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# 2002 Oxford University Press
Human Molecular Genetics, 2002, Vol. 11, No. 26
3309–3317
Molybdenum cofactor-deficient mice resemble the
phenotype of human patients
Heon-Jin Lee1, Ibrahim M. Adham1, Günter Schwarz2, Matthias Kneussel3, Jörn O. Sass4,
Wolfgang Engel1 and Jochen Reiss1,*
1
Institut für Humangenetik der Universität Göttingen, Göttingen, Germany, 2Institut für Pflanzenbiologie der
Technischen Universität Braunschweig, Braunschweig, Germany, 3Max-Planck-Institut für Hirnforschung, Frankfurt,
Germany and 4Zentrum für Kinderheilkunde und Jugendmedizin, Universitätsklinikum Freiburg, Freiburg, Germany
Received August 22, 2002; Revised and Accepted October 17, 2002
Human molybdenum cofactor deficiency is a rare and devastating autosomal-recessive disease for which no
therapy is known. The absence of active sulfite oxidase—a molybdenum cofactor-dependent enzyme—
results in neonatal seizures and early childhood death. Most patients harbor mutations in the MOCS1 gene,
whose murine homolog was disrupted by homologous recombination with a targeting vector. As in humans,
heterozygous mice display no symptoms, but homozygous animals die between days 1 and 11 after birth.
Biochemical analyis of these animals shows that molydopterin and active cofactor are undetectable. They do
not possess any sulfite oxidase or xanthine dehydrogenase activity. No organ abnormalities were observed
and the synaptic localization of inhibitory receptors, which was found to be disturbed in molybdenum
cofactor deficient-mice with a Gephyrin mutation, appears normal. MOCS1/ mice could be a suitable animal
model for biochemical and/or genetic therapy approaches.
INTRODUCTION
Molybdenum cofactor (MoCo) deficiency (MIM 252150) in
human results in untreatable neonatal seizures and other
neurological symptoms identical to those of sulfite oxidase
deficiency (MIM 272300) (1). The biosynthetic pathway of
MoCo has been discovered in humans and disease-causing
mutations have been identified in the conserved genes MOCS1
(2,3) and MOCS2 (4,5) as well as in the gene for gephyrin (6), a
protein that, besides its role in MoCo biosynthesis (7), has an
additional function in neurotransmitter receptor clustering
(8,9). Gephyrin-deficient mice resemble the phenotype of
humans with hereditary MoCo deficiency and additionally
display a failure in inhibitory neurotransmission (10). This
latter defect prevents suckling and the homozygous animals die
within the first day after birth.
In co-cultivation experiments with fibroblasts from different
MoCo-deficient patients two complementation groups (A and
B) have been found (11). The majority of MoCo-deficient
humans belong to complementation group A and harbor
mutations in the MOCS1 gene (7). This bicistronic gene (2)
encodes the two MoCo biosynthesis enzymes, MOCS1A and
MOCS1B, in two consecutive open reading frames and their
expression involves a conserved splicing pattern leading to a
functional MOCS1A protein without the MOCS1B domain and
activity and a fusion protein with MOCS1B activity and a
non-functional MOCS1A domain (12,13). To create an animal
model suitable for testing therapy approaches for the hitherto
incurable MoCo deficiency, we have removed exon 3 of the
murine MOCS1 gene by homologous recombination with a
targeting vector. Homozygous / mice show a severe
phenotype comparable to that of human MoCo-deficient
patients.
RESULTS
Generation of MOCS1-deficient mice
A murine MOCS1 cDNA was used to identify a genomic
cosmid clone containing the murine MOCS1 gene. This
revealed an exon/intron structure identical to that in the human
genome (3). We chose to eliminate exon 3 of the murine
MOCS1 gene by homologous recombination in embryonic
stem cells, since it encodes several amino acid residues which
are highly conserved in homologous proteins of mammals,
plants and a variety of prokaryotes (2). Substitution of this exon
by a neo-cassette should at least abolish the MOCS1A activity
and—depending on the stability of the resulting mRNA—the
MOCS1B activity could be affected as well (2,12,13). The
targeting strategy is outlined in Figure 1.
