Validation of abbreviated oral methionine-loading test.
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Adult↗
Biomedical subjects
J Selhub
Publications and source records attributed to J Selhub.
Global DNA hypomethylation increases progressively in cervical dysplasia and carcinoma.
BACKGROUND: Global DNA hypomethylation has been observed in some human neoplasms and has been implicated as an important factor in carcinogenesis. The current study was designed to assess whether DNA hypomethylation occurs in cervical dysplasia and cancer, and to determine the relationship between the degree of DNA hypomethylation and the grade of neoplasia. METHODS: Cervical biopsy specimens were obtained from colposcopically identifiable lesions in 41 patients with abnormal Pap smear results. The extent of global DNA methylation was assessed by incubating the extracted DNA with [3H]-S-adenosylmethionine and Sss1 methyltransferase, an enzyme that specifically catalyzes the transfer of methyl groups to cytosine residues in the cytosine-guanine doublet. The degree of exogenous 3H-methyl group incorporation into the DNA therefore is related reciprocally to the extent of endogenous DNA methylation. These data were compared with the histopathologic classification of the lesions. RESULTS: The extent of 3H-methyl group incorporation was increased threefold and sevenfold in the DNA from cervical dysplasia and cancer, respectively, compared with the DNA from normal cervical tissue (P = 0.006, analysis of variance). Significant incremental increases in DNA hypomethylation were observed in the progression from normal and low grade squamous intraepithelial lesions (SIL) to high grade SIL and to cancer (P < 0.0001, trend). CONCLUSIONS: These data show that global DNA hypomethylation is a significant epigenetic event in cervical carcinogenesis and that the degree of DNA hypomethylation increases with the grade of cervical neoplasia. These data suggest that global DNA methylation may serve as a biochemical marker of cervical neoplasia.
Cervix Uteri↗
Effect of chronic alcohol ingestion on hepatic folate distribution in the rat.
The mechanism by which ethanol impairs folate metabolism remains uncertain. In the present study, we used our new technique (affinity/HPLC) for folate analysis to study the effect of chronic alcohol ingestion on the content and distribution of folates in livers. Twelve male Sprague-Dawley rats (180 g) were divided into two groups, and fed for 4 weeks with Lieber-DeCarli semi-liquid isocaloric diets, with and without 5% ethanol. Livers were extracted in boiling, pH 9.3 borate buffers containing ascorbate/dithioerythritol. Folates in the supernatant fractions were purified by affinity chromatography and analyzed using ion pair high performance liquid chromatography. The data obtained showed that hepatic folate distribution in alcohol-treated rats differed from that of control animals in two ways. Livers from the ethanol-fed rats, when compared with those from control rats, exhibited increases in the percent concentrations of methylated tetrahydrofolates (21.46 +/- 2.21 vs 14.8 +/- 1.23), decreases in the percent concentrations of formylated tetrahydrofolates (25.62 +/- 4.02 vs 46.18 +/- 2.65) and higher concentrations of unsubstituted tetrahydrofolates (52.91 +/- 3.84 vs 38.88 +/- 2.50). In addition, alcohol ingestion was associated with longer glutamate chains of the folate molecules, characterized by lower relative concentrations of pentaglutamyl folates (29 vs 48%), and higher relative concentrations of hexa- and heptaglutamyl folates (55 vs 46% and 15 vs 6%) when compared with controls. The data are discussed in relation to the possibility that alcohol exerts its effect through: (1) inhibition of B12-dependent methyl transfer from methyltetrahydrofolate to homocysteine; (2) diversion of formylated tetrahydrofolates toward serine synthesis; and (3) interaction of acetaldehyde with tetrahydrofolates, thereby interfering with folate coenzyme metabolism.
5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗
Folate-deficiency-induced homocysteinaemia in rats: disruption of S-adenosylmethionine's co-ordinate regulation of homocysteine metabolism.
