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In mammalian liver, two intersecting pathways, remethylation and transsulfuration, compete for homocysteine that has been formed from methionine. Remethylation of homocysteine, employing either methyltetrahydrofolate or betaine as the methyl donor, forms a methionine cycle that functions to conserve methionine. In contrast, the transsulfuration sequence -- cystathionine synthase and cystathionase -- serves to irreversibly catabolize the homocysteine while synthesizing cysteine. The rate of homocysteine formation and its distribution between these two pathways are the sites for metabolic regulation and coordination. The mechanisms for regulation include both the tissue content and the kinetic properties of the component enzymes as well as the concentrations of their substrates and other metabolic effectors. Adenosylmethionine and adenosylhomocysteine are important regulatory metabolites and may use one or more mechanisms to affect the enzymes. Adenosylmethionine is a positive effector of its own synthesis, cystathionine synthase and glycine methyltransferase but impairs both homocysteine methylases. Thus, the concentration of adenosylmethionine may be self-regulatory in mammalian liver. By means of other enzymatic mechanisms, the hepatic concentration of adenosylhomocysteine, an index of homocysteine accumulation, is also self-regulated. These considerations pertain primarily to liver, which has the unique capacity to synthesize more adenosylmethionine in the presence of excess methionine. However, there are organ-specific patterns of methionine metabolism and its regulation. All tissues possess the methionine cycle with methyltetrahydrofolate as the methyl donor but only liver, kidney, pancreas, intestine and brain also contain the transsulfuration pathway. The limitation of adenosylmethionine concentrations may make adenosylhomocysteine a more significant metabolic regulator in extrahepatic tissues. However, estimates of regulatory changes based on determinations of the plasma concentrations of the two metabolites are of limited value and must be used with caution. In addition, the recent description of "cystathionine (CBS) domains" in proteins not involved with methionine metabolism raises the possibility that abnormal concentrations of the adenosyl metabolites may impact on other metabolic pathways.

Two superimposed metabolic sequences, transsulfuration and the methionine/homocysteine cycle, form the pathway for methionine metabolism in mammalian liver. This combined pathway was formulated first to explain observations in subjects with homocystinuria caused by cystathionine synthase deficiency. Since that time additional inborn errors have been discovered, and currently we know of human subjects with isolated defects in all of the reactions of the combined pathway with only one exception: betaine homocysteine methyltransferase. Studies of these inborn errors have contributed significantly to our knowledge of human methionine metabolism and to the clinical consequences of impaired metabolism. Transsulfuration appears to function primarily for the metabolism of excess methionine, and each of the 5 defects in this pathway results in the accumulation of 1 or more of the normal metabolites. Thus, studies of these disorders may provide insight into both the potential pathological sequelae of nutritional methionine excess as well as whether laboratory testing allows the detection of excess.

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 microM) without tyrosine elevation. Plasma total homocysteine was very slightly elevated for an infant to 14.5-15.9 microM. In plasma, S-adenosylmethionine was 30-fold and AdoHcy 150-fold elevated. Activity of AdoHcy hydrolase was approximately equal to 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.

This paper reports clinical and metabolic studies of two Italian siblings with a novel form of persistent isolated hypermethioninaemia, i.e. abnormally elevated plasma methionine that lasted beyond the first months of life and is not due to cystathionine beta-synthase deficiency, tyrosinaemia I or liver disease. Abnormal elevations of their plasma S-adenosylmethionine (AdoMet) concentrations proved they do not have deficient activity of methionine adenosyltransferase I/III. A variety of studies provided evidence that the elevations of methionine and AdoMet are not caused by defects in the methionine transamination pathway, deficient activity of methionine adenosyltransferase II, a mutation in methylenetetrahydrofolate reductase rendering this activity resistant to inhibition by AdoMet, or deficient activity of guanidinoacetate methyltransferase. Plasma sarcosine (N-methylglycine) is elevated, together with elevated plasma AdoMet in normal subjects following oral methionine loads and in association with increased plasma levels of both methionine and AdoMet in cystathionine beta-synthase-deficient individuals. However, plasma sarcosine is not elevated in these siblings. The latter result provides evidence they are deficient in activity of glycine N-methyltransferase (GNMT). The only clinical abnormalities in these siblings are mild hepatomegaly and chronic elevation of serum transaminases not attributable to conventional causes of liver disease. A possible causative connection between GNMT deficiency and these hepatitis-like manifestations is discussed. Further studies are required to evaluate whether dietary methionine restriction will be useful in this situation.

