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Articles |
1
Institutes of Neurology and Paediatrics, University Hospital Nijmegen, 6525 GC Nijmegen, The Netherlands.
2
University Marburg, Department of Neuropediatrics and
Metabolic Diseases, D-35037 Marburg, Germany.
3
University Paediatric Hospital Utrecht, Laboratory of
Metabolic Diseases, NL-3512 LK Utrecht, The Netherlands.
4
Laboratory for Genetic Metabolic Disease, Academic
Medical Centre, NL-1105 A2 Amsterdam, The Netherlands.
a Address correspondence to this author at: University Hospital Nijmegen, Institute of Neurology, Reinier Postlaan 4, 6525 GC Nijmegen, The Netherlands. Fax 31-24-3540297; e-mail r.wevers{at}ckslkn.azn.nl
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Abstract |
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Methods: We performed 1H-NMR spectroscopy on 500 and 600 MHz instruments with a standardized sample volume of 500 µL. We studied body fluids from 25 patients with nine inborn errors of purine and pyrimidine metabolism.
Results: Characteristic abnormalities could be demonstrated in the 1H-NMR spectra of urine samples of all patients with diseases in the pyrimidine metabolism. In most urine samples from patients with defects in the purine metabolism, the 1H-NMR spectrum pointed to the specific diagnosis in a straightforward manner. The only exception was a urine from a case of adenine phosphoribosyl transferase deficiency in which the accumulating metabolite, 2,8-dihydroxyadenine, was not seen under the operating conditions used. Similarly, uric acid was not measured. We provide the 1H-NMR spectral characteristics of many intermediates in purine and pyrimidine metabolism that may be relevant for future studies in this field.
Conclusion: The overview of metabolism that is provided by 1H-NMR spectroscopy makes the technique a valuable screening tool in the detection of inborn errors of purine and pyrimidine metabolism.© 1999 American Association for Clinical Chemistry
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The aim of this study was to collect the 1H-NMR spectra of
various intermediates in purine and pyrimidine metabolism. Furthermore,
we wanted to study the diagnostic performance of the technique in the
examination of body fluids from patients suspected to suffer from
inborn errors of metabolism in these pathways. The nine enzyme defects
studied and their places in the metabolic pathways are explained in
Fig. 1
.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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For quantification, trimethylsilyl-2,2,3,3-tetradeuteropropionic acid (TSP; sodium salt) was added as internal standard in a final concentration of 2.02 mmol/L (50 µL of TSP in D2O into 500 µL of urine). The sample volume in the NMR spectrometer was standardized at 500 µL. The water resonance was presaturated during the relaxation delay (6 s). For shimming of the magnetic field, the 29Si-1H long-range coupling of 3 Hz in the TSP resonance was used.
For data analysis, a Sine-Bell squared filter (SBB = 2) was used. Spectra were Fourier transformed after the free induction decay was zero-filled to 65 536 data points. The phase and baseline of the spectra were corrected manually. Resonances were fitted semi-automatically to a Lorentzian line shape model function. Integrals of these fits were used for metabolite quantification. Metabolites in urine are expressed per millimole of creatinine. For analysis of the spectra, 1D WinNMR and WinFit software were used (Bruker Analytische Messtechnik).
When certain compounds are described in this study as "NMR invisible", it means that in the relevant concentration range no characteristic signals could be observed under the 1H-NMR spectroscopy standard operating conditions used. The detection limit of 1H-NMR spectroscopy for each metabolite depends on the number of protons that contribute to a signal and on the multiplicity of the resonance (5)(7). When the molecule contains a methyl group, the estimated detection limit is in the range between 5 µmol/L for a singlet resonance and 10 µmol/L for a doublet resonance. Some purines and pyrimidines have only one proton contributing to the signal. The estimated detection limit of the technique in such a case is 15 µmol/L for a singlet resonance and 30 µmol/L for a doublet resonance. Some metabolites have no contributing proton in their chemical structure. For this reason, metabolites such as uric acid and 2,8-dihydroxyadenine are NMR invisible. It is conceivable that uric acid can be detected by 1H-NMR spectroscopy under different experimental conditions (e.g., at a different pH). Obviously, this would be of utmost importance for diagnosing purine inborn errors. However, because such conditions are likely to introduce other problems in the interpretation of the NMR spectra, the standard operating procedure that we have used for a long time in diagnosing inborn errors of metabolism was also used in the present study.