*To whom correspondence should be addressed at: Institut für Humangenetik der Universität Göttingen, Heinrich-Düker-Weg 12, D-37073 Göttingen,
Germany. Tel: þ49 5513912926; Fax: þ49 551399303; Email: [email protected]
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Figure 1. Disruption of the murine MOCS1 gene. (A) Targeting strategy. Wild-type MOCS1 gene (top), targeting vector (middle), and mutant locus (bottom) are
shown. Sites of primers for PCR genotyping (arrows) and external probe for Southern blot (‘probe’) are indicated. (B) Southern blot analysis of ES cell clones.
Genomic DNA extracted from ES clones was digested with HindIII and probed with the 50 external probe shown in panel (A). Homologous recombination events
yielded a 9.4 kb hybridizing band detected in heterozygous (þ/) cell lines, while the wild-type (þ/þ) cell line showed a 8.7 kb band. (C) PCR genotyping of
progeny. Wild-type (þ/þ), heterozygous (þ/) and homozygous (/) mice were identified by the amplication of PCR products specific for either the MOCS1
wild-type allele (650 bp) or the targeted allele (500 bp).
As a disruption vector the plasmid pPNT (14) was used. This
vector contains a neomycin phosphotransferase II resistance
gene driven by a PGK promoter (pgk-neo), which should
substitute for exon 3 of the MOCS1 gene and a herpes simplex
virus thymidine kinase gene (tk) cassette outside the recombination region, which allows for positive and negative selection.
Insertion of two genomic fragments (4.6 and 4.4 kb) resulted in
the targeting vector (Fig. 1A), which was linearized with
HindIII and transfected into RI ES cells (strain 129 Sv) (15).
Drug-resistant clones were selected, from which DNA was
isolated and screened by Southern blot analysis using a probe
upstream of the targeting vector (Fig. 1B). This external
probe detected the wild-type allele as an 8.7 kb fragment and the
recombinant allele as a 9.4 kb fragment. One of two MOCS1þ/
ES clones was injected into C57BL/6J blastocytes, which gave
rise to four male chimeric mice that transmitted the MOCS1
mutation in the germline. Chimeric mice were intercrossed to
C57BL/6J and 129/Sv females, respectively, to establish the
MOCS1-disrupted allele on a C57BL/6J 129/Sv hybrid and
on a 129/Sv inbred genetic background. Heterozygous animals
were identified by PCR analysis of DNA from mouse tails
(Fig. 1A and C). In both backgrounds, male and female mice
heterozygous for the MOCS1 mutation appeared normal and
fertile. Heterozygous animals were mated and 25% of the
offspring were homozygous for the mutant allele.
Homozygous (/) animals appeared normal at birth and
suckled. However, they failed to thrive (Fig. 2A and C) and
died between days 1 and 11 after birth (Fig. 2B) with a mean
life span of 7.5 days. MOCS1-deficient animals from the mixed
background strain (129/Sv C57BL6J) showed no phenotypic
differences as compared with those from the inbred line (129/
Sv) and were used for all subsequent analyses. Northern blot
analysis with a 30 flanking probe (corresponding to exon 10)
showed absence of MOCS1 RNA in the / animals,
indicating that, owing due to the integration of the neo
cassette, the expression of both the MOCS1A and the
MOCS1B proteins is hampered (Fig. 3).
Apart from their smaller size in comparison to their
littermates and curly whiskers, the homozygous animals
displayed no morphological abnormalities. Ataxia, convulsions
and ectopic ocular lenses were not observed. Since loss of
white matter has repeatedly been reported for MoCo-deficient
human patients (5,16,17), nuclear staining of neuronal tissue
was performed (Fig. 4). Hippocampus (Fig. 4A), spinal cord
(Fig. 4B) and the cortex (Fig. 4C) of homozygous animals
showed no macroscopic abnormalities in cell number or the
development of cellular layers. For the MoCo-deficient
Gephyrin knockout mouse, a disturbed postsynaptic clustering
of inhibitory receptors has been reported (9). Immunostainings
of MOCS1-deficient mice, in contrast, showed normal clusters
for both Gephyrin and glycine receptors (Fig. 5).
