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The human T developmental gene has been implicated in the etiology of neural tube defects (NTDs) on the basis both of mouse studies of its homologue, T (Brachyury), and of allelic association in a Caucasian population. We have investigated the frequency of the T allelic variant TIVS7-2 in 218 Irish NTD case-parent triads. This population showed the same trend as previously reported, with an excess of the TIVS7-2 allele among cases. Log-linear modeling of case and maternal genotypic effects within families indicated that TIVS7-2 was elevated in cases (relative risk, RR = 1.36) but not in mothers (RR = 0.91). The TIVS7-2 allele is markedly associated with cases born before 1980 (RR = 2.09; CI = 1.23-3.55; corrected p = 0.030), but not with more recent cases (RR = 0.92). Cases carrying a TIVS7-2 allele did not show any increased tendency to be homozygous for the thermolabile variant of the folate-dependent enzyme 5,10-methylene tetrahydrofolate reductase, which is an established genetic risk factor for NTDs. Since the incidence of NTDs has declined markedly in Ireland over the last few decades, we suggest that the T-associated risk is potentiated by nutritional or environmental risk factor(s), the impact of which have been diminishing over time.

Several new derivatives of sulphasalazine that make use of its active moiety, 5-aminosalicylic acid (5-ASA), have been introduced for the treatment of inflammatory bowel disease (IBD). In rats short term intravenous administration of 5-ASA has been associated with nephrotoxicity. A number of cases of nephrotoxicity have been reported recently in IBD patients taking oral maintenance treatment with 5-ASA compounds.To study the urinary and serum levels of acetylated 5-ASA (Ac-5-ASA) and the unacetylated 5-ASA (5-ASA) in patients with IBD maintained on sulphasalazine, olsalazine and mesalazine (pH dependent release form). We also sought correlation between levels of 5-ASA, clinical disease activity and extent of disease.We studied 79 patients (male, n = 30; female, n = 49) with established IBD [ulcerative colitis (UC), n = 48; Crohn's disease (CD), n = 31], 72 maintained on 5-ASA compounds (sulphasalazine = 27; olsalazine = 28; mesalazine = 17) and 7 patients were receiving no medication. Urinary and serum analysis of 5-ASA was performed by high performance liquid chromatography (HPLC). Clinical disease activity was quantified using simple index of Harvey and Bradshaw.Patients receiving mesalazine had significantly higher levels of serum free 5-ASA compared to those who were receiving olsalazine and sulphasalazine (mesalazine mean +/- SEM; range; 2.84 +/- 1.21 (0.00-16.00) vs olsalazine 0.45 +/- 0.18 (0.00-16.20); mumol/L; p0.04; sulphasalazine 0.37 +/- 0.25 (0.00-3.74); p0.03). Similarly levels of urinary free 5-ASA were significantly higher in patients maintained on mesalazine compared to those on olsalazine or sulphasalazine (mesalazine 219 +/- 80.43 (0.00-1050) vs olsalazine 33.3 +/- 17.23 (0.00-317) mumol/L; p0.01; and sulphasalazine 15 +/- 8.86 (0.00-192); p0.05). However, no significant difference was observed in the levels of urinary free 5-ASA between olsalazine and sulphasalazine. No significant difference was observed in the levels of free-5-ASA in UC patients with left sided disease and those with extensive disease. Furthermore, no significant difference was observed in the levels of serum and urinary 5-ASA in CD patients with ileo-colic disease and colonic disease. Urinary and serum free-5-ASA did not correlate with the clinical disease activity.Systemic absorption of 5-ASA from sulphasalazine and olsalazine is relatively low. However, pH-dependent mesalazine formulations may release their contents rapidly in the small intestine and proximal colon resulting in higher plasma and urinary concentrations of free 5-ASA. The effects of free 5-ASA on renal function in the human require further evaluation.

PUBLISHED, BACKGROUND: There is concern that the incidence of non-Hodgkin's 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.

