D. G. Weir

Homocysteine metabolism in pregnancies complicated by neural-tube defects

Abstract Folic acid taken around the time of conception can prevent many neural-tube defects. Women with low-normal vitamin B 12 values may also be at increased risk. We considered whether homocysteine metabolism via the enzyme methionine synthase, which requires both folate and B 12 , could be the critical defect in folate-related neural tube defects. Blood was obtained during pregnancies that produced 81 infants with neural-tube defects and 323 normal children. Samples were assayed for homocysteine, methylmalonic acid, plasma folate, red-cell folate, and B 12 . Mothers of children with neural-tube defects had significantly higher homocysteine values (8·62 [SD 2·8] μmol/L) than did B 12 -matched controls (7·96 [2·5] μmol/L, p=0·03). The difference was significant (p=0·004) in the lower half of the B 12 distribution after adjusting for plasma folate. Our study shows that an abnormality in homocysteine metabolism, apparently related to methionine synthase, is present in many women who give birth to children with neural-tube defects. Overcoming this abnormality is likely to be the mechanism by which folic acid prevents neural-tube defects. These findings suggest that the most effective periconceptional prophylaxis to prevent neural-tube defects may require B 12 as well as folic acid.

PATHOGENESIS OF SUBACUTE COMBINED DEGENERATION: A RESULT OF METHYL GROUP DEFICIENCY

Four pairs of monkeys were maintained in an atmosphere of nitrous oxide under conditions which had previously been shown to produce subacute combined degeneration (SCD) of the spinal cord. The diet of one of each pair was supplemented with methionine. In every case the monkey with the unsupplemented diet became ataxic at around 10 weeks and the disorder progressed over a period of 2-3 weeks until the animal was moribund. During this period there was no detectable clinical change in the monkeys receiving methionine supplementation. Microscopical examination of the spinal cord and peripheral nerves of the unsupplemented monkeys showed the classical changes of SCD. The histological changes correlated with the clinical observations. Sections form the methionine-supplemented monkeys showed no change or only slight changes. These results suggest that, in these animals, inability to resynthesise methionine from homocysteine leads to SCD. It seems probable that the primary lesion producing SCD in human beings with pernicious anaemia is also inability to maintain methionine biosynthesis.

The methyl folate trap. A physiological response in man to prevent methyl group deficiency in kwashiorkor (methionine deficiency) and an explanation for folic-acid induced exacerbation of subacute combined degeneration in pernicious anaemia.

It is suggested that in man the methyl folate trap is a normal physiological response to impending methyl group deficiency resulting from a very low supply of methionine. This decreases cellular S-adenosyl-methionine (SAM), which puts at risk important methylation reactions, including those required to maintain myelin. In order to protect these methylation reactions, the cell has evolved two mechanisms to maintain supplies of methionine and SAM as a first priority. (a) Decreased SAM causes the folate co-factors to be directed through the cycle involving 5-methyl-tetrahydrofolate (5-methyl-THF) and methionine synthetase and away from the cycles that produce purines and pyrimidines for DNA synthesis. This enhances the remethylation of homocysteine to methionine and SAM. In addition, by restricting DNA biosynthesis and with it cell, division, competition for methionine for protein synthesis is reduced. Thus, whatever methionine is available is conserved for the vital methylation reactions in the nerves, brain, and elsewhere. (b) 5-methyl-THF, the form in which almost all folate is transported in human plasma, must react with intracellular homocysteine before it can be retained by the cell as a polyglutamate. Since homocysteine is derived entirely from methionine, methionine deficiency will cause intracellular folate deficiency, and the rate of mitosis of rapidly dividing cells will be reduced. although these two processes have evolved as a response to methionine deficiency, they also occur in B12 deficiency, which the cell mistakenly interprets as lack of methionine. the resulting response is inappropriate and gives rise to a potentially lethal anaemia. In these circumstances the methylation reactions are also partly protected by the reduced rate of cell division. This explains why administration of folic acid, which induces cell division and use of methionine in protein synthesis, impairs methylation of myelin and precipitates or exacerbates subacute combined degeneration (SCD). During folate deficiency methionine biosynthesis is also diminished. As in methionine deficiency, the body responds to decreasing availability of SAM by diverting folate away from DNA biosynthesis towards the remethylation of homocysteine to methionine and SAM. The selective use pf available folate to conserve methionine, together with the ability of nerve tissue to concentrate folate form the plasma, explains the absence of SCD in folate deficiency.

