Short communication
309
J. Clin. Chem. Clin. Biochem.
Vol. 27, 1989, pp. 309-314
© 1989 Walter de Gruyter & Co.
Berlin · New York
SHORT COMMUNICATION
Phytanic Acid and Very Long Chain Fatty Acids
in Genetic Peroxisomal Disorders
By Brunhilde Molzer, Marina Kainz-Korschinsky, Regina Sundt-Heller and H. Bernheimer
Department of Neurochemistry, Neurological Institute of the University of Vienna, Viennat Austria
(Received October 14, 1988/February 3, 1989)
Summary: 1. Phytanic acid, phytanyl-triacylglycerols, and very
long chain fatty acids were analysed by gas chromatography
or thin-layer chromatography in blood and tissues of patients
with different genetic peroxisomal disorders (Refswrfs disease,
X-linked adrenoleukodystrophy, neonatal adrenoleukodystrophy, Zellweger syndrome).
2. We evaluated these analyses in the detection of patients with
Refsufrfs disease, X-linked adrenoleukodystrophy, neonatal adrenoleukodystrophy, and Zellweger syndrome, and of carriers
of X-linked adrenoleukodystrophy. In particular, the analysis
of phytanyl-triacylglycerols by thin-layer chromatography
proved to be a rapid and reliable method for the detection of
patients and the monitoring of their dietary treatment in Refsum's disease. In X-linked adrenoleukodystrophy, carrier de^·
tection may depend on very long chain fatty acid analysis in
more than one material (e. g. plasma and fibroblasts).
3. Analysis of phytanic acid showed that in patients with multiple impairments of peroxisomal functions (Zellweger syndrome, neonatal adrenoleukodystrophy) phytanic acid levels
may be increased not only in serüm, but also in the tissue (e. g.
brain, adrenals, kidney).
4. Analysis of very long chain fatty acids in cholesterol esters
from the brain, adrenals, kidney, and liver of patients with
peroxisomal disprders revealed fötir different types of very long
chain fatty acid patterns according to the behaviour of C 26:0
and of öther saturated and monounsätufated very long chain
fatty acids. These results led to the following conclusions:
1) accumulation of C 26:0 fatty acid in the brain seems not to
be obligatory in Zellweger syndrome;
2) different tissues seein to display different vulnerability to
very long chain fatty acid accumulation (e. g. adrenals ^
kidney > Hver);
3) in peripheral tissues accumulation of very long chain fatty
acids seems to develop more rapidly in disorders with multiple impairments of peroxisomal functions than in a disease
with a Singular peroxisomal defect;
4) in disorders with-multiple impainnents of peroxisomal functions accumulation of very long chain fatty acids seems to
develop more rapidly in peripheral tissues than in bfain.
J. Clin. Chem. Clin. Biochem. / Vol. 27,1989 / No. 5
Introduction
Accumulation of phytanic acid (C 20: br) and of very long chain
fatty acids are typical findings in certain peroxisomal disorders
(l, 2). Phytanic acid was found to accumulate in M. Refsum in
blood lipids (e. g. phytanyl-triacylglycerols) and lipids of the
liver, kidney, myelin and adipose tissue (3 — 5), due to a defect
of phytanic acid a-oxidase (3, 6). Since phytanic acid is of
exogenous origin, a diet low in phytanic acid and phytol is an
appropriate treatment for Refsum patients (3, 7).
An increase of phytanic acid was also detected in some peroxisomal disorders with multiple enzyme deficiencies, e. g. Zellweger syndrome, neonatal adrenoleukodystrophy (l, 2), infantile Refswrfs disease, (8) and rhizomelic chondrodysplasia punctata (9).
Accumulation of very long chain fatty acids (^ C24, particularly hexacosanoic acid, C26:0) is found in blood (10—12) and
tissue lipids (13 — 15) of X-linked adrenoleukodystrophy, due
to an enzyme deficiency of lignoceroyl-CoASH-ligase (16) or
hexacosanoyl-CöA synthetase (17), respectively. Accumulation
of very long chain fatty acids is also observed in several peroxisomal disorders with multiple enzyme deficiencies (2) such
äs Zellweger syndrome, neonatal adrenoleukodystrophy, and
infantile Refswn's disease (8), but not in rhizomelic chondrodysplasia punctata (9). Elevation of C 26:0 concentrations and
of C26:0/C22:0 ratios in plasma, white and red blood cells
or fibroblasts is an essential diagnostic marker in several peroxisomal disorders (e. g. X-linked adrenoleukodystrophy, Zellweger syndrome, neonatal adrenoleukodystrophy (10—13)) and
also in heterozygotes of X-linked adrenoleukodystrophy (18).
In the present account we report our studies on the diagnosis
of patients (M. Refsum, X-linked adrenoleukodystrophy/
adrenomyeloneuropathy; Zellweger syndrome, neonatal adrenoleukodystrophy) and carriers (X-linked adrenoleukodystrophy/adrenomyeloneuropathy) of peroxisomal disorders by the
assay of phytanic acid and/or very long chain fatty acids in
blood lipids. Furthermore, in order to study the distribution of
very long chain fatty acids and phytanic acid in different tissues
of patients with genetic peroxisomal disorders, we investigated
the fatty acid patterns of cholesterol esters in the brain and
visceral organs in Zellweger syndrome, neonatal adrenoleukodystrophy, and X-linked adrenoleukodystrophy.
Short communication
310
Mcthods
P h y t a n y l - t r i a c y l g l y c e r o l s and phytanic acid
Assay of phytanyl-triacylglycerols and of phytanic acid in
plasma or serum was pcrformed by thin-layer chromatography
and by gas chromatography, respectively.
