Trimethylaminuria Research Articles

A Mutation in the Flavin-containing Monooxygenase 3 Gene and its Effects on Catalytic Activity for N-oxidation of Trimethylamine In Vitro

To clarify the mutation of the flavin-containing monooxygenase (FMO) 3 gene causing fish-odor syndrome, we analyzed the FMO3 gene of a Thai subject who possibly suffered from fish-odor syndrome. A novel mutation, a single-base substitution from G to A at the position of 265 (G265A), was identified in exon 3. The mutation caused an amino acid substitution from valine to isoleucine at residue 58 (V58I). The mutated FMO3 protein with V58I exhibited the reduced trimethylamine N-oxidase activity when it was expressed in E. coli. The V(max)/K(m) value for the activity of the mutant-type FMO3 was about 5 times lower than that for the wild-type FMO3.

In Vivo Variability of TMA Oxidation Is Partially Mediated by Polymorphisms of the FMO3 Gene

Trimethylaminuria (TMAU) results from an accumulation of an excessive amount of unoxidized trimethylamine that is excreted in urine and body secretions. Mutations of the flavin-containing monooxygenase 3 (FMO3) gene (a hepatic phase I drug-metabolizing enzyme) account for the severe recessively encoded form of this condition. We have previously described a number of FMO3 polymorphisms which in vitro exhibit reduced substrate affinity for several FMO substrates. Here we show that three prevalent polymorphisms (E158K, V257M, and E308G) inherited in particular combinations confer a slight decrease in TMA oxidation under normal physiological conditions, which may be clinically “silent.” With the use of substrate loading or with the interaction of other known modulators of FMO3 activity such as hormonal influences, these genotypes may predispose to mild TMAU.

Compound heterozygosity for missense mutations in the flavin-containing monooxygenase 3 (FM03) gene in patients with fish-odour syndrome.

Fish-odour syndrome is a highly unpleasant disorder of hepatic trimethylamine (TMA) metabolism characterized by a body odour reminiscent of rotting fish, due to excessive excretion of the malodorous free amine. Although fish-odour syndrome may exhibit as sequelae with other conditions (e.g. liver dysfunction), many patients exhibit an inherited, more persistent form of the disease. Ordinarily, dietary-derived TMA is oxidized to the nonodorous N-oxide by hepatic flavin-containing monooxygenase 3 (FMO3). Our previous demonstration that a mutation, P153L (C to T), in the FMO3 gene segregated with the disorder and inactivated the enzyme confirmed that defects in FMO3 underlie the inherited form of fish-odour syndrome. We have investigated the genetic basis of the disorder in two further affected pedigrees and report that the three propositi are all compound heterozygotes for causative mutations of FMO3. Two of these individuals possess the P153L (C to T) mutation and a novel mutation, N61S (A to G). The third is heterozygous for novel, M4341 (G to A), and previously reported, R492W (C to T), mutations. Functional characterization of the S61, 1434 and W492 variants, via baculovirus-mediated expression in insect cells, confirmed that all three mutations either abolished, or severely attenuated, the capacity of the enzyme to catalyse TMA N-oxidation. Although 1434 and W492 were also incapable of catalysing the S-oxidation of methimazole, S61 was fully active with this sulphur-containing substrate. Since an asparagine is conserved at the equivalent position to N61 of FMO3 in mammalian, yeast and Caenorhabditis elegans FMOs, the characterization of the naturally occurring N61S (A to G) mutation may have identified this asparagine as playing a critical role specifically in FMO-catalysed N-oxidation.

Biochemical and molecular studies in mild flavin monooxygenase 3 deficiency.

