Growth depression and tissue reaction to the consumption of excess dietary methionine and S-methyl-L-cysteine

Similar depressions in growth were observed when rats consumed a 10% casein basal diet containing equal quantities of either methionine or S-methyl-L-cysteine. Supplemental glycine or serine partially alleviated the growth depression caused by the high levels of methionine but were ineffective in alleviating the growth depression caused by high levels of S-methylcysteine. Histological examination of five organs of rats fed the basal, high methionine or high S-methylcysteine diet for 6, 13 or 20 days revealed that only the spleens were affected in that there was erythrocyte engorgement and an accumulation of hemosiderin. The intensity of iron staining in spleens decreased from the second to the third week. The similarity in the depression of growth and splenic damage observed in rats consuming high levels of methionine or S-methylcysteine is consistent with an earlier suggestion that metabolism of the methionine or S-methylcysteine is consistent with an earlier suggestion that metabolism of the methyl group is in some way involved in the toxicity of methionine.     

Dietary betaine promotes generation of hepatic S-adenosylmethionine and protects the liver from ethanol-induced fatty infiltration

Previous studies have shown that ethanol feeding to rats alters methionine metabolism by decreasing the activity of methionine synthetase. This is the enzyme that converts homocysteine in the presence of vitamin B12 and N5-methyltetrahydrofolate to methionine. The action of the ethanol results in an increase in the hepatic level of the substrate N5-methyltetrahydrofolate but as an adaptive mechanism, betaine homocysteine methyltransferase, is induced in order to maintain hepatic S-adenosylmethionine at normal levels. Continued ethanol feeding, beyond 2 months, however, produces depressed levels of hepatic S-adenosylmethionine. Because betaine homocysteine methyltransferase is induced in the livers of ethanol-fed rats, this study was conducted to determine what effect the feeding of betaine, a substrate of betaine homocysteine methyltransferase, has on methionine metabolism in control and ethanol-fed animals. Control and ethanol-fed rats were given both betaine-lacking and betaine-containing liquid diets for 4 weeks, and parameters of methionine metabolism were measured. These measurements demonstrated that betaine administration doubled the hepatic levels of S-adenosylmethionine in control animals and increased by 4-fold the levels of hepatic S-adenosylmethionine in the ethanol-fed rats. The ethanol-induced infiltration of triglycerides in the liver was also reduced by the feeding of betaine to the ethanol-fed animals. These results indicate that betaine administration has the capacity to elevate hepatic S-adenosylmethionine and to prevent the ethanol-induced fatty liver.     

Importance of sarcosine formation in methionine methyl carbon oxidation in the rat

Experiments in vitro using rat liver slices indicated that the incorporation of the methionine methyl carbon into sarcosine and serine was dependent upon available glycine and most probably involves glycine methyltransferase. Although the sarcosine methyl carbon was rapidly oxidized to CO2, its formation accounted for only a small proportion of the oxidation of the methionine methyl carbon to CO2 under these conditions. In vivo experiments using a sarcosine trapping pool with 0.3% to 3.0% L-[methyl-14C]methionine in the diet indicated that from 5% to 14% of the absorbed methionine methyl carbon was metabolized via sarcosine, and that this accounted for only 10% to 20% of the observed oxidation of the methyl carbon to CO2. The adaptive response of the rat to high levels of dietary methionine, as indicated by greater oxidation of the methyl carbon to CO2, is in part due to increased sarcosine synthesis. The failure of supplemental glycine to stimulate oxidation of the methionine methyl carbon to CO2 in rats receiving 3% methionine plus 10% sarcosine may be due to sufficient glycine being produced from sarcosine metabolism.     

