Quantitation of circulating wild-type alpha-1-antitrypsin in heterozygous carriers of the S and Z deficiency alleles.

BACKGROUND: Alpha-1-antitrypsin (A1AT) deficiency disease results from mutations in the A1AT gene. Controversy exists in regards to treatment of heterozygous carriers of the S and Z deficiency alleles. Quantitation of allelic expression has not been possible with standard laboratory methods. Here we show that the recently described method for liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis of A1AT tryptic peptides can differentiate between mutated (S and Z) and wild-type (non-S and non-Z) proteins allowing for quantitation of circulating allelic expression in heterozygous patients. METHODS: Serum (244 M/M, 61 M/Z, and 63 M/S) was combined with isotopically labeled peptide standards, digested with trypsin, and quantitated by LC-MS/MS. Total and allele-specific A1AT quantitation was performed by comparison of peptide peak height ratios to a standard curve for each peptide. Linear regression was used to compare results and central 95(th) percentile intervals were calculated using parametric analysis. RESULTS: Quantitation of circulating wild-type A1AT based on the proteotypic and allelic (non-S and non-Z) peptides was validated in M/M patients. Proteotypic peptide concentrations correlated linearly with quantitation by non-Z and non-S peptides [slopes (Spearman correlation coefficient) of 1.09 (0.89) and 0.98 (0.80), respectively]. Allele-specific quantitation showed significant differences in wild-type protein expression in M/Z and M/S patients. Although average total A1AT concentration was lower for M/Z patients, the percentage of wild-type protein in M/Z patients was significantly higher at 82 % (55- > 95 %) compared to 63 % (43-83 %) for M/S heterozygotes. In a cohort of M/Z patients with sufficient total A1AT (≥80 mg/dL), half had insufficient wild-type protein that could have clinical implications for pulmonary dysfunction. CONCLUSIONS: For the first time, a method to quantitate A1AT allele protein expression is described. Given the wide range of circulating wild-type protein observed in heterozygous patients, this method has the potential to reveal correlations between allele concentration and development and/or severity of clinical symptoms.

Effect of colesevelam on faecal bile acids and bowel functions in diarrhoea-predominant irritable bowel syndrome.

BACKGROUND: About one-third of patients with IBS-diarrhoea (irritable bowel syndrome-D) have evidence of increased bile acid synthesis or excretion. AIMS: To assess effects of the bile acid sequestrant, colesevelam, on faecal excretion of BAs, hepatic BA synthesis and diarrhoea in IBS-D; to appraise whether individual or random stool samples accurately reflect 48-h total faecal bile acid excretion and proportions of the main bile acids excreted and to study the faecal fat excretion in response to colesevelam. METHODS: A single-centre, unblinded, single-dose trial of effects of colesevelam, 1875 mg [3 tablets (625 mg tablets)] orally, twice daily, for 10 days on total 48-h faecal bile acid excretion and fasting serum C4 (7α-hydroxy-4-cholesten-3-one; surrogate of hepatic bile acid synthesis). Stool diaries documented bowel functions for 8 days prior and 8 days during colesevelam treatment. Stool 48-h samples and fasting serum were collected for faecal fat, faecal bile acid and serum C4. RESULTS: Colesevelam was associated with significantly increased faecal total bile acid excretion and deoxycholic acid excretion, increased serum C4 and more solid stool consistency. There was a significant inverse correlation between number of bowel movements per week and the total bile acid sequestered into stool during the last 48 h of treatment. Random stool samples did not accurately reflect 48-h total or individual faecal bile acid excretion. Sequestration of bile acids by colesevelam did not increase faecal fat. CONCLUSIONS: Colesevelam increases delivery of bile acids to stool while improving stool consistency, and increases hepatic bile acid synthesis, avoiding steatorrhoea in patients with IBS-D. Overall effects are consistent with luminal bile acid sequestration by colesevelam.

Validating biomarkers of treatable mechanisms in irritable bowel syndrome.

BACKGROUND: A valid biomarker is 'an indicator of normal biologic or pathogenic processes, or pharmacological responses to a therapeutic intervention'. There is no validated biomarker for irritable bowel syndrome (IBS). The aim of the study was to assess ability of three quantitative traits to identify treatable processes to discriminate between IBS-diarrhea (IBS-D) patients, IBS-constipation (IBS-C) patients and healthy volunteers (HV). METHODS: In 30 HV, 30 IBS-C patients and 64 IBS-D patients, we characterized bowel symptoms and quantitated pathophysiological mechanisms: bile acid (BA) synthesis (serum C4 and FGF19), fecal BA and fat, colonic transit (CT), and intestinal permeability (IP). We used multiple logistic regression and receiver-operating characteristic (ROCAUC ) to appraise three factors (fecal BA, CT, and IP) individually and in combination to identify discriminant targets for treatment in IBS. KEY RESULTS: There were significant associations between the three subgroups and symptoms reflecting bowel function and the quantitative traits. There were significant associations between fecal BA and CT at 48 h (r = 0.43; p < 0.001) and between fecal BA and IP (r = 0.23; p = 0.015). Individually, fecal BA and CT48 (but not IP) were significant independent predictors for distinguishing HV from IBS. In combination, they discriminated HV from IBS-D patients (ROCAUC 0.70), HV from IBS-C patients (ROCAUC 0.73), and IBS-C patients from IBS-D patients (ROCAUC 0.86). Colonic transit and fecal BA excretion together discriminate between healthy volunteers and IBS-C patients or IBS-D patients, or between the IBS subgroups with 75-90% specificity at 60% sensitivity. CONCLUSIONS & INFERENCES: Colonic transit and fecal BA individually and together constitute useful biomarkers to identify treatable mechanisms in IBS and to differentiate subgroups of IBS.