Sulfite oxidase deficiency in man: demonstration of the enzymatic defect.

An infant who died with neurological abnormalities, mental retardation, and dislocated ocular lenses excreted in his urine abnormally large amounts of S-sulfo-L-cysteine, sulfite, and thiosulfate and virtulally no inorganic sutlfate. The present report establishes the occurrence of an ezymatic defect in this infant. His liver, brain, and kidney specifically lacked sulfite oxidase activity. Deficiency of sulfite oxidase, which has not apparently been described in man, provides a reasonable explanation for the abnormalities in this infant.

Cystathionine synthase in tissue culture derived from human skin: enzyme defect in homocystinuria.

Fibroblasts derived from normal human skin and from cells in amniotic fluid and grown in tissue culture have cystathionine synthase activity. Skin from homocystinuric patients gives rise to fibroblast lines with normal activities of methionine-activating enzyme, but with very low or undetectable cystathionine synthase activity. Thus, the enzyme lesion in homocystinuria is demonstrable in readily available human cells. Neither cystathionine synthase nor methionine-activating enzyme could be detected in intact normal skin.

Hypermethioninaemia due to methionine adenosyltransferase I/III (MAT I/III) deficiency: diagnosis in an expanded neonatal screening programme.

The Expanded Newborn Screening Program (MS/MS) in the region of Galicia (NW Spain) was initiated in 2000 and includes the measurement of methionine levels in dried blood spots. Between June 2000 and June 2007, 140 818 newborns were analysed, and six cases of persistent hypermethioninaemia were detected: one homocystinuria due to cystathionine ß-synthase (CßS) deficiency, and five methionine adenosyltransferase I/III (MAT I/III) deficiencies. The five cases of MAT I/III deficiency represent an incidence of 1/28 163 newborns. In these five patients, methionine levels in dried blood spots ranged from 50 to 147 µmol/L. At confirmation of the persistence of the hypermethioninaemia in a subsequent plasma sample, plasma methionine concentrations were moderately elevated in 4 of the 5 patients (mean 256 µmol/L), while total homocysteine (tHcy) was normal; the remaining patient showed plasma methionine of 573 µmol/L and tHcy of 22.8 µmol/L. All five patients were heterozygous for the same dominant mutation, R264H in the MAT1A gene. With a diet not exceeding recommended protein requirements for their age, all patients maintained methionine levels below 300 µmol/L. Currently, with a mean of 2.5 years since diagnosis, the patients are asymptomatic and show developmental quotients within the normal range. Our results show a rather high frequency of hypermethioninaemia due to MAT I/III deficiency in the Galician neonatal population, indicating a need for further studies to evaluate the impact of persistent isolated hypermethioninaemia in neonatal screening programmes.

Homocystinuria due to cystathionine synthase deficiency. Studies of nitrogen balance and sulfur excretion.

Apparent nitrogen balances and urinary sulfur excretions were determined for normal subjects, seven cystathionine synthase-deficient patients, and a single cystathioninuric patient on semisynthetic diets containing low-adequate amounts of methionine and very low amounts of methionine and very low amounts (12 mg daily, or less) of cystine. The amounts of supplemental cystine required to prevent abnormally high nitrogen or sulfur losses were determined. The five cystathionine synthase-deficient patients who had low residual activities of this enzyme detected in fibroblast and/or liver extracts did not lose more nitrogen or sulfur on diets virtually devoid of cystine than did the normal subjects. These results suggest that the widely expressed opinion that cystine is an essential amino acid for cystathionine syntase-deficient patients requires modification. Residual enzyme activity of only a few percent of normal may obviate such a cystine requirement. These results are compatible with, and lend support to, the working hypothesis which states that the pyridoxine response in cystathionine synthase-deficient patients is mediated by an increase in the residual activity of the affected enzyme.

Demyelination of the brain is associated with methionine adenosyltransferase I/III deficiency.

Individuals deficient in hepatic methionine adenosyltransferase (MAT) activity (MAT I/III deficiency) have been demonstrated to contain mutations in the gene (MATA1) that encodes the major hepatic forms, MAT I and III. MAT I/III deficiency is characterized by isolated persistent hypermethioninemia and, in some cases, unusual breath odor. Most individuals with isolated hypermethioninemia have been free of major clinical difficulties. Therefore a definitive diagnosis of MAT I/III deficiency, which requires hepatic biopsy, is not routinely made. However, two individuals with isolated hypermethioninemia have developed abnormal neurological problems, including brain demyelination, suggesting that MAT I/III deficiency can be deleterious. In the present study we have examined the MATA1 gene of eight hypermethioninemic individuals, including the two with demyelination of the brain. Mutations that abolish or reduce the MAT activity were detected in the MATA1 gene of all eight individuals. Both patients with demyelination are homozygous for mutations that alter the reading frame of the encoded protein such that the predicted MATalpha1 subunits are truncated and enzymatically inactive. The product of MAT, S-adenosylmethionine (AdoMet), is the major methyl donor for a large number of biologically important compounds including the two major myelin phospholipids, phosphatidylcholine and sphingomyelin. Both are synthesized primarily in the liver. Our findings demonstrate that isolated persistent hypermethioninemia is a marker of MAT I/III deficiency, and that complete lack of MAT I/III activity can lead to neurological abnormalities. Therefore, a DNA-based diagnosis should be performed for individuals with isolated hypermethioninemia to assess if therapy aimed at the prevention of neurological manifestations is warranted.