Flavin-containing monooxygenases (FMOs) are membrane-associated microsomal mixed-function oxidases that are required for the oxidation and detoxification of many nitrogen-, sulphur- and phosphorus-containing substances and are present in nearly all secretory cell types (Dolphin et al 1997; Treacy et al 1998). At least five different isoforms (1 to 5) have been identified in mammalian tissues (Lawton et al 1994). In humans, FMO3 is the major catalytic isoform expressed in the liver (Lomri et al 1993), although it is also present in the kidney and probably in other tissues. Substrates include endogenous amines (e.g. adrenaline), tyramine, nicotine, drugs (e.g. tricyclic antidepressants, ranitidine), as well as trimethylamine (TMA), a volatile malodourous substance that is present in fish or may be produced by the action of gut bacteria on choline, lecithin or carnitine (Lin et al 1996; Ziegler 1993). It has been shown that a deficiency of FMO3 is the cause of the recessive disorder trimethylaminuria (TMA-uria) or fish-odour syndrome' (Dolphin et al 1997; Treacy et al 1998). Individuals affected by this disease have a reduced capacity to oxidize free TMA to its odourless, nonvolatile metabolite TMA N-oxide (TMAO) and display a strong, fish-like odour due to the excretion of large amounts of TMA in urine, sweat and breath (Ayesh et al 1993). The diagnosis is made biochemically on the basis of elevated urinary free TMA; under physiological conditions, >95% of the total TMA (= free TMA + TMAO) is excreted as TMAO. In classical fish-odour syndrome, the TMAO/total TMA ratio is strongly decreased. The FMO3 gene is located on chromosome 1q23-25 and contains one noncoding and eight coding exons. A small number of severe, disease-causing mutations and several polymorphic variants in the gene have been reported (Akerman et al 1999; Dolphin et al 1997; Treacy et al 1998). We have recently found that a frequent variant allele carrying two amino acid polymorphisms, c.472G > A and c.923A > G [E308G; E158K], is associated with reduced N-oxidation capacity after oral trimethylamine challenge (Zschocke et al 1999). Patients previously reported to suffer from 'transient' trimethylaminuria (Mayatepek and Kohlmuller 1998) were compound heterozygous for the variant allele and a severe FMO3 mutation, corresponding to mild enzyme deficiency. Here we present the results of molecular analyses and biochemical studies under physiological conditions in patients with mild FMO3 deficiency and report the first symptomatic patient who is homozygous for the variant allele.

What's that smell?: Modern science puts its mark on a rare but ancient body‐odor disease

S hakespeare's description of the socially rejected island dweller, Caliban, aptly demonstrates that people have long been aware of a particularly offensive smell. Today, body odors are among the most sensitive of topics and figure into some of the deadliest insults. Inspired by advertising, most people avoid having a socially unacceptable smell thanks to frequent showers and deodorants, perfumes, or colognes. However, for people with a rare genetic disorder known as fish odor syndrome, none of these is enough. Since few health professionals are aware of this disease, also called trimethylaminuria, people experiencing its characteristic body odor may spend years seeking relief. They often acquire a long list of inaccurate diagnoses, ranging from poor hygiene to psychiatric problems. These people may withdraw from the outside world to avoid ridicule or try to mask their odor with strong perfumes and cigarettes. Many of those with the disorder also report symptoms of depression. People think this disease is a laughing matter, but for the people who have it, it is not. This syndrome can be highly destructive to a person's personal and social life and to their work and career, says Robert L. Smith, a molecular toxicologist at the Imperial College School of Medicine in London who recently cochaired the first-ever scientific meeting on trimethylaminuria. Held in late March at the National Institutes of Health in Bethesda, Md., the conference gathered from around the globe about 30 researchers studying trimethylaminuriamany of them without funding-and a few of the people who have been diagnosed with the disorder.

Quantitative determination of trimethylamine in urine by solid-phase microextraction and gas chromatography–mass spectrometry

Trimethylaminuria (fish odour syndrome) is diagnosed from an increase in urinary excretion of trimethylamine with decreased trimethylamine oxide. We report a new quantitative stable isotope dilution gas chromatography–mass spectrometry procedure for the analysis of these metabolites using solid-phase microextraction (SPME). Both polydimethylsiloxane and mixed Carboxen–polydimethylsiloxane SPME fibres were found to be suitable for the headspace extraction of TMA. This new sampling technique could have wide application for the analysis of volatile and semi-volatile compounds by metabolic screening laboratories.