This study demonstrates gluten-sensitive recurrent oral ulceration (ROU) in the absence of gastrointestinal abnormalities which is associated with a humoral response to wheat protein. Ten patients with severe ROU were investigated; all had normal small intestinal biopsies. Four patients had raised levels of antibodies to alpha gliadin, a wheat protein fraction; in three of these four, the ulceration remitted on treatment with a gluten-free diet (G.F.D.) and relapsed on gluten challenge. None of the remaining six patients had raised alpha gliadin antibody (AGA) levels and none responded to G.F.D. Thus raised AGA levels can be used to identify patients with ROU who are likely to respond to a GFD.

Carbon‐doped GaAs films have been grown by solid‐source molecular beam epitaxy using a graphite filament. The films were doped from 1×1015 cm−3 to 5×1019 cm−3 and the resulting mobilities are equivalent to beryllium‐doped films. A slight dependence of As4/Ga flux ratio on carbon doping was observed. The use of either As2 or As4 did not significantly affect the carbon doping concentrations. Lattice contractions were observed for films doped heavily with carbon or beryllium. For a given doping concentration the contraction is more significant for carbon doping which is consistent with the smaller tetrahedral covalent radius of carbon compared to beryllium. Good agreement between observed and calculated lattice contractions with carbon doping is obtained. Annealing studies on a film doped with carbon at 5×1019 cm−3 indicate that the electrical properties and lattice contraction are quite stable.

Objectives - Experimental models of vitamin B 12 deficient-neuropathy are characterized by central nervous system protein hypomethylation. The encephalitis/vacuolar myelopathy complicating HIV infection and subacute combined degeneration of the cord due to vitamin B 12 deficiency share similar biochemical and pathological abnormalities. Altered central nervous system methylation may be important in the pathogenesis of HIV encephalitis. To test this hypothesis we compared brain protein methylation of HIV-positive, and control, subjects. Materials and methods - Carboxymethyltransferase activity was assayed in postmortem cortical brain samples obtained from 16 control patients (9 males); mean age (59 ± 5.1 years, range 21-87 years), 9 HIV-positive patients (7 males, 6 IVDA, 3 homosexual, 4 with HIV encephalitis, mean age 37, range 23-45), and 3 patients with Alzheimer's disease (mean age 78 years). Results - The amount of radiolabelled SAM (S-adenosylmethionine) incorporated into carboxymethyl, and N-methylation sites within brain proteins from cortical white matter in vitro was significantly lower (P

BACKGROUNDInflammatory 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.AIMSTo assess the prevalence of the C677T MTHFR genotype and the contribution of this genotype to hyper-tHcy in patients with IBD.METHODSPatients 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).RESULTSPlasma 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.CONCLUSIONSThere 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.

Coeliac disease is a prevalent disorder but frequently remains undiagnosed because of varied modes of clinical presentation. In this study, methods for the detection of coeliac disease were evaluated in a clinical practice setting.Small intestinal histology, IgA anti-endomysial and IgG anti-gliadin antibody tests were performed on 441 unselected, consecutive patients under investigation for small intestinal disease. Response to treatment and other clinical events were monitored over the ensuing years.Untreated coeliac disease was diagnosed in 97 patients and was excluded in 344. At clinical presentation, the endomysial antibody test was positive in 84 of the 97 untreated coeliac patients (sensitivity 87%) and negative in 340 of the 344 non-coeliac patients (specificity 99%). A typical histological lesion was found in 83 of the 97 coeliac patients (sensitivity 86%) but was absent in all 344 non-coeliacs (specificity 100%). The sensitivity of the gliadin antibody test was 69% and the specificity was 71%.In unselected patients attending a gastroenterology clinic, small bowel histology and endomysial antibody serology show similar predictive value in the diagnosis of coeliac disease. These results emphasize that a combination of clinical, histological and serological criteria are required for effective diagnosis of this disorder. Exclusive reliance on histology or serology will result in failure to make a diagnosis in a significant proportion of patients.

