Developmental Brain Research, 45 (1989) 129-135 129 Elsevier

BRD 50856

Lipid peroxidation in developing fetal guinea pig brain during normoxia and hypoxia

Om Prakash Mishra and Maria Delivoria-Papadopoulos Department of Physiology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104 (U.S.A.)

(Accepted 13 September 1988)

Key words: Lipid peroxidation; Developing brain; Hypoxia; Guinea pig

Lipid peroxidation in the fetal guinea pig brain was studied at 30, 35, 40, 45, 50, and 60 days of gestation. Conjugated dienes and flu- orescent compounds, indices of lipid peroxidation, peaked at 35 days of gestation, decreased by 45 days, and remained at that level un- til birth. The higher levels of peroxidation products in early gestational periods (30-40 days) suggest that either the anti-oxidant mech- anisms for scavenging oxygen free-radicals and further metabolizing oxidation products are underdeveloped, or the rate of peroxida- tion is higher than periods near term. Prenatal hypoxia increased the levels of conjugated dienes and fluorescent products in the brains of preterm (50 days) and term (60 days) fetuses. Brain homogenates incubated in air at 37 °C underwent rapid lipid peroxidation as measured by the level of thiobarbituric acid (TBA)-reactive substances. However, term brain showed a higher rate of peroxidation and attained higher steady state levels of TBA-reactive substances than preterm brain. This may be due to the higher levels and de- grees of unsaturation in fatty acids in term brain. Following hypoxia, term brain showed 5 times the rate of lipid peroxidation and a 3- fold increase in total TBA-reactive substances over controls. These studies show that a significant degree of lipid peroxidation is occur- ring in the fetal brain during gestation and that the developing brain is more susceptible to lipid peroxidation near term. Furthermore, prenatal hypoxic stress further increases the susceptibility of the brain to peroxidative reactions.

INTRODUCTION drogenase, lactate dehydrogenase and citrate syn-

thetase, markedly increases 10-15 days before birth, A number of studies have shown that guinea pig whereas in the rat this occurs 10-15 days postnatally.

brain is morphologically, biochemically, and electro- The development of the enzymes of aerobic glycoly-

physiologically mature at birth 12. The brain is partic- tic metabolism correlates with the onset of neuro- ularly vulnerable to environmental and nutritional logical competence of newborns of guinea pig and stresses during its growth spurt period in all spe- rat 2.

cies 5'11'22. In guinea pigs, the period of rapid brain Brain tissue has a high metabolic rate and is highly

growth takes place before birth, in contrast to non- rich in polyunsaturated fatty acids which are highly precocial species, such as rat, in which this period oc- susceptible to oxidation 3s,4°. In addition, brain has

curs 10-15 days postnatally 9. low levels of anti-oxidant enzymes, such as catalase The development of guinea pig brain has been and glutathione peroxidase, and lacks cytochrome P

studied in detail and compared with the develop- 4503'6'31'34'35 . Therefore, by virtue of its biochemical

mental pattern in other species such as rat, pig, and composition and metabolic activity, brain is poten- man 1°. According to their studies in guinea pigs, the tially susceptible to peroxidation.

highest rate of increase in brain D N A and cholesterol In our previous studies 3°, we observed that the spe-

occurs during 7 -10 days before birth. The activity of cific activity of anti-oxidant enzymes: catalase, gluta- key enzymes of energy metabolism, such as hexoki- thione peroxidase, glutathione reductase, glucose-6- nase (mitochondrial and cytosolic), pyruvate dehy- phosphate dehydrogenase, is low until 45 days of ges-

Correspondence: O.P. Mishra, Department of Physiology, Room A201, Richards Bldg./G4, University of Pennsylvania, School of Medicine, Philadelphia, PA 19104, U.S.A.

