Methotrexate rescue by 5-methyltetrahydrofolate or 5-formyltetrahydrofolate in lymphoblast cell lines.

The rescue of lymphocytes from methotrexate (MTX) growth inhibition by 5-methyltetrahydrofolate (5-methyl-THF) and 5-formyltetrahydrofolate (5-formyl-THF) has been studied. Rescue by 5-methyl-THF is selective for cells with high levels of homocysteine:5-methyl-THF methyl-transferase (methyltransferase). At MTX concentrations which inhibited growth greater than or equal to 85% in both leukemic T-lymphocytes (CCRF-CEM) and Epstein-Barr-transformed B-lymphocytes (LAZ-007), 5 micro M 5-formyl-THF rescued more effectively than did 5-methyl-THF, in either the presence or absence of the methyltransferase inhibitor, nitrous oxide. At less inhibitory MTX concentrations, both reduced folates rescued equally, except when methyltransferase was inhibited by nitrous oxide in which case 5-formyl-THF was clearly superior. In the absence of nitrous oxide, both cell lines contained approximately equal amounts of methyltransferase. Some apparent differences in the rescue of these cell lines with 5-methyl-THF were attributable to their different sensitivity to MTX. When metabolism of reduced folates was severely impaired by MTX and nitrous oxide, lymphocytes were rescued with 5-[methyl-14C]methyl-THF, and the uptake of 14C into DNA was measured. In corporation was very low, indicating that cellular oxidation of 5-methyl-THF to 5,10-methylene-tetrahydrofolate is minimal even under forcing conditions. MTX selectively in vivo will be influenced by the level of methyltransferase in tumor and normal tissues.

Organophosphate inhibitors. The stereospecificity of hydrolysis of methyl n-butyl p-nitrophenyl phosphate by serum phosphotriesterases (EC 3.1.1.2) and by acetylcholinesterases (EC 3.1.1.7).

A chiral phosphotriester, methyl n-butyl p-nitrophenyl phosphate was used to determine the stereospecificity of hydrolysis catalysed by serum phosphotriesterases (aryl-ester hydrolases, EC 3.1.1.2) from horse, ox and rabbit. Each enzyme hydrolysed the (-)-enantiomer more quickly. The same phosphate was used to inhibit acetycholinesterase (acetycholine hydrolase, EC 3.1.1.7) from ox, rabbit and electric eel, and in each case, the (+)-enantiomer caused more rapid inhibition. Serum phosphotriesterase did not catalyse the dephosphorylation of dialkyphosphoryl-acetylcholinesterase or dialkylphosphoryl-carboxylesterase. Levels of serum phosphotriesterase in rabbits which received sub-lethal injections of the phosphotriester remained unchanged after one or several injections. In the same rabbits, the levels of blood acetylcholinesterase fell sharply following injections, but normal values were regained in 2-8 days. Serum phosphotriesterases seem incapable either of preventing acute phosphotriester poisoning or of regenerating active enzyme from phosphorylated acetylcholinesterase. However, phosphotriesterases would act in cases of chronic exposure by catalysing the hydrolysis of such organophosphate poisons as remain in the blood.

Characterisation of five missense mutations in the cystathionine beta-synthase gene from three patients with B6-nonresponsive homocystinuria.

Homocystinuria, due to a deficiency of the enzyme cystathionine beta-synthase (CBS), is an inborn error of sulphur-amino acid metabolism. This is an autosomal recessive disease which results in hyperhomocysteinaemia and a wide range of clinical features, including optic lens dislocation, mental retardation, skeletal abnormalities and premature thrombotic events. We report the identification of 5 missense mutations in the protein-coding region of the CBS gene from 3 patients with pyridoxine-nonresponsive homocystinuria. Reverse-transcription PCR was used to amplify CBS cDNA from each patient and the coding region was analysed by direct sequencing. The mutations detected included 3 novel (1058C-->T, 992C-->A and 1316G-->A) and 2 previously identified (430G-->A and 833C-->T) base alterations in the CBS cDNA. Each of these mutations predicts a single amino acid substitution in the CBS polypeptide. Appropriate cassettes of patient CBS cDNA, containing each of the above defined mutations, were used to replace the corresponding cassettes of normal CBS cDNA sequence within the bacterial expression vector pT7-7. These recombinant mutant and normal CBS constructs were expressed in Escherichia coli cells and the catalytic activities of the mutant proteins were compared with normal. All of the mutant proteins exhibited decreased catalytic activity in vitro, which confirmed the association between the individual mutation and CBS dysfunction in each patient.