Model compounds used in this study are available from Sigma Chemical Co., except for 5-acetyl-amino-6-formyl-amino-3-methyl uracil (kindly provided by Prof. H.A. Simmonds, Guy's Hospital, London, UK) and succinylaminoimidazole carboxamide (SAICA)-riboside and succinyladenosine (kindly provided by Prof. G. van den Berghe, Université Catholique de Louvain, Brussels, Belgium).
The diagnoses of all patients used in this study originally were made in the period 1981–1998 at various metabolic screening laboratories in The Netherlands, Germany, and Belgium. Samples were stored at -20 or -80 °C until analysis. These laboratories all used standard HPLC techniques (reversed-phase column, quantification by reading the absorbance at 260 nm) for measurement of purines and pyrimidines in body fluids.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. The HPLC results are the quantitative data on the sample that
were available from the laboratory that originally diagnosed the
patient. For some samples, this means that >10 years may have elapsed
between the HPLC and NMR measurements. Unfortunately, the limited
volumes of most of the samples made repeating the HPLC measurement
simultaneously with NMR spectroscopy impossible. The results of both
techniques compare rather well for most of the metabolites, although
rather large discrepancies were observed for uracil, thymine, inosine,
deoxyinosine, and deoxyguanosine.
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interpretation of the 1h-nmr spectrum
It is advantageous that many of the relevant compounds in purine
and pyrimidine metabolism have a chemical shift between 5.5 and 9.0
ppm, a region of the spectrum where only few metabolites present in
body fluids have resonances. Other known metabolites in this part of
the spectrum are tyrosine, phenylalanine, formic acid, indoxyl sulfate,
histidine, and hippuric acid. A list of 1H-NMR the
resonances of these compounds is available
(5)(7). Table 2
shows the resonances of the purine and pyrimidine metabolites
that are relevant under nondiseased physiological conditions or in
patients with inborn errors of metabolism in these pathways. The most
important resonances of a molecule for its identification in the
1H-NMR spectrum have been listed in Table 2
. Some molecules
have only one resonance, whereas others posses multiple resonances.
These can be recognized by their complete "fingerprints" in the
spectrum.
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Generally there is only minimal intersample variation in the chemical shift of a certain resonance (<0.005 ppm), which allows reliable identification of the various purines and pyrimidines in urine, plasma, and CSF. The only exception was the extremely variable chemical shift of the singlet resonance from the proton on carbon-2 of the imidazole ring of SAICA-riboside, which varied between 8.05 and 8.26 ppm between samples. The SAICA-riboside doublet resonance at 5.77 ppm has a stable resonance position, thus allowing reliable identification of this metabolite.
inborn errors of pyrimidine metabolism
Dihydropyrimidine dehydrogenase (EC 1.3.1.2) deficiency.
Dihydropyrimidine dehydrogenase is the initial enzymatic step in the
degradative pathway of the pyrimidine bases uracil and thymine (Fig. 1B
). The enzyme converts both compounds into their respective
5,6-dihydro derivatives. 1H-NMR spectra were obtained from
urine samples of four unrelated cases with dihydropyrimidine
dehydrogenase deficiency. An example of a 1H-NMR spectrum
of a urine sample of one of these cases has been published previously
(12). In all samples, uracil and thymine were clearly
increased (uracil, 95–626 µmol/mmol creatinine; thymine, 270–470
µmol/mmol creatinine). In nondiseased urine samples, thymine cannot
be detected with NMR spectroscopy, whereas uracil is observed in trace
amounts in some samples (<10 µmol/mmol creatinine).
5-Hydroxymethyluracil, a metabolite of thymine, was not detectable in
the samples.
Dihydropyrimidinase (EC 3.5.2.2) deficiency.