Phytanic acid was determined in the blood and tissues of
patients with multiple impairments of peroxisomal functions
(e.g. Zellweger syndrome, neonatal andrenoleukodystrophy;
tab. 1), using gas chromatography on a capillary column (DB17, J& W; 30m; 0.32 mm; film thickness 0.5 μηι; carrier gas
helium). Splitless injection was used with a temperature programme (injector 280 °C; Start temperature 100 °C for l minute;
temperature rise 24 °C/minute up to 210 °C, then 2 °C/minute
up to 225 °C, followed by 12 °C/minute up 290 °C; flame ionization detector). Phytanic acid was evaluated either s phytanic
acid concentration in comparison with an internal Standard
(C15:0) or s fraction of total fatty acids. The ratio of C 20: br/
C17:0 was also calculated. Comparison of C 20: br with C17:0
was chosen since C17:0 is a component of naturally occurring
lipids with near proximity to C 20: br in gas Chromatographie
analysis. Evaluation of the C20:br increase from the ratio
C20:br/C17:0 seems to have the same validity s the evaluation of C 26:0 increase from the ratio C 26:0/C 22:0 (13).
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For several years we have used gas chromatography to determine C26:0 in leukocytes (12), fibroblasts (23), and plasma.
Recently, sample injection directly onto the column (gas Chromatograph: Erba Science, Mega series) was introduced, using
a DB-1 capillary column (J & W, 30 m χ 0.32 mm, film thickness 0.25 μηι, carrier gas helium) and the following temperature
Programme: during sample application secondary cooling was
used; Start temperature 80 °C, hold for l minute, raise temperature at 31 °C/minute up to 210°C, then 6 °C/minute up to
300 °C, followed by 25 °C/minute up to 350 °C, hold 3 minutes.
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Results and Discussion
Phytanic acid in Refsum's disease
In untreated patients thin-layer chromatography of plasma
lipids reveals a triacylglycerol pattern composed of three fractions (19, 20):
1) non-phytanyl-triacylglycerols, the only triacylglycerol fraction occurring in normal controls,
2) monophytanyl-triacylglycerols with one phytanic acid residue, and
3) diphytanyl-triacylglycerols with 2 phytanic acid residues.
The proportions of diphytanyl-, monophytanyl- and non-phytanyl-triacylglycerols can be evaluated and compared by densitometry (21). In the course of an effective dietary treatment
(low in phytanic acid and phytol) the proportions of diphytanyland monophytanyl-triacylglycerols decrease, whereas non-phytanyl-triacylglycerols increase. In our experience with 7 patients
from 3 families, the evaluation of triacylglycerol patterns
proved to be a rapid, reliable, and sensitive method for monitoring the dietary treatment (fig. 1).
1984
Year
1985
1986
Triacylglycerol species and phytanic acid in serum samples of a patient with Refsum's disease treated with a
diet low in phytanic acid and phytol (for methods, see
I.e. (21)).
a) Triacylglycerol species: non-phytanyl-triacylglycerols
(o
·), monophytanyl-triacylglycerols (•^---«-^•),
diphytanyl-triacylglycerols (·
·) in percent of to^
tal triacylglycerols.
b) Phytanic acid, percent of total fatty acids in serum.
c) Phytanic acid, concentration in serum.
Phytanic acid in Zellweger syndrome and neonatal
adrenoleukodystrophy
the normal r nge. The ge-related increase of serum phytanic
acid in neonatal adrenoleukodystrophy is in line with recent
findings of Wanders et al. (22), showing a time-dependent ao
cumulation of phytanic acid in the serum of Zellweger patients.
Clearly, in both diseases, the accumulation of phytanic acid is
related to its dietary intake, since phytanic acid is an entirely
exogenous Substrate. In the Zellweger patients described by
Wanders et al. (22), the serum phytanic acid coneentration was
normal up to the age of 17 weeks; in our Zellweger case,
however, serum phytanic acid was already above normal at the
age of two weeks (tab. 1). These variations might be explained
by different feeding of the infants.
The highest increase of serum phytanic acid was found in a
two-year-old patient with neonatal adrenoleukodystrophy (tab.
1). In another case of neonatal adrenoleukodystrophy, phytanic
acid, determined at birth in umbilical cord plasma, was within
Phytanic acid was also investigated in cholesterol esters of
formalin fixed tissues (brain, adrenals, kidney, liver) obtained
at autopsy (tab, 1). Phytanic acid was increased in the brain,
adrenals and kidney of a case with Zellweger syndrome, and in
J. CHn. Chem. Clin. Biochein. / Vol. 27^ 1989 / No. 5
Short communication
311
Tab. 1 . Phytanic acid in blood and tissues
Serum, total lipids
Zellweger syndrome
Neonatal adrenoleukodystrophy
Neonatal adrenoleukodystrophy,
umbilical cord blood
Controls, (n = 11)
C20:br
μτηοΐ/ΐ
C20:br/C17:0
ratio
54.40
499.20
1.90
2.61
7.68
0.10
12.80 ± 12.50
1.90 - 39.90
Mean ± S.D.
Range
C20:br
Percentage of
total fatty acids
Brain white matter, cholesterol esters
Zellweger syndrome
Neonatal adrenoleukodystrophy
X-linked adrenoleukodystrophy, ri = 4;
0.24 + 0.12
0.04 - 0.38
C20:br/C17:0
ratio
0.16
0.30
0.05
0.02
0.08
0.05
+ 0.02
- 0.07
+ 0.03
- 0.11
Adrenal glands, cholesterol esters
Zellweger syndrome
Controls (n = 2)
0.84
0.02;
0.04
1.84
0.05;
Kidney, cholesterol esters
Zellweger syndrome
Neonatal adrenoleukodystrophy
X-linked adrenoleukodystrophy
Controls (n = 2)
0.18
0.04
0.09
0.06;
0.10
0.41
0.20
0.23
0.05; 0.11
Mean ± S. D.
Range
Mean ± S. D.
Range
Controls (n = 4)
Liver, cholesterol esters
Zellweger syndrome
Controls (n = 3)
0.11
0.11 ± 0.03
0.08 - 0.14
Mean ± S. D.
Range
the brain of a case of neonatal adrenoleukodystrophy. The
highest increase of phytanic acid was found in the adrenais of
the case with Zellweger syndrome. In the brain and kidney of
X-linked adrenoleukodystrophy n increase of phytanic acid
was found. Recently, elevation of phytanic acid was detected
in the liver total lipids of Zellweger syndrome and infantile
Refsum's disease (Ann Moser, private communication). These
results demonstrate that in diseases with multiple impairments
of peroxisomal functi ns, phytanic acid may be increased not
oniy in blood but also in tissues.
