs............................................................................61
2.3.3 Microsomal metabolism of 5-trans-AA ..................................................61
2.3.4 Hydrolysis of cis- and trans-5,6-EET by microsomal epoxidehydrolase........................................ 62
2.3.5 Conjugation of cis- and trans 5,6- EET with microsomalglutathione-S-transferase.................................................................................. 62
2.4 Methods for studies with polymorphonuclear leukocytes................... 63
2.4.1 Preparation of neutrophils from human b lood ............................63
2.4.2 Metabolism of 5-trans-AA by PMN or HL60 cells...............................63
2.5 M aterials................................................................................................ 64
2.6 Statistics..........................................................................................................64
3 RESULTS....................................................... 65
3.1 Interaction of trans-AA with platelets .................................................65
V
3.1.1 Metabolism of trans-AA by platelets .....................................................65
3.1.1.1 Co-incubation of platelets with cis- and 14-trans-AA.................................67
3.1.1.2 Effect of inhibitors on 14-trans-AA metabolism.........................................69
3.1.1.3 UV analyses of cis and 14-trans-AA metabolites.............. 72
3.1.1.4 Spectral analyses of unknown metabolites....................................................73
3.1.1.5 LC/MS analyses of the 14-trans-AA metabolites.................. 7 4
3.1.1.6 Gas chromatogram of metabolite A and TXB2 .............................................82
3.1.1.7 Identification of metabolites from 14-trans-AA by LC/MS/MS...................83
3.1.1.8 Identification of metabolite A .................................................. 85
3.1.1.9 Identification of metabolites B1 and B2 .........................................................88
3.1.1.10 Identification of metabolite C i................................................................ 89
3.1.1.11 Identification of metabolite C2 ......................... 90
3.1.1.12 Identification of metabolite D i .............................. 91
3.1.1.13 Identification of metabolite D2 ................................................................ 92
3.1.2 Effects o f 14-trans-AA on platelet function............................................93
3.1.2.1 Effect of 14-trans-AA on human platelet aggregation................................. 93
3.1.2.2 Effect of 14-trans-AA pre-incubation time on platelet aggregation............ 96
3.1.2.3 Concentration dependent inhibition of aggregation by 14-trans-AA............9 7
3.1.2.4 Thromboxane B2 formation by platelets....................................................... 98
3.1.2.5 Effect of 14-trans-AA on U-46619.............................................................100
3.1.2.6 Effect of other agonists..................................................................... . 102
3.1.2.7 Membrane incorporation of 14-trans-AA...................................................106
3.2 Metabolism of trans-AA by hepatic microsomes.............................107
3.2.1 Metabolism of \4-trans-AA by microsomes....................................... 107
3.2.2 Metabolism of 5-trans-AA by microsomes.......................................... 109
3.2.2.1 Hepatic microsomes-induced decay of 5,6-EET isomers........................... 120
3.2.2.2 Glutathione-dependent conjugation of 5,6-EET isomers........................... 121
VI
3.2.2.3 Mass spectrometric identification of 5,6-trans -EET..................................122
3.3 Metabolism of trans-AA by polymorphonuclear leukocytes andHL-60 cells ............................................................................................................. 124
4 DISCUSSION..................................................................................126
4.1 Interactions of trans-AA with platelets................................................. 126
4.1.1 Metabolism of trans-AA by platelets.............................................. 126
4.1.2 Effects of 14-trans-AA on platelet function........................................ 143
4.1.3 Relationship between metabolism and effects ................................. 145
4.2 Metabolism of trans-AA by hepatic microsomes................................. 147
4.2.1 Metabolism o f \ A-trans-AA by microsomes.........................................147
4.2.2 Metabolism of 5-trans-AA by microsomes ....................................150
4.3 Metabolism of trans-AA by polymorphonuclear leukocytes............152
4.4 General discussion...................................................................................... 153
4.5 Future w ork................................................ 156
4.6 Conclusion........................................... 157
5 LIST OF SUPPLIERS.......................................................................158
6 REFERENCES .............. 161
VII
LIST OF FIGURES:Page
Figure 1-1. Arachidonic acid cascade...................................................................1
Figure 1-2. Structure of AA and phospholipids.................................................4
Figure 1-3. Metabolism of AA ................................................................................9
Figure 1-4. CYP pathway of AA metabolism ............... 13
Figure 1-5. Structures of AA and different mono- trans-AAs.......................16
Figure 1-6. Formation of 14- trans-AA in vivo..................................................18
Figure 1-7. Mechanism for N 0 2-mediated formation of 14-trans-AA 20
Figure 1-8. Various sources of N 0 2 formation.................................................22
Figure 1-9. Mechanism of platelet adhesion and aggregation......................30
Figure 1-10. Mechanism of platelet aggregation............................................. 33
Figure 1-11. Terminal and sub-terminal hydroxylation of A A ....................39
Figure 1-12. Epoxidation of arachidonic acid............................................ .....40
Figure 1-13. Bisallylic hydroxylation of arachidonic acid................. 41
Figure 1-14. Hydroxylation with double bond migration of A A ................. 43
Figure 2-1. Chromatogram of the products of NQ2 reaction with AA........48
Figure 2-2. Block diagram showing the components of a H P L C ................ 50
Figure 2-3. Flow diagram showing GC/MS derivative preparation........... 51
Figure 2-4. Diagram showing the components of a GC/MS system ............ 52
Figure 2-5. Diagram showing the components of a LC/M S.......................... 54
Figure 2-6. Schematic diagram showing electro spray ionization............... 55
Figure 2-7. Diagram showing the function of an aggregometer...................58
Figure 2-8. TXB2 Standard curve........................................................................59
Figure 3-1. Metabolic products of radio-labeled AA and 14-trans-AA .......66
VIII
Figure 3-2. Metabolism of radio-labeled AA and in the presence of14-ftvzfis-AA................................ 67
Figure 3-3. Potentiation of biosynthesis of metabolites A, B, C, and Dby AA................ 68
Figure 3-4. Effects of inhibitors on AA metabolism........................................ 70
Figure 3-5. Metabolism of radio-labeled \4-trans-AA with nonradiolabeled AA and COX and LOX inhibitor........................................ 71
Figure 3-6. UV chromatogram at 234 nm of AA and 14-trans-AAmetabolites............................................. 72
Figure 3-7. UV spectrum of metabolite C and metabolite D ......................... 73
Figure 3-8. LC/MS analyses of metabolites of AA and 14-/mns-AAby human platelets................................. 75
Figure 3-9. Comparison of the negative-ion MS/MS spectra of variouseicosanoid standards with metabolites of \4-trans-A A .......................... 77
Figure 3-10. Comparison of the negative-ion LC/MS spectra ofmetabolites produced with COX inhibitor ......................................... 79
Figure 3-11. Comparison of the negative ion LC/MS spectra ofmetabolites produced with LOX inhibitor................................................80
Figure 3-12. Metabolism of dg-fm/zs-AA by platelets............... 81
Figure 3-13. GC/MS chromatograms of ion at m/z 585...... 82
Figure 3-14. Comparison of the MS/MS spectra of metabolite A ............... 86
Figure 3-15. Spontaneous transformation of metabolite A ........................... 87
Figure 3-16. Comparison of the MS/MS spectra of metabolites Biand B2..................................................................................................... 88
Figure 3-17. Comparison of the MS/MS spectra of metabolite C i.............. 89
Figure 3-18. Comparison of the MS/MS spectra of metabolite C2.............. 90
Figure 3-19. Comparison of the MS/MS spectra of metabolite D i .............. 91
Figure 3-20. Comparison of the MS/MS spectra of metabolite D2.............. 92
Figure 3-21. Effect of 14-*rafzs-AA on platelet aggregation.......................... 93
IX
Figure 3-22. Effect of 14-trans-AA on AA-mediated plateletaggregation......................... 95
Figure 3-23. Effect of 14-trans-AA incubation time on plateletaggregation............................................................................................ 96
Figure 3-24. Concentration-dependent inhibition of AA-inducedaggregation by 14-trans-AA ......................................................................... 97
Figure 3-25. Concentration-dependent inhibition of AA-inducedTXB2 formation by 14-trajfs-AA.............................................. 99
Figure 3-26. Effect of 14-trans-AA on platelet aggregation byTP receptor agonist................................................................ 101
Figure 3-27. Effect of ADP on platelet aggregation.................................... 102
Figure 3-28. Effect of epinephrine on platelet aggregation........................103
Figure 3-29. Effect of thrombin on platelet aggregation............................104
Figure 3-30. Effect of cPAF on platelet aggregation ........................... 105
Figure 3-31. Incorporation of radio-labeled-AA/14-tra«s-AA intoplatelet membrane phospholipids....................................................... 106
Figure 3-32. Metabolism of radiolabeled \4-trans-AA by rat livermicrosomes ................................................................................................ 107
Figure 3-33. Identification of peak t l and t2 ...................................................108
Figure 3-34. Metabolism of 5-trans-AA by rat liver microsomes 110
Figure 3-35. Mass spectra of four epoxides generated by microsomalmetabolism of 5-trans-AA .......................................................................... 112
Figure 3-36. Identification of 5-trans-EET...................................................... 113
Figure 3-37. Identification of diols........................................... 114
Figure 3-38. Identification of 5-HETE isomer................ 115
Figure 3-39. Identification of metabolite h i ...................................................116
Figure 3-40. GC/MS analysis of metabolite h i ...............................................117
Figure 3-41. The effect of the CYP2E1 antibody on the microsomalmetabolism of 5-trans-AA ...........................................................................118
Figure 3-42. Identification of metabolite h2 and h3......................................119
X
Figure 3-43. Comparison of hydrolysis rate for cis and trans isomersof 5,6-EET by hepatic microsomes............................................................120
Figure 3-44. Glutathione dependent conjugation of 5,6-EET isomers 121
Figure 3-45. Mass spectra of 5-cis and frafzs-EET........................................ 122
Figure 3-46. LC/MS analysis of cis and trans isomers of 5,6-EET............ 123
Figure 3-47. Determination of 5-trans-AA metabolic pattern inhuman PM Ns ...................................... 125
Figure 4-1. Trimethylsilyl ether derivative of thromboxane B2................ 129
Figure 4-2. Suggested structure of metabolite A and dS-metabolite A.... 131
Figure 4-3. Suggested structures of ions at m/z 127 and 155 ...................... 132
Figure 4-4. Suggested structure of keto form of metabolite A ....................132
Figure 4-5. Formation of ion at m/z 195 from ion m/z 225.......................... 133
Figure 4-6. Structures of LC/MS/MS cleavage products of TXB2 ............ 133
Figure 4-7. Ions of d8-metabolite A from dB-labeled 14-trans-AA ......134
Figure 4-8. Proposed mechanism of formation of metabolite A ........135
Figure 4-9. Formation of ion m/z 179 from Metabolite B i.......................... 136
Figure 4-10. Formation of ion m/z 179 from Metabolite B2........................ 137
Figure 4-11. LC/MS/MS cleavage of metabolite Q ...................................... 138
Figure 4-12. LC/MS/MS cleavage of metabolite C2...................................... 140
Figure 4-13. LC/MS/MS cleavage of metabolite D i ....... 141
Figure 4-14. LC/MS/MS cleavage of metabolite D2...................................... 142
Figure 4-15. Summary of metabolism of 14-trans-AA by humanplatelets....................................................... 143
Figure 4 -16 .14-trans-AA and platelet function..............................................146
Figure 4-17. Microsomal epoxidation of 14-trans-AA ..................................149
Figure 4-18. Summary of the key steps involved in the hepaticmicrosomal metabolism of 5-trans-AA .................................................... 151
XI
LIST OF TABLES:
Page
Table 1-1. Biological activities of eicosanoids...................................................15
Table 1-2. Geometrical isomers of 5,8,11,14-eicosatetraenoic acid............. 19
Table 1-3. Biological sources of the N 0 2 radical............................................. 23
Table 1-4. Receptors of the platelet plasma membrane............... 27
Table 3-1. Table showing LC elution time of different standardsdetected by mass spectrometer .......... 76
Table 3-2. The molecular weights (relative abundances) of fragmentions observed in the negative-ion MS/MS spectra ................... 84
Table 3-3. Comparison of retention times of commercial standards........109
Table 3-4. Identification of microsomal 5-trans-AA metabolites............... I l l
XII
PUBLICATIONS ARISING FROM THE WORK DESCRIBED IN THIS THESIS:
Uzzal Roy, Olivier Loreau, Michael Balazy. (2004). Cytochrome P450/NADPH-
dependent formation of trans epoxides from ZraTzs-arachidonic acids.
Bioorg.Med.Chem.Lett. 14, 1019-1022.
Uzzal Roy, Robert Joshua, Russell L. Stark, Michael Balazy. (2005a). Cytochrome
P450/NADPH-dependent biosynthesis of 5,6-/ra^-epoxyeicosatrienoic acid from
5,6-trans-AA. Biochem.J. 390, 717-727.
Uzzal Roy, Russell L. Stark, Robert Joshua, Michael Balazy. (2005b). Stereospecific
synthesis and mass spectrometry of 5,6-?raws-epoxy-8Z,llZ,14Z-eicosatrienoic acid.
Bioorg.Med.Chem.Lett. 15, 3029-3033.
Kavita Jain, Uzzal Roy, Barbara Ardelt, U.M. Krishna, J.R. Falck, Piotr Pozarowskil,
Jan Kunickil, Zbigniew Darzynkiewiczl, Michael Balazy (2005c) 5E, 8 Z, 11Z, 14Z-
eicosatetraenoic acid, a novel trans isomer of arachidonic acid causes Gi phase arrest
and induces apoptosis of HL-60 cells. Int.J.Oncol 27, 1177-1185.
XIII
LIST OF ABBREVIATIONS:
5-HT 5-hydroxytryptamine
AA arachidonic acid
ADP adenosine diphosphate
APCI atmospheric pressure chemical ionization
ATP adenosine triphosphate
BSTFA bis(trimethylsilyl)trifluoroacetamide
CAD collision assisted dissociation
CE collision energy
CEP collision cell entrance potential
CI chemical ionization
CoA coenzyme A
COOH carboxyl group
COX cyclooxygenase
CUR curtain gas
CXP collision cell exit potential
CYP cytochrome P450
DAG diacylglycerol
DIC disseminated intravascular coagulation
DHET dihydroxyeicosatetraenoic acid
DIPEA n,n-diisopropylethylamine
DP declaustering potential
EET epoxyeicosatrienoic acid
El electron ionization
EP entrance potential
ESI electro spray ionization
FFA free fatty acid
FIA flow injection analysis
FP focusing potential
GC-MS gas chromatography-mass spectrometry
GP glycoprotein
XIV
GSH glutathione
HETE hydroxyeicosatetraenoic acid
HHT hydroxyheptadecatrienoic acid
HPETE hydroperoxyeicosatetraenoic acid
HPLC high performance liquid chromatography
IS ion source
LA linoleic acid
LC-MS liquid chromatography-mass spectrometry
LOX lipoxygenase
LT leukotriene
LX lipoxin
MS mass spectrometry
MRM multiple reaction monitoring
NADPH nicotinamide adenine dinucleotide phosphate, reduced
NCI negative chemical ionization
NO nitric oxide
N02 nitrogen dioxide
NSAID non-steroidal anti-inflammatory drug
ODS octadecyl silica
PDGF platelet-derived growth factor
PF4 platelet factor 4
PFB pentafluorobenzylbromide
PG prostaglandin
PGI2 prostacyclin
PI phosphatidylinositol
PL phospholipase
PUFA polyunsaturated fatty acid
RNS reactive nitrogen species
SIRS systemic inflammatory response syndrome
TLC thin layer chromatography
IMS trimethylsilane
IX thromboxane
XV
1 INTRODUCTION
1.1 General - Arachidonic acid cascade
Arachidonic acid (AA) is a polyunsaturated fatty acid with four cis double bonds
found in animal and human fat as well as in the liver, brain, and glandular organs, and
is a main constituent of membrane phospholipids. It is formed by synthesis from
dietary linoleic acid and can be metabolized by three types of oxygenase enzymes:
cyclooxygenase (COX), lipoxygenase (LOX), and cytochrome P450 (CYP). The
COX pathway gives rise to prostaglandins (PCs), thromboxanes (TXs), and
hydroxyheptadecatrienoic acid (HHT); the LOX pathway gives rise to
hydroxyeicosatetraenoic acids (HETEs), lipoxins (LXs), and leukotrienes (LTs); and
the CYP pathway gives rise to epoxyeicosatrienoic acids (EETs), diols and other
metabolites. In addition, polyunsaturated fatty acids such as AA are susceptible to
biological autooxidation by free radicals and yield to isoprostaglandins (IsoPGs),
isoleukotrienes (IsoLTs), NO2AA and trans-AAs (Figure 1-1) (Wolfe, 1982; Oliw et
al, 1982; Samuelsson, 1983; Hammarstrom, 1983).
Cyclooxygenase Lipoxygenase
Cytochrome P450
Arachidonic acid
Prostaglandins (PGs) Thromboxane (TX)
Epoxy acids (EETs), 20-HETE, subterminal HETEs
Isoprostaglandins, Isoleukotrienes trans-AAs, N 0 2AA
Leukotrienes (LTs), Lipoxins (LXs) Hydroxy acids (HETEs)
Figure 1-1. Arachidonic acid cascadeArachidonic acid is converted enzymatically to PGs and TX by COX; to LTs, LXs and various hydroxy acids (HETEs) by LOX; and to EETs, terminal HETE (20-HETE) and subterminal HETEs (16-, 17-, 18- and 19-HETEs) by CYP P450. This fatty acid is also transformed to isoPGs, isoLTs, N02AA and trans-AAs by free radicals. The generic name eicosanoids was introduced by Corey et al (1980) to encompass the increasing number of these oxygenated products derived from 2 0 -carbon (eicosa) polyunsaturated fatty acids.
1
These oxidised arachidonate derivatives are collectively termed eicosanoids (from the
Greek eicosa = twenty; for twenty carbon fatty acid derivatives) (Corey et a l, 1980).
Eicosanoids are formed by virtually every tissue in the body and produce a wide range
of biological effects on inflammatory responses (predominantly those of the joints,
skin and eyes), on the intensity and duration of pain and fever, and on reproductive
function (including the induction of labour). Eicosanoids also play important roles in
the inhibition of gastric acid secretion, regulation of blood pressure through
vasodilatation or constriction, modulation of renal function, inhibition or activation of
platelet aggregation, and they also act as critical mediators of inflammatory diseases
such as bronchial asthma, arthritis, psoriasis, and thrombosis (Needleman et a l, 1986;
Smith, 1989).
Platelets are involved in normal and thrombosis. Thrombotic conditions such as
intravascular clotting in atherosclerosis, systemic inflammatory response syndrome
(SIRS), and haemorrhagic syndromes such as disseminated intravascular coagulation
(DIC) may be linked, at least partially, to hyper- and hypo-activity of platelets,
respectively. The mechanism(s) by which platelets contribute to the pathogenesis of
these conditions are not clearly understood. However, they involve the liberation of
AA (20:4n-6) from membrane phospholipids and its subsequent oxygenation by COX
into pro-aggregatory prostanoids (Samuelsson et al, 1976; Seeds & Bass, 1999;
Calabrese et a l, 2000).
This study involves the investigation of the metabolism(s) of AA in platelets in the
presence of trans-AA and the metabolism of trans-AA by platelets, microsomes and
polymorphonuclear leukocytes (PMNs). Trans-AA contains at least one double bond
in the trans configuration. Trans fatty acids are present in the diet. They result from
anaerobic bacterial fermentation in ruminant animals and are thereby introduced into
the food chain (Emken et al, 1994). Humans consume them in the form of meat and
dairy products. The reported intake of trans fatty acid ranges from 2.6 g/day to 12.8
g/day (Emken, 1995). Trans double bonds are also formed during the hydrogenation
of either vegetable or fish oils. Oils are hydrogenated to increase their plasticity and
chemical stability, hence their potential use in food products. Consumption of
hydrogenated oils also contributes considerable amounts of trans fatty acids into
human body. Hydrogenation results in a number of changes in the acyl chain of the
2
fatty acid moiety, all of which can influence physiological parameters. These changes
include the conversion of cis to trans double bonds, saturation of double bonds, and
migration of double bonds along the acyl chain resulting in multiple positional
isomers (van den Reek et al., 1986; Coulon et al, 2003; Balazy & Poff, 2004).
1.2 Arachidonic acid
Arachidonic acid is a 20-carbon essential polyunsaturated fatty acid (all c/5-5,8,11,14-
eicosatetraenoic acid) having four cz's-double bonds at the 5th, 8 th, 11th and 14th
carbon position from carboxyl (COOH) terminus, forming a molecule of
5Z,8Z,llZ,14Z-eicosatetraenoic acid (Figure 1-2). Essential means that the
mammalian cell cannot synthesize AA de novo from acetate, rather the precursor
linoleic acid is a direct biochemical precursor. This enzymatic capability exists within
most cells, to convert linoleic acid by chain elongation and desaturation into AA
(Cook, 1985). Nevertheless, AA is a very common polyunsaturated fatty acid. For
example, 10-20% of the total fatty acid content of most cells is AA (20:4 co-6 ).
Normally, AA is not found as a free carboxylic acid within most cells, but rather
exists esterified as a phospholipid in various lipid bilayers either at the plasma
membrane or in separate organelle membranes such as the nucleus, endoplasmic
reticulum, peroxisome and mitochondrion.
Arachidonic acid is typically found particularly on the 2nd carbon atom (sn-2) of
glycerol in the glycerophospholipid. Cellular phospholipids represent a very complex
mixture. Glycerophospholipids exist in various classes, with the four most common
being glycerophosphocholine (GPC) lipids, glycerophosphoethanolamine (GPE)
lipids, glycerophosphoserine (GPS) lipids and glycerophosphoinositol (GPI) lipids
(Figure 1-2).
There are many other minor phospholipid classes including mono- and bi-
phosphorylated forms of inositol, phosphatidic acid, phosphatidylglycerol and
cardiolipin. The essential fatty acid, AA, occupies a central position as precursor of
the widest variety of eicosanoids. In platelet phospholipids, AA is found mainly in
GPC and GPE (Dobner & Engelmann, 1998).
3
oIIHo-a
o1 iiCH2—O—C—R-j
O2c h - o -
o3 IICH2—O—P—O—X
O'
11
Arachidonic acid
14
Glycerophospholipid (general structure)
Name ofglycerophospholipid
Name of X Formula of X
Phosphatidic acid
Phosphatidylcholine
Phosphatidylethanolamine
Phosphatidylserine
Phosphatidylinositol
Choline
Ethanolamine
Serine
Inositol
H
CH2—CH2—N(CH3)3
CH2— CH2— NH3
— CH2— CH — NH3
COO"
OH
OH
OH OH
OH
Figure 1-2. Structure of AA and phospholipidsA phospholipid is composed of a glycerol backbone with a fatty acid (Ri) in sn-1 position, AA typically found in sn-2 position and a phosphatidyl group (phosphatidic acid / phosphatidylcholine / phosphatidylethanolamine / phosphatidylserine / phosphatidylinositol) in sn-3 position.
4
1.3 Metabolism of AA
Arachidonic acid is metabolized by all cells in human body but it has a significant
role in platelets, endothelium, smooth muscle, leukocytes, and parenchymal cells.
Blood platelets are amongst the most active blood elements that metabolize AA by
COX and LOX. The production of eicosanoids is a highly regulated process that is
dependent on the acylation and transfer of AA into specific phospholipid pools by
AA-selective acyl transferase and transacylase reactions, and the release from these
pools by a variety of phospholipases A2 (PLA2). The role for the widely distributed
cytosolic PLA2 in mediating stimulus-induced AA release for the production of
eicosanoids from the COX and LOX pathways is well established (Clark et a l, 1995;
Ichinose et a l, 2002). The levels of free AA available for eicosanoid production are
controlled by the rate of déacylation by PLA2 and by competing processes such as
reacylation into phospholipid and efflux from cells (Balsinde et al, 2002; Diaz &
Arm, 2003). Metabolism of free AA is mediated by a variety of oxygenases and
downstream synthases, which are differentially expressed in cells, and which
determine the types of metabolites produced. The eicosanoids are released from cells
and exert their biological effects in an autocrine or paracrine manner by binding to G-
protein-coupled receptors (Narumiya & FitzGerald, 2001).
1.3.1 Physical properties and their relevance to AA distribution
The lipids in the cell membrane have hydrophobic/non-polar sites and aqueous/polar
sites, therefore the fatty acids can be available in ionic forms. Like other fatty acids
AA is amphipathic, and its hydrophobic tail can remain in a lipid bilayer while its
polar carboxyl group (charged or uncharged) can emerge into the aqueous
environment outside the membrane. AA has a high pKa which is crucial as it is
responsible for the solubility and the possible distribution of the unesterified fatty acid
in cells (Spector, 1986). Though the fatty acids exist predominantly in anionic form
their solubility in water is poor. AA specifically binds with albumin through a
physical process that occurs when nonpolar and electrostatic interactions are
combined. The overall concentration of AA present in an aqueous environment can
increase by binding to albumin and decreasing the concentration of free molecules in the solution (Spector, 1975).
5
In human plasma the concentration of total AA is high (35 mg/ml, 0.6 mM), and
albumin (0.1 mM) greatly reduces the effective concentration of free AA molecules,
and permits millimolar concentrations to be stabilized in an aqueous environment.
Physiologically, this is critical to the presentation of low concentrations of the free
fatty acid (unbound) to cells. In the presence of albumin, the concentrations of free
fatty acids are likely kept well below 0.1 pM (Spector, 1986; McArthur et a l, 1999).
1.3.2 Cellular concentration of AA
The concentration of free AA in resting cells is universally described as low.
However, there are few reports of mass assay to determine the concentration of free
AA. In resting or inactivated human platelets, the concentration of esterified
arachidonate was measured using a mass per volume calculation and it was
approximately 5 mM (-30 pg arachidonate/109 platelets) (Neufeld & Majerus, 1983).
This calculated concentration represents an average for whole cells, but the level of
free AA is very low because of the rapid action of COX and LOX. The concentration
of AA is substantially higher in the plasma membrane as AA remains esterified in
membrane phospholipids. The release of 1% of this reserve of esterified arachidonate
could give up to 50 pM local concentrations prior to its release from the cell. In fact,
around up to 1 0 % of platelet arachidonate stores could be released upon activation
(Neufeld & Majerus, 1983). In resting leukocytes the concentration of AA is around
0.5-1 pM (-3 pmol arachidonate/106 cells) (Chilton et al., 1996).
1.3.3 Pathways of AA release
Cell stimulation by diverse agonists leads to the activation of a variety of intracellular
phospholipases, including phospholipases A2, C, and D. This triggers the production
of numerous lipid mediators and represents an important step in signal transduction.
6
1.3.3.1 Cytosolic AA and phospholipase A2
AA is most often esterified at the sn-2 position of the glycerophospholipid.
Phospholipase A2 (PLA2) catalyzes the hydrolytic cleavage of glycerophospholipid at
the sn-2 position yielding a free fatty acid and a lysophospholipid. More than 19
different PLA2 have been identified in the mammalian system (Balsinde et a l, 2002),
and different isoforms of PLA2 have been shown to participate in physiological events
related to cell injury, inflammation, and apoptosis. In vitro, purified cPLA2 is active at
concentrations of free Ca2+ (0.1-lpmol/l) similar to those reached intracellularly
during receptor-dependent Ca2+ responses (Cummings et a l, 2000; Sun et al, 2004).
Raising levels of Ca2+ from 100 to 300 nmol/1 causes the association of recombinant
cPLA2 with membrane vesicles, suggesting that, in response to receptor-stimulated
rises in Ca2+, cPLA2 may translocate from the cytosol to the cell membrane, where
both competent G proteins and phospholipid substrate are located (Channon & Leslie,
1990). Arachidonic acid may also be formed by metabolism of diacylglycerol by
diacylglycerol lipase (Siess et al, 1983).
1.3.3.2 Regulation of cPLA2 activity
Cytosolic phospholipase A2 is activated in response to various stimuli such as
cytokines, hormones, neurotransmitters, mitogens, antigens, endotoxins, and
extracellular matrix constituents (Clark et a l, 1995; Murakami et a l, 1997). Certain
physical and stressful stimuli including oxidation, hyperglycaemia, UV light and
shear stress may also induce activation of the enzyme (Kramer & Sharp, 1997). The
activation is mediated by several distinct, agonist- and cell-specific intracellular
signals that involve guanine nucleotide-binding proteins called G proteins (Winitz et
al, 1994; Burke et a l, 1997). The activation of stimulatory G protein increases the
concentration of Ca2+ ion, which activates kinases such as mitogen-activated protein
kinase (MARK) and protein kinase C (PKC), thus preferentially catalyses the
hydrolysis of AA from the sn-2 position of membrane phospholipids (Schalkwijk et
al, 1996; Qiu et a l, 1998; Hirabayashi et al, 1999).
7
1.3.3.3 Phospholipase C/diacylglycerol lipase
Activation of PLA2 provides a direct pathway of AA release. Alternatively, release
may be initiated by the activation of phosphoinositide-specific PLC, which cleaves
the phospholipid at the phosphate ester bond producing l,^-diacylglycerol (DAG).
This intermediate is in turn broken down by a DAG-lipase to yield free fatty acid and
monoacylglycerol. DAG activates protein kinase C, thus catalyses the hydrolysis of
AA from membrane phospholipids (Allen et ah, 1992).
1.3.4 Oxygenation of fatty acids
It is assumed that cellular oxygenases act upon AA only in their free form. The intra
cellular concentration of unesterified AA is very low, so their liberation from
phospholipids is the rate-limiting step. As in numerous cells, platelets contain active
COX and LOX, PMNs contain mainly LOX and hepatic microsomes contain CYP
P450 which can oxygenate most of the PUFAs (Samuelsson et al, 1978). When
cellular phospholipase (phospholipase A2) is activated, AA is released from
membrane phospholipids. Once AA is released from cell membranes, it may be
metabolized to eicosanoids via the COX or LOX pathway (Hamberg & Samuelsson,
1974). The COX pathway is mediated by two different enzymes, COX-1 and COX-2.
This pathway generates prostaglandins - PGE2, PGD2, PGF2a, PGI2 (prostacyclin),
and thromboxane - TXA2 (Hamberg et al, 1975) (Figure 1-3). The LOX pathway is
regulated by 5, 12, 15-LOX and other LOXs. This pathway generates different
HETEs, and leukotrienes - LTB4, LTC4, LTD4, LTE4 and lipoxins - LXA4 and LXB4
(Lapetina & Cuatrecasas, 1979; Dutilh et a l, 1980) (Figure 1-3). The major products
of cytochrome P450 (CYP)-catalyzed AA metabolism are regio- and stereo-specific
epoxyeicosatrienoic acids (EETs) and their corresponding dihydroxyeicosatrienoic
acids (DiHETEs), and 18, 19 and 20-HETE (Capdevila et al, 2000; Capdevila &
Falck, 2000) (Figure 1-3).