Dihydropyrimidinase (5,6-dihydropyrimidine amidohydrolase) is the
second enzyme in the breakdown of the pyrimidine bases uracil and
thymine. It catalyzes the conversion of 5,6-dihydrouracil to
3-ureidopropionic acid and of 5,6-dihydrothymine to 3-ureidoisobutyric
acid (Fig. 1B
). To our knowledge, only seven cases of this enzyme
defect have been described. We measured urine, plasma, and CSF in three
patients with this defect. Two of these patients have been described
clinically [case 1 by Putman et al. (9); case 2 by Assmann
et al. (10)], whereas case 3 was diagnosed recently.
Examples of 1H-NMR spectra of urine and CSF of these
patients have been published previously (9)(10)
and, therefore, are not shown here. Diagnostically high concentrations
of uracil, thymine, and dihydropyrimidines were found with
1H-NMR spectroscopy in the urine of all three patients
(concentrations of uracil, thymine, 5,6-dihydrouracil, and
5,6-dihydrothymine: 9–144, 44–230, 178–760, and 132–490 µmol/mmol
creatinine, respectively). The CSF of cases 1 and 2 showed obviously
increased concentrations for the dihydropyrimidines, but only slightly
increased concentrations for uracil and thymine (concentrations of
uracil, thymine, 5,6-dihydrouracil, and 5,6-dihydrothymine: not
detectable, trace to 10 µmol/L, 46–117 µmol/L, and 79–179
µmol/L, respectively). The CSF of the third patient was not
available. For all four compounds, the concentrations in plasma were
obviously lower than in CSF (9). The absence of high
concentrations of 3-ureidopropionic acid
(N-carbamyl-ß-alanine; model compound, Sigma cat. no.
C3750) in the urine or CSF of these patients excluded
ß-ureidopropionase deficiency as a possible defect. NMR findings were
compatible with dihydropyrimidinase deficiency, which was later
confirmed enzymatically in the liver of cases 1 and 2
(10)(13)(14). A liver biopsy of case
3 was not available.
ß-Ureidopropionase (EC 3.5.1.6) deficiency.
ß-Ureidopropionase catalyzes two reactions: the conversion of
3-ureidopropionic acid and 3-ureidoisobutyric acid into ß-alanine and
3-aminoisobutyric acid, respectively. A deficiency of the enzyme has
not yet been recognized in humans. However, resonances of both
substrates should be visible in the 1H-NMR spectra of body
fluids of a patient with this defect. Table 2
gives the characteristic
resonances of 3-ureidopropionic acid.
inborn errors of purine metabolism
Adenine phosphoribosyltransferase (APRT; EC 2.4.2.7)
deficiency.
APRT is a purine salvage enzyme that catalyzes the
conversion of adenine to AMP (Fig. 1A
). When the enzyme is deficient,
adenine is not salvaged to AMP, but instead is oxidized to
8-hydroxyadenine and 2,8-dihydroxyadenine by xanthine dehydrogenase
(XDH). APRT deficiency is characterized by 2,8-dihydroxyadenine
urolithiasis. Patients have increased concentrations of
2,8-dihydroxyadenine in the urine. The compound, however, is
1H-NMR invisible under the conditions used. We have
measured a urine sample of an adult case with this inborn error and
found no unusual resonances. The patient did not use allopurinol
medication. The lactic acid concentration in the sample was very high
(7 mmol/mmol creatinine). Adenine, 2-hydroxyadenine, and
8-hydroxyadenine may be increased in this disease. Detection of
2-hydroxyadenine and 8-hydroxyadenine is hampered by the fact that they
are not available as model compounds. They were not observed in the NMR
spectrum of the urine of our patient. Similar results were obtained
when the sample was measured at its native pH (pH 8.2). A plasma sample
of the patient showed high creatinine, indicating renal dysfunction.
However, no abnormal resonances that may be indicative for the
underlying metabolic defect could be observed.
Lesch-Nyhan disease/hypoxanthine-guanine phosphoribosyltransferase
(HGPRT; EC 2.4.2.8) deficiency.
