Regulation of the activity of ornithine decarboxylase and S-adenosylmethionine decarboxylase in mammary gland and liver of lactating rats. Effects of starvation, prolactin and insulin deficiency.

1. Starvation caused a marked decrease in the activity of ornithine decarboxylase in mammary gland, together with a lesser decrease in the activity of S-adenosylmethionine decarboxylase and a marked fall in milk production. Liver ornithine decarboxylase and S-adenosylmethionine decarboxylase activities were unaffected. 2. Refeeding for 2.5 h was without effect on ornithine decarboxylase in mammary gland, but it returned the S-adenosylmethionine decarboxylase activity in mammary gland to control values and elevated both ornithine decarboxylase and S-adenosylmethionine decarboxylase in liver. 3. Refeeding for 5 h returned the activity of ornithine decarboxylase in mammary gland to fed-state values and resulted in further increases in S-adenosylmethionine decarboxylase in mammary gland and liver and in ornithine decarboxylase in liver. 4. Prolactin deficiency in fed rats resulted in decreased milk production and decreased activity of ornithine decarboxylase in mammary gland. The increase in ornithine decarboxylase activity normally seen after refeeding starved rats for 5 h was completely blocked by prolactin deficiency. 5. In fed rats, injection of streptozotocin 2.5 h before death caused a decrease in the activities of ornithine decarboxylase and S-adenosylmethionine decarboxylase in mammary gland, which could be reversed by simultaneous injection of insulin. Insulin deficiency also prevented the increase in S-adenosylmethionine decarboxylase in liver and mammary gland normally observed after refeeding starved rats for 2.5 h.     

Role of pyridoxal phosphate in mammalian polyamine biosynthesis. Lack of requirement for mammalian S-adenosylmethionine decarboxylase activity.

1. Polyamine concentrations were decreased in rats fed on a diet deficient in vitamin B-6. 2. Ornithine decarboxylase activity was decreased by vitamin B-6 deficiency when assayed in tissue extracts without addition of pyridoxal phosphate, but was greater than in control extracts when pyridoxal phosphate was present in saturating amounts. 3. In contrast, the activity of S-adenosylmethionine decarboxylase was not enhanced by pyridoxal phosphate addition even when dialysed extracts were prepared from tissues of young rats suckled by mothers fed on the vitamin B-6-deficient diet. 4. S-Adenosylmethionine decarboxylase activities were increased by administration of methylglyoxal bis(guanylhydrazone) (1,1'-[(methylethanediylidine)dinitrilo]diguanidine) to similar extents in both control and vitamin B-6-deficient animals. 5. The spectrum of highly purified liver S-adenosylmethionine decarboxylase did not indicate the presence of pyridoxal phosphate. After inactivation of the enzyme by reaction with NaB3H4, radioactivity was incorporated into the enzyme, but was not present as a reduced derivative of pyridoxal phosphate. 6. It is concluded that the decreased concentrations of polyamines in rats fed on a diet containing vitamin B-6 may be due to decreased activity or ornithine decarboxylase or may be caused by an unknown mechanism responding to growth retardation produced by the vitamin deficiency. In either case, measurements of S-adenosylmethionine decarboxylase and ornithine decarboxylase activity under optimum conditions in vitro do not correlate with the polyamine concentrations in vivo.     

A new method for the assay of tissue. S-adenosylhomocysteine and S-adenosylmethione. Effect of pyridoxine deficiency on the metabolism of S-adenosylhomocysteine, S-adenosylmethionine and polyamines in rat liver.

The hepatic synthesis and accumulation of S-adenosylhomocysteine, S-adenosylmethionine and polyamines were studied in normal and vitamin B-6-deficient male albino rats. A method involving a single chromatography on a phosphocellulose column was developed for the determination of S-adenosylhomocysteine and S-adenosylmethionine from tissue samples. Feeding the rat with pyridoxine-deficient diet for 3 or 6 weeks resulted in a four- to five-fold increase in the concentration of S-adenosylhomocysteine, whereas that of S-adenosylmethionine was only slighly elevated. The concentration of putrescine was decreased to half, that of spermidine was somewhat decreased and that of spermine remained fairly constant. The activities of L-ornithine decarboxylase, S-adenosyl-L-methionine decarboxylase, L-methionine adenosyltransferase and S-adenosyl-L-homocysteine hydrolase were moderately increased. S-Adenosylmethionine decarboxylase showed no requirement for pyridoxal 5'-phosphate. The major effect of pyridoxine deficiency of S-adenosylmethionine metabolism seems to be a block in the utilization of S-adenosylhomocysteine, resulting in the accumulation of this metabolite to a concentration that may inhibit biological methylation reactions.     