1. The aim of this study was to determine the pattern of protein carboxymethylation in different regions of the human brain. 2. The availability of protein methylation sites was determined by measurement of the incorporation of methyl-3H groups into proteins isolated from postmortem brain tissue. The stability of protein carboxymethylation in post-mortem brain was determined by sampling post-mortem pig and human brain tissue at intervals during the first 24 h after death. This method has previously been used to demonstrate that decreased protein carboxymethylation occurred in post-mortem pig brain when methionine synthase was inhibited. 3. There were no significant differences in the protein carboxymethyltransferase activity in samples of pig brain obtained at the time of death compared with that obtained when the same tissue was maintained at room temperature for up to 24 h after death. Similarly, there were no significant differences in the protein carboxymethyltransferase activity in samples isolated from human brain 12 h after death compared with that obtained from the same human brain tissue maintained at room temperature for up to 24 h after death. These results suggest that the level of carboxymethylation of proteins from human post-mortem brain obtained within 24 h of death is not significantly different to the level present at the time of death. To characterize the distribution of protein carboxymethylation in human brain, nine regions of post-mortem brain were sampled from 16 human subjects. Protein carboxymethyltransferase activity was lowest in the cerebellum (P < 0.05) and highest in cortical white matter compared with other regions of the brain (P < 0.05). No significant differences in protein carboxymethyltransferase activity were noted between other regions of the cortex or the subcortical regions. 4. In human cortical white matter there are more available sites for protein carboxymethylation than other brain regions. This may explain the greater sensitivity of white matter to the adverse consequences of hypomethylation associated with vitamin B12 deficiency. Post-mortem brain tissue can serve as a tool for the study of physiological or pathological factors which influence human brain protein methylation in vivo.

Thermolabile 5,10-methylenetetrahydrofolate reductase (MTHFR) is the first folate-related variant to be associated with an increased risk of neural tube defects (NTDs). The variant causes high plasma homocysteine levels and reduced red cell folate (RCF) levels, both of which have also been linked to an increased risk of NTDs. We examined the relationship between folate status and presence of the common mutation MTHFR C677T in 82 NTD-affected and 260 control mothers. Homozygosity for the TT genotype was associated with very low folate status among both the cases (n = 13) and the controls (n = 21). However, after exclusion of TT homozygotes, only 10% of the remaining 240 controls had RCF levels less than 200 microg/L compared with 29% of the 69 cases (odds ratio, 3.67; 95% confidence interval, 1.88-7.18; P0.001), and those with RCF less than 150 microg/L had eight times higher risk of NTD than subjects with levels over 400 microg/L. Plasma homocysteine levels of non-TT cases were also higher than those of controls (P = 0.047). This study shows that homozygosity for the C677T MTHFR variant cannot account for reduced blood folate levels in many NTD-affected mothers. Thus, a strategy of genetic screening of all childbearing women for this variant would be ineffective as a method of primary prevention of NTDs. The data suggest that low maternal folate status is itself the major determinant of NTD risk, or else that other folate-dependent genetic variants confer risk through the reduction of folate levels. These results emphasize the importance of a food-fortification program as a population strategy for reducing the occurrence of NTDs.

Restricted folate supply is associated with the development of carcinoma, and folate supplements have a protective effect in colorectal carcinoma. This effect may be mediated through correction of local folate deficiency. The aim of this study was to define the folate content of neoplastic colonic epithelial cells and its relation to that of adjacent normal tissue and circulating levels.Epithelial cells were isolated from endoscopic biopsy specimens of normal, adenocarcinoma, adenoma, and adjacent normal colonic mucosa by ion chelation. Intracellular folate levels were determined by microbiological assay.Folate levels in carcinoma specimens were lower than in adjacent normal tissue (P0.02). Levels in adenoma epithelial cells were lower than in adjacent normal tissue, although this did not reach statistical significance (P0.06). Epithelial cells from normal tissue and mucosa adjacent to tumors and adenomata had similar folate contents. Blood folate and vitamin B12 indices for all groups were normal.Malignant colon epithelial cells show a relative localized folate deficiency. However, there is no evidence for the occurrence of generalized mucosal folate deficiency. This finding suggests that folate supplements do not inhibit carcinogenesis through correction of localized folate depletion.

