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
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
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
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
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.
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)
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
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.
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
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
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
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
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)
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
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
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.
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
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
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
Erythrocyte
Plasmalogens (= ether phospholipids)
CH3DH/HCl
80ºC; 4hr
Fatty acid methyl esters
+ dimethylacetals
extraction
Capillary GC
The PEX 7 spectrum
↓↑Severe RCDP
↓-n↑MR + cataract
N↑Refsum
PlasmalogensPhytanic
Clinical presentation Biochemical presentation
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
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
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)
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
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
Analysis of urine Organic acids
Urine (1-5 ml); acidified
Extraction (ethyl acetate)
Derivatisation (trimethylsilyl-)
Gas chromatography / mass spectrometry
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
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
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.)
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
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.