Interaction between adenosine generated endogenously in neocortical tissues,and homocysteine and its thiolactone

[¹⁴C]Adenine derivatives in normal guinea pig or rat neocortical tissues maintained by superfusion included ATP, ADP and AMP collectively forming some 98% of the acid-extracted ¹⁴C; adenosine, inosine and hypoxanthine each at less than 0.5% and S-adenosylhomocysteine at about 0.1%. l-Homocysteine and/or its thiolactone increased only a little the S-adenosylhomocysteine. The superfusion fluid carried from the tissue per minute about 0.1% of its acid-extractable [¹⁴C]adenine derivatives. Electrical stimulation of the superfused tissue increased 10-fold its output of [¹⁴C]adenine derivatives and diminished the 5′-nucleotides in the tissue to 94% of the acid-extractable [¹⁴C]adenine derivatives, the remainder being adenosine, inosine and hypoxanthine with little change in S-adenosylhomocysteine. Homocysteine in the superfusion fluids now caused large increases in tissue S-adenosylhomocysteine, which became the preponderant non-nucleotide ¹⁴C-derivative when homocysteine was 0.1 mM or greater. The total [¹⁴C]adenine conversion to non-nucleotide derivatives then increased and the 5′-nucleotides fell to 88% of the total. It is concluded that concentration relationships observed in the action of homocysteine make it feasible that convulsive conditions and mental changes associated with administered homocysteine and with homocystinuria are due to cerebral adenosine concentrations being diminished through formation of S-adenosylhomocysteine. Adenosine is preponderantly depressant in cerebral actions; effects of the S-adenosylhomocysteine produced may also be relevant.     

REDISTRIBUTION OF ADENINE DERIVATIVES AMONG SUBCELLULAR FRACTIONS FROM GUINEA PIG NEOCORTICAL TISSUES, ON INCUBATION IN VITRO

Abstract— After the brief in vitro exposure of guinea-pig neocortical tissue to [¹⁴C]adenine, synaptosomal fractions prepared from the incubated tissue contained about 6% of its retained ¹⁴C. On continued incubation and superfusion with or without stimulation, the synaptosomal proportion of the ¹⁴C increased, while the protein and K content of the fraction underwent smaller changes only. Colchicine, 0.5 m m , diminished the synaptosomal enrichment in [¹⁴C]adenine derivatives and also in some cases increased the ¹⁴C effluent from tissues to superfusates. Colchicine also diminished the uptake of adenosine, but not of adenine, to the neocortical tissues. It is concluded that nerve terminal regions receive adenine derivatives from other tissue components as part of their normal metabolism, and that much of this can arrive by extracellular fluids; transport cytoplasmically is not excluded.     

Metabolism of [14C]adenine and derivatives by cerebral tissues, superfused and electrically stimulated

1. Uptake of [(14)C]adenine and [(14)C]adenosine from surrounding fluids to guinea-pig cerebral tissues was measured during incubation in vitro. Output of (14)C-labelled compounds from the loaded tissues to superfusion fluids occurred on continued incubation, at about 0.2% of the tissue's content/min, and this rate was increased about fourfold by electrical excitation of the tissue. 2. The compounds released from the tissue to superfusion fluids included adenine, adenosine, inosine and hypoxanthine with small amounts of nucleotides. Output of all these compounds, except adenine, increased on excitation. Media depleted of oxygen or glucose also increased the output of (14)C-labelled derivatives from [(14)C]adenine-loaded tissues, and this augmented output was further increased by electrical stimulation. 3. [(14)C]Adenosine was found as the main product from [(14)C]ATP when this was added at low concentrations to fluids superfusing cerebral tissue. Metabolic and neurohumoural explanations of the liberation and action of adenosine derivatives in the tissue are discussed.     

The metabolism of adenine derivatives in different parts of the brain of the rat, and their release from hypothalamic preparations on excitation.