Evidence continues to confirm that periconceptional ingetion of folic acid can prevent neural tube defects, although the basis of this prevention has been unclear. To further elucidate the mechanism by which this occurs, blood samples were collected from more than 50,000 pregnant women and the samples analyzed for red cell folate, plasma folate, plasma vitamin B12, and plasma homocysteine levels. Based on the results, it appears that the beneficial effect of folic acid is in overcoming a metabolic block in a folate-dependent enzyme or transport process. It is likely that methionine synthase may be directly or indirectly involved.

It is now well-established that folic acid, taken peri-conceptionally, can reduce the risk of neural tube defects (NTDs). Recent work has demonstrated that an abnormality of homocysteine metabolism is a critical factor. The gene for 5,10 methylenetetrahydrofolate reductase, an enzyme important in homocysteine metabolism, was studied in relation to NTDs. To determine the frequency of the allele for the thermolabile form of the reductase, DNA samples were collected from people with NTDs, parents of people with NTDs, and normal controls. Of 82 people with NTDs, 15 (18.3%) were homozygous for the abnormal, thermolabile allele. This was significantly higher (p = 0.01) than the rate of 6.1% in the control population (odds ratio 3.47, 95% CI 1.28-9.41). This is the first specific genetic abnormality to be identified in NTDs. It explains the association between some NTDs and elevated homocysteine, given that the reductase is important in homocysteine metabolism. It also explains how folic acid supplementation prevents some NTDs, by overcoming a partial block in the conversion of 5,10 methylenetetrahydrofolate to 5 methyltetrahydrofolate. Genetic screening could identify women who will require folic acid supplements to reduce their risk of having a child with an NTD.

The amount of biologically active folate excreted in the urine corresponds to a small fraction of the recommended dietary allowance, suggesting that a large amount of folate is excreted as its catabolites. A strategy of assessing folate requirement by measuring the daily urinary levels of products of folate catabolism depends on the demonstration of an exclusive mechanism of breakdown as well as a suitable marker of the catabolic process. Rats were given [3H] and [14C]folic acid by gastric intubation daily for 10 d to simulate normal dietary intake of the vitamin. Total urine was collected throughout this period as well as for the following 10 d. Reverse-phase HPLC of the radiolabeled urinary products revealed the presence of a variety of intact folates as well as products of C9-N10 scission of the folate molecule, pteridines, para-aminobenzoylglutamate and para-acetamidobenzoylglutamate. We detected no other N10-containing catabolites, nor did we find the oxidized folate derivative '4 alpha-hydroxy-5-methyltetrahydrofolate'. Of all the urinary folate metabolites, only para-acetamidobenzoylglutmate persisted at high levels up to 10 d after radiolabel treatment was withdrawn. We conclude that folate catabolism occurs exclusively through C9-N10 cleavage and that measurement of urinary para-acetamidobenzoylglutmate provides a suitable indicator of daily folate turnover.

The pathogenesis of the neuropathy associated with vitamin B12 deficiency (subacute combined degeneration (SCD)) is now thought to be related to interference with the methylation reactions in the CNS. The methylation reactions are processed by S-adenosylmethionine (SAM), which is controlled by its product, S-adenosylhomocysteine (SAH). The relationship of these two compounds is termed the methylation ratio. It has been demonstrated that if the ratio falls, the methylation reactions are inhibited leading to a state of CNS hypomethylation. The ratio can fall either due to a rise in SAH or a fall in SAM. It is suggested that for clinical signs to develop in animals who are susceptible to the lesion, both events are usually required. Inhibition of the vitamin B12-dependent enzyme, methionine synthase, leads to a rapid fall in the ratio in the CNS, since unlike other organs such as the liver, it does not have an alternative method of re-methylating homocysteine to maintain the endogenous synthesis of SAM. The supply of methyl groups necessary for the re-methylation reactions is controlled by a series of enzymes, which include methionine synthase. The inborn errors of metabolism that produce deficiency or impairment of these enzymes are described. Neurological syndromes associated with deficiency of these enzymes have close associations with SCD. The other clinical evidence and animal experiments that support this hypothesis are also described.

