Lifestyle in patients at increased risk of colorectal cancer.

BACKGROUND: The present study aimed to assess modifiable risk factors in patients at high risk for colorectal cancer (CRC) and their experience of lifestyle advice. METHODS: A questionnaire study was conducted in high-risk CRC patients attending for surveillance colonoscopy. Current lifestyle behaviours [smoking, alcohol, diet (fruit and vegetables, wholegrains, red meat, processed meat), physical activity and bodyweight] related to CRC were ascertained, and experience on receiving, seeking and desire for advice was queried. RESULTS: In total, 385 study invitations were sent and 208 (54%) questionnaires were returned. The majority of participants (72%) were estimated to have a body mass index beyond the healthy range, 89% achieved a fibre score indicative of a low plant-based diet and 91% reported eating processed meat. Overall, 36% were achieving at least four recommendations and 2% were adhering to all recommendations examined. The main area in which participants reported receiving advice on was body weight (33%) and 31% reported that they had personally sought information on this topic, although the data suggest that 72% of people may benefit from such guidance. Fewer participants reported receiving (18-26%) and seeking (15-17%) dietary advice on fruits, vegetables and wholegrains. Many participants said they would find lifestyle information useful, notably in relation to body fatness (43%) and physical activity (38%). CONCLUSIONS: The development of a process for supporting lifestyle change in this patient group, comprising individuals who are already engaging in positive health practices (regular colonoscopy surveillance), could usefully be identified and tested.

Real-world Effectiveness and Safety of Vedolizumab for the Treatment of Inflammatory Bowel Disease: The Scottish Vedolizumab Cohort.

BACKGROUND & AIMS: Vedolizumab is an anti-a4b7 monoclonal antibody that is licensed for the treatment of moderate to severe Crohn's disease and ulcerative colitis. The aims of this study were to establish the real-world effectiveness and safety of vedolizumab for the treatment of inflammatory bowel disease. METHODS: This was a retrospective study involving seven NHS health boards in Scotland between June 2015 and November 2017. Inclusion criteria included: a diagnosis of ulcerative colitis or Crohn's disease with objective evidence of active inflammation at baseline (Harvey-Bradshaw Index[HBI] ≥5/Partial Mayo ≥2 plus C-reactive protein [CRP] >5 mg/L or faecal calprotectin ≥250 µg/g or inflammation on endoscopy/magnetic resonance imaging [MRI]); completion of induction; and at least one clinical follow-up by 12 months. Kaplan-Meier survival analysis was used to establish 12-month cumulative rates of clinical remission, mucosal healing, and deep remission [clinical remission plus mucosal healing]. Rates of serious adverse events were described quantitatively. RESULTS: Our cohort consisted of 180 patients with ulcerative colitis and 260 with Crohn's disease. Combined median follow-up was 52 weeks (interquartile range [IQR] 26-52 weeks). In ulcerative colitis, 12-month cumulative rates of clinical remission, mucosal healing, and deep remission were 57.4%, 47.3%, and 38.5%, respectively. In Crohn's disease, 12-month cumulative rates of clinical remission, mucosal healing, and deep remission were 58.4%, 38.9%, and 28.3% respectively. The serious adverse event rate was 15.6 per 100 patient-years of follow-up. CONCLUSIONS: Vedolizumab is a safe and effective treatment for achieving both clinical remission and mucosal healing in ulcerative colitis and Crohn's disease.

Probing the location of displayed cytochrome b562 on amyloid by scanning tunnelling microscopy.

