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Articles |
1
Laboratoire de Biochimie 1, Hôpital Bicêtre AP-HP, Paris, France.
2
Departments of Biochemistry and Molecular Biology, and
Medical Genetics, Mayo Clinic and Foundation, Rochester, MN 55905.
a Address correspondence to this author at: Institute of Neurology, PO Box 9101, 6500 HB Nijmegen, The Netherlands. Fax 31-24-3540297; e-mail R.Wevers{at}ckslkn.azn.nl
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Methods: We used 1H NMR spectroscopy to study serum and urine from the patient.
Results: The concentration of N,N-dimethylglycine (DMG) was increased ~100-fold in the serum and ~20-fold in the urine. The presence of DMG as a storage product was confirmed by use of 13C NMR spectroscopy and gas chromatography–mass spectrometry. The high concentration of DMG was caused by a deficiency of the enzyme dimethylglycine dehydrogenase (DMGDH). A homozygous missense mutation was found in the DMGDH gene of the patient.
Conclusions: DMGDH deficiency must be added to the differential diagnosis of patients complaining of a fish odor. This deficiency is the first inborn error of metabolism discovered by use of in vitro 1H NMR spectroscopy of body fluids.
© 1999 American Association for Clinical Chemistry
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). DMG is converted into sarcosine and sarcosine is converted
into glycine by two oxidative demethylation steps mediated by DMG
dehydrogenase (DMGDH; EC 1.5.99.2) and sarcosine dehydrogenase (EC
1.5.99.1), respectively. In these reactions, active one-carbon groups,
called "active formaldehyde", are formed. These active one-carbon
groups are used preferentially for the formation of serine from
sarcosine (1)(2). DMGDH and sarcosine
dehydrogenase are mitochondrial flavoproteins, requiring folate as a
cofactor. Both enzymes are among the major folate-binding proteins in
rat liver (2). Deficiencies of some of the enzymes and
cofactors involved in the choline metabolic pathway, such as sarcosine
dehydrogenase deficiency and electron transfer flavoprotein (ETF)
deficiency, have been described in the literature (2). Here,
we report a defect in the conversion of DMG into sarcosine that has not
been described previously. We had the opportunity to study body
fluidsof a patient with this defect by nuclear magnetic resonance (NMR)
spectroscopy. Metabolic investigations in the patient had been
requested because of the abnormal body odor of the patient, which
resembled the odor of fish. No abnormalities were found by conventional
metabolic screening techniques. To our knowledge, this is the first
time that a new inborn error of metabolism has been discovered by the
use of 1H NMR spectroscopy of body fluids.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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NMR measurements.
The 1H NMR measurements were
performed on a Bruker AMX-600 (Bruker Analytische Messtechnik)
spectrometer. The temperature during the measurements was 25 °C, and
NMR tubes with a diameter of 5 mm were used. The samples were spun (15
Hz) during the measurements.
For the 1H NMR measurements, a 60° radiofrequency pulse with a duration of 6 µs was used. The delay between two successive pulses was 6 s. The water resonance was presaturated during the relaxation delay (6 s). The number of scans for each experiment was 128, and 32 768 data points per scan were sampled. The sweep width was 6605 Hz. Shimming of the field homogeneity was supposed to be satisfactory when the 29Si-1H long-range coupling of 3 Hz in the TSP resonance could be observed.
For the 13C NMR measurements, a 60° radiofrequency pulse of 16 µs was used. The delay between two successive pulses was also 6 s, and 32 768 data points were sampled. The number of scans was 5191. The sweep width was 37 230 Hz.
NMR data analysis.
A Sine-Bell squared filter (SSB = 2)
was used, and the spectra were Fourier transformed after the free
induction decay was zero-filled to 64 000 data points. The chemical
shift of TSP was set at a position of 0.0 ppm. The phase and baseline
of the NMR spectra were corrected manually. The resonances in the
spectra were fitted semiautomatically to a Lorentzian lineshape model
function. The integrals of these fits were used for quantification of
the corresponding metabolites by comparing them with the fit integrals
of TSP or creatinine in serum and urine, respectively. For the analysis
of the NMR spectra, 1D WinNMR and WinFit software were used (Bruker
Analytische Messtechnik).
gas chromatography–mass spectrometry
Pure DMG (10 µmol) and urine from the patient (100 µL) were
dried by evaporation under a gentle stream of nitrogen and were
derivatized for 60 min at 55 °C with 200 µL of a solution of
N,O-bis(trimethylsilyl)trifluoroacetamide containing 10 mL/L
trimethylchlorosilane (Pierce Europe BV), diluted with chloroform (1:1,
by volume). For the gas chromatography (GC) analysis, 1 µL of this
solution was injected with a split ratio of 1:100 onto a 5890 series II
HP gas chromatograph (Hewlett Packard), equipped with a 25-m Chrompack
CP-SIL-8CB column (Chrompack). The initial oven temperature was
70 °C for 4 min; the temperature was then increased to 230 °C at
7 °C/min. This temperature was held for 1 min and then increased to
280 °C at 10 °C/min, which was held for 5 min. Helium was used as
a carrier gas. The injector temperature was 240 °C. For the mass
spectrometry (MS), a VG Trio-2 (Fisons Instruments) mass spectrometer
was used under positive electron ionization (70 eV). The source
temperature was 220 °C.