1. Pigs treated with nitrous oxide for periods of 1, 2 and 4 months demonstrated markedly reduced levels of methionine synthase and concomitant reduction in the ratio of S-adenosylmethionine to S-adenosylhomocysteine, the methylation ratio, at all time intervals. 2. Both ‘O’ and ‘N’ methylations were significantly reduced in pigs after 4 months in nitrous oxide but not after shorter periods. 3. Hypomethylation correlated with the development of clinical ataxia, but was absent when the pigs were clinically normal. It also only occurred when the S-adenosylmethionine level fell. 4. Rats maintained in nitrous oxide for 4 months showed a marked reduction of methionine synthase but no reduction in the methylation ratio or in brain hypomethylation. None of the rats became clinically ataxic. 5. Using an exogenous protein as a methyl group acceptor, it was demonstrated in an in vitro assay that the methyltransferase enzymes responsible for brain ‘O’ and ‘N’ methylation were not affected per se by nitrous oxide treatment. 6. It is concluded that reduction of the methylation ratio in the brain of pigs as a consequence of methionine synthase inhibition leads to brain hypomethylation. This hypomethylation could affect critical components of nerve tissue, inducing the vacuolar myelopathic changes seen in the spinal cord of these animals, which mimic those of subacute combined degeneration in man.

To determine whether tumour necrosis factor-alpha (TNF-alpha) is important in the pathogenesis of microalbuminuria in patients with inflammatory bowel disease (IBD).We measured serum TNF-alpha, interleukin (IL)-6, the erythrocyte sedimentation rate (ESR), C-reactive protein (CRP) levels and microalbuminuria in 48 patients with IBD. Serum TNF-alpha was measured by enzyme-linked immunosorbent assay and microalbuminuria was measured using an immunoturbiditimetric method. Clinical disease activity was quantified using the simple index of Harvey and Bradshaw.Microalbuminuria was more severe in patients with IBD than in controls, and in patients with active versus inactive disease. TNF-alpha levels were higher in patients with IBD than in controls (mean +/- SE 16.4 +/- 1.4 versus 6.6 +/- 1.3 pg/ml, respectively; P0.01) and in patients with active versus inactive IBD (means +/- SE 20.1 +/- 2 versus 12.8 +/- 2.7 pg/ml; respectively P = 0.056). Microalbuminuria correlated strongly with TNF-alpha (r = 0.60; P0.009), ESR (r = 0.67, P0.02) and CRP levels (r = 0.935, P0.001). TNF-alpha correlated significantly with CRP (r = 0.54, P0.01). IL-6 levels were raised significantly in patients with IBD (7 +/- 4 pg/ml, controls undetectable; P0.05). Patients with active IBD had higher IL-6 levels than those with inactive IBD (mean +/- SE 13 +/- 8 versus 0.90 +/- 0.35 pg/ml, respectively; P0.05). However, IL-6 levels did not correlate with microalbuminuria in patients with IBD (r = 0.105, P = 0.256).Our findings suggest that TNF-alpha may be important in the pathogenesis of microalbuminuria in patients with IBD, possibly through TNF-induced damage to the glomerular basement membrane. The mechanism for this has not been defined but may relate to TNF-induced disruption of sulphated glycosaminoglycans.

The relationship between growth rate and folate catabolism was determined using a catch-up growth model in weanling rats. A recently developed HPLC method was employed to compare the urinary excretion of the major end product of folate catabolism, acetamidobenzoylglutamate (apABGlu), in normally growing (n = 8) and age-matched growth-restricted (n = 8) weanling animals, during and after food restriction. During the 12-d period of food restriction in which restricted rats were fed (per gram of body weight) at a level of 50% of the required dietary intake for normal growth, apABGlu excretion was significantly higher in normally growing rats compared with growth-restricted rats. In the latter group after restriction, apABGlu concentrations increased markedly, tending to exceed those of normally growing rats of comparable age. Thus growth retardation resulted in a decreased rate of folate catabolism, whereas accelerated growth following dietary restriction was associated with an increase in catabolism. Because early growth in rats is predominantly hyperplastic, these results suggest that the rate of folate catabolism is related to folate utilization in cell division.