Relationships between polyamine metabolism and RNA synthesis in post-ischemic liver cell repair

In liver cells recovering from reversible ischemia the increase in RNA synthesis by isolated nuclei is preceded by activation of ornithine decarboxylase, leading in turn to an increase in putrescine concentration. Treatment of the animals with 1,3-diaminopropane and putrescine prevents ornithine decarboxylase activation but does not hinder the enhancement of RNA synthesis in post-ischemic liver nuclei; therefore, ornithine decarboxylase activation does not seem to be a necessary prerequisite for the increase in RNA synthesis. Hypophysectomy does not prevent the post-ischemic increases of ornithine decarboxylase and RNA synthesis; but pre-treatment of the animals with cycloheximide—which has a dual effect on the activity of ornithine decarboxylase—abolishes the post-ischemic enhancement of RNA synthesis. In contrast with regenerating liver, changes in ornithine decarboxylase activity and putrescine concentrations in reversible ischemia are not associated to changes in S-adenosylmethionine decarboxylase activity and in spermine and spermidine concentrations that seem to be characteristic of tissues where increases in RNA synthesis are followed by DNA synthesis and cell multiplication.     

Subcellular distribution of S-adenosylmethionine decarboxylase in rat liver. Evidence of decarboxylation of S-adenosylmethionine separate from synthesis of spermidine.

The activity of S-adenosylmethionine decarboxylase in rat liver homogenates is localized chiefly in the crude nuclear fraction, probably associated with membrane fragments, with the remainder in the supernatant fraction. This distribution is not paralleled by the activity of the cytoplasmic enzyme, lactate dehydrogenase. The spermidine-synthesizing activity of whole homogenate is recovered entirely in the supermidine-synthesizing activity of whole homogenate is recovered entirely in the supernatant fraction. Measurement of various kinetic parameters in crude fractions provided not positive evidence for isozymes of S-adenosylmethionine decarboxylase. Some species do not possess a sedimentable fraction of S-adenosylmethionine decarboxylase activity in liver. In those species all activity present in the whole homogenate of liver is released into the supernatant fraction.     

Effect of methylglyoxal bis(guanylhydrazone) on polyamine metabolism in normal and regenerating rat liver and rat thymus

1. Injections of sublethal doses of methylglyoxal bis(guanylhydrazone), a potent inhibitor of putrescine-activated S-adenosylmethionine decarboxylase in vitro, resulted after a few days in an immense increase in the activity of S-adenosylmethionine decarboxylase in normal and regenerating rat liver and in rat thymus. The increase in the activity of S-adenosylmethionine decarboxylase was at least partly due to a marked lengthening of the half-life of the enzyme. 2. In regenerating liver and thymus there was also a moderate stimulation of the activity of ornithine decarboxylase (EC 4.1.1.17) and a marked accumulation of tissue putrescine. 3. Injection of methylglyoxal bis(guanylhydrazone) into the rat also markedly decreased the activity of diamine oxidase (EC 1.4.3.6) in thymus. 4. In no cases where doses of methylglyoxal bis(guanylhydrazone) close to the LD(50) dose for the rat were used was it possible to lower tissue spermidine content to any significant extent. 5. Methylglyoxal bis(guanylhydrazone) seemed to act as a competitive inhibitor for the substrate S-adenosylmethionine and as an uncompetitive inhibitor for the activator putrescine in the decarboxylation of S-adenosylmethionine in vitro. 6. In the diamine oxidase reaction, with putrescine as the substrate, methylglyoxal bis(guanylhydrazone) was a non-competitive inhibitor for putrescine.     