Five enzymes concerned with the metabolism of adenine derivatives were assayed in seven regions of the rat brain. A region which included the hypothalamus had the highest AMP deaminase and adenosine deaminase activities, while its 5'-nucleotidase activities were relatively low. The enzymes named and also the uptake of [14C]adenine by incubated tissue samples were more active with hypothalamic than with neocortical tissues. On superfusion with glucose-bicarbonate saline after assimilating [14C]adenine, the hypothalamic tissues released about 0.2 per cent of their 14C content per minute. This release was increased fourfold with electrical excitation but the presence of 0.25 muM tetrodotoxin prevented most of this increase. The compounds released during superfusion and electrical stimulation were preponderantly hypoxanthine, inosine, and adenosine, with only small amounts of adenine nucleotides. The output of all these compounds increased during the period of stimulation and also the proportion of adenine nucleotides increased when stimulation was carried out in the presence of tetrodotoxin. The output of the nucleotides and adenosine increased more promptly when stimulated than did that of the other compounds named. The results are discussed in terms of the metabolic roles of the enzymes concerned. and in relation to whether the enzymes are acting on intracellular or extracellular substrates.     

5'-adenine mononucleotides in preparations from guinea pig cerebral cortex. Accumulation, translocation and release.

Isolated nerve terminals (synaptosome beds) were prepared from the neocortex of guinea pig and their ability to accumulate and release adenine nucleotides was studied. Synaptosome beds prelabelled with [14C]adenosine released newly synthesized [14C] adenine derivatives on superfusion. Electrical stimulation and K+ depolarization gave augmented output of both [14C] adenine derivatives and lactate from the preparations. Action of metabolic inhibitors on this output was examined. During incubation and superfusion, the synaptosomes displayed glycolysis and synthesis of ATP. Supply of adenine derivatives to the nerve terminals also occurred by translocation from other parts of the tissue.     

The metabolism of adenine derivatives in different parts of the brain of the rat,and their release from hypothalamic preparations on excitation

Five enzymes concerned with the metabolism of adenine derivatives were assayed in seven regions of the rat brain. A region which included the hypothalamus had the highest AMP deaminase and adenosine deaminase activities, while its 5'-nucleotidase activities were relatively low. The enzymes named and also the uptake of [¹⁴C]adenine by incubated tissue samples were more active with hypothalamic than with neocortical tissues. On superfusion with glucose-bicarbonate saline after assimilating [¹⁴C]adenine, the hypothalamic tissues released about 0.2% of their ¹⁴C content per minute. This release was increased fourfold with electrical excitation but the presence of 0.25 μUM tetrodotoxin prevented most of this increase. The compounds released during superfusion and electrical stimulation were preponderantly hypoxanthine, inosine, and adenosine, with only small amounts of adenine nucleotides. The output of all these compounds increased during the period of stimulation and also the proportion of adenine nucleotides increased when stimulation was carried out in the presence of tetrodotoxin. The output of the nucleotides and adenosine increased more promptly when stimulated than did that of the other compounds named. The results are discussed in terms of the metabolic roles of the enzymes concerned, and in relation to whether the enzymes are acting on intracellular or extracellular substrates.     

Selection and characterization of a murine lymphoid cell line partially deficient in S-adenosylhomocysteine hydrolase

The exact role of S-adenosylhomocysteine hydrolase (EC 3.3.1.1) in mediating the toxic effects of adenosine toward mammalian cells has not been ascertained. The selection and characterization of S-adenosylhomocysteine hydrolase-deficient cell lines offers a biochemical genetic approach to this problem. In the present experiments, a mutant clone (Sahn 12) with 11-13% of wild-type S-adenosylhomocysteine hydrolase activity was selected from the murine T lymphoma cell line R 1.1 after mutagenesis and culture in adenosine, deoxycoformycin, uridine and homocysteine thiolactone-supplemented medium. In the presence of 0.5 mM homocysteine thiolactone and 10-200 microM adenosine, wild-type and mutant cells synthesized S-adenosylhomocysteine intracellularly at markedly different rates, and excreted the compound extracellularly. Thus, at time points up to 10 h, the S-adenosylhomocysteine hydrolase-deficient lymphoblasts required 5-10-fold higher concentrations of adenosine in the medium to achieve the same intracellular S-adenosylhomocysteine levels as wild-type cells. Similarly, the Sahn 12 lymphoblasts were 5-10-fold more resistant than R 1.1 cells to the toxic effects of adenosine plus homocysteine thiolactone. These results establish that (i) 11-13% of wild-type S-adenosylhomocysteine hydrolase activity is compatible with normal growth, (ii) in medium supplemented with both adenosine and homocysteine thiolactone, intracellular S-adenosylhomocysteine is synthesized by S-adenosylhomocysteine hydrolase, (iii) the net intracellular level of S-adenosylhomocysteine is determined by both the rate of S-adenosylhomocysteine synthesis and its rate of excretion, (iv) under such conditions the accumulation of S-adenosylhomocysteine is related to cytotoxicity, (v) in the absence of an exogenous homocysteine source, S-adenosylhomocysteine derives from endogenous sources, and the accumulation of S-adenosylhomocysteine is not the primary cause of adenosine induced cytotoxicity.     