Microalbuminuria independently predicts the development of nephropathy and increased cardiovascular morbidity and mortality in diabetic patients, but it may be an indicator of the acute phase response. This study examined microalbuminuria as a marker of the acute phase response in patients with inflammatory bowel disease and correlated it with the disease activity in 95 patients with inflammatory bowel disease (ulcerative colitis (n = 52), Crohn's disease (n = 43)) determined by the simple index of Harvey and Bradshaw. Fifty patients were in complete clinical remission and 45 patients had active disease. Microalbuminuria was detected in all patients with inflammatory bowel disease (147 (17) v 18 (2) microgram/min, inflammatory bowel disease v controls mean (SEM), p < 0.007). Patients with active inflammatory bowel disease had higher concentrations of microalbuminuria compared with patients in remission (206 (19) v 65 (8) microgram/min, mean (SEM), p < 0.0001). Eight patients with active inflammatory bowel disease who were sequentially followed up with measurements of microalbuminuria had significantly lower values, when the disease was inactive (active inflammatory bowel disease 192 (44) v inactive inflammatory bowel disease 64 (14) microgram/min, p < 0.03). There was a significant correlation with the simple index of Harvey and Bradshaw (r = 0.818, p < 0.0001). Microalbuminuria values were significantly lower in inflammatory bowel disease patients in remission, maintained with olsalazine compared with those patients maintained with mesalazine and salazopyrine, but no significant difference was seen in values of microalbuminuria in active inflammatory bowel disease patients receiving different salicylates. This study also measured serum amyloid-A as an indicator of the acute phase response in the same patients. Serum amyloid-A was significantly increased in active disease compared with inactive disease (151 (43) v 33 (7) or controls 11 (2) micrograms/ml, p < 0.05). In conclusion microalbuminuria is present in abnormal amounts in all patients with active inflammatory bowel disease, and values fall when the disease is quiescent. Microalbuminuria is probably a consequence of an acute phase response and provides a simple, rapid, and inexpensive test, which has the potential to monitor inflammatory bowel disease activity and response to treatment.

Extrapulmonary small cell and small cell neuroendocrine tumors of unknown primary site are, in general, aggressive neoplasms with a short median survival. Like small cell lung cancer (SCLC), they often are responsive to chemotherapy and radiotherapy. Small cell lung cancer and well differentiated neuroendocrine carcinomas of the gastrointestinal tract and pancreas tend to express somatostatin receptors. These tumors may be localized in patients by scintigraphic imaging using radiolabeled somatostatin analogues. A patient with anaplastic neuroendocrine small cell tumor arising on a background of multiple endocrine neoplasia type 1 syndrome is reported. The patient had a known large pancreatic gastrinoma and previously treated parathyroid adenopathy. At presentation, there was small cell cancer throughout the liver and skeleton. Imaging with a radiolabeled somatostatin analogue, 111In-pentetreotide (Mallinckrodt Medical B. V., Petten, Holland), revealed all sites of disease detected by routine biochemical and radiologic methods. After six cycles of chemotherapy with doxorubicin, cyclophosphamide, and etoposide, there was almost complete clearance of the metastatic disease. 111In-pentetreotide scintigraphy revealed uptake consistent with small areas of residual disease in the liver, the abdomen (in mesenteric lymph nodes), and posterior thorax (in a rib). The primary gastrinoma present before the onset of the anaplastic small cell cancer showed no evidence of response to the treatment. The patient remained well for 1 year and then relapsed with brain, lung, liver, and skeletal metastases. Despite an initial response to salvage radiotherapy and chemotherapy with carboplatin and dacarbazine, the patient died 6 months later.

Fortification of food with folic acid to prevent neural-tube defects in babies also lowers plasma total homocysteine, which is a risk factor for vascular disease. We investigated the effect of folate and vitamin B12 on homocysteine concentrations. 30 men and 23 women received sequential supplementation with increasing doses of folic acid. After supplementation, the usual dependency of homocysteine on folate diminished, and vitamin B12 became the main determinant of plasma homocysteine concentration. This finding suggests that a fortification policy based on folic acid and vitamin B12, rather than folic acid alone, is likely to be much more effective at lowering of homocysteine concentrations, with potential benefits for reduction of risk of vascular disease.