Lipid peroxidation and activity of antioxidant enzymes in diabetic rats

We hypothesized that oxygen free radicals (OFRs) may be involved in pathogenesis of diabetic complications. We therefore investigated the levels of lipid peroxidation by measuring thiobarbituric acid reactive substances (TBARS) and activity of antioxidant enzymes [superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and catalase (CAT)] in tissues and blood of streptozotocin (STZ)-induced diabetic rats. The animals were divided into two groups: control and diabetic. After 10 weeks (wks) of diabetes the animals were sacrificed and liver, heart, pancreas, kidney and blood were collected for measurement of various biochemical parameters. Diabetes was associated with a significant increase in TBARS in pancreas, heart and blood. The activity of CAT increased in liver, heart and blood but decreased in kidney. GSH-Px activity increased in pancreas and kidney while SOD activity increased in liver, heart and pancreas. Our findings suggest that oxidative stress occurs in diabetic state and that oxidative damage to tissues may be a contributory factor in complications associated with diabetes.     

Modifications of major aspects of myocardial ribonucleic acid metabolism as a response to noradrenaline. Action of the hormone on cytoplasmic processing of ribonucleic acid after reserpine treatment.

Treatment of perfused rabbit heart with reserpine causes a decrease of incorporation of labelled precursors into RNA species of subcellular fractions and polyamines. Ornithine decarboxylase, S-adenosylmethionine decarboxylase and cytoplasmic Mn2+-stimulated polyadenylate polymerase activities are not modified. Addition of noradrenaline to reserpine-treated perfused hearts enhances, compared with the control, the incorporation of precursor into RNA in all subcellular fractions other than the nuclear one, restores incorporation of labelled putrescine into polyamines, enhances ornithine decarboxylase and S-adenosylmethionine decarboxylase activities and causes a 12-fold increase in cytoplasmic Mn2+-dependent polyadenylate polymerase activity. After treatment with noradrenaline the increase in radioactivity was found solely in AMP after hydrolysis of microsomal RNA to nucleoside monophosphates.     

Ornithine decarboxylase activity in insulin-deficient states

The activity of ornithine decarboxylase, the rate-controlling enzyme in polyamine biosynthesis, was determined in tissues of normal control rats and rats made diabetic with streptozotocin. In untreated diabetic rats fed ad libitum, ornithine decarboxylase activity was markedly diminished in liver, skeletal muscle, heart and thymus. Ornithine decarboxylase was not diminished in a comparable group of diabetic rats maintained on insulin. Starvation for 48h decreased ornithine decarboxylase activity to very low values in tissues of both normal and diabetic rats. In the normal group, refeeding caused a biphasic increase in liver ornithine decarboxylase; there was a 20-fold increase in activity at 3h followed by a decrease in activity, and a second peak between 9 and 24h. Increases in ornithine decarboxylase in skeletal muscle, heart and thymus were not evident until after 24–48h of refeeding, and only a single increase occurred. The increase in liver ornithine decarboxylase in diabetic rats was greater than in normal rats after 3h of refeeding, but there was no second peak. In peripheral tissues, the increase in ornithine decarboxylase with refeeding was diminished. Skeletal-muscle ornithine decarboxylase is induced more rapidly when meal-fed rats are refed after a period without food. Refeeding these rats after a 48h period without food caused a 5-fold increase in ornithine decarboxylase in skeletal muscle at 3h in control rats but failed to increase activity in diabetic rats. When insulin was administered alone or together with food to the diabetic rats, muscle ornithine decarboxylase increased to activities even higher than in the refed controls. In conclusion, these findings indicate that the regulation of ornithine decarboxylase in many tissues is grossly impaired in diabetes and starvation. They also suggest that polyamine formation in vivo is an integral component of the growth-promoting effect of insulin or some factor dependent on insulin.     

Comparative histopathological characteristics of highly pathogenic avian influenza (HPAI) in chickens and domestic ducks in 2008 Korea

We compared characteristic lesions occurring in chickens and domestic ducks naturally infected with H5N1 HPAI virus in April and May 2008. Infected chickens generally exhibited pale-green, watery diarrhoea, depression, neurological signs and cyanosis of wattles and combs, and infected ducks generally exhibited neurological signs and watery diarrhoea. Gross petechial or ecchymotic haemorrhage affected the heart, proventriculus, liver, muscle, fat, and pancreas in chickens, and muscle in ducks. Necrotic foci were primarily present in the pancreas of both species and in the heart of domestic ducks. Histopathologically, chickens exhibited multifocal encephalomalacia, multifocal lymphohistiocytic myocarditis, multifocal necrotic pancreatitis and haemorrhage of several organs and tissues; ducks exhibited lymphohistiocytic meningoencephalitis with multifocal haemorrhages, multifocal necrotic pancreatitis, and severe necrotic myocarditis with mineralisation. The characteristic histopathologic findings of 2008 HPAI were multifocal encephalomalacia and necrotic pancreatitis accompanied by lymphohistiocytic myocarditis, and haemorrhage in various organs and tissues in chickens, whereas in ducks, they were severe necrotic myocarditis with mineralisation and necrotic pancreatitis, accompanied with lymphohistiocytic meningoencephalitis. The high mortality of domestic ducks may be intimately associated with heart failure resulting from increased H5N1 HPAI viral cardiotropism.     