Increased incidence of non-Hodgkin's lymphoma in inflammatory bowel disease patients on immunosuppressive therapy but overall risk is low

BACKGROUND There is concern that the incidence of non-Hodgkins lymphoma (NHL) will rise with increasing use of immunosuppressive therapy. AIMS Our aim was to determine the risk of NHL in a large cohort of patients with inflammatory bowel disease (IBD), and to study the association between IBD, NHL, and immunosuppressive therapy. METHODS We studied 782 IBD patients (238 of whom received immunosuppressive therapy) who attended our medical centre between 1990 and 1999 (median follow up 8.0 years). Standardised incidence ratios (SIRs) and 95% confidence intervals (CI) were calculated. Expected cases were derived from 1995 age and sex specific incidence rates recorded by the National Cancer Registry of Ireland. RESULTS There were four cases of NHL in our IBD cohort (SIR 31.2; 95% CI 2.0–85; p=0.0001), all of whom had received immunosuppressive therapy: azathioprine (n=2), methotrexate (n=1), and methotrexate and cyclosporin (n=1). Our immunosuppressive group had a significantly (59 times) higher risk of NHL compared with that expected in the general population (p=0.0001). Three cases were intestinal NHL and one was mesenteric. Mean age at NHL diagnosis was 49 years, mean duration of IBD at the time of NHL diagnosis was 3.1 years, and mean duration between initiation of immunosuppressive therapy and diagnosis of NHL was 20 months. CONCLUSIONS Although underlying IBD may be a causal factor in the development of intestinal NHL, our experience suggests that immunosuppressive drugs can significantly increase the risk of NHL in IBD. This must be weighed against the improved quality of life and clinical benefit immunosuppressive therapy provides for IBD patients.

Accelerated folate breakdown in pregnancy

During pregnancy there is an increased requirement for folate. We studied pregnant women to determine whether the increased requirement might be due to enhanced catabolism of the vitamin. Six normal pregnant women provided 24 h urine samples during each trimester and postpartum while taking a defined diet. The urines were assayed for the folate breakdown products p-amino-benzoylglutamate (pABGlu) and its acetylated derivative p-acetamidobenzoylglutamate (apBGlu) by high-pressure liquid chromatography. Mean concentration of excreted apABGlu rose significantly in the second trimester but returned to baseline postpartum. This increased rate of folate catabolism produces an extra demand for dietary folate of about 200-300 micrograms per day in pregnant women, a considerably greater value than recent recommendations.

Increased prevalence of methylenetetrahydrofolate reductase C677T variant in patients with inflammatory bowel disease, and its clinical implications

BACKGROUND Inflammatory bowel disease (IBD) is associated with an increased incidence of thromboembolic disease. Hyperhomocysteinaemia (hyper-tHcy), a condition associated with the C677T variant of 5,10-methylenetetrahydrofolate reductase (MTHFR), is linked with an increased incidence of thromboembolic disease. Hyper-tHcy has been reported in patients with IBD. AIMS To assess the prevalence of the C677T MTHFR genotype and the contribution of this genotype to hyper-tHcy in patients with IBD. METHODS Patients with established IBD (n=174) and healthy controls (n=273) were studied. DNA samples were genotyped for the MTHFR (C677T) mutation. Subjects were categorised as homozygous for the thermolabile variant (TT), heterozygous for wild type and variant (CT), or homozygous for the wild type (CC). RESULTS Plasma homocysteine concentrations were significantly higher in patients with IBD than in healthy controls. A total of 17.5% of ulcerative colitis and 16.8% of Crohn’s disease patients were homozygous for the C677T variant compared with 7.3% of controls. Homozygosity (TT) for the variant was associated with higher plasma tHcy levels in patients with IBD and in healthy controls. When all subjects who were TT for the variant were excluded, median plasma tHcy was still significantly higher in IBD than controls. Plasma vitamin B12 levels were lower in patients with IBD irrespective of MTHFR genotype. CONCLUSIONS There is an association between the thermolabile MTHFR C677T variant and IBD. This accounts in part for the raised plasma tHcy found in patients with IBD and may contribute to the increased incidence of thromboembolic complications. All patients with IBD should receive low dose folic acid and vitamin B12 therapy to protect against the thromboembolic complications of raised tHcy.

Humoral response to wheat protein in patients with coeliac disease and enteropathy associated T cell lymphoma.