Amyloid fibres displaying cytochrome b562 were probed using scanning tunnelling microscopy (STM) in vacuo. The cytochromes are electron transfer proteins containing a haem cofactor and could, in principle, mediate electron transfer between the tip and the gold substrate. If the core fibres were insulating and electron transfer within the 3D haem network was detected, then the electron transport properties of the fibre could be controlled by genetic engineering. Three kinds of STM images were obtained. At a low bias (<1.5 V) the fibres appeared as regions of low conductivity with no evidence of cytochrome mediated electron transfer. At a high bias, stable peaks in tunnelling current were observed for all three fibre species containing haem and one species of fibre that did not contain haem. In images of this kind, some of the current peaks were collinear and spaced around 10 nm apart over ranges longer than 100 nm, but background monomers complicate interpretation. Images of the third kind were rare (1 in 150 fibres); in these, fully conducting structures with the approximate dimensions of fibres were observed, suggesting the possibility of an intermittent conduction mechanism, for which a precedent exists in DNA. To test the conductivity, some fibres were immobilized with sputtered gold, and no evidence of conduction between the grains of gold was seen. In control experiments, a variation of monomeric cytochrome b562 was not detected by STM, which was attributed to low adhesion, whereas a monomeric multi-haem protein, GSU1996, was readily imaged. We conclude that the fibre superstructure may be intermittently conducting, that the cytochromes have been seen within the fibres and that they are too far apart for detectable current flow between sites to occur. We predict that GSU1996, being 10 nm long, is more likely to mediate successful electron transfer along the fibre as well as being more readily detectable when displayed from amyloid.

Multicentre comparison of the Glasgow Blatchford and Rockall Scores in the prediction of clinical end-points after upper gastrointestinal haemorrhage.

BACKGROUND: The Glasgow Blatchford Score (GBS) is increasingly being used to predict intervention and outcome following upper gastrointestinal haemorrhage (UGIH). AIM: To compare the GBS with both the admission and full Rockall scores in predicting specific clinical end-points following UGIH. PATIENTS AND METHODS: Data on consecutive patients presenting to four UK hospitals were collected. Admission history, clinical and laboratory data, endoscopic findings, treatment and clinical follow-up were recorded. Using ROC curves, we compared the three scores in the prediction of death, endoscopic or surgical intervention and transfusion. Results A total of 1555 patients (mean age 56.7years) presented with UGIH during the study period. Seventy-four (4.8%) died, 223 (14.3%) had endoscopic or surgical intervention and 363 (23.3%) required transfusion. The GBS was similar at predicting death compared with both the admission Rockall (area under ROC curve 0.804 vs. 0.801) and full Rockall score (AUROC 0.741 vs. 0.790). In predicting endo-surgical intervention, the GBS was superior to the admission Rockall (AUROC 0.858 vs. 0.705; P<0.00005) and similar to the full Rockall score (AUROC 0.822 vs. 0.797). The GBS was superior to both admission Rockall (AUROC 0.944 vs. 0.756; P<0.00005) and full Rockall scores (AUROC 0.935 vs. 0.792; P<0.00005) in predicting need for transfusion. CONCLUSIONS: Despite not incorporating age, the GBS is as effective as the admission and full Rockall scores in predicting death after UGIH. It is superior to both the admission and full Rockall scores in predicting need for transfusion, and superior to the admission Rockall score in predicting endoscopic or surgical intervention.

Magnetic resonance follow-through imaging for evaluation of disease activity in ileal Crohn's disease: an observational, retrospective cohort study.

BACKGROUND: Magnetic resonance follow-through (MRFT) is a new cross-sectional imaging modality with the potential to accurately stage ileal Crohn's disease (CD), while avoiding ionizing radiation and the discomfort associated with enteroclysis. We aimed to assess the reliability of this technique in assessing the extent and activity of ileal CD, and to assess its influence on subsequent management. METHODS: Out of a total of 342 patients undergoing MRFT between 2004 and 2008, 221 were performed in 191 patients with confirmed CD. Case notes were reviewed in detail with documentation of all investigations pre- and post-MRFT. Agreement between inflammatory markers, histopathology, and MRFT findings was determined. RESULTS: Overall, 116/221 (52.5%) of MRFTs showed active ileal CD, and 76/221 (34.4%) quiescent CD, while 29/221 (13.1%) were suboptimal. Overall, 66 strictures and 18 fistulae were identified. There was substantial agreement between active ileal CD on MRFT and histopathology (n = 59; kappa = 0.66; P = 0.0006; sensitivity 85.1%, specificity 85.7%) and fecal calprotectin (n = 14; kappa = 0.72; P = 0.047), while C-reactive protein (CRP) showed moderate agreement (n = 107; kappa = 0.402; P = 0.00028). Management was influenced by MRFT reports following active (52/84, 62% treated medically) or quiescent (48/62, 77.4% managed conservatively) disease. Fibrotic strictures were predominantly treated surgically (7/14, 50%). In all, 13/32 (40.6%) patients with inflammatory ileal strictures required surgery, mostly due to steroid-resistant disease. Overall, 75 MR findings were documented in 221 MRFTs, including 1 renal cancer. CONCLUSIONS: MRFT provides accurate information on ileal CD activity, with close agreement to inflammatory markers and histopathology. It represents a substantial advance in the staging of CD, while avoiding painful enteroclysis and radiation exposure in young patients.