In a recent hypothesis [Selhub and Miller (1992) Am. J. Clin. Nutr. 55, 131-138], we proposed that homocysteinaemia arises from an interruption in S-adenosylmethionine's (AdoMet) coordinate regulation of homocysteine metabolism. The present study was undertaken to test a prediction of this hypothesis, that homocysteinaemia due to folate deficiency results from impaired homocysteine remethylation due to the deficiency and impaired synthesis of AdoMet, with the consequent inability of this metabolite to function as an activator of homocysteine catabolism through cystathionine synthesis. Rats were made folate-deficient by feeding them with a folate-free amino-acid-defined diet supplemented with succinylsulphathiazole. After 4 weeks, the deficient rats exhibited a 9.8-fold higher mean plasma homocysteine concentration and a 3.2-fold lower mean hepatic AdoMet concentration compared with folate-replete controls. Subsequent supplementation for 3 weeks of the folate-deficient rats with increasing levels of folate in the diet resulted in graded decreases in plasma homocysteine levels, accompanied by graded increases in hepatic AdoMet levels. Thus plasma homocysteine and hepatic AdoMet concentrations were inversely correlated as folate status was modified. In a second experiment, the elevation of plasma homocysteine in the deficient rats was found to be reversible within 3 days by intraperitoneal injections of ethionine. This effect of ethionine is thought to be exerted through S-adenosylethionine, which is formed in the liver of these rats. Like AdoMet, S-adenosylethionine is an activator of cystathionine beta-synthase and will effectively promote the catabolism of homocysteine through cystathionine synthesis. In crude liver homogenates of the rats treated with ethionine, cystathionine beta-synthase activity was 3-fold higher than that measured in homogenates from vehicle-treated controls.
Animals↗
High dose ascorbate supplementation fails to affect plasma homocyst(e)ine levels in patients with coronary heart disease.
Pharmacologic doses of folate, in the absence of clinical folate deficiency, can reduce plasma levels of the putatively atherothrombotic amino acid, homocysteine (H(e)). Data suggesting that H(e) may accumulate in experimental scurvy prompted us to explore the efficacy of high dose ascorbate supplementation as a H(e)-lowering treatment, in the absence of clinical ascorbate deficiency. A randomized, placebo-controlled trial of 12 weeks of high dose (4.5 g/day) ascorbate supplementation was completed by 44 patients with established coronary heart disease. No significant change in mean fasting total plasma H(e) levels was demonstrable despite a marked increase in mean fasting plasma ascorbate levels amongst those patients randomized to active treatment. Ascorbate supplementation to prevent the development of fasting hyperhomocysteinemia may only be relevant at scorbutic levels of plasma ascorbate.
Ascorbic Acid↗
Vitamin B-6 deficiency vs folate deficiency: comparison of responses to methionine loading in rats.
The plasma homocysteine response to methionine loading was assessed in vitamin B-6- and folate-deficient rats. Rats fed vitamin B-6- or folate-deficient diets for 4 wk were administered a gastric gavage of methionine (100 mg/kg body wt). Subsequent plasma analyses revealed a peak post-methionine load increase in plasma homocysteine concentration of > 300 mumol/L in the vitamin B-6-deficient rats. Folate-deficient rats exhibited no significant changes in plasma homocysteine after the load. These disparate responses can be explained by the observed increase in hepatic S-adenosylmethionine (SAM) concentration because of the load. In vitamin B-6 deficiency, increased SAM inhibits homocysteine remethylation, which, in conjunction with the impaired homocysteine catabolism due to the deficiency and the increased synthesis of homocysteine due to the methionine load, leads to a large elevation of homocysteine in the blood. In folate deficiency, increased SAM activates homocysteine catabolism, which compensates for the increased synthesis of homocysteine due to the load and thus no change in blood homocysteine is observed. These results have significant bearing on the interpretation of both positive and negative responses to methionine loading in humans.
Animals↗
Severe folate deficiency causes secondary depletion of choline and phosphocholine in rat liver.
It has previously been shown that choline deficiency causes depletion of hepatic folate concentration in rats. Two separate experiments were undertaken to investigate the converse phenomenon: whether folate deficiency would lead to depletion of hepatic choline. In Experiment 1, severe folate deficiency was induced in rats by feeding an amino acid-defined diet containing (per kg diet) 1.4 g choline, 0 mg folate and 10 g succinylsulfathiazole. Control rats were fed the same diet containing 8 mg folate/kg. After 4 wk, plasma and hepatic folate concentrations were significantly depleted in the severely folate-deficient rats compared with controls (P < 0.001), and hepatic choline and phosphocholine concentrations were 65 and 80% lower, respectively (P < 0.001). In Experiment 2, moderate folate deficiency was induced in rats by feeding the same diet as described above, but with the succinylsulfathiazole omitted. After 24 wk, significant systemic folate deficiency was present in the moderately folate-deficient rats compared with controls (P < 0.001). A modest reduction (36%, P = 0.087) in hepatic choline concentration was observed in the moderately folate-deficient rats compared with controls. No significant differences in hepatic phosphocholine concentrations were detected between the two groups. These results indicate that severe folate deficiency causes secondary hepatic choline deficiency in rats.
