Measurement of serum 7α‐hydroxy‐4‐cholesten‐3‐one (or 7αC4), a surrogate test for bile acid malabsorption in health, ileal disease and irritable bowel syndrome using liquid chromatography‐tandem mass spectrometry

Abstract

Background Bile acid malabsorption (BAM) is reported in up to 50% of patients with functional diarrhea and irritable bowel syndrome with diarrhea (IBS-D). Serum 7α-hydroxy-4-cholesten-3-one (7αHCO, or 7αC4), an indirect measurement of hepatic bile acid synthesis, has been validated as a measurement of BAM relative to the 75SeHCAT retention test. Aim To develop a serum 7αC4 assay, normal values, and compare results from healthy controls, patients with ileal Crohn’s disease or resection, and patients with IBS-D or IBS with constipation (IBS-C). Methods Stored serum samples were used from adult men and women in the following groups: 111 normal healthy controls, 15 IBS-D, 15 IBS-C, 24 with distal ileal Crohn’s disease, and 20 with distal ileal resection for Crohn’s disease. We adapted a published high pressure liquid chromatography, tandem mass spectrometry (HPLC-MS/MS) assay. Results The HPLC-MS/MS assay showed good linearity in concentration range 0–200 ng/mL, sensitivity (lowest limit of detection 0.04 ng/mL), and high analytical recovery (average 99%, range 93–107%). The 5th to 95th percentile for 111 normal healthy controls was 6–60.7 ng/mL. There were significant overall group differences (ANOVA on ranks p<0.001), with significantly higher values for terminal ileal disease or resection. There were significant differences between health and IBS (ANOVA p=0.043) with higher mean values in IBS-D relative to controls (rank sum test, p=0.027). Conclusions We have established a sensitive non-isotopic assay based on HPLC-MS/MS, determined normal 7αC4 values, and identified increased 7αC4 in IBS-D and in distal ileal resection and disease. This assay has potential as a noninvasive test for BAM in IBS.

Quantification of the 15 major human bile acids and their precursor 7α-hydroxy-4-cholesten-3-one in serum by liquid chromatography–tandem mass spectrometry

“…Extraction recoveries for bile acids were quite similar to those reported for other existing methods [16][17][18]20,[22][23][24]. The extraction recovery for C4 was found to be approximately 62%, which was markedly lower than for bile acids and for other C4 methods [12,14,[25][26]. This is presumably explained by its lipophilic properties and by the fact that the others methods used different sample preparation techniques (protein precipitation, liquid-liquid extraction followed by SPE, derivatisation and salting-out respectively).…”

“…Moreover, it is now well established that FXR activation by bile acids results in the regulation of various genes involved not solely in bile acid homeostasis but also in cholesterol, triglyceride and glucose metabolism [4][5][6][7][8][9]. Therefore, bile acids have gained much attention since they might be of interest as biomarkers or as pharmacological targets in several intestinal and metabolic conditions ranging from Crohn's disease to metabolic syndrome [5,[10][11][12].…”

“…In contrast to gas chromatography, which requires laborious sample preparation including hydrolysis and derivatisation, liquid chromatography coupled with mass spectrometry (LC-MS(/MS)) nowadays represents the most convenient alternative for this type of analysis, as demonstrated by the number of new quantification methods developed in the past years for bile acids [16][17][18][19][20][21][22][23][24] and C4 [12,14,[25][26].…”

“…Our new methods each required only 100μl of serum sample, which was less than for many of the existing methods [12,14,16,20,[22][23][24] although methods for quantification of C4 in even smaller sample volumes exist [25][26]. A low sample volume represents an important advantage if the method is to be adapted for analysis of mouse samples (muricholic acids), since mice have a very small blood volume.…”

Effect of cholic acid on colonic motility in mice

“…Bile acid malabsorption (BAM) is generally not regarded as a cause of IBS. However, recent improvements in the techniques employed for assessing BAM have demonstrated that it may promote and contribute to a low-grade mucosal inflammation in IBS (Camilleri et al, 2009) and diarrhea in patients with inflammatory bowel disease symptoms (IBD) (Surawicz, 2010). Although the cell types that mediate these actions of BAs have not been identified, studies using neurotoxins and neurotransmitter antagonists suggest that BAs control motility and secretion through effects on the enteric nervous system (Alemi et al, 2013).…”