Efficacy and complications of adalimumab treatment for medically-refractory Crohn's disease: analysis of nationwide experience in Scotland (2004-2008).

BACKGROUND: Adalimumab is a second generation humanized anti-tumour necrosis factor (TNF) monoclonal antibody with established efficacy in Crohn's disease (CD). AIMS: To evaluate the efficacy and safety of adalimumab on a nationwide clinical setting. METHODS: We used the Scottish Society of Gastroenterology network to identify and follow up the clinical outcomes of patients with CD treated with adalimumab over a 4-year period (2004-2008). RESULTS: A total of 98 patients received adalimumab - 100.5 patient follow-up years were recorded (64.3% females; median age at diagnosis of 20.7 years; 88.8% treated with 80/40 mg induction regimen. Eighty eight (89.8%) had previous infliximab with 29 (32.9%) primary nonresponders; 32 (32.6%) were corticosteroid-dependent; 47 (47.9%) were intolerant/resistant to most immunosuppressive therapies (two or more). In all, 60% of patients were in clinical remission at 1-year follow-up, with 30% and 55% requiring dose escalation to weekly therapy at 1-and 2-year follow-up respectively. Overall, 29 (29.6%) patients developed complications with eight nonfatal serious (8.2%) adverse events and 2 (2.0%) case fatalities (sepsis following perforation and disseminated colorectal cancer, respectively). CONCLUSIONS: Adalimumab is efficacious in severe and refractory CD in the clinical setting, although there remain significant therapy- and disease-related risks of serious complications.

Outpatient management of patients with low-risk upper-gastrointestinal haemorrhage: multicentre validation and prospective evaluation.

BACKGROUND: Upper-gastrointestinal haemorrhage is a frequent reason for hospital admission. Although most risk scoring systems for this disorder incorporate endoscopic findings, the Glasgow-Blatchford bleeding score (GBS) is based on simple clinical and laboratory variables; a score of 0 identifies low-risk patients who might be suitable for outpatient management. We aimed to evaluate the GBS then assess the effect of a protocol based on this score for non-admission of low-risk individuals. METHODS: Our study was undertaken at four hospitals in the UK. We calculated GBS and admission (pre-endoscopy) and full (post-endoscopy) Rockall scores for consecutive patients presenting with upper-gastrointestinal haemorrhage. With receiver-operating characteristic (ROC) curves, we compared the ability of these scores to predict either need for clinical intervention or death. We then prospectively assessed at two hospitals the introduction of GBS scoring to avoid admission of low-risk patients. FINDINGS: Of 676 people presenting with upper-gastrointestinal haemorrhage, we identified 105 (16%) who scored 0 on the GBS. For prediction of need for intervention or death, GBS (area under ROC curve 0.90 [95% CI 0.88-0.93]) was superior to full Rockall score (0.81 [0.77-0.84]), which in turn was better than the admission Rockall score (0.70 [0.65-0.75]). When introduced into clinical practice, 123 patients (22%) with upper-gastrointestinal haemorrhage were classified as low risk, of whom 84 (68%) were managed as outpatients without adverse events. The proportion of individuals with this condition admitted to hospital also fell (96% to 71%, p<0.00001). INTERPRETATION: The GBS identifies many patients presenting to general hospitals with upper-gastrointestinal haemorrhage who can be managed safely as outpatients. This score reduces admissions for this condition, allowing more appropriate use of in-patient resources.