8
Stimulus Phospholipids
1Activation of PLA., PLC, PLD
Lysophospholipid
Cyclooxygenasereaction
co x V
Arachidonic acidb
c
12-LOX 5-LOX 5 ,1 2 ,1 5LOXs
V 1r 1rPGG2 12-HPETE 5-HPETE DiHETEs
Peroxidase V reaction 5, 12-HETE Dehydrase
r NADPH
PGH, Leukotriene A,
CytochromeP450/
epoxygenase
EET16, 17, 18, 19, 20 HETES
Dihydroxyacids
GSH
▼ 1
TXsynthase r 1
Isomerasesr 1
Prostacyclinsynthaser '
GST
r i
Hydrolase
r ▼
Lipoxygenases
HHT TXA,
Spontaneous hydrolysis
TXB,
PGD,PGE,
PGL LTC4 LTB4 Lipoxins
PGF2aSpontaneous y-glutamyl transpeptidase hydrolysis ▼
LTD46-keto-PGF1a | Dipeptidase
LTE„
Figure 1-3. Metabolism of AAThree major pathways are involved in AA metabolism, (a) The cyclooxygenase (COX) pathway results in the formation of PGG2 from AA by a COX reaction. In a subsequent peroxidase reaction, PGG2 undergoes a two-electron reduction to PGH2. Both of these reactions are catalysed by COX (prostaglandin H synthase). PGH2 serves as a substrate for cell-specific isomerases and synthases, producing other eicosanoids such as prostacyclin (PGI2) and thromboxane A2 (TXA2). (b) The lipoxygenase (LOX) pathway forms hydroperoxyeicosatetraenoic acids (HPETEs) and dihydroxyeicosatetraenoic acid (DiHETEs) by LOX and subsequently converts these to (1) hydroxyeicosatetraenoic acids (HETEs) by peroxidases, (2) leukotrienes (e.g. LTB4) by hydrolase and glutathione S-transferase (GST), and (3) lipoxins by LOXs. (c) The epoxygenase pathway forms epoxyeicosatrienoic acid (EETs) and dihydroxyacids by cytochrome P450 epoxygenase.
9
1.3.5 Cyclooxygenase pathway
Cyclooxygenases (COXs), also known as prostaglandin H synthases or prostaglandin
endoperoxide synthases, are fatty-acid oxygenases of the myeloperoxidase
superfamily (Daiyasu & Toh, 2000). For many years, it was thought that the
constitutively active COX-1 protein was the only COX in eukaryotic cells. In 1991, a
second inducible enzyme was identified through studies of cell division; this second
enzyme is now called COX-2 (Miller et al, 1994). COX-1 is constitutively expressed
in many mammalian cells and tissues and is considered responsible for the formation
of PGs and TXs involved in the regulation of physiological fimctions. COX-2 is
considered to be an inducible enzyme that is expressed in low levels under basal
conditions, but is strongly induced in response to inflammatory stimuli (Smith &
Langenbach, 2001). A recent report from Chandrasekharan et al. (2000) describes a
third cyclooxygenase (COX-3) selectively inhibited not only by paracetamol but also
by low concentrations of some non-steroid anti-inflammatory drugs including aspirin.
COX-3 is a variant of COX-1 which has retained intron-1 during translation and
which is found in human tissues in a polyadenylated form. Selective inhibition of
COX-3 may lead to discovery of potent and valuable new drugs for controlling pain
and fever. Cyclooxygenases are important pharmacological targets for aspirin and
other non-steroidal anti-inflammatory drugs (Smith et al, 1996).
COX-1 is expressed in many tissues including platelets where the major end product
is TXA], a vasoconstrictor and platelet activator, and endothelial cells where the end
product of the AA pathway is prostacyclin (PGI2), a potent vasodilator and platelet
inhibitor. Unlike platelets which lack nuclei, endothelial cells have the capacity to
synthesize new COX-1 , regenerating their ability to synthesize PGI2 (de Gaetano et
al, 2003). Endothelial cells also have the inducible COX isoform COX-2, not present
in platelets, that can synthesize PGI2 in the presence of low dose of aspirin (Marcus et al, 2 0 0 2 ).
Much of the work has been done on platelets because of their patho-physiological
importance. Therefore, this study focused on the metabolic pathway mainly in the
platelets. The first step of AA metabolism is catalyzed by COX-1 which in platelets is
located in the dense tubular system (endoplasmic reticulum) that produces PGG2
10
(Gerrard et al, 1976). The conversion of AA to PGG2 by PGH synthases can also lead
to the formation of the side products 11R-HETE and 15-HETE (Thuresson et al,
2 0 0 0 ). PGG2 is a very short-lived compound that is rapidly converted into PGH2 via
PG-endoperoxidase activity of COX-1. The main oxygenated product of the COX-1
pathway in platelets is TXA2, formed by the enzyme TX synthetase (Needleman et
al, 1976). This enzyme’s activity is associated with the dense tubular system (Carey
et al, 1982) and catalyzes the formation of TXA2 and 12-HHT from PGH2. However,
some HHT may be produced from the nonenzymatic breakdown of PGH2 (Diczfalusy
eta l, 1977).
TXA2 is formed in response to local stimuli and exerts its effects within a short
distance of its biosynthesis. TXA2 is rapidly hydrolysed (ti/2 = 3 0 seconds) non-
enzymatically to form TXB2 , that is the chemically stable hydration product of pro-
aggregatory TXA2. In human, TXB2 undergoes two major pathways of metabolism,
resulting in the formation of 2,3-dinor-TXB2 and 1 l-dehydro-TXB2, respectively
(Ciabattoni et a l, 1989). 2 ,3 -dinor-TXB2 is an abundant metabolite that is found in
urine (Patrono et a l, 1986). Thus, TXA2 is a transient compound, so it is difficult to
measure accurately circulating levels in whole-animal experimental models.
Therefore, measurement of TXB2 metabolites such as 11-dehydro TXB2 and 2 ,3 -dinor
TXB2 in urine and plasma gives better estimates of in vivo TXA2 production
(Ciabattoni et a l, 1989; Murphy & FitzGerald, 1994). TX synthase can be inhibited
by imidazole (Needleman et al, 1977), substituted imidazoles such as dazoxiben
(FitzGerald et al, 1983), and PG analogues (Gorman, 1983). Primary PGs, PGD2, E2
and p2a, are produced in small amounts in platelets by enzyme PGD synthase (PGH-D
isomerase), PGE synthase, and PGF synthase, respectively (Osama et al, 1983;
Yamamoto, 1989).
PGF synthase (originally termed as PGD2 11-ketoreductase) synthesizes PGF2a and its
isomer (1 1 -epi- PGF2a) from PGH2 and PGD2, respectively (Watanabe et a l, 1985).
PGD synthase, long thought to be a cytosolic enzyme (Urade et al, 1985), proved to
be a membrane bound enzyme, as determined by molecular cloning (Urade et a l,
1989). PGE synthase (PGH-E isomerase) catalyzes the isomerization of PGH2 to
PGE2 (Ogorochi et al, 1987).
11
1.3.6 Lipoxygenase pathway
Lipoxygenases constitute a family of dioxygenases, which catalyze stereo specific
insertion of molecular oxygen into polyunsaturated fatty acids (Brash, 1999). In
mammals, the LOXs are divided into three major groups, 5-, 12-, and 15-LOXs,
depending on their positional specificity of AA oxygenation. In addition, an 8 -LOX
has been identified in mouse skin (Jisaka et a l , 1997).
In platelets, this pathway catalyzes the oxygenation of carbon 12 of AA. This 12-LOX
leads to the formation of 12-HPETE (Hamberg & Samuelsson, 1974), which further
reduced into 12-HETE (Grossi et al, 1989) via a glutathione-dependent peroxidase
requiring selenium at the active site (Bryant & Bailey, 1980). It is linked to the hexose
monophosphate shunt which provides nicotinamide adenine dinucleotide phosphate,
reduced (NADPH) to recycle the reduced glutathione (Bryant et al, 1982). Such a
peroxidase activity may be altered by high concentrations of aspirin-like drugs (Siegel
et al, 1979) or by glutathione depletion leading to the formation of trihydroxy-
eicosatrienoic acids (8,9,12- and 8,11,12-tri-OH) (Bryant & Bailey, 1979) via the 10-
OH-11,12-epoxy intermediate (Walker et al, 1979). Lipoxygenase activity was found
to be localized mainly in the cytosol (Nugteren, 1975; Chang et al, 1982) but around
one-fourth of the total activity seems to be tightly bound to membranes (Lagarde eZ
al, 1984). Unlike leukocyte 5-LOX, platelet LOX is not calcium-dependent (Chang et
af., 1982).
The activity of 5-LOX is found in leukocytes, mainly in neutrophils and macrophages.
Arachidonic acid is converted to 5 (S)-hydroperoxy-6 -Zra/«-8 ,11,1 A-cis-
eicosatetraenoic acid (5-HPETE) (Shimizu et a l, 1984) and subsequently to LTA4 by
the action of 5-LOX (Shimizu et al, 1986). LTA4 is, in turn, metabolized to LTB4 by
LTA4 hydrolase (Radmark et al, 1984) or LTC4 by the action of LTC4 synthase
(Izumi et al, 1988). LTC4 is converted to LTD4 and LTE4, and these three peptido-
leukotrienes constitute a major part of the so-called slow-reacting substance of
anaphylaxis. These LTs are synthesized primarily by mast cells, eosinophils, and
macrophages, and are potent bronchoconstrictors and also increase microvascular
permeability, and induce airway secretion (Dahlen et al, 1980). 12-LOX is the
principal LOX in the brain (Shimizu et a l, 1987). 15-LOX synthesizes 15(S)-
12
hydroperoxy-5,8 ,11 -cis-13-fraH.s-eicosatetraenoic acid (15-HPETE) (Bryant et al,
1985), a key intermediate for synthesis of lipoxins (Serhan & Samuelsson, 1988).
Three LOXs (5-, 12-, and 15-) were purified to homogeneity, and 5-LOX (Dixon et
al, 1988), 12-LOX (Izumi et a l, 1990) and 15-LOX (Balcarek et a l, 1988) cDNAs
were cloned. 5-LOX, 12-LOX and 15-LOX have many sequences homologous to
each other, and, moreover, they have regions homologous to a reticulocyte LOX and
plant LOX, such as soybean LOX (Zhao & Funk, 2004).
1.3.7 Cytochrome P450 pathway
Every cell contains GYP, but hepatic microsomes are the main storehouse of this
enzyme. CYPs metabolize AA to HETEs and EETs. These eicosanoids are formed in
a tissue and cell-specific manner and have numerous biological functions. CYPs can
oxygenate AA by one or more reactions shown in Figure 1-4.
Cytochrome P450
Hydroxylation with double bond migration
Bisallylichydroxylation
Terminal hydroxylation Epoxidation
Arachidonic acid
7-HETE10-HETE13-HETE
5,6-EET8,9-EET
11,12-EET14,15-EET
16-HETE17-HETE18-HETE19-HETE20-HETE
5-HETE8-HETE9-HETE11-HETE12-HETE 15-HETE
Figure 1-4. CYP pathway of AA metabolismOnly the primary oxygenation products are shown, HETE-hydroxyeicosatetraenoic acid; EET-epoxyeicosatrienoic acid.
13
1.4 Biological activities of AA metabolites
Arachidonic acid is a precursor of eicosanoids, which are formed by most human cells
and functions as autocrine and paracrine lipid mediators (i.e., they signal at or
immediately adjacent to their site of synthesis). Eicosanoids have a broad spectrum of
biological activities and have been implicated in the pathogenesis of inflammatory
and atherothrombotic processes. Their synthesis is increased at sites of inflammation,
and they can be found in inflammatory exudates. The key actions of eicosanoids are
summarized in the Table 1-1 .
14
Table 1-1. Biological activities of eicosanoids
Eicosanoid Biological activityp g d 2 Growth inhibition, anti-aggregation, smooth muscle constriction, hypothermia,
sleep induction, narcolepsy, acetylcholine release, inhibition of prolactin release,
inhibition of LH-RH release, LH release (at pituitary level), increase of serotonin
turnover
p g e 2 Smooth muscle dilatation, Na+ excretion, plasma exudation, inhibition of gastric
secretion, hyperthermia (fever), awake state, stimulation of LH-RH release,
regulation of catecholamine release, inhibition of natural killer cells
PGF2a Bronchoconstriction, vasoconstriction, acetylcholine release
p g i2 Vasodilatation, anti-aggregation, inhibition of gastric secretion
t x a 2 Bronchoconstriction, vasoconstriction, aggregation
l t b4 Chemotaxis, adhesion of leukocytes to endothelial cells, lysosomal release,
superoxide generation, activation of natural killer cells, induction ofIL-2 receptor
l t c4 Bronchoconstriction, vasoconstriction, plasma exudation, LH release, LH-RH
release, depolarization of Purkinje cells, opening of atrial K+ channel
l t d 4 Bronchoconstriction, vasoconstriction, plasma exudation
l t e4 Vasoconstriction, plasma exudation, bronchoconstriction
l x a 4 Chemokinesis, TX formation, bronchoconstriction, inhibition of natural killer
cells, activation of protein kinase C, antagonism of LTD4 receptor in kidney
l x b 4 Inhibition of natural killer cells
5-HPETE Stimulation of steroidogenesis in adrenal gland
12-HPETE Migration of vascular smooth muscle cells, vasoconstriction
5,6-EET Secretion of somatostatin, LH, TSH, prolactin, vasodilatation, Na+, K+-ATPase
inhibition ; inhibits platelet aggregation in vitro
8,9-EET Secretion of oxytocin, inhibition of COX
11,12-EET Secretion of vasopressin, anti-aggregation
14,15-EET Glucagon secretion, anti-aggregation, inhibition of COX
12-HETE Inhibition of Na+-K+-ATPase; proinflammatory mediator
16-HETE Inhibition of neutrophil aggregation and adhesion
18-HETE Weak vasodilator; inhibitors of ATPase
19-HETE Weak vasoconstrictor; stimulates Na+-K+-ATPase
20-HETE Regulation of membrane ion transport in the medullary thick ascending limb and
proximal tubules; inhibition of platelet aggregation in vitro and thromboxane
biosynthesis; stimulation of stem cell growth in the human bone marrow
11,12-DHET Na+-K-ATPase inhibition
Summary of the general biological activity of the eicosanoids adapted from(Shimizu & Wolfe, 1990; Spector et a l, 2004; Benatti et a l, 2004).
15
1.5 Trans fatty acids
Trans unsaturated fatty acids, or trans fats, are fats produced artificially by heating
liquid vegetable oils in the presence of metal catalysts and hydrogen (Katan et al,
1995). The carbon/carbon double bonds in unsaturated fatty acids can exist in either
cis or trans configuration. In the double bond of the unsaturated fatty acid, the two
hydrogen atoms on the carbon atoms that form the double bond may be located on the
same side of the carbon chain in which case they are termed cis, and on the opposite
side of the carbon chain in which case they are termed as trans. Isomers of AA are
shown in Figure 1-5.
COOH
14
Arachidonic acid [all c/s-AA]
COOH / = =
S-frans-arachidonic acid [5E-AA]
COOH
8-frans-arachidonic acid [8E-AA]
.COOH
11-fraas-arachidonic acid [11E-AA]
14-frans-arachidonic acid [14E-AA]
Figure 1-5. Structures of AA and different mono- trans-AAsThis figure represents polyunsaturated fatty acids having one trans- and three cis- double bonds and the double bonds are present between 5th and 6th, 8th and 9th, 11th and 12th, and 14th and 15th carbons.
Trans fatty acids are those unsaturated fatty acids having at least one double bond in
the trans configuration instead of having all cis bonds. In contrast to typical cis
16
isomeric configuration, the double bond angle of the trans fatty acid is smaller and the
acyl chain is more linear, resulting in a more rigid molecule with a higher melting
point. In cis configuration, the hydrogen atoms are on the same side of the carbon
chain, resulting in a ‘kink’ in the acyl chain and a more flexible phospholipid
molecule as opposed to trans which makes it more rigid (Steinhart et al, 2003;
Stender & Dyerberg, 2004; Wahrburg, 2004).
Trans fatty acids are found in dairy fats and, mainly in products of hydrogenated fat,
such as margarine. Hydrogenation of fatty acids is done to increase their plasticity and
chemical stability, thus making transportation and storage easier. Food manufacturers
also use partial hydrogenation of vegetable oil to destroy some fatty acids, such as
linolenic and linoleic acid, which tend to oxidize, causing fat to become rancid with
time. Commercial baked goods frequently include trans fats to protect against
spoilage. During the hydrogenation process, variable amounts of the unsaturated fatty
acids that are not hydrogenated are converted from the cis to the trans configuration
(Figure 1-6). This process of isomerization from cis to trans also occurs naturally in
the rumen of sheep and cattle; for this reason, trans fatty acids are found in the milk
and fat of these animals (Emken et a l , 1994).
The common hydrogenation process uses a nickel catalyst and results in both the
reduction and migration of double bonds. Because hydrogenation raises the melting
point of the fat, the usual product is a fat that is solid at 25°C, although there is wide
variation in physical properties depending on the starting fat and the degree of
hydrogenation (Steinhart et al, 2003). Most of the trans fats produced commercially
are monoenes. Trans fatty acids are also formed in human by the oxidation of fatty
acids (Jones et a l, 1978; Ascherio & Willett, 1997; Nelson, 1998). An increased level
of trans fatty acids can alter the biological membrane rigidity, permeability and
asymmetry (Patel & Block, 1986; Mann, 1994).
17
c/s-arachidonic acid
Isomerization by •N 0 2,N20 3,H N 02
COOH
14-frans-arachidonic acid
Elongation and desaturation
.COOH
12-fra/7S-Linoleic acid
Diet
COOH
Linoleic acid
Figure 1-6. Formation of 14- trans-AA in vivoDietary ds-linoleic acid may be converted to 12-/ram-linoleic acid by N 0 2 or it may be supplied as 12-/nms-linoleic from diet. The elongation and desaturation process converts 12- frvms-linoleic acid to 14-/rûm-arachidonic acid.
1.5.1 eN02-dependent cis-trans isomerization of AA
•N02 efficiently isomerizes A A to trans-A A, and both mono trans and poly trans
isomers have been detected (Jiang et a l, 1999). Other unsaturated fatty acids are
also readily isomerized by "NOz. Such isomerization is significant in vivo during
pathological conditions like inflammation and endotoxic shock where the
18
formation of NO and its oxidation product NO2 are increased (Balazy & Lopez-
Femandez, 2003).
The isomerization of AA is mediated by "NOz. Other free radicals (OH, peroxyl,
and superoxide) do not isomerize AA. Peroxynitrite (ONOO-) also causes the
isomerization of AA in addition to oxidation. Arachidonic acid reacts readily with
NO2 in a dose-dependent manner, generating a mixture of products. The major
product obtained from the NO2 AA reaction is a mixture of four isomers having
one trans and three cis bonds (trans-AA). The reaction also produces isomers
having more than one trans double bond (Balazy, 1994; Jiang et a i, 1999; Boulos
etal., 2000).
Overall formation of 14 isomers of 5,8,11,14-eicosatetraenoic acid having non
conjugated cis and trans bonds is possible (Table 1-2).
Table 1-2. Geometrical isomers of 5,8,11,14-eicosatetraenoic acid
Isomer of AA Number of isomersAll cis 11 trans, 3 cis 42 trans, 2 cis 6
3 trans, 1 cis 4All trans 1
Of these, the four mono trans isomers appear to be the most abundant products
generated by NO2. The isomerization is likely to involve the reversible binding of
NO2 and the formation of a nitroarachidonyl radical followed by the elimination of
NO2 and generation of a trans bond (Figure 1-7) (Prutz et al., 1985).
19
Arachidonic acid
•N0o
Isomerization
OgNElimination Hydroxylation
14-fraf7s-arachidonic acid 14-nitro-15-hydroxyeicosatrienoic acid
Figure 1-7. Mechanism for NC^-mediated formation of 14-trans-KAWhen N02 reacts with AA, it forms c/s-nitro-AA that spontaneously isomerizes to trans- nitro-AA. 7nms-nitro-AA is converted by the process of elimination to /raws-arachidonic acid and by the process of hydroxylation to nitro-hydroxy-AA.
NO is also responsible for cis-trans isomerization of AA. In endotoxaemic rat
where the formation of NO is increased, the plasma levels of trans-AA are
increased two-fold and trans-AA esterifled in the heart phospholipids increase 5-
fold (Balazy, 2000). Because the trans-AA isomers are not readily metabolized by
COX and LOX, but appear to be better metabolized by CYP (Berdeaux et a l,
1998), they could function as unique markers of nitro oxidative processes related to inflammation (Balazy, 2000).
20
1.5.2 Origin of frans-AAs - Summary
Trans-AA may originate from two sources: the diet and endogenous cis-trans
isomerization. \A-trans-AA and 5-trans-AA are the models of trans fatty acid to be
studied in this thesis. 14-trans-AA can originate either from diet via
elongation/desaturation of 12-trans-LA or by 'NOz-mediated isomerization. Dietary
intake of trans-LA and in diet containing nitrite may also contribute to the formation
of trans-AA (Balazy, 2000). Trans-AAs are formed in vivo via a reversible addition
of NO2 to the olefinic cis bond of fatty acids. Thus, trans-AAs are likely to be
formed in conditions characterized by increased exposure to "NO] such as
inflammation, sepsis, smoking, exposure to polluted air in which the formation of
NO and its oxidation product, "NO] is increased (Rudrum, 1995).
1.5.3 Origin of reactive nitrogen species
NO] is a potent toxic oxidant gas (Tewari & Shukla, 1989) and a major air pollutant
that originates from the combustion of fossil fuels and other sources. The
concentration of NO] in urban areas can reach levels up to 1500 pg/m3 (0.8 ppm),
which varies seasonally. NO] concentrations in homes with combustion appliances
are higher than homes without such appliances. Average indoor concentrations of
NO] in excess of 100 pg/m3 have been measured in some homes with fuel heaters.
Virtually all of the emitted NO] is in the form of NO, which is gradually oxidized
into NO]. Environments containing cigarette smoke and other types of smoke may
have higher concentrations of both NO and NO] (Jones et al., 1978; Elsayed, 1994;
Postlethwait & Bidani, 1994). Various sources of NO] formation are shown in Figure
1-8.
21
2N0+0; NO/NO2(Nitrite)
NO+Of-> ONOO enzymes — ►Lipid peroxidation(Peroxynitrite) i -
Air Pollution (Fuel Combustion)
Figure 1-8. Various sources of NO2 formationNO2 forms mainly by the oxidation of nitric oxide (NO) in cells and also found to be in nature as a product of fuel combustion.
Platelets contain NO synthase in their cytoplasm, which upon activation generate
NO. Nitric oxide modulates platelet reactivity by increasing cyclic GMP (Radomski
et al., 1990). The NO% radical is a common intermediate in the NO oxidation
pathways to nitrite. A reaction of NO with superoxide, which generates an
intermediate ONOO", has a rate comparable to the superoxide scavenging rate of
superoxide dismutase (SOD), and is a significant pathway by which NO% is
generated in vivo (Koppenol, 1998). Intracellular CO2 plays an important role in the
decomposition of ONOO" to NO2 (Goldstein et a l, 2001; Meli et al, 2002; Uppu et
al, 1996). More recent work has identified novel sources of NO2, which involve
enzymatic and non-enzymatic oxidation of nitrite by hydrogen peroxide (H2O2)
(Table 1-3).
Myeloperoxidase (MPO) has been identified as an effective catalyst for the
oxidation of nitrite to 'NO2 in addition to its role in the oxidation of chloride to
hypochlorous acid (Kraemer et a l, 2004). The MPO/H2O2/NO2 system can induce
the nitration of fatty acids via formation of the NO2 radical (Byun et a l, 1999;
Eiserich et a l, 2002). A recent study demonstrated that heme catalyzes the oxidation
of nitrite to NO2 by H2O2 without the aid of the prosthetic group of peroxidases. It is
22
thus likely that in addition to protein nitration, heme and other iron complexes, in
the presence of nitrite and H2O2, could potentially induce lipid nitration by a
mechanism similar to the one that leads to the formation of nitro-tyrosine in albumin
(Bian et ah, 2003).
Thus, significant amounts of N 02 can be generated in vivo by multiple pathways.
However, the processes involving the reactions of NO2 with lipids have not been
fully understood (Carr & Frei, 2001). N 0 2 could be an important mediator of lipid
metabolism because it is highly lipophilic. The results obtained by Liu et al, show
that the oxidation of NO to N 0 2 in aqueous solutions is greatly accelerated by
phospholipids and the NO oxidation occurs predominantly in the hydrophobic
bilayer of phospholipid vesicles (Liu et a l, 1998). This study indicates that the
biological membrane may play an important role in the oxidation of NO. In
addition, the hydrolysis rate of NO2 in the lipid phase is minimal (Goss et a l, 1999).
N 02 may remain in membrane lipids long enough to react with fatty acids and
phospholipids. The reaction of N 02 with NO produces dinitrogen trioxide (N2O3), a
potent nitrating/nitrosating compound. In the aqueous phase, N2O3 rapidly
hydrolyzes to form two NO2 molecules. Thus, because some nitrite may be
converted to N 0 2 by heme or peroxidases (Schmitt et a l, 1999), there may be a
steady concentration of N 0 2 in tissues. Additional nitrite may be supplied in the
diet, as nitrite is a common and abundant food preservative (Cammack et a l, 1999;
Oldreive & Rice-Evans, 2001).
Table 1-3. Biological sources of the NO2 radical
Reaction References
2NO + 0 2-> 2 -N02 (Koppenol, 1998)NO + O2 -> ONOO" + H+ ONOOH -> NO2 + -OH (Bian et al, 2003a)Myeloperoxidase + nitrite + H2O2 —» 'NO 2 (Byun et al, 1999)Cu/Zn-SOD + nitrite + H2O2 -> 'NO2 (Singh e ta l, 1998)Peroxidases + nitrite + H2O2 —> 'NO2 (Gebicka, 1999)Cytochrome P450 + ONOO" —» 'NO2 (Mehl et a l, 1999)Lactoperoxidase + nitrite + H2O2 —> 'NO2 (Reszka et a l, 1999)Heme / iron + nitrite + H2O2 —> 'NO2 (Bian et al, 2003a)
Mechanism of N02 generation in cells (Balazy, 2000)
23
1.5.4 Biological role of trans-fatty acids
Despite numerous epidemiological and experimental feeding studies, evidence
regarding the health risk of tmns-FA as causative factors in the development of
cardiovascular disease and cancer is not conclusive (Ascherio & Willett, 1997;
Nelson, 1998; Mozaffarian et a l, 2004). However, it has been established that
increased levels of trans-FA alter biological membrane rigidity, permeability and
asymmetry. Additionally, none of the previous studies have considered the
possibility that at least some trans-FA may originate from the endogenous sources
(Patel & Block, 1986; Mann, 1994).
In endotoxaemia or inflammation, significant amounts of trans-FA isomers may be
generated, possibly within the cell membrane phospholipids, increasing the pool of
phospholipids containing trans-FA (Szabo et al., 1995). The trans-FA generated
by endogenous isomerization may differ from the ones ingested with diet. The
identification of the trans-FA that originate solely from NOz-dependent reactions
may provide a useful index to assess the damage done to the biological membrane
via reactive nitrogen species (RNS), and therefore, may be complementary to the
marker importance of isoPGs, which reflect the damage to membrane mediated by
OH radical (Balazy, 2000).
In this regard, the RNS-dependent trans-FA may function as specific lipid
mediators of inflammation and potentially other pathologies. The trans-AA may
remain bound within membrane phospholipids for prolonged periods and released
following stimulation by phospholipases. Additionally, because biological
membranes enriched with trans-FA appear to be more rigid and permeable and
have altered phospholipid asymmetry, they may provide less protection against
smaller molecules such as certain carcinogens. These molecules may easily cross
membranes composed of increased amounts of the diet-independent trans-FA.
Studies of cells exposed to trans-AA may provide supporting evidence for a novel
mechanism involved in the association between inflammation and cancer ( Andre
et a i, 2002; Coussens & Werb, 2002; Ip & Marshall, 1996; Slattery et al., 2001).
24
In addition to specific lipid markers, nitro-eicosanoids possess some biological
activity. Nitrohydroxyarachidonic acid causes relaxation of vascular smooth
muscle at micromolar concentrations via the activation of guanylate cyclase by a
novel mechanism involving the release of NO. The mechanism of this NO release
is unknown but, it may involve glutathione (Balazy et a l, 1998; Lufrano &
Balazy, 2003).Berdeaux et al. (1996) found that the addition of a \A-trans isomer of AA to rat
platelet suspensions inhibited the aggregation induced by AA. However, the addition
of its structural homolog 5,8,11-eicosatrienoic acid (20:3n-9) did not induce any
modification of the platelet aggregation. By contrast, the 12-lipoxygenase pathway
was stimulated by \A-trans-AA, by the increased production of 12-HETE. The 5,8,11-
eicosatrienoic acid (20:3n-9), not being a substrate of the COX, did not induce any
significant modification in the formation of TXB2 and 12-HHT. The 1 A-trans-AA
alone did not induce any platelet aggregation. However, this fatty acid was
metabolized to a limited extent into two products that have not been identified yet. It
was suggested that one of them could be a product of the 1 2 -lipoxygenase pathway
(Berdeaux et a l, 1996).
1.6 Inhibition of AA metabolism
Biosynthesis of eicosanoids is increased and sustained in inflammation and other
pathological conditions, which have potent and detrimental effects on the functions of
various organs. Anti-inflammatory steroids like dexamethasone inhibit the release of
AA and its metabolites by cells, and inhibit the formation of eicosanoids by down-
regulation of COX-2 expression (Hong & Levine, 1976; Schleimer et a l, 1986).
Steroids inhibit cell phospholipases that are responsible for the release of AA from
phospholipid stores, by stimulating the biosynthesis of an inhibitory protein called
macrocortin in macrophages (Flower & Blackwell, 1979) and lipomodulin in
neutrophils (Schoene & lacono, 1976; Hirata et al, 1980).
COXs are important targets for non-steroidal anti-inflammatory drugs (NSAIDs).
Aspirin acetylates serine-530 of COX-1, thereby blocks TXA2 synthesis in platelets
and reduces platelet aggregation (Vane & Botting, 2003). This mechanism of action
25
accounts for the effect of aspirin on prevention of coronary artery and cerebrovascular
thrombosis. Aspirin is less effective in inhibiting COX-2 activity, whereas celecoxib
and rofecoxib selectively inhibit COX-2 activity as they contain a side chain to anchor
to the side pocket of COX-2 substrate channel (Brune & Hinz, 2004). Aspirin and
salicylate at therapeutic concentrations inhibit COX-2 protein expression through
interference with binding of CCAAT/enhancer binding protein beta (C/EBPbeta) to its
cognate site on COX-2 promoter/enhancer (Wu, 2003). COX-3 is selectively inhibited
by analgesic/antipyretic drugs such as acetaminophen, phenacetin, antipyrine, and
dipyrone, and is potently inhibited by some NSAIDs. Thus, inhibition of COX-3
represent a primary central mechanism by which these drugs could decrease pain and
possibly fever (Chandrasekharan et al, 2002; Brune & Hinz, 2004). Ridogrel, a
combined TXA2 synthase inhibitor and TX-receptor antagonist, inhibits platelet
function (McNicol & Israels, 2003).
There are five active LOXs found in humans: 5-LOX, 12S-LOX, 12R-LOX, 15-LOX-
1 and 15-LOX-2. Several drugs have now been developed that block either
leukotriene formation or their receptors. Baicalein and esculetin are naturally
occurring flavonoids that inhibit 12-LOX, and thus inhibit LTB4 and LTC4
biosynthesis at micromolar concentrations (Sekiya & Okuda, 1982; Kimura et al,
1987). Zileuton is both potent and selective as a 5-LOX inhibitor; it inhibits the
formation of all 5-LOX-derived lipids (Szczeklik et a l, 2001). Lukasts (i.e.,
montelukast, zafirlukast, pranlukast, pobilukast) are a group of drug that inhibit the
function of leukotrienes by acting as leukotriene receptor antagonists (Spector, 1996;
Suzuki et al, 2003). New specific lipoxygenase inhibitors are directed towards
inhibition of tumour growth and potentially other effects (Poff & Balazy, 2004b).
Finally, a third pathway of AA metabolism involves oxidation by the inducible
cytochrome P450 isozymes, and although no clinically useful drugs are yet available,
promising research in this area (Sato et al, 2001) is directed toward yielding valuable
drugs that block generation and symptoms of stroke (Bednar et al, 2000b).