Failure to detect homocysteine in the acid-hydrolysed plasmas of recent myocardial infarct patients.

We have sought the very high levels of homocysteine-containing compounds in the plasma hydrolysates of myocardial infarct patients reported by Olszewski and Szostak (Atherosclerosis, 69 (1988) 109-113). We studied 6 adult males with recent myocardial infarcts and 6 healthy adult males. We found that after hydrolysis of their plasmas for 5 h in 4 mol/l HCl at 110 degrees C, the amino acid chromatographs contained several small peaks in addition to the expected substantial peaks of the protein-constituent amino acids. However, the small peaks which eluted at the same times as homocysteine, homocystine and the mixed disulphide of homocysteine and cysteine, were in each case shown not to represent these compounds. Furthermore no homocysteine thiolactone was found in the chromatographs. We found no significant differences in the size of the small peaks between the patients and the controls. Prolonged hydrolysis resulted in decreased size of all but one of the small peaks suggesting that they were hydrolytic intermediates in the breakdown of plasma proteins. Electrophoresis at pH 10.39 of the unhydrolysed plasmas showed that the proteins were not significantly more homocysteinylated in patients than in controls. Thus we have been unable to substantiate some key observations made by Olszewski and Szostak.

Homocysteine catabolism: levels of 3 enzymes in cultured human vascular endothelium and their relevance to vascular disease.

Elevated plasma homocysteine enhances the risk of thrombosis and premature arteriosclerosis. We have assessed the activity of the 3 prime enzymes of homocysteine metabolism in cultured human venous endothelial cells, in a study of their possible protective roles. In cells from 4 individuals, cultured in Dulbecco's modified Eagle medium, the mean activity +/- S.D. of cystathionine beta-synthase (nmol of product/h per mg of cell protein, at 37 degrees C) was 3.58 +/- 3.11 at pH 8.6. The assay used was our newly developed amino acid analyser-based procedure. The activity of 5-methyltetrahydrofolate:homocysteine methyltransferase at pH 7.4 was 4.12 +/- 1.25 and betaine:homocysteine methyltransferase (BHMT) was undetectable (< 1.4 nmol/h per mg protein). Cells were also cultured in a medium aimed at stimulating methionine biosynthesis, containing methionine-deficient Dulbecco's modified Eagle medium to which L-homocystine (100 mumol/l) and methylcobalamin (1 mumol/l) had been added. In these cells 5-methyltetrahydrofolate:homocysteine methyltransferase activity increased to 7.95 +/- 1.45, P < 0.001, there was a non-significant decrease in cystathionine beta-synthase activity to 2.16 +/- 1.52 and BHMT activity was still undetectable. These cells were more resistant to in vitro homocysteine-induced detachment than were cells from the same line cultured in Dulbecco's modified Eagle medium alone. Our findings establish that human endothelial cells express 2 of the 3 primary enzymes of homocysteine catabolism. They suggest that persons who are deficient in cystathionine beta-synthase or 5-methyltetrahydrofolate:homocysteine methyltransferase activity may not only develop homocysteinemia, but also have vascular endothelium which is more susceptible to damage by homocysteine than persons with normal enzyme levels.

Human arterial endothelial cell detachment in vitro: its promotion by homocysteine and cysteine.

We have explored earlier evidence that premature atherosclerosis in homocystinuria is triggered by homocysteine-induced loss of vascular endothelium. We used a reproducible sluicing assay to test in vitro detachment of human arterial endothelial cells. Cell detachment was induced by exposure of cultured endothelial cells to the sulphydryl-containing amino acids homocysteine and cysteine, whereas methionine, alanine, valine and isoleucine at comparable concentrations were ineffective. This cellular detachment was greatly diminished by growth of the endothelial cells on fibronectin coated- rather than plain tissue culture dishes. Considerably higher concentrations of homocysteine were required for in vitro effects than are associated with atherogenesis in homocystinuria, and despite the cysteine associated changes, cysteine itself is not known to be related to atherogenesis. These data suggested that in vitro detachment of cultured endothelial cells, induced by sulphydryl-containing amino acids, may have marginal relevance to mechanisms of atherogenesis in homocystinuria.