ACTIONS OF NEUROHUMORAL AGENTS AND CEREBRAL METABOLITES ON OUTPUT OF ADENINE DERIVATIVES FROM SUPERFUSED TISSUES OF THE BRAIN

—Adenine nucleotides of guinea-pig neocortical tissues were labelled by prior incubation with [¹⁴C]adenine and excess of adenine was then removed by superfusion with precursor-free media. During continued superfusion labelled adenine derivatives were released at a stable rate of about 0·05 per cent of the tissue ¹⁴C/min and this rate was increased about five-fold by electrical stimulation. Various compounds, including some known to increase the cyclic AMP content of cerebral tissues, were examined for action on the release of [¹⁴C]adenine derivatives from the tissue and also on the rates of lactate production by the tissue, both before and during electrical excitation. The tissue content of adenine nucleotides following exposure of the tissue to these compounds was also determined. Noradrenaline, γ-aminobutyrate and acetylcholine together with carbamoylcholine at the concentrations examined were without effect on the release of ¹⁴C compounds from the tissue. Also, noradrenaline and γ-aminobutyrate caused no alteration in lactate production but brought about some decrease in the adenylate energy charge of the tissue. Histamine, 100 μm , brought about a small but consistent increase (35 per cent) both in release of ¹⁴C-compounds and lactate output, while reducing the adenylate energy charge of the tissues. l -Glutamate at 5 mm decreased the tissue adenylate energy charge to a greater extent than did histamine; it increased the release of ¹⁴C-compounds seven to eight-fold and similarly increased the tissues' rates of lactate production. Lower concentrations of glutamate had smaller effects. In those cerebral tissues whose cyclic AMP content is increased by l -glutamate, the increase is probably brought about by intermediation of released adenosine.     

Adenine derivatives as neurohumoral agents in the brain. The quantities liberated on excitation of superfused cerebral tissues

Adenine nucleotides of guinea-pig neocortical tissues were labelled by incubation with [(14)C]adenine and excess of adenine was then removed by superfusion with precursor-free medium. Adenine derivatives released from the tissue during continued superfusion, including a period of electrical stimulation of the tissue, were collected by adsorption and examined after elution and concentration. The stimulation greatly increased the (14)C output, and material collected during and just after stimulation had a u.v. spectrum which indicated adenosine to be a major component. The additional presence of inosine and hypoxanthine was shown by chromatography and adenosine was identified also by using adenosine deaminase. Total adenine derivatives released from the tissue during a 10min period of stimulation were obtained as hypoxanthine, after deamination and hydrolysis of adenosine and inosine, and amounted to 159nmol/g of tissue. This corresponded to the release of approx. 7pmol/g of tissue per applied stimulus. The hypoxanthine sample derived from superfusate hypoxanthine, inosine and adenosine was of similar specific radioactivity to the sample of inosine separated chromatographically, and each was of higher specific radioactivity than the adenine nucleotides obtained by cold-acid extraction of the tissue.     

Output of [14C]adenine nucleotides and their derivatives from cerebral tissues. Tetrodotoxine-resistant and calcium ion-requiring components

1. Neocortical tissues, exposed briefly to [(14)C]adenine and containing over 98% of their (14)C as adenine nucleotides, when superfused with glucose-bicarbonate salines released about 0.1% of their (14)C content/min to the superfusate. 2. Addition of unlabelled adenosine to the superfusing fluid increased the (14)C output three- to four-fold; half-maximal increase was given by about 40mum-adenosine, and reasons are adduced for considering the activity of adenosine kinase to be a major factor in conditioning the (14)C output. Adenosine similarly increased the enhanced (14)C output caused by electrical excitation of the superfused tissue; it brought about only a small increase in tissue glycolysis. 3. Output of (14)C from the [(14)C]adenine-labelled tissues was increased when Ca(2+) was omitted from the superfusing fluids, but electrical stimulation did not then liberate more (14)C. Nevertheless, such tissues still responded to electrical stimulation by increased glycolysis, and their (14)C output again became susceptible to increase by electrical stimulation when Ca(2+) was restored. 4. The six-fold increase in tissue glycolysis caused by electrical excitation was almost completely inhibited by tetrodotoxin at 0.1mum and above, but this was associated with about 50% inhibition only in the output of (14)C from tissues preincubated with [(14)C]adenine. The (14)C-labelled compounds of which output was most inhibited by tetrodotoxin were adenosine, inosine and hypoxanthine whereas output in a nucleotide fraction was little affected.     