Molecular mechanisms of an inborn error of methionine pathway. Methionine adenosyltransferase deficiency.

Methionine adenosyltransferase (MAT) is a key enzyme in transmethylation, transsulfuration, and the biosynthesis of polyamines. Genetic deficiency of alpha/beta-MAT causes isolated persistent hypermethioninemia and, in some cases, unusual breath odor or neural demyelination. However, the molecular mechanism(s) underlying this deficiency has not been clearly defined. In this study, we characterized the human alpha/beta-MAT transcription unit and identified several mutations in the gene of patients with enzymatically confirmed diagnosis of MAT deficiency. Site-directed mutagenesis and transient expression assays demonstrated that these mutations partially inactivate MAT activity. These results establish the molecular basis of this disorder and allow for the development of DNA-based methodologies to investigate and diagnose hypermethioninemic individuals suspected of having abnormalities at this locus.

Homocystinuria due to cystathionine synthase deficiency: the effect of pyridoxine.

We investigated the effect of pyridoxine administration in three patients with homocystinuria due to cystathionine synthase deficiency. The drug decreased the plasma concentration and urinary excretion of methionine and homocystine and the urinary excretion of homolanthionine and the homocysteine-cysteine mixed disulfide. Urinary cystine rose somewhat. Oral methionine tolerance tests before and during the patients' response to pyridoxine indicated that during response they remained deficient in their capacity to convert the sulfur of methionine to inorganic sulfate, although this capacity increased somewhat. During pyridoxine response only, the methionine loads caused increased homocystinuria. There was no indication that pyridoxine stimulated an alternate pathway of metabolism. The values for specific activity of cystathionine synthase in liver biopsy specimens from two patients in pyridoxine response were 3 and 4% of the mean control value. When these patients were not receiving pyridoxine, comparable values were 2 and 1%, respectively. The hepatic enzyme activity of the mutant patients was similar to normal enzyme activity with respect to trypsin activation, heat inactivation, and stabilization by pyridoxal phosphate. Approximate estimates were made of the relation between total body capacity to metabolize methionine and hepatic cystathionine synthase activity. These estimates suggested that because of the large normal reserve capacity of cystathionine synthase, a few per cent residual activity is sufficient to metabolize the normal dietary load of methionine. Thus, small increases in residual capacity may be of major physiological importance. However, many liver biopsies would be required to establish unequivocally that such changes were due to the administration of a particular therapeutic agent rather than to biological variation. All the data in the present study are consistent with the interpretation that pyridoxine does act by causing an increase in residual cystathionine synthase activity.

Transsulfuration in an adult with hepatic methionine adenosyltransferase deficiency.

We investigated sulfur and methyl group metabolism in a 31-yr-old man with partial hepatic methionine adenosyltransferase (MAT) deficiency. The patient's cultured fibroblasts and erythrocytes had normal MAT activity. Hepatic S-adenosylmethionine (SAM) was slightly decreased. This clinically normal individual lives with a 20-30-fold elevation of plasma methionine (0.72 mM). He excretes in his urine methionine and L-methionine-d-sulfoxide (2.7 mmol/d), a mixed disulfide of methanethiol and a thiol bound to an unidentified group X, which we abbreviate CH3S-SX (2.1 mmol/d), and smaller quantities of 4-methylthio-2-oxobutyrate and 3-methylthiopropionate. His breath contains 17-fold normal concentrations of dimethylsulfide. He converts only 6-7 mmol/d of methionine sulfur to inorganic sulfate. This abnormally low rate is due not to a decreased flux through the primarily defective enzyme, MAT, since SAM is produced at an essentially normal rate of 18 mmol/d, but rather to a rate of homocysteine methylation which is abnormally high in the face of the very elevated methionine concentrations demonstrated in this patient. These findings support the view that SAM (which is marginally low in this patient) is an important regulator that helps to determine the partitioning of homocysteine between degradation via cystathionine and conservation by reformation of methionine. In addition, these studies demonstrate that the methionine transamination pathway operates in the presence of an elevated body load of that amino acid in human beings, but is not sufficient to maintain methionine levels in a normal range.

Affective disorders and S-adenosylmethionine: a new hypothesis.