S-adenosylmethionine metabolism and DNA methylation in hydrazine-treated rats

The treatment of rats with hepatotoxic doses of hydrazine (NH2-NH2) induces the rapid formation of 7-methylguanine and O6-methylguanine in liver DNA. The methyl moiety in these reactions might be derived from the cellular S-adenosylmethionine pool because radioactivity administered to these rats as methionine rapidly appears in the DNA as methylated guanine. An increased incorporation of radioactivity into 5-methylcytosine was previously reported followed by subsequent suppression. This increased radiolabeling of 5-methylcytosine coincided with time of maximal DNA guanine methylation. To determine the nature of S-adenosylmethionine metabolism during the period of DNA methylation induced by hydrazine treatment, and to determine if the increased radiolabeling of 5-methylcytosine at this time reflected an actual increase in 5-methylcytosine synthesis, liver DNA synthesis and S-adenosylmethionine levels and turnover were assayed. Liver S-adenosylmethionine concentrations varied slightly between control rats and hydrazinetreated rats during the first five hours after hydrazine administration, and no difference was detectable in the incorporation of administered [3H]methionine into S-adenosylmethionine. Because S-adenosylmethionine specific radioactivity in hydrazine-treated rats was not different from control rats, the previously observed increased radiolabeling of 5-methylcytosine appeared to represent an actual increase in synthesis. This conclusion was supported by finding that incorporation of radioactive thymidine into DNA was also accelerated immediately following hydrazine administration, again followed by a decrease. 5-Methylcytosine sythesis, therefore, appears to follow DNA synthesis during hydrazine toxicity, and formation of 7-methylguanine and O6-methylguanine in liver DNA of hydrazine-treated rats occurs during a short period of increased DNA sythesis and 5-methylcytosine formation very early in hydrazine toxicity.     

Altered metabolism of the methionine methyl group in the leukocytes of patients with schizophrenia

Previous work has indicated that abnormal methylation processes may be associated with schizophrenia. In this study, leukocytes from patients with schizophrenia were incubated with methyl-14C-L-methionine and the evolved 14CO2 measured. With increasing concentration of methionine, the evolved 14CO2 was lower in the patients than in normal control subjects. The incorporation of 14C into protein was the same in both groups, and when carboxyl-14C-L-methionine was used the evolved 14CO2 was the same in both groups, thus excluding the possibility that altered incorporation into protein or oxidation of the methionine molecule as a whole were responsible. The observed differences in methionine-methyl metabolism suggest that an abnormality in transmethylation processes or in oxidation of the methyl group to CO2 is associated with schizophrenia. That this occurs in a peripheral tissue indicates that the abnormality is not restricted to the central nervous system.     

Interaction of liver methionine adenosyltransferase with hydroxyl radical

Liver methionine adenosyltransferase (MAT) plays a critical role in the metabolism of methionine converting this amino acid, in the presence of ATP, into S-adenosylmethionine. Here we report that hydrogen peroxide (H2O2), via generation of hydroxyl radical, inactivates liver MAT by reversibly and covalently oxidizing an enzyme site. In vitro studies using pure liver recombinant enzyme and mutants of MAT, where each of the 10 cysteine residues of the enzyme subunit were individually changed to serine by site-directed mutagenesis, identified cysteine 121 as the site of molecular interaction between H2O2 and liver MAT. Cysteine 121 is specific to the hepatic enzyme and is localized at a "flexible loop" over the active site cleft of MAT. In vivo studies, using wild-type Chinese hamster ovary (CHO) cells and CHO cells stably expressing liver MAT, demonstrate that the inactivation of MAT by H2O2 is specific to the hepatic enzyme, resulting from the modification of the cysteine residue 121, and that this effect is mediated by the generation of the hydroxyl radical. Our results suggest that H2O2-induced MAT inactivation might be the cause of reduced MAT activity and abnormal methionine metabolism observed in patients with alcoholic liver disease.     