SUBCELLULAR LOCALIZATION OF [14C]ADENINE DERIVATIVES NEWLY-FORMED IN CEREBRAL TISSUES AND THE EFFECTS OF ELECTRICAL EXCITATION

Abstract— Guinea pig neocortical tissues were incubated with [¹⁴C]adenine, dispersed in cold isotonic sucrose and subcellular fractions prepared by centrifugation. Some 98 per cent of the assimilated ¹⁴C was found as acid-soluble nucleotides in the incubated tissues. In primary fractions obtained by differential centrifugation, about 60 per cent of the [¹⁴C]-nucleotides were in supernatant fractions, in distinction to ATP of which the greatest molar quantity (61 per cent of that in the dispersion) was in the crude mitochondrial fraction. When the crude mitochondrial fraction was separated by density gradient centrifugation, most ¹⁴C was found in synaptosomal fractions and about 85 per cent of this ¹⁴C was adenine nucleotides.
Electrical stimulation of incubating tissues immediately prior to their dispersion and centrifugation greatly diminished the proportion of ¹⁴C subsequently found in nucleotides (collectively) in the supernatant fraction, and increased their inosine and hypoxanthine. Stimulation increased the tissue's cyclic AMP but a preferential localization for this was not established. Results are tentatively interpreted in terms of liberation of an adenine derivative on excitation, and its action or reuptake at a tissue component different from that from which it was liberated. Fractionation of tissues which had been incubated with both [¹⁴C]-adenine and [³H]adenosine suggested that of the two compounds, more adenosine was taken up by synaptic regions in preference to other cellular regions of the tissue.     

Inhibition of equine S-adenohomocysteine hydrolase by 2'-deoxyadenosine

2'-Deoxyadenosine and 9-beta-D-arabinofuranosyladenine (ARA) are apparent suicide inhibitors for equine S-adenosylhomocysteine hydrolase. In initial velocity studies of the synthetic reaction converting adenosine and homocysteine to S-adenosylhomocysteine, adenine, adenosine 5'-triphosphate, and 9-beta-D-arabinofuranosyladenine were found to be competitive inhibitors with Kis of 3.8 microM, 1.1 mM, and 30 microM, respectively. In contrast, linear mixed inhibition was observed for 2'-deoxyadenosine, indicating that 2'-deoxyadenosine must bind in more than one fashion to the enzyme.     

Glutathione changes occurring after S-adenosylhomocysteine hydrolase inhibition

Freshly isolated rat hepatocytes, which metabolize methionine through the cystathionine pathway, and cultured L5178Y cells, which do not, were compared for their response to the inhibition of S-adenosylhomocysteine (SAH) hydrolase (EC 3.3.1.1). When cells were incubated in Fischer's medium lacking cystine but containing 0.67 mM methionine and 10% serum, the addition of periodate-oxidized adenosine (POA), an inhibitor of SAH hydrolase, increased the level of SAH approximately 4-fold in L5178Y cells (5 mM POA) and 30-fold in hepatocytes (1 mM POA). POA treatment also decreased the amount of intracellular glutathione (GSH) in hepatocytes by 6-fold, and in L5178Y cells by 3-fold. Incubation of hepatocytes with adenosine plus homocysteine, 2-chloroadenosine, or 2',3'-acyclic adenosine increased intracellular SAH and also lowered GSH levels. Neither GSH oxidation nor efflux of GSH or GSH conjugates appeared to account for the GSH loss. Intracellular GSH, covalently bound to proteins as mixed disulfides, increased when hepatocytes were incubated with POA, but the increase was insufficient to account for the total GSH loss. In hepatocytes with prelabeled [35S]GSH, POA caused the cellular GSH content to decrease while the specific activity of [35S]GSH remained constant, suggesting that inhibitor treatments that caused elevated SAH levels may have increased the degradation of GSH while GSH synthesis was inhibited.     