26
1.7 Platelets and arachidonic acid
Resting platelets are small, round, disk shaped cells, which lack nuclei and are found
in the blood. Platelets originate from the cytoplasm of megakaryocytes. They
distinguish between normal endothelial cell lining and areas with lesions in order to
maintain a crucial balance between anti- and pro-coagulative actions within the body.
Platelets display several cell surface receptors. These receptors include (a) the major
serpentine receptors for thrombin, ADP, TXA2 and epinephrine; (b) the glycoprotein
receptor for von Willebrand factor (vWF); (c) (33 and pi integrin receptors for
fibrinogen and collagen; and (d) the immunoglobin superfamily receptors, Fc
receptor, FcyRIIA, and GP VI ( Andrews & Bemdt, 2004; Gibbins, 2004; Hourani &
Cusack, 1991) (Table 1-4).
Table 1-4. Receptors of the platelet plasma membrane
Ligand Receptor
von Willebrand factor GPIbap-IX-V
Fibrinogen GPHb-IIIa or 0 ^ 3
Collagen GPIa-IIa or a2piGP VI (Immunoglobin superfamily)
P-selectin
Laminin a6PiFibronectin asPsImmunoglobulin G FCyRIIA (Immunoglobin superfamily)
Thrombin PAR-1, -2, -3, -4
GPIpa-V
ADP P2Yi
P2 Yi2
P2Xi
Thromboxane TP receptor
PAF PAF
Epinephrine a2
Receptors present in the membrane o f platelets.
27
Glycosylated proteins are the main receptors that are found on the cell surface. The
most important surface glycoprotein receptor is an integrin, GP Ilb-IIIa (<xllbp3)
which has the ability to bind both fibrinogen and vWF (Shattil & Brass, 1987). GP
Ilb-IIIa is a heterodimeric complex composed of a platelet-unique a and p integrin
subunit (allbpS). The alpha subunit is made up of a heavy and light chain covalently
linked by a single disulfide bond. Both chains are formed from a pro-IIb molecule that
is enzymatically cleaved and processed in the megakaryocyte (Shattil et a l, 1998).
GP Ilb-IIIa however, is inactive on the surface of the resting cell. Intracellular signals
produced by other receptor-agonist triggers its ligand binding activity. These
intracellular signals have the ability to trigger a conformational change in the
molecule causing it to open its ligand-binding site (Ginsberg et al, 1992). The ligand-
binding site is specific that identifies and responds to all proteins having an Arg-Gly-
Asp sequence present in the two major platelet ligands, fibrinogen and vWF. A
platelet contains approximately 50,000 molecules of GP Hb-IIIa; 80% to 90% of those
molecules are expressed on the cell surface (Shattil et a l, 1985).
The cytoplasm of platelet contains granules, organelles, the specialized membrane
systems of the cell and the actin cytoskeleton. There are three types of storage
granules in the platelet cytoplasm, a granules, dense granules and lysosomal granules.
When platelets become activated, these granules can be released into the surrounding
environment. The a granules are the most prevalent granules which contain platelet
factor 4, g-thromboglobulin, platelet-derived growth factor and fibrinogen (Harrison
& Cramer, 1993). These proteins serve to facilitate the adhesive process, support cell-
to-cell communication, trigger vascular restoration, and can influence blood vessel
function and clotting cascade. The dense granules are composed of large amounts of
serotonin (5HT), adenosine di- and tri-phosphate (ADP and ATP), and Ca2+ (McNicol
& Israels, 1999). If released, they can modulate vascular tone, recruit additional
platelets to sites of injury and enhance the thrombus-forming ability of other platelets.
The lysosomal granules can be released after stimulation by aggregating agents like
thrombin and high concentrations of collagen (Berger et a l, 1998; Diacovo et al,
1996; Frenette et a l, 2000; Hourani & Cusack, 1991).
Upon activation, the platelet microtubules contract and allow the platelet to change its
shape. Activated platelets become sticky and change their shape from smooth to
28
spiny, thus causing an increase in surface area. Their round shape is maintained by an
actin-hased cytoskeleton, as they circulate throughout the vascular system. This
cytoskeleton prevents the cell from being distorted by fluid shear forces. At the core
of the cytoskeleton is a three-dimensional support system of actin and tubulin
polymers. Activated and loosely connected platelets can revert to passive and free
platelets respectively, until a certain point. When the granules are released from the
platelets, they can no longer return to their original phase. As the activation process
continues, granules within the platelet are pulled outwards, and they release their
contents into the surrounding environment. This causes platelets to adhere to exposed
surfaces (Zucker-Franklin, 1970).
The sub-endothelium contains collagen that can function as a platelet activator. If the
endothelium is damaged, platelets attach themselves to the exposed surface and
become activated drawing the attention and responses of other platelets to the affected
area. Activated platelets can activate other platelets and thus the response is highly
intensified. Platelets are able to fuse to surfaces because the platelet membrane is
being exposed to protein binding sites on the contacting surface. Fibrinogen is the
chief plasma protein that is drawn to and fuses with these binding sites. Binding can
only occur if a platelet is activated (Blockmans et al, 1995; Nachmias, 1980).
In physiological conditions, platelets can contribute to hemostasis, whereby
haemorrhage is stopped when tissue trauma occurs. However, in unhealthy arteries,
platelet accumulation can result in thrombotic occlusion of the vessel lumen along
with the flow of blood being impeded resulting in ensuing tissue damage. Thus, one
of the main functions of endothelial cells is maintaining equilibrium between blood
fluidity and swift thrombus development in response to injury. They manage vascular
tone, synthesize inhibitors and activators of platelet function and play a role in blood
clotting. When there is subsequent vascular damage and the extracellular matrix is
free to mix with flowing blood because the endothelial cell barrier is destroyed,
haemostatic thrombus formation occurs. Platelets respond to this occurrence in three
phases involving adhesion, activation and aggregation (Gross & Aird, 2000; Ware &
Heistad, 1993).
29
1.7.1 Platelet adhesion
Initial platelet adhesion is mediated by membrane receptors that function regardless of
cellular activation. These membrane receptors cause the first layer of activated
platelets to be attached to the affected area within seconds. The type and seriousness
of the damage determines which extracellular matrix components will be in contact
with the responding platelets. The means by which platelets adhere to the endothelium
is demonstrated in Figure 1-9.
Platelet
GPI b/llla
/C o lla g e n in subendothelium
Fibrinogen
GPIb/IX
Von Willebrand factor
Endothelial cell
Figure 1-9. Mechanism of platelet adhesion and aggregationThis mechanism is found in the normal arteriolar circulation or in larger arteries with stenotic lumen. Tethering to immobilized vWF allows circulating platelets to contact a thrombogenic surface or adherent platelets that have been activated. Interaction o f activated platelets with collagen in the extracellular matrix is responsible for adhesion, and interaction with fibrinogen is responsible for aggregation.
These matrix components can also interact with secreted molecules such as collagen
or bound vWF. When the endothelium is damaged the main basement membrane
components, such as proteoglycans, collagen type IV, nidogen (entactin) laminin and
fibulin, are allowed to mix with the circulating blood.
30
Although proteoglycans are vital in coagulation modulation, they are not necessarily
directly associated with platelet adhesion. Similarly, while collagen type IV and most
other collagen types, can trigger platelet responses it is not as efficient as other
collagens that are located within the vascular wall (types I, HI and VI) (Saelman et al,
1994). During high blood flow, collagen receptors cannot function to trigger thrombus
formation thus, glycoproteins are needed (Savage et al., 1998). There are two platelet
membrane glycoproteins (GPs), integrin-c^Pi (GPIa-IIa) and GPVI, which interact
directly with collagen (Savage et al, 1996). The communication between GPIb and
vWF allows the initial layer of platelet to adhere to the surface. The interaction
between GPIb and vWF is a requirement for typical platelet bonding (Doggett et al,
2002).
1.7.2 Platelet activation and procoagulant activity
Platelet activation is initiated when adhesive ligands and excitatory agonists bind to
cognate receptors located on the platelet’s membrane. It is further carried out through
cell signalling reactions involving enzymes, substrates and co-factors engaged in
specified protein-protein and protein-lipid interactions. Prostacyclin and nitric oxide
are some of the inhibitory substances that aid in restricting major thrombus formation
in disruptions within the vessel wall. Among those substances that stimulate platelet
action are collagen, vWF, a-thrombin, ADP, serotonin (5-HT), epinephrine and TXA2
(Heemskerk et a l, 2 0 0 2 ).
a-thrombin induces platelet activation through G protein-linked protease-activated
receptors (PARs) which trigger intracellular signalling (Sambrano et a l, 2001). There
have been four specific PARs identified, they are PARI, PARS, PAR4, which respond
to thrombin and PAR2, which is activated by trypsin (Coughlin, 1999). The response
formulated by PARs cause a series of events to occur. It causes the activation of
phospholipase C which hydrolyzes the membrane-associated phosphatidylinositol-
4,5-bisphosphate into inositol-tris-phosphate and diaclyglycerol. Inositol-tris-
phosphate promotes mobilization of calcium (Ca++) from endoplasmic reticulum and
diaclyglycerol activates protein kinase C. Increased intracellular calcium leads to
platelet aggregation by p38 MAP (mitogen-activated protein) kinase-dependent
31
activation of phospholipase A2 (Coulon et a l, 2003). The latter hydrolyzes membrane
phospholipids and this result in AA being released from phospholipid and TX being
formed. During platelet activation, there have been two occurrences that have been
linked with thrombin receptors and calcium levels. The first signal was mediated by
PARI with a subsequent calcium signal (Covic et a l, 2000). Apart from being able to
activate platelets through PARs, a-thrombin can also trigger platelet responses by
binding to the GPIb-DC-V complex (Mazzucato et a l, 1998).
Although ADP is vital to platelet function because it magnifies the responses of other
agonists and it is considered to be a weak agonist itself. By itself, it only causes the
modification in shape and reversible platelet aggregation. Further aggregation and
secretions are the outcomes of ADP-induced synthesis of TXA2. ADP affects calcium
and adenylyl cyclase by interacting with specific platelet receptors. It causes a rise in
cytoplasmic calcium through interaction with the Gq-coupled P2Yi receptor, and
inhibits adenylyl cyclase, by communicating with the Gi-coupled P2 Yi2 receptor. To
cause normal ADP-induced platelet aggregation, both the Gq and Gi pathways need to
be activated. Epinephrine and TXA2 can also activate platelets through specific G
protein-coupled receptors (GPCRs) (Gachet, 2000).
Activated platelets exhibit the actin polymerization with cytoskeletal reorganization,
secretion from storage granules and aggregation dependent on the soluble ligand
binding to integrin-allbp3. The process of secretion involves granular contents being
released into the cytoplasm and the extracellular space and it includes the membrane
proteins being transferred to the cell surface (for example, P-selectin). These
occurrences improve platelet activation and aggregation. Platelet activation causes the
regulation of surface exposure for phosphatidylserine, which facilitates the assembly
of enzymes into functional complexes by providing docking sites on the surface of the
anionic phospholipids. The procoagulant activity of activated platelets leads to the
formation of thrombin and helps to deposit fibrin within the aggregates and contribute
to the stability of the developing thrombus (McNicol & Israels, 2003).
32
1.7.3 Platelet aggregation
When platelets aggregate, this triggers the accumulation of platelets into the
haemostatic thrombus. The aggregation step is mediated by adhesive substrates that
are found on the membranes of activated platelets.
AA release
R elease of ADP 5-HT etc.
PGG
TXA.
GPIIb/lllaexposure
CollagenThrombin
AGGREGATION
ADP5HT
Epinephrine
Figure 1-10. Mechanism of platelet aggregationExposure and activation of the allbpS (GPIIb/IIIa) receptor, the final common pathway leading to aggregation, can be realized via different mechanisms and via different stimuli (Herman, 1998). The impact of the various pathways, which finally lead to the exposure of the GPIIb/IIIa receptor, can thus vary according to the type and/or the concentration of the agonists present. The mediators released during the process of platelet aggregation can potentiate each other and, for example, small subthreshold doses of 5-HT markedly diminish the amount of ADP required to induce aggregation.
33
When aiibPs or GPIIb-IIIa is activated, there is a change in the ligand-binding ability
(Woodside et a l, 2001). Activated GPIIb-IIIa aids in stable adhesion and
immobilization of soluble proteins such as vWF, fibrinogen and fibronectin, on the
surface of activated platelets and more platelets are recruited on this substrate (Savage
et al, 2002). The mechanism of platelet aggregation is shown in Figure 1-10.
1.7.4 Platelet and the development of atherosclerosis
Two of the most important characteristics of atherosclerosis are the deposition of
lipids and the local infiltration of leukocytes (Lusis, 2000). The resulting environment
affects endothelial cell function and increases the possibility of communication with
other cells including platelets (Sachais, 2001). When platelets are adhered to
atherosclerotic plaques, they become activated and then cause more aggregation
which influences the plug progression (Ross, 1999).
A deficiency of vWF provides some protection from atherosclerosis (Methia et al,
2001). Enhanced secretion of vWF can result in platelet stimulation and can lead to
activation and accumulation (Theilmeier et a l, 2002). More features such as infection
from different pathogens can trigger inflammation and lead to atherosclerosis
(Willerson, 2002). When platelets are exposed to microorganisms, antibodies bound
to these microorganisms can activate the platelets through the immunoglobulin G
receptor, FCyRIIA. Therefore, recurrent infection may be the cause of the accelerated
development of an atherosclerotic plaque because platelets are being activated and
drawn to the affected region (Sjobring et al, 2002).
Activated platelets that have adhered to an atherosclerotic lesion can manipulate the
development of the plaque by releasing adhesive ligands, binding molecules from
plasma and providing a reactive surface for monocytes and lymphocytes. Among the
molecules affected is a ligand called P-selectin, which after release becomes
expressed on the platelet membrane (Cell et a l, 1997). Lymphocytes can attach to the
damaged vessel regardless of the high blood flow whereas they may not have been
able to do so if there were no interactions between activated platelets. Platelets also
contain and release growth factors which have been known to play a role in cellular
34
proliferation (Ross, 1993). Current studies place platelets in a more direct role in the
development of the atherosclerotic lesion because platelets play a role in the cellular
metabolism of low-density lipoproteins (Sachais et al, 2002). Similarly, when 13-
amyloid, derived from ingested platelets, is processed, this can activate macrophages
in an atherosclerotic plaque. These reactions further demonstrate the idea that the
attachment of platelets to the vesicular wall contributes to chronic lesions that
precede, typically by many years, the acute onset of arterial thrombosis (Tedgui & Mallat, 2002).
1.7.5 Platelets and arterial thrombosis
The normal arterial wall and an atherosclerotic plaque vary in structure. Triggering a
platelet response can be the result of susceptible unstable atherosclerotic plaques. The
resulting thrombosis can also be increased due to dysfunction of the endothelial cells,
exposure of tissue and highly reactive collagens (Ruberg et al, 2002). The area
surrounding an atherosclerotic lesion triggers shear stress due to a rise in blood flow (
Mailhac et a l, 1994; Strony et al, 1993). This stress also has the ability to increase
platelet reactivity through the vWF-GPIIb pathway (Goto et a l, 1999).
1.7.6 Platelet inhibition
Platelet inhibitors are derived mostly from endogenous anti-thrombotic compounds.
The platelet inhibitory function is mediated by NO and PGI2 generated from platelets,
endothelial cells and leukocytes. Nitric oxide inhibits platelet adhesion, aggregation,
and stimulates disaggregation of preformed platelet aggregates. These effects of NO( !
are mediated by guanylate cyclase stimulation and the formation of cyclic GMP and
its subsequent transduction mechanism. Prostacyclin can prevent and reverse platelet
aggregation similar to NO but via a different mechanism by stimulation of adenylate
cyclase and the formation of cyclic AMP (Radomski et al, 1990). This is important
because inhibiting the formation of prostacyclin by COX-2 can result in thrombosis
and may in fact increase the risk of heart attacks (McNicol & Israels, 2003). COX-2
inhibitors recently have withdrawn from market due to this effect.
35
The endothelium discharges a far greater amount of NO than platelets. By stimulating
guanylyl cyclase, NO can prevent platelets from being activated. Additionally,
ADP/ATP can be converted to adenosine through enzymatic processes and this too
prevents platelet aggregation. Thus, the immediate stimulus to platelet activation is
followed after a delay by inhibition. The cycle can be quite quick experimentally,
with the decay occurring over minutes; locally decay may be much faster. Adenosine
is also released by all cells if they run out of oxygen. Therefore, if a platelet
aggregation blocks a small blood vessel, downstream tissue releases adenosine which
starts to break up the aggregation and this is pathologically important (Sogo et al, 2000).
Some inhibitors have direct effects on blood vessels. These compounds cause the
vessels to relax. In a manner comparable to adenosine, NO stimulation results in the
relaxation of blood vessels. Platelets communicating with endothelial cells have some
effects on blood vessels. Depending on the signals emitted, small aggregates can be
broken up or more platelets can be activated and accumulate at the affected region. If
there is no damage to the endothelium, endothelial cells release PGI2 and NO which
inhibit aggregation and prevents the constriction of blood vessels (Hankey & Eikelboom, 2003).
Clopidogrel and ticlopidine, the thienopyridine derivatives, are metabolised in the
liver to active compounds which covalently bind to the ADP receptor on platelets and
dramatically reduce platelet activation (Savi & Herbert, 2005). Dipyridamole inhibits
phosphodiesterase, which inactivates cyclic AMP. Increased intraplatelet
concentrations of cyclic AMP reduce the activation of cytoplasmic second
messengers. Dipyridamole also stimulates prostacyclin release and inhibits TXA2
formation, also block adenosine uptake and therefore enhances effect of adenosine (Liu ef a/., 2004).
Glycoprotein Hb/IIIa receptor antagonists block the final common pathway for
platelet aggregation. Abciximab is a humanised mouse antibody fragment with a high
binding affinity for the glycoprotein Ilb/IIIa receptor. Tirofiban (a non-peptide
derivative of tyrosine) and eptifibatide (a synthetic heptapeptide) mimic part of the
structure of fibrinogen that interacts with the glycoprotein Ilb/IIIa receptor and thus
36
compete with ligand binding of fibrinogen to the glycoprotein Ilb/IIIa receptor
(Conde & Kleiman, 2004).
1.8 Hepatic microsomes and arachidonic acid
Beside platelets, AA is also extensively metabolised by microsomes. A hepatic
microsome is a small vesicle that is derived from fragmented endoplasmic reticulum
produced when hepatocytes are homogenised. Purified microsome preparations are
routinely used to study the role of cytochrome P450s and other enzymes involved in
drug metabolism. These preparations are a rich source of membrane-bound,
hydrophobic enzymes originating from smooth endoplasmic reticulum and rough
endoplasmic reticulum that catalyze phase I reactions of metabolism. The CYP
monooxygenases constitute a major metabolic pathway for AA in the hepatic
microsomes.
1.8.1 Cytochrome P450
Cytochrome P450 was discovered in the late 1950’s (Klingenberg, 1958).
Cytochrome P450 refers to a super family of heme-thiolate enzymes whose Fe2+-
carbon monoxide complex shows an absorption spectrum with a maximum at 450 nm.
There is enormous diversity between members of the CYP family. The CYP enzymes
can be found in virtually all organisms including bacteria, fungi, yeast, plants, insects,
fish and mammals. All CYP genes have probably evolved from one single ancestral
gene, which existed before the time of prokaryote/eukaryote divergence (Nelson et
al., 1996).
Due to the large number of CYPs, a standardised nomenclature system has been
developed. The enzymes have been organised based on identities in protein sequence.
The enzymes are named CYP, representing cytochrome P450, followed by a number
denoting the family, a letter designating the subfamily and a second numeral
representing the individual enzyme. The corresponding gene name is written in
italics. Enzymes that have more than 40% amino acid sequence identity are
37
considered to belong to the same family, whereas enzymes with more than 55%
sequence identity are in the same subfamily (Nelson et al, 1996),
The CYP enzymes have two major functions. They are involved in the biosynthesis,
bio-activation and catabolism of endogenous compounds, such as fatty acids, steroid
hormones, vitamins, bile acids, and eicosanoids. They are also involved in the
metabolism of foreign chemicals and drugs. Drugs and other chemicals are often
converted by CYP to more polar compounds that can be directly excreted or further
conjugated by other enzymes. The conjugate makes the modified compound more
water-soluble so it can be excreted into the urine. Some of these CYP reactions can
lead to the bio-activation of drugs or to the formation of more toxic compounds
(Klaassen, 1996; Guengerich & Shimada, 1998).
Within the cell, the CYPs are mainly located in the endoplasmic reticulum (ER) and
in the inner membrane of the mitochondria. The mitochondria house several CYP
enzymes that are involved in the biosynthesis and metabolism of steroid hormones,
bile acids, and vitamin D (Klaassen, 1996). CYPs metabolize AA by four pathways.
1.8.1.1 Terminal and sub-terminal hydroxylation of AA
The 20th carbon of AA is called terminal carbon and 16th, 17th, 18th and 19th carbon of
AA is called sub-terminal carbons. Arachidonic acid terminal and sub-treminal
hydroxylations have been demonstrated in microsomes isolated from many different
tissues (Oliw, 1994). Many studies have shown that CYPs belonging to the CYP4A
subfamily are the predominant fatty acid terminal/sub-terminal-hydroxylases in most
mammalian tissues including the kidney and liver (McGiff & Quilley, 1999).
However, the CYP4A enzymes usually metabolize saturated fatty acids such as lauric
acid at much higher rates than AA (Hoch et a l, 2000). Thus, the CYP4A enzymes
probably play an important role in cellular fatty acid homeostasis. The formation of
20-HETE and 19-HETE is best characterized in the kidney, as they are most prevalent
there and have several postulated functional roles in renal physiology (Capdevila et
al, 2 0 0 0 ).
38
Microsomes from the human kidney cortex convert AA mainly to 20-HETE, which
has been proposed to be involved in renal ion transport and vascular tone. 20-HETE
and a glucuronide conjugate of 20-HETE have been identified in human urine
(Prakash et a l, 1992). It has been reported that 16R-, 18R-, and 19-HETE exert
vasodilator effects and that 16S-, and 17S-HETE inhibit renal proximal tubular
Na+/K+-ATPase activity (Carroll et a l, 1996). Recently, it was shown that human
PMNs contain endogenous pools of 16-HETE produced via a cytochrome P450
pathway, and some of it was stored within PMNL membrane phospholipids (Bednar
et al, 2000a). The products of hydroxylation of the terminal and sub-terminal carbons
of AA by CYP are shown in Figure 1-11.
sub-terminalhydroxylation terminal
hydroxylation
-COOK •COOH COOH
OHOH16-HETE
OH18-HETE 20-HETE
COOH
OHOH17-HETE
Figure 1-11. Terminal and sub-terminal hydroxylation of AA
Terminal hydroxylation generates 20-HETE and sub-terminal hydroxylation generates 16-,17-, 18-, and 19-HETE.
1.8.1.2 Epoxidation of AA
Arachidonic acid has four double bonds and CYP can oxygenate all of these to cis-
epoxides (EETs). These EETs can be further hydrated by epoxide hydrolases to
39
dihydroxyeicosatrienoic acids (DiHETs). The EETs/DHETs have been detected in
many tissues and body fluids, including human liver, kidney, heart, plasma and urine
(Capdevila et a l, 2000).
Racemic EETs can be formed non-enzymatically, but strong evidence for the
enzymatic formation of endogenous EETs has been provided by chiral analysis of
EETs in phospholipids. It has been proposed that EETs play a role in the structure and
hence the function of cellular membranes (Oliw, 1994). Many studies have indicated
that EETs are involved in regulation of vascular tone and might serve as an
endothelium derived hyperpolarization factor (Fisslthaler et ah, 1999). EETs may also
play a role in vascular inflammation (Node et al, 1999) and be involved in
transcriptional regulation of PGH synthase-2 and CYP (Peri et al, 1997; Peri et al,
1998). The products of epoxidation of AA by CYP are shown in Figure 1-12.
8 9 5 6COOH
11 12 14 15
c/s-AA
epoxidation
COOH
COOH
11,12-EET5,6-EET
COOHCOOH
8,9-EET14,15-EET
Figure 1-12. Epoxidation of arachidonic acid
CYPs oxidise AA at the double bond regions by epoxidases and generates 5,6-EET, 8,9-EET, 11,12-EET, and 14,15-EET.
40
1.8.1.3 Bisallylic hydroxylation of AA
In addition to four double bonds AA has three bisallylic carbons in positions 7, 10 and
13. These bisallylic carbons can be hydroxylated by CYPs (Oliw et a l, 1996). The
bisallylic hydroxyl metabolites are chemically unstable at acidic pH, which is
commonly used for the extractive isolation of fatty acid metabolites. At acidic pH the
bisallylic HETEs will undergo a non-enzymatic rearrangement to cis-trans conjugated
HETEs ( Brash et al, 1995; Oliw et al, 1993). The products of bisallylic
hydroxylation of AA by CYP are shown in Figure 1-13.
Figure 1-13. Bisallylic hydroxylation of arachidonic acidAA has three bisallylic carbons in position 7, 10 and 13 and CYP generates 7-HETE, 10- HETE and 13-HETE by bisallylic hydroxylation from AA.
The formation of bisallylic HETEs and 11-hydroxyoctadecadienoic acid (HODE) (the
corresponding bisallylic product of linoleic acid) has been studied in liver microsomes
of rats treated with different kinds of CYP inducers (Oliw et a l, 1996). Liver
microsomes prepared from rats treated with phénobarbital converted AA to 7-HETE,
10-HETE and 13-HETE, and linoleic acid to 11-HODE (Brash et a l, 1995). Steric
analysis revealed that these metabolites were almost racemic. Dexamethasone, which
is an inducer of the CYP3A isoforms, efficiently and selectively increased the
bisallylic hydroxylation activity in rat liver microsomes (Homsten et a l, 1996). Rats
treated with inducers of other CYP isoforms, e.g. CYP1A, also increased the bisallylic
COOH10* '
13’c/s-AA
*bisallylic carbons
bisallylic hydroxylation
OH ▼•COOHCOOH
OHOH
7-HETE 10-HETE 13-HETE
41
hydroxylation activity. This indicates that the formation of bisallylic hydroxy
metabolites can be catalyzed by rat CYP3A isoforms, but that other CYP isoforms
may also possess this activity (Homsten et al, 1996).
The formation of 13-HETE and 11-HODE has been detected in human liver
microsomes (Oliw et a l, 1996). The human CYP isoforms that are involved in the
formation of bisallylic hydroxy fatty acids, have not been studied (Capdevila et al,
2000). However, it has been reported that human CYP1A2, CYP2C8 and CYP2C9
formed large amounts of cis-trans conjugated HETEs following acidic isolation
(Rifkind et al, 1995).
1.8.1.4 Hydroxylation with double bond migration of AA
Hydroxylation with double bond migration leads to the enzymatic biosynthesis of cis-
trans conjugated hydroxy fatty acids. Arachidonic acid can be converted by CYP to
six cis-trans conjugated HETEs (5-, 8 -, 9-, 11-, 12-, and 15-HETE), which are
structurally similar to the HETEs generated by LOX, but their biosynthesis does not
involve formation of a hydroperoxide (HPETE) (Capdevila et al, 2000). Cis-trans
conjugated HETEs can also be formed non-enzymatically by acid-catalyzed
rearrangement of bisallylic HETEs (Brash et a l, 1995), by the LOX pathway (Brash,
1999) or by PGH synthase (Thuresson et a l, 2000). The human CYP isoforms that
catalyze the formation of these metabolites have not been well characterized. Previous
studies on the formation of HETEs by CYP, which have used acidic extractions, may
have partly overlooked the stereo selectivity and the quantitative importance of the
cis-trans conjugated HETEs. Cis-trans conjugated hydroxy fatty acids have been
implicated as signal molecules involved in the regulation of gene expression
(Capdevila et a l, 2002). The products of hydroxylation with double bond migration
of AA by CYP are shown in Figure 1-14.
42
c/s-AA
Hydroxylation with double bond migration
.COO"COOHOHCOOH
OHHO 11-HETE15-HETE
5-HETE
OHCOOHCOOH
OH12-HETE8-HETE
Figure 1-14. Hydroxylation with double bond migration of AACis-trans conjugated hydroxy fatty acids are synthesized from AA by hydroxylation with double bond migration. Arachidonic acid can be converted by CYP to six cis-trans conjugated HETEs (5-, 8-, 9-, 11-, 12-, and 15-HÈTE).
CYP enzymes of human liver microsomes form 12-HETE with stereo specificity
(>90% 12R-HETE) (Oliw et a l, 1996). Studies have demonstrated that 12R-HETE is
an agonist for the LTB4-receptor and might exert inhibitory effects on Na+/K+-ATPase
activity (Masferrer et al., 1990; Tanaka et al, 1991).
1.8.2 Relation of cytochrome P450 with COX and LOX pathway
The biological significance of the AA metabolites formed by the CYP pathway are
not fully understood (Capdevila et al, 2000). Many studies have suggested that the
metabolites have a wide range of physiological implications. They appear to act
primarily within the cells of origin and do not need to be extruded into the
extracellular space to stimulate membrane receptors. Within the cells, they have been
proposed to be involved in the regulation of ion channels and transporters, and are
known to act as mitogens. In whole animal physiology, they have been implicated in
the mediation of peptide hormone release, regulation of vascular tone and volume
homeostasis. Several studies have indicated that some of the biological effects
43
mediated by CYP metabolites of AA are PGH synthases dependent. Some of these
effects might be due to modulation of the biosynthesis of prostanoids (Oliw, 1994).
CYP metabolites of AA can be substrates for PGH synthases. 20-HETE
(Schwartzman et al., 1989) and 5,6-EET can be converted by PGH synthase to 20-
hydroxy-PGHz (Carroll et al, 1993) and 5,6-epoxy-PGHi (Oliw, 1984), respectively.
Both of these compounds can be further metabolized into bioactive prostanoids. 8,9-,
11,12-, and 14,15-EET lack the necessary double bonds to be converted to
prostanoids by PGH synthases, but they might undergo abortive PGH synthase
reactions or can inhibit the enzyme (McGiff & Quilley, 1999). It has been shown that
8,9-EET can be hydroxylated at C ll and C l5 by PGH synthase (Zhang et a l, 1992).
18-HETE and 19-HETE can be converted into prostanoids by PGH synthase, but they
are considered to be poor substrates (Capdevila et a l , 2000).
The enzymes catalyzing the formation of TXA2 and PGH are two CYP enzymes.
CYP5A (TXA2 synthase) of platelets and CYP8A (PGI2 synthase) of the vascular
endothelium rearrange PGH2 into TXA2 and PGI2, respectively. These two CYP
enzymes catalyze isomerization reactions and do not require NADPH (Hecker &
Ullrich, 1989). CYP5A also converts PGH2 to 12-HHT and malondialdehyde.
Recently, it was shown that human drug metabolizing CYP and liver microsomes
could also catalyze the formation of 12-HHT and malondialdehyde from PGH2
(Plastaras et al, 2000).
1.9 Polymorphonuclear leukocytes and arachidonic acid
Stimulation of human PMNs by soluble agonists and phagocytic stimuli increases the
levels of intracellular-free calcium and causes cytosolic phospholipase A2 (cPLA2)-
mediated release of AA. Although AA is considered an important PMN activator, the
molecular mechanisms through which it regulates PMN functions are still poorly
understood and contentious. The major AA metabolites synthesized by activated
PMN are the products of the stereospecific biotransformation of AA by the 5-LO. 5-
HpETE, the first metabolite of the 5-LO pathway, can be reduced to 5-HETE and/or
metabolized further by the 5-LO to leukotriene A4 (LTA4) (Borgeat & Naccache,
44
1990). LTA4 is then rapidly converted to LTB4 by the LTA4 hydrolase. LTB4 is a
biologically active compound that acts through interaction with the specific cell-
surface receptors (Yokomizo et al., 2001).