0165-3806/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)

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tation and increases rapidly during the last two weeks termined that under the above conditions of maternal of gestation, achieving almost adult levels at term. hypoxia, fetal brain ATP level was reduced by ap- These results indicated the possibility of higher levels proximately 50%, indicating that severe hypoxic in- of lipid peroxidation products during a 30-45 days sult had been induced in the fetal brain. period followed by a sharp decrease in the level of

these products by term. In this study, we have deter- Brain lipid extraction and determination of lipid per- mined the status of lipid peroxidation in the develop- oxidation products ing fetal guinea pig brain and examined the suscepti- Lipids were extracted by the method of Folch-Pi et bility of the fetal brain to hypoxia during the prenatal a1.13. Brain tissue stored in liquid nitrogen was period, weighed under frozen conditions and homogenized

in a chloroform:methanol (2:1) mixture containing MATERIALS AND METHODS 0.5 mM EDTA and allowed to oscillate for 1 h under

N2 at 30 °C in a shaking incubator. The samples were All reagents and chemicals used were of the high- filtered through glass wool and mixed with 0.2 vol-

est purity available. Thiobarbituric acid was pur- umes of 0.88% NaCI. Following centrifugation, the chased from Sigma. Chloroform, methanol, n-buta- lower phase was collected and dried under a stream nol, and n-heptane were of high-performance liquid- of nitrogen. The samples were redissolved in chloro- chromatography (HPLC) grade and purchased from form and dried under nitrogen. This process was re- J.T. Baker, Inc. Malonaldehyde bis-dimethylacetal peated three times. Finally, the samples were dis- was purchased from Pfaltz and Bauer Inc. Pregnant solved in spectrophotometric grade n-heptane to pro- guinea pigs of 30, 35, 40, 45, 50, and 60 days of gesta- vide 0.5 mg lipid/ml heptane solution and absorbance tion were purchased from Charles River, Boston. spectra were taken between 200 and 300 nm. Differ-

For each gestational age, fetuses were obtained ence spectrum was obtained with the help of comput- from 6 normoxic (control group) guinea pigs. Conju- er software purchased from SLM Aminco, Conju- gated dienes and fluorescent compounds were deter- gated dienes were calculated from the difference mined as indices of lipid peroxidation in fetal guinea spectrum using a molar extinction coefficient of 26,000 pig brain of 30, 35, 40, 45, 50, and 60 days of gesta- M -l.cm -l (ref. 4). The conjugated diene data was de- tion. To obtain control fetuses, the mother was care- termined in terms of per mg lipid. In addition, the fully laid down on the operating table and the abdo- amount of total lipid per g brain was determined and men anesthetized by infiltration of 5.0 ml lidocaine finally, the amount of conjugated dienes was ex- (1%). An incision was made in the lower abdomen pressed in terms of ~moi per g brain. Fluorescent and the fetuses were removed and quickly decapitat- products were determined spectrophotofluorome- ed. Their brains were frozen in liquid nitrogen within trically at excitation/emission wavelengths (360/435 4-10 s of decapitation and kept in liquid nitrogen un- nm) according to the method of Dillard and Tappel ~ til chemical analyses. For experimental group fe- and expressed as relative fluorescence intensity at tuses, hypoxia was induced individually in pregnant 360/435 nm per g brain. guinea pigs carrying 50 day (preterm) and 60 day (term) fetuses by allowing them to breathe 7% O 2 for Lipidperoxidation in vitro 40 min in a specially designed chamber fitted with a To determine the susceptibility of the preterm and probe to monitor oxygen tension. The bottom of the term fetal brain to oxidative reactions, lipid peroxi- chamber was layered with Baralyme to remove CO 2. dation was carried out in vitro by using homogenates ArterialpO 2 of the mother was determined at 10 min obtained from preterm and term fetal brains. Brain intervals during exposure. ThepO2 of the mother de- homogenates were allowed to peroxidize at 37 °C creased to approximately 20 mm Hg within 10 min and the lipid peroxides formed were analyzed as and was maintained at that level for the duration of TBA-reactive substances. Tissue samples were ho- the experiment. Following hypoxia, fetuses were re- mogenized in 1.15% KCI containing 0.5 mM EDTA moved and treated in the manner previously de- and a 0.5 ml aliquot was mixed with 0.5 ml distilled scribed. In a separate series of experiments, we de- water and incubated in air at 37 °C for 0, 5, 10, 20,

131

and30min Fo owingincubation the eve of BA 018, reactive substances was determined as described be-

low i]- °'°I i Similarly, the susceptibility of the hypoxic brain to 0.12 I-

lipid peroxidation was determined in brain homoge- ~ = nates obtained from control and hypoxic term fe- ~ o.09