A study of the safety of current gastrointestinal endoscopy (EGD).

BACKGROUND AND STUDY AIMS: Previous attempts at assessing the safety of upper gastrointestinal endoscopy have been hampered by incomplete data collection. We aimed to assess the 30-day mortality associated with esophagogastroduodenoscopy (EGD) and assess the important risk factors. PATIENTS AND METHODS: A retrospective cohort study was conducted of patients who underwent endoscopy at Ninewells Hospital in Dundee between 1 June 2000 and 31 May 2003. A total of 11 501 EGDs were performed in 8926 patients. These patients were record-linked to the death registry and the database of hospital admissions in order to calculate the all-cause 30-day mortality. An expert panel judged whether EGD had caused or contributed to the deaths. Logistic regression analysis was performed on outcomes of all-cause and EGD-contributed mortality. RESULTS: The median age of the patients was 62 years (interquartile range 48 - 74 years), 54 % were women, and 94 % of procedures were diagnostic. A total of 395 patients died within 30 days (all-cause 30-day mortality rate 4.4 %). One patient death was caused directly by the EGD (procedure-caused mortality rate 1 in 9000). EGD was judged to have contributed to patient deaths at a rate of 1 in 182, based on majority agreement of experts: some factors associated with these deaths were percutaneous endoscopic gastrostomy insertion (odds ratio [OR] 18.39, 95 % confidence interval [CI] 5.71 - 59.22), melena or hematemesis indications (OR 9.01, 95 % CI 3.53 - 22.99), and esophageal varices (OR 6.28, 95 % CI 1.54 - 25.60). CONCLUSIONS: A causal death rate of 1 in 9000 suggests that EGD is very safe. However, certain patient groups have an increased mortality, and the risks and benefits of EGD should be carefully evaluated in each patient.

A retrospective analysis of the efficacy and safety of infliximab as rescue therapy in acute severe ulcerative colitis.

BACKGROUND: Forty per cent of patients with acute severe ulcerative colitis will not respond to intravenous corticosteroids and require second-line medical therapy or colectomy. A recent controlled trial has suggested that infliximab may be effective as rescue therapy. AIM: To assess the value of infliximab as rescue therapy for acute severe colitis in a retrospective cohort of ulcerative colitis patients in Scotland. METHODS: All patients satisfied Truelove and Witts criteria on admission, failed to respond to intravenous corticosteroids and received infliximab (5 mg/kg) as rescue therapy. Response was defined as need for colectomy at hospital discharge and by 90 days. RESULTS: A total of 39 patients (median age 31.7 years) were treated. 26/39 (66%) responded, avoiding colectomy during the acute admission, and were followed up for a median of 203 days (Interquartile range = 135.5-328.5). Hypoalbuminaemia was a consistent predictor of non-response on univariate and multivariate analysis. At day 3 of intravenous steroids, 9/18 (50.0%) with serum albumin <34 g/L had urgent colectomy vs. 1/13 (7.7%) >or=34 g/L (P = 0.02, OR = 12.0, C.I. 1.28-112.7). Two serious adverse events occurred - one death due to Pseudomonas pneumonia, and one post-operative fungal septicaemia. CONCLUSIONS: Infliximab represents a moderately effective rescue therapy for patients with acute severe ulcerative colitis. Serious adverse events, including death, do occur and should be discussed with patients prior to therapy.

Predicting the outcome of severe ulcerative colitis: development of a novel risk score to aid early selection of patients for second-line medical therapy or surgery.