0.38
0.39
0.08
0.03
0.14
0.04
± 0.04
-0.11
H- 0.08
- 0.21
0.09
0.19
0.12 + 0.03
0.09 - 0.14
Very long chain fatty acids in peroxisomal disorders
Clinical chemical investigations
Results of investigations in plasma are compiled in table 2. On
average, C26:0 concentrations and C26:0/C22:0 ratios
showed a 5^fold increase in patients of X-linked adrenoleukodystrophy/adrenomyeloneuropathy, and a 9—14-fold increase
in a patient with neonatal adrenoleukodystrophy, compared
with controls. In heterozygotes of X-linked adrenoleukodystrophy, a more than 2-fold increase of mean C 26:0 concentrations
Tab. 2. C26:0 concentrations and C22:0 ratios in plasma.
Group
C 26 : 0 concentration
μιηοΐ/g lipid
μιηοΐ/ΐ
C26:0/C22:0
ratio
X-linked adrenoleukodystrophy/
adrenomyeloneuropathy
8
0.189 + 0.050
(0.131 - 0.277)
1.006 ± 0.416
(0.534 - 1.588)
0.280 -f 0.091
(0.185 - 0.424)
Heterozygotes of X-linked
adrenoleukodystrophy
5
0.096 ± 0.015
(0.078 - 0.113)
0.466 H- 0.144
(0.292 - 0.650)
0.133 ± 0.035
(0.105 - 0.191)
Neonatal adrenoleukodystrophy
1
0.552
1.795
0.853
0.040 ± 0.013
(0.018 - 0.076)
0.202 4- 0.076
(0.113 - 0.378)
0.058 ± 0.020
(0.020-0.110)
Controls
28
With the exception of neonatal adrenoleukodystrophy, all values are means ± S. D. (Range in parentheses).
J. Clin. Chem. Clin. Biochem. / Vol. 27,1989 / No. 5
Short communication
312
and C26:0/C22:0 ratios was detected. However, s judged
from investigations in obligate heterozygotes (18, 24), not all
heterozygotes may be detectable by assay of very long chain
fatty acids in plasma alone. Analysis in more than one material
(e. g. plasma and leukocytes or fibroblasts) may be important
for a reliable detection of heterozygotes (10, 12, 18, 24).
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Type 2: Patterns with a moderate increase of C26:0 and of
other even numbered, saturated, very long chain fatty acids,
mainly C 24:0; saturated odd numbered and monounsaturated
even and odd numbered very long chain fatty acids may be
increased s well. Examples of this type are brain in a case of
neonatal adrenoleukodystrophy, kidney in a case of X-linked
adrenoleukodystrophy, and liver in a case of Zellweger syndrome.
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Using gas chromatography (OV-1 packed column; (15)) we
investig ted very long chain fatty acid patterns in cholesterol
esters of brain white matter (Zellweger syndrome, neonatal
adrenoleukodystrophy, X-linked adrenoleukodystrophy and Xlinked adrenoleukodystrophy heterozygotes; (25)), adrenals
(Zellweger syiidrome, X-linked adrenoleukodystrophy), kidney
(Zellweger syndrome, neonatal adrenoleukodystrophy, X^
linked adrenoleukodystrophy), and liver (Zellweger syndrome,
X-linked adrenoleukodystrophy).
Type 1: Patterns without accumulation of C26:0, b t with
moderate increase of various other saturated or monounsaturated very long chain fatty acids, even or odd numbered. This
type was found in the brain pf a case of Zellweger syndrome
s well s iii the liver of a case of X-linked adrenoleukodystr phy.
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^r.y long chain fatty acid patterns in tissues
According to the behaviour of C26:0 and other very long
chain fatty acids, ((25, 26) and data to be published), 4 types
of very long chain fatty acid patterns may be characterized in
these tissues (tab. 3):
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In total we detected 23 cases of X-linked adrenoleukodystrophy/adrenomyeloneuropathy (originating from Austria, Federal Republic of Gennany and CSSJR), 2 cases of neonatal
adrenoleukodystrophy, 2 cases of Zellweger syndrome and 13
heterozygotes of Xrlinked adrenoleukodystrophy.
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Type 3: Patterns with a marked to very marked increase of
C26:0 and a moderate to very marked increase of other even
numbered (C24:0, C28:0) and odd numbered (C25:0,
C27:0) saturated very long chain fatty acids. Even numbered
monounsaturated (C26:l, C28:l, C24:l) very long chain
fatty acids are moderately elevated; odd numbered monounsaturated very long chain fatty acids may be elevated too. This
type was detected in the brain in X-linked adrenoleukodystrophy and in the adrenals and kidney in a case with Zellweger
syndrome.
Type 4: Patterns with a very marked increase of C 26:0 and a
moderate to very marked increase of other even and odd numbered saturated very long chain fatty acids. In comparison with
type 3, the increase of odd numbered saturated very long chain
fatty acids appears to be more pronounced. Monounsaturated
very long chain fatty acids, even and odd numbered, are prac^
tically absent, with the exception of a minimal increase of
C28: l in 2 of 3 tissues (see tab. 3). Examples of this type are
the brain of a heterozygote of X-linked adrenoleukodystrophy
(case M. M.; (25))> the adrenals in a case of X-linked adrenoleukodystrophy, and the kidney in a case of neonatal adreno^
leukodystrophy.
The following conclusions may be drawn frbni these findings:
l. Accumulation of C 26:0 in the brain seems not to be oblig^
atory in Zellweger syndrome, since no elevation of C 26:0 (the
crucial marker of very long chain fatty acid accumulation) was
J. Clin. Chem. Clin. Biochem. /Vol. 27,1989 / Να. 5
Short communication
found in this tissue. This finding is in contrast to reports of
other groups (27, 28) and might be due to the short course of
tbe disease in our patient (deceased at the age of 16 days).
However, some other very long chain fatty acids were elevated
in the present case (type l pattern; tab. 3).