Gender differences and other determinants of the rise in plasma homocysteine after L-methionine loading.

The methionine load test is widely used to identify subjects with mild homocysteinemia. We studied factors influencing the rise in plasma homocysteine (tHcy) levels following oral methionine loading, after taking into account determinants of the basal level. The study population comprised 522 persons (mean age 53, range 17 to 95; 63% male; 60% with coronary disease). tHcy was measured fasting and 4 h after administration of L-methionine (dose 4 g/m2). The percentage rise in tHcy was greater in females, at younger ages, and with lower serum creatinine. Basal tHcy was lower in those with high levels of serum folate, red cell folate and serum B12; they also showed a greater percentage rise with loading. After adjustment for age, creatinine and serum vitamins the percent rise was greater in females (mean 230%, S.D. 92) than in males (mean 172%, S.D. 71, P < 0.0001). About 1/4 of this effect was explained by the methionine dose, but important independent gender effects remained. When the results of the loading test were compared with an overall reference range, females were systematically labelled as 'high' but comparison with age and gender-specific reference ranges normalised the sex distribution. Our data show that a single L-methionine loading protocol for both sexes is not appropriate. Gender-specific reference ranges should be used to define post-load homocysteinemia and previous studies which have identified a high proportion of females with this trait probably reflect the need for gender specific reference ranges.

Early expression of the apolipoprotein (a) gene: relationships between infants' and their parents' serum apolipoprotein (a) levels.

The serum concentration of apo(a), the unique apolipoprotein of lipoprotein (a), reflects serum lipoprotein (a) levels. High concentrations are associated with increased cardiovascular risk. Inasmuch as atherogenesis may begin in childhood, the early expression of the apo(a) gene and relationships between serum levels in infants and their parents were explored. Serum apo(a) and lipid profiles were measured in 51 infants when aged 8.5 +/- 2 months. They were from among 1032 consecutively born babies in whom apo(a) levels had been measured on day 2 to 5. Levels in 18 infants were in the top 5% of the neonatal apo(a) distribution and in 33 from below the 95th percentile. Parental values were also assessed. Infants' apo(a) levels (n = 51) at the ages of 2 to 5 days and 8.5 +/- 2.3 months were highly correlated (r = .73, P less than .0001) and increased from an initial median value of 48 U/L (range 1 to 462 U/L) to 100 U/L (5 to 969 U/L) at 8.5 months, and they were then not different from parental levels. Measurements at both times were closely correlated with parental levels. Regression coefficients between 8.5-month levels, and the levels of fathers, of mothers, and the average level of both parents were 0.439, 0.521 and 0.93, respectively (P less than .0001 for each). It is concluded that the gene for the regulation of apo(a) is fully expressed before the age of 1 year. The apo(a) levels in infants during this time track closely and are predictive of parental values.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of homocysteine and related compounds on prostacyclin production by cultured human vascular endothelial cells.

Elevated plasma homocysteine is associated with an increased risk of intravascular thrombosis. Platelet aggregation and thrombosis are inhibited by prostacyclin produced by the vascular endothelium. Our aim was to investigate whether homocysteine and related metabolites inhibit endothelial prostacyclin production. We used a radioimmunoassay for 6-ketoprostaglandin-F1 alpha to assay medium which had been in contact with confluent cultured endothelial cells. In medium containing 20% human serum, endothelial prostacyclin production was not specifically inhibited by homocysteine, S-adenosylhomocysteine or protein-bound homocysteine. Further, there was no consistent difference in prostacyclin production by cells cultured in medium containing sera from homocystinuria patients, compared with medium containing normal healthy sera. We conclude that vascular disorder in homocystinuria is unlikely to result from effects of homocysteine or related metabolites on endothelial prostacyclin production. By contrast, S-adenosylhomocysteine and protein-bound homocysteine specifically inhibited prostacyclin production by cells cultured in medium containing 20% fetal calf serum.