Trimethylamine and Foetor Hepaticus

In patients with various degrees of hepatocellular failure and portosystemic shunting of blood, the breath may acquire a sweet, musty, or even slightly faecal aroma, termed foetor hepaticus, which has been attributed mainly to volatile sulphur compounds with contributions from various nitrogenous substances. Not infrequently in such patients, unusual body odours are also encountered and, by analogy with the 'fish-odour syndrome' known to be caused by excessive trimethylamine, the availability of this latter graveolent amine to potentially contribute to odours associated with hepatic disease was investigated. Urine (0-24 h) was collected from 63 patients with various liver diseases previously confirmed in hospital by means of various biochemical, immunologic, pathologic, and radiologic investigations. Total trimethylamine and trimethylamine N-oxide levels in urine were measured with head-space gas chromatography. In total, 50% (32 of 63) of the patients (primary liver disease, 25 of 47, or 53.2%; secondary liver disease, 7 of 16, or 43.8%) had urinary trimethylamine levels greater than the upper end of the range considered normal (0.08-1.84 microg/ml). Seventeen patients excreted large amounts of free trimethylamine (more than 10 microg/ml), above the threshold usually associated with the appearance of a 'fish-like' body odour and tainted breath. Excessive amounts of trimethylamine may well contribute to the overall body odour problems encountered among patients with severe hepatic disease, precipitating in these individuals a secondary form of the 'fish-odour syndrome'.

Missense mutation in flavin-containing mono-oxygenase 3 gene, FMO3, underlies fish-odour syndrome.

Individuals with primary trimethylaminuria exhibit a body odour reminiscent of rotting fish, due to excessive excretion of trimethylamine (TMA; refs 1-3). The disorder, colloquially known as fish-odour syndrome, is inherited recessively as a defect in hepatic N-oxidation of dietary-derived TMA and cannot be considered benign, as sufferers may display a variety of psychosocial reactions, ranging from social isolation of clinical depression and attempted suicide. TMA oxidation is catalyzed by flavin-containing mono-oxygenase (FMO; refs 7,8), and tissue localization and functional studies have established FMO3 as the form most likely to be defective in fish-odour syndrome. Direct sequencing of the coding exons of FMO3 amplified from a patient with fish-odour syndrome identified two missense mutations. Although one of these represented a common polymorphism, the other, a C-->T transition in exon 4, was found only in an affected pedigree, in which it segregated with the disorder. The latter mutation predicts a proline-->leucine substitution at residue 153 and abolishes FMO3 catalytic activity. Our results indicate that defects in FMO3 underlie fish-odour syndrome and that the Pro 153-->Leu 153 mutation described here is a cause of this distressing condition.

Structural Organization of the Human Flavin-Containing Monooxygenase 3 Gene (FMO3), the Favored Candidate for Fish-Odor Syndrome, Determined Directly from Genomic DNA

The inherited metabolic disorder trimethylaminuria (fish-odor syndrome) is associated with defective hepaticN-oxidation of dietary-derived trimethylamine catalyzed by flavin-containing monooxygenase (FMO). AsFMO3encodes the major form of FMO expressed in adult human liver, it represents the best candidate gene for the disorder. The structural organization ofFMO3was determined by sequencing the products of exon-to-exon and vectorette PCR, the latter through the use of vectorette libraries constructed directly from genomic DNA. The gene contains one noncoding and eight coding exons. Knowledge of the exon / intron organization of the humanFMO3gene enabled each of the coding exons of the gene, together with their associated flanking intron sequences, to be amplified from genomic DNA and will thus facilitate the identification of mutations inFMO3in families affected with fish-odor syndrome.