Biochemical analysis of MOCS1-deficient mice
Molybdopterin was determined using the very sensitive nit-1
reconstitution assay. Figure 6A shows that molybdopterin (free
Human Molecular Genetics, 2002, Vol. 11, No. 26
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Figure 2. MoCo-deficient mice. (A) MOCS1/ mouse (left) and healthy littermate (right) on day 8 after birth. (B) Survival of homozygous MoCo-deficient mice
in days. No MOCS1/ mouse so far has survived beyond d11. (C) Weight development of þ/þ, þ/ and / mice during the first 5 days post partum.
or in active cofactor form) in control animals is in the range
30–60 units/mg protein and virtually absent in the homoyzgous
animals. Owing to the loss of MoCo all molybdenumdependant enzymes are inactive, as demonstrated for sulfite
oxidase and xanthine dehydrogenase by direct measurement of
enzyme activity from liver extracts (Fig. 6B and C). In control
animals sulfite oxidase activity is increased in an agedependent manner. Because of the total loss of molybdopterin,
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Figure 3. Northern blot analysis of liver RNA from the three genotypes.
Hybridization with the murine MOCS1 exon 10 probe (top) revealed a 3.6 kb
mRNA prominent in MOCS1þ/þ, reduced in MOCS1þ/, and absent in
MOCS1/ mice. Rehybridization with human elongation factor (hEF, bottom)
confirms equal amounts of mRNA.
residual sulfite oxidase activities in homozygous mice are due
to unspecific cytochrome c reductase activities in crude liver
extracts (18).
Semiquantitative determination of sulfite (Merckoquant) in
fresh urine confirmed elevated sulfite levels in MOCS1/
animals as compared to wild-type and heterozygotes (data not
shown). Amino acid analysis showed elevated taurin levels as
compared to other amino acid concentrations in / mice. All
urine samples revealed a signal coeluting with authentic
sulfocystein reference compound, which was impaired by
interfering components. This peak was higher in urine of /
animals than in urine of wild-type or heterozygous animals.
Cystin was clearly elevated in MoCo-deficient mice (Fig. 7A).
The loss of xanthine dehydrogenase activity results in an
elevation of the substrate xanthine and undetectable levels of
the product uric acid in the urine (Fig. 7B and C). Wild-type as
well as heterozygous mice show similar levels of xanthine
oxidase educt and product. Hypoxanthine was undetectable in
all samples.
DISCUSSION
In most MoCo-deficient patients severe mutations such as
frameshift mutations or substitutions of conserved positions are
found in the genes MOCS1, MOCS2 or Gephyrin (7). These
mutations result in undetectable levels of molybdopterin,
MoCo and sulfite oxidase activity with the consequence of
considerable neurological damage and mental retardation. A
few milder cases of isolated sulfite oxidase deficiency have
been described (19,20), and Johnson et al. (5) have reported the
first mutation in an unusual mild case of molybdenum cofactor
deficiency. One of the two mutations identified in the MOCS2
gene apparently results in residual MoCo activity, thus leading
to a milder phenotype.
In this study, we have set out to create an animal model for
the more common form of the disease, which is devastating and
untreatable by any known therapy. We therefore eliminated a
complete exon of the MOCS1 gene, removing a conserved
domain of the MOCS1A ORF. Additionally, MOCS1B
expression is hampered by the integration of the neo cassette.
The presented data corroborate our assumption that thereby the
synthesis of MoCo is completely abolished. As in humans, this
results in a complete inactivation of the MoCo-dependant
enzymes sulfite oxidase and xanthine dehydrogenase. It has
been firmly established that sulfite oxidase deficiency alone is
responsible for the severe neurological damages in MoCo
deficiency (1). The phenotypic variance of the MoCo-deficient
mice, as expressed in a lifespan of 1–11 days, is very similar to
that observed in human patients, who show a survival of 1 week
up to 10 years, but usually do not reach adolescence. The
absence of any phenotypic differences between knockout mice
with either a genetic background from the inbred strain 129/Sv
or the mixed background C57BL/6J 129/Sv excludes any
additional genetic elements in the mouse with a potentially
compensating or modifying effect. As in humans, murine
MoCo (MOCS1) deficiency is inherited as a monogenetic
autosomal-recessive trait, for which our statistical data indicate
complete penetrance.
Various brain dysmorphologies have been described in
MoCo-deficient patients and explained by both sulfite toxicity
to the CNS and sulfate deficiency leading to neuronal loss (1).