Homocysteine does not occur in the diet but it is an essential intermediate in normal mammalian metabolism of methionine. Each compound, methionine or homocysteine, is the precursor of the other. Similarly, the synthesis of one is the mechanism for the detoxification of the other. The ubiquitous methionine cycle is the metabolic basis for this relationship. In some tissues the transsulfuration pathway diverts homocysteine from the cycle and provides a means for the synthesis of cysteine and its derivatives. Methionine, (or homocysteine) metabolism is regulated by the disposition of homocysteine between these competing sequences. Both pathways require vitamin-derived cofactors, pyridoxine for transsulfuration and both folate and cobalamin in the methionine cycle. The clinical consequences of disruption of these pathways was apparent first in rare inborn errors of metabolism that cause homocystinuria, but recent studies focus on "hyperhomocysteinemia"--a lesser metabolic impairment that may result from genetic variations, acquired pathology, toxicity and nutritional inadequacy. Hyperhomocysteinemia is an independent risk factor for thrombovascular diseases however it is not clear whether the minimally increased concentration of the amino acid is the causative agent or merely a marker for the pathology. Until we resolve that question we cannot predict the potential efficacy of therapies based on folate administration with or without additional cobalamin and pyridoxine.

Two intersecting pathways, the methionine cycle and the transsulfuration sequence, compose the mechanisms for homocysteine metabolism in mammals. The methionine cycle occurs in all tissues and provides for the remethylation of homocysteine, which conserves methionine. In addition, the cycle is essential for the recycling of methyltetrahydrofolate. The synthesis of cystathionine is the first reaction in the irreversible pathway for the catabolism of homocysteine by means of the sequential conversion to cysteine and sulfate. This pathway has a limited distribution and is found primarily in the liver, kidney, small intestine and pancreas. Regulation of homocysteine metabolism is achieved by changes in the quantity of homocysteine distributed between the two competing pathways. Two mechanisms are basic to the regulatory process. Changes in tissue content of the relevant enzymes are the response to sustained perturbations. The inherent kinetic properties of the enzymes provide an immediate response to alterations in the tissue concentrations of substrates and other metabolic effectors. S-adenosylmethionine, S-adenosylhomocysteine, and methyltetrahydrofolate are of particular importance in that context.

Two pathways, the methionine cycle and transsulfuration, account for virtually all methionine metabolism in mammals. Every tissue possesses the methionine cycle. Therefore, each can synthesize AdoMet, employ it for transmethylation, hydrolyze AdoHcy, and remethylate homocysteine. Transsulfuration, which occurs only in liver, kidney, small intestine and pancreas, is the means for catabolizing homocysteine. Liver has a unique isoenzyme of MAT that allows the utilization of excess methionine for the continued synthesis of AdoMet. Metabolic regulation is based on the distribution of available homocysteine between remethylation and conversion to cystathionine. The tissue content of the enzymes and their inherent kinetic properties provide the basis for the regulatory mechanism. The effector properties of the metabolites AdoMet, AdoHcy and methylTHF are of particular relevance.

The maternal vitamin status, especially of folate, is involved in the pathogenesis of neural-tube defects (NTDs). Maternal folate administration can prevent these malformations. The precise metabolic mechanism of the beneficial effect of folate is unclear. In this study we focus on homocysteine accumulation, which may derive from abnormalities of metabolism of folate, vitamin B12, and vitamin B6. We studied nonpregnant women, 41 of whom had given birth to infants with NTDs and 50 control women who previously had normal offspring. The determinations included the plasma total homocysteine both in the fasting state and 6 hours after the ingestion of a methionine load. In addition, we measured the fasting blood levels of folate, vitamin B12, and vitamin B6. The mean values for both basal homocysteine and homocysteine following a methionine load were significantly increased in the group of women who previously had infants with NTDs. In nine of these subjects and two controls, the values after methionine ingestion exceeded the mean control by more than 2 standard deviations. Cystathionine synthase levels in skin fibroblasts derived from these methionine-intolerant women were within the normal range. Our findings suggest a disorder in the remethylation of homocysteine to methionine due to an acquired (ie, nutritional) or inherited derangement of folate or vitamin B12 metabolism. Increased homocysteine levels can be normalized by administration of vitamin B6 or folate. Therefore, we suggest that the prevention of NTDs by periconceptional folate administration may effectively correct a mild to moderate hyperhomocysteinemia.