A technique for preparing viable, single cell suspensions of the epithelial layer of small intestinal tissue obtained endoscopically is described. Constant agitation of four biopsies for 60 min in the presence of chelating and reducing agents gave yields of 1.2-6.7 x 10(6) cells, of which 11-30% were intraepithelial lymphocytes (IEL). Passage through a nylon wool column removed dead cells. This preparation was suitable for flow cytometric analysis. Using this technique, surface MHC class II molecule expression was studied in 14 patients with normal small intestinal mucosa. Fluorescence labelling of these cells showed strong HLA-DR expression by epithelial cells (EC), DP was expressed less strongly, while little DQ expression could be detected. This technique demonstrates that small intestinal biopsies taken during routine endoscopy can yield adequate numbers of viable epithelial cells to perform flow cytometric analysis.

Folate plays a crucial and indispensible role in cell division so it is not surprising that it is important in pregnancy. This importance was first highlighted by the historical observation made by Lucy Wills in India1 that the latter stages of pregnancy are frequently associated with a megaloblastic anaemia that is folate responsive, i.e. in pregnancy not only is extra folate needed but it is frequently lacking. Subsequent studies over the years have found that folate deficiency in the second and particularly the third trimester is common in some countries and quite rare in others. The determining feature was found to be adequate folate nutrition for the mother not only during pregnancy but before the pregnancy began2. The most often suggested mechanism for folate deficiency in pregnancy is increased requirement for the rapidly growing fetus and placenta. However the mechanism is unclear and certainly cannot be accounted for by transfer of the vitamin to the fetus. One possible explanation is that the events of fetal/placental growth cause an increase in the rate of catabolism of the vitamin. Early studies in our laboratory demonstrated that the mechanism of folate catabolism in the rat was cleavage of the C9-N10 bond with excretion of a mixture of pteridines and p-aminobenzoylglutamate (pABGlu) with the latter being largely acetylated to acetamidobenzoylglutamate (apABGlu)3. Subsequent studies by us in the rat4 and by Krumdieck5 in man confirmed this mechanism. These studies used radioactive tracers. They had the disadvantage that they assumed that the exogenous tracer would equilibrate with the endogenous pool given sufficient time. In addition while they could be used to compare rates of catabolism in specific circumstances between animals, say treated with convulsant drugs and controls6, they did not measure true endogenous rates of catabolism. Furthermore, since they used radioactive tracers they were unsuitable for routine studies on humans.

The accuracy of non-invasive radionuclide angiography in detecting portal vein occlusion was assessed in 61 patients--10 with portal vein occlusion confirmed by conventional portography, 25 with chronic liver disease and a patent portal vein (mild = 12, severe = 13), and 26 with normal liver function, who served as controls. The median percentage portal venous flow for the portal vein occlusion group was 8% (range 1-30) (consistent with negligible flow) compared with 78% (52-87) for control subjects (p < 0.005) and 68% (61-80) and 49% (23-59) respectively for patients with mild and severe liver disease (p < 0.001 and p < 0.005). At a portal venous inflow of < 20%, the procedure had a specificity of 100% and sensitivity of 90% in diagnosing portal vein occlusion. Non-invasive radionuclide angiography provides a safe and accurate screening method for evaluating portal vein patency or occlusion in the investigation of portal hypertension or before liver transplantation.

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.

