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A discrepancy has been identified between numbers of expected and identified patients with homocystinuria due to cystathionine beta-synthase (CBS) deficiency. Patients homozygous for the frequent c.833T>C (p.I278T) are most often responsive to vitamin B6, and can present with a total-homocysteine (tHcy) T polymorphism in patients with tHcy ≥50μM and the association with clinical manifestations. We studied a cohort of patients with intermediate and severe hyperhomocysteinemia (tHcy ≥50μM) determined between 1996 and 2011. Among the 413 eligible patients, 184 (45%) patients agreed to participate in the present follow-up study. A MTHFR(3)c.677TT genotype was found in 49% of the patients. Eight patients were found to have mutations in CBS(2). Of those, two were homozygous for c.833T>C (p.I278T), and four were compound heterozygous for c.833T>C. One c.833T>C (p.I278T) compound heterozygote was identified by lowering the threshold for sequencing from tHcy at 100μM to 50μM. The most prominent clinical presentation among patients with a CBS(2) mutation was thrombosis presenting at a median age of 25years. In case of arterial or venous thrombosis without any explanation in individuals below 40years, tHcy should be part of the thrombophilia screening. When tHcy is between 50 and 100μM genotyping for the MTHFR(3) c.677TT is relevant, and when tHcy >100μM CBS should be genotyped.

This paper reports studies of two patients proven by a variety of studies to have mitochondrial depletion syndromes due to mutations in either their MPV17 or DGUOK genes. Each was initially investigated metabolically because of plasma methionine concentrations as high as 15-21-fold above the upper limit of the reference range, then found also to have plasma levels of S-adenosylmethionine (AdoMet) 4.4-8.6-fold above the upper limit of the reference range. Assays of S-adenosylhomocysteine, total homocysteine, cystathionine, sarcosine, and other relevant metabolites and studies of their gene encoding glycine N-methyltransferase produced evidence suggesting they had none of the known causes of elevated methionine with or without elevated AdoMet. Patient 1 grew slowly and intermittently, but was cognitively normal. At age 7 years he was found to have hepatocellular carcinoma, underwent a liver transplant and died of progressive liver and renal failure at age almost 9 years. Patient 2 had a clinical course typical of DGUOK deficiency and died at age 8 ½ months. Although each patient had liver abnormalities, evidence is presented that such abnormalities are very unlikely to explain their elevations of AdoMet or the extent of their hypermethioninemias. A working hypothesis is presented suggesting that with mitochondrial depletion the normal usage of AdoMet by mitochondria is impaired, AdoMet accumulates in the cytoplasm of affected cells poor in glycine N-methyltransferase activity, the accumulated AdoMet causes methionine to accumulate by inhibiting activity of methionine adenosyltransferase II, and that both AdoMet and methionine consequently leak abnormally into the plasma.

This review covers briefly the major conditions, genetic and non-genetic, sometimes leading to abnormally elevated methionine, with emphasis on recent developments. A major aim is to assist in the differential diagnosis of hypermethioninemia. The genetic conditions are: (1) Homocystinuria due to cystathionine β-synthase (CBS) deficiency. At least 150 different mutations in the CBS gene have been identified since this deficiency was established in 1964. Hypermethioninemia is due chiefly to remethylation of the accumulated homocysteine. (2) Deficient activity of methionine adenosyltransferases I and III (MAT I/III), the isoenzymes the catalytic subunit of which are encoded by MAT1A. Methionine accumulates because its conversion to S-adenosylmethionine (AdoMet) is impaired. (3) Glycine N-methyltrasferase (GNMT) deficiency. Disruption of a quantitatively major pathway for AdoMet disposal leads to AdoMet accumulation with secondary down-regulation of methionine flux into AdoMet. (4) S-adenosylhomocysteine (AdoHcy) hydrolase (AHCY) deficiency. Not being catabolized normally, AdoHcy accumulates and inhibits many AdoMet-dependent methyltransferases, producing accumulation of AdoMet and, thereby, hypermethioninemia. (5) Citrin deficiency, found chiefly in Asian countries. Lack of this mitochondrial aspartate-glutamate transporter may produce (usually transient) hypermethioninemia, the immediate cause of which remains uncertain. (6) Fumarylacetoacetate hydrolase (FAH) deficiency (tyrosinemia type I) may lead to hypermethioninemia secondary either to liver damage and/or to accumulation of fumarylacetoacetate, an inhibitor of the high K(m) MAT. Additional possible genetic causes of hypermethioninemia accompanied by elevations of plasma AdoMet include mitochondrial disorders (the specificity and frequency of which remain to be elucidated). Non-genetic conditions include: (a) Liver disease, which may cause hypermethioninemia, mild, or severe. (b) Low-birth-weight and/or prematurity which may cause transient hypermethioninemia. (c) Ingestion of relatively large amounts of methionine which, even in full-term, normal-birth-weight babies may cause hypermethioninemia.

A child with severe S-adenosylhomocysteine hydrolase (AHCY) deficiency (AHCY c.428A > G, p.Tyr143Cys ; c.982 T > G, p.Tyr328Asp) presented at 8 months of age with growth failure, microcephaly, global developmental delay, myopathy, hepatopathy, and factor VII deficiency. Plasma methionine, S-adenosylmethionine (AdoMet), and S-adenosylhomocysteine (AdoHcy) were markedly elevated and the molar concentration ratio of AdoMet:AdoHcy, believed to regulate a myriad of methyltransferase reactions, was 15% of the control mean. Dietary therapy failed to normalize biochemical markers or alter the AdoMet to AdoHcy molar concentration ratio. At 40 months of age, the proband received a liver segment from a healthy, unrelated living donor. Mean AdoHcy decreased 96% and the AdoMet:AdoHcy concentration ratio improved from 0.52 ± 0.19 to 1.48 ± 0.79 mol:mol (control 4.10 ± 2.11 mol:mol). Blood methionine and AdoMet were normal and stable during 6 months of follow-up on an unrestricted diet. Average calculated tissue methyltransferase activity increased from 43 ± 26% to 60 ± 22%, accompanied by signs of increased transmethylation in vivo. Factor VII activity increased from 12% to 100%. During 6 postoperative months, head growth accelerated 4-fold and the patient made promising gains in gross motor, language, and social skills.