A clinically benign form of persistent hypermethioninaemia with probable dominant inheritance was demonstrated in three generations of one family. Plasma methionine concentrations were between 87 and 475 mumol/L (normal mean 26 mumol/L; range 10-40 mumol/L); urinary methionine and homocystine concentrations were normal. Plasma homocystine, cystathionine, cystine and tyrosine were virtually normal. The concentrations in serum and urine of metabolites formed by the methionine transamination pathway were normal or moderately elevated. Methionine loading of two affected family members revealed a diminished ability to catabolize methionine, but the activities of methionine adenosyltransferase and cystathionine beta-synthase were not decreased in fibroblasts from four affected family members. Fibroblast methylenetetrahydrofolate reductase activity and its inhibition by S-adenosylmethionine were also normal, indicating normal regulation of N5-methyltetrahydrofolate-dependent homocysteine remethylation. Serum folate concentrations were not increased. The findings in this family differ from those previously described for known defects of methionine degradation. Since the hepatic and fibroblast isoenzymes of methionine adenosyltransferase differ in their genetic control, this family's biochemical findings appear consistent with a mutation in the structural gene for the hepatic methionine adenosyltransferase isoenzyme.

Cystine supplementation of adequate diets resulted in significantly higher hepatic levels of betaine-homocysteine methyltransferase. Other changes occurred but were a function of the basal diet. When the latter contained 0.25% methionine + 0.5% cystine, the additional cystine caused a markedly lower hepatic cystathionine synthase activity and lower levels of both adenosylmethionine and serine. The metabolic effect of these changes may be enhanced methionine retention and diminished transsulfuration.

We have evaluated factors which regulate the content of methionine, adenosylmethionine, adenosylhomocysteine, cystine, cysteine and acid-soluble thiols in rat tissues. In liver the concentration of methionine appears relatively insensitive to changes in dietary protein intake. In contrast the hepatic levels of adenosylmethionine, adenosylhomocysteine, cystine, cysteine and soluble thiols increased with augmented dietary protein. The ratio of adenosylmethionine: adenosylhomocysteine approximated 6.0 in livers, brains, kidneys and skeletal muscles from rats fed the stock diet. Independent variation in the concentrations of these two metabolites did occur. However, the ratios in livers of animals maintained on diets with varying casein content equaled or exceeded a value of 5.0. We conclude that the maintenance of the concentration of methionine is the primary result of the various homeostatic mechanisms. In addition, most previous reports have overestimated the tissue content of adenosylhomocysteine.

A randomised, double-blind, controlled trial has been undertaken to compare the efficacy of hepatitis B immune globulin (H.B.I.G.) with that of immune serum globulin (I.S.G.) for the prophylaxis of viral hepatitis. Participants in the trial were individuals exposed accidentally to material infectious for hepatitis (primarily viral B hepatitis). Preliminary evaluation of the first 302 of the 561 individuals entered into the study indicates that H.B.I.G. significantly reduced the frequencies of both clinical and subclinical hepatitis during the first 3--4 months after the injection. Less than 10% of H.B.I.G. recipients had detectable anti-HBs at the sixth month after the injection, suggesting that H.B.I.G. might need to be given every 3--4 months to continually exposed individuals. Further long-term evaluation is required in order to define more clearly those most likely to benefit from H.B.I.G.

