the laboratory diagnosis of peroxisomal disease · analysis of very long-chain fatty acids and...
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The Laboratory DiagnosisOf
Peroxisomal Disease
Ries Duran, AMC, AmsterdamRies Duran, AMC, Amsterdam
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Relevant Analyses for the Primary Diagnosis of Peroxisomal Disorders
ES-MSMSAcylcarnitines
TechniquePlasmaGC/MS, (ES-MS/MS)Very long-chain fatty acids
GC/MSPhytanic + pristanic acids
ES-MS/MSBile acids (C27)
ES-MS/MSPipecolic acid
GCPolyunsaturated fatty acids (Acylcarnitines)
ErythrocytesGCPlasmalogens
GCPolyunsaturated fatty acids
UrineES-MS/MSBile acids
GC/MSDicarboxylic acids
ES-MS/MS, (AAA)Pipecolic acid
ICOxalic acid
GC/MS/ICGlycolic acid
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Peroxisomal ParametersCSF
Plasma
Urine
Erythrocytes
Bile
Pipecolic acid
VLCFA
Phytanic acid
Pristanic acidC27-bile acids
Pipecolic acid
DHA
Acylcarnitines
C27-bile acids
Pipecolic acid
Dicarboxylic acids
Oxalic acid
PlasmalogensDMA
C27-bile acids
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Analysis of very long-chain fatty acids and Phytanic / Pristanic acid
plasma (100 μl)
add 2H-internal standards (100 μl)
add 2 ml 0.5 M HCl
add 2 ml 1.0 M NaOH
add – extract 4 ml hexane
remove sterols – extract 4 ml 1 M KOH
add – extract 4 ml hexane
derivatise MTBSTFA
analyse GC/MS (stable isotope dilution)
110º, 45 min
110º, 45 min
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Control
Refsum
Zellweger
prist
prist
prist
phyt
phyt
phyt
22:0
22:0
22:024:0
24:0
24:0
26:0
26:0
26:0
VLCFA,
phytanic
and
pristanic
acid by
GC/MS
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Are the VLCFA levels constant?C26 of Zellweger and X-ALD
0
1
2
3
4
5
6
7
8
ABCDEF
The upper normal level of C26 is 1.32 μmol/L; otherwise quite large variations occur and the most severely affected patients do not necessarily have the highest C26 values.
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Procedure• 100 µl plasma + 100 µl IS (2H4-labeled C22:0,
C24:0 and C26:0)• hydrolysis with 1 ml acetonitrile / 37% HCl (4:1)• incubate at 90ºC for 2 hours• extract free fatty acids with hexane• reconstitute in chloroform-methanol-water
(50:45:5) + 0.01% ammonia• analyze on mass spectrometer in negative ion
mode
Valianpour et al, Mol Genet Metab, 2003
Electrospray ionization mass spectrometryof VLCFA (ESI-MS)
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VLCFA analysis with ESI-MS
0
100
%
C22:0
d4C22:0
C24:0
d4C24:0
340 345 350 355 360 365 370 375 380 385 390 395 400 405m/z0
100
%0
100
%
393 394 395 396 397 398 399 400m/z
0
100
% d4C26:0C26:1 C26:0
control
X-ALD
C22:0
d4C22:0
C24:0
d4C24:0
d4C26:0C26:1
C26:0
d4C26:0
d4C26:0
C24:1
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Peroxisomal Fatty Acid ß-oxidation in Human Peroxisomes and its Deficiency
C26:0 Pristanic acid THCA
Cholesterol
AOX1 AOX2
DBP
pTH1 pTH2
Acyl CoA oxidase def. D-bifunctional protein def.
Racemase def.
X-ALD
ALDP racemase
CoASH
CoASH
ATP
SCPα def.
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Fatty acid oxidation in peroxisomes
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Neonatal evolution of D-bifunctional protein deficiency
0123456789
10
7 14 34
PristC27C26Pip
A neonate with extreme hypotonia was subjected to selective screening of peroxisomal disease at the age of one week. VLCFA were strikingly abnormal, whereas pristanic acid and C27-bile acids increased with age.The pattern was consistent with D-BP deficiency, including normal plasmalogens.