Cloning and mapping of a cDNA for methionine synthase reductase, a flavoprotein defective in patients with homocystinuria

Methionine synthase catalyzes the remethylation of homocysteine to methionine via a reaction in which methylcobalamin serves as an intermediate methyl carrier. Over time, the cob(I)alamin cofactor of methionine synthase becomes oxidized to cob(II)alamin rendering the enzyme inactive. Regeneration of functional enzyme requires reductive methylation via a reaction in which S-adenosylmethionine is utilized as a methyl donor. Patients of the cblE complementation group of disorders of folate/cobalamin metabolism who are defective in reductive activation of methionine synthase exhibit megaloblastic anemia, developmental delay, hyperhomocysteinemia, and hypomethioninemia. Using consensus sequences to predicted binding sites for FMN, FAD, and NADPH, we have cloned a cDNA corresponding to the "methionine synthase reductase" reducing system required for maintenance of the methionine synthase in a functional state. The gene MTRR has been localized to chromosome 5p15.2-15.3. A predominant mRNA of 3.6 kb is detected by Northern blot analysis. The deduced protein is a novel member of the FNR family of electron transferases, containing 698 amino acids with a predicted molecular mass of 77,700. It shares 38% identity with human cytochrome P450 reductase and 43% with the C. elegans putative methionine synthase reductase. The authenticity of the cDNA sequence was confirmed by identification of mutations in cblE patients, including a 4-bp frameshift in two affected siblings and a 3-bp deletion in a third patient. The cloning of the cDNA will permit the diagnostic characterization of cblE patients and investigation of the potential role of polymorphisms of this enzyme as a risk factor in hyperhomocysteinemia-linked vascular disease.     

Wünderlich syndrome caused by metastatic gastric sarcoma in the kidney. Report of a case

Two of the most important biochemical hepatic pathways in the liver are those that synthesize methionine and S-adenosylmethionine (SAM) through the methylation of homocysteine. This article reviews some recent findings in this laboratory, which demonstrate that ethanol feeding to rats impairs one of these pathways involving the enzyme methionine synthetase (MS), but by way of compensation increases the activity of the enzyme betaine:homocysteine methyl transferase (BHMT), which catalyzes the second pathway in methionine and SAM biosynthesis. It has been shown that despite the inhibition of MS, the enhanced BHMT pathway utilizes hepatic betaine pools to maintain levels of SAM. Subsequent to the above findings, it has been shown that minimal supplemental dietary betaine at the 0.5% level generates SAM twofold in control animals and fivefold in ethanol-fed rats. Concomitant with the betaine-generated SAM, ethanol-induced hepatic fatty infiltration was ameliorated. In view of the fact that SAM has already been used successfully in the treatment of human maladies, including liver dysfunction, betaine, shown to protect against the early stages of alcoholic liver injury as well as being a SAM generator, may become a promising therapeutic agent and a possible alternative to expensive SAM in the treatment of liver disease and other human maladies.     

Inhibition of DNA methylation by S-adenosylethionine with the production of methyl-deficient DNA in regenerating rat liver

Ethionine, a liver carcinogen, was administered p.o. (300 mg/kg) to rats 17 hr after partial hepatectomy. At 6 hr after administration of the ethionine, hepatic S-adenosylethionine levels were 30- to 40-fold greater than the hepatic level of S-adenosylmethionine. A 10-fold ratio of S-adenosylethionine to S-adenosylmethionine still persited at 24 hr after ethionine administration. When given at 17 hr after partial hepatectomy, ethionine produced a 30% inhibition of DNA synthesis, measured by the incorporation of [methyl-3H]thymidine at 23 to 24 hr after partial hepatectomy (6 to 7 hr after ethionine administration). DNA synthesized during this interval was methyl deficient as judged by the reduced incorporation of radioactivity from L-[methyl-3H]methionine into 5-methylcytosine residues of DNA. In an assay for DNA methylation in vitro using whole nuclei, the methyl-deficient DNA was methylated by S-adenosylmethionine 8 times more than was control DNA; the DNA methylation was competitively inhibited by S-adenosylethionine. These data suggest that S-adenosylethionine, formed in vivo from ethionine, competitively inhibits the methylation of DNA in vivo by S-adenosylmethionine, resulting in the production of methyl-deficient DNA.     