BACKGROUND: The failure rate of medical therapy in severe ulcerative colitis is high. A risk index, to aid the identification of patients of not responding at an early stage to intravenous corticosteroid therapy, would be useful to facilitate second-line treatment or surgery. METHODS: We recruited 167 consecutive patients with severe ulcerative colitis between January 1995 and March 2002; and employed multiple logistic regression to analyse parameters within the first 3 days of medical therapy. We applied statistical modelling to formulate a risk score according to the likelihood of medical failure. RESULTS: Sixty-seven (40%) patients failed to respond to medical therapy. Multiple logistic regression analysis identified mean stool frequency and colonic dilatation within the first 3 days and hypoalbuminaemia as independent predictors of outcome (P < 0.001, 0.001 and 0.002 respectively). A numerical risk score was formulated based on these variables. Patients with scores of 0-1, 2-3 and > or =4 had a medical therapy failure rate of 11%, 43% and 85% respectively. Receiver-operator characteristic analysis of this score yielded area under curve of 0.88, with a sensitivity of 85% and specificity of 75% using score > or =4 in predicting non-response. CONCLUSION: This risk score allows the early identification of patients with severe ulcerative colitis who would be suitable for second-line medical therapy or surgery.

Randomised trial of endoscopy with testing for Helicobacter pylori compared with non-invasive H pylori testing alone in the management of dyspepsia.

OBJECTIVE: To compare the efficacy of non-invasive testing for Helicobacter pylori with that of endoscopy (plus H pylori testing) in the management of patients referred for endoscopic investigation of upper gastrointestinal symptoms. DESIGN: Randomised controlled trial with follow up at 12 months. SETTING: Hospital gastroenterology unit. PARTICIPANTS: 708 patients aged under 55 referred for endoscopic investigation of dyspepsia, randomised to non-invasive breath test for H pylori or endoscopy plus H pylori testing. MAIN OUTCOME MEASURE: Glasgow dyspepsia severity score at one year. Use of medical resources, patient oriented outcomes, and safety were also assessed. RESULTS: In 586 patients followed up at 12 months the mean change in dyspepsia score was 4.8 in the non-invasive H pylori test group and 4.6 in the endoscopy group (95% confidence interval for difference -0.7 to 0.5, P=0.69). Only 8.2% of patients followed up who were randomised to breath test alone were referred for subsequent endoscopy. The use of non-endoscopic resources was similar in the two groups. Reassurance value, concern about missed pathology, overall patient satisfaction, and quality of life were similar in the two groups. The patients found the non-invasive breath test procedure less uncomfortable and distressing than endoscopy with or without sedation. No potentially serious pathology requiring treatment other than eradication of H pylori was missed. CONCLUSION: In this patient group, non-invasive testing for H pylori is as effective and safe as endoscopy and less uncomfortable and distressing for the patient. Non-invasive H pylori testing should be the preferred mode of investigation.

In vitro studies indicate that acid catalysed generation of N-nitrosocompounds from dietary nitrate will be maximal at the gastro-oesophageal junction and cardia.

BACKGROUND: Dietary nitrate increases saliva nitrite levels and swallowed saliva is the main source of nitrite entering the acidic stomach. In acidic gastric juice, this nitrite can generate potentially carcinogenic N-nitrosocompounds. However, ascorbic acid secreted by the gastric mucosa can prevent nitrosation by converting the nitrite to nitric oxide. METHODS: To study the potential for N-nitrosocompound formation in a model simulating salivary nitrite entering the acidic stomach and the ability of ascorbic acid to inhibit the process. Concentrations of ascorbic acid, total vitamin C, nitrite, nitrosomorpholine, oxygen and nitric oxide were monitored during the experiments. RESULTS: The delivery of nitrite into HCl containing thiocyanate resulted in nitrosation of morpholine, with the rate of nitrosation being greatest at pH 2.5. Under anaerobic conditions, ascorbic acid converted the nitrite to nitric oxide and prevented nitrosation. However, in the presence of dissolved air, the ascorbic acid was ineffective at preventing nitrosation. This was due to the nitric oxide combining with oxygen to reform nitrite and this recycling of nitrite depleting the available ascorbic acid. Further studies indicated that the rate of consumption of ascorbic acid by nitrite added to natural human gastric juice (pH 1.5) was extremely rapid with 200 micromol/l nitrite consumed 500 micromol/l ascorbic acid within 10 s. CONCLUSIONS: The rapid consumption of ascorbic acid in acidic gastric juice by nitrite in swallowed saliva indicates that the potential for acid nitrosation will be maximal at the GO junction and cardia where nitrite first encounters acidic gastric juice. The high incidence of mutagenesis and neoplasia at this anatomical location may be due to acid nitrosation arising from dietary nitrate.