Studies were carried out to identify the cause of combined severe hypermethioninemia and moderate hyperhomocysteinemia in a cluster of 10 infants ascertained between 1999 and early 2001. Although several were thought initially to have cystathionine beta-synthase (CBS) deficiency and treated accordingly, CBS deficiency and other known genetic causes of hypermethioninemia were ruled out by assay of CBS activity in fibroblasts of four patients and by assays of plasma cystathionine and S-adenosylmethionine. Retrospective data on dietary methionine intakes and plasma concentrations of methionine and related metabolites established that the hypermethioninemia in nine of the 10 babies was related to ingestion of an infant protein hydrolysate formula, the methionine content of which had been increased from May 1998 to February 2001. The formula in question has now been reformulated and is no longer available. The 10th infant manifested similar metabolic abnormalities while receiving TPN containing excessive methionine. Brain MRI abnormalities indicative of cerebral edema, most marked in the cerebral cortex and posterior brainstem, occurred in two patients near times of extreme hypermethioninemia. Metabolic and MRI abnormalities resolved when the methionine intake decreased. A third infant had a normal MRI 1 day after the formula was changed. The possible relationship between extreme hypermethioninemia and cerebral edema is discussed and a working hypothesis offered to explain the relative sensitivity of the inferior colliculi, based upon the facts that this is the region most active in glucose utilization and that Na(+),K(+)-ATPase is inhibited by methionine and related metabolites.

Cystathionine beta-synthase (CBS) deficiency, the most common form of homocystinuria, is an autosomal recessive inborn error of homocysteine metabolism. Treatment of B6-nonresponsive patients centers on lowering homocysteine and its disulfide derivatives (tHcy) by adherence to a methionine-restricted diet. However, lifelong dietary control is difficult. Betaine supplementation is used extensively in CBS-deficient patients to lower plasma tHcy. With betaine therapy, methionine levels increase over baseline, but usually remain below 1,500 micromol/L, and these levels have not been associated with adverse affects. We report a child with B6-nonresponsive CBS deficiency and dietary noncompliance whose methionine levels reached 3,000 micromol/L on betaine, and who subsequently developed massive cerebral edema without evidence of thrombosis. We investigated the etiology by determining methionine and betaine metabolites in our patient, and several possible mechanisms for her unusual response to betaine are discussed. We conclude that the cerebral edema was most likely precipitated by the betaine therapy, although the exact mechanism is uncertain. This case cautions physicians to monitor methionine levels in CBS-deficient patients on betaine and to consider betaine as an adjunct, not an alternative, to dietary control.

Methionine adenosyltransferase (MAT) catalyzes the synthesis of S-adenosylmethionine (AdoMet), the main alkylating agent in living cells. Additionally, in the liver, MAT is also responsible for up to 50% of methionine catabolism. Humans with mutations in the gene MAT1A, the gene that encodes the catalytic subunit of MAT I and III, have decreased MAT activity in liver, which results in a persistent hypermethioninemia without homocystinuria. The hypermethioninemic phenotype associated with these mutations is inherited as an autosomal recessive trait. The only exception is the dominant mild hypermethioninemia associated with a G-A transition at nucleotide 791 of exon VII. This change yields aMAT1A-encoded subunit in which arginine 264 is replaced by histidine. Our results indicate that in the homologous rat enzyme, replacement of the equivalent arginine 265 by histidine (R265H) results in a monomeric MAT with only 0.37% of the AdoMet synthetic activity. However the tripolyphosphatase activity is similar to that found in the wild type (WT) MAT and is inhibited by PPi. Our in vivo studies demonstrate that the R265H MAT I/III mutant associates with the WT subunit resulting in a dimeric R265H-WT MAT unable to synthesize AdoMet. Tripolyphosphatase activity is maintained in the hybrid MAT, but is not stimulated by methionine and ATP, indicating a deficient binding of the substrates. Our data indicate that the active site for tripolyphosphatase activity is functionally active in the monomeric R265H MAT I/III mutant. Moreover, our results provide a molecular mechanism that might explain the dominant inheritance of the hypermethioninemia associated with the R264H mutation of human MAT I/III.

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/III-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.

Background There is not much information on established standard therapy for patients with severe methionine adenosyltransferase (MAT) I/III deficiency. Case presentation We report a boy with MAT I/III deficiency, in whom plasma methionine and total homocysteine, and urinary homocystine were elevated. Molecular genetic studies showed him to have novel compound heterozygous mutations of the MAT1A gene: c.191T>A (p.M64K) and c.589delC (p.P197LfsX26). A low methionine milk diet was started at 31 days of age, and during continuing dietary methionine restriction plasma methionine levels have been maintained at less than 750 μmol/L. He is now 5 years old, and has had entirely normal physical growth and psychomotor development. Conclusions Although some severely MAT I/III deficient patients have developed neurologic abnormalities, we report here the case of a boy who has remained neurologically and otherwise normal for 5 years during methionine restriction, suggesting that perhaps such management, started in early infancy, may help prevent neurological complications.

We describe a family illustrating the diagnostic difficulties occurring when pyridoxine-responsive cystathionine beta-synthase (CBS) deficiency presents with thrombotic disease without associated ocular, skeletal, or CNS abnormalities, a situation increasingly recognized. This family had several thromboembolic episodes in two generations with apparently inconstant elevations of plasma total homocysteine (tHcy). When taking (sometimes even low amounts) of pyridoxine, the affected family members had low-normal tHcy and normal values for cystathionine, methionine, and cysteine. Withdrawal of vitamin therapy was necessary before lower cystathionine, elevated methionine, and decreased cysteine became apparent, a pattern suggestive of CBS deficiency, leading to the finding that the affected members were each compound heterozygotes for CBS p.G307S and p.P49L. To assist more accurate diagnosis of adults presenting with thrombophilia found to have elevated tHcy, the patterns of methionine-related metabolites in CBS-deficient patients are compared in this article to those in patients with homocysteine remethylation defects, including inborn errors of folate or cobalamin metabolism, and untreated severe cobalamin or folate deficiency. Usually serum cystathionine is low in subjects with CBS deficiency and elevated in those with remethylation defects. S-Adenosylmethionine and S-adenosylhomocysteine are often markedly elevated in CBS deficiency when tHcy is above 100 umol/L. We conclude that there are likely other undiagnosed, highly B6-responsive adult patients with CBS deficiency, and that additional testing of cystathionine, total cysteine, methionine, and S-adenosylmethionine will be helpful in diagnosing them correctly and distinguishing CBS deficiency from remethylation defects.

Methionine adenosyltransferases (MAT's) are central enzymes in living organisms that have been conserved with a high degree of homology among species. In the liver, MAT I and III, tetrameric and dimeric isoforms of the same catalytic subunit encoded by the gene MAT1A, account for the predominant portion of total body synthesis of S-adenosylmethionine (SAM), a versatile sulfonium ion-containing molecule involved in a variety of vital metabolic reactions and in the control of hepatocyte proliferation and differentiation. During the past 15 years 28 MAT1A mutations have been described in patients with elevated plasma methionines, total homocysteines at most only moderately elevated, and normal levels of tyrosine and other aminoacids. In this study we describe functional analyses that determine the MAT and tripolyphosphatase (PPPase) activities of 18 MAT1A variants, six of them novel, and none of them previously assayed for activity. With the exception of G69S and Y92H, all recombinant proteins showed impairment (usually severe) of MAT activity. Tripolyphosphate (PPPi) hydrolysis was decreased only in some mutant proteins but, when it was decreased MAT activity was always also impaired.