Serial measures of plasma homocyst(e)ine after acute myocardial infarction.

To determine whether plasma levels of homocyst(e)ine are affected by the acute phase response, we studied 10 subjects serially after acute myocardial infarction. Our data indicate that measurement of homocyst(e)ine in patients with myocardial infarction should ideally be deferred for 7 days if spuriously low levels are to be averted.

Interrelations between plasma free and protein-bound homocysteine and cysteine in homocystinuria.

To study the interrelations between plasma free and protein-bound homocysteine and cysteine, we measured the levels of these four variables in 167 samples from 17 patients with homocystinuria during different treatment regimens, 14 heterozygotes for cystathionine B-synthase deficiency, and 17 normal subjects. There was a strong positive correlation between free and protein-bound homocysteine, and between free and protein-bound cysteine over a wide range of values in varying clinical situations, but homocysteine and cysteine had differing binding characteristics. At low concentrations homocysteine bound more tightly than cysteine to plasma protein, while at high concentrations of the free amino acids more cysteine than homocysteine was bound to plasma protein. In patients with homocystinuria due to cystathionine B-synthase deficiency, levels of protein-bound homocysteine after an overnight fast were eight to 12 times higher than mean levels +/- SD in the normal subjects of 0.15 +/- 0.03 mumol/g of plasma protein, n = 17, and levels of protein-bound cysteine were lower than mean normal levels +/- SD of 2.30 +/- 0.23 mumol/g, n = 17. In the patients before treatment the proportions of both plasma thiols that were protein-bound were approximately half those in the normal subjects. For homocysteine the proportion was 35% in a representative patient and 78% in normal subjects and in heterozygotes, while for cysteine the corresponding proportions were 26% and 59%. In all blood samples the sum of the plasma free and protein-bound homocysteine and cysteine remained relatively constant (mean +/- SD = 270.6 +/- 68.6 mumol/L of plasma, n = 142).(ABSTRACT TRUNCATED AT 250 WORDS)

Homocysteinemia: depressed plasma serine levels.

Plasma serine levels were found to be lower than normal (mean +/- SD, 91 +/- 18 mumol/L, n = 16) in homocystinuria patients with a deficiency of cystathionine B-synthase on folate therapy, compared with healthy adults (121 +/- 25 mumol/L, n = 25, P less than 0.001). Of 13 other patients with elevated plasma total homocysteine, two patients with homocystinuria due to remethylation defects had normal serine levels, while 11 renal transplant recipients with mild elevations of serum creatinine had lower than normal serine levels (100 +/- 28 mumol/L, P less than .05). Treatment of both the pyridoxine responsive and nonresponsive cystathionine B-synthase-deficient patients with betaine, which lowered plasma homocysteine, also normalized plasma serine levels. In the two patients with remethylating defects however, betaine lowered plasma homocysteine levels without changing plasma serine levels. By contrast, treatment of the renal transplant patients with pyridoxine, folic acid, and vitamin B12 (cofactors required for homocysteine metabolism), caused falls in plasma homocysteine levels, with a concurrent decline in plasma serine levels. These findings may be explained in terms of the requirements for serine in homocysteine metabolism, both as a source of methyl carbon atoms in the methylation of homocysteine by N5-methyltetrahydrofolate and as a substrate in the cystathionine B-synthase reaction. During periods of elevated plasma total homocysteine in man, increased amounts of serine may be diverted to lowering plasma homocysteine.

Free and protein-bound homocysteine and cysteine in cystathionine beta-synthase deficiency: interrelations during short- and long-term changes in plasma concentrations.

Homocysteine interacts in a complex way in the plasma with cysteine and plasma proteins. To explore the interrelations between free and protein-bound homocysteine and cysteine during short- and long-term changes in plasma levels, free and bound homocysteine and cysteine were measured in 13 patients with homocystinuria due to cystathionine beta-synthase deficiency. Levels were measured during oral methionine loads (4 g/m2 body surface area) and after oral betaine (3 g twice daily). In six pyridoxine-responsive patients, free and bound levels of homocysteine and cysteine, measured 4 to 24 hours after oral methionine, changed in a parallel manner. Similar close tracking occurred in fasting plasma samples collected from two pyridoxine-nonresponsive patients before and during betaine therapy. Oral betaine given to seven pyridoxine-nonresponsive patients decreased free and bound homocysteine and increased free and bound cysteine toward normal levels during monitoring periods of up to 300 days. In these studies as the level of homocysteine decreased, the proportion of protein-bound homocysteine and cysteine increased. The present study establishes that changes in bound and free levels of either homocysteine or cysteine track closely in the short-term (four hours or less) and generally also in the long-term (up to 300 days).