Regulation of S-adenosyl-L-methionine decarboxylase by 1-aminooxy-3-aminopropane: enzyme kinetics and effects on the enzyme activity in cultured cells

The kinetics of inactivation of adenosylmethionine decarboxylase of rat liver and of baby hamster kidney cells (BHK21/C31) by 1-aminooxy-3-aminopropane was studied. The apparent dissociation constants (Ki) for the hepatic and BHK21/C13 enzymes were 1.5 and 2.0 mM and the times of half-inactivation at infinite concentration of the inhibitor (tau 1/2) were 1.2 and 3.8 min, respectively. Treatment of BHK21/C13 with 0.5 mM 1-aminooxy-3-aminopropane prevented cell growth and depleted the cells of putrescine and spermidine within 1 day. The depletion of spermidine resulted in increased activity of S-adenosylmethionine decarboxylase which was due, at least partly, to the increase in the half-life of the enzyme activity. Because spermine levels were not significantly affected, it appears that spermidine is the principal feedback regulator of S-adenosylmethionine decarboxylase. So, 1-aminooxy-3-aminopropane is a very weak inhibitor of S-adenosylmethionine decarboxylase and the cellular effects can be correlated primarily with its inhibitory effects on ornithine decarboxylase and spermidine synthase. In cell-free systems, however, 1-aminooxy-3-aminopropane is likely to find use in unraveling the reaction mechanism of S-adenosylmethionine decarboxylase.     

S-adenosylmethionine decarboxylase degradation by the 26 S proteasome is accelerated by substrate-mediated transamination

The short-lived enzyme S-adenosylmethionine decarboxylase uses a covalently bound pyruvoyl cofactor to catalyze the formation of decarboxylated S-adenosylmethionine, which then donates an aminopropyl group for polyamine biosynthesis. Here we demonstrate that S-adenosylmethionine decarboxylase is ubiquitinated and degraded by the 26 S proteasome in vivo, a process that is accelerated by inactivation of S-adenosylmethionine decarboxylase by substrate-mediated transamination of its pyruvoyl cofactor. Proteasome inhibition in COS-7 cells prevents the degradation of S-adenosylmethionine decarboxylase antigen; however, even brief inhibition of the 26 S proteasome caused substantial losses of S-adenosylmethionine decarboxylase activity despite accumulation of S-adenosylmethionine decarboxylase antigen. Levels of the enzyme's substrate (S-adenosylmethionine) increased rapidly after 26 S proteasome inhibition, and this increase in substrate level is consistent with the observed loss of activity arising from an increased rate of inactivation by substrate-mediated transamination. Evidence is also presented that this substrate-mediated transamination accelerates normal degradation of S-adenosylmethionine decarboxylase, as the rate of degradation of the enzyme was increased in the presence of AbeAdo (5'-([(Z)-4-amino-2-butenyl]methylamino]-5'-deoxyadenosine) (a substrate analogue that transaminates the enzyme); conversely, when the intracellular substrate level was reduced by methionine deprivation, the rate of degradation of the enzyme was decreased. Ubiquitination of S-adenosylmethionine decarboxylase is demonstrated by isolation of His-tagged AdoMetDC (S-adenosylmethionine decarboxylase) from COS-7 cells co-transfected with hemagglutinin-tagged ubiquitin and showing bands that were immunoreactive to both anti-AdoMetDC antibody and anti-hemagglutinin antibody. This is the first study to demonstrate that AdoMetDC is ubiquitinated and degraded by the 26 S proteasome, and substrate-mediated acceleration of degradation is a unique finding.     