Features that might distinguish uncomplicated coeliac disease from enteropathy associated T cell lymphoma were investigated. Of 76 patients with coeliac disease, 71 (93%) had raised levels of alpha gliadin antibody and all responded clinically and histologically to treatment with a gluten free diet. In contrast, none of 16 patients with enteropathy associated T cell lymphoma had raised levels of alpha gliadin antibody, and treatment with a gluten free diet resulted in histological improvement in one and transient clinical improvement in six patients. The ratio of women to men was 2.2:1 in the group with coeliac disease and 1:1.6 in the patients with enteropathy associated T cell lymphoma. Thus patients with enteropathy associated T cell lymphoma do not display a humoral immune response to wheat protein (alpha gliadin), rarely respond to a gluten free diet, and are often men. Patients with uncomplicated coeliac disease usually have raised levels of alpha gliadin antibody, always respond to a gluten free diet, and are frequently women. These findings suggest the presence of two separate forms of enteropathy: one is benign and sensitive to wheat protein whereas the other runs a malignant course.

Are common mutations of cystathionine β-synthase involved in the aetiology of neural tube defects?

Mildly elevated maternal plasma homocysteine (Hcy) levels (hyperhomocysteinemia) have recently been observed in some neural tube defect (NTD) pregnancies. Plasma levels of Hcy are governed by both genetic and nutritional factors and the aetiology of NTDs is also known to have both genetic and nutritional components. We therefore examined the frequency of relatively common mutations in the enzyme cystathionine β‐synthase (CBS), which is one of the main enzymes that controls Hcy levels, in the NTD population. Neither the severely dysfunctional G307S CBS allele nor the recently reported 68 bp insertion/I278T CBS allele was observed at increased frequency in the cases relative to controls. We therefore conclude that loss of function CBS alleles do not account for a significant proportion of NTDs in Ireland.

Randomised comparison of olsalazine and mesalazine in prevention of relapses in ulcerative colitis

Sulphasalazine extends remissions and lessens disease activity during relapses of ulcerative colitis, but it also causes many adverse side-effects. The adverse reactions are mostly attributable to the sulphapyridine carrier moiety rather than the active principle 5-aminosalicylic acid (5-ASA), so agents to deliver 5-ASA to the colon by other means have been designed. We have compared the efficacy and tolerability of two such agents, olsalazine and mesalazine, in maintenance therapy of ulcerative colitis. 100 patients with ulcerative colitis in remission were recruited at one centre and assigned randomly to treatment with olsalazine (Dipentum; 1.0 g daily) or mesalazine (Asacol, with Eudragit-S coating; 1.2 g daily). Compliance, biochemical and haematological variables, and clinical evidence of disease activity were assessed every 3 months for 12 months by observers unaware of treatment allocation. In intention-to-treat analysis, which included as treatment failures patients withdrawn for protocol violations, adverse reactions, intercurrent illness, or non-compliance as well as those with relapses of ulcerative colitis, the olsalazine group had a significantly lower rate of treatment failure than the mesalazine group (12/49 [24%] vs 23/50 [46%]; p = 0.025). Analysis restricted to 64 patients still in remission at 1 year and 18 with relapses also showed a significant difference in relapse rate (olsalazine 5/42 [12%] vs mesalazine 13/40 [33%]; p = 0.024). Both drugs were well tolerated; only 9 patients reported substantial side-effects. Olsalazine was clearly superior to mesalazine in prevention of relapses in ulcerative colitis, especially in patients with left-sided disease.

Alpha gliadin antibody levels: a serological test for coeliac disease.

The diagnostic value in coeliac disease of circulating antibodies to casein, crude gliadin, and alpha gliadin was assessed using an adaption of the enzyme linked immunosorbent assay system. alpha Gliadin was the only antigen which consistently separated 26 patients with untreated coeliac disease from 26 normal controls and 13 patients with chronic inflammatory bowel disease. The mean assay index for the 26 patients was 3.1 (SD 1.2) compared with 1.05 (0.5) for the normal controls and 1.1 (0.6) for patients with chronic inflammatory bowel disease. The alpha gliadin antibody levels of six patients with coeliac disease who had maintained a gluten free diet for at least two years were not significantly higher than normal (1.0 (0.4)). The validity of the test was determined in 90 consecutive patients who were being investigated for the presence of coeliac disease. Levels of alpha gliadin antibody were raised in 36 out of 44 patients found to have histologically proved coeliac disease and in six out of 46 subjects whose jejunal mucosa was normal. Serial alpha gliadin concentrations were measured in 12 patients with coeliac disease who had repeat jejunal biopsies performed six months after starting a gluten free diet. The levels of antibody fell in seven of the eight patients whose jejunal mucosa improved on maintaining the diet. They remained raised in four patients who did not adhere to the diet and whose mucosa did not improve. Although a test measuring alpha gliadin antibodies is unlikely to replace jejunal biopsy in the diagnosis of coeliac disease it may be useful in screening for the disease among outpatients.