HGPRT also is a purine salvage
enzyme, converting hypoxanthine and guanine into IMP and GMP,
respectively (Fig. 1A
). In the deficiency state, guanine and
hypoxanthine are instead converted into xanthine and finally into uric
acid. Uric acid overproduction is a characteristic of Lesch-Nyhan
disease. Uric acid is a trioxypurine without protons on its carbon
atoms; therefore, it is 1H-NMR invisible. Increased
hypoxanthine and xanthine are expected to be the only characteristic
abnormalities in the NMR spectrum in body fluids of untreated
Lesch-Nyhan patients. Urine samples of three classical Lesch-Nyhan
cases were measured. Two of these patients were on allopurinol
medication. In the 1H-NMR spectrum of the patient without
allopurinol, xanthine and hypoxanthine were clearly increased
(xanthine, 37 µmol/mmol creatinine; hypoxanthine, 198 µmol/mmol
creatinine; reference values for both compounds, <4.1 µmol/mmol
creatinine). In other cases of Lesch-Nyhan disease, xanthine in urine
may be within the reference interval, but hypoxanthine will always be
increased. In the patients that used medication, the excretion was
higher (xanthine, 312–827 µmol/mmol creatinine; hypoxanthine,
855-1363 µmol/mmol creatinine) because feedback inhibition of de novo
purine synthesis by IMP and GMP does not take place because of the
HGPRT deficiency. Guanine was not detectable, whereas orotidine was
clearly increased in both samples (112 and 63 µmol/mmol creatinine).
This can be explained by the inhibitory effect of oxypurinol, which is
generated from allopurinol by the enzymatic action of XDH or aldehyde
oxidase (AO; Fig. 2
) on orotate phosphoribosyl transferase. Resonances from
allopurinol-1-ribonucleotide and allopurinol-1-riboside were not
observed because the conversion of allopurinol into these metabolites
requires the action of HGPRT, which is deficient in these patients
(Fig. 2
). In the CSF of the untreated patient, xanthine and
hypoxanthine were both increased (22 and 94 µmol/L respectively). In
most CSF samples, both compounds are not observed in the NMR spectrum
because the reference intervals for both are below the detection limit
of the technique under the conditions used.
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Adenosine deaminase (ADA; EC 3.5.4.4) deficiency.
ADA
catalyzes the conversion of adenosine into inosine. The enzyme also
accepts deoxyadenosine as a substrate, converting it to deoxyinosine
(Fig. 1A
). Deoxyadenosine and adenosine therefore are the metabolites
that are of diagnostic importance. In urine, deoxyadenosine generally
is present in higher concentrations than adenosine in patients with ADA
deficiency. We investigated the urine of one patient with this disease.
The NMR spectrum showed the presence of high concentrations of
deoxyadenosine (205 µmol/mmol creatinine; reference value, <1
µmol/mmol creatinine). Adenosine was not detectable with NMR
spectroscopy, but was within the reference interval when the sample was
analyzed with HPLC. Orotic acid was not observed in the NMR spectrum. A
complication in the NMR spectrum was the presence of an unknown doublet
resonance at 6.54 ppm, which interfered with the 6.54 triplet resonance
from deoxyadenosine. Determining whether the doublet resonance is a
characteristic feature for this metabolic defect requires measurement
of additional samples from ADA-deficient cases.
Purine-nucleoside phosphorylase (PNP; EC 2.4.2.1) deficiency.