2. DifTerent tissues seem to display different degrees of vulnerability to very long chain fatty acid accumulation. With respect
to visceral organs it appears that adrenals (types 3 and 4; tab.
3) show an equal or even a higher vulnerability than kidney
(type 2—4), whereas liver (type l and 2) seems to be less
vulnerable.
3. Very long chain fatty acid accumulation in peripheral tissues
seems to develop more rapidly in disorders with multiple impairments of peroxisomal functions (Zellweger syndrome, neo-
313
natal adrenoleukodystrophy) than in a disease with a single
peroxisomal defect (X-linked adrenoleukodystrophy). This conclusion is based on the following facts: Kidney tissue of a
patient with Zellweger syndrome (deceased at the age of 16
days) and of a case of neonatal adrenoleukodystrophy (age at
death l year) displayed higher very long chain fatty acid accumulation (types 3 and 4) than the kidney of a patient with
X-linked adrenoleukodystrophy (deceased at the age of 12
years; type 2). In liver tissue, moderate elevation of C26:0
(type 2) was found in Zellweger syndrome, whereas no elevation
(type 1) was observed in X-linked adrenoleukodystrophy.
4. In disorders with multiple impairments of peroxisomal functions very long chain fatty acid accumulation seems to develop
more rapidly in peripheral tissues than in brain.
References
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19. Karlsson, K.-A., Nilsson, K. & Pascher, I. (1967) Separation of lipids containing phytanic acid by thin-layer chromatography. Lipids 3, 389-390,
20. Laurell, S. (1968) Separation and characterization of phytanic acid-containing plasma triglycerides from a patient
with Refsum's disease. Biqchim. Biophys. Acta 752,75—79.
21. Molzer, B., Bernheimer, H., Barolin, G. S., Höfinger, E. &
Lenz, H. (1979) Di-, mono-, and nonphytanyl triglycerides
in the serum: a sensitive parameter of the phytanic acid
accumulation in Refsum's disease. Clin. Chim. Acta 97,
133-140.
22. Wanders, R. J. A., Smit, W., Heymans, H. S. A., Schutgens,
R. B. H., Barth, P. G., Schierbeek, H., Smit, G. P. A.,
Berger, R., Przyrembel, H., Eggelte, T. A., Tager, J. M.,
Maaswinkel-Mooy, P. D., Peters, A. C. B., Monnens, L.
A. H., Bakkeren, J. A. J. M., Trijbels, J. M. F., Lommen,
E. J. P. & Beganovic, N. (1987) Age-related accumulation
of phytanic acid in plasma from patients with the cerebrohepato-renal (Zellweger) syndrome. Clin. Chim. Acta 166,
45-56.
314
23. Molzer, B., Korschinsky, M., Bernheimer, H., Schmid, R.,
Wolf, C. & Röscher, A. (1986) Very long chain fatty acids
in genetic peroxisomal disease fibroblasts: differences between the cerebro-hepato-renal (Zellweger) syndrome and
adrenoleukodystrophy variants. Clin. Chim. Acta 161,
81—90.
24. Watkins, P. A., Naidu, S. & Moser, H. W. (1987) Adrenoleukodystrophy: biochemical procedures in diagnosis, prevention and treatment. J. Inher. Metab. Dis. 10 (Suppl. I),
46—53.
25. Schlote, W., Molzer, B., Peiffer, J., Pofemba, M., Schumm,
F., Harzer, K., Schnabel, R. & Bernheimer, H. (1987) Adrenoleukodystrophy in an adult female. A clinical, morphological and neurochemical study. J. Neurol. 235, 1—9.
Short communication
26. Molzer, B., Kainz, M., Budkä, H., Grabisic, A., Gräfin
Vitzthum* H., Bernheimer, H. (1988) Very long chain fatty
acids and phytanic acid in genetic peroxisomal diseases. In:
Lipid Storage Disorders (Salvayre, R^., Douste-Blazy, L. &
Gatt, S., eds.) Plenum Press, New York, pp. 423—428.
27. Brown III, F. R., MC Adams, A. J., Cummins, J. W,,
Konkol, R., Singh, I., Moser, A. B. A Möser, H. W. (1982)
Cerebro-hepato-renal (Zellweger) syndrome and neonatal
adrenoleukodystrophy: Similarities in phenötype and accumulation of very long chain fatty acids. Johns Hopkins
Med. J. 757, 344-361.
28. Moser, A. E., Singh, L, Brown III, F. R., Solish, G. L,
Kelley, R. I., Benke, P. J. & Moser, H. W. (1984) The
cerebrohepatorenal (Zellweger) syndrome. Increased levels
and impaired degradation of very-long^chain fatty acids
and their use in prenatal diagnosis. N. Engl. J. Med. 310,
1141-1146.
Dr. Brunhilde Molzer
Dept. of Neurochemistry
Neurological Institute
University of Vienna
Schwarzspanierstraße 17
A-1090 Wien
J. Clin. Chem. Clin. Biochem. / Vol. 27,1989 / No. 5
i
\
Short communication
315
J. Clin. Chem. Clin. Biochem.
Vol. 27, 1989, pp. 315-317
© 1989 Walter de Gruyter & Co.
Berlin · New York
SHORT
COMMUNICATION
Plasmalogen Biosynthesis in the Diagnosis of Peroxisomal Disorders
By K. Kremser
Institut Jur Medizinische Chemie der Universität Wien and
, A. Röscher
'. Abteilung för Klinische Chemie und Biochemie, Dr. v. Haunersches Kinderspital der Universität München
(Received October 14, 1988//February 15/MarchlO, 1989)
Summary: Fibroblasts of patients sufTering from peroxisomal
disorders such äs chondrodysplasia punctata (rhizomelic type),
neonatal adrenoleukodystrophy, Zellweger syndrome and control fibroblasts were used for the evaluation of a procedure
suitable for pre- and postnatal diagnosis.
This technique is based on the detection of impaired peroxisomal plasmalogen synthesis by means of a double Substrate,
double labelling technique using 14C-labelled hexadecanol and
?