Animals↗
DNA methylation as an intermediate biomarker in colorectal cancer: modulation by folic acid supplementation.
Several studies have suggested that DNA hypomethylation is an early step in colorectal carcinogenesis. However, it is not clear at which stage in carcinogenesis this hypomethylation occurs, what promotes it, the extent to which it can be reversed and the consequences of such reversal in affecting tumour development. In an attempt to address some of these questions, we studied three groups of subjects with similar age and gender distributions: a group of 12 patients with colorectal carcinomas; a group of 12 patients with colorectal adenomas; and a group of eight healthy control subjects. Two experimental protocols were employed. In the first protocol, intrinsic DNA methylation was evaluated in neoplastic and in normal-appearing rectal mucosa of patients with colonic carcinomas or adenomas, compared with a group of healthy controls. In the second protocol, we examined, in a prospective and controlled fashion, the effect of folic acid supplementation (10 mg/day) on the degree of DNA methylation of rectal mucosa from those same patients after removal of the neoplasms. The degree of intrinsic DNA methylation was assessed on the basis of the capacity of the DNA isolates to serve as methyl acceptors in in vitro incubations that contained DNA methylase and [3H-methyl] S-adenosylmethionine. Intrinsic DNA methylation was significantly lower in carcinomas than in adenomas (P < 0.005). In addition, normal-appearing rectal mucosa from patients with carcinomas was significantly less methylated than in healthy controls (P < 0.005); the mean value found in the latter was also greater than the value observed in patients with adenomas, but not significantly so (P > 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)
Adenoma↗
Folate binding proteins. Mechanisms for placental and intestinal uptake.
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Carrier Proteins↗
Vitamin status and intake as primary determinants of homocysteinemia in an elderly population.
OBJECTIVE: To describe the distribution of plasma homocysteine concentrations in an elderly population and to analyze the relationship between homocysteine level and intake of vitamins and serum levels of vitamins that serve as coenzymes in homocysteine metabolism. DESIGN: Cross-sectional analysis of homocysteine levels and vitamin blood levels and intake in elderly participants in the Framingham Study. SETTING: Population-based cohort in Framingham, Mass. PARTICIPANTS: A total of 1160 adult survivors, aged 67 to 96 years, from the original Framingham Heart Study cohort. MAIN OUTCOME MEASURES: Plasma homocysteine concentration correlated with plasma folate, vitamin B12, pyridoxal-5'-phosphate (PLP), and oral intakes of these vitamins, and the contribution of these vitamins to the prevalence of elevated homocysteine in the population. RESULTS: Homocysteine levels were positively correlated with age after controlling for vitamin concentrations. After controlling for age, sex, and levels of other vitamins, homocysteine exhibited a strong inverse association with plasma folate. When subjects were grouped by deciles of plasma folate, mean homocysteine was significantly higher in the lowest two folate deciles (15.6 and 13.7 mumol/L, respectively) than in the highest decile (11.0 mumol/L). Homocysteine demonstrated weaker, inverse associations with plasma vitamin B12 and PLP. Similar inverse associations were demonstrated between homocysteine and intakes of folate and vitamin B6, but not vitamin B12. Prevalence of high homocysteine (> 14 mumol/L) was 29.3% in this cohort, and was greatest among subjects with low folate status. Inadequate plasma concentrations of one or more B vitamins appear to contribute to 67% of the cases of high homocysteine. CONCLUSIONS: These results indicate a strong association between homocysteine concentration and folate, vitamin B12, and vitamin B6 status, as well as age. It is possible that a substantial majority of the cases of high homocysteine in this older population can be attributed to vitamin status.
Aged↗
Elevations in total plasma homocysteine in premature coronary artery, cerebrovascular and peripheral vascular disease.
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Coronary Disease↗
Internalization and intracellular transport of folate-binding protein in rat kidney proximal tubule.