1.9.1 Role of AA on apoptosis
Apoptosis is a tightly regulated form of cell death, also called the programmed cell
death by which cells undergo death to control cell proliferation or in response to DNA
damage. Morphologically, it is characterized by chromatin condensation and cell
shrinkage in the early stage and then the nucleus and cytoplasm fragment, forming
membrane-bound apoptotic bodies that can be engulfed by phagocytes. Apoptotic
messages may be from outside the cell (called extrinsic inducers) like NO2 or from
inside the cell (intrinsic inducers) are a response to stress, such as nutrient deprivation
or DNA damage (Chiarugi & Moskowitz, 2002). Both extrinsic and intrinsic
pathways have in common the activation of central effectors of apoptosis, a group of
cysteine proteases called caspases, which carry out the cleaving of both structural and
functional elements of the cell (Nicholson, 2000).
Schonberg et al. observed that n-3 fatty acids may inhibit the growth of tumour cells
both in vivo and in vitro (Schonberg et ah, 1997). AA blocked proliferation of the
promyelocytic leukemic HL-60 cells and this blockage which is a predecessor of both
apoptosis and necrosis was unrelated to oxygenase metabolism or lipid peroxidation
(Finstad et al., 1994). AA stimulates neutral sphingomyelinase activity (Jayadev et
al., 1994), which catalyzes the hydrolysis of sphingomyelin to generate ceramide.
Ceramide acts as second messenger that activates the cellular apoptotic machinery
(Jayadev et a l, 1997). Mechanisms of the anti-proliferative or pro-apoptotic actions
of AA remain to be clarified and may vary among different cell types (Cao et al,
2000). AA metabolism hold the potential basis for several new therapeutic strategies
for treating neoplasia (Zarebska et a l , 2005).
45
1.10 Aims of the study
Platelets metabolize AA and causes formation of TXA2, which causes platelet
aggregation. However, it has been found that the \A-trans isomer of AA is not an
agonist for rat platelet aggregation. Indeed, the proaggregant effects of AA are
inhibited by \A-trans-AA through unknown mechanism(s) and that the trans-AA was
metabolized into products that they have not identified (Berdeaux et ai., 1996). Jiang
et al, (1999) also demonstrated that 14-trans-AA is one of the main products of
oxygen free radical mediated oxidation of AA in platelet membrane phospholipids.
The aim of this study was to examine the metabolism and effects of \4-trans-AAs in
human platelets where AA is mainly metabolized by COX. The metabolic study of
trans-AA by hepatic microsomes and PMNs were also observed as AA is metabolized
in these cells by CYP and LOX respectively. The study was designed to identify and
characterize any new metabolites formed from trans-AA using chromatography,
radioactivity detector and mass spectrometry and for functional studies aggregometry
was used. Studies using platelets, hepatic microsomes and PMNs were used as a
model for trans-AA metabolism in humans. These will assist in establishing if these
pathways exist in other tissues and in vivo.
The specific aims of this study are as follows:
1. To profile the metabolism of lA-trans-AA in platelets with special emphasis is
on the metabolism of \A-trans-AA by COX and the generation of PG/TX
analogues.
2. To study the effect of inhibitors (indomethacin, baicalein) on \A-trans-AA metabolism in platelets.
3. To study the effect of lA-trans-PA on AA metabolism and to compare it with
platelet AA metabolism.
4. To compare AA estérification with \A-trans-PA. into platelet membrane phospholipids.
46
5. To study the effects of \A-trans-AA. on platelets and the inhibitory effects of
\A-trans-AA. on AA-induced aggregation.
6 . To profile the metabolism of trans-AA by hepatic microsomes and compare
with metabolism of AA.
7. To evaluate the role of trans-AA metabolism in PMNs in relation to the effect
of the trans-AA isomers on the proliferation and viability of HL-60 cells.
47
2 M ETHODS
2.1 General and analytical methods
2.1.1 Synthesis and purification of trans-AAs
Trans-AAs were synthesized by passing NO2 over AA (Jiang et al, 1999).
Arachidonic acid (100 p,g) was dissolved in 1 ml of hexane, and 100 pg sodium
arachidonate was dissolved in 1 ml of 0.5 mM phosphate buffer (pH 7.4). Deuterium
labeled AA (10 pg) was mixed with [1-I4C] AA (10 pCi) and was used to prepare the
internal standards for quantitative analyses.
„ 1600
I“ 1200cino
cm 800
AA14-fra/7S-AA+ other trans-AAs8
-2Nitro-AAs
400
30 40 502010Time [min]
Figure 2-1. Chromatogram of the products of NO2 reaction with AA
100 pg AA in 1 ml of hexane was reacted with 43 pM NO2 and products were extracted with ethyl acetate, dissolved in methanol and separated using a HPLC. Chromatogram shows that the NO2 radical mediated cis-trans isomerization of AA along with the formation of nitro- AAs.
Nitrogen dioxide was prepared shortly before the reaction with AA as previously
described (Jiang & Balazy, 1998). In short, approximately 1 ml of liquid NO2 was
collected from a tank via narrow teflon tubing inserted into a tightly capped, helium-
filled glass vial (5 ml) through a teflon-lined silicon rubber seal. NO2 was delivered
into arachidonate solution via bubbling and helium was used as a carrier gas at a rate
of 0.1 ml/min. The final concentration of NO2 was 43-430 pM. The reaction was
48
carried out for 5 min, and the lipids were isolated by extraction with ethyl acetate. The
extracts were dried under nitrogen, dissolved in a small volume of methanol and were
analyzed and purified by reverse phase HPLC (Krishna et a l, 2001). A chromatogram
shows the products of the NO2 reaction with AA in Figure 2-1.
2.1.2 HPLC analyses
In chromatography, the sample is dissolved in a mobile phase, and the mobile phase is
forced through an immobile, immiscible stationary phase. The phases are chosen in a
way that the components of the sample have different solubilities in each phase. A
component, which is quite soluble in the stationary phase, will take longer to travel
through it, than a component that is not very soluble in the stationary phase but very
soluble in the mobile phase. Because of these differences in mobilities, sample
components become separated from each other as they travel through the stationary
phase along with mobile phase (Parvez et al, 1985). The schematic of HPLC is
shown in Figure 2-2.
Reverse phase (RP) HPLC was used to separate various metabolites. In RP HPLC,
compounds stick to the RP HPLC column containing C l8 octadecyl silica in high
aqueous mobile phase and are eluted from RP HPLC column with a highly organic
mobile phase. In RP HPLC, compounds are separated based on their hydrophobic
character. Lipids were separated by running a linear gradient of the organic solvent.
HPLC analyses were performed on a HPLC system (HP 1050, Hewlett-Packard, Palo
Alto, CA) equipped with a quaternary pump, a variable-wavelength UV detector and
using a C l8 octadecyl silica (ODS) column (250 X 4.6 mm, Beckman Instruments,
CA). Analyses were performed by eluting the sample with a linear gradient of
acetonitrile in water (pH 4, adjusted with acetic acid). The initial concentration of
acetonitrile was 25% and it gradually increased to 100% (v/v) within 60 minute at a
flow rate of 1 ml/min by a gradient program. The effluent was analyzed using an in
line UV variable wave length detector (Jiang et al, 1999).
49
Separated materials elute from the column
Packed Column
Mobilephase
Collect I WasteInjection of sample mixture containing materials A, B & C
InjectorSolventDeliverySystem
Detector
RecorderTime
Chart record
Figure 2-2. Block diagram showing the components of a HPLC systemA HPLC system consists of solvent delivery system (pumps), injector, packed column and detector. After injection, the sample is dissolved in a mobile phase, and the mobile phase is forced through an immobile stationary phase packed in the column. The detector detects the samples and the data is sent to a computer.
An on-line radio-chromatography detector (Radoimatic Flo-One\Beta Series A-100)
was used to detect radiolabeled products (Radiomatic Instruments, Tampa, FL) using
Sigma-fluor scintillation fluid (Sigma Chemical Co.). Fractions were collected using a
Gilson FC 203B (Gilson Inc., WI) fraction collector and used for gas chromatographic
analysis (Boulos et a l , 2000).
2.1.3 UV Spectroscopy
Some purified sample fractions by HPLC system (HP 1050, Hewlett-Packard, Palo
Alto, CA) described in section 2.2 were injected into a HPLC (HP 1100, Hewlett-
Packard, Palo Alto, CA) equipped with a C l8(2) octadecyl silica (ODS) column (150
X 2.00 mm, Phenomenex, Torrance, CA) and the spectra were recorded using a diode
array detector (Hewlett-Packard G1315A). The spectra were taken from 200 to 400
50
nm at a scan rate of 250 nm/s. Specific absorbance at 234 nm was for 12- HETE and
the absorbance maxima (Imax) for the unknown metabolites were measured.
2.1.4 Mass spectrometry
For mass spectrometric analysis of the samples, GC/MS and LC/MS was used.
Samples were derivatized before injection into GC/MS in order to make more
volatile.
2.1.4.1 Preparation of derivatives for GC/MC
For GC/MS analyses, pentafluorobenzyl (PFB) esters were prepared. In short,
samples were dried completely under nitrogen, and dissolved in acetonitrile (1 0 0 pi).
Ten percent PFB (30 pi) and diisopropylethylamine (DIPEA) (30 pi) were added to a
sample and the derivatization was continued at room temperature for 30 min. The
samples were dried completely under nitrogen, dissolved in 50 pi of isooctane, and
analyzed by gas chromatograph (Agilent network GC system 6890N)/mass
spectrometer (Agilent mass selective detector 5973N).
Figure 2-3. Flow diagram showing GC/MS derivative preparation
All samples were derivatized with pentafluorobenzyl, but the samples containing hydroxyl group were further derivatized with trimethylsilyl and injected into a GC/MS.
TMS derivatization was done for samples having a hydroxyl group. Trimethylsilyl
(TMS) (80 pi) and pyridine (20 pi) were added to the completely dried PFB ester
samples to convert the hydroxyl groups into the TMS derivative. After 30 min at
room temperature the reagents were evaporated completely under nitrogen and the
GC/MS
Samples — ► PFB esters
TMS derivative — ► GC/MS
51
TMS esters were dissolved in 50 |il of isooctane and subjected to analysis by GC/MS
(Balazy, 1991a). The schematic of sample derivatization is shown in Figure 2-3.
2.1.4.2 Gas chromatography/mass spectrometry
Gas chromatography was used for the separation and identification of volatile organic
compounds. A gas chromatograph consists of a flowing mobile phase, an injection
port, a separation column containing the stationary phase, and a detector. The organic
compounds were separated due to differences in their partitioning behaviours between
the mobile gas phase and the solid stationary phase in the column. The schematic of
GC/MS is shown in Figure 2-4.
InjectorportFlow
controller
Recorder
DetectorColumn
Column oven
Carrier gas
Figure 2-4. Diagram showing the components of a GC/MS systemA gas chromatograph consists of a flowing mobile phase, an injection port, a separation column containing the stationary phase, and a detector. The samples are separated by the column and the detector sends the data to a computer.
Inert gas helium was used as a mobile phase. The injection port consisted of a rubber
septum through which a syringe needle was inserted to inject the sample. The
injection port was maintained at a higher temperature than the boiling point of the
least volatile component in the sample mixture. Since the partitioning behaviour
depends on temperature, the separation column was contained in a thermostat-
controlled oven. Separating components with a wide range of boiling points was
52
accomplished by starting at a low oven temperature and increasing the temperature
gradually with time to elute the high-boiling point components.
Mass spectrometry was carried out on a quadrupole gas chromatograph 6890N
network GC system (Agilent, Wilmington, DE) directly interfaced with a mass
spectrometer 5973 mass selective detector (Agilent, Wilmington, DE). TMS
derivatives of metabolites and standards were analyzed by GC/MS in an electron
ionization mode (70 eV) or in a positive ion chemical ionization (Cl) mode (120 eV),
whereas PFB derivatives were analyzed by GC/MS using a negative ion chemical
ionization (NCI) mode. In chemical ionization experiments, methane (40 psi) was
used as a reagent gas at a flow rate 40 1/min resulting in a pressure of 0.9 torr in the
ion source.
The source and transfer line temperatures were 250 °C and 280 °C, respectively. The
samples were dissolved in iso-octane and analyzed on a DB-1 fused silica gas
chromatographic column (50 m, 0.25-mm internal diameter, 0.25-mm film thickness),
(J/V Scientific, Folsom, CA). The initial temperature of the column (150 °C) was
maintained for 1 min and then increased to 280 °C at a rate of 25 °C/min. The injector
was used in a splitless mode at a temperature of 300 °C. Helium was used as a carrier
gas at the pressure of 29.57 psi, delivering a total flow of 30.1 ml/min and resulting in
a column flow of 1.3 ml/min. The relative retention time for each derivatized
metabolite or standard was expressed as a carbon number equivalent. The measured
retention times, under the above conditions of individual standards derivatized as PFB
esters, were correlated with the number of carbons in a fatty acyl chain. The linear
function (r = 0 .9 9 9 ) was used to calculate the carbon number equivalent for each
metabolite derivative (Zghibeh et al.9 2004; Tsikas, 2001).
2.1.4.3 Liquid chromatography/mass spectrometry
Electrospray (ES) was used as it is a well-established technique allowing mass
spectrometric (MS) analysis of various compounds without derivatization and on-line
53
analysis in conjunction with liquid chromatography (LC). Schematic shows different
components of mass spectrometer in Figure 2-5.
IonSource
DataSystem
MassAnalyzer
IonDetector
Vacuum System
Electronics
Inlet (HPLC/
Syringe Pump) Mass Spectrum
Figure 2-5. Diagram showing the components of a LC/MSA liquid chromatograph / mass spectrometer consist of an online HPLC system, ion source, mass analyzer and ion detector. After detection, data is sent to a computer and analyzed by a software system.
Electrospray is one of the interface in LC/MS that imparts very little internal energy
into the analyte for ion generation process (Homing et al, 1974). Ions in solution
from the eluent of the LC migrate from the bulk to the surface of the liquid under the
influence of the high electric field strength near the tip of the needle (Figure 2-6). The
characteristic "cone jet" geometry is the result of the liquid accelerating toward the
collection electrode. When the electrostatic forces exceed the inward forces from the
surface tension, a cone jet is created. The highly charged surface of the liquid jet
immediately disperses into droplets that accelerate toward the collector electrode
(Iribame & Thomson, 1975).
54
The charged droplets evaporate in the region between the needle and the collector.
The excess charge on the droplet concentrates on the surface as the droplet
evaporates. This increases the field around the droplet to the point where the surface
deforms into tear-shaped emitters of smaller particles (Ito et a l, 1986). With
continued evaporation, this tear-shaped geometry is stable until the charge is depleted,
the analyte crystallizes into solid particles, or the droplets completely evaporate. The
significance of this process is that it occurs at relatively low temperatures (Gamero-
Castano & Mora, 2000). Electro spray ionization in shown in Figure 2-6.
Curtain Gas
High Voltage
1
1. Formation of 3. Coulomb- charged droplets explosion
VACUUM A _______
LC* r tNebulizer gas Ion Spray
— Orifice plate
Mass Analyzer
0 )e C D d D
2. Evaporation
Curtain Plate —
Œ
Orifice
J
Curtain Gas
Figure 2-6. Schematic diagram showing electro-spray ionization (ESI)Schematic diagram of electrospray shows the motion of the charged droplets completing the mass analyzer. The high voltage applied to the needle electrode (in this case) drives the deformation of the liquid into a conejet-geometry. All variations of electrospray including ion spray, and turbo spray conform to this basic circuit.
Samples were injected in the HPLC system (HP 1100, Hewlett-Packard, Palo Alto,
CA) equipped with a binary pump, a diode array UV detector using C l8 Ultrasphere
ODS column (150 x 2.00 mm, 5 \i, Phenomenex prodigy, Phenomenex, Torrance,
CA). Samples were eluted at a flow rate of 0.3 ml/minute with a linear gradient of
acetonitrile in water (25% to 100% in 25 minute) and the effluent was analyzed by an
online MS. Electrospray ionization MS was performed in negative ion mode on an
API 2000 triple quadrupole LC/MS/MS mass spectrometer (Applied
55
Biosystems/MDS Sciex, Forest City, CA) and data was analyzed by software package
Analyst 1.4 (Applied Biosystems, Forest City, CA) to identify the structure of the
unknown metabolites. The curtain gas was 35 psi, ion source voltage was -4200 volts,
temperature of the source was 325° C, and flows of gas 1 and 2 were 35 and 50
L/minute, respectively. The collision assisted dissociation (CAD) gas flow was 6
L/min, declaustering potential (DP), focusing potential (FP), entrance potential (EP)
and collision energy (CE) was -20, -350, -10 and -26 (arbitrary units), respectively.
Samples were analyzed in Q1 MS, product ion (MS/MS) and multiple reaction
monitoring (MRM) mode.
2.1.5 Trans-AA metabolism by LOX
A 12-LOX (200 pi) solution in 20 mM in phosphate buffer was preincubated at 37°C
for 15 min and mixed with 14-trans-AA (100 pM). Control experiments were done
with AA (100 pM). The incubations were carried for 10, 20 and 30 min at 37 °C and
were terminated by the addition of methanol (1 ml). The solution was thoroughly
mixed using a vortex and then centrifuged at 4000 rpm for 10 min. The supernatant
was decanted and evaporated under nitrogen. Fatty acids were extracted using ethyl
acetate (0.5 ml) and acidified water (0.5 ml) at pH 4 (containing 10% acetic acid). The
solvent of extracts were evaporated under a stream of nitrogen. The lipid residue was
dissolved in 100 pi of acetonitrile/water (1:1) and subjected to analysis by LC/MS as
described in section 2.1.4.3.
2.2 Methods for studies with platelets
2.2.1 Platelet preparation
Fresh platelet concentrate (within 24 hours of collection) from human donors, who
has not ingested any drugs known to interfere with platelet function for at least 15
days, was purchased from Hudson Valley Blood Service (Elmsford, NY). Washed
platelets were prepared using a method described previously (Gerrard, 1982). Briefly,
10 ml of fresh platelet concentrate was centrifuged at 1100 g for 15 minutes at room
56
temperature. After discarding the supernatant, platelets were resuspended in 5 ml
buffer A containing 140 mM NaCl, 2 mM KC1, 14 mM NaHCOg, 5.5 mM glucose, 1
mM MgClz, 5 mM Hepes buffer, 0.2% BSA, 1.5 pg/ml apyrase, 0.2 |_iM PGE% and the
pH was adjusted to 6.4. Platelets in buffer A were centrifuged again at 1100 g for 15
minutes at 21°C and resuspended in buffer B (same as buffer A, but without BSA,
PGEi and apyrase, pH, 7.4). Platelets were counted using a Coulter Counter PS 540
(Coulter Electronics, Hialeah, FL) and the density of platelets was adjusted to 1x108
cells/ml.
2.2.2 Methods for observation of metabolism of trans-fldk by platelets
Metabolites were produced by incubating platelets with different amount of fatty
acids for 30 minutes. To 1 ml of platelet suspension in buffer B, AA (100 pM) or 14-
trans-AA (100 pM) or both were added and incubated while shaking at 1200 rpm in a
37° C water bath for 30 minutes. In some experiments, platelets were preincubated
with COX or LOX inhibitor for 5 minutes. To this mixture, 5 ml of methanol was
added, vortexed, and centrifuged for 5 minutes at 1100 g. The supernatant containing
fatty acids was transferred and dried completely under vacuum. Ethyl acetate (1 ml)
and water (1 ml) were added to the tube, the pH adjusted to 4.0 by adding 10% acetic
acid, and the tube was vortexed, and centrifuged again as before. The top phase was
transferred to another tube and evaporated to dryness under nitrogen. Methanol (100
pi) was added, mixed and 20 pi of the sample was injected into a HPLC system as
described in section 2 .1 .2 .
2.2.3 Methods for observation of effects of trans-AA on platelet function
The aggregation study was performed according to the turbidimetric method of Bom
(Bom, 1962; Bom & Cross, 1963). Figure 2-7 shows the function of an aggregometer.
57
LightTransmission
A
Figure 2-7. Diagram showing the function of an aggregometerThe platelet aggregometer measures platelet function by measuring light transmission through a suspension of stirred platelets. As shown in the figure above, the initial suspension is opaque and the resting discoid platelets give baseline oscillations. After the addition of an aggregating agent as shown of point A in the figure, platelets form larger clumps and the solution clears until maximum aggregation is achieved forming the aggregation curve.
Platelets were screened before the aggregation study for the ability to produce TXA2,
which eliminated donors on NSAID medication or having a genetic defect of AA
oxidase enzymes (Pareti et al, 1980). In a siliconized glass tube, 400 [iL platelet
suspension was placed and warmed to 37°C for three minutes in a dual-channel
aggregometer (Payton, NY). A small magnetic stirring bar was placed into the tube
after 30 seconds and the suspension was stirred at 900 rpm. After 2 minutes, platelet
activation was started by adding different amounts of AA, 14-trans-AA, U46619,
ADP, epinephrine or cPAF. Aggregation was also observed with AA or other
compounds in presence 14-trans-AA. The aggregation was monitored for 6 minutes
with a strip chart recorder. The measurement of platelet aggregation was the output
from turbidimetric aggregometer on a strip chart recorder and expressed in percentage
terms as described earlier (Bom & Hume, 1967). This was achieved by calibrating the
aggregometer in such a way that the optical density of the unstimulated platelet
58
suspension represented 0% aggregation, while the optical density of the buffer B
without any platelets represented 1 0 0 % aggregation.
2.2.3.1 Thromboxane B2 assay
The levels of TXB2, the stable metabolite of TXA2, in the supernatants were measured
using LC/MS (as described in section 2.1.4.3) against a standard curve (Figure 2-8)
made by injecting different known amount of commercial TXB2 standard. This
approach provided quantitative measurement of TXB2 in platelets.
9.5e5
7.5e5
3.5e5
1.5e5
40 50302010
Concentration [ng/ml]50 ng/ml
100
25 ng/ml
8CCTJTJC3JDro§
12.5 ng/ml
6.3 ng/ml
3.1 ng/ml
1.6 ng/mls
0.0
Time [min]
Figure 2-8. TXB2 Standard curve
The relative abundance of TXB2 in calibration standards was determined by tandem mass spectrometry (MRM mode). The bottom panel shows the relative abundance of fragment ion m/z 169 that originated from the deprotonated TXB2 at m/z 369. Each peak results from a discrete injection of 5 pi by the autosampler. The interval between each injection was 1.5 min. The average integrated peak area of duplicate 5 pi injections of the calibration standards was plotted versus concentration to generate the linear calibration curve. The correlation coefficient is 0.992 (top).
59
2.2.3.2 Membrane incorporation study
Washed human platelet suspension (1 ml) was preincubated at 37°C for 5 min and
0.25 pCi of [1-14C]-AA (specific activity of 50 mCi/mmol, radiochemical purity >
99%) or [l-14C]-14-rra7zs-AA (specific activity of 50 mCi/mmol, radiochemical purity
> 99%), was added. The incubations were carried for 3 minutes at 37 °C with constant
stirring at 1200 rpm. Then the vials were centrifuged at 2000 g for 5 min. The
supernatant was decanted, and in the precipitate 1 ml buffer B was added and an
aliquot (10 pi) was counted by a liquid scintillation counter. For 0-minute
measurement, 1 0 pi of platelet suspension was counted by a liquid scintillation
counter just after addition of radiolabeled AA or \A-trans-AA.
2.3 Methods for studies with hepatic microsomes
2.3.1 Generation, isolation, purification and analysis of microsomal 14-frans-AA metabolites
Rat liver microsomes (cellzDirect, Tuscon, AZ) were suspended in phosphate buffer
(1 mg/mL), added to trans-AA isomers (final concentration 15 pM), and incubated
for 30-180 min at 37 °C. The metabolism was stimulated by the addition of NADPH
(1 mM) and NADPH regenerating system (CellzDirect, Tuscon, AZ). In experiments
involving 14-tmns-AA, its radiolabeled analog, [l-u C]-l4-trans-AA (specific activity
of 50 mCi/mmol, radiochemical purity > 99%), was used (0.25 pCi of [1-14C]14-
irans-AA per incubation). Some experiments involved co-incubation of [1-14C]-14-
trans-AA with equimolar amounts of [1-14C] AA. Standards of trans-EET, and
deuterium-labeled EET were prepared by reacting w-chloroperoxybenzoic acid
(MCPBA) with trans-AA and d8 - AA, respectively as previously described (Balazy &
Nies, 1989). Standards of threo- and e^/zro-diHETEs were prepared by a mild acidic
hydrolysis of cis- and fraws-EETs, respectively or by incubation with microsomes
respectively as previously described (Balazy & Nies, 1989). The epoxides obtained
from the reaction of \4-trans-AA with MCPBA were fractionated and incubated with
microsomes in phosphate buffer (1 mg/mL) for 30 min at 37 °C. This procedure
yielded 14,15-eotf/zro-DiHETE. The microsomal incubations were terminated by the
60
addition of 5 volumes of cold methanol, vortexed, and centrifuged for 5 min at 1100 g
to precipitate microsomal debris and proteins. The supernatant, which contained
>90% of radioactivity was transferred to a glass tube and the solvent was evaporated
under vacuum. The residue was dissolved in 1 mL of ethyl acetate and mixed with 1
mL of acidified water (pH was adjusted to 4.0 with 10% acetic acid). The organic
phase was separated by centrifugation, transferred to a glass tube and evaporated to
dryness. The residue was dissolved in methanol and an aliquot (20 pi) was analyzed
by HPLC as described in section 2.1.2. An on-line radio-chromatography detector
(Radiomatic Flo-One/Beta Series A-100) detected radioactivity with a Sigma-Fluor
scintillation fluid. Gas chromatography was performed on a HP-5MS capillary
column (30 m, 0.25 mm i.d., 0.25 pm film thickness) using an Agilent 5973 GC-MS
instrument as described in section 2.1.4.
2.3.2 Isomerization of HETEs
19-HETE (1 pg) was dissolved in hexane (100 pi) and mixed with 100 pi of a 1%
solution of NO2 in hexane. The reaction was carried out for 1-10 minutes. The solvent
was evaporated under nitrogen, and the lipids were dissolved in acetonitrile and
subjected to HPLC purification on a Phenomenex Luna CIS column (150 x 2 mm,
particle size 5 pm). The fraction at 6 -8 min was collected and analyzed by LC/MS and
GC/MS. The isomerization produced four isomers of 19-HETE after treatment with
NO2 for 5 min. Other HETE molecules (20-HETE, 18-HETE, 16-HETE) were also
isomerized by NO2 and the trans isomers were isolated by HPLC and characterized
by GC/MS as described in section 2.1.4.
2.3.3 Microsomal metabolism of S-frans-AA
A suspension of rat liver microsomes in phosphate buffer (200 pi, 1 mg/ml) was
preincubated at 37°C for 15 min and mixed with 5-trans-AA (final concentration 73-
736 pM). Control experiments were done with AA (73-736 pM). A NADPH-
regeneration system solution was mixed with the tetra-sodium salt of reduced P-NAD
phosphate (0.3 mg) and added to the microsomal mixture containing fatty acid
substrate to stimulate the CYP-dependent metabolism. The final concentrations for
61
the components of the NADPH-regeneration system were as follows: 1.3 mM
NADP+, 3.3 mM glucose-6 -phosphate, 0.4 U/ml glucose-6 -phosphate dehydrogenase,
and 3.3 mM MgCla. The incubations were carried out for 10 and 20 min at 37 °C and
were terminated by adding ethyl acetate (0.5 ml) and water (0.5 ml). The solution was
thoroughly mixed using a vortex and then centrifuged at 4000 rpm for 10 min. The
supernatant was decanted and the remaining solution was re-extracted with ethyl
acetate two more times, and the extracts were combined. The solvent was evaporated
under a stream of nitrogen. The lipid residue was dissolved in 100 pi of
acetonitrile/water (1:1) and subjected to analysis by LC/MS (section 2.1.4.3). Acid
was not used in the extraction to minimize hydrolysis of the epoxides to diols. Control
experiments were done without microsomes and/or NDPH-generating system and did
not show any metabolite formation. In some experiments, the microsomes were
preincubated with anti-CYP450 2E1 IgG protein (5 and 10 pg) for 20 min at 37 °C.
The effect of microsomal epoxide hydrolase was tested by pretreatment of
microsomes with a solution of dicyclohexyl urea (1 pi in DMSO, final concentration
10 mM) (Goldfarb et al., 1993).
2.3.4 Hydrolysis of c/s- and frans-5,6-EET by microsomal epoxide hydrolase
5,6-trans-EEY, 5,6-cw-EET and their mixture (100 ng each) were incubated with rat
microsomes (100 pg in 100 pi) for 10, 20, 60, 180 and 360 min. The incubations were
terminated by addition of 100 pi of methanol. The mixture was vortexed, centrifuged
and aliquots (20 pi) were analyzed by LC/MS as described in section 2.1.4.3.
2.3.5 Conjugation of c/s- and trans 5,6- EET with microsomal glutathione-S-transferase
Glutathione-S-transferase (GST) from rat liver (Sigma, 5 mg, 18 U/mg protein) was
suspended in 500 pi of distilled water. The GST solution contained 18 U/100 pi along
with 1 pmol of reduced glutathione (pH 6.5). A mixture of 5,6 EET isomers (100 ng
each, final concentration 3.12 pM) were incubated with 100 pi of GST solution for 1,
62
6 and 12 hrs at 37 °C with shaking. The incubations were terminated by centrifugation
and aliquots (20 gl) were analyzed by LC/MS (section 2.1.4.3).
2.4 Methods for studies with polymorphonuclear leukocytes
2.4.1 Preparation of neutrophils from human blood
Human PMNs were isolated as previously described (Gronert et al, 1999). Briefly,
blood from a healthy human donor (60 ml) was collected in Vacutainer tubes
containing acid-citrate-dextrose (ACD). Blood was centrifuged at 800 rpm (134 g)
and the pellet was gently resuspended in a Dulbecco's phosphate-buffered saline
(DPBS) solution (1 ml) containing 6 % dextrin. After sedimentation for 30 min, the
supernatant was transferred to test tubes containing 0.3 ml of Histopack-1077.
Samples were spun at 1500 rpm (514 g). The sedimented erythrocytes were lysed with
water and the PMNs were resuspended in DPBS to a final concentration of 2.5 x 106
cells.
2.4.2 Metabolism of 5-trans-AA by PMN or HL60 cells
5-trans-AA (10 nM -10 pM) was incubated with freshly isolated PMNs or HL-60
cells (5 x 105 cells) stimulated with calcium ionophore (A23187 1 pM) for 2 hours at
37° C. The incubations were terminated by adding methanol and centrifuging. Lipids
were then extracted from the sample by the addition of 5 volumes of cold methanol,
and the internal standard, dg-12-HETE (10 ng), was added before the supernatant was
transferred. The methanol extracts were evaporated, resuspended in acetonitrile and
the aliquots (10 pi) were analyzed quantitatively by LC/MS (section 2 .1.4.3.) via
monitoring of ions at m/z 319 and 335 to detect HETE isomers and LTB4,
respectively. The MS/MS spectra were acquired by using the collision energy o f-30
volts with nitrogen in the second quadrupole, which resulted in the decrease of the
precursor ion intensity by about 50%. The detection limit was 100 pg of HETE
injected on column.
63
2.5 Materials
Arachidonic acid (purity >99%), thrombin, indomethacin, NaCl, KC1, NaHCOs,
MgCb, glucose, hepes buffer, bovine serum albumin (BSA), apyrase, meta-
chloroperoxybenzoic acid, calcium, ionophor, dicyclohexyl urea, dimethyl sulfoxide
(DMSO), pentafluorobenzyl bromide (PFB), diisopropylethylamine (DIPEA),
pyridine and O-bis (trimethylsilyl) trifluoroacetamide were purchased from Sigma-
Aldrich Chemical Co. (St. Louis, MO). TXB2, PCs, HETEs, 12-HHT, EETs,
DiHETrEs, 12-LOX and U46619 were purchased from Cayman Chemical Co. Inc.