~E tuses. The homogenates were incubated at 37 °C for ~ 0, 10, 20, and 30 min and the level of TBA-reactive ~ 0.06 substances was determined as described below.

o.oa ½ {

Determination of TBA-reactive substances 0 i i ; 0 i i I I

Lipid peroxidation in vitro was measured in terms 30 35 45 50 5s 60 of malonaldehyde content according to the methods Days of Gestation of Uchiyama and Mihara and Ohkawa 32,43, with Fig. 1. Level of conjugated dienes in guinea pig fetal brain at

different ages of gestation. Values are expressed as #mol/g slight modification. Following the incubation of brain brain. Each point represents the mean + S.D. of 12 animals. homogenate as described above, 3.0 ml of 1% phos- phoric acid and 1 ml of 0.6% TBA were added and

the mixture was kept in a boiling water bath for 45 fluorescent products (expressed as fluorescence in- min. Samples were removed, cooled and extracted tensity at 360/435 nm/g tissue) in the fetal brain were with 4 ml n-butanol. Samples were centrifuged at 5.00 + 0.72 and 5.65 _+ 0.54 at 30 and 35 days of ges- 2000 g for 10 min and the butanol layer was removed, tation. The level of fluorescent compounds sharply The optical density was then measured at 535 nm. decreased to 2.72 + 0.27 by 40 days, followed by a The amount of TBA-reactive substances was calcu- further decrease to 1.11 _+ 0.25 by 45 days. This level lated using malonaldehyde bis-dimethylacetal stan- was maintained until 60 days of gestation. The level dards run in parallel and expressed as nmol of maiD- of fluorescent products at 45 and 60 days were signifi- naldehyde per g brain. Statistical analysis was carried cantly lower than at 35 days (P < 0.005, when com- out by using Student's t-test. Data with P value less pared with 45 or 60 days). than 0.05 were considered significant. Following 40 min of hypoxia, preterm (50 days)

and term (60 days) fetuses showed an increased level RESULTS of conjugated dienes. The level of conjugated dienes

in hypoxic preterm and term fetal brain increased sig- The level of conjugated dienes in developing fetal nificantly and ranged between 0.27 and 1.04 #mol/g

brain during gestation is shown in Fig. 1. The level of brain. conjugated dienes in the fetal brain were 0.096 + 0.034 and 0.137 + 0.037 pmol/g brain at 30 and 35 days of gestation, respectively. The level of conju- ~ 7 8.0

gated dienes at 35 days was not significantly different ~ ~ T [ Guinea Pigs ; 6 o - 1 from that at 30 days (P > 0.05). The level of conju- ~ ~ /

gated dienes showed a sharp decrease to 0.068 + ~ 4.0 I " 0.015 #mol/g brain by 40 days and a further decrease ~ to 0.035 + 0.005/zmol/g brain where by 45 days it re- ~ .-~ 2.0 mained unchanged until 60 days. The level of conju- \

gated dienes at 45 days and 60 days were significantly t~ r, , l L I I I t I I

lower than that at 35 days (P < 0.005, when com- 0 :30 35 40 45 50 55 60

pared with 45 or 60 days). Days of Gestation

In guinea pig fetal brain during gestation, the pat- Fig. 2. Level of fluorescent compounds in guinea pig fetal brain at different ages of gestation. Values are expressed as fluores- tern of fluorescent compounds was similar to that of cent intensity at 360/435 nm per g brain. Each point represents

conjugated dienes, as shown in Fig. 2. The level of the mean _+ S.D. of 12 animals.

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Following 40 min of maternal hypoxia, a signifi- 100

cant increase in the level of fluorescent products in ~ o- o .o preterm and term fetal brain was observed. The level ~ °5 80 HyPo×ic

of fluorescent compounds in preterm and term fetal .~ brain increased significantly from 0.59 _+ 0.11 to 0.88 ~ ~ 60

+ 0.12 (P < 0.005) and 0.45 _+ 0.09 to 0.75 +_ 0.08 (P < 0.005) fluorescence intensity (arbitrary units) at 40

360/435 nm per g tissue, respectively. The measure- ~ "~ t e • ment of TBA-reactive substances in the brains of pre- ~ ~ 20 e ~ / Control

term and term fetuses did not show a significant dif- ference after hypoxia. The level of malonaldehyde in 0 t t L I