Capsular management and refractive error in pediatric intraocular lenses

The effects of dietary protein types and methionine supplementation on phospholipid metabolism were investigated to clarify the mechanism of the hypocholesterolemic action of soybean protein in rats fed a cholesterol-free diet. The effect of switching from a casein diet to a soybean protein diet was also investigated. Rats were fed casein, soybean protein or soybean protein + methionine diet for 14 d. Compared with casein diet, feeding of soybean protein diet led to significantly higher proportions of linoleic acid and linoleic acid-containing molecular species, especially 16:0-18:2, in plasma and liver microsomal phosphatidylcholine (PC). In addition, significantly lower plasma cholesterol concentration, hepatic S-adenosylmethionine concentration and liver microsomal PC:phosphatidylethanolamine ratio resulted. These alterations caused by the soybean protein diet were significantly suppressed by supplementing methionine to the level of the casein diet (3.4 g/kg diet). The proportion of the sum of certain plasma PC molecular species, which contain 18:1 or 18:2 in the sn-2 position, increased in response to the switch from the casein diet to the soybean protein diet at a rate similar to the decrease in plasma cholesterol concentration; there was a significant correlation between the two variables (r = -0.992, P < 0.001). These results indicate that about 40% of the hypocholesterolemic action of soybean protein is due to the low methionine content of the protein and might be associated with alterations of the plasma phospholipid molecular species profile.     

The structure of the C-terminal domain of methionine synthase: presenting S-adenosylmethionine for reductive methylation of B12

BACKGROUND: In both mammalian and microbial species, B12-dependent methionine synthase catalyzes methyl transfer from methyltetrahydrofolate (CH3-H4folate) to homocysteine. The B12 (cobalamin) cofactor plays an essential role in this reaction, accepting the methyl group from CH3-H4folate to form methylcob(III)alamin and in turn donating the methyl group to homocysteine to generate methionine and cob(I)alamin. Occasionally the highly reactive cob(I)alamin intermediate is oxidized to the catalytically inactive cob(II)alamin form. Reactivation to sustain enzyme activity is achieved by a reductive methylation, requiring S-adenosylmethionine (AdoMet) as the methyl donor and, in Esherichia coli, flavodoxin as an electron donor. The intact system is controlled and organized so that AdoMet, rather than methyltetrahydrofolate, is the methyl donor in the reactivation reaction. AdoMet is not wasted as a methyl donor in the catalytic cycle in which methionine is synthesized from homocysteine. The structures of the AdoMet binding site and the cobalamin-binding domains (previously determined) provide a starting point for understanding the methyl transfer reactions of methionine synthase. RESULTS: We report the crystal structure of the 38 kDa C-terminal fragment of E.coli methionine synthase that comprises the AdoMet-binding site and is essential for reactivation. The structure, which includes residues 901-1227 of methionine synthase, is a C-shaped single domain whose central feature is a bent antiparallel betasheet. Database searches indicate that the observed polypeptide has no close relatives. AdoMet binds near the center of the inner surface of the domain and is held in place by both side chain and backbone interactions. CONCLUSIONS: The conformation of bound AdoMet, and the interactions that determine its binding, differ from those found in other AdoMet-dependent enzymes. The sequence Arg-x-x-x-Gly-Tyr is critical for the binding of AdoMet to methionine synthase. The position of bound AdoMet suggests that large areas of the C-terminal and cobalamin-binding fragments must come in contact in order to transfer the methyl group of AdoMet to cobalamin. The catalytic and activation cycles may be turned off and on by alternating physical separation and approach of the reactants.     