PNP catalyzes the reversible phosphorylation of inosine and
deoxyinosine or guanosine and deoxyguanosine to give either
hypoxanthine or guanine plus the corresponding ribose-1-phosphate (Fig. 1A
). Therefore, inosine, guanosine, and their deoxy forms are increased
in body fluids in the PNP-deficient state. Fig. 3
A shows the 1H-NMR spectrum of a urine of a patient
with this deficiency. Urine samples from two cases were available. Both
patients had the typical clinical signs and symptoms of PNP deficiency
(15)(16). The enzyme defect was shown in
erythrocytes and fibroblasts in both cases. We found increased
concentrations for inosine (case 1, 49 µmol/mmol creatinine; case 2,
1110 µmol/mmol creatinine), guanosine (case 1, 35 µmol/mmol
creatinine; case 2, 588 µmol/mmol creatinine) and deoxyinosine [case
1, trace (<5 µmol/mmol creatinine); case 2, 522 µmol/mmol
creatinine]. In controls, these compounds are found in the urine in
very low concentrations (<1 µmol/mmol creatinine). Deoxyguanosine
was not observed in the urine of case 1, but could clearly be observed
in the urine of case 2 (322 µmol/mmol creatinine; reference, <1
µmol/mmol creatinine). Xanthosine was not found in the urine samples
of the two patients. The metabolite concentrations between the two
patients showed remarkable differences. In addition, other differences
between the patients were observed. Increased orotic acid (95
µmol/mmol creatinine; reference, <10 µmol/mmol creatinine) was
observed in case 1, but not in case 2. Orotidine was not detectable in
these samples. The patients had not used allopurinol medication. Very
high, as yet unexplained, resonances (doublet resonances at 5.33 and
5.40 ppm and singlet resonances at 5.60, 5.63, 6.08, and 6.63 ppm) were
found in the sample of case 1. In case 2, the 5.40 ppm doublet
resonance and the 6.63 ppm singlet resonance were also observed,
although these resonances were considerably lower than in the urine of
case 1. The 6.08 ppm resonance may be present in the urine of case 2,
but this resonance would overlap with the doublet resonance deriving
from the very high concentration of inosine in this sample. The urine
sample of case 2 contained very high additional unknown resonances
(singlet resonance at 2.26 ppm and doublet resonance at 2.96 ppm) that
were not present in the urine of case 1. A plasma sample from another
patient had clearly increased inosine (case 3, 40 µmol/L; reference,
<0.1 µmol/L), whereas guanosine and both deoxy compounds were not
detectable. Except for the 6.08 ppm singlet resonance, the other
unknowns observed in the urine of the other two patients were not
detectable in this blood sample. It would require measurement of urine
samples from other patients with this defect to determine whether these
unknown resonances are caused by the metabolic block.
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Xanthinuria.
In classical xanthinuria, a deficiency of XDH (EC
1.1.1.204) exists. The enzyme converts hypoxanthine into xanthine and
xanthine into uric acid (Fig. 1A
). Furthermore, a combined XDH/AO
deficiency is known. Patients with the combined defect have an
inability to convert allopurinol to oxypurinol (Fig. 2
). In both
diseases, xanthine is the predominant purine excreted. Urine samples of
three patients from two families with isolated XDH deficiency could be
measured both before and during allopurinol therapy. In one patient,
isolated XDH deficiency was confirmed enzymatically. In urines from
three patients not on medication, the increased xanthine concentration
was the only characteristic feature in the 1H-NMR spectrum
(154–890 µmol/mmol creatinine; Fig. 3B
), and hypoxanthine was
slightly increased (37–217 µmol/mmol creatinine). In four additional
urine samples of two of these patients while on allopurinol treatment,
a similar picture was observed. The xanthine concentrations tended to
be even higher than before treatment (507–941 µmol/mmol creatinine).
This may relate to the competitive inhibition of oxypurinol deriving
from allopurinol on the residual XDH activity. Resonances from
allopurinol (14–1176 µmol/mmol creatinine) and oxypurinol (255–367
µmol/mmol creatinine) were observed in the 1H-NMR
spectrum of these four samples (Fig. 3B
). The presence of oxypurinol
illustrates that the patients have isolated XDH deficiency because
oxypurinol cannot be formed in the combined XDH/AO deficiency because
this requires AO enzymatic activity (Fig. 2
). Allopurinol-1-riboside
could also be detected (31–161 µmol/mmol creatinine). In the urine
of one patient while on allopurinol, the orotidine concentration was 30
µmol/mmol creatinine. We were unable to demonstrate the presence of
oxypurinol-1-riboside or oxypurinol-7-riboside while the patients were
on allopurinol. Their detection with 1H-NMR spectroscopy is
hampered by the fact that they are not available as model compounds.
Molybdenum cofactor deficiency.
