The Laboratory DiagnosisOf

Peroxisomal Disease

Ries Duran, AMC, AmsterdamRies Duran, AMC, Amsterdam

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.

Fatty acid oxidation in peroxisomes

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

Fatty acid oxidation in peroxisomes

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

Fatty acyl-CoA

Perox.β-oxidation

Mitochondria

Carnitine esters

Dicarboxylic 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.