(Ann Arbor, MI). Baicalein and cPAF were from Biomol Research Laboratories
(Plymouth Meeting , PA). [1-14C] Arachidonic acid (54.2 mCi/mmol) was purchased
from Moravek Biochemical, fric (Brea, Ca). Acetonitrile, methanol, ethyl acetate,
acetic acid and water (Fisher Scientific, Inc. Suwance, GA) were all glass distilled and
used as received. ADP and epinephrine were from Chronolog, Inc., (Havertown, PA).
Nitrogen, helium, nitric oxide and nitrogen dioxide were obtained from TechAir
(White plains, NY). Rat liver microsomes CYP2E1 and NADPH-regeneration
solution were purchased from CellzDirect (Tuscon, AZ). The NADPH-regeneration
solution was composed of NADP+ and glucose-6 -phosphate (solution A, 100 |il) and
glucose-6 -phosphate dehydrogenase (solution B, 20 (il) diluted 20- and 100-fold,
respectively. Carbon 14-labeled lA-trans-AA was a kind gift from Dr. Oliver Loreau
from CEA Saclay, Service des Molécules Marquées, Gif-sur-Yvette, France.
2.6 Statistics
Experimental results are expressed as the means ± standard error of mean (SEM) and
are accompanied by the number of observations. Student’s t-tests were done to
determine the descriptive statistics of this study. One way or two way analysis of
variance (ANOVA) was done to determine the group and concentration differences. A
value of p<0.05 was considered statistically significant. The data analyses were
performed using the software package StatView (Version 5).
64
3 RESU LTS
3.1 Interaction of trans-AA with platelets
3.1.1 Metabolism of trans-AA by platelets
Consistent with previous reports (O'Keefe et al., 1990), arachidonic acid (15 pM) was
metabolized to TXB2, 12-HHT and 12-HETE by human platelets, which was
confirmed by injecting standards (Figure 3-1). The next series of experiments describe
the characterization of 14-trans-AA metabolism by human platelets. Initial
experiments were done with radiolabeled 14-trans-AA. The following sections
describe an in depth analysis of metabolism by LC/MS. Platelets were incubated with
[1-14C] radiolabeled AA/14-trans-AA for 30 minutes and the metabolites were
extracted and injected into the HPLC equipped with an on-line radio-chromatography
detector. However, 14-trans-AA did not produce metabolites like AA, but gave rise to
four unique metabolites. These novel metabolites were named metabolite A, B, C, and
D. It was observed that 84 ±7% AA was metabolized by human platelets compared to
the 42 ±5% of 14-trans-AA which was metabolized as shown in chart C of Figure 3-
1 .
65
12-HHT,COOH 12-HETE90
AAAA
TXB,
§
4010 20 30 40 50
08OyCOa:
80 j ,COOH
14-trans-AA
10 20 30 40 50
Time [min]
C100 -,
ea a a
m 14-trans-A A90 -
80 -
70 -
o-QCO 60 -
QE 50 -
co40 -o
‘o 30 -
20 -
10 -
Amount of substrates Total amountafter metabolism of metabolites
Figure 3-1. Metabolic products of radio-labeled AA and 14-trans-AA
Washed platelet suspension (1 ml) was incubated with A. 0.5 pCi (15 pM) [1-14C] AA; and B. 0.5 pCi (15 pM) [1-14C] 14-trans-AA for 30 minutes at 37° C with constant stirring at 1200 rpm. The reaction was stopped by adding methanol, and fatty acids were extracted using ethylacetate and injected into HPLC equipped with an on-line radio-chromatography detector to identify the metabolic products. Figure C showed that 14-frcms-AA was not extensively metabolized like AA. Total metabolites equal to the sum of peak areas for TXB2,12-HHT and 12-HETE from AA and metabolites A, B, C, D from \4-trans-AA. {n = 5, *P< 0.05, relative to data of AA metabolism)
66
3.1.1.1 Co-incubation of platelets with c/s- and 14-trans-AA
To observe the metabolic pattern of trans-AA in the presence of cis-A A, platelets
were incubated with an equal amount of radiolabeled cis-AA and 14-trans-AA.
Radio-chromatograms presented in Figure 3-2 show increased formation of
metabolites A, B, C, and D in the presence of cis-AA. Further experiments were done
for quantification of increased production of unidentified metabolites due to co
oxidation.
90
oo
o. 40SLO
orooT3
5
14-frans-AA
AmaA.
10
14-trans-AA
B j v A* 4 &W* L20 30 40 50
14-frans-AA12-HHT9012-HETE
AA
TXB.
40
30 40 502010Time [min]
Figure 3-2. Metabolism of radio-labeled 14-trans-AA and in the presence of AA
Washed platelet (1 ml) was incubated with A. 0.5 pCi (15 pM) [1-14C] 14-trans-AA; and B. 0.5 pCi (15 pM) [1-14C] AA and 0.5 pCi (15 pM) [1-14C] 14-£r<m5,-AA AA for 30 minutes at 37° C with constant stirring at 1200 rpm. Fatty acids were extracted and injected into the HPLC equipped with an on-line radio-chromatography detector to compare the metabolic products that formed from 14-trans-AA alone and those that form in the presence of cis-AA.
67
From the previous experiments shown in Figure 3-2, it was unclear if the increased
production of metabolites A, B, C and D was due to utilisation of cis or trans-AA.
Thus, non-radiolabeled AA was mixed with an equal amount of radiolabeled 14-trans-
AA and incubated with platelets. The result was compared with the metabolites from
radiolabeled 14-trans-AA alone by platelets (Figure 3-3). The formation of
metabolites A, B, C, and D was increased in the presence of non-radiolabeled AA,
suggests that \4-trans-AA metabolism increases in the presence of AA.
Metabolite DMetabolite CMetabolite BMetabolite A
Figure 3-3. Potentiation of biosynthesis of metabolites A, B, C, and D by AAPlatelet suspension (1 ml) was incubated with 0.5 pCi (15 pM) [1 -14C] -14-trans-AA in the presence or absence of non-radiolabeled AA (15 fiM) in a water bath with shaker at 1200 rpm for 30 minutes at 37°C. Fatty acids were extracted and injected into RP-HPLC equipped with an on-line radio-chromatography detector to compare the metabolic products formed by trans-AA alone and in the presence of AA (n = 5, *P< 0.05, relative to data of only \A-trans- AA metabolism).
68
3.1.1.2 Effect of inhibitors on 14-trans-AA metabolism
To determine the enzymatic origin of metabolites A, B, C and D, platelets were
preincubated with inhibitors of the oxidative metabolism of AA, indomethacin, a
potent COX inhibitor (Salari et al, 1984)y or baicalein, a potent LOX inhibitor
(Sekiya & Okuda, 1982), prior to incubation with AA and \A-trans-P±A. Metabolites
of AA and trans-AA. derived from COX and LOX were identified by reversed-phase
high performance liquid chromatography (RP-HPLC) equipped with an on-line radio
chromatography detector. Radio-chromatograms presented in Figure 3-4 show that
indomethacin, inhibited formation of TXB2, 12-HHT and metabolites B and C. In the
presence of baicalein, there was decreased formation of 12-HETE along with
metabolites A and D. These data confirm that TXB2 and 12-HHT are COX products
and that 12-HETE is a LOX product, and suggest that metabolite B and C are formed
by COX and metabolites A and D are formed by LOX (Figure 3-4).
69
90-
70-
50-i
o
ECL90 :
o7 0 -i
&ëro0
0150^
A Control AA14-trans-AA
TXB2
- J hJL12-HHT
12-HETE
i k j10 20 30 40 50
B Indomethacin AA
12-HETE14-trans-AA
10
C Baicalein 14-trans-AA
12-HHT
TXB; jJ l12-HETE
a A .20 30
Time (min)40 50
Figure 3-4. Effects of inhibitors on AA metabolism
Washed platelet suspensions (1 ml) were preincubated (A) alone or with (B) 10 pM indomethacin (COX inhibitor), or (C) 10 pM baicalein (LOX inhibitor) prior to stimulation with 0.5 pCi (15 pM) radiolabeled AA and 14-trcms-AA. Then the platelet suspensions were incubated in a water bath with shaker at 1200 rpm for 30 min at 37 °C. Fatty acids were extracted and injected into RP-HPLC equipped with an on-line radio-chromatography detector to observe the metabolites formed from cis- and 14-trans-AA in the presence of inhibitors.
70
This experiment was designed to distinguish trans-AA metabolites according to the
enzyme responsible for their production without the potentially confounding presence
of AA. Radio chromatograms presented in Figure 3-5 show the metabolism of
radiolabeled trans-AA in the presence of non-radiolabeled AA and a COX or LOX
inhibitor. Indomethacin inhibited the formation of metabolites B and C, while the
baicalein inhibited formation of metabolites A and D. These results confirm that
metabolite B and C are the COX products and metabolites A and D are the LOX
products.
90
o oES01 ™I% 505
90
70
50 i
14-trans-AA9 0 i Control
30 50402010
B Indomethacin
AD
À
14-trans-AA
10
C Baicalein
20 30 40 5014-trans-AA
BC
L10 20 30 40 50
Time [min]
Figure 3-5. Metabolism of radio-labeled 14-trans-AA with non-radiolabeled AA and COX and LOX inhibitor
Washed platelet suspensions (1 ml) were preincubated with (B) 10 pM indomethacin (COX inhibitor), (C) 10 pM baicalein (LOX inhibitor) for 3 minutes and (A) 0.5 pCi (15 pM) radiolabeled 14-trans-AA and 14 pM non-radiolabeled AA was added for 30 minutes at 37° C in a water bath with shaker. Fatty acids were extracted and injected into HPLC to observe the metabolic products. This figure is a representative of three experiments.
71
3.1.1.3 UV analyses of cis and *\4-trans-AA metabolites
In order to obtain preliminary structural data about metabolites A, B, C, and D, the
UV absorption properties of these metabolites were determined. Platelets were
incubated with 100 pg of cis and 14-trans-AA for 30 minutes, and fatty acids were
extracted and injected into HPLC. Figure 3-6 shows the UV chromatogram at 234 nm
of 12-HHT, 12-HETE as well as two unknown metabolites C and D. As AA and TX
have absorption maxima Àmax = 205, those were not detected in this chromatogram. It
was observed that metabolites C and D elute around the same time and have similar
UV absorption as 12-HETE. These results suggest that metabolites C and D may have
structural similarity to HETE metabolites. Thus, the application of advanced
spectroscopic UV analysis (diode array) and LC/MS were used to determine structure
of these metabolites.
!EcsCN8cCO-2
<
12-HETE
60
40
12-HHT20
0
30 50402010
Time [min]
Figure 3-6. UV chromatogram at 234 nm of AA and 14-trans-AA metabolitesPlatelet suspension (1 ml) was incubated with 100 pg AA and 100 pg 14-/nms-AA for 30 minutes in a water bath with shaker at 1200 rpm for 30 minutes at 37° C. Fatty acids were extracted and injected into HPLC and UV analysis was done at 234 nm to observe the metabolic products. This figure is a representative of four experiments.
72
3.1.1.4 Spectral analyses of unknown metabolites
A complete UV spectrum from 205 to 300 nm of metabolites C and D were measured
on a Diode Array Detector. The maximum absorbances (X max) for metabolites C and
D were 236 nm, and for 12-HETE was 234 nm (Figure 3-7). This again suggests
structural similarities among these metabolites and with the unsaturated hydroxy fatty
acids (HETEs).
XMax=236 Compound C
0.4
300280260240220
1 .4 " B
XMax=236 Compound D8§■e
0.8 -
° 0.4_oro>ZD
300280260240220
1.5
A,M =234 12-HETE1.0-
0.5
300280260
Wavelength [nm]
240220
Figure 3-7. UV spectrum of metabolite C and metabolite D
Washed platelets (1 ml) were incubated with 100 pg 14-rnms-AA for 30 minutes in a water bath with shaker at 1200 rpm at 37° C. Fatty acids were extracted and injected to HPLC and metabolites C and D were isolated. Isolated (A) metabolites C (B) metabolite D and (C) 12- HETE standards were injected into a HPLC with diode array detector and spectral analyses were done from 205 to 300 nm.
73
3.1.1.5 LC/MS analyses of the 14-trans-AA metabolites
Figures 3-8 to 3-12 show LC/MS analyses of metabolites from lA-trans-AA,
comparison with standards, and effect of inhibitors. Use of LC/MS provided a better
resolution than initial experiments with radiolabeled \A-trans-AA. Metabolites
formed by platelets from AA and \A-trans-AA, were injected into HPLC equipped
with an on-line MS. Selected ion monitoring was done for ion 369 (TXB2), 279 (12-
HHT) and 319 (HETEs).
Metabolites formed by platelets from lA-trans-AA were injected into a HPLC system
equipped with an online mass spectrometer by using an auto injector. These
metabolites generated from lA-trans-AA. by platelets, showed seven peaks (Figure 3-
8) as compared to radio chromatogram where there were four peaks (Figure 3-1 B).
As LC/MS has better resolution and identification capacity, it detected all the
metabolites and these were named metabolite A, Bi, B2, C%, C2, Di and D2. Further
LC/MS analyses of the samples of experiments with COX and LOX inhibitor were
done to identify the enzymatic origin of the metabolites.
74
17.1
1.8e7 A. AA
1.0e77.7
14.0
8.8
16.8>> 2.0e6 14.3
O 1.8e6 B. 14-trans-AA
1.0e6
TXB2
2.0e5
Time [min]
Figure 3-8. LC/MS analyses of metabolites of AA and 14-/ra«s-AA by human plateletsMetabolites generated by human platelets incubated with (A) AA or (B) 14-trans-AA, were separated by gradient elution RP-HPLC system. Metabolites eluted from the RP-HPLC column were detected by an on-line mass spectrometer. Under negative electrospray ionization (ESI) mode, selected ion chromatography of m/z 369 (for TXB2), 279 (for 12- HHT) and 319 (for HETEs) were done to identify the metabolites. The chromatogram shows the total ion chromatogram (TIC).
75
For the identification of the peaks obtained by LC/MS, the elution times of the
metabolites formed by \A-trans-AA (Figure 3-8) was compared with the elution times
of LC/MS of the commercial standards (Figure 3-9). Table 3-1 shows molecular
weight and RP-HPLC elution time of standards.
Table 3-1. LC elution time of different standards detected by mass spectrometer
Eicosanoid standard Molecular weightRP-HPLC elution time
[min]
t x b 2 370 7.7
12-HHT 280 14.0
15-HETE 320 16.5
12-HETE . 320 17.0
11-HETE 320 17.3
5-HETE 320 17.8
Different HPLC elusion times for commercial standards.
76
100% 1 A. Platelets* '\ 4-trans-AA
o%3
1 0 0 % 3 B. TXB.
0% J 1 0 0 % 3 C. 12-HHT
o%mocro
T3cZ3
X I<CD>
] D. 15-HETE
V. . ....... ,............W4Wa) 0%
E. 11-HETE
F. 12-HETE
1 0 0 % 3 G. 5-HETE
o%Time [min]
Figure 3-9. Comparison of the negative-ion MS/MS spectra of various eicosanoid standards with metabolites of 14-trans-AAMetabolites produced by human platelets incubated with 14-trans-AA and different commercial standards were injected into RP-HPLC equipped with an on-line mass spectrometer. In linear gradient RP-HPLC, commercial standards eluted at different times and their elution time were compared with the 14-trans-AA derived metabolites.
77
The metabolic pattern of different metabolites generated from AA or \4-trans-PJ± in
presence of a COX inhibitor (indomethacin) was characterized. In control
experiments with AA and indomethacin, the formation of TXB2 and 12-HHT were
inhibited. Formation of metabolites Bi, B2, Ci and C2 were decreased in the
experiment with \4-trans-AA and indomethacin in comparison with 14-trans-AA
only experiment, suggesting that these are COX products. While the formation of
TXB2 was inhibited in the control experiment, the formation of metabolite A was not
inhibited. This suggested that metabolite A is not formed by the COX pathway. It was
also observed that the formation of metabolite D% was increased in the experiment
with \4-trans-AA and indomethacin in comparison with 14-trans-AA only
experiment. The metabolites formed by platelets from 14-trans-AA in presence of
indomethacin are shown in Figure 3-10.
78
12-HETE2.0e7
1.2e7
4.0e6
A. AA+lndomethacin
11 13 15 17 19 21 23 25
2.2e6 B. 14E-AA+lndomethacin
6.0e5
C11.8e6 C. 14E-AA
1.0e6
TXB2
2.0e5
11 13 15 17 19 21 23 25
Time [min]
Figure 3-10. Comparison of the negative-ion LC/MS spectra of metabolites produced with indomethacinIndomethacin (10 (iM)-preincubated human platelet (1 ml) were incubated with (A) AA (100 pg) and (B) \4-trans-AA (100 pg) for 30 minutes in a water bath with shaker at 37°C. Figure C is the control incubation of platelet with 100 pg 14-trans-AA. Metabolites were extracted and injected into linear gradient elution RP-HPLC equipped with an on-line triple-quadrupole mass spectrometer. Under negative electrospray ionization (ESI) mode, selected ion chromatography of m/z 369, 279 and 319 were done.
79
Incubation of platelets with AA in presence of a LOX inhibitor (baicalein) partially
inhibited the formation of 12-HETE. When platelets were incubated with \A-trans-
AA in presence of baicalein, it inhibited the formation of metabolite A and D% but the
formation of metabolite Ci and C2 was increased (Figure 3-11). This experiment
suggested metabolite A and D% were the LOX products.
1 .5 e7
1 .1 e7
7 .0 e 6
3 .0 e 6
12-HETE
A. AA+Baicalein TXB,
12-HHT
TXB,
u9 11 13 15 17 19 21 2 3 25
2 .5 e 6B. 14E-AA+Baicalein
1 .8 e6
1 .0 e6
2 .0 e 5 _____23 25
1 .8 e6C. 14E-AA
1 .0 e6
TXB;
j x b 2Z.OeS .....
21 2 3 25Time [min]
Figure 3-11. Comparison of the negative ion LC/MS spectra of metabolites produced with baicaleinBaicalein (10 pM)-preincubated washed human platelet (1 ml) was incubated with A. AA (100 pg) and B. 14-trans-AA (100 pg) C. 14-trans-AA. (100 pg) only for 30 minutes in a water bath with a shaker at 37°C. Metabolites were extracted and injected into liner gradient elution RP-HPLC equipped with an on-line triple-quadrupole mass spectrometer. Under negative electrospray ionization (ESI) mode, selected ion chromatography of m/z 369, 279 and 319 were done.
80
To eliminate any endogenous AA metabolites that might interfere with the
understanding of the metabolic origin of unknown metabolites, platelets were
incubated with d%-\A-trans-AA alone and with indomethacin or baicalein. It was
observe that the formation of metabolite A and Di was inhibited by the LOX inhibitor
(Figure 3-12C), which confirms their LOX-mediated origin. Metabolites Bi, Bi, Ci,
C2 and D2 formation was completely inhibited by the COX inhibitor (Figure 3-12B),
confirming their COX-mediated origin.
D,1 .2 9 e6
9 .0 0 e5
5 .0 0 e5
A. d8-14-trans-AA
Bi B
11 13 15 17 19 21
Di2 3 25
3 .4 e6B. d8-14-frans-AA+lndomethacin
2 .5 e6
c 1 .5 e6
5 .0 e5
257 11 13 15 17 19 231 5 9 213
4 .5 e5
2 .5 e5
5 .0 e4
C. d8-14-frans-AA+Baicalein
Time [min]
Figure 3-12. Metabolism of à%-trans-AA by plateletsWashed platelets (1 ml) were incubated with A. d8-14-/nms,-AA (lOOpg) and B. indomethacin (10 pM) and C. baicalein (10 pM) for 30 minutes in a water bath with a shaker at 37°C. Metabolites were extracted and injected into linear gradient elution RP-HPLC equipped with an on-line triple-quadrupole mass spectrometer. Under negative electrospray ionization (ESI) mode, selected ion chromatography of m/z 377 and 327 were done.
81
3.1.1.6 Gas chromatogram of metabolite A and TXB2
Washed platelets were incubated with \A-trans-AA, and metabolite A was extracted
and isolated from other metabolites by HPLC. BSTFA derivatization of the metabolite
was done and injected into a GC/MS system. The result shows that metabolite A has
same MW (585) as of TXB2 but the elution time is 10.8 minute in comparison with
TXB2 which is 10.4 minutes (Figure 3-13). The experiment confirms that metabolite
A is a different compound and more polar than TXB2.
Metabolite A10000
5000
10CO10.g 0
A
t x b 21
10 12 14 16 18 20TXB
ç 10000co
5000
Time [min]
Figure 3-13. GC/MS chromatograms of ion at m/z 585Metabolite A, synthesized by platelets from \4-trans-AA, was separated and purified by RP- HPLC and derivatized to TMS ester. Sample (1 jil) in iso-octane was injected into GC/MS (negative ions) and (A) there was separation of metabolite A from TXB2 as metabolite A eluted at 11.4 min in comparison TXB2 which eluted at 10.9 min. (B) elution time for TXB2 was confirmed by injecting commercial TXB2 standard.
82
3.1.1.7 Identification of metabolites from 14-frans-AA by LC/MS/MS
Fragment ions of individual eicosanoids were determined from the LC/MS/MS
analysis of commercial standards. Product ion MS/MS spectra of the eicosanoid
standards were acquired by passing the (M-H)" of individual eicosanoids, which had
been mass-selected with the first quadrupole, into the second quadrupole where they
were dissociated by collision with ultra pure helium gas. Fragment ions generated by
these collisions then were mass analyzed by the third quadrupole, and detected. The
third quadrupole was scanned in 0.1-a.m.u. (atomic mass unit) increments from 15
a.m.u. up to and including the mass of the parent ion in less than 10 seconds, and at
least 10 scans were averaged for each MS/MS spectrum. Collision energies were
adjusted to 15-25 eV with respect to the laboratory reference for this study.
Representative data are shown in Figure. 3-15 to 3-21. Complete fragmentation data
of each of the eicosanoids characterized in this study are summarized in Table 3-2.
The product ion that was indicative of each eicosanoid is in bold type.
83
Table 3-2. The molecular weights (relative abundances) of fragment ions observed in the negative-ion MS/MS spectra
Eicosanoid
Parent ion
[M-H"]
Fragment ions
(MS/MS)
Arachidonic acid 303 (100) 285 (3), 259 (3), 231 (3), 205 (3), 191 (2), 177 (3), 163 (2),
83 (2), 59 (13)
t x b 2 369(2) 289 (9), 195 (50), 191 (3), 177 (6), 169 (100), 151 (4), 125
(3)
p g d 2 351 (3) 333 (2), 315 (11), 297 (3), 271 (100), 233 (11), 217 (4),
203 (7), 189 (69)
p g e2 351 (7) _ 333 (14), 315 (16), 271 (100), 235 (10), 217 (7), 206 (4),
189 (36), 175 (10), 163 (3), 159 (2), 113 (3), 109 (2)
PGF2a 353 (92) 335 (8), 317 (11), 309 (100), 299 (11), 291 (42), 281 (6),
273 (16), 263 (8), 255 (9), 247 (26),235 (8), 217 (8), 209
(12), 193 (45), 171 (13), 165 (12), 111 (4)
6-Keto PGFia 369 (100) 351 (21), 333 (23), 315 (20), 307 (13), 289 (23), 271 (12),
263 (11), 245 (39), 233 (12), 207 (36), 163 (40), 89 (15)
12-HHT 279 (40) 261 (20), 217 (15), 189 (22), 179 (100), 163 (5), 153 (6),
135 (60), 113 (18), 107 (5), 99 (4), 81 (8), 59 (20)
5-HETE 319(45) 301 (20), 283 (4), 257 (25), 203 (22), 115 (100)
8-HETE 319(35) 301 (20), 283 (4), 257 (25), 163 (45), 155 (100), 127 (10)
11-HETE 319 (40) 301 (4), 291 (5), 167 (100)
12-HETE 319(32) 301 (49), 291 (4), 283 (7), 275 (8), 257 (89), 229 (19), 215
(5), 208 (17), 203 (48), 189 (8),179 (100), 177 (15), 163
(43), 153 (22), 139 (18), 135 (33), 107 (12), 59 (22)
15-HETE 319(35) 301 (80), 283 (8), 275 (11), 257 (100), 229 (7), 219 (36),
203 (52), 175 (62), 163 (13), 149(11), 147 (14), 139 (13),
133 (8), 121 (40), 113 (50), 99 (8), 59 (22)
Note: Ion abundances are normalized to the most abundant ion in each MS/MS spectrum.
84
3.1.1.8 Identification of metabolite A
Metabolites generated from \A-trans-PiA by platelets were injected into a HPLC
system equipped with an online mass spectrometer by using an auto injector.
Negative-ion MS/MS of the different ion were done. The MS/MS spectra of novel
metabolites were compared with spectra of commercial standards and their structures
were confirmed.
Negative ion MS/MS were done on metabolite A, that gave rise to ion m/z 169, m/z
195 and m/z 127 (Figure 3-14 A). When MS/MS were done on metabolite A
generated from à%-\A-trans-AA by platelets, it showed a prominent ion m/z 129 as
well as ion m/z 158 and m/z 200 (Figure 3-14 B). Incubation of lA-trans-AA. with 12-
LOX also gave rise to metabolite A that gave ion m/z 127 (Figure 3-14 C). For the
confirmation of TXB2 structure, MS/MS analysis of commercial TXB2 standard were
done and ions m/z 169 and m/z 195 was observed (Figure 3-14 D).
85
100%
0%
100%
sc(0"Oc3<
0%
B
169127
85 96 12® 139 155
1 95 2 2 5 xr ,.1 5 5
\ OH \ O H /
- 1 2 7M-H]-369
COOH
221
80 120 160 200 24 0 2 8 0 320 36 0 4 0 0
129
88112
[M-H]-37 7
C O O H
138158 20 0 2 1 8
J I 226 35 880 120 160 200 24 0 2 8 0 32 0 360
100%169
0%
OH
4 0 0
ro 100%1 27 OHOH
.COOH
155113 139 165 183 195 221 3510%
4 0 036 02 8 0 3 202 4 02001601208 0
D 1 95 / k ^ _____r ' y ' ' v / ' c o o
HO O
OH [M-H]-36 9
3 25I
80 12 0 160 2 0 0 2 4 0 2 8 0 3 2 0 36 0 4 0 0
m/z
Figure 3-14. Comparison of the MS/MS spectra of metabolite AMetabolite A, was generated by 1 ml platelets incubating with A. \A-trans-AA (100 jig); B. d8-14-/nms-AA (100 pg); and C. 12-LOX (100 units) with \4-trans-AA (100 jig), extracted with ethyl acetate and water, injected into RP-HPLC for separation and purification. Samples were injected into a mass spectrometer by auto injector using flow injection analysis (FIA) mode. Negative-ion MS/MS of ion A. 369 m/z, B. 377 m/z and C. 369 m/z were done for metabolite A generated by platelets from \A-trans-AA, d8-14-/r<ms-AA and by 12-LOX from \A-trans-kk respectively, D. Negative-ion MS/MS of ion 369 m/z were done for TXB2 standard.
86
In the last experiment (Figure 3-14), it was confirmed that ion m/z 127 is specific for
metabolite A. Multiple reaction monitoring (MRM) analysis of transition m/z
369—>127 was done for compound A (Figure 3-16 C. and D.) and its spontaneous
transformation was observed. This experiment shows that ion 195 disappears from
experiment A to C and transformed from A[l] to A[2] from experiment B to D, which
suggested metabolite A is an unstable compound which transformed at room
temperature.
A[1]477 1079
400 900
TXB2[2] +A[2]700300
500200
A[2]300100
369->127100
Q. 5.0 6.0 7.0Time [min]
TXB2[2] +A[2]
8.0 5.0 6.0 7.0Time [min]
2116c 1100
t x b 2 [1]1800
900
1400700
1000500
600300
369->1272001000
5.0 6.0 7.0 5.0 6.0 7.0 8.0Time [min]
Figure 3-15. Spontaneous transformation of metabolite AIsolated metabolite A was kept at room temperature (24°C) for 72 hours and injected to a LC/MS system. Chromatogram A and C show transition 369—>195 and 369—>127 of freshly prepared metabolite whereas chromatograms B and D show transition 369—>195 and 369—>127 of metabolite 72 hours after preparation. Metabolite A was detected by transition 369—>127 (only from metabolite A). For fresh metabolite A the transition 369—>195 shows a peak A[l].
87
3.1.1.9 Identification of metabolites Bi and B2
The results from the previous experiment (Figure 3-12) confirmed that metabolites Bi
and B2 are COX products. Negative ion MS/MS analysis of ion m/z 279 of metabolite
B generated from \A-trans-AA by human platelets was done to confirm their
structure. This spectrum was compared with the negative ion MS/MS analysis of ion
m/z 279 of commercial 12-HHT standard (Figure 3-16).
179100%
135179
189113 217
99 1070% 300260220180140100
"O.COO"
[M-H]-279179
1 100% J 135 179 ''oh217
113 189163
107 1390% 300260220180140100
m/z
Figure 3-16. Comparison of the MS/MS spectra of metabolites Brand B2Metabolite B was generated by washed platelets (1 ml) incubating with A. 14-/nm?-AA (100 jig), extracted with ethyl acetate and water, injected into RP-HPLC for separation and purification. Samples were injected into a mass spectrometer by auto injector using flow injection analysis (FIA) mode. Negative-ion MS/MS of ion A. 279 m/z was done for metabolite B generated from \A-trans-AA and B. 279 m/z was done for commercial 12-HHT standard.
88
3.1.1.10 Identification of metabolite C1
Metabolite Ci was a COX product as confirmed in a previous experiment (Figure 3-
12). Negative ion MS/MS analysis of ion m/z 319 of metabolite Ci generated from
\A-trans-AA by human platelets was done to confirm the structure and compared with
the negative ion MS/MS analysis of ion m/z 319 from commercial 15-HETE standard
(Figure 3-17).
.COO" [M-H]-319
100% +H219 219
113
121
107 203139 147 2750% 320240 280200160120
226D8= 100% "O
D DDCOO"
188+H !0H 182226
115 154 2120% 320240 28020016012080
-H20301■COO"
+H ! OH113 219 [M-H]-319219175
80 120 160 200 240 280 320
m/z
Figure 3-17. Comparison of the MS/MS spectra of metabolite QMetabolite Cl, was generated by washed platelets (1 ml) incubated with A. \A-trans-AA (100 pg) and B. d8-14-/r<ms-AA (100 pg), extracted with ethyl acetate and water, injected into RP- HPLC for separation and purification. Samples were injected into a mass spectrometer by auto injector using flow injection analysis (FIA) mode. Negative-ion MS/MS of ion A. m/z 319 and B. m/z 327 were done for metabolite Ci generated from 14-fnms-AA and dg-14- trans-AA respectively and C. m/z 319 was done for 15-HETE standard.
89
3.1.1.11 Identification of metabolite C2
Metabolite C2 was a COX product as confirmed in a previous experiment (Figure 3-
12). Negative ion MS/MS analysis of ion m/z 319 of metabolite C2 generated from
\4-trans-Ah by human platelets was done to confirm the structure and compared with
the negative ion MS/MS analysis of ion m/z 319 from commercial 11-HETE standard (Figure 3-18).