Parasite sulphur amino acid metabolism

This paper reviews current knowledge regarding the metabolism of the sulphur-containing amino acids methionine and cysteine in parasitic protozoa and helminths. Particular emphasis is placed on the unusual aspects of parasite biochemistry which may present targets for rational design of antiparasite drugs. In general, the basic pathways of sulphur amino acid metabolism in most parasites resemble those of their mammalian hosts, since the enzymes involved in (a) the methionine cycle and S-adenosylmethionine metabolism, (b) the trans-sulphuration sequence, (c) the transminative catabolism of methionine, (d) the oxidative catabolism of cysteine and (e) glutathione synthesis have been demonstrated variously in several helminth and protozoan species. Despite these common pathways, there also exist numerous differences between parasite and mammalian metabolism. Some of these differences are relatively subtle. For example, the biochemical properties (and primary amino acid structures) of certain parasite methionine cycle enzymes and S-adenosylmethionine decarboxylases differ from those of the corresponding mammalian enzymes, and nematodes and trichomonads possess a novel, non-mammalian form of the trans-sulphuration enzyme cystathionine beta-synthase. The most profound differences between parasite and mammalian biochemistry relate to a number of unusual enzymes and thiol metabolites found in parasitic protozoa. In certain protozoa the pathway for methionine recycling from 5'-methylthioadenosine differs markedly from the mammalian route, and involves 2 exclusively microbial enzymes. Trypanosomatid protozoa contain the non-mammalian antioxidant thiol compounds ovothiol A and trypanothione, together with unique trypanothione-linked enzymes. Specific anaerobic protozoa possess another exclusively microbial enzyme, methionine gamma-lyase, which catabolises methionine (and homocysteine); the physiological significance of these non-mammalian activities is not fully understood. These unusual features offer opportunities for chemotherapeutic exploitation, and in some cases represent metabolic similarities with bacteria. Additionally, some anaerobic protozoa contain unidentified thiols and this implies the presence of further unusual enzymes/pathways in these organisms. So far, no truly unique targets for chemotherapy have been found in helminth sulphur amino acid metabolism, and to some degree this reflects the relative lack of detailed study in the area.     

S-adenosylmethionine synthesis: molecular mechanisms and clinical implications

Methionine adenosyltransferase (MAT) is an ubiquitous enzyme that catalyzes the synthesis of S-adenosylmethionine from methionine and ATP. In mammals, there are two genes coding for MAT, one expressed exclusively in the liver and a second enzyme present in all tissues. Molecular studies indicate that liver MAT exists in two forms: as a homodimer and as a homotetramer of the same oligomeric subunit. The liver-specific isoenzymes are inhibited in human liver cirrhosis, and this is the cause of the abnormal metabolism of methionine in these subjects.     

Effect of methionine on the metabolism of formate and histidine by rats fed folate/vitamin B-12-methionine-deficient diet

The metabolism of formate and histidine were compared in rats and in perfused livers of rats on diets deficient in vitamin B-12, methionine, and folic acid. Excretion of formate and formiminoglutamic acid, and the oxidation of [2-14C]histidine and [14C]formate to 14CO2 were measured. Liver folate levels decreased to 40% of normal on the vitamin B-12- and methionine-deficient diets but the rate of oxidation of histidine to CO2 in the whole animal decreased to 15% of normal. This indicated a reduction in the metabolic activity of the liver folates in vitamin B-12deficiency. Comparison of formate and histidine catabolism in folic acid deficiency showed that the oxidation of histine was decreased to 5% of normal but formate oxidation was decreased to only 30% of normal. This indicates that 25% of formate oxidation normally proceeds by a non-folate-dependent pathway.     