This paper reports the clinical and metabolic findings in two sibling sisters born with fetal hydrops and eventually found to have deficient S- adenosylhomocysteine hydrolase (AHCY) activity due to compound heterozygosity for two novel mutations, c.145C>T ; p.Arg49Cys and c.257A>G ; p.Asp86Gly. Clinically, the major abnormalities in addition to fetal hydrops (very likely due to impaired synthetic liver function) were severe hypotonia/myopathy, feeding problems, and respiratory failure. Metabolic abnormalities included elevated plasma S-adenosylhomocysteine, S-adenosylmethionine, and methionine, with hypoalbuminemia, coagulopathies, and serum transaminase elevation. The older sister died at age 25 days, but the definitive diagnosis was made only retrospectively. The underlying genetic abnormality was diagnosed in the second sister, but treatment by means of dietary methionine restriction and supplementation with phosphatidylcholine and creatine did not prevent her death at age 122 days. These cases extend the experience with AHCY deficiency in humans, based until now on only the four patients previously identified, and suggest that the deficiency in question may be a cause of fetal hydrops and developmental abnormalities of the brain.

Over a four-year period, we collected clinical and biochemical data from five Amish children who were homozygous for missense mutations in 5,10-methylenetetrahydrofolate reductase (MTHFR c.1129C>T). The four oldest patients had irreversible brain damage prior to diagnosis. The youngest child, diagnosed and started on betaine therapy as a newborn, is healthy at her present age of three years. We compared biochemical data among four groups: 16 control subjects, eight heterozygous parents, and five affected children (for the latter group, both before and during treatment with betaine anhydrous). Plasma amino acid concentrations were used to estimate changes in cerebral methionine uptake resulting from betaine therapy. In all affected children, treatment with betaine (534+/-222 mg/kg/day) increased plasma S-adenosylmethionine, improved markers of tissue methyltransferase activity, and resulted in a threefold increase of calculated brain methionine uptake. Betaine therapy did not normalize plasma total homocysteine, nor did it correct cerebral 5-methyltetrahydrofolate deficiency. We conclude that when the 5-methyltetrahydrofolate content of brain tissue is low, dietary betaine sufficient to increase brain methionine uptake may compensate for impaired cerebral methionine recycling. To effectively support the metabolic requirements of rapid brain growth, a large dose of betaine should be started early in life.

Various questions have been raised about labile methyl balance and total transmethylation fluxes, and further discussion has been encouraged. This report reviews and discusses some of the relevant evidence now available. The fact that, if needed, labile methyl balance is maintained by methylneogenesis appears to be established, but several aspects of transmethylation remain uncertain: definitive measurements of the rate of total transmethylation in humans of both sexes on various diets and at various ages; the extent to which synthesis of phosphatidylcholine has been underestimated; and the relative contributions of the 2 pathways for the formation of sarcosine (ie, N-methylglycine). The available evidence indicates that the quantitatively most important pathways for S-adenosylmethionine-dependent transmethylation in mammals are the syntheses of creatine by guanidinoacetate methyltransferase, of phosphatidylcholine by phosphatidylethanolamine methyltransferase, and of sarcosine by glycine N-methyltransferase. Data presented in this report show that S-adenosylmethionine and methionine accumulate abnormally in the plasma of humans with glycine N-methyltransferase deficiency but not of those with guanidinoacetate N-methyltransferase deficiency or in the plasma or livers of mice devoid of phosphatidylethanolamine N-methyltransferase activity. The absence of such accumulations in the latter 2 conditions may be due to removal of S-adenosylmethionine by synthesis of sarcosine. Steps that may help clarify the remaining issues include the determination of the relative rates of synthesis of sarcosine, creatine, and phosphatidylcholine by rapid measurement of the rates of radiolabel incorporation into these compounds from L-[methyl-3H]methionine administered intraportally to an experimental animal; clarification of the intracellular hepatic isotope enrichment value during stable-isotope infusion studies to enhance the certainty of methyl flux estimates during such studies; and definitive measurement of the dietary betaine intake from various diets.

Recently, we have described the first human case of AdoHcyase (S-adenosylhomocysteine hydrolase) deficiency. Two point mutations in the AdoHcyase gene, the missense mutation p.Y143C (AdoHcyase in which Tyr143 is replaced by cysteine) and the truncation mutation p.W112stop (AdoHcyase in which Trp112 is replaced by opal stop codon) were identified [Barić, Fumić, Glenn, Ćuk, Schulze, Finkelstein, James, Mejaški-Bošnjak, Pažanin, Pogribny et al. (2004) Proc. Natl. Acad. Sci. U.S.A. 101, 4234–4239]. To elucidate the molecular and catalytic properties of AdoHcyase, we have made recombinant wild-type and mutant p.Y143C (AdoHcyase in which Tyr143 is replaced by cysteine) enzymes for a comparative analysis. The catalytic rates of p.Y143C protein in the directions of S-adenosylhomocysteine synthesis or hydrolysis are decreased from 65% to 75%. Further, the oxidation states of coenzyme NAD differ between mutant and wild-type protein, with an increased NADH accumulation in the mutant p.Y143C enzyme of 88% NADH (wild-type contains 18% NADH). Quantitative binding of NAD is not affected. Native polyacrylamide gel electrophoresis showed, that mutant p.Y143C subunits are able to form the tetrameric complex as is the wild-type enzyme. CD analysis showed that the p.Y143C mutation renders the recombinant protein thermosensitive, with an unfolding temperature significantly reduced by 7 °C compared with wild-type protein. Change of Glu115 to lysine in wild-type protein causes a change in thermosensitivity almost identical with that found in the p.Y143C enzyme, indicating that the thermosensitivity is due to a missing hydrogen bond between Tyr143 and Glu115. We emphasize involvement of this particular hydrogen bond for subunit folding and/or holoenyzme stability. In summary, a single mutation in the AdoHcyase affecting both the oxidation state of bound co-factor NAD and enzyme stability is present in a human with AdoHcyase deficiency.

Contains fulltext : 49036.pdf (Publisher’s version ) (Closed access) (1)H-NMR spectroscopy at 500 MHz was used to confirm that a previously unidentified singlet resonance at 3.14 ppm in the spectra of cerebrospinal fluid and plasma samples corresponds to dimethyl sulfone (DMSO(2)). A triple resonance inverse cryogenic NMR probe, with pre-amplifier and the RF-coils cooled to low temperature, was used to obtain an (1)H-(13)C HSQC spectrum of CSF containing 8 microM (753 ng/ml) DMSO(2). The (1)H-(13)C correlation signal for DMSO(2) was assigned by comparison with the spectrum from an authentic reference sample. In plasma and CSF from healthy controls, the concentration of DMSO(2) ranged between 0 and 25 micromol/l. The concentration of DMSO(2) in plasma from three of four patients with severe methionine adenosyltransferase I/III (MAT I/III) deficiency was about twice the maximum observed for controls. Thus, DMSO(2) occurs as a regular metabolite at low micromolar concentrations in cerebrospinal fluid and plasma. It derives from dietary sources, from intestinal bacterial metabolism and from human endogenous methanethiol metabolism.