Metabolism of adenosine, AMP and ATP in rats

Metabolism of [14C]adenosine in a dose of 100 mg per 1 kg of mass and [14C]ATP in the equimolar quantity was studied in rats after intraperitoneal administration. Adenosine is shown to enter tissues of the liver, spleen, thymus, heart and erythrocytes where it phosphorylates into adenine nucleotides (mainly ATP) and deaminates into inosine. The content of adenosine increases for a short period in the above tissues, except for erythrocytes and plasma. The latter accumulates a considerable amount of inosine and hypoxanthine, but only traces of uric acid, xanthine and adenine nucleotides. ATP administered to rats catabolizes through the adenosine formation. The exogenic adenosine and ATP replace in tissues and erythrocytes only a slight part (1-12%) of their total adenine nucleotide pool. The content of these metabolites and ADP in the blood plasma does not change essentially under the effect of adenosine, ATP and AMP. It is shown on rats whose adenine nucleotide pool of cells is marked by the previous administration of [14C]adenine that injections of adenosine, ATP and inosine do not accelerate catabolism of adenine nucleotides in tissues and erythrocytes as well as do not increase the level of catabolism products in the blood plasma. Adenosine enhances and ATP lowers the content of cAMP in spleen and myocardium, respectively.     

Role of S-adenosylhomocysteine hydrolase in adenosine metabolism in mammalian heart.

S-Adenosylhomocysteine hydrolase of mammalian hearts from different species is exclusively a cytosolic enzyme. The apparent Km for the guinea-pig enzyme was 2.9 microM (synthesis) and 0.39 microM (hydrolysis). Perfusion of isolated guinea-pig hearts for 120 min with L-homocysteine thiolactone (0.23 mM) and adenosine (0.1 mM), in the presence of erythro-9-(2-hydroxynon-3-yl)adenine to inhibit adenosine deaminase, caused tissue contents of S-adenosylhomocysteine to increase from 3.5 to 3600 nmol/g. When endogenous adenosine production was accelerated by perfusion of hearts with hypoxic medium (30% O2), L-homocysteine thiolactone (0.23 mM) increased S-adenosyl-homocysteine 17-fold to 64.3 nmol/g within 15 min. In the presence of 4-nitro-benzylthioinosine (5 microM), an inhibitor of adenosine transport, S-adenosylhomocysteine further increased to 150 nmol/g. L-Homocysteine thiolactone decreased the hypoxia-induced augmentation of adenosine, inosine and hypoxanthine in the tissue and the release of these purines into the coronary system by more than 50%. Our findings indicate that L-homocysteine can profoundly alter adenosine metabolism in the intact heart by conversion of adenosine into S-adenosylhomocysteine. Adenosine formed during hypoxia was most probably generated within the myocardial cell.     

Metabolism of S-adenosylhomocysteine and S-tubercidinylhomocysteine in neuroblastoma cells.

The metabolism of the methylase product inhibitor S-adenosylhomocysteine and its 7-deaza analogue S-tubercidinylhomocysteine has been studied in cultured N-18 neuroblastoma cells. The latter compound, designed to resist metabolic degradation, has been shown to be inert under the same conditions where S-adenosylhomocysteine is rapidly and extensively degraded. The product analyses elucidated by high-performance liquid chromatography indicate that the primary route of S-[8-(14)C]adenosylhomocysteine metabolism in these cells leads to adenosine. This product does not accumulate but is rapidly converted to nucleotides or oxypurines by the action of adenosine kinase and adenosine deaminase, respectively. The presence of the potent adenosine deaminase inhibitor coformycin leads to a pronounced inhibition of oxypurine formation, an increase in nucleotide formation, and a slight accumulation of the primary metabolic products adenosine and adenine.     

Inactivation of liver S-adenosylhomocysteine hydrolase in vitro of rats treated with erythro-9-(2-hydroxynon-3-yl)adenine.

S-Adenosylhomocysteine hydrolase activity decreased in vitro time-dependently in liver homogenates obtained from rats treated in vivo with erythro-9-(2-hydroxynon-3-yl)adenine, a potent inhibitor of adenosine deaminase. The inhibitor in itself had no effect on the stability of the hydrolase. The inactivation of S-adenosylhomocysteine hydrolase was irreversible, proceeded fairly rapidly at a low temperature (0 degrees C) and showed first-order reaction kinetics. Adenosine was found to accumulate in these tissue homogenates during storage. Several lines of evidence suggest that adenosine caused the observed suicide-like inactivation post mortem. Pre-incubation of purified S-adenosylhomocysteine hydrolase at 0 degrees C with adenosine showed a half-maximal inactivation rate at 33 microM substrate concentration; the rate constant of inactivation was 0.01 min-1. Inactivation during tissue preparation and storage complicates the assay of S-adenosylhomocysteine hydrolase activity in samples that contain an inhibitor of adenosine deaminase. These results also suggest that the decrease of S-adenosylhomocysteine hydrolase activity reported to occur in several disturbances of purine metabolism should be re-examined to exclude the possibility of inactivation of the enzyme in vitro.     