100%167
0%
100%
SII1m 0%
100%
149
167
+HCOO'
HO
195-H20301
[M-H]-319
80 120 160 200 240 280 320
171
BCOO"
D OH D D
264
[M-H]- -H20 327309 I
80 120 160 200 240 280167
0%
167
+Hj COO"
HO
320
[M-H]-319
80 120 160 200 m/z
240 280 320
Figure 3-18. Comparison of the MS/MS spectra of metabolite C2Metabolite C2, was generated by platelets (1 ml) incubated with A. \A-trans-AA (100 pg) and B. d^-lA-trans-AA (100 pg), extracted with ethyl acetate and water, injected into RP-HPLC for separation and purification. Samples were injected into a mass spectrometer by auto injector using flow injection analysis (FIA) mode. Negative-ion MS/MS of ion A. m/z 319 and B. m/z 327 were done for metabolite C2 generated from \A-trans-AA and d8-14-/ra«5- AA, respectively and C. m/z 319 was done for 11-HETE standard.
90
3.1.1.12 Identification of metabolite D,
Metabolite Di was a LOX product as confirmed in a previous experiment (Figure 3-
12). Negative ion MS/MS analysis of ion m/z 319 of metabolite D% generated from
XA-trans-AK by human platelets was done to confirm the structure and compared with
the negative ion MS/MS analysis of ion m/z 319 from commercial 12-HETE standard
(Figure 3-19).
100%
0%59 81i
80
179
135
107
J2L
163
139 155I .li
179 ; o h ..•••
208-"' -(H20 + C 0 2) 257
203 208
120 160 200 240 280
[M-H]-319
-H20301
3201-H]-
327184COO"100% :
140214
1110% 320280240200160120
179100% i COO"
1 7 9 OH
208
163
135 208107 1,39 153 203
0% 320240 280200160120
m/z
Figure 3-19. Comparison of the MS/MS spectra of metabolite DiMetabolite Di, was generated by platelets (1 ml) incubated with A. \A-trans-AA (100 pg) andB. d8-14-/nms-AA (100 pg), extracted with ethyl acetate and water, injected into RP-HPLC for separation and purification. Samples were injected into a mass spectrometer by auto injector using flow injection analysis (FIA) mode. Negative-ion MS/MS of ion A. m/z 319 and B. m/z 327 were done for metabolite Di generated from \A-trans-AA and &%-\4-trans- AA, respectively and C. m/z 319 was done for 12-HETE standard.
91
3.1.1.13 Identification of metabolite D2
Metabolite D2 was a COX product as confirmed in a previous experiment (Figure 3-
12). Negative ion MS/MS analysis of ion m/z 319 of metabolite D2 generated from
\A-trans-AA by human platelets was done to confirm the structure and compared with
the negative ion MS/MS analysis of ion m/z 319 from commercial 5-HETE standard
(Figure 3-20).
g 100%
roc3<
I0)% 0%
B
115100% : 1.115
OH203
.COO'-H20301 [M-H]-
319203
0%120 160 200 240 280 320
116210
.COO"
D D
210 265i80 120
115
160
100%
0%
200
2 0 3 115' OH
240
COO"
280
-(H20+ C 0 2)257
203
80 120 160 200 m/z
240 280
[M-H]-327
-H20309
320
[M-H]-319
-H20301
320
Figure 3-20. Comparison of the MS/MS spectra of metabolite D2Metabolite D2, was generated by platelets (1ml) incubated with A. \A-trans-AA (100 pg) andB. ds-M-Znms-AA (100 pg), extracted with ethyl acetate and water, injected into RP-HPLC for separation and purification. Samples were injected into a mass spectrometer by auto injector using flow injection analysis (FLA) mode. Negative-ion MS/MS of ion A. m/z 319 and B. m/z 327 were done for metabolite D2 generated from lA-trans-AA and dg-M-Zra/w-AA Respectively and C. m/z 319 was done for 5-HETE standard.
92
3.1.2 Effects of 14-frans-AA on platelet function
In order to demonstrate the effect of \4-trans-AA on platelets, washed platelets were
treated as described in section 2.2.2.1 with either AA or \A-trans-Pd± and aggregation
was analysed using an aggregometer.
3.1.2.1 Effect of 14-frans-AA on human platelet aggregation
Consistent with previous reports, AA (15 pM) induced aggregation of human platelets
(Figure 3-21). In order to see the effect of \A-trans-AA on platelet aggregation,
platelets were initially incubated with various amounts (15, 50 and 100 pM) of 14-
trans-AA. Data presented in Figure 3-21 show that \A-trans-AA did not cause
aggregation of platelet at concentrations of up to 100 pM. No aggregation or shape
change was observed after increasing concentration and time of incubation with 14-
trans-AA. It was concluded that 14-trans-AA does not cause platelet aggregation;
therefore, it does not activate platelets.
►4
Figure 3-21. Effect of 14-trans-AA on platelet aggregationThis figure is a representative of five experiments. The indicated concentrations of 14-trans- AA (15, 50, 100 pM) were added to 0.5 ml of washed platelet and incubated for 4 min at 37° C with constant stirring at 1200 rpm. Aggregation was measured with an aggregometer and the results were compared to control (platelet aggregation with 15 pM AA).
JZO)
100 H
50 -
0
Time [Min] 014-frans-AA 15 pM
c/s-AA 0 pM
050 pM
0 pM
0 4100 pM
0 pM
change
0 pM 15 pM
93
The results from experiments shown in Figure 3-21 stimulated further experiments
designed to study whether \A-trans-AA has platelet inhibitory activity.
Data presented in Figure 3-22 show that AA (15 pM) caused aggregation of platelets.
Preincubation of platelets with lA-trans-kK (5, 10 and 15 pM) for 4 minutes
inhibited AA-induced aggregation. Analyses of the data indicated a 73±6% decrease
of aggregation with 5 pM \A-trans-AA, 83.5±3% decrease with 10 pM \A-trans-AA,
and 92±4% decrease with 15 pM \A-trans-AA as compared to the control platelets.
This data suggest that \A-trans-AA inhibits the aggregation of platelets stimulated by
AA in a concentration-dependent manner. The data in Figure 3-22 suggest that 14-
trans-AA did not inhibit platelet shape change induced by AA.
94
A 100-1
Oî0
Time [Min] o 4'14-trans-AA
All c/s-AA0>jM 15 jjM
5 |JM 15 (jM
10 |JM 15 pM
15 pM 15 pM
B
0 30 -
0) 20 -
14-/rans-AA(|jM) 10 15
Figure 3-22. Effect of 14-trans-AA on AA-mediated platelet aggregationPlatelet suspension (500 pL) was preincubated with the indicated amounts of 14-trans-AA for 4 min at 37° C with constant stirring at 1200 rpm and then 15 pM AA was added. Aggregation was measured with an aggregometer and the results were compared to control (platelet aggregation with 15 pM AA alone) (n = 5, *P < 0.05 relative to data of only AA- induced aggregation).
95
3.1.2.2 Effect of 14-frans-AA pre-incubation time on platelet aggregation
Platelet suspensions were incubated with M-Znms-AA (5 pM) for various periods of
time (0-5 min). \A-trans-AA exhibited a time-dependent inhibition of platelet
aggregation by AA (5 pM). Within the first minute the inhibitory effect of \A-trans-
AA increased to 75%. The maximal inhibition (88±6%) was observed after a 3-min
preincubation as compared to the control (without 14-trans-AA) (Figure 3-23). For
this reason, this period of incubation was used for the subsequent experiments, which
aimed to study the effects of 14-trans-AA on AA- and other agonist-induced
aggregation.
100
co
O)8» 5 0 .I
CL
3 4 5210Time [min]
Figure 3-23. Effect of 14-trans-AA incubation time on platelet aggregationWashed platelets (0.5 ml) were pre-incubated with 14-trans-AA (5 pM) for 7 different times: 15 s, 30 s, 1 min, 2 min, 3 min, 4 min and 5 min at 37 °C with constant stirring at 1200 rpm and then AA (5 pM) was added. Aggregation was measured with an aggregometer and the results were compared to control (platelet aggregation with 5 pM AA) (n = 3, *P < 0.05 relative to aggregation in 3 min).
96
3.1.2.3 Concentration-dependent inhibition of aggregation by 14- trans-AA
In order to define the concentration-response properties of \4-trans-AA more
carefully (Figure 3-24), platelets were pre-incubated with different amounts of 14-
trans-AA (0, 3, 5, 7, 9 and 11 pM) and AA (3 pM). The observed results showed that
the inhibitory effect of 14-trans-AA is concentration-dependent and the IC50
(concentration causing 50% inhibition) of 14-trans-AA was found to be at 7.2 pM as
calculated from the chart.
100 n
co
50 -
10)CL
6 8 10 124214-trans-AA [pM]
Figure 3-24. Concentration-dependent inhibition of AA-induced aggregation by 14-trans-AAPlatelet suspension (0.5 ml) was preincubated with different amounts (0, 3, 5, 7, 9, and 11 pM) of 14-trans-AA for 3 min at 37 °C with constant stirring at 120 rpm and then 3 pM AA was added. Aggregation was measured with an aggregometer and the results were compared to control (platelet aggregation with 3 pM AA alone). The results suggest that the \4-trcms- AA-mediated inhibition of AA induced platelet aggregation is concentration dependent and the ID5o of 14-trans-AA was 7.2 pM (n = 3, *P < 0.05).
97
3.1.2.4 Thromboxane B2 formation by platelets
The measurement of TXB2, a stable metabolite of TXA2, in supernatants of platelet
suspension was used as index of TXA2 generation. TXA2 and PGH2 are pro-
aggregatory metabolites of AA in platelets. Resting platelets produced below 200
ng/ml TXB2 but in the presence of AA (3 pM), there was a significant elevation of
TXB2 formation. Furthermore, various concentrations of \A-trans-PJv (3 to 11 pM)
inhibited TXB2 formation in comparison with the platelets that were stimulated only
with AA (Figure 3-25). 14-£r<ms-AA did not inhibit TXB2 formation completely even
at llpM although at this concentration it inhibited AA-induced platelet aggregation
completely. Figure 3-25 shows that \4-trans-kK (3 pM) inhibited TXB2 synthesis by
about 35% relative to the control (3 pM of AA). Increasing the amount of lA-trans-
AA to 7 pM had little effect on TXB2 inhibition, but a further increase in the
concentration resulted in additional TXB2 inhibition, which at 11 pM was about 85%.
Comparison of data in figures 3-4 and 3-5 suggests that the effect of 14-trans-AA on
platelet aggregation can only partially be explained by the inhibition of COX by 14-
trans-AA. In these two sets of experiments, the same concentration of AA (3 pM)
was used and while aggregation was inhibited by \A-trans-AA at concentrations
ranging between 3 to 7 pM, the TXB2 formation was partially inhibited. This
suggested that \A-trans-AA is a partial inhibitor of TXB2 biosynthesis while it is a
good inhibitor of platelet aggregation. This observation prompted the exploration of
other mechanisms that may be affected by \A-trans-AA.
98
10000
M-trans-A/K
Figure 3-25. Concentration-dependent inhibition of AA-induced TXB% formation by 14-trans-AAPlatelet suspension (0.5 ml) was pre-incubated with different amounts (0, 3, 5, 7, 9, and 11 pM) of 14-trans-AA for 3 min at 37 °C with constant stirring at 1200 rpm and then 3 pM AA was added and incubated for 3 min. The suspension was centrifuged as 1000 g for 5 minutes and 20 pi supernatant was injected into LC/MS. TXB2 was measured by LC/MS as the area of the peak generated from ion 369 and compared with the standard curve (n = 3, *P < 0.05 relative to aggregation with AA only).
99
3.1.2.5 Effect of 14-frans-AA on U-46619
In the next experiment, U-46619 (a stable TXA2 analogue) was used as the platelet
agonist that stimulates the TP receptor. Platelets were pre-incubated with different
amounts of \A-trans-AA and U-46619 was added to observe the effect of lA-trans-
AA on U-46619-mediated aggregation. Inhibition of U-46619-mediated aggregation
by 14-trans-AA was concentration dependent and the IC5o was found to be 2.2 pM as
calculated from the chart (Figure 3-26). The effects of 14-trans-AA on other
pathways of platelet activation were also explored.
100
Control 14-frans-AA100 i [MM]
1.5U46619 0.2 pM
U46619 0.2 pM
CD
2.5
3.5
63r0
Time [Min]
100-,
co
5 0 .
0)Q_
3 4210
14-frar?s-AA [pM]
Figure 3-26. Effect of 14-fra«s-AA on platelet aggregation by TP receptor agonistPlatelet suspension (0.5 ml) was preincubated with the indicated amounts of 14-/r<ms-AA for 3 min at 37 °C with constant stirring at 1200 rpm and then 0.2 pM U-46619 was added. Aggregation was measured with an aggregometer and the results were compared to control (platelet aggregation with 0.2 pM U46619 alone) {n = 3, *P < 0.05 relative to aggregation with AA only).
101
3.1.2.6 Effect of other agonists
To observe the effects of \A-trans-AA on other agonists, platelets were incubated
with ADP, epinephrine, thrombin and a stable analogue of platelet activating factor
(PAF) called cPAF. \A-trans-AA (5 pM) inhibited ADP (50 pM)-mediated
aggregation by 87±8% (Figure 3-27).
100
8c(0E(OcIJZ0 5
0
Time [Min] o 3 614-trans-AA
ADP
cf?O)0 5ra
I0)Q_
L
ADP
^ - 4
14-trans-AA+ADP
0 pM 50 fjM
3
5 |jM 50 pM
Figure 3-27. Effect of ADP on platelet aggregationPlatelet suspension (0.5 ml) was preincubated with the indicated amounts of 14-trans-AA for 3 min at 37 °C with constant stirring at 1200 rpm and then 50 pM ADP was added. Aggregation was measured with an aggregometer and the results were compared to the control (platelet aggregation with 50 pM ADP alone) (n=3, *P < 0.05 relative to aggregation with AA only).
102
14-fnms-AA (5 fiM) inhibited epinephrine (50 pM)-induced aggregation by 85 ± 12%
(Figure 3-28).
100 - ,
8croEwc!cn_ i
0 - 1
Time [Min] o 63
14-trans-AAEpinephrine
0 pM 50 pM
rogo>O) ,CD 50
1
Epinephrine 14-trans-AA+ Epinephrine
3
5 pM 50 pM
Figure 3-28. Effect of epinephrine on platelet aggregationPlatelet (0.5 ml) suspension was preincubated with the indicated amounts of 14-/nms-AA for 3 min at 37 °C with constant stirring at 1200 rpm and then 50 pM epinephrine was added. Aggregation was measured with an aggregometer and the results were compared to the control (platelet aggregation with 50 pM epinephrine alone) (n=3, *P < 0.05 relative to aggregation with AA only).
103
\4-trans-AA (5 piM) inhibited thrombin (0.5 unit/ml)-mediated aggregation by 75 ±
10% (Figure 3-29).
Thrombin 14-frar?s-M+ Thrombin
Time [Min] o 3 6 0 3 614-trans-AA 0 jjM 5 pM
Thrombin 0.5 unit/ml 0.5 unit/ml
Figure 3-29. Effect of thrombin on platelet aggregationPlatelet suspension (0.5 ml) was preincubated with the indicated amounts of \A-trans-AA for 3 minutes at 37° C with constant stirring at 1200 rpm and then 0.5 units/ml thrombin was added. Aggregation was measured with an aggregometer and the results were compared to the control (platelet aggregation with 0.5 units/ml thrombin alone) (n=3, *P < 0.05 relative to aggregation with AA only).
104
Carbamyl platelet activating factor (cPAF) is a non-hydrolyzable PAF agonist, that
possesses full agonist activity approaching PAF in potency and completely resists
inactivation by acetylhydrolase (Tessner et a l, 1989). \4-trans-P±A (5 pM) inhibited
cPAF (50 nM)-mediated platelet aggregation by 80 ± 8% (Figure 3-30).
cPAF 14-trans-AA+ cPAF
i--------------1-------------1 i-------------1------------ 1Time [Min] o 3 6 0 3 6
14-trans-AA 0 pM 5 pMcPAF 50 nM 50 nM
Figure 3-30. Effect of cPAF on platelet aggregationPlatelet suspension (0.5 ml) was preincubated with the indicated amounts of 14-trans-AA for 3 min at 37 °C with constant stirring at 1200 rpm and then 50 nM cPAF was added. Aggregation was measured with an aggregometer and the results were compared to the control (platelet aggregation with 50 nM cPAF alone) (n=3, *P < 0.05 relative to aggregation with AA only).
Data shown in figures 3-27 to 3-30 suggest that at the concentration of 5 pM 14-
trans-AA was an effective inhibitor of aggregation induced by ADP, epinephrine,
thrombin and PAF, which resulted in 75-87% inhibition. Thus, a trans fatty acid, 14-
trans-AA was able to inhibit many other pathways of platelet activation in addition to
that induced by AA.
105
3.1.2.7 Membrane incorporation of 14-trans-AA
To assess changes in the AA metabolism, the incorporation of l*C-\4-trans-AA (10
pM) into platelet and compared with 14C-AA (10 pM). The platelets were incubated
with cis- or \A-trans-AA for 3 minutes and the radioactivity in the platelet precipitate
was measured by liquid scintillation counter and expressed as counts per minute
(cpm). Figure 3-31 shows that \A-trans-AA was incorporated by platelets about 3
times more than AA. At least part of \A-trans-AA was likely to be esterified in
platelet phospholipids membrane. This experiment demonstrates that avid binding of
\A-trans-AA by platelets could result in changes in phospholipids of the membrane
near various receptors that could decrease the affinity of receptors for their ligands.
■ 14CA A
0 14C 14-trans-AA
after 3 min incubation
Figure 3-31. Incorporation of radio-labeled-AA/14-/ra«.s-AA into platelet membrane phospholipidsWashed platelet suspension (1ml) was incubated with (A) 14C-AA (10 (iM) (B) ™C-\A-trans- AA (10 pM) for 3 min at 37 °C with constant stirring at 1200 rpm then centrifuged at 2000 g for 5 min and the precipitate containing platelets was counted by a liquid scintillation counter (n=3). To standardise the counts of AA and \A-trans-AA both counts were expressed as 100% in the control from 3 different experiments and relative percentage to control after 0 and 3 minutes of incubation (0 min = aliquot removed immediately after adding), (n = 3, *P < 0.05 relative to control at 0 min).
106
3.2 Metabolism of trans-AA by hepatic microsomes
3.2.1 Metabolism of 14-frans-AA by microsomes
It was observed that 14-trans-AA was not readily metabolized by human platelets
(Figure 3-1). Dietary and endogenous 14-trans-AA enter hepatic circulation, therefore
a study of its metabolism by CYP450 in hepatic microsomes was designed. Rat liver
microsomes fortified with a NADPH-regenerating system were incubated with 14-
trans-AA and metabolites were analyzed by radio-activity detector showing the
formation of metabolite tl, t2 and t3 (Figure-3-32). Comparison with synthetic
standards allowed structural identification of cl-c4 and tl-t3 (Table 3-3).
AA60-
56
52-
c4 el-e4
504030201 0trans-AA
65 i
60-:
55t2
J jb u s .50 tel-te4
5040302010
trans-AAAA70-
65
60-
55-
50
5040302010tim e [m in]
Figure 3-32. Metabolism of radiolabeled 14-trans-AA by rat liver microsomesComparison of radio-chromatograms obtained by HPLC analysis of products extracted from incubation of rat hepatic microsomes (180 min, 37 °C) with 14C-radiolabled forms of: AA (A), 14-/ra7w-AA (B) and AA plus \A-trans-AA (C). Metabolites cl-c4 was generated from the metabolism of AA in comparison to \4-trans-AA gave rise to metabolites tl-t3.
107
GC-MS analysis oîtl-tS revealed ions at m/z 483 (Figure 3-33), which corresponded
to a diTMS 14C-labeled DiHETE carboxyl anion. Fraction tl contained two closely
eluting peaks tla and tlb whereas t2 was a single peak and t3 was a relatively minor
product to do mass spectrometric analysis.
COCOxrsN
s
1co
t l
9.0
::r=r1T— r=¥=>p-rSL-9.4
I10.2
i i i i — 10.6
T “
10.610.29.89.49.0
time [min]
Figure 3-33. Identification of peak t l and t2Chromatograms of ion at m/z 483 obtained from analysis by GC-MS (negative ions) of products tl and t2 isolated as described in Figure 3-32B. Ion m/z 483 corresponds to a diTMS derivative of a 14C-labeled DiHETE carboxylic anion.
108
AA and \A-trans-AA was metabolized by CYP450 epoxygenase with reduced NADP
and metabolites were extracted with ethyl acetate, derivatized and injected into a
GC/MS. Values are retention times in minute obtained by GC-MS analysis on a HP-
5MS column. Ions at m/z 481 and 483 were recorded for standards and metabolites,
respectively and the retention times were compared in Table 3-3.
Table 3-3. Comparison of retention times of standards and metabolites from experiments illustrated by Figures. 3-32 and 3-33
Standards AAmetabolites
14,15-*ra«s-AAmetabolites
14,15-DiHETE 9.84 cl 9.83 tlb 9.80
11,12-DiHETE 9.74 c2 9.73 t2 9.76
8,9-DiHETE 9.72 c3 9.70 tla 9.72
5,6-DiHETE 9.73 c4 9.69 t3 9.68
3.2.2 Metabolism of 5-trans-AA by microsomes
It has been shown by Balazy et al (2005) that the rate of 5-trans-AA metabolism by
microsomes appeared to be higher than that described for 14-trans-AA. This
observation stimulated the present study designed to observe the metabolic pattern of
5-trans-AA by CYP of hepatic microsomes. CYP metabolized 5-trans-AA into a
complex mixture of oxidized lipid products (Figure 3-34). The metabolism required
oxygen and NADPH. Mass spectrometric analysis based on an LC/MS with negative
ion detection was done to establish the complete microsomal metabolic profile of 5-
trans-AA.
109
h58.8
9.3h2 h46.7 8.1
10.^ '^ 1 4 .5 16.917.87.35.8
e419.9
0.05 10 15
1.9e7
C/3CLO
tncsc
Time, mina 14,15 DiHETrE b 11,12 DiHETrE c 8,9 DiHETrE d 5,6 DiHETrE e 5,6 EET f 14,15 EET 9 8,9 EET h 11,12 EET
0.0
J i 19-HETEC m n |D j 20-HETE
k k 18-HETE0 n 116-HETE
H ? m 15-HETE
ft n 11-HETEo 8-HETE
1 1 p 12-HETE
- 6 . ■■ A I ! ) L L l A / I M L ..q 5-HETE
.-J10 15 20 25
Figure 3-34. Metabolism of 5-trans-AA by rat liver microsomesLC/MS analysis of a lipid extract obtained from incubation of 5-trans-AA with hepatic microsomes fortified with a NADPH-regenerating system (A). Structural assignment of metabolites hl-h8 and el-e4 is summarized in Table 3-4. For comparison shown are the standards derived from microsomal metabolism of AA (B and C) analyzed under the same conditions. Chromatograms were generated from selected monitoring of ion at m/z 319, with the exception of DiHETEs (B), which were detected by monitoring of ion at m/z 327. Lipids were analyzed on a Cl8 column using a solution composed of water/acetonitrile/acetic acid (37.5:62.5:0.01 v/v/v) at a rate of 300 pl/min.
110
Table 3-4. Identification of microsomal 5 -tra n s-A A metabolites
RetentionTime [min] 5,6-?ra«5-Product
5.8 hi 19-HETE
6.7 h i 13-HETE
7.3 h3 10-and 7-HETE
8.1 h4 15-HETE
8.8 h5 11-HETE
9.3 h6 12-HETE
10.2 h7 8-HETE
11.0 h8 5-HETE
13.2 X
14.5 el 14,15-EET
16.9 e2 11,12-EET
17.8 e3 8,9-EET
19.9 e4 5,6-EET
The retention time and assignment of metabolites corresponds to the labelling on
Figure 3-34; x - indicates a compound showing a parent ion at m/z 319 that did not
produce identifiable fragments following collisional activation and thus remained
unidentified.
I l l
The mass spectral analysis revealed that four epoxides were formed (products el-e4
on Figure 3-34A, Figure 3-35a-d, Table 3-4). Each of these products eluted from the
microbore HPLC column shortly after the corresponding synthetic standard (Figure 3-
34b), which was consistent with the presence of a single trans bond in the microsomal
epoxides. The mass spectra of metabolites el-e4 contained prominent ions at m/z 319,
which upon collisional activation generated characteristic fragments that allowed
structural identification of four epoxides (Figure 3-35a - 3-35d). These spectra were
qualitatively similar to those that detected the cis-EETs, yet subtle differences were
noted.
191319
‘c o o -'co o -127
1375 ,6-frans-8,9-c/s-EET5,6-frans-EET
tIi
t
$1 1912137
219 275 301340
m/z m/z
-COO-
167100 167.+H179 .
319100 •COO-208 ' --113
5,6-frans-l 1,12-c/s-EET219-* °
5,6-frans-14,15-c/s-EET
ÇS179
!113
219175 257 301301
300 340140 180 220 260100 100 220
m/z m/z
Figure 3-35. Mass spectra of four epoxides generated by microsomal metabolism of 5-trans-AAThe mass spectra correspond to the following structures of the fmMj-isomers of epoxides (metabolites el-e4): 5,6-EET (a), 8,9-EET (b), 11,12-EET (c) and 14,15-EET (d) obtained by collisional activation of carboxylate anion at m/z 319.
112
Additional mass spectrometric analysis involved a reaction monitoring technique to
reveal only the compounds that contained a precursor ion at m/z 319 and a fragment
ion at m/z 191 (transition m/z 319 -> 191) during analysis of the microsomal lipid
extract (Figure 3-36). This analysis showed a peak at 20.7 min (Figure 3-36 b) that
had the retention time identical to the peak generated from the 5,6-trans-EET standard
(Figure 3-36 a). Thus, this experiment confirmed that liver microsomes oxidized 5,6-
trans-AA to an epoxide having the oxirane ring of trans configuration.
20.7
5,6-frans-EET
19.0CD
5,6-c/s-EETfCD
•wI 18.7 20.7cco
m/z 319-»191
IW m»,II A
time [min]
Figure 3-36. Identification of S-trans-EJLTMultiple reaction monitoring (LC/MS/MS) was used to detect specific products based on mass transition and retention time. The chromatograms show the relative intensity of the peaks for the equimolar mixture of cis and trans isomers of 5,6-EET (m/z 319 —> 191)(panel a), and the microsomal extract (panel b). A metabolite at 20.7 min has the same transition and retention time as 5,6-/ra?M-EET standard. The LC/MS conditions were as in Figure 3-34, except the mobile phase flow rate was 250 pl/min.
113
Another group of microsomal 5,6-trans-AA metabolites were dihydroxyeicosatrienoic
acids (DiHETrEs), which likely originated from hydrolysis of the corresponding
epoxides (dl-d4, Figure 3-37). The mass spectra revealed that four such compounds
were detected at retention times slightly longer than those for respective all ds-EET
standards (Figure 3-34 b). The structures were assigned based on tandem mass spectra
obtained by collisional activation of carboxylate anion at m/z 337. This result
suggested that because one bond of the precursor substrate fatty acid was of trans
configuration and one of the precursor epoxide had trans configuration, the resulting
5,6-DiHETrE eluting at 6.1 min (Figure 3-37, product d4) had the erythro
configuration.
COOH
HO OH
COOH5.4e5
HO OH
HO OHè*(/)cB
COOH
cco d2
d3HO
d4 COOHOH
0,02 4
m/z 337
14iff > m'lWJH
18 206 8 10 12 16 22
time [min]
Figure 3-37. Identification of diolsMass spectrometric identification of metabolites dl-d4 as diols (DiHETrEs) resulting from hydrolysis of the corresponding epoxides by hepatic microsomes. Full scan spectra were acquired and extracted ion profile is shown for m/z 337.
114
The tandem mass spectra also revealed that 5,6-fraw-AA was metabolized to a group
of products (hl-h8 on Figure 3-34 a, Table 3-4) identified as isomers of HETEs.
These compounds originated from CYP-dependent oxygenation by three mechanisms
allylic, foj-allylic, and subterminal hydroxylations. Among these lipids, metabolite h8
(Figure 3-34a) showed a mass spectrum having fragment ions at m/z 203 and 115 that
was very similar to the spectrum of 5-HETE standard obtained from AA metabolism
by 5-LOX (Figure 3-38). However, although metabolite h8 showed a UV spectrum
typical for a conjugated diene with an a hydroxyl, its maximum absorbance was at
236 nm, i.e. 2 nm more than in the UV spectrum of the 5-HETE standard (Figure 3-
38). This suggested that compound h8 had a different double bond configuration than the 5-HETE standard.
5-HETE from 5,6-trans-AA
5-HETE standard12
10 = 236 nm
= 234 nm8
64
20
200 220 240 260 280 300
wavelength [nm]
115100COO"
OH
80
tB 60
c
I 31940<5
2 257
20 203 301
80 120 160 200 240 280 320
m/z
Figure 3-38. Identification of 5-HETE isomerComparison of UV spectra of metabolite h8 and 5-HETE standard (top). Tandem mass spectrum of metabolite h8 shows characteristic fragments for 5-HETE (bottom).
115
A HETE metabolite eluting at 6.7 min (hi, Figure 3-34 a) showed a mass spectrum
(Figure 3-39a) that was nearly identical to the spectrum of 19-HETE, a known
product of AA metabolism by microsomal co-l hydroxylase. The ions at m/z 59 and
231 were characteristic for metabolite hi and 19-HETE and corresponded to
fragments indicated on Figure 3-39.
319100
275
231 OH(/>c£ 5-fraA7S-19-HETE 301c
Iro2
135
231121
257
60 100 140 180 220 260 300 340m/z
Figure 3-39. Identification of metabolite hiTandem mass spectra of metabolites hi identified as trans isomers of 19-HETE, having characteristic ion m/z 59 and 231.
116
Metabolite hi was further analyzed by GC/MS as a pentafluorobenzyl, trimethylsilyl
derivative and revealed two components (Figure 3-40b). The retention time of these
two metabolites were compared with the retention time of the all cis HETE standards
(Figure 40a) and the products obtained by isomerization of 19-HETE, 18-HETE and
20-HETE induced by the reaction with the N 02 radical (Figure 3-40c). This
experiment demonstrated that a component eluting at 8.12 min (Figure 3-40b)
matched precisely one of the four isomers of 19-HETE generated from the N 02-
mediated isomerization (Figure 3-40c).
a 18-HETE 20-H ETE
16-H ETE 17-HETE
8 0 0 0
19-HETE
4 0 0 0
8.808.408.007.607 .20
5-trans -19-HETE
8 0 0 0
5-trans- 18-HETE4 0 0 0 5-trans-
20-HETE
8 .808 .408.007 .607 .20
trans -19-HETE isomers19-HETE
8 0 0 0
4 0 0 0
8.808.408.007 .607 .20
time [min]
Figure 3-40. GC/MS analysis of metabolite h iChromatograms were obtained by monitoring of ion at m/z 391 generated from the pentafluorobenzyl, trimethylsilyl derivatives and represent analysis of (a) a mixture of five all-ds HETE standards; (b) metabolite hi, and (c) products of 19-HETE treatment with N02 showing coelution (dotted line) of one of the isomers with a microsomal metabolite.
117
A minor component eluting at 7.92 min (Figure 3-40b) was tentatively characterized
as 5,6-trans-l 8-HETE because it coeluted with a product of 18-HETE isomerization
(data not shown). Because this metabolic pattern of HETEs resembled the one that
was observed previously with CYP2E1-mediated metabolism of AA (Laethem et ah,
1993), it was tested whether this isoform of CYP was involved in the microsomal
metabolism of 5,6-trans-PJ±. Pre-incubation of microsomes with a CYP2E1 antibody
reduced formation of metabolite hi (5,6-trahs forms of 19- and 18-HETE) by 57%
(Figure 3-41). Biosynthesis of allylic HETEs was reduced by 28-36% and Ms-allylic
HETEs (see below) by 40-48%. The CYP2E1 antibody had minimal effect on
biosynthesis of epoxides. The data provided evidence that hepatic CYP2E1 isozyme
participated in oxidation of 5,6-trans-AA. at carbons C l9 and Cl 8.