The regulation of folate and methionine metabolism

1. The isolated perfused rat liver and suspensions of isolated rat hepatocytes fail to form glucose from histidine, in contrast with the liver in vivo. Both rat liver preparations readily metabolize histidine. The main end product is N-formiminoglutamate. In this respect the liver preparations behave like the liver of cobalamin- or folate-deficient mammals. 2. Additions of L-methionine in physiological concentrations (or of ethionine [2-amino-4-(ethylthio)butyric acid]) promotes the degradation of formiminoglutamate, as is already known to be the case in cobalamin of folate deficiency. Added methionine also promotes glucose formation from histidine. 3. Addition of methionine accelerates the oxidation of formate to bicarbonate by hepatocytes. 4. A feature common to cobalamin-deficient liver and the isolated liver preparations is taken to be a low tissue methionine concentration, to be expected in cobalamin deficiency through a decreased synthesis of methionine and caused in liver preparations by a washing out of amino acids during the handling of the tissue. 5. The available evidence is in accordance with the assumption that methionine does not directly increase the catalytic capacity of formyltetrahydrofolate dehydrogenase; rather, that an increased methionine concentration raises the concentration of S-adenosylmethionine, thus leading to the inhibition of methylenetetrahydrofolate reductase activity [Kutzbach & Stokstad (1967) Biochim. Biophys. Acta 139, 217-220; Kutzbach & Stokstad (1971) Methods Enzymol. 18B, 793-798], that this inhibition causes an increase in the concentration of methylenetetrahydrofolate and the C1 tetrahydrofolate derivatives in equilibrium with methylenetetrahydrofolate, including 10-formyltetrahydrofolate; that the increased concentration of the latter accelerates the formyltetrahydrofolate dehydrogenase reaction, because the normal concentration of the substrate is far below the Km value of the enzyme for the substrate. 6. The findings are relevant to the understanding of the regulation of both folate and methionine metabolism. When the methionine concentration is low, C1 units are preserved by the decreased activity of formyltetrahydrofolate dehydrogenase and are utilized for the synthesis of methionine, purines and pyrimidines. On the other hand when the concentration of methionine, and hence adenosylmethionine, is high and there is a surplus of C1 units as a result of excess of dietary supply, formyltetrahydrofolate dehydrogenase disposes of the excess. When ample dietary supply causes an excess of methionine, which has to be disposed of by degradation, the increased activity of formyltetrahydrofolate dehydrogenase decreases the supply of methyltetrahydrofolate. Thus homocysteine, instead of being remethylated, enters the pathway of degradation via cystathionine. 7. The findings throw light on the biochemical abnormalities associated with cobalamin deficiency (megaloblastic anaemia), especially on the 'methylfolate-trap hypothesis'. This is discussed. 8...     

Substrate and cofactor reactivity of a carbon monoxide dehydrogenase-corrinoid enzyme complex: stepwise reduction of iron-sulfur and corrinoid centers, the corrinoid Co2+/1+ redox midpoint potential, and overall synthesis of acetyl-CoA

Cleavage of the acetyl carbon-carbon bond of acetyl-CoA in Methanosarcina barkeri is catalyzed by a high molecular mass multienzyme complex. The complex contains a corrinoid protein and carbon monoxide dehydrogenase and requires tetrahydrosarcinapterin (H4SPt) as methyl group acceptor. Reactions of the enzyme complex with carbon monoxide and with the methyl group donor N5-methyltetrahydrosarcinapterin (CH3-H4SPt) have been analyzed by UV-visible spectroscopy. Reduction of the enzyme complex by CO occurred in two steps. In the first step, difference spectra exhibited peaks of maximal absorbance decrease at 426 nm (major) and 324 nm (minor), characteristic of Fe-S cluster reduction. In the second step, corrinoid reduction to the Co1+ level was indicated by a prominent peak of increased absorbance at 394 nm. Spectrophotometric analyses of the corrinoid redox state were performed on the intact complex at potentials poised by equilibration with gas mixtures containing different [CO2]/[CO] ratios or by variation of the [H+]/[H2] ratio. The corrinoid Co2+/1+ midpoint potential was -426 mV (+/- 4 mV, n = 1.16 electrons, 24 degrees C), independent of pH (pH 6.4-8.0). The results indicated a significant fraction of Co1+ corrinoid at potentials existing in vivo. The reduced corrinoid reacted very rapidly with CH3-H4SPt. Reaction with methyl iodide was slow, and methylation by S-adenosylmethionine was not observed. Tne rate of methyl group transfer from CH3-H4SPt greatly exceeded the rate of CO reduction of enzyme centers. The enzyme complex catalyzed efficient synthesis of acetyl-CoA from coenzyme A, CO, and CH3-H4SPt. During acetyl-CoA synthesis, demethylation of CH3-H4SPt was monitored by the absorbance increase at 312 nm.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of S-adenosyl-L-methionine and dilinoleoylphosphatidylcholine on liver lipid composition and ethanol hepatotoxicity in isolated perfused rat liver