H-labelled hexadecylglycerol äs precursors for peroxisomal and
microsomal plasmalogen synthesis. Pathological cells are characterized by a decreased utilization of hexadecanol, thus resulting in an increased 3H/14C ratio within plasmalogens. Sensitivity and reproducibility of this method were improved by
changing both the Chromatographie cpnditions and the calculation of the diagnostic parameters.
The simplified scheme in figure l shows the initial reactions
involved in plasmalogen synthesis in aerobic cells (for details
see I.e. (2)). Two enzyme activities responsible for the introduction of the ether bond in ether-phospholipids are localized
in peroxisomes. First, acyl-CoA: dihydroxyacetone phosphate
Dihydroxyacetone-P
Acyl-CoA
Fatty acid
l -Acyl-dihydroxyacetone-P
Fatty Acid
l
Acyl-CoA
Introduction
A method for the post- and pfenatal diagnosis of cerebrohepato-renal syndrome (Zellweger syndrome) was devised by
Schrakamp et al. (1) and by Röscher et al. (2), based on the
detection of impaired plasmalogen synthesis in fibroblasts. This
method can also be used for the diagnosis of the rhizomelic
form of chondrodysplasia punctata, the neonatal form of adrenoleukodystrophy and probably for some other, not yet fully
characterized, peroxisomaL defects.
• Long chain alcohol
(e. g. hexadecanol)
1-Alkyl-dihydroxyacetone-P "
l-Alkyl-glycerol-3-P
In fibroblasts and in amniocytes the differences between normal
and pathological cell-lines, äs well äs within the different peroxisomal disease entities, have been shown to be quantitative
rather than qualitative.
Furthermore, the results were affected by non^-standardized
variables such äs growth conditions, washing Steps and unknown absolute amounts of precursor substances during incubation and Separation.
In this study the method is reevaluated in order to provide
optimal conditions for reliable prenatal diagnosis.
J. Clin. Chem. Cliia. Biochem* /Vol. 27,1989 / No. 5
Ether glycerolipids
(plasmalogens)
Fig. 1. Initial reactions in plasmalogen biosynthesis
1 == Acyl-CoA: glycerpne-phosphate O-acyltransferase,
EC 1.3.1.42
2 = Alkylglyeerone-phosphate synthase, EC 2.5.1.26
Short communication
316
acyltransferase catalyses the acylation of dihydroxyacetone
phosphate in the Cl position. Thcn an acyl-CoA is reduced in
two steps, using NADPH, to a long chain alcohol. The second
enzyme, alkyl-dihydroxyacetone phosphate synthase, catalyses
the replacement of the fatty acid by the alcohol. The subsequent
Steps common to the formation of both acyl- and alkyl-glycerolipids occur in the microsomes.
tion. Spots were visualized by exposure to iodine vapor, labelled
and decolorized. All the spots äs well äs the background were
scraped off, transferred into counting vials and counted in a
liquid scintillation counter (Beckman). The same results were
obtained if only the aldehyde and the hexadexylglycerol spots
were scraped off and counted. In this case the total radioactivity
incorporated was calculated from the original lipid extracts.
In the experiments described, a long chain alcohol
([l4C]hexadecanol) is utilized to monitor the peroxisomal biosynthetic Steps.
Six control cell lines (skin biopsies were taken on the occasion
of minor surgery from otherwise healthy patients), 3 cell lines
of chondrodysplasia punctata (rhizomelic type) and cell lines
of the neonatal form of adrenoleukodystrophy and Zellweger
syndrome — diagnosed by the typical clinical and biochemical
criteria — were incubated with radioactively labelled precürsors
äs described (2).
Furthermore, an alkylglycerol ([3H]hexadecylglycerol) serves äs
a precursor for the microsomal formation of plasmalogens.
Material and Methods
Human fibroblasts were grown to dififerent degrees of confluency in Dulbecco^ minimal essential medium, containing
fetal calf serum (volume fraction 0.1) and 2mmol/l glutamine
at 37 °C. The medium was changed the day before experiments
were carried out. Incubations were carried out with fibroblast
cultures that had been equilibrated for 30 minutes in incubation
medium, (serum-free, containing 0.5 g/l bovine serum albumin).
RadiolabeUed Substrates were prepared in absolute ethanol and
diluted with the incubation medium to give the desired amount
of 3H/14C. This solution was sonicated for 30 seconds and an
aliquot containing 11 kBq of [l-14C]hexadecanol and 111 kBq
of [3H]labelled. hexadecylglycerol was added to fibroblast cultures. Sixteen hours later the cells were washed, scraped off
and collected by centrifugation. Washed fibroblast pellets were
disintegrated by sonication and total cellular lipids were extracted according to Folch et al. (3). Aliquots of lipid extracts
were applied to thin layer plates coated with 0.25 mm silica gel
60 (Merck, Darmstadt, FRG). Unlabelled phosphatidylethanolamine, phosphatidylcholine, containing about 50% plasmalogens and unlabelled hexadecylglycerol were added. The plates
were developed twice with ether/water (100 + 0.25, by vol.) to
remove labelled neutral lipids and to separate unconverted
[3H]hexadecylglycerol from all the other spots. Then the plate
was exposed to HCl-fumes for 12 minutes and developed twice
with light petroleum/ether (95 + 5, by vol.) in the same direc-
Results and Discussion
Cell lines of neonatal adrenoleukodystrophy and Zellweger
syndrome were used äs pathological controls because of theif
well known peroxisomal dysfunction (2, 4—13). Only cells at
low passage number (< 12) were used for incubations.
Two different calculations of diagnostic parameters were compared:
1. After acid treatment of total phospholipids the radioactivity
associated with aldehydes was measured and related to the
total radioactivity incorporated (for both aldehyde^pH] and
aldehyde-[14C]). Then the 3H/14C ratio of the counts within
aldehydes was determined (ratio 1) äs described by Röscher
et al. (2).
2. Aiternatively the residual amount of uüconverted
[3H]hexadecylglycerol was subtracted from the total 3H-radioactivity, and the resulting value was used to calculate
ratio 2. The use of ratio 2 resulted in higher absolute values
äs well äs in a lower Standard deviation of pathological
states (äs determined for Zellweger syndrome and chondrodysplasia punctata).