0 5 10 15 20 25 30 term fetal brain following prenatal hypoxia was 49.65 + 3.58 nmol malonaldehyde/g brain as compared to Time (rain)

control levels of 48.05 + 4.04 nmol malonaldehyde/g Fig. 4. The time course of formation of TBA-reactive sub- brain, stances in control and hypoxic term fetal brain. Each point rep-

resents the mean of 4 experiments conducted in duplicate with The formation of TBA-reactive substances by pre- standard deviations ranging from 5 to 10% of the mean.

term and term fetal brain during incubation at differ- ent time intervals is shown in Fig. 3. The level of

TBA-reactive substances increased rapidly during at a much faster rate than normoxic brain tissue with- the first 10 min of incubation and reached a steady in the first 10 min of incubation. The rate of forma- state level after 20 min in both the preterm and term tion of TBA-reactive substances by hypoxic brain fetal brain tissue. However, the rate of formation of was 5 times faster than normoxic brain. In addition,

TBA-reactive substances was higher in the term fetal the steady-state level of TBA-reactive substances brain during 0-10 min as well as 10-20 min of incu- produced by hypoxic brain samples was 3 times bation. The level as well as the amount of TBA-reac- higher than that produced by controls. tive substances formed were higher in term fetal brain at all time intervals of incubation in comparison DISCUSSION to preterm brain.

Data presented in Fig. 4 show that the brain tissue The deleterious effects of peroxidation of mem- obtained following hypoxia undergoes peroxidation brane lipids on membrane functions have been well

recognized and excellently reviewed by several authors 19'2°'41. In this study, we present evidence that

1 O0 a significant degree of lipid peroxidation occurs in the o - - o T e r m .= fetal guinea pig brain during gestation. The level of

8 0 0 - - 0 4 5 clay the peroxidation products, conjugated dienes and

fluorescent compounds (as shown in Figs. 1 and 2), • ~® 6 0 j o ~ are twice as high at 35 days of gestation in compari- • o o / son to the 7-10 day period before birth. These results / ______--o

____.__-~ o ~ 4 0 ° / o / O ~ indicate that either the rate of lipid peroxidation is • J relatively high or the activity of the anti-oxidant en-

20 zymes superoxide dismutase, catalase, glutathione peroxidase, and glutathione reductase are underde-

E 0 l z A I L I I veloped during 30-40 days of gestation and increase ¢= 0 5 10 15 20 25 30

rapidly during 45-60 days of gestation. In our stud- T ime (min) ies 3°, we have observed that the activity of anti-oxi-

Fig. 3. The time course of formation of TBA-reactive sub- dant enzymes, catalase, glutathione peroxidase, glu- stances in preterm and term fetal brain. Each point represents

tathione reductase, and glucose-6-phosphate dehy- the mean of 4 experiments conducted in duplicate with stan- dard deviations ranging from 5 to 10% of the mean. drogenase, r ema i n at low level during 30-40 days of

133

gestation and increase significantly during 45-60 brane lipid peroxidation in hypoxic/ischemic brain: days of gestation. In fact, these studies indicated (a) reactions generating superoxide anion radical in that, depending on the development of anti-oxidant arachidonic acid degradation via cyclooxygenase and enzymatic mechanisms and the rate of lipid peroxida- iipoxygenase pathways45; (b) Fe 2+ ion-catalyzed tion, the level of peroxidation products in the devel- flee-radical-mediated lipid peroxidation, especially oping brain and its susceptibility to such reactions by low molecular weight chelate complexes of Fe 2+. during earlier and later periods of gestation could be Free intracellular iron may be released from ferritin different, and increased significantly during ischemialS'21'44; (c)

Fetal hypoxia significantly increased the level of increase in hypoxanthine concentration as a result of lipid peroxidation products in the brain of preterm increased degradation of ATpT; (d) ion-activated (50 days of gestation) and term (60 days of gestation) protease-mediated conversion of xanthine dehydro- fetuses. We selected the preterm (50 days) and term genase to xanthine oxidase during ischemia 33. (60 days) ages because of the same starting level of The level of TBA-reactive substances in the brain lipid peroxidation products prior to hypoxic expo- homogenates of preterm and term fetuses did not sure. change significantly after hypoxia. However, it has