The myelopathy associated with human immunodeficiency virus (HIV) infection closely resembles that in subacute combined degeneration, a disorder of vitamin B12 metabolism. To investigate whether the disorders share a pathogenetic mechanism, S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) were measured in the cerebrospinal fluid (CSF) of 20 HIV-seropositive patients and 30 HIV-negative patients who were undergoing lumbar puncture for other medical reasons. The HIV-seropositive patients had significantly lower CSF concentrations of SAM (mean 77 [SD 25] vs 131 [35] nmol/l; p less than 0.001) and significantly higher concentrations of SAH (30.5 [6.8] vs 19.0 [7.1] nmol/l; p less than 0.001) than the controls. There was therefore a significant difference between the groups in the SAM/SAH (methylation) ratio (HIV 2.7 [1.0] vs control 7.6 [3.4]; p less than 0.001). There were no correlations between SAM or SAH concentrations or methylation ratio and age or sex in both groups, or serum B12 and folate concentrations, CSF folate, serum or CSF methylmalonic acid, risk factors, body mass index, specific drug treatment received, or disease stage in the HIV group. This finding suggests that HIV affects the brain from a very early stage of the infection. We suggest that, as in the pig, the CSF methylation ratio closely reflects that in the brain. In HIV-infected patients a reduced brain methylation ratio would inhibit methyltransferase enzymes, which would lead to hypomethylation in the central nervous system and ultimately to neurological lesions. In a pig model of subacute combined degeneration and in vitamin-B12-deficient human beings, the primary cause of the low methylation ratio is impaired recycling of SAH back to SAM, a process which requires vitamin-B12-dependent methionine synthase. The HIV patients in this study were vitamin B12 and folate replete, which suggests a different cause for the low methylation ratio.

We investigated polymorphism of HLA-DP genes in three DR3 related diseases, confirming an association of coeliac disease with a Bgl II DP alpha polymorphism (a restriction fragment sized 3.5 kb present in 75% of patients compared to 34% of control subjects, p less than 0.001), and finding a weaker association with dermatitis herpetiformis (57% v 34%, p = 0.01) and no association with insulin dependent diabetes mellitus. The association with coeliac disease was further investigated. Msp I DP beta polymorphism was studied in 52 healthy subjects and 59 patients: a 4.9 kb fragment was present in 51% of patients with coeliac disease compared to 11.5% of control subjects (p less than 0.001). Furthermore, nearly all subjects with the DP alpha 3.5 kb fragment also had the DP beta 4.9 kb fragment. However, disease frequency was still increased in the DP alpha 3.5 positive/DP beta 4.9 negative group. In seven families, each with at least two affected members, while the DP alpha 3.5 fragment was frequently present in patients it did not preferentially segregate with any particular HLA haplotype--for example, those associated with DR3 or DR7--and therefore is not part of an extended haplotype associated with coeliac disease. We therefore conclude that a gene(s) in the HLA-DP region predisposes to coeliac disease independently of the HLA-DR/DQ regions.

Chemiluminescence (CL) is a simple quantitative assay of polymorphonuclear leucocyte (PMNL) oxidative metabolism. PMNL CL was found to be significantly higher in patients with chronic inflammatory bowel disease than in normal controls (167 +/- 60 vs. 139 +/- 50 mV/10(5) cells, p less than 0.05). There were no significant differences between patients with ulcerative colitis and Crohn's disease. Disease controls with rheumatoid arthritis and with bronchiectasis also demonstrated elevated CL. These results were obtained using a two-step gelatin/Ficoll-Hypaque procedure for PMNL separation. However when PMNLs were prepared using a one-step Ficoll-Hypaque procedure PMNL CL was found to be depressed in chronic inflammatory bowel disease (CIBD). It was demonstrated that this disparity was caused by the elimination of low-density neutrophils with high CL production by the one-step procedure. These data indicate that reports of abnormal in vitro neutrophil function in CIBD should be interpreted with caution since separation techniques which are satisfactory in normal individuals may significantly influence results in patients with inflammatory diseases. Furthermore these data indicate the presence of a subpopulation of activated low-density PMNL in patients with CIBD.

GENETIC FACTORS. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . .. . . . . . .. . . . . . . .. . . . . . . . . 278 ENVIRONMENTAL FACTORS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . 279 Nutrient Excess 282 Nutrient Deficiency...... 283

A 48 year old patient with resistant coeliac disease developed prolonged unexplained pyrexia after surgery for small bowel volvulus. Despite extensive investigations and intensive antibiotic therapy, he deteriorated and died eight weeks postoperatively and significant isolated pulmonary valve endocarditis was discovered at autopsy. This diagnosis should be considered in all critically ill patients with unexplained pyrexia even in the absence of clinical features of endocarditis and transoesophageal echocardiography performed to exclude or confirm this lesion.

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.