Table l shows the 3H/14C diagnostic ratios within alkenyl groups
of plasmalogens.
Tab. l. 3H/14C diagnostic ratios within alkenyl groups of plasmalogens
Control
Cell-line
Number of
incubations
1
2
3
4
5
6
3
3
3
3
3
9
Ratio 2
± s
Ratio 1
± s
0.91
0.53
0.92
0.60
0.81
1.21
±
±
±
±
±
±
0.04
0.07
0.13
0.04
0.04
0.19
= 0.83
Zellweger syndrome
Neonatal adrenoleukodystrophy
1
2
±
±
±
±
±
±
0.1
0.5
0.3
0.3
0.2
0.4
x ± = 2.7
6
5.0 ± 1.7
18.4 ± 4.6
7
4
3.1 ± 0.8
3.7 ± 1.0
8.2 ± 1.7
9..2 ± 1.9
3.4
Chondrodysplasia punctata
2.7
1.8
2.8
2.6
3.7
3.6
23.8 ± 7.6
21.3 ±6.0
20.0 ± 5.0
21.7
x = 8.6
84
85
94
± 22
± 17
± 18
x = 88
The values shown represent means ± 1SE and were achieved with one stock solution of labelled precursors at a constant
incubation time of 16 hours. Ratio l and Ratio 2 were calculated äs described in the text.
J. Clin. Chem. Clin. Biqchem. / Vol. 27,1989 / No. 5
317
Short communication
Incorporation of [3H]hexadecylglycerol into the l'-alkenyl
groups of plasmalogens was similar (9—11% of total 3H inco O ated into the lipid) in pathological cells and controls.
This is consistent with the hypothesis that the biosynthetic Steps
located in the microsomes are normal in these cell lines. In
contrast, utilization of [14C]hexadecanol (the precursor of peroxisomal biosynthetic Steps) was variably diminished in pathological cells. Cell lines were evaluated at different degrees" of
confluency. This resulted in a rather large variance in absolute
values of incorporation rates making distinction between different genotypes diflicult. However, this drawback is circumvented by using the normal 3H-incorporation into plasmalogens
äs an "internal Standard" and by calculation of 3H/14C diag-
nostic ratios. The ratios determined for chondrodysplasia punctata difiered greatly from those determined for controls, the
neonatal form of adrenoleukodystrophy and Zellweger syndrome; this was due to a decreased
[14C]hexadecanol utilization,
3 14
resulting in markedly elevated H/ C diagnostic ratios. Ratio
2 was more sensitive and more reproducible for the purpose of
distinguishing between pathological states.
The double Substrate, double labelling technique described here
provides a sensitive tool for the pre- and postnatal diagnosis
of chondrodysplasia punctata, Zellweger syndrome and neonatal adrenoleukodystrophy in amniocytes and fibroblasts.
References
1. Schrakamp, G., Schutgens, R. B. H., Wanders, R. J. A.,
Heymans, H. S. A., Tager, J. M. & van den Bosch, H.
(1985) Biochim. Biophys. Acta 833, 170-174.
2. Röscher, A., Molzer, B., Bernheimer, H., Stöckler, S., Mutz,
I. & Paltauf, F. (1985) Pediat. Res. 19, 930-933.
3. Folch, J., Lees, M. & Stanley, G. H. S. (1957) J. Biol.
Chem. 226, 497-511.
4. Schutgens, R. B. H., Wanders, R. J. A., Nijenhuis, A., van
den Hoek, C. M., Heymans, H. S. A., Schrakamp, G.,
Bleeker-Wagenmakers, E. M., Dellemann, J. W., Schräm,
A. W., Tager, J. M. & van den Bosch, H. (1987) Enzyme
38, 161-176.
5. Kaiser, E. & Kramar, R. (1988) Clin. Chim. Acta 773,
57-80.
6. Monnens, L. & Heymans, H. (1987) J. Inher. Metab. Dis.
70,23-32.
7. Besley, G. T. N. & Broadhead, D. M. (1987) J. Inher.
Metab. Dis. 10, 236-238.
8. Sakai, T., Antoku, Y. & Goto, I. (1986) Exp. Neurol. 94,
149-154.
9. Schutgens, R. B. H., Heymans, H. S. A., Wanders, R. J.
A., van den Bosch, H. & Tager, J. M. (1986) Eur. J. Pediatr.
144, 430-440.
10. Heymans, H. S. A., Schutgens, R. B. H., Tan, R., van den
Bosch, H. & Borst, P. (1983) Nature 306, 69-70.
11. Goldfischer, S., Collins, J., Rapin, U., Coltoff-Schiller, B.,
Chang, C.-H., Nigro, M., Black, V. H., Javitt, N. B.,
Moser, H. W. & Lazarow, P. B. (1985) Science 227, 67-70.
12. Goldfischer, S. & Reddy, J. K. (1984) Int. Rev. Exp. Pathol.
26, 45-84.
13. Goldfischer, S., Moore, C. L., Johnson, A. B., Spiro, A. J.,
Valsamis, M. P, Wisniewski, H. K., Ritch, R. H., Norton,
W. T, Rapin, L & Gärtner, L. (1973) Science 182, 62-64.
Prof. Dr. A. Röscher
Dr. v. Haunersches Kinderspital
Abt. f. Klin. Biochemie
Lindwurmstr. 4
D-8000 München 2
J. Clin. Chem. Clin. Biochem. / Vol. 27,1989 / No. 5
Short communication
319
J. Clin. Chem. Clin. Biochem.
Vol. 27, 1989, pp. 319-321
© 1989 Walter de Gruyter & Co.