In previous studies, we have observed that in- been previously recognized by other investigators creased peroxidation in term fetal brain following hy- that the measurement of malonaldehyde is not a poxia is associated with a 50% decrease in the activi- good index of in vivo lipid peroxidation as malonal- ty of Na+,K+-ATPase 29, whereas the activity in the dehyde is fairly soluble in aqueous medium and can preterm brain is unaffected. These studies indicate move away from the site of production into circula- that the term fetal brain cell membrane is more sus- tion 25. ceptible to hypoxic stress. We speculate that several The susceptibility of the preterm and term fetal other membrane-bound enzyme systems would be af- brain to lipid peroxidation was carried out in vitro by fected by hypoxia, using homogenates obtained from preterm and term

Under normal conditions, oxygen flee-radicals fetal brain. The level of TBA-reactive substances in- which are generated in aerobic cells can initiate oxy- creased rapidly during the first 10 min of incubation gen free radical dependent lipid peroxidation. Super- and attained a steady state level after 20 min in both oxide dismutase, the enzyme responsible for convert- the preterm and term brain, as shown in Fig. 3. A ing these superoxide radicals to hydrogen peroxide, rapid increase in the level of TBA-reactive sub- is known to be present in all types of aerobic cells 17. stances during 0-20 min of incubation with a similar The hydrogen peroxide produced is further degraded time course curve was reported in cerebral cortex to water by the action of catalase, slices in vitro 24, brain homogenates 1, and microsomal

Evidence suggesting an increase in the production lipids 26,z7. The rate of formation of TBA-reactive of superoxide anions and hydrogen peroxide under substances was higher in the term brain than in pre- hypoxic conditions has been adequately discussed ~5 term brain at both 0-10 min and 10-20 min of incu- and mechanisms for these reactions have been pro- bation. The steady-state level as well as the amount posed 16. Under the condition of decreased oxygen of TBA-reactive substances formed were higher in availability at the oxygen reduction site, cytochrome term fetal brain at every time interval of incubation. oxidase, an increase in the level of reduced forms of The higher level of TBA-reactive substances in term electron transport chain components o c c u r s 28'36'37. fetal brain might be due to higher levels of unsatu- One of these components, the reduced form of ubi- rated fatty acids in the term fetal brain than in pre- quinone (semiubiquinone), undergoes autoxidation term brain. The higher rate of peroxidation in term and could be a major source of production of oxygen fetal brain in comparison to preterm brain could be free-radicals 14'42. By measuring chemiluminescence, ascribed to the presence of fatty acids of higher de- the formation of flee-radicals was demonstrated dur- grees of unsaturation, since highly unsaturated fatty ing hypoxia 23,39. acids have a higher rate of peroxidation 41.

Several other possible mechanisms could contrib- The susceptibility of hypoxic brain tissue to lipid ute to the formation of free-radicals leading to mem- peroxidation using brain homogenates obtained from

134

control and hypoxic term fetuses is shown in Fig. 4. membranes resulting in an increased number of sites

The hypoxic brain tissue undergoes peroxidat ion at a available for lipid peroxidat ion.

rate which is 5 times faster than normoxic tissue. In summary, this study shows that a significant de-

Moreover , the steady-state level of TBA-reac t ive gree of lipid peroxidat ion occurs in the fetal guinea

substances produced by hypoxic brain samples was 3 pig brain during gestation. The fetal brain is more

times higher than controls, The higher rate and the susceptible to hypoxia near term periods when com-

higher steady-state level of TBA-reac t ive substances pared to pre term brain. The susceptibili ty of the term

formed may be due to the presence of higher levels of fetal brain to lipid peroxidat ion is further increased precursor molecules such as free fatty acid, conju- following hypoxia.

gated dienes, and hydroperoxides produced during

hypoxia which are rapidly converted to TBA-reac- ACKNOWLEDGEMENTS

tive substances during in vitro incubation. The higher

amounts of total TBA-reac t ive substances produced This study was suppor ted by NIH Grant HD-

by hypoxic tissue during incubation might be due to 20337-01 and completed with the technical assistance

the effect of hypoxia on the microenvironment of the of Chris topher F. Cameron.

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