Folate-binding protein (FBP) is involved in folate reabsorption in the renal proximal tubule. Immunocytochemical studies have located FBP to the brush-border membrane, endocytic vacuoles, and dense apical tubules. We applied the same polyclonal antibody (anti-FBP) against FBP to investigate the dynamic relationship between FBP in the different compartments by microinjecting the antibody into rat kidney proximal tubules in situ. Specific binding of anti-FBP in vivo to the brush-border membrane was followed by fixation at various times. Protein A-gold labeling shows that anti-FBP is transported from endocytic invaginations into vacuoles followed by transport into dense apical tubules within 15 s. Thus FBP is rapidly internalized, and together with previous studies this study strongly suggests recycling of FBP back to the luminal plasma membrane through dense apical tubules. The results are consistent with reabsorption of folate through endocytosis of the FBP-folate complex followed by dissociation and recycling of FBP. When time is allowed there is a steady accumulation of FBP in dense apical tubules combined with an increase in surface density of the same compartment. A possible explanation involves partial inhibition of the fusion between dense apical tubules and plasma membrane because of the anti-FBP labeling of the receptor.
Animals↗
Folate deficiency enhances the development of colonic neoplasia in dimethylhydrazine-treated rats.
In patients with ulcerative colitis, epidemiological work has suggested an association between low folate status and an increased risk of colonic neoplasia. The aim of the present study was to determine if experimental folate deficiency increases the likelihood of developing neoplasia in rats treated with the carcinogen dimethylhydrazine. Weanling male Sprague-Dawley rats were fed with an amino acid-defined diet containing either 8 or 0 mg/kg folic acid. After 5 weeks of defined diet, weekly s.c. injections of dimethylhydrazine (20 mg/kg) were administered to both groups. Serum, whole blood, liver, and colonic folate concentrations at the time of sacrifice were significantly lower in folate-depleted animals (P less than 0.001). There were significant differences in the incidence of colonic neoplasia between the two groups after 20 weeks of dimethylhydrazine exposure: folate-deficient rats had a greater incidence of dysplasia (6 of 7 versus 2 of 7 animals; P less than 0.05) and carcinoma (6 of 7 versus 1 of 7 animals; P less than 0.01). Furthermore, a significantly greater proportion of folate-replete rats than folate-deficient rats were free of neoplastic lesions (5 of 7 versus 0 of 7 animals; P less than 0.05). These results suggest that, in this animal model, folate deficiency increases the risk of malignancy when there is an underlying predisposition to colorectal cancer.
Animals↗
Folate binding protein from kidney brush border membranes contains components characteristic of a glycoinositol phospholipid anchor.
A number of cell surface proteins have been shown to be anchored to the plasma membrane by a covalently attached glycoinositol phospholipid (GPL) in amide linkage to the C-terminus of the mature protein. We applied several criteria to establish that folate binding protein (FBP) in brush border membranes of rat kidney contains a GPL anchor. Brush border membranes were isolated and labeled with [3H]folate, and the complex of FBP and [3H]folate was shown to be released to the supernatant by incubation with purified bacterial phosphatidylinositol-specific phospholipase C (PIPLC) but not by incubation with a purified bacterial phosphatidylcholine-specific phospholipase C. The FBP-[3H]folate complex both in crude extracts and after FBP purification by ligand-directed affinity chromatography interacted with Triton X-114 micelles, and prior incubation with PIPLC prevented this detergent interaction. Individual residues characteristic of GPL anchors were found to be covalently associated with FBP following polyacrylamide gel electrophoresis in sodium dodecyl sulfate. These included glucosamine and ethanolamine, which were radiolabeled by reductive methylation and identified by chromatography on an amino acid analyzer, and inositol phosphate, which was inferred by Western blotting with an anti-CRD antisera. This antisera gave positive immunostaining only after FBP had been cleaved by PIPLC, a reliable diagnostic of a GPL anchor. The relationship between GPL-anchored FBP in biological membranes and soluble FBP in biological fluids also is discussed.
Animals↗
The pathogenesis of homocysteinemia: interruption of the coordinate regulation by S-adenosylmethionine of the remethylation and transsulfuration of homocysteine.
A unified, biochemical hypothesis is proposed to explain the pathogenesis of homocysteinemia. This hypothesis is based on the existence of coordinate regulation by S-adenosylmethionine (SAM) of the partitioning of homocysteine between de novo methionine synthesis and catabolism through cystathionine synthesis. This coordination, which serves to modulate the cellular concentration of homocysteine based on the requirements for methionine, is impaired in homocysteinemia. This hypothesis is evaluated in the context of the conditions known to be associated with homocysteinemia, including enzymatic defects and vitamin deficiencies. The novelty of the hypothesis is the assertion that impairment of one homocysteine metabolic pathway must lead to the impairment of the other homocysteine metabolic pathway to cause homocysteinemia. This extends the simplistic view that a block of only one of the pathways is sufficient to cause homocysteinemia.