S-Adenosyl-L-methionine (AdoMet) is a safe and probably effective antidepressant agent in certain forms of clinical depression. This article presents a new hypothesis to account for the mechanism of action of S-adenosylmethionine in such illnesses, based upon the known biochemistry of this compound, and upon current knowledge of clinical and genetic aspects of affective disorders. Giulio Cantoni, S. Harvey Mudd and V. Andreoli postulate that at least some major mood disorders are due to abnormalities affecting the AdoMet-dependent methylation of a substance in the CNS. For convenience and without prejudging the chemical structure of this substance, they call it 'barinine'. The model requires that barinine be subject to AdoMet-dependent methylation and that methylbarinine be subject to metabolic demethylation to regenerate the original barinine. Methylbarinine should be mood elevating, whereas barinine itself should not be. Depression is a result of abnormalities lowering the normal steady-state concentration of methylbarinine, whereas mania results from an abnormal elevation of methylbarinine.

S-adenosylhomocysteine hydrolase deficiency in a 26-year-old man.

This paper reports the third proven human case of deficient S-adenosylhomocysteine (AdoHcy) hydrolase activity. The patient is similar to the only two previously reported cases with this disorder in having severe myopathy, developmental delay, elevated serum creatine kinase (CK) concentrations, and hypermethioninaemia. Although he has been followed from infancy, the basic enzyme deficiency was established only at age 26 years. The diagnosis was based on markedly elevated plasma concentrations of both AdoHcy and S-adenosylmethionine, some 20% of the mean control activity of AdoHcy hydrolase activity in haemolysates of his red-blood cells, and two missense mutations in his gene encoding AdoHcy hydrolase. He had low values of erythrocyte phosphatidylcholine and plasma free choline and marginally elevated excretion of guanidinoacetate, suggesting that the elevated AdoHcy may have been inhibiting methylation of phosphatidylethanolamine and guanidinoacetate. His leukocyte DNA was globally more methylated than the DNA's of his parents or the mean extent of methylation measured in age-matched control subjects.

Labile methyl balances for normal humans on various dietary regimens.

Normal young adult male and female subjects were maintained on fixed dietary regimens which were either essentially normal or were semisynthetic and curtailed in methionine and choline intakes and virtually free of cystine. The subjects maintained stable weights and remained in positive nitrogen balance or within the zone of sulfur equilibrium. Choline intakes were calculated, and urinary excretions of creatinine, creatine, and sacrosine were measured. Creatinine excretions of male subjects on essentially normal diets outweighed the total intakes of labile methyl groups. Taking into account the excretions of additional methylated compounds, as judged from published values, it appears that methyl neogenesis must normally play a role in both males and females. When labile methyl intake is curtailed, de novo formation of methyl groups is quantitatively more significant than ingestion of preformed methyl moieties. On the normal diets used in these experiments, the average homocysteinyl moiety in males cycled between methionine and homocysteine at least 1.9 times before being converted to cystathionine. For females, the average number of cycles was at least 1.5. When labile methyl intake was curtailed, the average number of cycles rose to 3.9 for males and 3.0 for females under the conditions employed.

Isolated hypermethioninemia: measurements of S-adenosylmethionine and choline.

The concentrations of methionine and S-adenosylmethionine (AdoMet) in plasma and free choline and phospholipid-bound choline in both plasma and red blood cells from individuals with isolated hypermethioninemia have been measured. The only genetic abnormalities identified in these individuals have been inactivating mutations in MAT1A, the gene that encodes the subunit of the isozymes of methionine adenosyltransferase (MAT), MAT I, and MAT III, expressed only in adult liver. These measurements were performed to learn more about AdoMet metabolism and to test the working hypotheses that inadequate delivery of AdoMet, or of choline or a choline derivative, from liver to brain might be a cause of the neurologic disease often found in humans with the most severe losses of MAT I/III activity. In striking contrast to the elevations of plasma AdoMet reported in control humans with hypermethioninemia resulting from methionine loading, plasma AdoMet levels were generally below the mean reference value in the MAT I/II-deficient hypermethioninemic patients. This is interpreted as a result of subnormal formation of AdoMet in liver due to the deficient activity of MAT I/III and resultant lower-than-normal delivery of AdoMet from liver to plasma. A low plasma AdoMet concentration in the presence of an elevated methionine provides a useful diagnostic tool that pinpoints the cause of a case of hypermethioninemia as defective MAT I/III activity. Plasma-free choline concentrations were also generally somewhat below normal in the hypermethioninemic patients. However, neither plasma AdoMet nor plasma choline concentrations were strikingly lower in MAT I/III-deficient individuals with neurologic abnormalities than in those without. These results thus fail to provide support for the working hypotheses in question.

Methionine transamination in patients with homocystinuria due to cystathionine beta-synthase deficiency.