We report studies of a Croatian boy, a proven case of human S -adenosylhomocysteine (AdoHcy) hydrolase deficiency. Psychomotor development was slow until his fifth month; thereafter, virtually absent until treatment was started. He had marked hypotonia with elevated serum creatine kinase and transaminases, prolonged prothrombin time and low albumin. Electron microscopy of muscle showed numerous abnormal myelin figures; liver biopsy showed mild hepatitis with sparse rough endoplasmic reticulum. Brain MRI at 12.7 months revealed white matter atrophy and abnormally slow myelination. Hypermethioninemia was present in the initial metabolic study at age 8 months, and persisted (up to 784 μM) without tyrosine elevation. Plasma total homocysteine was very slightly elevated for an infant to 14.5–15.9 μM. In plasma, S -adenosylmethionine was 30-fold and AdoHcy 150-fold elevated. Activity of AdoHcy hydrolase was ≈3% of control in liver and was 5–10% of the control values in red blood cells and cultured fibroblasts. We found no evidence of a soluble inhibitor of the enzyme in extracts of the patient's cultured fibroblasts. Additional pretreatment abnormalities in plasma included low concentrations of phosphatidylcholine and choline, with elevations of guanidinoacetate, betaine, dimethylglycine, and cystathionine. Leukocyte DNA was hypermethylated. Gene analysis revealed two mutations in exon 4: a maternally derived stop codon, and a paternally derived missense mutation. We discuss reasons for biochemical abnormalities and pathophysiological aspects of AdoHcy hydrolase deficiency.

Abnormal elevation of plasma methionine may result from several different genetic abnormalities, including deficiency of cystathionine [beta ]-synthase (CBS) or of the isoenzymes of methionine adenosyltransferase (MAT) I and III expressed solely in nonfetal liver (MAT I/III deficiency). Classically, these conditions have been distinguished most readily by the presence or absence, respectively, of elevated plasma free homocystine, detected by amino acid chromatography in the former condition, but absent in the latter. During the present work, we have assayed methionine, S-adenosylmethionine, S-adenosylhomocysteine, total homocysteine (tHcy), cystathionine, N-methylglycine (sarcosine), and total cysteine (tCys) in groups of both MAT I/III- and CBS-deficient patients to provide more evidence as to their metabolite patterns. Unexpectedly, we found that MAT I/III-deficient patients with the most markedly elevated levels of plasma methionine also had elevations of plasma tHcy and often mildly elevated plasma cystathionine. Evidence is presented that methionine does not inhibit cystathionine [beta ]-synthase, but does inhibit cystathionine gamma-lyase. Mechanisms that may possibly underlie the elevations of plasma tHcy and cystathionine are discussed. The combination of elevated methionine plus elevated tHcy may lead to the mistaken conclusion that an MAT I/III-deficient patient is instead CBS-deficient. Less than optimal management is then a real possibility. Measurements of plasma cystathionine, S-adenosylmethionine, and sarcosine should permit ready distinction between the 2 conditions in question, as well as be useful in several other situations involving abnormalities of methionine and/or homocysteine derivatives.

In recent years, there has been an upsurge of interest in elevation of the plasma concentration of homocysteine and closely related metabolites as an independent risk factor for cardiovascular disease (reviewed, for example, in References 1 through 3). Homocysteine itself is a thiol(sulfhydryl-) containing amino acid, but in normal human plasma and other tissues, a variety of related disulfide derivatives may be present. Different authors have written about these compounds and their effects by using differing terminologies. To promote clarity of meaning and to minimize uncertainty, perhaps even confusion, it is important that each article discussing these compounds either defines explicitly the terms and/or abbreviations used or cites a prior publication in which such definitions are provided. Optimally, a more uniform consensus terminology will be developed and adopted by the field. This article describes very briefly the structures of the relevant compounds and sets forth terms and abbreviations that, it is hoped, may provide a basis

A search was made for compounds that would inhibit methionine biosynthesis in Lemna paucicostata Hegelm. 6746. dl-Propargylglycine (0.15 micromolar) produced growth inhibition and morphological changes which were prevented by exogenous methionine. Also, dl-propargylglycine inhibits cystathionine gamma-synthase activity. l-Aminoethoxyvinylglycine (0.05 micromolar) produced growth inhibition and morphological changes partially preventable by exogenous methionine. l-Aminoethoxyvinylglycine impairs the cleavage of cystathionine to homocysteine. Lysine and threonine, at concentrations which individually had little effect on growth or morphology of Lemna, together produced growth inhibition and morphological changes preventable by exogenous methionine. The resulting metabolic block prevented conversion of cysteine to cystathionine, presumably secondary to depletion of the supply of O-phosphohomoserine.Inhibition of Lemna growth resulted when the molybdate:sulfate ratio in the medium was increased to 20:1 or more. Such inhibition was prevented by lowering this ratio to 0.3 or less. A non-steady-state experiment (molybdate:sulfate, 20:1) showed that molybdate inhibited sulfate uptake, but it provided no evidence of a further impairment in the organification of sulfate. Molybdate-induced growth inhibition of Lemna was prevented by cystine but not by cystathionine or methionine. Cystathionine is not converted by Lemna to cysteine rapidly enough to sustain growth.

Lemna perpusilla 6746, grown photoautotrophically at a series of sulfate concentrations ranging from 0.32 to 1,000 μm, was labeled to radioisotopic equilibrium with 35 SO 4 2− . Sulfur-containing compounds were isolated and purified from the colonies. Radioactivity in each compound was a measure of the amount of that compound present in the tissue. The following compounds were identified and quantitated: inorganic sulfate, glutathione, homocyst(e)ine, cyst(e)ine, methionine, S-methylmethionine sulfonium, S-adenosylmethionine, S-adenosylhomocysteine, cystathionine, chloroformsoluble (presumed to be sulfolipid), protein cyst(e)ine, and protein methionine. γ-Glutamylcyst(e)ine, erythro - and threo -thiothreonine, and S-methylcysteine were not detected. No volatile 35 S compounds were formed during plant growth at 1,000 μm sulfate, nor were significant amounts of 35 S compounds excreted into the medium. The amount of each component present in colonies grown over the 3,000-fold range of medium sulfate was relatively constant except for inorganic sulfate. This increased about 30-fold from the lowest to the highest medium sulfate concentration. The total soluble sulfur amino acids increased about 1.5- to 2-fold, due primarily to an increased amount of glutathione. Protein cyst(e)ine and protein methionine were the major organic sulfur compounds in Lemna , and the amounts of these compounds remained virtually constant despite the variation in external sulfate concentration. Procedures for the analysis of S-adenosylmethionine, S-methylmethionine sulfonium, and S-adenosylhomocysteine are presented.