Monoamine and diamine oxidase activities in uremic rats

The activities of monoamine and diamine oxidases in various organs and tissues and the amine levels in plasma and urine were determined in chronically uremic and pair-fed control rats. Plasma amine levels were elevated in uremic animals while the urinary excretion of amines was decreased. In uremic as compared to control animals, monomaine oxidase activity was decreased in kidney and muscle, increased in heart and plasma and not altered in liver and cerebrum. Diamine oxidase activity in uremic rats was decreased in kidney, increased in plasma and unchanged in liver and muscle. These alterations of amine oxidase activities in renal failure may affect the metabolism of many amines and thus contribute to the pathogenesis of the uremic syndrome.     

Regulation of S-adenosylmethionine decarboxylase activity in rat liver and prostate

Two methods were used for the quantitation of S-adenosylmethionine decarboxylase protein. The first involved titrating the active site of the enzyme by reduction of the Schiff base between 3H-decarboxylated S-adenosylmethionine and the pyruvate prosthetic group with sodium cyanoborohydride. The second method was radioimmunoassay with rabbit antiserum which was used to determine the total immunoreactive enzyme protein. It was found that the increased S-adenosylmethionine decarboxylase activity produced in rat prostate by treatment with alpha-difluoromethylornithine and in both prostate and liver by methylglyoxal bis(guanylhydrazone) were due entirely to increases in the amount of enzyme protein. The ratio of enzyme activity to protein (measured by either method) remained constant in rats treated with the drugs. Treatment with 2% alpha-difluoromethylornithine in the drinking water for 3 days increased prostatic S-adenosylmethionine decarboxylase protein by 5-fold. A substantial part, but not all, of this increase could be accounted for by a slowing of the rate of degradation of the enzyme. The half-life for loss of activity and titratable protein after inhibition of protein synthesis by cycloheximide was increased from 35 to 108 min by treatment with alpha-difluoromethylornithine. However, the half-life for loss of immunoreactive protein which was considerably longer was only increased from 139 to 213 min. The molecular weight of the S-adenosylmethionine decarboxylase subunit determined by immunoblotting was 32,000, and no smaller immunoreactive fragments were detected. These results indicate that spermidine depletion produced by alpha-difluoromethylornithine affects the degradation of S-adenosylmethionine decarboxylase at an early step involving the loss of the active site without substantial breakdown of the protein.     

Effect of diabetes on the induction of ornithine decarboxylase by refeeding.

Refeeding of starved rats that had previously been schedule-fed increased ornithine decarboxylase activity 140-fold in liver and six-fold in skeletal muscle within three hours. In diabetic rats, refeeding caused a smaller increase in enzyme activity in liver and none at all in muscle. When insulin was administered together with food to the diabetic rats, ornithine decarboxylase in muscle increased to levels greater than those observed in refed controls. The activity of the enzyme in liver also increased; however, the increase was still less than that observed in refed control rats. The data indicate that the induction of ornithine decarboxylase in liver and muscle following food ingestion is altered in diabetes. In addition, they suggest that insulin, or a factor dependent on insulin, modulates the activity of ornithine decarboxylase in skeletal muscle.     

Effects of potassium deficiency on potassium,polyamines and amino acids in mouse tissues

Sexual dimorphism in potassium content was found in plasma, kidney, heart and skeletal muscle of CD1 mice. We observed that feeding mice with a K(+)-deficient diet had an uneven and gender-dependent effect on organ weight and tissue potassium concentrations. Treatment produced a marked decrease in plasma, pancreas and skeletal muscle K(+) levels in both sexes, and a reduction in kidney, liver and heart potassium concentrations in females. Moreover, K(+) deficiency produced a 2-3-fold increase in the concentrations of cationic amino acids, such as arginine and lysine in both heart and skeletal muscle of the two sexes, a slight increase ( approximately 37%) in renal arginine in the male mice. The concentrations of these amino acids in plasma and other tissues in both sexes remained unaltered. Polyamine levels in heart, liver, skeletal muscle and pancreas from male and female mice were not affected by K(+) deficiency. However, in the male kidney potassium deficiency was accompanied by an increase of putrescine and spermidine concentration, and a reduction of putrescine excretion into the urine, even though renal K(+) concentration was not significantly affected and ornithine decarboxylase activity was dramatically decreased. The general lack of correlation between tissue potassium decrease and the increase in organic cations suggests that it is unlikely that the changes observed could be related with an attempt of the tissues to compensate for the reduction in cellular positive charge produced by the fall in K(+) content. The mechanisms by which these changes are produced are discussed, but their physiological implications remain to be determined.     