Berlin · New York
SHORT COMMUNICATION
Peroxisomal Oxidation of Pipecolic Acid in the Rat1)
By R. Kramar, K. Kremser and H. Schön
Institut jur Medizinische Chemie der Universität Wien
(Received October 14, 1988/January 24, 1989)
Summary: Postnuclear fractions from rat liver and kidney oxidize L-pipecolic acid, a by-product of lysine catabolism, in a
hydrogen
peroxide-producing reaction. This pipecolate
oxidase2) activity is enhanced in preparations from animals
treated with clofibrate and thyroxine, substances known to act
äs peroxisome proliferators and inducers of the peroxisomal
fatty acid ß-oxidation. The enzymatic activity co-purified with
the peroxisomal marker fatty acyl-CoA oxidase2) rather than
with the mitochondrial marker glycerol-3-phosphate
dehydrogenase2). Thus the degradation of L-pipecolic acid may
Start in the rat with a peroxisomal oxidase comparable to other
hydrogen peroxide-producing oxidases found in peroxisomes.
These Undings provide indirect evidence that the marked hyperpipecolinaemia described in a group of human genetic disorders connected with peroxisomal defects such äs Zellweger
syndrome might be due to the absence of peroxisomal Lpipecolate oxidase.
Like its lower homologue, L-proline, L-pipecolic acid might
also be dehydrogenated by an integral flavoprotein tightly
bound to the inner mitochondrial membrane and closely connected to the respiratory chain (5). In Pseudomonas putida Lpipecolate is indeed dehydrogenated in this way (6). In purified
rat liver mitochondria, we were not able to detect this reaction.
In mammalian liver L-pipecolate seems to be dehydrogenated
by a hydrogen peroxide-forming peroxisomal oxidase: Zaar et
al. (7) demonstrated that peroxisomes from rat and beef kidney
metabolize L-pipecolate very sluggishly with the formation of
hydrogen peroxide.
The aim of the present study was to demonstrate that at least
in the rat, L-pipecolate is dehydrogenated specifically and at
äppreciable rates by a peroxisomal hydrogen peroxide-producing oxidase. This is important, since it suggests that the hyperpipecolinaemia observed in some human peroxisomal diseases
might be due to a genetic defect of a peroxisomal pipecolate
oxidase.
Introduction
Pipecolic acid arises frpm the metabolism of lysine (1). In rat
liver (fig. l, A) the mainstream of lysine degradation does not
involve pipecolate (2), whereas in rat brain (fig. l, B) it is the
main product (3).
The degradation of pipecolic acid is impaired in a group of
human genetic disorders which are characterized by a total loss
of peroxisomal functipns (Zellweger syndrome, neonatal
adrenoleukodystrophy and infantile Refsum disease; for review
seel.c. (4).
f
)
2
Supported by the Anton Dreher Gedächtnisschenkung
) Enzymes
(Fatty) acyl-CoA oxidase, acykCqA: pxygen oxidoreductase
(EC 1.3.3.?)
D-amino acid oxidase, D-amino acid: pxygen oxidoreductase
(deaminating) (EC l .4.3.3)
Glycerol-3-phosphate dehydrogenase, jw-glycerol-3-phosphate-.(acceptor) oxidoreductase (EC 1.1.99.5)
L-pipecolate oxidase, L-pipecolate: oxidoreductase (EC
1.5.3.?)
J. Clin. Chem. Clin. Biochem. /Vol. 27,1989 /No. 5
[
—COOH
JH 2
NH
* NH 2
(B)
(A)
Lysine
"Alph>^ C lr-0
COOH^"~
deamination"
lination" NH?
NH 2 II
NH 2
deominotion"
Allysine
2 - Oxo- 6-aminocaproate
it
CXcOOH
• 1-
earboxylate
II
Pipecolate
oxidase
NADH
aNAD
H
Pipecolate
Fig. 1. Pathway of lysine degradation
(A) ... pathway in rat liver
(B) ... pathway in rat brain
OOH
A'-Piperideine-Zcarboxylate
Short communication
320
Tab. l. Effect of clofibrate and thyroxine feeding on the activity of L-pipecolate oxidase, fatty acyl-CoA oxidase and mitochondrial
glycerol-3-phosphate dehydrogenase in cell fractions of liver and kidney homogenates.
Ten animals per group were assayed individually. Activities are given in nmol Substrate (leuko-2,7-dichlorofluorescein, or
2-0>-iodophenyl)3-3(p-nitrophenyl)-5-phenyltetrazoliurachIoride) turned over per min (mU).
Values represent means ± SD.
Protein
mg/g liver
L-Pipecolate oxidase
mU/ing protein
mU/g liver
GIycerol-3phosphate
dehydrogenase
mU/mg protein
Fatty acylCoA oxidase
mU/mg protein
Liver
Postnuclear supernatant
Control
Clofibrate
Thyroxine
143 ± 7
157 ± 8
145 ± 7
Liver
Large particle fraction
Control
Clofibrate
Thyroxine
37.4 -h 2.0
49.8 + 2.5
40.2 ± 3.5
Kidney
Large particle fraction
Control
Clofibrate
Thyroxine
44.8 + 3.0
45.5 + 3.5
48.7 ± 3.5
79 + 10
151 + 23*
125 ± 19*
0.55 + 0.07
0.95 + 0.14*
0.87 ± 0.14*
1.2 + 0.2
7.8 + 1.2*
3.4 ± 0.5*
3.2 ± 0.4
19.6 ± 2.7*
29.5 ± 3.8*
63 ± 8
137 ± 19*
108 ± 15*
1.64 + 0.23
2.74 + 0.40
2.66 ± 0.38
3.5 ± 0.4
23.4 ± 3.0*
10.0 ± 1.4*
9.3 ± 1.0
56.8 ± 10.6*
91.0 ± 11.5*
0.36 ± 0.06
1.75 + 0.26
1.17 + 0.17
2.1 + 0.3
13.0 -f 1.7
5.6 + 0.8
10.5 ± 1.5
—
41.0 ± 5.5
16.6 ± 2.5
81.4+ 11
58.3 + 8.3
p < 0.01
Material and Methods
Feeding of the peroxisome proliferators clofibrate (8) and thyroxine (9) to male Sprague-Dawley rats, the preparation of cell
fractions (postnuclear supernatant and "large particles"), assays
of protein and of the mitochondrial marker glycerol-3-phosphate dehydrogenase were performed s described previously
(10). Peroxisomal fatty acyl-CoA oxidase and L-pipecolate oxidase were assayed by measuring the formation of H2O2 during
the oxidation of palmitoyl-CoA or L-pipecolic acid. Leuko-2,7dichlorofluorescein was used s a peroxidase Substrate in a
photometric assay at 502 nm (11). The reaction mixture contained 230 μηιοΐ/ΐ leuko-2,7-dichlorofluorescein, 23 mmol/1 3amino-lH-l,2,4-triazole, 40 mg/1 horse radish peroxidase, 114
mmol/1 potassium phosphate, pH 7.5 and 50 μιηοΙ/1 palmitoylCoA or 500 μιηοΐ/ΐ L-pipecolic acid. For the assay of pipecolate
oxidase 0.2 g/l Triton X-100 was added. After a preincubation
of three minutes at 30 °C the reaction was started by adding
the Substrate.