Extracts of phylogenetically diverse plans were surveyed for their ability to synthesize the following homoserine esters which are potential precursors for methionine and threonine synthesis in green plants: O -acetyl-, O -oxalyl-, O -succinyl-, O -malonyl-, and O -phosphohomoserine. Synthesis of O -acylhomoserine esters was detected only in Pisum sativum L. and Lathyrus sativus L. Extracts of P. sativum , a plant known to accumulate O -acetylhomoserine, catalyzed the specific synthesis of this ester from homoserine and acetyl-CoA. Extracts of L. sativus , a plant known to accumulate O -oxalylhomoserine, catalyzed the specific synthesis of this ester from homoserine and oxalyl-CoA. None of the other plants surveyed, including representatives of the green algae, horsetails, gymnosperms, and angiosperms, catalyzed the synthesis of any of the O -acylhomoserine esters studied. In contrast, synthesis of O -phosphohomoserine by the reaction catalyzed by homoserine kinase was demonstrated in extracts of all plants examined, including the two exceptional legumes. These results suggest that, among the five homoserine esters studied, O -phosphohomoserine is the major activated homoserine derivative in plants. Direct confirmation of the dominant physiological role of O -phosphohomoserine in the synthesis of cystathionine in the transsulfuration pathway of methionine biosynthesis in plants has recently been provided (Datko, A. H., Giovanelli, J., and Mudd, S. H. 1974. J. Biol. Chem. 249: 1139-1155).

Optimal conditions for biosynthesis of cystathionine with crude extracts of green plants were determined. The specific activities observed under these conditions are considerably higher than those initially detected (Giovanelli, J., and Mudd, S. H. (1966) Biochem. Biophys. Res. Commun. 25, 366), and compare favorably with those of bacterial extracts. At near saturating concentrations of homoserine esters, crude extracts of tissues representing most of the major phylogenetic divisions of green plants generally catalyze the highest rates of cystathionine synthesis in the presence of O-malonylhomoserine, intermediate rates in the presence of O-oxalyl-, O-succinyl-, and O-phosphorylhomoserine, and very low rates with O-acetylhomoserine. Green plants are unique among the organisms studied by us or other workers in their capacity to use O-phosphorylhomoserine in cystathionine synthesis. Enzymic assay systems permitting the detection of compounds capable of serving as α-aminobutyryl donors in the synthesis of cystathionine were developed, taking advantage of the fact that crude preparations of plant cystathionine γ-synthase are active with a wide range of compounds. An analogous system designed to detect O-acetylhomoserine with greater sensitivity was developed through the use of an enzyme preparation from Bacillus subtilis. An α-amino-butyryl donor was purified by ion exchange chromatography and paper electrophoresis from tissues of green plants ranging from the green alga Chlorella to the higher flowering plants. Only a single α-aminobutyryl donor was detected among these green plants. This compound was characterized as O-phosphorylhomoserine on the basis of its base stability, its behavior during ion exchange chromatography and paper electrophoresis, and a rate of hydrolysis by highly purified alkaline phosphatase that was indistinguishable from that of authentic O-phosphorylhomoserine. It is proposed that O-phosphorylhomoserine is the dominant physiological precursor of cystathionine in green plants. Some of the implications of this finding are discussed. During the course of this work, studies on the relative activity of cystathionine γ-synthase with various substrates were extended to a number of lower organisms. The patterns of substrate activity with crude extracts of a blue-green alga and Escherichia coli are very similar, and resemble the pattern for cystathionine γ-synthase of Salmonella typhimurium (Kaplan, M. M., and Flavin, M. (1966) J. Biol. Chem. 241, 4463). O-Succinylhomoserine is the most active substrate for all three organisms. Extracts of B. subtilis are most active with O-acetylhomoserine, a pattern not previously observed with bacterial cystathionine γ-synthases.

Lemna paucicostata Hegelm. 6746 possesses specific systems for uptake of choline and ethanolamine. Each is distinct from the six other systems for uptake of organic compounds so far identified in this plant. Both systems show biphasic kinetics, so that uptake by them can be described as the composite result of two Michaelis-Menten processes. Inhibitor studies are reported which indicate the very strict structural specificity of each system. The kinetic constants of choline uptake are such that, at an external concentration of 0.65 micromolar, the total requirement of the plant for this compound would be met, 41% via the high affinity system and 59% via the lower. At an external concentration of 2.4 micromolar ethanolamine, an amount of this compound sufficient to form the total choline of the plant would be supplied, 59% via the high affinity system and 41% via the lower. These, and other observations, strongly support the physiological importance of these systems under natural conditions.

Extract: The intracellular content and proportional distribution of B12 (cobalamin) derivatives in fibroblasts cultured from patients with various forms of methylmalonic aciduria, as well as from normal control subjects, has been determined by a two-dimensional chromatobioautographic technique. Each line of fibroblasts was grown in the presence of four concentrations of cobalamin, ranging from the 0.04–0.07 pmol/ml contained in the basal medium to 74 pmol/ml (100 ng/ml), added in form of hydroxocobalamin (OHCBI). Control cells grown in the basal medium contained substantial proportions of both methylcobalamin (MeCbl) and adenosyl-cobalamin (AdoCbl), with the former predominating. As increasing concentrations of OH-CBI were added to the growth medium, the total cellular cobalamin content increased without marked changes in the relative proportions of MeCbl, AdoCbl, and OHCbl. Three different patterns were discernable in the cobalamin distributions of the cells cultured from patients with methylmalonic aciduria (Table 1 and Fig. 1). Speculation: Information about the cobalamin contents of cultured fibroblasts yields further understanding of cobalamin metabolism. Such information, obtained by the technique of two-dimensional chromatobioautography, permits definition of genetically determined abnormalities in the handling of cobalamins, as well as examination of possible correlations between cellular responses to increased concentrations of these compounds and the effectiveness of cobalamins in the clinical treatment of patients.