Cystine can replace approximately 70% of the dietary requirement for methionine. We used standard enzyme assays, determinations of the hepatic concentrations of metabolites and an in vitro system which simulates the regulatory site formed by the enzymes which utilize homocysteine in this study of the mechanism for this adaptation. A significant alteration in the pattern of hepatic homocysteine metabolism occurs following the substitution of cystine for methionine. The major change is a marked reduction in the synthesis of cystathionine. Decreases in both the level of cystathionine synthase and in the concentration of adenosyl-methionine, a positive effector of the enzyme, explain this finding. Despite significant increases in the hepatic levels of betaine-homocysteine methyltransferase and methyltetrahydrofolate-homocysteine methyltransferase, flow through these reactions remains relatively constant. The betaine enzyme may be essential for efficient methionine conservation. In the absence of choline, cystine cannot replace methionine in an adequate diet limited in the latter amino acid.

The level of betaine-homocysteine methyltransferase increases in the livers of rats fed diets supplemented with betaine or choline. The increase occurs within 3 days following the change in diet. When we administered betaine by intraperitoneal injection to rats fed choline-free diets, we observed a similar increase within 24 hours. Since betaine-homocysteine methyltransferase catalyzes a reaction which is essential for the catabolism of betaine, these changes provide a means for adaptation to excessive levels of dietary choline and betaine.

Hepatitis B immune globulin (HBIG) and immune serum globulin (ISG) were examined in a randomized, double-blind trial to assess their relative efficacies in preventing type B hepatitis after needle-stick exposure to hepatitis B surface antigen (HBsAG)-positive donors. Clinical hepatitis developed in 1.4% of HBIG and in 5.9% of ISG recipients (P = 0.016), and seroconversion (anti-HBs) occurred in 5.6% and 20.7% of them respectively (P less than 0.001). Mild and transient side-effects were noted in 3.0% of ISG and in 3.2% of HBIG recipients. Available donor sera were examined for DNA polymerase (DNAP) and e antigen and antibody (HBeAg; anti-HBE). Both DNAP and HBeAg showed a highly statistically significant correlation with the infectivity of HBsAg-positive donors. Hepatitis B immune globulin remained significantly superior to ISG in preventing type B hepatitis even when the analysis was confined to these two high-risk subgroups. The efficacy of ISG in preventing type B hepatitis cannot be ascertained because a true placebo group was not included.

To test the response to increased utilization of methyl groups, we administered large dosages of nicotinamide to rats fed an adequate diet that contained limited amounts of methionine and choline. During the 4 d after the injection, we observed several significant effects on the hepatic concentrations of the enzymes and metabolites of methionine metabolism. Methionine and S-adenosylmethionine remained at control levels; the concentrations of S-adenosylhomocysteine exceeded the control values from 4 to 16 h; and the levels of serine and betaine were lower after 16 h. Treatment with nicotinamide resulted in higher hepatic levels of methionine adenosyltransferase (after 4 h) and cystathionine synthase (after 16 h). These data indicate that increases in both homocysteine methylation and S-adenosylmethionine synthesis may be components of the response to excessive methyl group consumption. An increased synthesis of cystathionine would provide for the removal of S-adenosylhomocysteine (and homocysteine) derived from the adenosylmethionine-dependent methylation of nicotinamide.

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.

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.

Preincubation of betaine-homocysteine methyltransferase, prepared from rat liver, with either S-adenosylmethionine or S-adenosylethionine results in a marked loss of enzyme activity. Gel filtration did not restore activity. However both S-adenosylhomocysteine and L-homocysteine, when added to the preincubation medium, inhibited the inactivation of betaine-homocysteine methyltransferase.

A double blind, randomized, controlled trial has been conducted in 11 Veterans Administration hospitals during a 49-month period to compare the relative efficacies of immune serum globulin (ISG) and an albumin placebo for the prevention of post-transfusion hepatitis (PTH). A total of 2204 patients, of whom 1094 received ISG, participated in the study. The results indicate that ISG significantly reduced the incidence of icteric type non-B hepatitis only (inferred to be also type non-A hepatitis). Adverse reactions were rare, and the ISG did not significantly alter the incubation period or duration of the disease. The data suggest, however, that a similar reduction in type non-A, non-B hepatitis would have occurred had commercial blood been excluded from use. Analysis of the 241 patients who developed hepatitis indicates that type B hepatitis constituted less than 20% of the cases each year of the study. Furthermore, the efficacy of the ISG, manufactured in 1944, against apparent type non-A, non-B hepatitis suggests that this overlooked disease has existed from at least that time. Host- and transfusion-related factors that might have modified the development of PTH were examined. The use of commercial blood was observed to be the most important risk factor. It is concluded that the PTH incidence can be most effectively reduced by eliminating commercial donor blood, and continuing to screen volunteer donors for hepatitis B surface antigen (HBsAg) by sensitive procedures. Of prime importance is the need to define the agent(s) responsible for type non-A, non-B hepatitis.