When the large particle fraction is separated into mitochondria
and peroxisomes L-pipecolate oxidase (specific activity about
4 nmol/min · mg protein, tab. 2) copurifies with the peroxisomal
enzyine, fatty acyl-CoA oxidase, rather than with the mitochondrial marker, glycerol-3-phosphate dehydrogenase. However, some L-pipecolate oxidase activity (about 0.4 nmol/min
• mg) is found in the mitochondrial fraction so that an addi^
tional mitochondrial metabolisra cannot be fully excluded,.
Tab. 2. Specific activities of pipecolate oxidase, fatty acyl-CoA
oxidase (peroxisomal marker) and glycerol-3-phosphate
dehydrogenase (mitochondrial marker) in peroxisomes
and mitochondria (fatty acyl-Co A oxidase and glycerol3-phosphate dehydrogenase, resp., peak fractions), purified on a sucrose gradient froin the pooled large particles used for the experiment reported in table 1.
Gradient: 50 ml 0.25 mol/1 sucrose, large particles in 50
ml l mol/1 sucrose, 70 ml 1.4 mol/1 sucrose, 70 ml 1.6
mol/ l sucrose and 2 mol/1 sucrose to a total volume of
365 ml. BECKMAN L8-55 ultracentrifuge, Z-60 rotor
at 50000 min"·1 for 4 hours.
Results and Discussion
The large particle fraction of rat liver and kidney consists
essentially of mitochondria, peroxisomes and lysosomes; it oxidizes L-pipecolate with the production of hydrogen peroxide.
The extent of this L-pipecolate oxidase activity is comparable
to that of peroxisomal acyl-CoA oxidase (0.55 vs 1.2 mU/mg
protein).
A peroxisomal location for the enzymatic activity of L-pipecolate oxidase in liver is likely, since it is raised by the peroxisome proliferators (8, 9), clofibrate and thyroxine, by more
than 60% (tab. 1).
In this respect L-pipecolate oxidase behaves like the fatty acylCoA oxidase (12, 13) of the peroxisomal -oxidation.
Peroxisomes
Control
Clofibrate
Mitochondria
Control
Clofibrate
L-Pipecolate
oxidase
Fatty
acyl-CoA
oxidase
m /nig
protein
mU/mg
protein
3.8
20
25
190
0.35
1.84
1.3
17.1
<:
Glycerol-3phosphate
dehydrogenase
mU/mg
protein
2.1
14.8
39.0
281
J. Clin. Chem. Clin. Biochem. /Vol. 27,1989 / No. 5
Short communication
Contrary to our fmdings Zaar et aL (7) detected only very low
oxidation of L-pipecolate by peroxisomes from rat and beef
kidney (60—80 pmol/min · mgprotein). Liver peroxisomes were
found to be completely inactive. D-pipecolate was oxidized at
much higher rates, presumably due to Z)-amino acid oxidase2)
activity.
Zaar et al. ascribe the capacity of kidney peroxisomes to oxidize
L-pipecolate to this active D-amino acid oxidase which is known
to also attack L-proline, the homologue of L-pipecolic acid,
whereas other L-amino acids remain unaffected (14).
321
Some months ago Trijbels et al. (18) presented another type of
experiment in order to demonstrate the participation of liver
peroxisomes in pipecolate catabolism. Using Z),L-pipecolic acid
14
C-labelled in the carboxylic group äs Substrate they measured
the production of 14CO2 by purified rat liver peroxisomes. 2Aminoadipate, which is a potential catabolite of pipecolic acid
arising from allysine (see fig. 1), did not influence the CO2
production, thus indicating the existence of another pathway
of pipecolate degradation. But the reaction measured might be
due solely to the D-moiety of pipecolate present in the added
racemate, which is dehydrogenated by peroxisomal Z>-amino
acid oxidase. This would result in the formation of
A1piperideine-2-carboxylic acid. After hydrolytic ring opening
the 2-oxo-6-aminocaproate might undergo spontaneous decarboxylation with the formation of 14CO2. This experiment therefore does not prove the peroxisomal degradation of L-pipecolic
acid.
Nevertheless, on the basis of our data we prefer the view that
in mammais L-pipecolate is dehydrogenated by a distinct Lspecific peroxisomal oxidase.
Under the conditions of our pipecolate oxidase assay, we failed
to detect any L-pipecolate oxidase activity in purified kidney
D-amino acid oxidase (delivered from SIGMA, St. Louis).
Furthermpre, D-amino acid oxidase is known to be markedly
decreased by clofibrate feeding (15), whereas the activity of Lpipecolate oxidase is enhanced by this drug (tab. 1). Thus we
suggest that in the rat L-pipecolate is degraded by a specific Lpipecolate oxidase rather than by Z>-amino acid oxidase. This
also seems to be true in man: Liver D-amino acid oxidase is
quite active in Zellweger patients (16) and even elevated in In summary, our data from rat liver peroxisomes support the
neonatal adrenoleukodystrophy (17). Nevertheless, L-pipeco- hypothesis that a defect of a peroxisomal L-pipecolate oxidase
late is not degraded in these disorders.
is responsible for the accumulation of L-pipecolate in some
human peroxisomal disorders.
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Prof. Dr. R. Kramar
Institut für Medizinische Chemie
der Universität Wien
Währingerstraße 10
A-1090 Wien