Patients with molybdenum
cofactor deficiency have an inability to synthesize the pteridyl moiety
of the cofactor essential for the enzymatic activity of XDH, sulfite
oxidase, and AO. Xanthinuria, hypouricemia, and increased urinary
sulfite, S-sulfocysteine, and thiosulfate concentrations are
characteristic for the disease. Xanthinuria can be demonstrated easily
in the 1H-NMR spectrum. Serum and CSF samples were
available from one patient with this defect. Xanthine was increased in
both body fluids (case 1: serum xanthine, 136 µmol/L; CSF xanthine,
24 µmol/L). Hypoxanthine was not clearly increased in these samples.
Urine was available of four patients with this defect (cases 2–5). In
three of these patients without medication (cases 2–4), xanthine was
clearly increased (450–498 µmol/mmol creatinine), whereas
hypoxanthine was only slightly increased (47–56 µmol/mmol
creatinine). Xanthosine was detected in only one of these urine
samples, in a low concentration (80 µmol/mmol creatinine). An
additional patient (case 5) described by van Gennip et al.
(17) was on allopurinol treatment because of the existence
of renal xanthine stones. In this case, clearly increased xanthine was
found in three urine samples during allopurinol treatment (xanthine,
275–726 µmol/mmol creatinine), whereas hypoxanthine was only
slightly increased (22–67 µmol/mmol creatinine). Allopurinol was
found in two of the three samples, whereas all three samples contained
allopurinol-1-riboside (314–834 µmol/mmol creatinine). No oxypurinol
was found because allopurinol cannot be converted into oxypurinol in
this disease (Fig. 2
). Sulfite and thiosulfate are 1H-NMR
invisible, and S-sulfocysteine could not be demonstrated in
any of the urines of the patients with molybdenum cofactor deficiency.
For S-sulfocysteine, this may relate to its instability
under certain pH conditions or to the fact that NMR spectroscopy under
the conditions used is too insensitive for its detection. This relative
insensitivity is caused by the multiplicity of the resonance (4.26
doublet-doublet resonance, four peaks) and by the fact that only one
proton contributes to this signal. Orotidine was not increased in any
of these samples, as expected.
Adenylosuccinate lyase (EC 4.3.2.2) deficiency.
Adenylosuccinate lyase, or adenylosuccinase, catalyzes two reactions in
the biosynthesis of purine nucleotides. This concerns the conversion of
SAICA-ribotide into aminoimidazole carboxamide ribotide and of
adenylosuccinate into AMP (Fig. 1A
). A defect in the enzyme leads to
accumulation of two usually undetectable compounds: SAICA-riboside,
deriving from SAICA-ribotide; and succinyladenosine, deriving from
adenylosuccinate. Both compounds can be detected readily in the urine
of affected patients (Fig. 3C
). The 1H-NMR spectrum of
urine from two unrelated patients showed increased succinyladenosine
(101 and 188 µmol/mmol creatinine) and SAICA-riboside (162 and 214
µmol/mmol creatinine). Recognition of SAICA-riboside in urine with
1H-NMR spectroscopy may be hampered by the use of the
antiepileptic drug Sabril® (Vigabatrin), which produces a
characteristic resonance profile that overlaps with the doublet
resonance of SAICA-riboside. In such a case, the compound can still be
found in the spectrum through its singlet resonance. In the CSF of one
of the patients, both SAICA-riboside and succinyladenosine were present
in detectable amounts (198 and 216 µmol/L, respectively).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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of the
present study, we have compared HPLC and NMR results for several
metabolites in urine that are of diagnostic importance in inborn errors
of purine and pyrimidine metabolism. The sometimes very long time
interval (up to 17 years) between the HPLC and the NMR measurements of
course is a serious drawback of this approach. Table 1
is no more than
a rough indication that for most metabolites the two techniques will
give similar quantitative results. The larger discrepancies found for
uracil, thymine, inosine, deoxyinosine, and deoxyguanosine in part are
in line with the differences found in our earlier study
(12). The addition of uracil and thymine to urine samples
gave excellent recovery values with NMR spectroscopy (12).