Homocystinuria due to cystathionine beta-synthase deficiency--the effects of betaine treatment in pyridoxine-responsive patients.

Homocystinuria due to cystathionine beta-synthase deficiency may be responsive to pyridoxine, a precursor of the cofactor pyridoxal phosphate, and the amount of residual enzyme activity present is the probable determinant of this. In six treated pyridoxine-responsive patients whose biochemical control of fasting plasma amino acid levels appeared optimal, we assessed the effects on plasma amino acids of standard oral methionine loads (4g/m2 of body area) before and after adding betaine (trimethylglycine) 6 g/d, to the treatment regimen of pyridoxine and folic acid. Our aim was to define the capacity of these patients to metabolize methionine and to determine whether betaine would effect a reduction in postload homocysteine levels. During the 24 hours after the methionine challenge all patients had higher plasma methionine and homocysteine and lower cysteine than did 17 normal subjects. After betaine these homocysteine responses were reduced to near normal, and there was a trend toward increased methionine. There was a direct correlation between premethionine fasting homocysteine and mean homocysteine responses during the 24 hours following the methionine load, both before (r = 0.79) and after betaine (r = 0.71). Betaine also increased plasma cysteine levels in patients with the more severe biochemical abnormalities. After betaine there were modest increases in plasma serine (mean increase 25%; P less than 0.025). Since the vascular complications of homocystinuria are related to increased plasma homocysteine, betaine therapy may reduce this risk in patients receiving a standard pyridoxine and folic acid regimen in whom there are abnormal homocysteine responses after a standard methionine load.

Folic acid lowers elevated plasma homocysteine in chronic renal insufficiency: possible implications for prevention of vascular disease.

To explore interrelations between folic acid and methionine metabolism in chronic renal insufficiency, we measured plasma amino acids in 21 patients with mean serum creatinine +/- SD of 560 +/- 240 mumol/L, after a ten-hour overnight fast, before and after administration of 5 mg of oral folic acid daily for 15 +/- 6 days. Mean plasma homocysteine was 12.9 +/- 6.8 mumol/L in the patients and 4.2 +/- 0.8 mumol/L in 24 normal controls (P less than .001), and after folic acid administration it declined in the patients to 6.8 +/- 2.8 mumol/L (P less than .0001) in linear proportion (r = .92) to the prefolate homocysteine level. Methionine concentrations were normal in the patients and did not change after folate administration, nor did elevated cysteine and creatinine. Plasma serine was lower (88.3 +/- 17.2 v 121 +/- 25 mumol/L, P less than .41) and declined further to 67.8 +/- 16.4 (P less than .0001) after folate, while prefolate glycine levels increased from 273.3 +/- 61.2 to 313.2 +/- 97.5 mumol/L (P less than .01). Serum and red-cell folate levels were normal in the patients before treatment. The results show that homocysteine levels are increased in chronic renal insufficiency, but may be lowered by folate enhancement of remethylation of homocysteine to methionine. Since elevated plasma homocysteine is associated with premature vascular disease, folic acid may reduce cardiovascular risk in chronic renal insufficiency.

Identification and characterization of a cystathionine beta/gamma-lyase from Lactococcus lactis ssp. cremoris MG1363.

This paper describes the nucleotide sequence of a gene encoding cystathionine beta/gamma-lyase from Lactococcus lactis ssp. cremoris MG1363, its overexpression in Escherichia coli and some functional characteristics of the purified recombinant protein.

Increased plasma copper in patients with homocystinuria due to cystathionine beta-synthase deficiency.