Comprehensive studies were made with Lemna paucicostata Hegelm. 6746 of the effects of combinations of lysine, methionine, and threonine on growth rates, soluble amino acid contents, aspartokinase activities, and fluxes of 4-carbon moieties from aspartate through the aspartokinase step into the amino acids of the aspartate family. These studies show that flux in vitro through the aspartokinase step is insensitive to inhibition by lysine or threonine, and confirm previous in vitro data in establishing that aspartokinase in vivo is present in two orders of magnitude excess of its requirements. No evidence of channeling of the products of the lysine- and threonine-sensitive aspartokinases was obtained, either form of the enzyme alone being more than adequate for the combined in vivo flux through the aspartokinase step. The marked insensitivity of flux through the aspartokinase step to inhibition by lysine or threonine strongly suggests that inhibition of aspartokinase by these amino acids is not normally a major factor in regulation of entry of 4-carbon units into the aspartate family of amino acids. Direct measurement of fluxes of 4-carbon units demonstrated that: (a) Lysine strongly feedback regulates its own synthesis, probably at the step catalyzed by dihydrodipicolinate synthase. (b) Threonine alone does not regulate its own synthesis in vivo, thereby confirming previous studies of the metabolism of [(14)C]threonine and [(14)C]homoserine in Lemna. This finding excludes not only aspartokinases as an important regulatory determinant of threonine synthesis, but also two other enzymes (homoserine dehydrogenase and threonine synthase) suggested to fulfill this role. Complete inhibition of threonine synthesis was observed only in the combined presence of accumulated threonine and lysine. The physiological significance of this single example of apparent regulation of flux at the aspartokinase step, albeit under unusually stringent conditions of aspartokinase inhibition, remains to be determined. (c) Isoleucine strongly inhibits its own synthesis, probably at threonine dehydratase, without causing compensatory reduction in threonine synthesis. A fundamentally changed scheme for regulation of synthesis of the aspartate family of amino acids is presented that has important implications for improvement of the nutritional contents of these amino acids in plants.

The pathway for synthesis of phosphatidylcholine, the dominant methyl-containing end product formed by Lemna paucicostata, has been investigated. Methyl groups originating in methionine are rapidly utilized by intact plants to methylate phosphoethanolamine successively to the mono-, di-, and tri-methyl (i.e. phosphocholine) phosphoethanolamine derivatives. With continued labeling, radioactivity initially builds up in these compounds, then passes on, accumulating chiefly in phosphatidylcholine (34% of the total radioactivity taken up by plants labeled to isotopic equilibrium with l-[(14)CH(3)]methionine), and in lesser amounts in soluble choline (6%). Radioactivity was detected in mono- and dimethyl derivatives of free ethanolamine or phosphatidylethanolamine only in trace amounts. Pulse-chase experiments with [(14)CH(3)]choline and [(3)H] ethanolamine confirmed that phosphoethanolamine is rapidly methylated and that phosphocholine is converted to phosphatidylcholine. Initial rates indicate that methylation of phosphoethanolamine predominates over methylation of either phosphatidylethanolamine or free ethanolamine at least 99:1. Although more studies are needed, it is suggested this pathway may well turn out to account for most phosphatidylcholine synthesis in higher plants. Phosphomethylethanolamine and phosphodimethylethanolamine are present in low quantities during steady-state growth (18% and 6%, respectively, of the amount of phosphocholine). Radioactivity was not detected in CDP-choline, probably due to the low steady-state concentration of this nucleotide.

To provide information upon the ways in which Lemna paucicostata uses the methyl group of methionine, plants were grown for various periods (from 1 minute to 6.8 days) in the presence of a tracer dose of radioactive methyl-labeled methionine. Protein methionine accounted for approximately 19% of the accumulated methyl moieties; other methylated products, about 81%. The latter group included (percent of total methyl in parentheses): methylated ethanolamine derivatives (46%); methyl esters of the pellet (chiefly, or solely, pectin methyl esters) (15%); chlorophyll methyl esters (8%); unidentified neutral lipids (6%); nucleic acid derivatives (2-5%); methylated basic amino acids (2%). No other major methylated compounds were observed in any plant fraction. Available evidence suggests that little, if any, oxidation of the methyl group of methionine, directly or indirectly, occurs in Lemna. Our results indicate that S-methyl-methionine sulfonium is formed relatively rapidly, but does not accumulate at a commensurate rate, probably being reconverted to methionine. To our knowledge, this is the first time a reasonably complete accounting of the metabolic fate of methionine methyl has been obtained for any plant. The extent to which the results with Lemna may be representative of the situation for other higher plants is discussed.

The methylation steps in the biosynthesis of phosphatidylcholine by tissue culture preparations of carrot (Daucus carota L.) and soybean (Glycine max), and by soybean leaf discs, have been studied. Preparations were incubated with tracer concentrations of l-[(3)H(3)C]methionine and the kinetics of appearance of radioactivity in phosphomethylethanolamine, phosphodimethylethanolamine, phosphocholine, phosphatidylmethylethanolamine, phosphatidyldimethylethanolamine, phosphatidylcholine, methylethanolamine, dimethylethanolamine, and choline followed at short incubation times. With soybean (tissue culture or leaves), an initial methylation utilizes phosphoethanolamine as substrate, forming phosphomethylethanolamine. The latter is converted to phosphatidylmethylethanolamine, which is successively methylated to phosphatidyldimethyethanolamine and to phosphatidylcholine. With carrot, again, an initial methylation is of phosphoethanolamine. Subsequent methylations occur at both the phospho-base and phosphatidyl-base levels. Both of these patterns differ qualitatively from that previously demonstrated in Lemna (SH Mudd, AH Datko 1986 Plant Physiol 82: 126-135) in which all three methylations occur at the phospho-base level. For soybean and carrot, some added contribution from initial methylation of phosphatidylethanolamine has not been excluded. These results, together with those from similar experiments carried out with water-stressed barley leaves (WD Hitz, D Rhodes, AD Hanson 1981 Plant Physiol 68: 814-822) and salinized sugarbeet leaves (AD Hanson, D Rhodes 1983 Plant Physiol 71: 692-700) suggest that in higher plants some, perhaps all, phosphatidylcholine synthesis occurs via a common committing step (conversion of phosphoethanolamine to phosphomethylethanolamine) followed by a methylation pattern which differs from plant to plant.

Inorganic phosphate (Pi) inhibits threonine synthase of Lemna, and cystathionine gamma-synthase less strongly. AMP is an extremely potent and structurally specific inhibitor of threonine synthase. Each inhibition progressively decreases with increasing concentrations of O-phosphohomoserine (OPH). To study the in vivo effects of these inhibitions, Lemna was grown with a range of Pi concentrations. A 25,000-fold increase in Pi concentration in the culture medium caused an increase of only 6-fold in total phosphorus of the plants. This is explained by the fact that a high affinity Pi uptake system is selectively down-regulated during growth with high concentrations of Pi. Pi and AMP in plants grown with various Pi concentrations were determined, and concentrations estimated for chloroplasts, the organelle containing threonine synthase and cystathionine gamma-synthase. Calculations indicated that for growth at standard external Pi (0.4 millimolar) or above, if total OPH were uniformly distributed within the plants, activities of the two enzymes in question would be severely inhibited, and each would fall two orders of magnitude below the amount required to provide threonine (plus isoleucine) or methionine adequate for growth. If OPH were restricted to chloroplasts, these inhibitions would be much less severe, resulting in enzyme activities approaching the required physiological amounts. Evidence is presented that even up to 50 millimolar external Pi, this ion does not limit production of threonine or methionine sufficiently to retard growth, consistent with the postulated localization of OPH within chloroplasts.