We conducted a systematic evaluation of the effects of increasing levels of dietary methionine on the metabolites and enzymes of methionine metabolism in rat liver. Significant decreases in hepatic concentrations of betaine and serine occurred when the dietary methionine was raised from 0.3 to 1.0%. We observed increased concentrations of S-adenosylhomocysteine in livers of rats fed 1.5% methionine and of S-adenosylmethionine and methionine only when the diet contained 3.0% methionine. Methionine supplementation resulted in decreased hepatic levels of methyltetrahydrofolate-homocysteine methyltransferase and increased levels of methionine adenosyltransferase, betaine-homocysteine methyltransferase, and cystathionine synthase. We used these data to simulate the regulatory locus formed by the enzymes which metabolize homocysteine in livers of rats fed 0.3% methionine, 1.5% methionine, and 3.0% methionine. In comparison to the model for the 0.3% methionine diet group, the model for the 3.0% methionine animals demonstrates a 12-fold increase in the synthesis of cystathionine, a 150% increase in flow through the betaine reaction, and a 550% increase in total metabolism of homocysteine. The concentrations of substrates and other metabolites are significant determinants of this apparent adaptation.

A 31-year-old man with hepatic methionine adenosyltransferase (MAT) deficiency was evaluated for an odd odor to his breath. He had no other symptoms. Plasma methionine was 716 microM (normal, 15-40 microM), and plasma methionine-oxidation products were 460 microM (normal, 0). Hepatic MAT activity was 28% of normal. Unlike the control human enzyme, the patient's residual MAT activity was not stimulated by 10% dimethylsulfoxide and the velocity was not increased by high substrate concentration; at 1.0 mM methionine, the patient's MAT activity was only 7% of normal. These biochemical findings are consistent with a deficiency of the high-Km isoenzyme of MAT. Despite this enzyme deficiency, liver histology and clinical tests of hepatic and other organ function were normal. The patient, who is 25 years older than the oldest reported individual with MAT deficiency, provides evidence that partial MAT deficiency is a benign disorder and that chronic hypermethioninemia (less than 1 mM) is not by itself detrimental to health.

Acetylation is the major route of metabolism of many drugs including the antiarrhythmic agent procainamide. Coadministration of para-aminobenzoic acid was observed to decrease the biotransformation of procainamide to N-acetylprocainamide in a patient with rapid acetylation kinetics. In view of the distinct antiarrhythmic and toxic properties of procainamide and N-acetylprocainamide, the observed drug interference may have great clinical relevance in long-term oral antiarrhythmic therapy and in instances where other drugs converge for acetylation.

Using an in vitro system which contained enzymes, substrates, and other reactants at concentrations which approximated the in vivo conditions in rat liver, we measured the simultaneous product formation by three enzymes which utilize homocysteine. In the control system, 5-methyltetrahydrofolate homocysteine methyltransferase, betaine homocysteine methyltransferase, and cystathionine beta-synthase accounted for 27, 27, and 46%, respectively, of the homocysteine consumed. Subsequent studies demonstrated that the adaptation from a high protein diet to a low protein diet is achieved by a significant increase in betaine homocysteine methyltransferase, and 83% reduction in cystathionine synthase, and a total decrease of 55% in the consumption of homocysteine. S-Adenosylmethionine, by activating cystathionine synthase, contributes significantly to the regulation of the pathway.

A deficiency, or absence, of cystathionine synthetase activity has been demonstrated in liver obtained from a mentally retarded child with homocystinuria.

Vitamin D(3) placed directly into loops of rat duodenum in vitamin D deficient animals increases markedly the subsequent transport of calcium by slices of the duodenal loop in vitro. Under similar conditions the same dose of vitamin given intravenously or placed in a jejunal loop has little or no effect on the duodenal tissue. Thus the vitamin acts directly on the small intestine without prior activation in another organ.