Because the here described MoCo-deficient animals die within
their first days in life without any significant changes in CNS
morphology, our observations put more emphasis on the critical
role of sulfite toxicity. The precise mechanisms of this toxicity
are not completely understood. The reaction of sulfite with
disulfide bonds or with sulfhydryl groups would be a general
process without pronounced sensitivity of the brain. Graf et al.
(17) already pointed out that one effect of elevated sulfite levels
might be the depletion of glutathione, which itself is necessary
for cell membrane resistance to oxidative injury. Salman et al.
(21) investigated a deceased MoCo-deficient patient in detail
and concluded that the sulfite-induced toxic insult to the brain
causes excitotoxic neuronal injury in the presence of excess
magnesium.
The connection of MoCo biosynthesis and postsynaptic
receptor clustering, the two roles of the bifunctional protein
Gepyhrin, remains elusive for the time being. One might
speculate that these two biological processes interact with each
other and that a MoCo deficiency other than Gephyrin
deficiency also disturbs the postsynaptic architecture. We
therefore examined the distribution of Gephyrin itself as well
as that of the inhibitory glycine receptor in MOCS1-deficient
mice. However, we did not find any abnormalities at the
molecular level (Fig. 5) and these findings are in accordance
with the absence of motor defects as described for the Gephyrin
knockout mouse (10).
The phenotype of the MoCo-deficient mice described in this
study corresponds to that found in the majority of human
patients, a MOCS1 mutation abolishing the formation of precursor Z. However, no morphological changes in brain tissue
and no ectopic lenses were observed. These symptoms usually
manifest after the neonatal period in human patients and a
maximal life span of 11 days of the homozygous animals might
be too short to develop such gross abnormalities. Although we
did not observe any signs of convulsions or ataxia, it is difficult
to exclude these symptoms in mice of such limited age. Clearly,
further work is required here to determine the myelinization
state of neurons.
The homozygous animals truly reflect the biochemical
abnormalities typical and diagnostic of MoCo deficiency,
which finally result in premature death. These animals therefore
can now be used for testing of therapeutical concepts for this
hitherto incurable disease. Two options are available to this end.
Human Molecular Genetics, 2002, Vol. 11, No. 26
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Figure 4. Nuclear staining of neuronal tissue derived from MOCS1A-deficient mice. The hippocampus (A), the spinal cord (B) and a cortical detail (C) are shown.
As compared to wild-type tissue sections no macroscopic abnormalities in cell number or the development of cellular layers are detectable upon the loss of
MOCS1A protein.
One is the purification of precursor Z, which is the product of
the MOCS1-encoded proteins and more stable than molybdopterin or active MoCo, and its delivery in different therapeutical
regimes. The second option is gene therapy, delivering one or
two types of cDNAs to various organs by the use of viral or
plasmid vectors. Although these protocols in attempts to cure
other diseases often lack a sufficient expression level to reverse
or at least improve the clinical phenotype, the apparently very
low level of expression of all MOCS genes (2,22) renders
MoCo deficiency a promising candidate for this approach. A
correction of the biochemical abnormalities of MoCo deficiency by these means will not necessarily translate into a
reversal of neurological symptoms. The described murine
model will therefore also be helpful to determine how early an
effective treatment regime will have to start.
MATERIALS AND METHODS
Construction and transfection of the targeting vector
A murine MOCS1 cDNA (GenBank AF214016) was isolated
by reverse transcription followed by PCR amplification and
used to screen a murine cosmid library (RZGPD). A genomic
clone (MPMGc121C15173Q2) was identified and further
characterized by restriction mapping and sequence analysis.
A 4.6 kb XbaI fragment containing the 50 -flanking region of the
MOCS1 gene exon 3 (including exon 2) was isolated and
ligated with XbaI-digested pPNT vector (clone MOCS1/1).
A 4.4 kb fragment containing a 30 -flanking region of the
MOCS1-gene exon 3 (including exons 4–7) was amplified by
high-fidelity PCR (Clontech), subcloned into pGEM-Teasy
(Promega) and isolated again using two NotI sites flanking the
cloning site. This 30 -flanking region was inserted into the NotI
digested clone MOCS1/1 and the resulting targeting construct
(Fig. 1A) was linearized with BamHI before transfection.