case report
The patient was a 38-year-old man of African ancestry. The man was
in good health and has normal intelligence. He had complained about a
fish odor since the age of 5 years, which had led to severe
psychological and professional problems. The fish odor increased under
stress and effort. Furthermore, the patient had complained about
unusual muscular fatigue. No data were available on consanguinity in
the family. The plasma creatine kinase was consistently approximately
fourfold higher than the upper limit of the reference interval (1066
U/L; reference interval, 30–270 U/L). The routine clinical chemical
and hematological determinations, including serum cobalamin (222
pmol/L; reference, 150–400 pmol/L) and urea (5 mmol/L; reference,
2.5–7.5 mmol/L), were unremarkable. In addition, the plasma folate
(9.7 nmol/L) and homocysteine (12 µmol/L) were within the
health-related reference intervals (4–15 nmol/L and 8–18 µmol/L,
respectively). Analysis of the very-long-chain fatty acids and a
carnitine ester profile in serum showed results within the
health-related reference intervals. The results of amino acid and
organic acid analyses of blood and urine from the patient were
unremarkable. The concentration of methionine showed serum and urine
values within the reference intervals. The brothers, sisters, and
the two sons of the patient were without signs or symptoms. The patient
did not use any dietary supplements containing DMG. The patient was
given riboflavin (10 mg/day) for 3 months, but the clinical symptoms
did not change.
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Results |
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A; which shows only the relevant part of the spectrum, a region
containing resonances of many amines. The urine trimethylamine (<2
mmol/mol creatinine) and trimethylamine N-oxide (55 mmol/mol
creatinine) concentrations were within the reference ranges (<2
mmol/mol creatinine and 20–125 mmol/mol creatinine, respectively). In
addition, after the patient ate fresh fish, his urine did not contain
substantial quantities of trimethylamine (<2 mmol/mol creatinine).
This excluded the diagnosis of trimethylaminuria or fish odor syndrome.
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Two unusually high singlets at 2.93 and 3.80 ppm can be seen in
the spectrum of the patient's urine (Fig. 2A
). In nondiseased urines
only very low resonances can be observed at these positions, as shown
in Fig. 2B
. 1H NMR measurements of model compounds
revealed that the resonances were caused by DMG. The characteristic 3:1
ratio of the two singlet resonances of pure DMG was also observed in
the 1H NMR spectrum of the patient's urine. Addition of
pure DMG to the urine sample confirmed that the two singlets were
caused by DMG. A high DMG concentration was found consistently in all
urine samples from the patient. Pure DMG smells like fish.
confirmation of the accumulation of dmg with
13c nmr spectroscopy and gc-ms
The presence of DMG as a storage product in the patient's urine
was confirmed by independent techniques. The mass spectrum of the DMG
peak in the GC-MS chromatogram of his urine is shown in Fig. 3
A. No extractions were performed before the samples were
measured by GC-MS. The high concentration of DMG in the patient's
urine made it possible to detect this metabolite without extraction.
However, the DMG could not be detected when the urine was extracted
with ethyl acetate according to the routine procedure for analysis of
organic acids. The mass spectrum, with characteristic fragments at
m/z 58 and 160 (Fig. 3A
), and the retention time of
the supposed DMG peak in the GC-MS chromatogram corresponded to those
obtained for pure DMG. The total-ion chromatogram of the patient's
urine, without extraction with ethyl acetate, is shown in Fig. 3B
. In
Fig. 3B
, a peak caused by DMG can be observed at a retention time of
3.50 min (peak 29).
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The presence of high concentrations of DMG in the patient's urine was
also confirmed by 13C NMR. The 13C NMR
spectra of the patient's urine and pure DMG are shown in Fig. 4
. The resonances at 172.28, 62.20, and 45.82 ppm are present in
both spectra. These resonances were not be observed in the spectra of
urine samples from healthy individuals.
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concentrations of relevant metabolites in body fluids
The concentration of DMG in urine is age-dependent (Fig. 5
). Especially in the first 2 months of life, values up to 550
mmol DMG/mol creatinine may be found. The DMG concentration was
increased ~20-fold in the patient's urine (Fig. 5
). The DMG
concentrations in urine from the two sons of our patient were within
the reference intervals. The relative concentrations of relevant
metabolites in serum and urine samples from our patient as well as from
control subjects are presented in Table 1
. The DMG concentration was increased in both the serum and the
urine of the patient, whereas the betaine concentrations were slightly
above the upper limit of the reference interval. in the patient's
urine but not in his serum.