We investigated whether S-adenosyl-L-methionine (SAMe), dilinoleoylphosphatidylcholine (DLPC), or SAMe + DLPC influence liver lipid composition as well as acute ethanol hepatotoxicity in the isolated perfused rat liver (IPRL). SAMe (25 mg/kg intramuscularly three times a day) was administered for five consecutive days, while DLPC was administered intraperitoneally for five days. The liver was then isolated, perfused with taurocholate to stabilize bile secretion, and exposed to 0.5% ethanol for 70 min. SAMe, without changing total phospholipid (PL) content, induced an increase in the phosphatidylcholine/phosphatidylethanolamine (PC/PE) molar ratio in both liver homogenate and microsomes and a significant enrichment of 16:0-20:4 and 18:0-20:4 PC molecular species. DLPC induced a significant enrichment of PL in liver homogenate and microsomes due to a contemporary increase in PC and PE. The PC enrichment specifically involved 16:0-20:4 and 18:0-20:4 PC molecular species besides the HPLC peak containing the administered 18:2-18:2 PC species. DLPC + SAMe increased the concentration of PC in liver homogenate and microsomes due to a specific enrichment of 16:0-22:6, 16:0-20:4, and 18:0-20:4 PC molecular species, and the HPLC peak containing the administered 18:2-18:2 PC species. Ethanol acute exposure in the control IPRLs for 70 min induced a depletion of cholesterol in both liver homogenate and microsomes without significant changes in the composition of PL classes and PC molecular species. SAMe, DLPC, or SAMe + DLPC counteracted the cholesterol depletion induced by ethanol, indicating that phospholipid changes promoted by these treatments all induce a major resistance of liver membranes to the effect of ethanol. Ethanol administration in control IPRLs induced a fivefold increase of AST and LDH release in the perfusate, depletion of glutathione in homogenates and mitochondria, decreased oxygen liver consumption, and inhibition of bile flow. These effects of ethanol were significantly antagonized by SAMe. In contrast, DLPC alone only minimally attenuated enzyme release in the perfusate and the inhibitory effect of ethanol on bile flow, but it failed to influence the depletion of total and mitochondrial glutathione or the depressed oxygen consumption induced by ethanol. DLPC, administered together with SAMe, added nothing to the protective effect of SAMe against ethanol hepatotoxicity and cholestasis. In conclusion, this study demonstrates that both SAMe and DLPC induced marked modifications in the lipid composition of liver membranes with a similar enrichment of polyunsaturated PC molecular species. Only SAMe, however, significantly protected against the hepatotoxic and cholestatic effect of acute ethanol administration, an effect associated with maintained normal glutathione mitochondrial levels and oxygen liver consumption. This indicates that the protective effect of SAMe against ethanol toxicity is linked to multiple mechanisms, the maintenance of glutathione levels probably being one of the most important.     

Feedback loops involving Spo0A and AbrB in in vitro transcription of the genes involved in the initiation of sporulation in Bacillus subtilis