Colonies resistant to G418 (400 mg/ml) and ganciclovir
(GANC) (2 mM) were selected, genomic DNA extracted,
digested with HindIII and blotted onto Hybond N membrane
(Amersham).
PCR genotyping
Genomic DNA was extracted from mouse tails and PCR was
carried out for 35 cycles using the following conditions: 45 s at
94 C, 45 s at 60 C and 75 s at 72 C. The following primers
were used to discriminate wild-type and mutant alleles:
1(MOCS1 antisense), 50 -GGCAGAGGCTGTTCAACATGG-3;
2(MOCS1 sense), 50 -CTGGGTTCCTGTGCCATCTAG-3;
3(Pgk sense), 50 -TCTGAGCCCAGAAAGCGAAGG-30 . The
amplification products were analysed on 1.5% agarose gels. A
500 bp fragment of the mutant allele was amplified with
primers 1 and 3 whereas primers 1 and 2 amplified 650 bp
wild-type product with template DNA from both heterozygous
and wild-type animals (Fig. 1).
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Figure 5. Postsynaptic immunoreactivities in spinal cord sections derived from wildtype (þ/þ) and MOCS1 knockout (/) mice. Immunostainings using antibodies specific for the postsynaptic clustering protein gephyrin (A–D) and the inhibitory glycine receptor (E and F) are shown. Sections derived from MOCS1deficient mice display normal gephyrin clusters in number and size as compared to wild-type sections (A and B). At higher magnification gephyrin immunoreactivity is detectable in dendritic neuronal processes of both genotypes (C and D). Glycine receptor immunoreactive hotspots are concentrated along spinal dendrites
of both genotypes as shown in representative projections (E and F).
Northern blot analysis
Total RNA (40 mg) was extracted from mouse liver using ‘Total
RNA Reagent’ according to a protocol provided by the
manufacturer (Biomol). RNA samples were electrophoresed
on 1% denaturing agarose gels containing formaldehyde (5%)
and subsequently transferred onto nitrocellulose membrane
(Hybond-C extra, Amersham). This filter was hybridized with
32
P-dCTP-labeled (3000 Ci/mmol) MOCS1 exon 10 fragment.
To check for integrity and equal amounts of RNA, the filter was
Human Molecular Genetics, 2002, Vol. 11, No. 26
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Figure 6. Biochemical analysis of MOCS1-deficient mice. (A) Molybdopterin content (including active molybdenum cofactor) of wild-type and MOCS1-deficient
mice at various days after birth. (B) Sulfite oxidase activity of the same animals as investigated in (A). (C) In gel detection of xanthine dehydrogenase activity. As
positive control (C) 0.75 mU of bovine xanthine oxidase were used.
rehybridized with a human elongation factor-2 (hEF) cDNA
probe (23).
Histology
Cryostat sections were fixed for 20 min in 4% (w/v)
paraformaldehyde and stained with 0.1% (w/v) cresylviolet
for 10 min to visualize nuclei. Sections were washed in water
twice for 2 min followed by the sequential incubation in 70%
(v/v), 90% (v/v), 100% (v/v), 100% (v/v) ethanol for 2 min
each. For delipidation sections were then incubated in xylol
twice for 2 min before being covered in entellan mounting
solution (Merck). Stained sections were visualized using a
Zeiss Axiovert microscope equipped with a cooled CCD
camera and the imaging software MetaMorph (Visitron
Systems).
Immunochemistry and confocal microscopy
Cryostat sections were fixed for 5 min in 4% (w/v) paraformaldehyde and processed for immunofluorescence as previously
described (9). In brief, cells were permeabilized in 0.5% (v/v)
Triton-X-100, blocked in 5% (v/v) goat serum for 20 min and
processed for immunofluorescence. Gephyrin and glycine
receptor were visualized using the monoclonal antibodies mAb7
and mAb4, respectively (24). As secondary antibodies Alexa 488/
594 were used (Molecular Probes). Confocal microscopy was
performed using a Leica TCS-SP confocal laser scanning
microscope equipped with Leica TCS-NT image software.