Review article: spontaneous bacterial peritonitis--diagnosis, treatment and prevention.

Spontaneous bacterial peritonitis is a serious complication of cirrhotic ascites, arising most frequently in those with advanced liver disease. Its development leads to a further reduction in the effective arterial blood volume, and it has a mortality rate equivalent to that of a variceal bleed. However, problems remain with regard to the identification and optimal treatment of spontaneous bacterial peritonitis. Several important studies and consensus documents on the condition have recently been published which aid in the identification of patients at risk and help to guide therapy. In this review, we discuss these publications and address the issues of diagnosis, treatment and both primary and secondary prophylaxis of spontaneous bacterial peritonitis in the light of recent data.

Structural and spectroscopic analysis of the F393H mutant of flavocytochrome P450 BM3.

In the preceding paper in this issue [Ost, T. W. B., Miles, C. S., Munro, A. W., Murdoch, J., Reid, G. A., and Chapman, S. K. (2001) Biochemistry 40, 13421-13429], we have established that the primary role of the phylogenetically conserved phenylalanine in flavocytochrome P450 BM3 (F393) is to control the thermodynamic properties of the heme iron, so as to optimize electron-transfer both to the iron (from the flavin redox partner) and onto molecular oxygen. In this paper, we report a detailed study of the F393H mutant enzyme, designed to probe the structural, spectroscopic, and metabolic profile of the enzyme in an attempt to identify the factors responsible for causing the changes. The heme domain structure of the F393H mutant has been solved to 2.0 A resolution and demonstrates that the histidine replaces the phenylalanine in almost exactly the same conformation. A solvent water molecule is hydrogen bonded to the histidine, but there appears to be little other gross alteration in the environment of the heme. The F393H mutant displays an identical ferric EPR spectrum to wild-type, implying that the degree of splitting of the iron d orbitals is unaffected by the substitution, however, the overall energy of the d-orbitals have changed relative to each other. Magnetic CD studies show that the near-IR transition, diagnostic of heme ligation state, is red-shifted by 40 nm in F393H relative to wild-type P450 BM3, probably reflecting alteration in the strength of the iron-cysteinate bond. Studies of the catalytic turnover of fatty acid (myristate) confirms NADPH oxidation is tightly coupled to fatty acid oxidation in F393H, with a product profile very similar to wild-type. The results indicate that gross conformational changes do not account for the perturbations in the electronic features of the P450 BM3 heme system and that the structural environment on the proximal side of the P450 heme must be conformationally conserved in order to optimize catalytic function.

Kinetic and crystallographic analysis of the key active site acid/base arginine in a soluble fumarate reductase.