To assess the ability of patients with homocystinuria due to cystathionine beta-synthase (CBS) deficiency to perform the reactions of the methionine transamination pathway, the concentrations of the products of this pathway were measured in plasma and urine. The results clearly demonstrate that CBS-deficient patients develop elevations of these metabolites once a threshold near 350 micromol/L for the concurrent plasma methionine concentration is exceeded. The absence of elevated methionine transamination products previously reported among 16 CBS-deficient B6-responsive patients may now be attributed to the fact that in those patients the plasma methionine concentrations were below this threshold. The observed elevations of transamination products were similar to those observed among patients with isolated hypermethioninemia. Plasma homocyst(e)ine did not exert a consistent effect on transamination metabolites, and betaine appeared to effect transamination chiefly by its tendency to elevate methionine. Even during betaine administration, the transamination pathway does not appear to be a quantitatively major route for the disposal of methionine.

Epileptiform ocular movements with methylmalonic aciduria and homocystinuria.

A 7 1/2-year-old girl with a rare defect in cobalamin (vitamin B12) metabolism ("cobalamin C" type) developed epileptiform ocular and eyelid movements as the major clinical manifestation of the disease. One of three other patients who have been described with congenital syndrome was similarly noted to have "fluttering" of the eyelids interpreted as epileptic discharges. The metabolic abnormality produced a defect in synthesis of cobalamin coenzymes. It is characterized biochemically by the excreation of methylmalonic acid and homocystine in the urine.

Cystathioninuria and homocystinuria.

Three circumstances prompted us to reexamine the relationship between abnormal cystathionine accumulation and possible homocystinuria resulting from this condition: (a) discovery of an infant girl with apparently alternating massive cystathioninuria and homocystinuria; (b) the presence of homocystinuria in some, but not all, previously reported cases of cystathioninuria probably due to gamma-cystathionine deficiency; and (c) the recent demonstration that mammalian cystathionine beta-synthase can cleave cystathionine to homocysteine. The following conclusions were reached: (a) Homocystine may arise as a result of bacterial contamination of a urine sample initially containing cystathionine, but not homocystine. (b) After a methionine load, a cystathioninuric patient may excrete readily detected amounts of homocystine. (c) However, homocystinuria is not a necessary concomitant of even massive cystathioninuria. These findings and some of their implications are briefly discussed.

Radioactive methionine: determination, and distribution of radioactivity in the sulfur, methyl and 4-carbon moieties.

A simple and inexpensive method is described for isolation and determination of [14C]methionine in the non-protein fraction of tissues extensively labeled with 14C. The effectiveness of the method was demonstrated by isolation of non-protein [14C]methionine (as the carboxymethylsulfonium salt) of proven radiopurity from the plant Lemna which had been grown for a number of generations on [U-14C]sucrose and contained a 2000-fold excess of 14C in undefined non-protein compounds. To our knowledge, this is the first reported assay for radioactive methionine under these demanding conditions. This method also offers an attractive alternative to the use of more expensive and sophisticated equipment for assay of radioactive methionine under less demanding conditions. An advantage is that the isolated methioninecarboxymethylsulfonium salt is readily degraded to permit separate determination of radioactivity in the 4-carbon, methyl and sulfur moieties of methionine. During this work, a facile labilization of 3H attached to the (carboxy)methylene carbon of methioninecarboxymethylsulfonium salt was observed. This labilization is ascribed to formation of a sulfur ylid.

Phosphatidylcholine synthesis in the rat: the substrate for methylation and regulation by choline.

Two lines of evidence led us to reexamine the possibility that methylation of phosphoethanolamine and its partially methylated derivatives, in addition to methylation of the corresponding phosphatidyl derivatives, plays a role in mammalian phosphatidylcholine biosynthesis: (a) Results obtained by Salerno and Beeler with rat [Salerno, D.M. and Beeler, D.A. (1973) Biochim. Biophys. Acta 326, 325-338] appear to strongly support such a role for methylation of phosphobases; (b) Such reactions have recently been shown to play major roles in phosphatidylcholine synthesis by higher plants [see Datko, A.H. and Mudd, S.H. (1988) Plant Physiol. 88, 854-861 and references therein]. We found that, following continuous labeling of rat liver with L-[methyl-3H]methionine for 10.4 min (intraperitoneal administration) or for 0.75 min (intraportal administration), virtually no 3H was detected in methylated derivatives of phosphoethanolamine, but readily detectable amounts of 3H were present in the base moiety of each methylated derivative of phosphatidylethanolamine. Thus, there was no indication that phospho-base methylation makes a significant contribution. Studies of cultured rat hepatoma cells showed definitively for the first time in a mammalian system that choline deprivation up-regulates the rate of flow of methyl groups originating in methionine into phosphatidylethanolamine and derivatives. Even under these conditions, methylation of phosphoethanolamine bases appeared to play a negligible role.