1H-NMR analysis of trimethylamine in urine for the diagnosis of fish-odour syndrome

This paper reports the use of proton NMR spectroscopy for the analysis of trimethylamine in the urine of a patient with trimethylaminuria. Analysis of this compound was also performed for other members of his family. Qualitative and quantitative determination of trimethylamine and trimethylamine- N-oxide was simultaneously performed on untreated urine within a few minutes. The application of the method is discussed.

Fish-odour syndrome and impaired N-oxidation.

Fish-odour syndrome and impaired N-oxidation.

Discontinuous distribution of N-oxidation of dietary-derived trimethylamine in a British population.

1. Whilst the majority of individuals within a British white population are able to convert > 90% of their dietary-derived trimethylamine to its N-oxide, outliers exist who show varying degrees of decreased metabolism. Such individuals, excrete unoxidized trimethylamine in their urine and, if N-oxidation is sufficiently low, may experience malodour problems (Fish-Odour syndrome). 2. Such observations have now been extended to a much larger group (n = 421; 221 males) of British white volunteers recruited from staff and students of Imperial College Medical School at St. Mary's, London. Each subject collected a 0-24-h urine sample, which was subsequently analysed for total trimethylamine and trimethylamine N-oxide content. 3. Sixteen subjects (3.8% population; seven male, nine female) excreted < 90% of their total trimethylamine output as N-oxide. All six subjects who excreted < 80% as N-oxide (indicative of potential heterozygous status for deficient N-oxidation-fish odour syndrome) were female.

Fish odour syndrome: verification of carrier detection test.

An oral trimethylamine challenge test has been used to confirm the heterozygous status of patients with 'fish-odour syndrome'. By measuring the percentage of total urinary trimethylamine-related material excreted as the N-oxide, no discrimination could be made between obligate heterozygotes (parents of 'fish-odour syndrome' patients) (n = 15; 96 +/- 2%, range 92-98%) and control individuals (parents of unaffected children) (n = 16; 96 +/- 2%, range 93-99%) on a normal diet. However, after ingesting a trimethylamine load (600 mg base) the obligate heterozygotes were clearly distinguishable (76 +/- 3%, range 71-79%) from controls (95 +/- 2%, range 91-99%) (t-test; p <0.001). One of a hundred apparently normal volunteers who were subsequently challenged with trimethylamine had a N-oxidation capacity which fell within the range found among the obligate heterozygotes.

The N-oxidation of trimethylamine in a Jordanian population.

The ability to oxidise trimethylamine (TMA) to trimethylamine N-oxide (TMAO) is distributed polymorphically within a British white population with the majority of individuals excreting greater than 90% of total urinary TMA as TMAO. The opposite extreme is characterised by a rare inborn error of TMA N-oxidation known as the fish-odour syndrome. However there is a lack of information regarding inter-individual variability in the N-oxidation of TMA in other ethnic groups. In this study the urinary excretion of TMA and TMAO was determined over a period of 24 h in 82 Jordanian subjects. A frequency distribution histogram of % of total urinary TMA excreted as TMAO revealed that the majority of subjects excreted greater than 80% of the total urinary TMA as TMAO, however eight subjects (9.7%) excreted less than 80% of the total TMA as TMAO. In a previous study of 169 white British subjects only one (0.6%) excreted less than 80% of the total TMA as TMAO. The results suggest that the prevalence of compromised ability to N-oxidise TMA may be higher in a Jordanian population than in a British population.

4f-conduction electron hybridization in ternary CeTMAl compounds

Abstract We present an investigation of CeTMAl phase diagrams with TMRu, Pd, Pt and Au. Several new compounds have been characterized by measurements of the electrical resistivity ϱ ( T ), the do susceptibility χ ( T ) and, in some cases, the specific heat C ( T ). We find that in the compounds with TMRu, the hybridization strength between 4f and conduction electrons is strong, leading in most compounds to an intermediate valent (IV) state. By contrast, most of the investigated compounds with TMPd, Pt and Au order magnetically indicating a much weaker hybridization strength. However, some of the Pt and especially Pd compounds are very near to the crossover from the magnetic to the non-magnetic regime as deduced from a Kondo-type maximum in the resistivity and a large electronic specific heat at low temperatures.