There is now overwhelming evidence supporting a common mechanism for fumarate reduction in the respiratory fumarate reductases. The X-ray structures of substrate-bound forms of these enzymes indicate that the substrate is well positioned to accept a hydride from FAD and a proton from an arginine side chain. Recent work on the enzyme from Shewanella frigidimarina [Doherty, M. K., Pealing, S. L., Miles, C. S., Moysey, R., Taylor, P., Walkinshaw, M. D., Reid, G. A., and Chapman, S. K. (2000) Biochemistry 39, 10695-10701] has strengthened the assignment of an arginine (Arg402) as the proton donor in fumarate reduction. Here we describe the crystallographic and kinetic analyses of the R402A, R402K, and R402Y mutant forms of the Shewanella enzyme. The crystal structure of the R402A mutant (2.0 A resolution) shows it to be virtually identical to the wild-type enzyme, apart from the fact that a water molecule occupies the position previously taken by part of the guanidine group of R402. Although structurally similar to the wild-type enzyme, the R402A mutant is inactive under all the conditions that were studied. This implies that a water molecule, in this position in the active site, cannot function as the proton donor for fumarate reduction. In contrast to the R402A mutation, both the R402K and R402Y mutant enzymes are active. Although this activity was at a very low level (at pH 7.2 some 10(4)-fold lower than that for the wild type), it does imply that both lysine and tyrosine can fulfill the role of an active site proton donor, albeit very poorly. The crystal structures of the R402K and R402Y mutant enzymes (2.0 A resolution) show that distances from the lysine and tyrosine side chains to the nearest carbon atom of fumarate are approximately 3.5 A, clearly permitting proton transfer. The combined results from mutagenesis, crystallographic, and kinetic studies provide formidable evidence that R402 acts as both a Lewis acid (stabilizing the build-up of negative charge upon hydride transfer from FAD to fumarate) and a Brønsted acid (donating the proton to the substrate to complete the formation of succinate).

Alterations in intragastric nitrite and vitamin C levels during acid inhibitory therapy.

Most nitrite entering the healthy acid-secreting stomach is derived from dietary nitrate. The latter is absorbed from the small intestine, 25% then being secreted by the salivary glands into the mouth. Buccal organisms subsequently convert 20% of this nitrate to nitrite. When this nitrite is swallowed, the ascorbic acid in the acidic gastric juice reduces it to nitric oxide, which is absorbed by the mucosa. In the process, the ascorbic acid is oxidized to dehydroascorbic acid. When the intragastric pH is elevated by powerful anti-secretory agents, this gastric chemistry is profoundly modified. At a neutral pH, the swallowed nitrite does not react with ascorbic acid but accumulates in the stomach. The level of nitrite in the gastric juice during treatment with anti-secretory medication is particularly high after a nitrate-containing meal. Powerful anti-secretory medication also lowers the intragastric concentration of ascorbic acid and total vitamin C, probably because of the relative instability of the vitamin at a higher pH. These changes in the intragastric concentrations of nitrite and ascorbic acid are most marked in Helicobacter pylori -infected subjects on proton pump inhibitor therapy. It is recognized that an elevated nitrite-to-ascorbic acid ratio predisposes to the formation of potentially carcinogenic N -nitroso compounds. It is, however, unclear at present whether such compounds are formed within the human stomach.

Changing the heme ligation in flavocytochrome b2: substitution of histidine-66 by cysteine.

Substitution by cysteine of one of the heme iron axial ligands (His66) of flavocytochrome b2 (L-lactate:cytochrome c oxidoreductase from Saccharomyces cerevisiae) has resulted in an enzyme (H66C-b2) which remains a competent L-lactate dehydrogenase (kcat 272+/-6 s(-1), L-lactate KM 0.60+/-0.06 mM, 25 degrees C, I 0.10, Tris-HCl, pH 7.5) but which has no cytochrome c reductase activity. As a result of the mutation, the reduction potential of the heme was found to be -265+5 mV, over 240 mV more negative than that of the wild-type enzyme, and therefore unable to be reduced by L-lactate. Surface-enhanced resonance Raman spectroscopy indicates similarities between the heme of H66C-b2 and those of cytochromes P450, with a nu4 band at 1,345 cm(-1) which is indicative of cysteine heme-iron ligation. In addition, EPR spectroscopy yields g-values at 2.33, 2.22 and 1.94, typical of low-spin ferric cytochromes P450, optical spectra show features between 600 and 900 nm which are characteristic of sulfur coordination of the heme iron, and MCD spectroscopy shows a blue-shifted NIR CT band relative to the wild-type, implying that the H66C-b2 heme is P450-like. Interestingly, EPR evidence also suggests that the second histidine heme-iron ligand (His43) is displaced in the mutant enzyme.

Omeprazole, Helicobacter pylori status, and alterations in the intragastric milieu facilitating bacterial N-nitrosation.