Amino Acid Metabolism, Inborn Errors↗
Effect of vitamin B-6 deficiency on fasting plasma homocysteine concentrations.
The catabolism of homocysteine through cystathionine synthesis requires pyridoxal-5'-phosphate, thus the effect of vitamin B-6 deficiency on plasma homocysteine concentrations was evaluated. Total fasting plasma homocysteine concentrations were measured in 11 elderly subjects aged 64.4 +/- 1.7 y (mean +/- SE) who consumed a vitamin B-6-deficient diet for less than or equal to 20 d. Only 1 of the 11 subjects was found to have elevated homocysteine concentrations even though all subjects exhibited high urinary xanthurenic acid concentrations after a tryptophan load, a measure indicative of vitamin B-6 deficiency. In a supporting study, fasting plasma homocysteine concentrations were measured in 3- and 23-mo-old rats fed vitamin B-6-deficient diets and were compared with those of vitamin B-6-replete, pair-fed controls. There was no difference in homocysteine concentrations between deficient and pair-fed animals after 6 wk of the dietary regimen for either age group; after 9 wk a modest elevation was observed in the 3-mo-old deficient rats whereas no difference was observed for the 23-mo-old rats. It is concluded that fasting plasma homocysteine concentrations are not initially elevated in vitamin B-6 deficiency and therefore fasting plasma homocysteine concentrations are not a good indicator of vitamin B-6 status.
Aged↗
Long-term folate deficiency alters folate content and distribution differentially in rat tissues.
We fed rats a defined amino acid, folate-free diet that did not contain sulfathiazole for 25 wk and examined the effects upon the content and distribution of folates in various tissues, based on the glutamic acid chain length and pteridine ring distribution. Compared with controls, rats fed the folate-deficient diet had lower folate concentrations in liver (11.10 +/- 1.04 vs. 27.11 +/- 2.72 nmol/g, P less than 0.001), kidney (4.79 +/- 0.65 vs. 11.69 +/- 1.40 nmol/g, P less than 0.01) and spleen (1.29 +/- 0.12 vs. 3.74 +/- 0.52 nmol/g, P less than 0.001), but not in brain (0.60 +/- 0.09 vs. 0.65 +/- 0.04 nmol/g, P greater than 0.1). The folate-deficient diet also resulted in changes in folate distribution reflected mostly by longer glutamate chains. In livers of the animals fed the folate-deficient diet the relative concentration of pentaglutamyl folates was lower and that of hexaglutamyl folates was higher, and there were also considerable amounts of hepta and octaglutamyl derivatives. Similar changes in folate distribution were observed in kidney and spleen. Brain folate distribution, however, was the same in the folate-deficient and folate-replete rats. The folate-deficient diet was without effect on the pteridine ring structure distribution in the various tissues except for small changes in the kidney. These results are discussed with respect to the relationship between tissue folate depletion and glutamic acid chain elongation and the possibility that folate conservation is different in the brain than in other tissues.
Animals↗
Plasma homocysteine in vascular disease and in nonvascular dementia of depressed elderly people.
Depression among elderly people with reversible cognitive loss often manifests with concomitant vascular disease and can also precede the development of nonvascular degenerative dementia. Little is known about etiological factors for reversible or irreversible dementias in older depressed people. The amino acid homocysteine (HC), which is both a vascular disease risk factor and a precursor of the excitotoxic amino acids cysteine and homocysteic acid, could play a role in the pathophysiology of such individuals. Twenty-seven depressed elderly acute inpatients by DSM-III-R criteria had significantly higher plasma homocysteine levels and lower cognitive screening test scores than did 15 depressed young adult inpatients. HC was highest in the older patients who had concomitant vascular diseases (n = 14). HC was lowest in the older depressives who had neither vascular illnesses nor dementia (n = 8), comparable to the young adult depressives. Higher HC correlated significantly with poorer cognition only in the nonvascular geriatric patients (rs = -0.53). The findings extend earlier work showing higher HC in vascular patients from general medical populations, and also suggest a possible metabolic factor in certain dementias associated with late-life depression.
Adult↗