5,6-frans-HETEs2.0e6
controlwith CYP2E1 Ab
COCM1.8e6
1.6e6
SI s
io m1.4e6
$c/)c£
in1.2e6 HI CM CM T v- COcCD h- O
HI 5"1.0e6COCO
ico
8.0e5 co
h-6.0e5 m o
4.0e5
T ■ I ■ I —I
2.0e5
0.0
time [min]
Figure 3-41. The effect of the CYP2E1 antibody on the microsomal metabolism of 5-trans-AAThe number in parenthesis indicates the percent inhibition by antibody treatment for a metabolite represented by a chromatographic peak. The largest inhibitory effect is seen for the formation of trans isomers of 19-HETE and 18-HETE.
118
Mass spectra of metabolites h2 and h3 (Figure 3-34) showed close similarity to the
spectra obtained by Bylund et al. for the three Zus-allylic HETEs formed by liver
microsomes (Bylund et ah, 1998). Metabolite h2 showed a prominent ion at m/z 193
and m/z 149 {m/z 193-C02) and a fragmentation pattern similar to that in the spectrum
of 13-HETE (Figure 3-42). Metabolite h3 showed characteristic ions at m/z 137, 153,
181, 193 and 203 suggesting that this metabolite could be a mixture of 5,6-trans
isomers of 10-HETE, 7-HETE and perhaps some 13-HETE . Consistent with previous
observations, the Zw-allylic HETEs eluted before allylic HETEs on the reverse-phase
HPLC and did not show UV absorbance above 205 nm.
319193100C O G -
80
5-frar7S-13-HETE301
■■5 40
2,57202,03149
140 180m/z
220 260 300100 34060
Q) 60
59 8.1180 220
m/z
Figure 3-42. Identification of metabolite h2 and h3Tandem mass spectra of metabolites h2 (top) and h3 (bottom) identified as trans isomers of 13-HETE and a mixture of 10- and 7-HETE.
119
3.2.2.1 Hepatic microsomes induced decay of 5,6-EET isomers
Incubation of equal amounts (100 ng) of 5,6-trans-EET and 5,6-cw-EET with hepatic
microsomes revealed that the trans epoxide was significantly more resistant to
microsomal metabolism (Figure 3-43). The initial rate of microsomal metabolism, a
significant portion of which was hydrolysis, was about 2 -fold faster for the cis isomer.
Following incubation for 60 min there was 3.6-fold more of the trans isomer
remaining in solution than of the cis isomer.
100 f
80 -5.6-c/s-EET
5.6-frans-EET
«E5D) 60 -o
S 40 't .LLl 20 -
0 20 40 60
9.0e560 min
05
CO
-NE& 14 15 16 17 18 19 20 21
■p 2.8e6
0 min
14 15 16 17 18 19 20 21
4.0
8.5-tDUJto
?cCO
3.0-
2.5-
2 . 0 -o
E
1.0 *0 10 20 30 40 50 60
time [min] time [min]
Figure 3-43. Comparison of hydrolysis rate for cis and trans isomers of 5,6-EET by hepatic microsomesA quantitative LC/MS assay was used to determine the amount of each isomer (a) and the changes of their ratio (b) during incubation. After 60 min 32% of trans(t) and only 6.5 % of cisfcj epoxide remained in solution in an intact form (c) starting from initial equal amount (100 ng) of each isomer (d).
120
3.2.2.2 Glutathione-dependent conjugation of 5,6-EET isomers
Incubation of equal amounts of 5,6-trans-'EEY and 5,6-czj-EET with glutathione-S-
transferase (GST) (Figure 3-44) also revealed that the epoxide was significantly
more resistant to glutathione conjugation than its cis isomer. After 360 min incubation
with hepatic GST, the cis isomer was undetectable whereas 39.2% of the 5,6-trans-
EET was detected in the intact form (Figure 3-44).
co80-
oO)cO)c'c'ro
0)■ o
IQ) 40-
o
20 -0)0_
0 100 200 300 400
Time [Min]
Figure 3-44. Glutathione dependent conjugation of 5,6-EET isomersConcentration of intact 5,6-EET cis and trans isomers (100 ng) remaining in solution after incubation with hepatic glutathione-S-transferase ( 1 8 units) and glutathione ( 1 jllM ) .
121
3.2.2.3 Mass spectrometric identification of 5,6-trans -EET
A method developed by Corey (Corey et a l, 1979) was used with modifications
(Balazy, 1991) to prepare 5,6-trans-EET, 5,6-cz>EET and d^-5,6-cis-EET from 5,6-
trans-AA, AA and dg-AA, respectively. The LC/MS and GC/MS properties were
compared with its cis isomer that was synthesized by the same protocol. Synthetic
5,6-trans-EET wsa used as a standard for metabolic studies with its precursor, 5,6-
trans-AA (Figure 3-45).319
100
191143
163
115137
177203 219 2 4 7 257 275 301129
50 100 150 200 250 300 350
191100
31959.
V)c0) 163cco 137 177
219257 275
247 301
100 150 200 25050 300 350
197100168 327
100
0100 20050 150 250 300 350
m/z
Figure 3-45. Mass spectra of 5-cis and trans-EETComparison of tandem mass spectra obtained by collisional activation at -30 V of the carboxylic anion at m/z 319 generated from 5,6-/ra?zs-EET (top), 5,6-c/s-EET (middle) and of ion m/z 327 from octadeuterium-labeled 5,6-c/s-EET (bottom), which were prepared by iodolactionization of 5,6-trans-AA, AA and dg-AA, respectively. The spectra were averaged and background noise was subtracted.
122
Multiple reaction monitoring (MRM) analysis of m/z 319 and 143 were done. Both
cis and Zrcms-5,6-EET were detected by m/z 319 ion monitoring but only 5 ,6 -m-EET
gave rise to m/z 143 (Figure 3-46).
18.0 19.5
5 ,6 - tran s-E E T
6 8 10 12 14 16 18 20 22 24
tim e [min]
Figure 3-46. LC/MS analysis of cis and trans isomers of 5,6-EETAcquisition of selected ions at m/z 319 and 143 was done. The latter appears only in the cis isomer. Lipids were analyzed on a Phenomenex Luna C18 column and eluted with a solution composed of water/acetonitrile/acetic acid (37.5:62.5:0.01 v/v/v) at a rate of 300 pl/min. Lipids were ionized and detected by the API 2000 mass spectrometer operating in a turbo-ion spray negative mode.
123
3.3 Metabolism of trans-AA by polymorphonuclear leukocytes and HL 60 cells
Metabolism of 5-trans-AA in freshly isolated PMNs and in HL-60 cells, and the
metabolism of 5-trans-AA by 5-LOX in these cells were studied. 5-trans-AA (10"8 to
10'5 M) was incubated with PMNs and with HL-60 cells (DMSO-differentiated and
non-differentiated) stimulated with calcium ionophore (A23187 1 pM) for 2 hours.
PMNs metabolized 5-trans-AA by a concentration-dependent mechanism (Figure 3-
47 B). At submicromolar concentration, 5,6-frYms-8-HETE was the dominant
metabolic product of 8 -LOX, whereas at micromolar concentrations, more of 5,6-
fraws-15-HETE was observed suggesting involvement of 15-LOX. The identity of
these products was confirmed by tandem mass spectra following collisional activation
of ion at m/z 319. Trace amounts of 5,6-trans-5-HETE were also detected (Figure 3-
47 A). However cells, whether differentiated or not, the HL-60 did not produce
detectable LOX products from endogenous and exogenous AA and 5-trans-AA (data
not shown).
124
B
(/)cscC T >
CO
Co
V)c
105x— CO•s
15-frar?s-HETE4.5e5
10 mM 5E-AA4.0e5
3.5e5
3.0e5AZ-trans-HETE
2.5e5
2.0e5
8-trans-HETE
5.0e4
1 3 5 7 9 11 13 15 17 19 21 23 25
Time [min]1.0e7
8.0e6
6.0e6
4.0e6-
2.0e6-ro0CE
0.0
15-frans-HETE12-frans-HETE8-trans-HETE5-trans-HETE
---------- -4— — -------------------------- -------------------------
-------A
0 1 0 -8 1 0*? 1 0 -8 1 0 -5
5-trans-AA concentration [M]
Figure 3-47. Determination of 5-trans-AA metabolic pattern in human PMNsIncubation of 5-trans-AA (10"8-10‘5 M) resulted in a series of trans HETE isomers (A), of which IS-fram-HETE was the most prominent component at higher concentrations; (B). Identification of isomers was accomplished via analysis of tandem mass spectra.
125
4 D ISC U SSIO N
Arachidonic acid is the precursor for a number of biologically active metabolites
formed by oxygenation through the COX, LOX and CYP pathways (Peters-Golden,
1998; Brash, 1999; Smith & Langenbach, 2001). The eicosanoids (PCs and LTs) play
important roles in regulating many physiological processes such as platelet
aggregation and acute inflammatory responses (Funk, 2001). Berdeaux et al. (1996)
observed that the addition of a trans isomer of AA (\A-trans) to rat platelet
suspensions inhibited the aggregation induced by AA. However, the addition of its
structural homolog 5,8,11-eicosatrienoic acid (20:3n-9) or the natural isomer (20:4n-
6) did not induce any modification of the platelet aggregation. Addition of \A-trans-
AA to the platelet significantly decreased TXBz and 12-HHT production but 12-
HETE production was increased 55%. They observed that \A-trans-PJ± alone did not
induce any platelet aggregation, rather this fatty acid was metabolized to a limited
extent into two unidentified products; one of those was a LOX product.
4.1 Interaction of trans-AA with platelets
4.1.1 Metabolism of trans-AA by platelets
When platelets are incubated with AA, the enzyme COX causes endoperoxidation of
AA leading to the formation of PGH2, by the reduction of PGG2 (Hamberg &
Samuelsson, 1974). Isomerization of PGH2 leads to the generation of TXA2, as well
as 12-HHT (Hamberg et al, 1975). In addition to the COX pathway, platelets also
metabolize AA via 12-LOX to form 12-HpETE, followed by the reduction of the
hydroperoxide to form the stable end product 12-HETE (Hamberg et al, 1975;
Nugteren, 1975; Hamberg & Hamberg, 1980). To observe the metabolites of AA
production, platelets were incubated with different concentrations of radio labeled [1-
14C] AAs for various length of time. After incubation, the fatty acids were extracted
and HPLC analysis was performed. Results were obtained using either radioactivity
detector or variable wavelength UV detector.
126
AA metabolism by platelets produced TXB2, 12-HHT and 12-HETE (Figure 3-1).
The identity of these metabolites was confirmed by chromatography and UV spectral
analysis, comparing with the standards. \A-trans-AA did not produce such
metabolites and showed less metabolism by platelets in comparison to AA (Figure 3-1
C). However, this metabolism was shown to give rise to four different metabolites, as
their elution time was different from the conventional metabolites. These novel
metabolites were labeled as metabolite A, B, C, and D (Figure 3-1). Further LC/MS
analyses were done to characterize these novel metabolites.
When indomethacin, a potent COX inhibitor, was used in the test system, there was
no formation of TXB2, 12-HHT or metabolite B or C. This suggested that the
metabolites B and C are the product of the COX pathway (Figure 3-4 B). Baicalein, a
potent LOX inhibitor, was found to cause a decrease in the production of 12-HETE,
metabolite A and D. This suggested that these metabolites were the product of the
LOX pathway (Figure 3-4 C).
Non-radiolabeled AA was used along with radiolabeled \4-trans-AA to observe the
influence of AA on metabolism of trans-AA. It was found that metabolism of 14-
trans-AA was increased in the presence of AA as all the novel metabolite production
was increased (Figure 3-5). It suggests that AA has an influence on metabolism of 14-
trans-AA by human platelets. The \4-trans-AA metabolism was increased in the
presence of AA because of co-oxidation of trans-AA in the presence of AA that
caused formation of increased amounts of unidentified metabolites. Co-oxidation of
xenobiotics by AA, COX, and LOX has been described (Nadin & Murray, 2000).
Then the COX inhibitor was used and it was found that there was no formation of
metabolites B and C (Figure 3-5 B). The LOX inhibitor inhibited the formation of
metabolites A and D (Figure 3-5 D). These experiments further confirmed that
unknown metabolites B and C are COX products, and metabolites A and D are LOX
products.
In order to characterize metabolites C and D, they were subjected to spectral analysis
using an HPLC system equipped with a multiple wavelength detector. It was observed
that both metabolite C and D showed absorption maxima at 236 nm (Figure 3-7)
however, both were distinct from the absorption maxima of 12-HETE at 234 nm.
127
Metabolites formed by platelets from lA-trans-kA showed seven peaks while using
LC/MS (Figure 3-8 B) as compared to using radio-chromatogram where there were
four peaks (A, B, C and D) (Figure 3-1 B). As LC/MS has better resolution and
identification capacity, it detected all the metabolites and those were named
metabolite A, B%, B2, Ch C2, D% and D2. These metabolites were injected into a HPLC
system equipped with online mass spectrometer by using an auto injector and
negative-ion MS/MS were done for identification of their structures. The MS/MS
spectra of novel metabolites were compared with spectra of commercial standards.
A. Identification of metabolite A
a. Molecular mass of metabolite A
Metabolite A was detected during the LC/MS analysis of lipids from the incubation of
\4-trans-KK with platelets. Metabolite A showed a prominent negative ion at m/z
369. It eluted between two isomers of TXB2 (Figure 3-8 B). The retention times were
as follows (on a standard HPLC and LC/MS system): TXB2 (peak 1), 7.7 min,
metabolite A 8.1 min, and TXB2 (peak 2) 8.7 min (Figure. 3-9). Metabolite A was a
chromatographically distinguishable metabolite having a molecular mass of 370.
Analysis of fractions 8 and 9 (Figure 3-8) containing TXB2 isomers and metabolite A
by GC/MS (negative ion chemical ionization) as a pentafluorobenzyl (PFB) ester,
trimethylsilyl (TMS) ether (Figure 4-1) revealed two peaks (Figure 3-13 A) by
monitoring of an ion m/z 585. This ion corresponded to a carboxylic anion of the
trimethylsilyl derivative of TXB2 (Figure 3-13 B) shown below for the TXB2
derivative:
128
5 8 5 — 1OTMS
TMSO'OTMS
çh3TMS = — Si— CH3
c h 3
Figure 4-1. Trimethylsilyl ether derivative of thromboxane B2
Figure shows the structure of TMS derivative of PFB ester of TXB2 (above) and the structure of TMS (below).
Metabolite A showed the same ion suggesting a structure isomeric with TXB2.
Additional analysis involved the preparation of all TMS derivatives of fractions 7-9
(Figure 3-13) and analysis by GC/MS via electron ionization. Two spectra were
obtained. One was interpreted as corresponding to the tetra TMS derivative of TXB2
and the other to the unknown metabolite A. Both spectra showed a molecular ion
(M+) at m/z 585. The GC/MS analyses also revealed that metabolite A had 3
hydroxyl groups and one non-hydroxyl oxygen similar to TXB2. In both GC/MS
analyses, TXB2 derivatives of the two isomers observed by HPLC and LC/MS
analyses eluted as a single peak, distinguishable from the peak generated by
metabolite A. Thus mass spectrometric analysis provided evidence that metabolite A
had molecular mass of 370 and was likely to be an isomer of TXB2 or a prostaglandin.
b. Biosynthesis of metabolite A
Two inhibitors were used to probe the biosynthetic pathways of \A-trans-KK
metabolites in platelets; indomethacin, which inhibits platelet COX at IC50 0.1 pM
and baicalein, a plant flavonoid that inhibits platelet 12-LOX at IC50 0.12 pM.
Experiments with inhibitors confirmed preliminary observations with 14C-labeled 14-
trans-AA that metabolite A is a metabolite of 12-LOX. Preincubation of platelets with
129
indomethacin (10 iM) removed all COX metabolites as tested with AA metabolism
(Figure 3-10), which revealed only 12-HETE. However, the formation of metabolite
A was not affected by indomethacin (Figure 3-10 B) as compared to control
experiments without an inhibitor (Figure 3-10 C). The control experiment also
showed peaks corresponding to TXB2 resulting from adventitious activation of
platelets during platelet preparation and incubation. This TXB2 was likely to be
formed from the endogenous AA.
Treatment of cells with baicalein (10 pM) caused partial inhibition of 12-LOX and the
formation of its metabolite 12-HETE. TXB2 levels were increased relative to the
control (Figure 3-11 A vs. Figure 3-8 A). Baicalein inhibited the formation of the 12-
HETE isomer from \4-trans-hA metabolism and also inhibited the biosynthesis of
metabolite A (Figure 3-11 B vs. Figure 3-11 C). The results with the inhibitors
suggest that metabolite A is likely to be a product of 12-LOX. The results were
confirmed by the analysis of octadeuterium-labeled \A-trans-AA {à%-\A-trans-AA)
metabolism in platelets. Compared to control experiments (Figure 3-12 A),
indomethacin caused the increase of metabolite A and the 12-HETE analogue (Figure
3-12 B) whereas baicalein completely inhibited the formation of the deuterium
analogue of metabolite A (Figure 3-12 C). Analysis of &%-\4-trans-AA metabolism
and specific ions corresponding to deuterated metabolites was useful because it was
not confused with AA metabolites that lacked the deuterium label. Experiments with
both labeled and non-labeled \A-trans-AA revealed a novel unique lipid product
generated by 12-LOX having similar chromatographic polarity (by GC, LC) as TXB2
and possibly being its isomer. Such a prostaglandin-like metabolite generated by 12-
LOX has not been observed previously.
c. Structure of metabolite A
Data obtained so far suggested that metabolite A is a 12-LOX metabolite having the
molecular mass of 370 identical to TXB2 and containing 3 hydroxyls, one non
hydroxyl oxygen and a carboxylic group. Metabolite A was formed in amounts
comparable to TXB2 from endogenous AA (Figure 3-11 C). However, mass spectra of
metabolite A showed a lack of similarity with mass spectra of TXB2 obtained by
LC/MS/MS and GC/MS.
130
The LC/MS/MS spectrum of metabolite A obtained by collisional activation of its
carboxylic anion at m/z 369 (Figure 3-14 A) showed a prominent ion at m/z 127
which was shifted by 2 a.m.u. in the spectrum of deuterium-labeled metabolite A
(Figure 3-14 B) indicating that it had one of the original double bond configuration.
Additionally, ions of m/z 155 and 158 were observed in the spectra of unlabeled and
dg-labeled metabolite A respectively, suggesting that it had three carbons that were
bound to the deuterium in the precursor substrate \A-trans-AA. In trying to identify
the structure of metabolite A (Figure 4-2), it was noticed that prominent ions at m/z
127 and 155 are also found in the spectrum of isoprostaglandin Fia-IV (Morrow et al,
1990), therefore it is likely that a similar structural fragment is in metabolite A.
,,155225 V /— 127
X * '
OH OH
COOH
Suggested structure of metabolite A MW 370
OH OH
HO
Suggested structure of dS-metabolite A MW 378
Figure 4-2. Suggested structure of metabolite A and dg-metabolite AIn LC/MS/MS study metabolite A shows ions at m/z 127, 155 and 225 (above) and structure of d8-metabolite A below.
131
The structures of ions at m/z 127 and 155 (Figure 4-3) for metabolite A, are proposed
as follows:
.COO"
m/z 127
COO"
m/z 155
Figure 4-3. Suggested structures of ions at m/z 127 and 155Figure shows the proposed structures of ion at m/z 127 and ion at m/z 155 originated from the a cleavage of metabolite A.
Both ions are generated from a bond cleavage at the hydroxyl group at carbon C8 .
The ion at m/z 127 originated from a cleavage of bond C7-C8 followed by the
transfer of a hydrogen to C7. Ion at m/z 155 originated from the a cleavage of the C8-
C9 bond with the abstraction of hydrogen generating an aldehyde. Additional
fragments can be generated from the upper chain of metabolite A structure. Ions at
m/z 181 and 183 (Figure 4-4) originated from the keto-enol tautomeric form that can
exist for metabolite A.— 183
OH
COOH
HO
Keto form of metabolite A
COO"
m/z 183
Figure 4-4. Suggested structure of keto form of metabolite ALC/MS/MS of keto form of metabolite A gives rise to ion at m/z 183 (above) and the structure of ion m/z 183 is shown (below).
132
Additional evidence to support the proposed structure of metabolite A comes from the
detection of ions at m/z 225 and 195 (Figure 4-5). Ion 225 is likely to originate from
the cleavage of the pyrene ring.
COO"
m/z 225
HCO
COO"
m/z 195
Figure 4-5. Formation of ion at m/z 195 from ion m/z 225Figure shows the formation of ion m/z 195 from ion m/z 225 that is likely to originate from the cleavage of the pyrene ring.
While the presence of the ion at m/z 195 could be confused with the same ion that
originates from the analysis of TXB2 (Figure 4-6) and because metabolite A and
TXB2 eluted closely, there was same uncertainty about this ion.
OH
OH Thromboxane B2
LC/MS/MS
0▼ ! ^ v
m/z 169 m/z 195
Figure 4-6. Structures of LC/MS/MS cleavage products of TXB2Figure shows the structures of LC/MS/MS cleavage products of thromboxane B2 (ions at m/z 169 and m/z 195)
133
This concern was removed by the analysis of metabolite A biosynthesized by platelets
from octadeuterium-labeled \A-trans-AA. Deuterium-labeled metabolite A produced
a carboxylic anion at m/z 377 suggesting that it originated from the deuterium-labeled
precursor \A-trans-AA. The spectrum of dg-metabolite A showed a prominent ion at
m/z 129 (Figure 4-7), which suggested that it contained two deuterium atoms as
compared to ion 1 at m/z 127 from non-labeled metabolite (Figure 3-14).
Figure 4-7. Ions of d8-metabolite A from dg-labeled \4-trans-AAFigure shows LC/MS/MS fragments of dg-metabolite A, showed a prominent ion at m/z 129 and ions at m/z 158 and m/z 2 0 0 .
Similar reasoning leads to structure of ions at m/z 158 and 200, which contained three
and five deuterium atoms, respectively.
d. Proposed mechanism of metabolite A biosynthesis
The data suggested that metabolite A is a unique metabolite that was a product of 12-
LOX likely having a structure similar to that of thromboxane. A key experiment was
performed with purified 12-LOX enzyme, which was incubated with \A-trans-hh.
This experiment yielded a metabolite that had a spectrum showing ions at m/z 127,
155 and 183 from the activation of the ion at 369 (Figure 3-14 C). Metabolite A was
likely to be formed by the following mechanism (Figure 4-8).
D D
m/z 129
O
m/z 158
OH d D
D m/z 200
134
12-LOX
COOH
14-trans-12-HpETE radical
0 0
00COOH
IsomerizationOH
COOH
ReductionOH OH t
COOH
HO'
Iso-thromboxane (Metabolite A)
Figure 4-8. Proposed mechanism of formation of metabolite A14-trans-AA is metabolized by platelet 12-LOX to 14-trans-12-HpETE radical, which by isomerization and reduction forms iso-thromboxane.
In summary, metabolite A is a product of 12-LOX in platelets and obtained from a
purified enzyme. The biosynthesis of metabolite A in platelets was inhibited by
baicalein. Ions characteristic for metabolite A (m/z 127, 155, 183) were also found in
an iso-prostaglandin F2a type IV (Morrow et al, 1990), which strongly supported the
configuration of the upper chain containing the carboxyl group of metabolite A.
Metabolite A has a structure similar to TX. Many other possibilities was considered
135
for structure of this metabolite that included prostacyclin, PGs, iso-PGs and LT
structures. None of these structures fit the proposed structure of metabolite A. It is
interesting to note that a structure similar to metabolite A was reported by Morrow et
al., (1996) who reported iso-thromboxane B2 type III (Morrow et a l, 1996) from
spontaneous oxidation of AA in vitro. In contrast to the report by Marrow et al.,
metabolite A described here is an enzymatic product of platelet 12-LOX.
Thromboxane synthase may be involved in the formation of metabolite A in platelets.
B. Identification of metabolite B
Metabolite B contained two isomers (B% and B2, on Figure 3-8) that show LC/MS/MS
spectra almost identical to the spectrum of standard 1 2 -hydroperoxyeicosatrienoic
acid (12-HHT). Metabolite Bi (Figure 4-9) was the authentic 12-HHT resulting from
spontaneous decomposition of TXA2 and loss of malonyldialdehyde (Figure. 3-9).
Metabolite B2 was likely to have the structure shown on the Figure 3-16 with the bond
C1 0 -C1 1 having trans configuration.
Figure 4-9. Formation of ion m/z 179 from MetaboliteFigure shows the suggested structure of metabolite Bi (12-HHT) and LC/MS/MS product of metabolite Bi which formed ion m/z 179.It is unlikely that metabolite B2 (Figure 4-10) originated from spontaneous
decomposition of metabolite A, which was a LOX product.
COO
OH12-HHT
(Suggested structure of metabolite
LC/MS/MS
COO"
m/z 179
136
8-ZraA7S-12-HHT(Suggested structure of metabolite B2)
LC/MS/MS
m/z 179
Figure 4-10. Formation of ion m/z 179 from Metabolite B2
Figure shows the suggested structure of metabolite B2 (%-trans-12-HHT) and LC/MS/MS product of metabolite B2 which formed ion m/z 179.
Metabolite B2 was probably a by-product of endogenous AA metabolism by
COX/TXS (thromboxane synthase).
C. Identification of metabolite C
a. Metabolite Ci
Metabolite Ci (Figure 3-8, 3-10) was a major product of \A-trans-AA metabolism.
Biosynthesis of this metabolite was inhibited more than 80% by indomethacin but not
by baicalein suggesting that metabolite Ci was a product of COX. This metabolite
showed maximal U V absorbance at Xmax 236 (Figure 3-7) and retention time close to
15-HETE (Figure 3-9). In the co-incubation experiments with equimolar amounts of
AA, the formation of metabolite 0% was accelerated by about 5-fold (Figure 3-3).
LC/MS analysis showed abundant carboxylate anion at m/z 319, which when
collisionally fragmented showed a pattern of ions characteristic for a HETE. The
LC/MS/MS spectrum of metabolite Ci (Figure 3-17 A) showed fragments
corresponding to loss of H2O (m/z 301), CO2 (m/z 275) and H2O plus CO2 (m/z 257)
137
which one typically found in the spectra of HETE molecules. An abundant fragment
at m/z 219 (Figure 4-11) was likely to correspond to a fragment anion (C13H19COO")
resulting from the cleavage at the hydroxyl group (Figure 3-17 A). Loss of 44 a.m.u.
(CO2) yielded anion at m/z 175. Both ions are also found in the LC/MS/MS spectrum
of a standard sample of 15-HETE. Ion at m/z 113 is also typical for 15-HETE and
results from the cleavage of the C6-C7 bond with a hydrogen shift yielding anion
C5H9COO". The key question about the structure of metabolite Ci is the configuration
of the double bond next to the hydroxyl group. In 15-HETE the configuration of this
bond (but carbons C13-C14) is trans. In metabolite C% this bond is likely to be c/s.
This conclusion is supported by detection of ion at m/z 121 in the spectrum of
metabolite Ci. This ion is absent in the spectrum of 15-HETE. Ion at m/z 121 is likely
to originate from cyclization induced alcoxyl anion and attack C8 yielding an eight
carbon anion.
COO
OHall-c/s-15-HETE
(Suggested structure of metabolite C.,)
LC/MS/MS
Figure 4-11. LC/MS/MS cleavage of metabolite CiLC/MS/MS metabolite C% (all-c/s-15-HETE) formed ions m/z 219 and m/z 175.
Ion 121 can be formed only if the C13-C14 bond is cis, if this bond is trans as in 15-
HETE, ion 121 is not formed. Thus the data are consistent with the metabolite Ci
being an isomer of 15-HETE, the all-c/j-15-HETE. Incubation of platelets with
138
deuterium-labeled \A-trans-AA (dg-14-rmMJ-AA) as shown in Figure 3-12 resulted in
octadeuterium-labeled metabolite C% having molecular mass of 328 (Figure 3-17 B).
Ion at m/z 226 in this spectrum was at 7 a.m.u. higher than ion 219 in unlabeled
metabolite Ci, thus confirming the structure of 15-HETE. There was an ion analogous
to ion 121 at m/z 125, and it was very small probably because of isotopic effects
involved in its formation. Thus \4-trans-AA was metabolized mainly to 15-HETE by
COX, and the LOX inhibitor stimulated its formation. The UV absorbance maxima of
metabolite Ci shifted to the chronically by 2 nm in comparison to the maxima of the
UV absorbance for 15-HETE standard. 15-HETE which has a trans-cis configuration
of the conjugated bonds in the a position to the hydroxyl ion and its absorbance
maximum is at 234 nm. The absorbance at Xmax= 236 nm would be consistent with a
cis-cis configuration. Thus the structure of metabolite Ci, is likely to be
5Z,8Z, 11Z, 13Z-15-hydroxyeicosatetraenoic acid (all-cz's-15 -HETE). The
stereochemistry of the hydroxyl was not determined because of the limited
availability of the material.
b. Metabolite C2
Metabolite C2 was also a product of COX and was a HETE molecule. Its LC/MS/MS
spectrum showed a characteristic ion at m/z 167 (Figure 3-18 A) that was also found
in the spectrum of 11-HETE standard. This ion was shifted by 4 a.m.u. to m/z 171 in
the spectrum of octadeuterium-labeled metabolite C2 (Figure 3-18 B). This fragment
ion likely originated from the cleavage of the C10-C11 bond and contains a
transformed proton, and has the structure of a carboxylate anion C9H15COO" or
C9H11D4COO' in deuterium analogue. Thus the mass spectrum and work with
inhibitors suggested that metabolites C2 was likely to be an isomer of 11-HETE. The
spectrum of this metabolite showed a lot of similarity with the mass spectrum of 1 1 -
HETE standard (Figure 3-18 C).
139
HO14-fraA7S-11-HETE
(Suggested structure of metabolite C2)
LC/MS/MS
m/z 167
Figure 4-12. LC/MS/MS cleavage of metabolite C2
LC/MS/MS metabolite C2 ( 14-trans-11 -HETE) formed ion m/z 167.
The double bond configuration of the C14-C15 bond was unlikely to be changed as a
result of incubation with platelets and metabolism of 14-trans-AA by COX, and thus
the data support the structure of 14-trans-11 -HETE (5Z,8Z,12E,14E-11-
hydroxyeicosa-tetraenoic acid) (Figure 4-12) for metabolite C2. \4-trans-\ 1-HETE
contains a hydroxyl ion that is in a position to the two conjugated trans double bonds.
The maximal absorbance of such configuration is at >umax = 236 nm, identical to
metabolite Ci.
D. Identification of metabolite D
a. Metabolite D%
Metabolite D was a product of LOX as demonstrated in preliminary results on Figure
3-4 using radiolabeled 14C-14-/nms-AA. More detailed analysis using LC/MS
revealed two components in metabolite D (Figure 3-8, 3-10). Biosynthesis of
metabolite Di was almost completely inhibited by treatment of platelets with baicalein
suggesting that this metabolite was a product of 12-LOX (Figure 3-11, 3-12). The
LC/MS/MS spectrum of metabolite Di (Figure 3-19) showed a prominent ion at m/z
179 resulting in a form that is typical for HETEs fragmentation at carbon bond next to
the hydroxyl group.
140
OH14-frans-12-HETE
(Suggested structure of metabolite D.,)
LC/MS/MS
m/z 179
Figure 4-13. LC/MS/MS cleavage of metabolite DiLC/MS/MS metabolite D% ( 14-trans-12-HETE) formed ion m/z 179.