Adenosine induces endothelial apoptosis by activating protein tyrosine phosphatase: a possible role of p38alpha

Endothelial cell (EC) apoptosis is important in vascular injury, repair, and angiogenesis. Homocysteine and/or adenosine exposure of ECs causes apoptosis. Elevated homocysteine or adenosine occurs in disease states such as homocysteinuria and tissue necrosis, respectively. We examined the intracellular signaling mechanisms involved in this pathway of EC apoptosis. Inhibition of protein tyrosine phosphatase (PTPase) attenuated homocysteine- and/or adenosine-induced apoptosis and completely blocked apoptosis induced by the inhibition of S-adenosylhomocysteine hydrolase with MDL-28842. Consistent with this finding, the tyrosine kinase inhibitor genistein enhanced apoptosis in adenosine-treated ECs. Adenosine significantly elevated the PTPase activity in the ECs. Mitogen-activated protein kinase activities were examined to identify possible downstream targets for the upregulated PTPase(s). Extracellular signal-regulated kinase (ERK) 1 activity was slightly elevated in adenosine-treated ECs, whereas ERK2, c-Jun NH(2)-terminal kinase-1, or p38beta activities differed little. The mitogen-activated protein kinase-1 inhibitor PD-98059 enhanced DNA fragmentation, suggesting that increased ERK1 activity is a result but not a cause of apoptosis in adenosine-treated ECs. Adenosine-treated ECs had diminished p38alpha activity compared with control cells; this effect was blunted on PTPase inhibition. These results indicate that PTPase(s) plays an integral role in the induction of EC apoptosis upon exposure to homocysteine and/or adenosine, possibly by the attenuation of p38alpha activity.     

Isoprenylcysteine carboxyl methyltransferase activity modulates endothelial cell apoptosis

Extracellular ATP, adenosine (Ado), and adenosine plus homocysteine (Ado/HC) cause apoptosis of cultured pulmonary artery endothelial cells through the enhanced formation of intracellular S-adenosylhomocysteine and disruption of focal adhesion complexes. Because an increased intracellular ratio of S-adenosylhomocysteine/S-adenosylmethionine favors inhibition of methylation, we hypothesized that Ado/HC might act by inhibition of isoprenylcysteine-O-carboxyl methyltransferase (ICMT). We found that N-acetyl-S-geranylgeranyl-L-cysteine (AGGC) and N-acetyl-S-farnesyl-L-cysteine (AFC), which inhibit ICMT by competing with endogenous substrates for methylation, caused apoptosis. Transient overexpression of ICMT inhibited apoptosis caused by Ado/HC, UV light exposure, or tumor necrosis factor-alpha. Because the small GTPase, Ras, is a substrate for ICMT and may modulate apoptosis, we also hypothesized that inhibition of ICMT with Ado/HC or AGGC might cause endothelial apoptosis by altering Ras activation. We found that ICMT inhibition decreased Ras methylation and activity and the activation of the downstream signaling molecules Akt, ERK-1, and ERK-2. Furthermore, overexpression of wild-type or dominant active H-Ras blocked Ado/HC-induced apoptosis. These findings suggest that inhibition of ICMT causes endothelial cell apoptosis by attenuation of Ras GTPase methylation and activation and its downstream antiapoptotic signaling pathway.     

Specific enzymatic assay method for homocysteine and its application to the screening of neonatal homocystinuria

A simple and specific enzymatic assay method for homocysteine is described. The method is based on the formation of S-adenosylhomocysteine from adenosine and homocysteine through the catalysis of S-adenosylhomocysteine hydrolase (EC 3.3.1.1), followed by its separation by high-performance liquid chromatography. The results for human blood, serum, and urine and those extracted from filter paper showed good correlation with those obtained on measurement with a conventional amino acid analyzer, which demonstrates that the method is useful for neonatal screening for homocystinuria.