Abnormal ornithine decarboxylase activity in transgenic mice increases tumor formation and infertility

A transgenic mouse line carrying ornithine decarboxylase cDNA as the transgene under the control of a mouse mammary tumor virus long terminal repeat (MMTV LTR) promoter was generated in order to study whether ornithine decarboxylase transgene expression will have any physiological or pathological effect during the entire life of a transgenic mouse. The high frequency of infertile animals and the loss of pups made the breeding of homozygous mice unsuccessful. However, a colony of heterozygous transgenic mice was followed for 2 years. In adult heterozygous transgenic mice, ornithine decarboxylase activity was significantly increased in the testis, seminal vesicle and preputial gland when compared to non-transgenic controls. In contrast, ornithine decarboxylase activity was decreased in the kidney and prostate of transgenic mice. No significant changes in ornithine decarboxylase activity were found in the ovary and mammary gland and only moderate changes in ornithine decarboxylase activity were detected in the heart, brain, pancreas and lung. The most common abnormalities found in adult animals (12 males and 20 females) of the transgenic line were inflammatory processes, including pancreatitis, hepatitis, sialoadenitis and pyelonephritis. Spontaneous tumors were observed in eight animals, including two benign tumors (one dermatofibroma, one liver hemangioma) and six malignant tumors (one lymphoma, one intestinal and three mammary adenocarcinomas and one adenocarcinoma in the lung). No significant pathological changes were found in 17 nontransgenic controls.     

Regulation of polyamine biosynthesis in tobacco. Effects of inhibitors and exogenous polyamines on arginine decarboxylase, ornithine decarboxylase, and S-adenosylmethionine decarboxylase

Treatment of tobacco liquid suspension cultures with methylglyoxal bis(guanylhydrazone) (MGBG) an inhibitor of S-adenosylmethionine decarboxylase, resulted in a dramatic overproduction of a 35-kDa peptide on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Malmberg, R.L., and McIndoo, J. (1983) Nature 305, 623-625). MGBG treatment also resulted in a 20-fold increase in the activity of S-adenosylmethionine decarboxylase. Purification of S-adenosylmethionine decarboxylase from MGBG-treated cultures revealed that the overproduced 35-kDa peptide and S-adenosylmethionine decarboxylase are identical. Precursor incorporation experiments using [3H] methionine and [35S]methionine revealed that MGBG does not induce any increased synthesis of S-adenosylmethionine decarboxylase but rather stabilizes the protein to proteolytic degradation. The half-life of the enzyme activity was increased when MGBG was present in the growth medium. In addition to stabilizing S-adenosylmethionine decarboxylase, MGBG also resulted in the rapid and specific loss of arginine decarboxylase activity with little effect ornithine decarboxylase. The kinetics of this effect suggest that arginine decarboxylase synthesis was rapidly inhibited by MGBG. Exogenously added polyamines had little effect on ornithine decarboxylase, whereas S-adenosylmethionine and arginine decarboxylase activities rapidly diminished with added spermidine or spermine. Finally, inhibition of ornithine decarboxylase was lethal to the cultures, whereas inhibition of arginine decarboxylase was only lethal during initiation of growth in suspension culture.     

Formation of CO2 from the carboxyl group of S-adenosylmethionine by liver membrane-associated enzymes involves the demethylation-transsulphuration pathway

The activity released from membrane fragments into the supernatant fraction of rat liver homogenate by Triton X-100 and forming ¹⁴CO₂ from carboxyl-labeled S-adenosylmethionine (1) is not a true S-adenosylmethionine decarboxylase. It did not produce decarboxylated S-adenosylmethionine but was also able to use S-adenosylhomocysteine as a substrate. The formation of CO₂ from these two substrates was absolutely dependent on the presence of cytosol proteins and low-molecular weight compounds and it accounted for 5 to 10% of the total S-adenosylmethionine degrading activity of the supernatant fraction. The reaction showed abn initial lag period and was inhibited by every intermediate of the transsulphuration pathway. It is concluded that the formation of CO₂ from S-adenosylmethionine involves the demethylation-transsulphuration route from S-adenosylmethionine to α-ketobutyric acid which is finally decarboxylated.