Age days
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Peroxisomal ParametersCSF
Plasma
Urine
Erythrocytes
Bile
Pipecolic acid
VLCFA
Phytanic acid
Pristanic acid
C27-bile acidsPipecolic acid
DHA
Acylcarnitines
C27-bile acids
Pipecolic acid
Dicarboxylic acids
Oxalic acid
PlasmalogensDMA
C27-bile acids
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Fatty acid oxidation in peroxisomes
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Peroxisome Biogenesis DefectBile Acids in urine (tandem-MS)
400 420 440 460 480 500 520 540 560 580 600 620 640m/z0
100
%
530.3
443.3
423.4
415.0
403.3
441.2
431.0
480.4
464.4459.2
457.2479.3
481.2
523.2
505.3
497.0489.2
506.3
539.3
572.3
541.2
555.1
570.4
627.4
611.2
588.3573.2 607.3
589.2
613.2 641.3
629.3643.3
Tau-
OH-CA
Tau-
OH-THCA
inte
nsity
mass
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Analysis of Plasma C27-bile acids
plasma (50 μl)
+ I.S. (2H)
deproteinise (acetonitril)
evaporate 100 μl H2O / CH3OH
HPLC / MS/MS
RP-C8
50 x 1.0 mm
A. formate buffer pH 8.1 + CH3OH
B. acetonitril / 10% H2O
MRM Tau-conj.
C29-dic.
Gly-conj.
Free acids
MS/MS
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3.00 3.25 3.50 3.75 4.00 4.25 4.50 4.75 5.00 5.25 5.50 5.75 6.00 6.25 6.50 6.75 7.00 7.25 7.50 7.75 8.00Time0
100
%
0
100
%
0
100
%
0
100
%
0
100
%
4.645728
5.2623452
4.722207
6.4099815
4.8540101
LC-MS/MS of plasma bile acids
C29 Bile dicarboxylic acid
Tauro-THCA
Int. St. Tauro-CA
Int. St. CA (free)
DHCA (free)
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Peroxisomal ParametersCSF
Plasma
Urine
Erythrocytes
Bile
Pipecolic acid
VLCFA
Phytanic acid
Pristanic acid
C27-bile acids
Pipecolic acidDHA
Acylcarnitines
C27-bile acids
Pipecolic acid
Dicarboxylic acids
Oxalic acid
PlasmalogensDMA
C27-bile acids
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D.M. Danks, P. Tippett, C. Adams, P. Campbell
Cerebro-hepato-renal syndrome of Zellweger.A report of eight cases with comments upon theincidence, the liver lesion, and a fault in pipecolicacid metabolism.
Journal of Pediatrics 86 (1975), 382-387
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There is a wide variation of plasma Pipecolic acid in Zellweger
0
50
100
150
200
250
300
350
ABCDE
The upper normal level of plasma pipecolic acid is approx. 5 μmol/L. PBD patients generally range from 15 μmol/L upwards, although mild patients may be as low as 8 μmol/L.A steady increase with age accompanies a bad clinical evolution.
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Is there a relationship between phytanic and pipecolic acid?
0
50
100
150
200
250
300
350
A
B
Two PBD-patients showed a steady increase of pipecolic acid over a three-year-period. Only one patient had a concomitant phytanic acid increase.Phytanic acid is of minor importance in most mild PBD-patients
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Pipecolic acid my be increased in
• Peroxisome biogenesis defects• Vitamin B6-responsive convulsions (antiquitin
defects)• Hyperlysinemias• Hyperprolinemia type 2• Unexplained conditions
but NOT in• Isolated peroxisomal enzyme defects
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Peroxisomal ParametersCSF
Plasma
Urine
Erythrocytes
Bile
Pipecolic acid
VLCFA
Phytanic acid
Pristanic acid
C27-bile acids
Pipecolic acid
DHA
Acylcarnitines
C27-bile acids
Pipecolic acid
Dicarboxylic acids
Oxalic acid
PlasmalogensDMA
C27-bile acids
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Erythrocyte
Plasmalogens (= ether phospholipids)
CH3DH/HCl
80ºC; 4hr
Fatty acid methyl esters
+ dimethylacetals
extraction
Capillary GC
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The PEX 7 spectrum
↓↑Severe RCDP
↓-n↑MR + cataract
N↑Refsum
PlasmalogensPhytanic
Clinical presentation Biochemical presentation