Experiments with deuterium-labeled 14-trans-AA yielded an octadeuterium-labeled
metabolite D showing an ion at m/z 184 (Figure 3-19 B) that is consistent with a
fragment having 5 deuterium. Loss of 44 a.m.u. (CO2) from ions 179 (Figure 4-13)
and 184 produced ions at m/z 135 and 140, respectively. The data suggest that
metabolite D% was a 14-trans-12-HETE. The spectrum of metabolite Di was similar to
the spectrum of 12-HETE standard. The trans bond from precursor substrate was not
likely to change the configuration as a result of the reaction with 12-LOX and this
trans bond had no effect on the fragmentation. However, 14-trans-12-HETE eluted
after the peak of 12-HETE (Figure 3-9).
b. Metabolite D2
A minor component of metabolite D designated as metabolite D2 (Figure 3-8, 3-10, 3-
11) was likely a product of COX as experiments with d^-14-trans-AA showed
complete inhibition of formation by indomethacin (Figure 3-12). Baicalein also had
some inhibitory effect on its formation (Figure 3-11). The mass spectrum revealed the
structure of metabolite D2 to be a 14-/r<ms-5-HETE (Figure 4-14).
141
OH
14-frans-5-HETE (Suggested structure of metabolite D2)
LC/MS/MS
▼O
m/z 115
Figure 4-14. LC/MS/MS cleavage of metabolite D2
LC/MS/MS metabolite D2 ( 1 A-trans-5-HETE) formed ion m/z 115.
In summary, \A-trans-AA was metabolized by two set of enzymes (COX and LOX)
in the platelets. The metabolites of COX pathway were 12-HHT, all-ds-15-HETE,
\A-trans-\ 1-HETE, and 14-/ra«5-5-HETE and the LOX pathway were iso
thromboxane, 12-HHT and 14-trans-12-HETE. The metabolites are shown in
Figure 4-15.
142
Cyclooxygenase
14-frans-AA
Lipoxygenase
HO
OH14-frans-11-HETE (C2)
12-HHT (BJ OH
OH
all-c/s-15-HETE (CJ 14-frans-5-HETE (D2)
HO
OH OHCOOH
Iso-thromboxane (A) 8-frans-12-HHT (B2) 14-frans-12-HETE (D^
Figure 4-15. Summary of metabolism of 14-trans-AA by human platelets14-/nms-AA was metabolized by COX and LOX in platelets. The COX products were 12- HHT, 13-trans-15-HETE, M-frcws-l 1-HETE, and 14-£r<ms-5-HETE and the LOX products were iso-thromboxane, %-trans-12-HHT and 14-trans-12-HETE.
4.1.2 Effects of 14-trans-AA on platelet function
In order to determine whether the effects of trans-AA on rat platelets showed by
Berdeaux et al, exist in human, the current study looked at the same experiments
using human platelets. Arachidonic acid (15 pM) induced aggregation of human
platelets and different concentrations (15, 50 & 100 pM) of 14-trans-AA were used to
induce platelet aggregation, but there was no evidence of platelet aggregation or shape
change (Figure 3-21). This led to the idea that 14-trans-AA does not cause platelet
aggregation or shape change by itself. Indeed, aggregation of platelets by AA was
decreased by pre-incubation with 14-trans-AA (Figure 3-22), consistent with the
143
previous result (Berdeaux et ah, 1996). These results suggest that the aggregation of
platelets by AA was inhibited or antagonized by \A-trans-PsA or by novel metabolites
o f \A-trans-AA.
In order to observe the concentration-response aggregation shape (Figure 3-24),
platelets were pre-incubated with different amounts of \A-trans-AA (0, 3, 5, 7, 9 & 11
pM) and AA (3 pM). The observed results showed that the inhibitory effect of 14-
trans-AA is concentration-dependent and the IC50 (inhibitory concentration at 50%)
of lA-trans-AA was found to be at 7.2 pM.
Since TXA2 is known to cause platelet aggregation and the level of TXB2 (non-
enzymatic hydrolyzed product of TXA2) reflects the level of TXA2, experiments were
performed with measurement of TXB2 level to determine the effects of TXB2 on
platelets treated with \A-trans-AA. While measuring aggregation levels, the level of
TXB2 was measured using LC/MS against a standard curve (Figure 2-8). Platelets
were pre-incubated with lA-trans-AA and results showed that the levels of TXB2, as
seen in Figure 3-25, decreased in the presence of increasing amounts of lA-trans-AA.
At the highest concentration of lA-trans-AA (11 pM), approximately 1700 ng/ml of
TXB2 was found. So, while the formation on TXB2 was not completely inhibited,
platelet aggregation was completely prevented. This experiment confirmed that the
inhibition of platelet aggregation by lA-trans-AA occurs in the presence of TXB2
production.
Tests were also performed to determine whether lA-trans-AA inhibits TP receptor-
mediated aggregation. A TP receptor agonist (U46619) was used to observe platelet
aggregation. Platelets were pre-incubated with different amounts of lA-trans-AA and
U46619 was added to observe the effect of lA-trans-AA on U46619-mediated
aggregation. After careful observation and data interpretation, it was determined that
the inhibition of U46619-mediated aggregation by lA-trans-AA is concentration
dependent and the IC50 was found to be 2.2 pM (Figure 3-26). This experiment
confirms that lA-trans-AA inhibits TP receptor aggregation with U46619. Platelet 14-
trans-AA metabolism may result in the formation of novel products that could inhibit
platelet aggregation. This is why the structural characterization of lA-trans-AA
metabolites was an important part of understanding its action on platelets.
144
The effects of 14-trans-AA on other pathways of platelet activation were also
explored. To observe the effects of \A-trans-AA on other agonists, platelets were
incubated with ADP, epinephrine, thrombin and cPAF. It was observed that lA-trans-
AA (5 pM) inhibited ADP-mediated (50 pM) aggregation by 87%±8 (Figure 3-27),
epinephrine-induced (50 pM) aggregation by 85%±12 (Figure 3-28), thrombin ( 0.5
unit/ml) mediated aggregation by 75% ± 1 0 (Figure 3-29) and cPAF (50 pM)
mediated platelet aggregation by 80% ± 10 (Figure 3-30). These experiments show
that the inhibitory effect is not specific for the TP receptor. lA-trans-AA is a non
specific inhibitor that inhibits ADP-, epinephrine-, thrombin- and cPAF-mediated platelet aggregation.
Experiments were also performed to determine the amount of trans-AA and AA that
goes into the membrane phospholipids. Platelets were incubated with 14C-AA or 14C-
14-trans-AA to assess the incorporation into membrane phospholipids. The data
presented in Figure 3-31 shows that 14-trans-AA was transported into platelets about
3 times faster than AA. At least part of 14-trans-AA was likely to be esterified in
platelet phospholipids membrane. This experiment demonstrates that avid binding of
14-trans-AA by platelets could result in changes in the phospholipid membrane near
various receptors that could decrease the affinity of receptors to their ligands.
4.1.3 Relationship between metabolism and effects
It is very well known that metabolism of AA has profound effect on platelet function
as the COX-mediated product (TXA2) causes receptor-mediated aggregation. It was
anticipated that trans-AA. could be metabolized into oxidized lipids that may have
modulatory effects on platelets. The study revealed that lA-trans-AA is metabolized
into novel products and AA stimulates trans-AA metabolism, trans-AA metabolites
were structurally profiled and a complete profile of metabolism, which is original and
unique was established. But the amount of generated metabolites were very low,
therefore it was not feasible to isolate and purify sufficient quantity for biological
activity study. However, this study established the structures and enzymatic origins of
the metabolites even though it was not possible to synthesize these metabolites in
larger quantities for testing with platelet. Particularly the observation of the
145
production of metabolite A is very interesting because it is an analogue of TX and
therefore, it is possible that it may be relevant and may modulate platelet function via
interaction with TP receptor. Further study is required to establish the role of
metabolite A as a mediator of the inhibitory effect of \A-trans-KA. Figure 4-16.
shows the formation o f \A-trans-AA and it’s effects on platelet function.
NO Nitro oxidation N 0 2"
NO,
trans-AA
Partial inhibition of TXAo formation
iPlatelets
Metabolism
Unknownmechanism inhibition of
aggregation
COX
8-frans-12-HHTall-c/s-15-HETE14-frans-11-HETE14-frans-5-HETE
LOX
14-frans-12-HETEv
Iso-TXA,
? effect unknown
Figure 4-16.14-trans-AA and platelet functionIllustration shows the basic concept of interaction of 14-trans-AA and platelet. The key expected effect of 14-trans-AA is that it will reduce platelet aggregation induced by AA. It is also expected that while aggregation will be greatly reduced in the presence of AA, the biosynthesis of PGHa endoperoxidase is not efficiently reduced therefore some of PGH2 may be available for endothelial cells to be converted into PGI2. Thus, \4-trans-AA may favourably affect the balance between TXA2 and PGI2.
146
4.2 Metabolism of frans-AA by hepatic microsomes
Previous experiments with \4-trans-AA and platelets have found that \4-trans-P±A is
not readily metabolized by platelet COX and LOX as compared to AA (Figure 3-1 C).
Thus, it was thought that perhaps trans-AA isomers function more like xenobiotics
amenable to hepatic microsomal metabolism that could be of importance in their
activation into more reactive oxidized lipids. Most of what has been known in the area
of polyunsaturated fatty acid metabolism by hepatic microsomes involves studies of
CYP) 45O/NADPH-dependent monooxygenation of the cis double bonds into
epoxides of cis configuration. Such a process generates four epoxides (EETs) of AA
(Capdevila et a l, 1981). Further metabolism of the EETs involves hydrolysis to
vicinal diols, dihydroxyeicosatrienoic acids (DiHETEs), by cytosolic or microsomal
epoxide hydrolase (Poff & Balazy, 2004b). In contrast, microsomal metabolism of
fatty acids that have both cis and trans bonds has not been well characterized. Studies
of the trans-AA metabolism could be important in providing a better understanding of
their biological effects (Jiang et al, 1999; Balazy & Lopez-Femandez, 2003) as these
fatty acids have been suggested to contribute to cardiovascular disorders, cancer and
other pathologies (Hunter, 1982; Ascherio et al, 1994). Therefore, trans-AAs were
incubated with hepatic microsomes to understand the metabolic profile of their CYP
metabolism.
4.2.1 Metabolism of 14-frans-AA by microsomes
Hepatic microsomes were incubated with AA and \A-trans-AA. Metabolism of AA
and trans-AA required oxygen and reduced NADP indicating that these fatty acids
were oxidized by a CYP450 epoxygenase, a monooxygenase enzyme. At incubation
time of 90 min or longer, vicinal diols (DiHETEs) were the major products (Figure 3-
32). They originated from hydrolysis of the initially formed epoxides (EETs) of trans-
AA and AA by microsomal epoxide hydrolase.
However, the metabolic profile of 14-trans-AA was much different from that of AA
(Figure 3-32 A and B). While AA produced four major metabolites (cl-c4), lA-trans-
147
AA produced three metabolites at a comparable rate of metabolism (Figure 3-
32). The retention times of tl-t3 were longer than cl-c4 by about 1 min suggesting
that they were likely to be Zrcms-isomers of cl-c4, because a similar difference was
seen between trans-AA and AA. Incubations of equimolar amounts of AA and trans-
AA generated a complex mixture of metabolites and indicated that 14-trans-AA
modulated the metabolism of AA (Figure 3-32 C).
GC-MS analysis confirmed that the four AA metabolites cl-c4 were DiHETE
molecules. Each fraction produced a single chromatographic peak and a spectrum
with an abundant ion at m/z 483, which corresponded to a diTMS 14C-labeled
DiHETE carboxyl anion. Comparison with synthetic standards allowed structural
identification of cl-c4 (Table 3-3). Thus, metabolites cl-c4 corresponded to four
DiHETEs, which were the hydrolysis products of the respective EETs (el-e4). GC-
MS analysis of tl-t3 also revealed ions at m/z 483 (Figure 3-33). Fraction tl
contained two closely eluting peaks tla and tlb (Figure 3-33) whereas t3 was a
relatively minor product. This suggested that tl-t3 were DiHETEs and thus 14-trans-
AA, like AA, was metabolized primarily by epoxygenase and epoxide hydrolase
system. Comparison of retention times (Table 3-3) revealed that metabolite tlb had
similar retention time as 14,15-DiHETE and thus was likely to be 14,\5-erythro-
DiHETE resulting from hydrolysis of 14,15-fnms-EET. This observation is consistent
with another study which showed that the erythro isomer tlb eluted before the threo
isomer cl (Abalain et a l, 1983).
Further confirmation of this observation was obtained by analysis of products of the
reaction of lA-trans-AA. with m-chloroperoxybenzoic acid (MCPBA), which
produced a mixture of epoxides. These epoxides were separated and hydrolyzed by
incubation with microsomes. The most polar and thus earlier eluting epoxide by
HPLC was isolated and following hydrolysis produced a DiHETE that had identical
retention time as the microsomal product tlb (Figure 3-33). This procedure provided
fairly pure standards of 14,15-rra?zs-EET and 14,15-e^/zra-DiHETE for comparison
with microsomal products. Other 14-frYzws-AA-derived microsomal metabolites had
retention times similar to those of other standard DiHETEs (Table 3-3). Thus, because
three cis bonds in lA-trans-P^A were available for epoxidation, it suggests that 14-
trans-AA was metabolized via a CYP P450/NADPH system to four epoxides, which
148
were hydrolyzed by epoxide hydrolase to one erythro diol and three threo diols each
having a trans double bond (Figure 4-17).
COOH
1 4 ,1 5 -fra n s-A A
C Y P450 NAD PH
COOH COOH
+ 8 ,9 a n d 5 ,6 is o m e rs
1 4 ,1 5 - f r a n s -E E T 1 4 ,1 5 - tr a n s -1 1 ,1 2 -c /s -E E T
e p o x id eh y d ro la s e
COOH COOH
OH
HO
1 4 ,1 5 -e ry fh ro -D i H E I E
</ = X / = x / x / vu'
1 4 ,1 5 - tr a n s -1 1 ,1 2 -fftreo -D i H E T E
+ 8 ,9 a n d 5 ,6 is o m e rs
Figure 4-17. Microsomal epoxidation of 14-trans-AAThe epoxidation of 14-trans-AA by hepatic microsome produced four epoxides one of which was a trans epoxide and 3 other were cis epoxides. Microsomal epoxide hydrolase converted the epoxides into diols, one of which was an erythro diol and 3 other were threo diols with one trans double bond.
Hydrolysis of epoxides by microsomal and cytosolic epoxide hydrolases is highly
stereoselective. Thus, c/s-EETs generate exclusively Z/zreo-DiHETEs by an SN2
mechanism described by Zeldin et al, (1993) and while Znms-EETs have not been
observed before, other trans epoxides such as a Zmw-epoxide of stearic acid are
hydrolyzed exlusively into erythro diols (Gill & Hammock, 1979).
The metabolic profile shown in Figure 3-32 C indicated that many metabolites of 14-
trans-AA coeluted with metabolites of AA. Even more complex mixture of
metabolites was observed from microsomal metabolism of all four trans-AA isomers
and AA. Such mixture might have contained up to 20 EETs, including four trans-
EETs. It is thus possible that past measurements of endogenous CYP450-AA
metabolites might have inadvertently detected EETs and DiHETEs derived from
trans-AA. More advanced analytical methods will be needed to isolate and analyze
these isomers.
4.2.2 Metabolism of 5-trans-AA by microsomes
5-tmns-AA is a recently identified trans fatty acid that originates from the cis-trans
isomerization of AA initiated by the NO2 radical. This trans fatty acid has been
detected in blood circulation and it has been shown that it functions as a lipid
mediator of the toxic effects of NO2 (Zghibeh et al, 2004). To understand its role of a
lipid mediator, the metabolism of 5,6-trans-AA by liver microsomes stimulated with
NADPH was studied. Profiling of metabolites by LC/MS revealed a complex mixture
of oxidized products among which were four epoxides, their respective hydrolysis
products (DiHETEs), and several HETEs resulting from allylic, bis-allylic and
terminal/subterminal hydroxylations. It was found that the C5-C6 trans bond
competed with the three cis bonds for oxidative metabolism mediated by CYP
epoxygenase and hydroxylase. This was evidenced by the detection of 5,6-trans-EET,
5.6-erythro-DiHETE, and an isomer of 5-HETE (Figure 3-34). Additional lipid
products originated from the metabolism involving the cis bonds and thus these
metabolites had the trans C5-C6 bond. CYP2E1 metabolizes AA to 18- and 19-HETE
(Bylund et a l, 1998). The 5,6-trans isomers of 18- and 19-HETE were likely to be
products of the CYP2E1 because a neutralizing antibody partially inhibited their
formation without having an effect on the formation of the epoxides (Figure 3-41).
The experiments of microsomal hydrolysis and GST-mediated conjugation when both
epoxides were incubated demonstrated that the major product of CYP metabolism,
5.6-trans-'EEY was significantly more stable than its cis isomer (Figure 3-43 and 3-
44).
Thus, 5,6-trans-YEY was more resistant to metabolism by key enzymes active at the
site of its formation in the liver. These two enzymes represent efficient pathways that
inactivate exogenous and endogenous epoxides, many of which can cause toxicity by
binding to DNA and proteins. While it appears that both 5,6-EET isomers can be
formed by hepatic microsomes, the trans isomer is more likely to escape hepatic
metabolic transformations and perhaps enter circulation in the intact form (Figure 4-
150
18). These experiments have provided the first indication that such an epoxide may
have a longer half-life than its opposite isomer. While many studies have detected the
biological activity of the cis epoxides of linoleic and arachidonic acids (Capdevila et
al, 1986; Capdevila et a l, 1988), comparative biological data for fatty acid trans
epoxides are rare (Katz, 2002).
OH
5-HETE
.COOH COOH
19-HETE OH
COOH
18-HETE19-HETE
COOH
OH
hepatic CYP2E1
COOH5,6-trans-AA
hepatic microsomal CYP epoxygenase
COOH
plus 5,6-trans isomers of 8,9-, 11,12-, 14,15-EET
microsomal hydrolases
vicinal dihydroxy eicosatrienoic acids (1 erythro 3 threo diols)
Figure 4-18. Summary of the key steps involved in the hepatic microsomal metabolism of 5-trans-AA5-trans-AA was metabolized into three classes of lipid products: EETs, DiHETEs and HETEs. Allylic, bis-allylic and sub-terminal (co-1 plus co-2) were the three forms of HETE molecules derived from 5-trans-AA. Allylic hydroxylation gave rise to 5-, 8-, 12-, 11- and 15-HETE, bis-allylic hydroxylation gave rise to 13-, 10- and 7-HETE and sub-terminal hydroxylation gave rise to 19- and 18-HETE. CYP epoxygenase, a monooxygenase that was capable of epoxidation of the 5-trans-AA double bonds and generated 5,6-/nm?-EET and 5,6- trans-S,9-, 11,12-, 14,15-c/j-EETs.
151
These experiments do not exclude the possibility that the differences in the rates of
hydrolysis and GST conjugation were caused by the inhibitory effect of the trans
isomer. The lipids characterized in this study constitute a novel class of lipids that
could function as lipid markers of the N 02 radical, originating from steps of AA
isomerization followed by CYP-mediated monooxygenation. The findings of 5-trans-
AA metabolism by CYP could be further developed to detect 5,6-frYms-EET
stereospecifically in vivo along with other products of 5,6-trans-AA metabolism.
4.3 Metabolism of trans-fiJK by polymorphonuclear leukocytes
Treatment ofHL-60 cells with AA and its mono trans isomers revealed that only one
trans-AA isomer, 5-trans-AA, have a potent apoptotic effect. Cells treated with 5-
trans-AA had significantly less DNA than corresponding control cells. 5-trans-AA
increased the degradation of DNA to nucleosomal subunits that appeared as a
subGo/Gi peak beginning within 24-48 hours post-treatment. Other trans-AA isomers
and AA did not modulate the growth and apoptosis HL-60 cells.
A number of studies have shown that AA metabolism is linked to HL-60 cell
proliferation and survival during myeloid maturation (Steinhilber et al, 1993a;
Dittmann et al, 1998; Hoemlein et a l, 1999; Nie et al, 2001; Arita et a l, 2001). An
important role in triggering apoptosis in HL-60 (Brungs et a l, 1994; Ghosh & Myers,
1999; Poff & Balazy, 2004a) and other cells (Miller et al, 1990; Ghosh & Myers,
1998) have been found for 5-LOX, which metabolizes AA to 5-HETE and LTB4. It
was initially thought that because 5-trans-AA is a unique AA isomer having the trans
at the critical site for 5-LOX catalytic activity, which causes hydroxylation at the
carbon 5 double bond, it might inhibit 5-LOX and 5-HETE formation by competing
with AA metabolism. However, 5-LOX products in HL-60 cells were not detected.
Other reports have shown that HL-60 cells do not express 5-LOX mRNA
constitutively, and although HL-60 cells can exert 5-LOX activity after differentiation
(Steinhilber et al, 1993b), they frequently fail to form 5-LOX products when cultured
in 0.5 % DMSO for 48 hrs (Hoemlein et al, 1999b). Activation of 5-LOX in HL-60
appears to require a complex system of cofactors and cytokines (Steinhilber et al.
152
1993c; Brungs et al, 1994a). In contrast to HL-60 cells, control experiments with
PMN showed a very active metabolism of 5-trans-AA into a group of novel lipids
among which was a trans isomer of 15-HETE. Thus, 5-trans-AA induced apoptosis in
HL-60 cells via a mechanism that was 5-LOX independent and may be mediated
through esterification/deesterification within cell membrane, which is consistent with
a relatively long time required for induction of the antiproliferative effect.
4.4 General discussion
It has been established that trans-AA isomers might originate from two sources:
dietary fat (Sébédio & Christie, 1998) and endogenous cis-trans isomerization (Jiang
et al, 1999). 14-trans-AA is an AA isomer that can originate from both sources and
has been detected in human blood plasma (Zghibeh et a l, 2004). Rats fed with
partially saturated canola oil that contained \2-trans-\mo\Qic acid (\2-trans-LA), 14-
trans-AA appeared in blood, suggesting that elongation and desaturation of 12-trans-
LA leads to formation of \A-trans-AA (Ratnayake et al, 1994). On the other hand,
reaction of AA with nitrogen dioxide, nitrite (acidified), N2O3, and peroxynitrite
causes AA isomerization and formation of trans-AAs, among which \A-trans-AA was
identified (Jiang et a l, 1999). Conditions that result in elevated levels of NO2, such as
sepsis and smoking have also resulted in elevated levels of trans-AA (Balazy, 2000b).
Thus, \A-trans-AA is an interesting trans fatty acid that could be a marker of
inflammation and excessive exposure to NO2, but can also be due to excessive dietary
trans-linoleic acid (Balazy & Poff, 2004). The study of its effects on platelets was
therefore justified.
This study found unique properties of 14-trans-AA. This isomer:
a) inhibited platelet aggregation stimulated by AA, ADP, epinephrine, U46619,
thrombin and PAF at low micro-molar concentrations.
b) inhibited platelet aggregation induced by AA that was not correlated with
inhibition of COX, TXA2 or PGH2.
153
c) was metabolized to unique set of metabolites, a major component being an isomer
of 15-HETE.
d) The metabolism of \A-trans-AA was faster in the presence of AA, an effect known as AA-induced co-oxidation.
It was noticed that a compound having properties of \4-trans-AA would make an
interesting anti-platelet agent.
1) \A-trans-AA inhibited platelet aggregation, but the levels of TXB2 were only
partially inhibited. Thus if this property holds in vivo, lA-trans-KA is not likely
to inhibit PGH formation from platelet-derived PGH2. All COX inhibitors such
as aspirin inhibit PGI2. Therefore, the dose of aspirin is important. Lower doses
are aimed to inhibit platelet aggregation without inhibition of PGI2. With 14-
trans-AA, this concern will be removed. 14-trans-AA will inhibit platelets, but
PGI2 will be formed from platelet-derived PGH2. More studies are needed to
establish this postulate.
2) 14-trans-AA inhibited platelet aggregation induced by U46619 with IC50 of 2.2
uM, which suggests that \ 4-trans-AA inhibited TP receptor. Other work
supports that 14-trans-AA also inhibits TP receptor in micro vessels (Chemtob,
2005). Because 14-trans-AA is structurally different from TXA2 and U46619, it
is not likely to be competing with these two ligands, but it is likely to either
modify the TP receptor directly or change the receptor environment either by
allosteric interaction or by modifying surrounding membrane possibly via
incorporation into sn-2 position of phospholipids.
3) 14-trans-AA protects platelets from pro-aggregatory activity of other agonists.
More studies will be needed to establish whether this property is maintained in
vivo, at what concentrations and for how long.
4) Formation of 15-HETE isomer from \4-trans-AA could be an important
positive feature. 15-HETE is a precursor for lipoxins those are anti
inflammatory. Work by Serhan et al. was shown that aspirin-induces formation
154
of aspirin triggered 15-epi-lipoxin A4 (ATL) (Chiang et a l, 2004). So all-cw-15-
HETE, a product of \A-trans-AA, is likely to be a substrate for 5-LOX to
generate a novel class of anti-inflammatory lipoxins. Additional studies will be
needed to establish this property in vivo.
In summary, 14-trans-AA is likely to interact with platelets by several mechanisms -
one is via metabolism but that will require isolation of unstable metabolites and the
other is via incorporation into the platelet membrane. Many interesting properties of
14-trans-AA appear to make it a candidate template for a novel anti-platelet drug;
new analogues are being developed to address this possibility.
The lipid metabolites characterized in the study with trans-AA and hepatic
microsomes constitute a novel class of lipids that could function as lipid markers of
the NO2 radical, originating from steps of AA isomerization followed by CYP-
mediated mono-oxygenation. Many studies explored various aspects of microsomal
epoxidation and hydroxylation of AA and other PUFAs; however, considerably less is
known about such a metabolism of trans fatty acids. Detection of oxygenated
metabolites derived from tans-AA would require specific assays that would involve
chromatographic separation of cis- and frafzs-isomers. 5,6-EET has been detected in
vivo in the rats, human platelets, and rat erythrocytes (Ozawa et al, 1988; Katz, 2002;
Goodfriend et al, 2004). Because a special analytical approach needs be applied for
the separation and simultaneous detection of the two 5,6-EET isomers (Corey et al,
1980), previous studies (Ozawa et al, 1988; Katz, 2002; Goodfriend et al, 2004)
were likely to detect both of these isomers. The findings of this study could be further
developed to detect S-trans-YET stereospecifically in vivo along with other products
of 5-trans-AA metabolism (summarized in Figure 4-18). While many studies have
addressed concerns regarding the adverse health effects of trans fatty acids (Slattery
et al, 2001; Lemaitre et a l, 2002; Stender & Dyerberg, 2004), mechanisms of their
activity are not well understood. Oxidative microsomal metabolism described in the
present study could be an important aspect in the activation of trans-AA and possibly
of other trans fatty acids into unique metabolites, among which the fraMs-epoxides
could be a distinct and characteristic group although it is unclear what cellular role
they may play in the formation of biologically active metabolites.
155
5-trans-AA appears to be a toxic trans-fatty acid that causes apoptosis ofHL-60 cells
via Gi phase arrest and also apoptosis and death of microvascular endothelial cells
(Kermovant et a l, 2004). Cigarette smoking, hypoxia, and sepsis induce AA
isomerization and increase the levels of trans-AA, including 5-trans-AA, in blood
circulation. 5-trans-AA and other trans-AA are likely to be formed via N 02-mduced
isomerization within cell membrane as phospholipid esters from which they can be
released by phospholipases. NO2 required for such isomerization may originate from
exogenous sources such as urban air or cigarette smoke, but also from endogenous
sources such as oxidation of nitrite (Bian et a l, 2003). The levels of endogenous
trans-AA as free acids can reach 0.5 pM (Balazy, 2000), however, the levels of
esterified trans-AA within cells are unknown, but probably unlikely to reach
concentrations required to induce apoptosis. The effects of 5-trans-AA may be
mediated through esterification/deesterification within cell membrane, which is
consistent with a relatively long time required for induction of the antiproliferative
effect.
4.5 Future work
Further work relating to this project is being actively persued. Novel water-soluble
analogues are being synthesized and they will be tested for biological activity because
the most important aspect of this project is the finding that \A-trans-AA can
dissociate between aggregation and TXA2 formation. This may have profound effects
on platelet interaction with vascular wall. Therefore, the future studies will focus on
the effect of trans-AA on TXA2 and PGI2 biosynthesis following exposure to trans-
AA. The studies will be performed in vitro and in vivo. Eventually I envision that
trans-AA could serve as a template for the development of drugs to rival the NSAID
family and spare PGI2 biosynthesis.
Another important conclusion from performed studies is the identification for the first
time of the Zrarzs-epoxides from trans-AA. Future studies could be designed to
identify trans-AA in vivo and they can be used as biomarkers of hepatic trans-AA
metabolism. One study also revealed a huge difference between cis and trans epoxide
156
in term of hydrolysis and GSH conjugation and it will be fascinating to compare these
properties in vivo. This could be accomplished by the use of LC-MS.
Another important aspect of this study is the effect of 5-trans-AA on cell
proliferation. Future work will establish in greater details the role of 5-trans-AA in 5-
LOX mediated mechanisms of neoplastic cell proliferation and tumorigenesis.
4.6 Conclusion
This dissertation explored a new and original aspect of trans-FA biochemistry.
Despite a great interest in the effect of trans-FA on human health, very little is known
about their metabolism in the cardio-vascular system. Although recent studies show
the trans-FA have profound effect in this system, almost nothing is known about the
mechanism by which the trans-FA exerts their effects. The hypothesis is that many of
the effects of trans-FA are related to their oxidative metabolism. This dissertation is
the beginning of the exploration and established numerous novel oxidative
metabolites and novel biological effects. As a pioneering study it opens doors for
additional studies in many directions: biological; structural; and analytical. In this
respect it could be viewed as a preliminary study of a new area of research regarding
trans-FA. This study will be continued and definitely provide answers to many
questions that arise from the data presented in this dissertation.
157
5 LIST OF S U P P L IE R S
Names and addresses of the manufacturers or suppliers of instruments and chemicals.
Agilent Technologies
2850 Centerville Road Wilmington, DE 19808
USA
Alltech Associates
2051 Waukegan Road
DeerFieldJL 60015-1899
USA
Applied Biosystems
850 Lincoln Centre Drive
Foster City, CA 94404
USA
Beckman Instruments
1050 Page Mill Road Palo Alto, CA 94304
USA
Becton Dickinson
P.O. Box 243 Cockeysville, MD 21030
USA
Biomol Research Lab. Inc
5120 Butler PikePlymouth Meeting, PA 19462-1202
USA
158
Cayman Chemical
1180 E. Ellsworth Road
Ann Arbor, MI 48108
USA
Chrono-Log
2 West Park Road
Havertown, PA 19083
USA
Coulter Corporation
11800 South West, 147 Avenue
Miami, FL 33196-2500
USA
Dell Corporation Co.
1 Dell way, P.O. Box 8723
Round Rock, TX 78682
USA
Fisher Scientific Company
1970 John Creek Court, Suit 500
Suwance, GA 30024
USA
Gilson, Inc.
3000 W. Beltline Hwy.
P.O. Box 620027
Middleton, WI 53562-0027
USA
Hudson valley Blood Bank
Valhalla, NY 10595
USA
159
MDS Sciex
71 Four Valley Drive
Concord, ON, L4K 4V8
Canada
Moravek Biochemals, Inc.
577 Mercury Lane
Brea, California 92821-4890
USA
Pharmacia
800 Centennial Avenue
Piscataway, NJ 08855-1327
USA
Shimadzu Scientific Inc.
7102 Riverwood Drive
Columbia, MD 21046
USA
Sigma Aldrich
P.O. Box 14508
St. Louis, MO 63178
USA
TechAir
465 Knollwood Road
White Plains, NY 10603-1914
USA
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