BACKGROUND & AIMS: Omeprazole produces greater acid inhibition in Helicobacter pylori-positive than -negative subjects. We investigated whether this is accompanied by more profound changes in the intragastric milieu that facilitates bacterial synthesis of N-nitroso compounds. METHODS: Gastric juice pH; nitrite, ascorbic acid, and total vitamin C concentrations; and colonization by other bacteria were examined before and during omeprazole treatment in subjects with and without H. pylori infection. Studies were performed in the fasting state and after consumption of 2 mmol nitrate (equivalent to a salad meal). RESULTS: Before omeprazole, H. pylori-positive and -negative subjects were similar for all parameters. During omeprazole, H. pylori-positive subjects had a higher intragastric pH (7.8 vs. 3.0; P < 0.00001) and greater colonization with non-H. pylori species (5 x 10(7) vs. 5 x 10(5) CFU/mL; P < 0.05). These bacteria included nitrosating species. During omeprazole treatment, H. pylori-positive subjects had higher intragastric nitrite levels after the nitrate meal (median area under the concentration/time curve, 12,450 vs. 4708 micromol/L. min; P = 0.04). Omeprazole lowered intragastric vitamin C levels in H. pylori-positive but not -negative subjects (1.8 vs. 3.4 microg/mL, respectively; P = 0.02). CONCLUSIONS: In H. pylori-positive subjects, omeprazole produces disturbances in intragastric nitrite, vitamin C, and bacterial colonization that facilitate bacterial N-nitrosation. This may place them at increased risk of mutagenesis and carcinogenesis.

Kinetic and crystallographic studies on the active site Arg289Lys mutant of flavocytochrome b2 (yeast L-lactate dehydrogenase).

Flavocytochrome b(2) from Saccharomyces cerevisiae couples L-lactate dehydrogenation to cytochrome c reduction. The crystal structure of the native yeast enzyme has been determined [Xia, Z.-X., and Mathews, F. S. (1990) J. Mol. Biol. 212, 837-863] as well as that of the sulfite adduct of the recombinant enzyme produced in Escherichia coli [Tegoni, M., and Cambillau, C. (1994) Protein Sci. 3, 303-313]; several key active site residues were identified. In the sulfite adduct crystal structure, Arg289 adopts two alternative conformations. In one of them, its side chain is stacked against that of Arg376, which interacts with the substrate; in the second orientation, the R289 side chain points toward the active site. This residue has now been mutated to lysine and the mutant enzyme, R289K-b(2), characterized kinetically. Under steady-state conditions, kinetic parameters (including the deuterium kinetic isotope effect) indicate the mutation affects k(cat) by a factor of about 10 and k(cat)/K(M) by up to nearly 10(2). Pre-steady-state kinetic analysis of flavin and heme reduction by lactate demonstrates that the latter is entirely limited by flavin reduction. Inhibition studies on R289K-b(2) with a range of compounds show a general rise in K(i) values relative to that of wild-type enzyme, in line with the elevation of the K(M) for L-lactate in R289K-b(2); they also show a change in the pattern of inhibition by pyruvate and oxalate, as well as a loss of the inhibition by excess substrate. Altogether, the kinetic studies indicate that the mutation has altered the first step of the catalytic cycle, namely, flavin reduction; they suggest that R289 plays a role both in Michaelis complex and transition-state stabilization, as well as in ligand binding to the active site when the flavin is in the semiquinone state. In addition, it appears that the mutation has not affected electron transfer from fully reduced flavin to heme, but may have slowed the second intramolecular ET step, namely, transfer from flavin semiquinone to heme b(2). Finally, the X-ray crystal structure of R289K-b(2), with sulfite bound at the active site, has been determined to 2.75 A resolution. The lysine side chain at position 289 is well-defined and in an orientation that corresponds approximately to one of the alternative conformations observed in the structure of the recombinant enzyme-sulfite complex [Tegoni, M., and Cambillau, C. (1994) Protein Sci. 3, 303-313]. Comparisons between the R289K-b(2) and wild-type structures allow the kinetic results to be interpreted in a structural context.