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Early increase of Plasma Phytanic acid in RCDP-patients
0.04-5.30-4 monthsControls *
9.33 weeks613.22 weeks59.92 weeks44.41 week35.83 days20.71 day1
Phytanic acid (μmol/L)
AgePatient
H.J. ten Brink et al – J Lipid Res, 1992
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Peroxisomal ParametersCSF
Plasma
Urine
Erythrocytes
Bile
Pipecolic acid
VLCFA
Phytanic acid
Pristanic acid
C27-bile acids
Pipecolic acid
DHA
Acylcarnitines
C27-bile acids
Pipecolic acid
Dicarboxylic acids
Oxalic acid
PlasmalogensDMA
C27-bile acids
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Fatty acyl-CoA
Perox.β-oxidation
Mitochondria
Carnitine esters
Dicarboxylic acids
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Analysis of Acylcarnitines
plasma (50μl)
add internal standards (2H-labelled)
deproteinise / extract with acetonitril
make butyl esters (butanol /acetyl chloride)
electrospray MS/MS (parents of 85)
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450 460 470 480 490 500 510 520 530 540 550 560 570 580 590m/z0
100
%
459.6456.4
454.6
482.6
461.5
472.7
462.4
463.4
471.1
480.6
474.0
542.5
484.6
514.4
500.5
486.7
490.6
498.4 502.6
512.5
528.4
515.5
523.5520.3
540.4
596.7
568.4543.5
556.5544.2
547.4 565.3
594.9
570.6
572.4588.5
580.6 597.5
I.S.18:1
18:0
16:0 DC
24:026:1
26:0
Peroxisome Biogenesis DefectAcylcarnitines in plasma (tandem-MS)
inte
nsity
mass
OH-prist
OH-phyt
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Peroxisomal ParametersCSF
Plasma
Urine
Erythrocytes
Bile
Pipecolic acid
VLCFA
Phytanic acid
Pristanic acid
C27-bile acids
Pipecolic acid
DHA
Acylcarnitines
C27-bile acids
Pipecolic acid
Dicarboxylic acidsOxalic acid
PlasmalogensDMA
C27-bile acids
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Analysis of urine Organic acids
Urine (1-5 ml); acidified
Extraction (ethyl acetate)
Derivatisation (trimethylsilyl-)
Gas chromatography / mass spectrometry
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Urine Organic acids in Zellweger
1. ω-Oxidation
C6-dicarboxylicC8, C8:1-dicarboxylicC10, C10:1-dicarboxylic3-OH-C10-dicarboxylic2-OH-C10-dicarboxylic
C7-dicarboxylicC9-dicarboxylic
2. Other
3,6-epoxy-C14-dicarboxylic4-OH-phenyllactic2-OH-isovaleric
S.H. Korman et al. – J. Inherit. Metab. Dis. 2000; 23:425-428
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Characteristic Biochemical Genetic Findings in the Various Disorders of Peroxisomal Function
nn↑n- ↑↑nRacemase
nn↓↑ ↑nnRefsum
nnnnnn- ↑X-ALD female
nnnnn↑X-ALD male
nn↑↑↑↑Bifunctional protein
↓n↓↑nnRCDP
n↑↑↑↑↑PBD mild
↓↑↑↑↑↑PBD severe
Plasmalogens (ery’s)
Pipecolic acid
Pristanic acid
Phytanic acid
C27 bile acid
C24 + C26fatty acid
Disorder
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Follow-up studies for the confirmation of peroxisomal disease
1. Fibroblast studies
- β-oxidation of VLCFA / pristanic acid- α-oxidation of phytanic acid- Catalase staining- Plasmalogen biosynthesis- Individual enzymes (DBP, AMACR, etc.)
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Follow-up studies for the confirmation of peroxisomal disease
2. Molecular genetic studies
- Complementation groups (PEX 1, PEX 2, etc.)
- mutation screening of the PEX gene(s)
- mutation screening related to individual enzymes
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Conclusion
1. The assay of VLCFA alone is not sufficient2. Never analyse phytanic acid without pristanic acid3. Plasma bile acids have a diagnostic significance
(urine less so)4. Pipecolic acid is only increased in PBD-patients5. Pipecolic acid may be increased in non-
peroxisomal patients6. The value of urine organic acids and plasma
acylcarnitines remains to be established7. One patient had normal plasma parameters, but
fibro’s were diagnostic.