The keto acid 2-oxo-4[methylthio]butanoic acid (OMTB) is an intermediate in the conversion of synthetic feed grade methionine sources to L-methionine in vivo in poultry and other animals. Because methionine sources are utilized by the chick with considerably less than 100% efficiency as sources of L-methionine, it is important to determine what metabolic process may limit the utilization of these sources. Because OMTB is converted to L-methionine by transamination, a study was conducted to determine which amino acids might serve as nitrogen donors in the conversion of OMTB to L-methionine in the chicken. Dialyzed tissue homogenates, mitochondria, and cytosol from liver, kidney, intestine, and skeletal muscle were incubated with OMTB and individual L-amino acids (isoleucine, leucine, valine, glutamic acid, aspartic acid, alanine, glutamine, asparagine, and phenylalanine) and the methionine that accumulated was determined by ion exchange chromatography. Tissues differed in the conversion of OMTB to methionine: kidney was most active, liver and intestinal mucosa were intermediate, and skeletal muscle had lowest activity. All amino acids supported methionine synthesis. Branched-chain amino acids and glutamic acid were the most effective substrates in tissue cytosols except in intestinal mucosa, in which asparagine was also effective. The preferred substrates in mitochondria were glutamate in liver mitochondria, isoleucine and alanine in kidney mitochondria, and branched-chain amino acids and glutamic acid in skeletal muscle mitochondria. All amino acids except alanine supported methionine synthesis from OMTB in mitochondria of intestinal mucosa. We conclude that a wide variety of amino acids can serve as substrates for transamination of OMTB in the chicken, and that the availability of nitrogen donors is unlikely to be a limiting factor in the conversion of OMTB to methionine.     

Nutrition and metabolic studies of methyl esters of dimeric fatty acids in the rat

Methyl esters of dimeric fatty acids were prepared by fractionating a mixture of conjugated linoleic and oleic acids that was heated for 24 hr at 300 C in the absence of air. Rats fed diets containing less than 1% dimers showed no significant difference (P less than 0.05) in the growth rate, feed efficiency, liver:body weight ratio, and lipid:liver weight ratio from those fed normal diets. A lymph cannulation study using 14C labeled dimers showed that ca. 0.4% of the dimers fed were absorbed within 12 hr and were transported as free acids in the lymph. Within a 28 hr period, 2% of the labeled dimers fed by gastric intubation were oxidized to 14CO2, and 1% radioactivity was recovered from the urine. The metabolism of methyl oleate appeared normal for rats prefed diets containing dimers.     

Prophylaxis of influenza

Mechanisms of selenium methylation and toxicity were investigated in the liver of ICR male mice treated with selenocystine. To elucidate the selenium methylation mechanism, animals received a single oral administration of selenocystine (Se-Cys; 5, 10, 20, 30, 40, or 50 mg/kg). In the liver, both accumulation of total selenium and production of trimethylselenonium (TMSe) as the end-product of methylation were increased by the dose of Se-Cys. A negative correlation was found between production of TMSe and level of S-adenosylmethionine (SAM) as methyl donor. The relationship between Se-Cys toxicity and selenium methylation was determined by giving mice repeated oral administration of Se-Cys (10 or 20 mg/kg) for 10 days. The animals exposed only to the high dose showed a significant rise of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities in plasma. Urinary total selenium increased with Se-Cys dose. TMSe content in urine represented 85% of total selenium at the low dose and 25% at the high dose. The potential of Se-methylation and activity of methionine adenosyltransferase, the enzyme responsible for SAM synthesis, and the level of SAM in the liver were determined. The high dose resulted in inactivation of Se-methylation and decrease in SAM level due to the inhibition of methionine adenosyltransferase activity. To learn whether hepatic toxicity is induced by depressing selenium methylation ability, mice were injected intraperitoneally with periodate-oxidized adenosine (100 mumol/kg), a known potent inhibitor of the SAM-dependent methyltransferase, at 30 min before oral treatment of Se-Cys (10, 20, of 50 mg/kg). Liver toxicity induced by selenocystine was enhanced by inhibition of selenium methylation. These results suggest that TMSe was produced by SAM-dependent methyltransferases, which are identical with those involved in the methylation of inorganic selenium compounds such as selenite, in the liver of mice orally administered Se-Cys. Depression of selenium methylation ability resulting from inactivation of methionine adenosyltransferase and Se-methylation via enzymic reaction was also found in mice following repeated oral administration of a toxic dose of Se-Cys. The excess selenides accumulating during the depression of selenium methylation ability may be involved in the liver toxicity caused by Se-Cys.