We measured blood copper-containing proteins and plasma total copper in 15 patients with homocystinuria (14 with cystathionine beta-synthase deficiency and one with abnormal cobalamin metabolism), in 13 heterozygotes for cystathionine beta-synthase deficiency, and in 44 normal subjects. Plasma total copper was increased in patients with cystathionine beta-synthase deficiency compared with age- and sex-matched controls; the ratio was 1.41 +/- 0.14 for females and 1.39 +/- 0.15 for males (means +/- SD). This was due to corresponding increases in caeruloplasmin concentrations, but levels were unrelated to total plasma homocysteine. Erythrocyte superoxide dismutase levels were normal. The heterozygotes had normal plasma copper and caeruloplasmin levels. The increased copper and caeruloplasmin may contribute to the precocious atherogenesis occurring in homocystinuria by decreasing the adhesion of vascular endothelial cells to the intima. It is unlikely that decreased lysyl oxidase activity due to chelation of copper by homocysteine is important for the pathogenesis of the connective tissue defect in homocystinuria.

Betaine:homocysteine methyltransferase--a new assay for the liver enzyme and its absence from human skin fibroblasts and peripheral blood lymphocytes.

Chronic elevation of plasma homocysteine is associated with increased atherogenesis and thrombosis, and can be lowered by betaine (N,N,N-trimethylglycine) treatment which is thought to stimulate activity of the enzyme betaine:homocysteine methyltransferase. We have developed a new assay for this enzyme, in which the products of the enzyme-catalysed reaction between betaine and homocysteine are oxidised by performic acid before being separated and quantified by amino acid analysis. This assay confirmed that human liver contains abundant betaine:homocysteine methyltransferase (33.4 nmol/h/mg protein at 37 degrees C, pH 7.4). Chicken and lamb livers also contain the enzyme, with respective activities of 50.4 and 6.2 nmol/h/mg protein. However, phytohaemagglutinin-stimulated human peripheral blood lymphocytes and cultured human skin fibroblasts contained no detectable betaine:homocysteine methyltransferase (less than 1.4 nmol/h/mg protein), even after cells were pre-cultured in media designed to stimulate production of the enzyme. The results emphasize the importance of the liver in mediating the lowering of elevated circulating homocysteine by betaine.

An indirect sandwich ELISA for LP(a) in serum and dried blood spots.

We have established a reproducible and inexpensive indirect sandwich ELISA for Lp(a) quantitation in both serum and dried blood spot samples. All reagents used in the assay are available commercially. The intra-assay CVs were 3.8 +/- 0.9% for serum and 4.5 +/- 1.7% for dried blood spots on filter paper. The inter-assay CVs were 6.3 +/- 2.3% and 4.5 +/- 0.1% for serum and dried blood spot, respectively. Lp(a) concentrations measured by the ELISA and a commercial RIA were highly correlated (r = 0.989, n = 60, P less than 0.001). However concentrations measured by RIA were 34.3% +/- 9.7% higher than those by ELISA. Lp(a) concentrations in serum and in dried blood spots were also highly correlated (r = 0.966, n = 40, P less than 0.001). This indirect ELISA is suitable for assaying large numbers of serum or dried blood spot samples. However, the differences between the concentrations measured by ELISA and RIA stress the need for standardization of Lp(a) measurements.

Changes in the immunoreactivity of Apo A-I during storage.

We assayed apolipoprotein (Apo) A-I levels in re-constituted standard sera, and in serum samples and dried blood samples spotted onto filter-paper, taken from 4 healthy adults. An ELISA method was used to study changes in immunoreactivity after storage at different temperatures, for periods of up to 2 yr. For standard Apo A-I sera, immunoreactivity increased significantly with both time and temperature of storage after reconstitution. Immunoreactivity of Apo A-I in sera of healthy adults was stable on storage at -16 degrees C and -70 degrees C for 43 days, at 4 degrees C for 21 days, and at room temperature for only 10 days, after which there was a considerable increase. Dried blood spot Apo A-I immunoreactivity was also stable at -16 degrees C for at least 43 days, but not at 4 degrees C. Storage at room temperature caused significantly increased immunoreactivity from day 2. These changes could result from temperature and time-dependent structural or compositional changes in high density lipoprotein particles leading to exposure of more Apo A-I antigenic sites. The results define the conditions of time and temperature of storage of samples and standards necessary for Apo A-I assays using ELISA methodology.