Molybdopterin analysis
Neurospora crassa nit-1 extract was prepared as described (25)
and desalted prior to use by gelfiltration using Nick columns
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Human Molecular Genetics, 2002, Vol. 11, No. 26
Figure 7. Metabolic analysis of MOCS1-deficient mice. (A) Cystin concentration of healthy (þ/þ), heterozygous (þ/) and homozygous (/) animals. Urine of
at least three animals was collected on day 3 or 4 post partum and pooled. (B) Xanthine concentration in the urine of wild-type, heterozygous and / mice at day
5 post partum. (C) Uric acid levels in the urine of the same animals as investigated in (B).
(Amersham/Pharmacia). Crude protein extracts from mouse
liver were prepared using 2 vol nit-1 buffer (50 mM sodium
phosphate, 200 mM NaCl, 5 mM EDTA, pH 7.2), sonication and
centrifugation. According to the linear range of the assay 2 ml
of different dilutions of liver extracts were co-incubated with
20 ml of nit-1 extract containing 5 mM sodium molybdate and
2 mM reduced gluthatione. Complementation was carried out
overnight at 4 C under anaerobic conditions. After addition of
20 mM NADPH for 10 min, reconstituted NADPH-nitrate
reductase activity was determined. One unit of molybdopterin
activity is defined as reconstituted nit-1 nitrate reductase
sufficient to produce an increase at 540 nm of 1.0 absorbance
units per 20 min reaction time. Activity is expressed as units
per mg protein of the crude extract.
increase of 1.0 absorbance at 550 nm per min at 25 C. Activity
is expressed as units per mg protein of the crude extract.
Sulfite oxidase assay
Metabolite analysis
Sulfite oxidase activity was determined according to Johnson
et al. (26). Crude protein extracts from mouse liver were
prepared using one volume of 0.1 M Tris–HCl, 0.1 mM EDTA,
pH 8.5 by sonication. Enzyme activity was assayed in a
reaction volume of 300 ml containing 0.5 M Tris/HCl, 0.16 mM
sodium deoxycholic acid, 2 mM potassium cyanide, 0.25 mg
cytochrome c and 1 mM sodium sulfite. One unit sulfite oxidase
activity is defined as enzyme activity needed to produce an
Concentrations of the purines uric acid, xanthine and
hypoxanthine in urine were determined by reversed-phase
HPLC using a modification of a method described by
Simmonds et al. (29). In brief, separation of purines was
achieved by gradient elution on a Spherisorb 5 mm ODS-2
analytical column (250 4.6 mm; Phenomenex, Aschaffenburg,
Germany) at a flow rate of 1.3 ml/min. Eluent A consisted
of 40 mM ammonium acetate in HPLC grade water adjusted
Xanthine dehydrogenase assay
Xanthine dehydrogenase activity was determined based on the
methods of Mendel and Müller (27) and Koshiba et al. (28).
Crude protein extracts from mice liver were prepared by
sonication in 3 vol 100 mM potassium phosphate, 2.5 mM
EDTA, 5 mM dithiothreitol, pH 7.5. After centrifugation protein
concentrations was determined and 280 mg protein were loaded
on each lane of a 6% native polyacrylamide gel. Xanthine
dehydrogenase activity was detected by activity staining using
300 mM hypoxanthine as substrate. As positive control 0.75 mU
XO from cow buttermilk (Sigma) was used.
Human Molecular Genetics, 2002, Vol. 11, No. 26
to pH 5.00 with acetic acid and eluent B of 80% methanol:10%
acetonitrile:10% tetrahydrofuran (v/v/v). Urine was diluted
with a 9-fold volume of eluent A. Fifty microliters of the
diluted urine were directly injected into the HPLC system.
Purines were quantitated based on peak area units obtained by
UV detection at 254 nm using calibration with authentic
standard compounds. Peak identity was confirmed by comparison of both the retention times and of the spectra obtained with
a photodiode array detector between 220 and 320 nm. The
determination of the creatinine concentration was based on
the Jaffé reaction (30). Amino acid concentrations in urine were
determined with a Biotronik LC 3000 aminoacid analyzer
using two detection wavelenghts (440 and 570 nm).
ACKNOWLEDGEMENTS
This work was supported by the Deutsche Forschungsgemeinschaft (Re768/5-4). We thank Ina Bartnik, Sigrid GrossHardt, Tanja Otte, Ilona Paprotta, H. Riedesel, Monika
Schindler and Stefan Wolf with his blue angels for excellent
technical assistance.
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