Individual rates of metabolism of the sulfur, methyl, and 4-carbon moieties of methionine were estimated in Lemna paucicostata Hegelm. 6746 growing under standard conditions, and used to quantitate pathways of methionine metabolism. Synthesis of S-adenosylmethionine (AdoMet) is the major pathway for methionine metabolism, with over 4 times as much methionine metabolized by this route as accumulates in protein. More than 90% of AdoMet is used for transmethylation. Methyl groups of choline, phosphatidylcholine, and phosphorylcholine are major end products of this pathway. Flux through methylthio recycling is about one-third the amount of methionine accumulating in protein. Spermidine synthesis accounts for at least 60% of the flux through methylthio recycling. The results obtained here, together with those reported for methionine-supplemented plants (Giovanelli, Mudd, Datko 1981 Biochem Biophys Res Commun 100: 831-839), indicate that methionine supplementation reduced methylneogenesis by no more than the small amount expected from the reduced entry of sulfate sulfur into methionine (Giovanelli, Mudd, Datko, 1985 Plant Physiol 77: 450-455). Methionine supplementation had no significant effect on transmethylation or methylthio recycling. The combined data provide the first comprehensive estimates of the quantitative relationships of major pathways for methionine metabolism and their control in plants.

This paper constitutes the first description of a previously unreported disorder of metabolism of sulfur-containing compounds. The patient was born with neurological abnormalities and deteriorated to a virtually decorticate state by nine months. Bilateral ectopia lentis was discovered at 1 year. We studied him at age 30 months and found his urine to contain abnormally increased amounts of S-sulfo- l -cysteine, sulfite, and thiosulfate. His urinary excretion of inorganic sulfate was markedly reduced and, in contrast to normal controls, it did not increase after administration of l -cysteine. These chemical abnormalities are best explained by the presence of a block in the conversion of sulfite to sulfate secondary to a deficiency of sulfite oxidase activity. Studies from tissues obtained from the patient postmortem reveal such a defect (5). The pathology could have been caused by toxic effects of accumulation of sulfite or perhaps other metabolites. Possibly, deficiency of inorganic sulfate could also have caused pathological changes. Although three of the patient's seven siblings died in infancy with neurological disease, the remaining siblings and the parents do not bear clinical stigmata suggestive of the patient's disease, and as yet we have detected no chemical abnormalities to suggest they are carriers of a trait. Thus, we suspect, but have no definite evidence, that the disease is an hereditary disorder of metabolism. We have named it sulfite oxidase deficiency.

The clinical and biochemical findings in two brothers each of whom has homocystinuria and methylmalonic aciduria are described. Studies of fibroblasts grown from skin biopsies of these patients demonstrated that their homocystinuria is due to decreased activity of N5-methyltetra-hydrofolate methyltransferase, rather than due to decreased activity of cystathionine synthase. These siblings are the second and third reported cases of homocystinuria due to a defect in homocysteine methylation. Both the means and importance of differentiating homocystinuria due to cystathionine synthase deficiency from that due to defects in methylation of homocysteine are discussed. The cause of the methylmalonic aciduria has not been finally established, although the data are compatible with a partial decrease in activity of methylmalonyl-CoA isomerase. Together, the abnormalities in these brothers may tentatively be ascribed to a defect in the uptake or early metabolism of B12, the condition most likely being inherited as an autosomal recessive trait.

The possibility of a functionally significant sulfate deficiency in a patient with sulfite oxidase deficiency has been evaluated by study of sulfate esters in tissues and urine of this patient. Sulfatides in brain and kidney were normal qualitatively and normal in concentration. Since the brain weight was decreased, the total brain sulfatide was lower than normal. The daily urinary excretions of tyrosine-O-sulfate and indoxyl-3-sulfate were comparable to excretions found in control patients.

Inactivation of spinach β-cystathionase by rhizobitoxine is an example of an active-site-directed irreversible inhibition, and may be represented by the following mechanism: E P + R ⇌E PR →E PR I E P , the holoenzyme form of β-cystathionase (P, pyridoxal phosphate); R, rhizobitoxine; E PR , a dissociable enzyme-rhizobitoxine complex; E PR I , inactivated enzyme. This proposed mechanism is based on the following observations: 1. 1. The inactivation is progressive and ultimately complete as would be expected for an irreversible process. Irreversibility of the inactivation wa demonstrated directed by gel filtration. 2. 2. The rate of inactivation is first order with respect to active enzyme. 3. 3. A plot of the pseudo first-order rate constant as a function of rhizobitoxine concentration shows saturation kinetics, consistent with the formation of a dissociable complex E PR . The equilibrium constant (K i ) for the dissociation of E PR is 8.0 · 10 −5 M ; the rate of the inactivation at infinite rhizobitoxine concentration is equivalent to a minimum half-time of inactivation (T 1 2 ) of 0.2 min. 4. 4. The rate of dissociation of E PR is rapid relative to the rate of inactivation. 5. 5. The substrates cystathionine and djenkolate, as well as the competitive inhibitor β-cyanoalanine, protect the enzyme from inactivation by rhizobitoxine. The kinetics of this protection are consistent with a competition between these amino acids and rhizobitoxine for the active enzyme. 6. 6. The rhizobitoxine-inactivated enzyme can be reactivated specifically by incubation with pyridoxal phosphate, suggesting that the irreversible step in inactivation involves formation of a bond between rhizobitoxine and pyridoxal phosphate.

An infant is described who died at seven and one half weeks of age and whose clinical manifestations included poor feeding, hematemesis, melena, normocytic anemia and failure to gain weight. He was found to accumulate abnormally large amounts of cystathionine, homocystine and homocysteine-cysteine mixed disulfide and to have low concentrations of methionine in blood and urine. In addition he manifested a marked methylmalonic aciduria that did not respond to the intramuscular administration of vitamin B, (cyanocobalamin). Enzymatic analyses of liver, kidney and brain obtained at autopsy revealed specifically deficient activity in the B,-dependent enzyme, N,-methyltetrahydrofolate-homocysteine methyltransferase. Studies of fibroblasts grown from skin obtained ante mortem showed that the activity of the B--dependent enzyme, methylmalenyl-CoA (coenzyme A) isomerase, also was deficient, but this activity could be restored by growing the cells in medium containing excess hydroxocobalamin. Analysis of liver and kidney revealed that the concentration of deoxyadenosyl cobalamin (coenzyme-B,) was abnormally low whereas the concentration of total B, was high in serum and normal in liver. Apparently this infant could not adequately metabolize B, and as a result failed to accumulate coenzymatically-active derivatives of vitamin B, in normal amounts. It is suggested that therapeutic benefit may derive from the administration of forms of 6, that could circumvent the metabolic block, by giving substances that could stimulate the non-B, -dependent methionine-synthesizing system or by the administration of methionine in large doses.