Currently we are preparing a separate validation study on NMR
spectroscopy in urine samples where NMR technical aspects (length of
relaxation time and other factors) and NMR performance (within- and
between-run coefficients of variation, accuracy, recovery, and
correlation with other techniques) will be addressed. NMR spectroscopy measurements in urine samples were informative in all patients with inborn errors in pyrimidine metabolism. The 1H-NMR spectra of urine samples from patients with dihydropyrimidine dehydrogenase and dihydropyrimidinase deficiency led directly to the diagnosis in all cases. Blood plasma and CSF were not investigated in dihydropyrimidine dehydrogenase deficiency; however, in dihydropyrimidinase deficiency, the diagnosis could be found with 1H-NMR spectroscopy in both body fluids.
The absence of 3-ureidopropionic acid in the urine spectra of the patients with dihydropyrimidinase deficiency excluded the theoretical possibility of ß-ureidopropionase deficiency. In this case, 1H-NMR spectroscopy helped to pinpoint the enzyme defect. The defect in these patients was later confirmed at the enzyme level in a liver biopsy (10)(13)(14). In future cases the use of 1H-NMR spectroscopy of urine may be considered to confirm this particular enzyme defect at the metabolite level, omitting the liver biopsy and the confirmation at enzyme level and going directly to the DNA level for mutation analysis. Primary ß-ureidopropionase deficiency has as yet not been found in humans. Secondary ß-ureidopropionase deficiency has been reported in propionic acidemia (18). Characteristic resonances of 3-ureidopropionic acid, one of the expected metabolites in this deficiency, can be observed with 1H-NMR spectroscopy. In this way, the technique may help in diagnosing patients with as yet unknown inborn errors in purine or pyrimidine metabolism. Whether such resonances in a spectrum would be properly assigned depends only on the quality of the available model compound 1H-NMR database. This is unlike the situation with conventional techniques, where special and often elaborate sample preparation in combination with thin-layer chromatography or HPLC is required to identify 3-ureidopropionic acid (19)(20). To find as yet unknown inborn errors of metabolism with 1H-NMR spectroscopy, the spectra of the model compounds of as many intermediates relevant in human metabolism as possible should be measured. This is a topic for further research.
In the patients with inborn errors of purine metabolism, 1H-NMR spectroscopy of urine samples led to the diagnosis in all patients with PNP deficiency, xanthinuria, molybdenum cofactor deficiency, Lesch-Nyhan disease, ADA deficiency, and adenylosuccinate lyase deficiency. After allopurinol loading, the NMR spectra of the urine could discriminate between isolated XDH deficiency and combined XDH/AO deficiency through the presence of oxypurinol. Increased urinary xanthine and/or hypoxanthine formed the only characteristic in the 1H-NMR spectrum in all samples from patients with XDH deficiency, molybdenum cofactor deficiency, and Lesch-Nyhan disease. It, therefore, was impossible to discriminate on the basis of the 1H-NMR spectrum between these diagnoses without the use of other additional tests. In this respect it is a major disadvantage that uric acid is 1H-NMR invisible and therefore cannot aid discrimination between these inborn errors of purine metabolism. APRT deficiency was the only enzyme defect in purine metabolism where no abnormality could be observed in the urinary 1H-NMR spectrum. This is mainly because the characteristic metabolite 2,8- dihydroxyadenine is NMR invisible. The diagnosis of this patient would have been missed with 1H-NMR spectroscopy of urine.
For some of the inborn errors in purine and pyrimidine metabolism, no 1H-NMR spectra were recorded. Patients with myoadenylate deaminase deficiency and patients with pyrimidine-5'-nucleotidase deficiency have no characteristic metabolite profile in body fluids, although increased concentrations of pyrimidine nucleotides have been demonstrated in erythrocytes with 1H-NMR spectroscopy of pyrimidine-5'-nucleotidase-deficient patients (11). No samples were available of patients with orotic aciduria type I or II. These defects can be diagnosed on basis of the increased concentrations of orotic acid in urine. Orotic acid can be detected easily in the 1H-NMR spectrum (singlet resonance at 6.22 ppm); therefore, these conditions cannot be missed with 1H-NMR spectroscopy. In addition, no sample was available from patients with phosphoribosyl pyrophosphate synthase superactivity. Because hypoxanthine in urine is characteristically increased in this disease, we expect no difficulties in diagnosing this defect with 1H-NMR spectroscopy.