Change of the Yb valence in YbTMAl compounds

We present an investigation of YbTMAl phase diagrams with TMPd, Ag and Au. Several new compounds have been characterized by measurements of the magnetic susceptibility, the electrical resistivity and, in some cases, the specific heat. It seems that the crossover from the stable-trivalent to the intermediate-valent behavior occurs much more abruptly in Yb- than in Ce-based compounds on changing the composition. A comparison of the CeTMAl compounds with their Yb homologs suggests that replacement of Ce by Yb in an intermediate-valent Ce compound and in a trivalent Ce compound results in an Yb3+ and in an Yb2+ state, respectively.

Medical genetics: advances in brief: The fish odour syndrome: biochemical, familial, and clinical aspects

Medical genetics: advances in brief: The fish odour syndrome: biochemical, familial, and clinical aspects

Acid soaps in the fish odour syndrome

Acid soaps in the fish odour syndrome

The fish odour syndrome: biochemical, familial, and clinical aspects.

To study the biochemical, familial, and clinical features of the fish odour syndrome among subjects with suspected body malodour. Subjects who responded to a newspaper article were screened for the fish odour syndrome by interview and biochemical tests. Families of subjects with the syndrome were tested if possible. St Mary's Hospital, London, and some interviews at subjects' homes. 187 subjects (28 males) with suspected body malodour, of whom 156 (19 males) underwent biochemical tests. Five families of six of the subjects with the fish odour syndrome agreed to further tests. Amounts of trimethylamine and trimethylamine N-oxide in urine collected over 24 hours under normal dietary conditions and for eight hours after oral challenge with 600 mg trimethylamine. The fish odour syndrome was diagnosed in 11 subjects: the percentage of total trimethylamine excreted in their urine samples that was oxidised to trimethylamine N-oxide was < 55% under normal dietary conditions and < 25% after oral challenge with trimethylamine (in normal subjects > 80% of trimethylamine was N-oxidised). Parents of six of the subjects with the syndrome were tested: all showed impaired N-oxidation of excreted trimethylamine (< 80%) after oral challenge, indicating that they were heterozygous carriers of the allele for the syndrome. The syndrome was associated with various psychosocial reactions including clinical depression. The fish odour syndrome can be inherited in an autosomal recessive fashion. It should be considered as a possible causative factor in patients complaining of body malodour.

Metabolic polymorphisms

Polymorphisms have been detected in a variety of xenobiotic-metabolizing enzymes at both the phenotypic and genotypic level. In the case of four enzymes, the cytochrome P450 CYP2D6, glutathione S-transferase μ, N-acetyltransferase 2 and serum cholinesterase, the majority of mutations which give rise to a defective phenotype have now been identified. Another group of enzymes show definite polymorphism at the phenotypic level but the exact genetic mechanisms responsible are not yet clear. These enzymes include the cytochromes P450 CYP1A1, CYP1A2 and a CYP2C form which metabolizes mephenytoin, a flavin-linked monooxygenase (fish-odour syndrome), paraoxonase, UDP-glucuronosyltransferase (Gilbert's syndrome) and thiopurine S-methyltransferase. In the case of a further group of enzymes, there is some evidence for polymorphism at either the phenotypic or genotypic level but this has not been unambiguously demonstrated. Examples of this class include the cytochrome P450 enzymes CYP2A6, CYP2E1, CYP2C9 and CYP3A4, xanthine oxidase, an S-oxidase which metabolizes carbocysteine, epoxide hydrolase, two forms of sulphotransferase and several methyltransferases. The nature of all these polymorphisms and possible polymorphisms is discussed in detail, with particular reference to the effects of this variation on drug metabolism and susceptibility to chemically-induced diseases.