1. Hepatic levels of cystathionine synthase and methionine-activating enzyme are significantly lower in rats fed a diet low in methionine and supplemented with cystine than in rats growing at the same rate while maintained on a diet adequate in methionine, with or without cystine supplementation. Cystathionase levels are also decreased, but to a smaller extent. Betaine-homocysteine methyltransferase is not affected. 2. The enzymatic activities which are lowered are restored toward normal by injections of l-methionine or l-homocysteine. 3. Methionine-activating enzyme and cystathionine synthase are inhibited in vitro by l-cystine. However, the decreased enzyme levels in the livers of rats fed the low methionine, cystine-supplemented diet cannot be attributed to either a dissociable inhibitor or cystine binding by the enzyme proteins. It seems likely that the cystine effect represents repression of enzyme synthesis. 4. The physiological meaning of these changes in enzymatic activity is briefly discussed. The changes are such that they may well explain the known "methionine-sparing" effect of cystine. 5. A possible application of these findings to the treatment of patients with homocystinuria due to cystathionine synthase deficiency is mentioned.

Studies are reported of fibroblasts cultured from the skin of a patient with abnormalities of sulfur amino acid and of methylmalonic acid metabolism. These cells are unable to grow as do normal cells if methionine in the medium is replaced by homocystine. Addition of hydroxy-B12, 1 μg/ml, to the medium allowed the cells of the patient to grow slowly. Extracts of the cells contained a normal concentration of cystathionine synthase, but an abnormally low concentration of holoenzyme for N5-methyltetrahydrofolate methyltransferase activity. N5-Methyltetrahydrofolate methyltransferase apoenzyme was present, as indicated by assay in the presence of appropriate B12-containing compounds. Growth of control cells in media containing hydroxy-B12, 1 μg/ml, led to a several-fold increase in total methyltransferase activity and a decrease of dependence of activity upon in vitro supplementation with B12-containing compounds. Growth of the patient's cells under these conditions led to formation of a small, but detectable, amount of holoenzyme. However, the major part of the methyltransferase activity in extracts of his cells grown on hydroxy-B12 remained dependent upon in vitro addition of a B12-containing compound. The patient's cells converted propionate-1-14C to 14CO2 at a slow rate compared to control cells. During incubation with propionate-1-14C his cells accumulated abnormally great amounts of methylmalonate-14C but failed to form the usual amounts of radioactive succinate, lactate, glutamate, or aspartate. Growth of the patient's cells with supplemental hydroxy-B12 in the medium partially restored the rate at which these cells converted propionate-1-14C to 14CO2. The specificity of this restorative effect as well as the effects of variation in concentration and time of exposure to hydroxy-B12 are reported. All the available data are consistent with the hypothesis that cells from the patient are deficient in their ability to accumulate coenzymatically active derivatives of B12 with resultant deficits in the activities of N5-methyltetrahydrofolate methyltransferase and methylmalonyl-CoA isomerase, the two enzymes known in mammals to depend upon B12 derivatives.

Summary Methionine adenosyltransferase (MAT) I/III deficiency, caused by mutations in the MAT1A gene, is characterized by persistent hypermethioninemia without elevated homocysteine or tyrosine. Clinical manifestations are variable and poorly understood, although a number of individuals with homozygous null mutations in MAT1A have neurological problems, including brain demyelination. We analyzed MAT1A in seven hypermethioninemic individuals, to provide insight into the relationship between genotype and phenotype. We identified six novel mutations and demonstrated that mutations resulting in high plasma methionines may signal clinical difficulties. Two patients—a compound heterozygote for truncating and severely inactivating missense mutations and a homozygote for an aberrant splicing MAT1A mutation—have plasma methionine in the 1,226–1,870 μM range (normal 5–35 μM) and manifest abnormalities of the brain gray matter or signs of brain demyelination. Another compound heterozygote for truncating and inactivating missense mutations has 770–1,240 μM plasma methionine and mild cognitive impairment. Four individuals carrying either two inactivating missense mutations or the single-allelic R264H mutation have 105–467 μM plasma methionine and are clinically unaffected. Our data underscore the necessity of further studies to firmly establish the relationship between genotypes in MAT I/III deficiency and clinical phenotypes, to elucidate the molecular bases of variability in manifestations of MAT1A mutations.

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.

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.

This review will focus on certain aspects of vitamin-responsive genetic disorders. To be included, a condition must be gentically determined, and its characteristic chemical and/or biochemical manifestations must be alleviated by larger than physiological amounts of a particular vitamin or by use of an unusual route of administration of that vitamin. Such conditions have recently attracted much attention from human geneticists and from those concerned clinically and biochemically with inborn errors of metabolism. Previous reviews provide adequate coverage of the clinical features of these conditions and of many details concerning the history, structure, and the biochemical role of the particular vitamins to be discussed (Frimpter et al., 1969; Mudd, 1971, 1974a, 1977; Scriver, 1973; Rosenberg, 1976). In general, these matters will be beyond the scope of this chapter. Here, emphasis will be on the general properties that characterize vitamin-responsive conditions, the mechanism or mechanisms underlying vitamin responsiveness, and the implications of our present understanding of these matters both for those concerned with individual patients with metabolic diseases and for those concerned with more general aspects of human nutrition.

Homocystinuria and homocystinemia without hypermthioninemia, but with reccurent episodes of folate responseive schizophrenic-like behavior, was documented in a mildly retarded adolescent girl who lacked the habitus associated with cystathionine synthase deficiency. Enzymes involved in homocysteine-methionine metabolism were demonstrated to be normal. A defect in the ability to reducte N-5-10--methylenetetrahydrofolate to 5-methyltetrahydrofolate was demonstrated. Methylenetetrahydrofolate reductase was 18 per cent of control values. Methyltetrahydrofolate is used for the methylation of homocysteine to methionine, and a deficiency of this compound could explain the homocystinemia and homocystinuria.

A survey of the capacity of Lemna paucicostata to take up organic compounds such as might be present in the natural environment of this plant has identified eight discrete transport systems. Reciprocal inhibition studies defined the preferred substrates for these systems as follows: (a) neutral l-alpha-amino acids, (b) basic amino acids, (c) purine bases, (d) choline, (e) ethanolamine, (f) tyramine, (g) urea, and (h) aldohexoses. Each of these systems takes up its preferred substrates at high rates. At low concentrations, each Lemna frond during each minute takes up amounts which would be found in volumes ranging from 0.4 (tyramine) to 3.9 (urea) times its own volume. The two systems for amino acid transport both showed kinetics of the biphasic type, so that uptake by each can be described as the composite result of two Michaelis-Menten processes. The neutral amino acid system neither transports basic amino acids nor is inhibited by these compounds. The basic amino acid system does not transport neutral amino acids but is strongly inhibited by some, but not all, of these compounds. It is argued that the maintenance of these active, specific, and discrete systems in Lemna suggests they play important roles permitting this plant to utilize organic compounds occurring naturally in its environment.