In PNP deficiency, as yet unidentified resonances were demonstrated in all body fluid samples investigated. We never have observed these unknowns in >400 other body fluid samples (urine, plasma, and CSF) of controls or patients suspected to have inborn errors of metabolism. The resonances are likely to derive from several compounds. No other metabolites are known to occur in increased concentrations in body fluids of patients with PNP deficiency. Furthermore, there are to our knowledge no published reports indicating high concentrations of unknown metabolites in this disease. The urines of cases 1 and 2 were stored at -80 °C for several years; therefore, storage artifacts should be considered. However, because some of these unknowns were also found in the serum of case 3, we consider this option unlikely because this sample was measured with NMR spectroscopy immediately after venipuncture. There were obvious differences in the NMR spectrum of the urine of cases 1 and 2. The concentrations of the four characteristic metabolites in this disease varied considerably between the two patients, and orotic aciduria was present only in case 1. Some of the unknown resonances were present in the urine of both patients, but again obvious concentration differences were present. Other unknowns were present in only one of the two patients. These data allow various explanations. Theoretically, the unknowns can be caused by medication, derive from the food, or may actually be a characteristic of the underlying enzyme deficiency. It would be conceivable that the patients may suffer from different metabolic diseases with partially overlapping metabolite profiles. However, this seems unlikely because the enzyme deficiency was shown convincingly in the patients (15)(16). The occurrence of unknown resonances should be checked in urine samples from other patients with the disease. Because PNP deficiency is very rare, we would appreciate if urine, plasma, or CSF samples of affected patients would be made available for 1H-NMR spectroscopy. In case our findings can be confirmed in other cases, additional NMR techniques (COSY, J-resolved, and long-range spectra) may help to solve the chemical structure of the unknown metabolites involved.
Orotic aciduria is a more or less controversial finding in PNP
deficiency. It was described for the first time by Cohen et al.
(21) in two unrelated patients with the disease and
confirmed by van Gennip et al. (22). Simmonds et al.
(23), however, could not confirm it with three independent
methods. The mechanism of the orotic aciduria is unknown. It could be
caused by the inhibition of orotate phosphoribosyltransferase by
accumulating abnormal metabolites. The enzyme converts orotic acid and
5-phospho-
-D-ribosyl-1-pyrophosphate into orotidine
5-phosphate. It is known that inosine does not inhibit the enzyme in
vitro. Perhaps one of the unknown metabolites found in our patients can
cause this inhibitory effect. Another explanation for the presence of
orotic acid is the higher availability of
5-phospho--D-ribosyl-1-pyrophosphate, which leads to
increased pyrimidine synthesis de novo and overflow at orotate
phosphoribosyl transferase, the rate-limiting step in this pathway. NMR
spectroscopy of urine of affected patients may uncover the role that
the as yet unknown metabolites play in causing orotic aciduria in these
patients.
The overall view on metabolism that 1H-NMR spectroscopy provides is nicely illustrated by the various inborn errors of metabolism discussed here. The technique allows investigation of a body fluid sample from a patient clinically suspected to suffer from an inborn error of metabolism without focusing on a specific group of metabolites. Conventional techniques in metabolic screening provide information on specific groups of metabolites (amino acids, organic acids, polyols, and purines). Each of these requires specific methodology such as derivatization, extraction, chromatography steps, or detection techniques. The 1H-NMR spectrum provides quantitative information on representatives from all of these metabolite groups without the use of derivatization or extraction steps. This is the major advantage of 1H-NMR spectroscopy. Unfortunately, the spectrometer required for the technique is still very expensive. Another disadvantage is that the software required for the interpretation of the spectra cannot yet automatically recognize, assign, and quantify the many resonances present in a spectrum of complex matrices such as body fluids. Therefore, the interpretation of the spectra remains the most laborious part of the analysis. This aspect together with the high price of high-field strength spectrometers severely discourages the routine application of the technique in clinical chemistry.
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