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molecular genetic analysis
Analysis of the DMGDH gene of the patient revealed a
homozygous missense mutation. Details about the human gene and about
the mutation in the patient will be described separately.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mitochondrial oxidation of several amino acids and fatty acids involves acyl CoA intermediates, which are oxidized by flavin-containing acyl CoA dehydrogenases. Nine flavoprotein dehydrogenases, including DMGDH, transfer their electrons via ETF and ETF-QO into the respiratory chain. A defect in the biosynthesis or the transport of FAD would bring about many metabolic disturbances, with obvious abnormalities in the organic acid profile in the urine. The same is true for defects in the ETF or ETF-QO proteins, in which various combinations of short-chain volatile acids (e.g., isovaleric, isobutyric, and 2-methylbutyric acid), glutaric, ethylmalonic, 3-hydroxyisovaleric, 2-hydroxyglutaric, 5-hydroxyhexanoic, adipic, suberic, sebacic, and dodecanedioic acid, and isovalerylglycine, isobutyrylglycine, and 2-methylbutyrylglycine are found (6). The organic acid profile in the urine from our patient was normal and, therefore, excludes primary defects in biosynthesis or transport of FAD and ETF or ETF-QO.
All known and theoretical explanations for the accumulation of DMG in our patient have been excluded; therefore, it is likely that he suffers from a deficiency of the enzyme DMGDH. Enzymatic activity of DMGDH cannot be detected in human control blood cells or fibroblasts (R. Brandsch, personal communication). A liver biopsy of our patient was not available. Therefore, the enzyme defect in our patient could not be confirmed enzymatically. A homozygous missense mutation found in the DMGDH gene of the patient was confirmed by molecular genetic analysis. The mutation will be described separately.
Our patient's most remarkable symptom is the fish odor. Another disease with this characteristic has been described (7). This disease, the "fish odor syndrome" or trimethylaminuria, can be diagnosed using 1H NMR spectroscopy in vitro (8). As a diagnostic option for our patient, this disease could be excluded by the absence of trimethylamine under routine dietary conditions and also after the consumption of fresh fish. DMGDH deficiency must be added to the differential diagnosis of patients who smell like fish. The clinical signs and symptoms of the disease seem to be relatively mild. The central nervous system was not clearly affected. The increased creatine kinase in the blood suggests muscle involvement, which may explain our patient's fatigue. Unfortunately, a muscle biopsy from our patient was not available.
If there is some residual DMGDH activity in a patient, one theoretical therapeutic option would be to give the patient high doses of the cofactors of the enzyme. Our patient was given riboflavin; however, this did not change the clinical symptoms. Giving the patient high doses of both riboflavin and folate might also be considered.
An 1H NMR spectrum of a body fluid provides an overall view of almost all proton-containing metabolites in the micro- and millimolar concentration range. 1H NMR has been used previously for diagnosing known inborn errors of metabolism (9)(10)(11)(12). In addition, analysis of betaine and DMG in urine by 1H NMR spectroscopy has been reported (13). In vivo NMR spectroscopy of the central nervous system has already been used successfully to demonstrate guanidinoacetate-methyltransferase deficiency as a new inborn error of creatine biosynthesis (14). To our knowledge, this is the first time that a new inborn error of metabolism was found by use of in vitro 1H NMR. This will encourage additional work on the application of in vitro 1H NMR spectroscopy in diagnosing patients with inborn errors of metabolism.
An unusual body odor in a patient suspected to suffer from an inborn error of metabolism may be one reason to consider additional studies with in vitro 1H NMR spectroscopy on body fluids from the patient. The measurements can be performed on urine, serum, plasma, and cerebrospinal fluid (8)(15). Another clinical indication that we use for performing NMR spectroscopy on body fluids is the presence of two or more children in the same family with similar clinical signs and symptoms if infectious and toxic causes have already been excluded and metabolic disease is suspected.
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Acknowledgments |
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Footnotes |
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1 Nonstandard abbreviations: DMG, N,N-dimethylglycine; DMGDH, dimethylglycine dehydrogenase; ETF, electron transfer flavoprotein; NMR, nuclear magnetic resonance; TSP, trimethylsilyl-2,2,3,3-tetradeuteropropionic acid; GC, gas chromatography; and MS, mass spectrometry. 
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The following articles in journals at HighWire Press have cited this article:
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  N. Shanaiah, M. A. Desilva, G. A. Nagana Gowda, M. A. Raftery, B. E. Hainline, and D. Raftery Class selection of amino acid metabolites in body fluids using chemical derivatization and their enhanced 13C NMR PNAS, July 10, 2007; 104(28): 11540 - 11544. [Abstract] [Full Text] [PDF]
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 N. D. Ferrari III and L. S. Nield Smelling Like Dead Fish: A Case of Trimethylaminuria in an Adolescent Clinical Pediatrics, November 1, 2006; 45(9): 864 - 866. [PDF]
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, control; 
, patient; 
, patient's son.