Altered bile acid metabolism in childhood functional constipation: inactivation of secretory bile acids by sulfation in a subset of patients.

OBJECTIVE: An elevated concentration in the colon of the primary bile acid chenodeoxycholic acid (CDCA) or the secondary bile acid deoxycholic acid (DCA) is known to induce water secretion, causing diarrhea. We hypothesized that of the many fecal bile acids, only CDCA and DCA function as endogenous laxatives; therefore, a decrease in their proportion may be a cause of childhood functional constipation. To test this possibility, fecal bile acid composition was determined in children with functional constipation and in nonconstipated control children. PATIENTS AND METHODS: Fecal samples were obtained from 207 children, 103 with functional constipation and 104 with normal bowel habits. Bile acid classes were determined by use of electrospray ionization-single ion monitoring-mass spectrometry (ESI-SIM-MS), and individual bile acids were measured by gas chromatography (GC)-MS (GC-MS). The structure of individual sulfated bile acids was obtained by use of liquid chromatography (LC)-MS (LC-MS). RESULTS: By ESI-SIM-MS, the proportions of DCA did not differ in constipated children (n = 73) from that in control children (n = 92), but monosulfated dihydroxy bile acids were greater (P < 0.05). The difference was attributable to 6 patients in the constipated group whose major fecal bile acid by LC-MS was the 3-sulfate of CDCA. Sulfation of CDCA is known to abolish its secretory activity. By GC-MS, the bile acid profile was identical in the 2 groups. CONCLUSIONS: In most children with functional constipation, the fecal bile acid profile seems to be normal. There is a small subset of children, however, whose dominant fecal bile acid is the 3-sulfate of CDCA, indicating a novel disturbance in bile acid metabolism. Such sulfation abolishes the secretory activity of CDCA and may contribute to constipation.

Interference with hemoglobin A(1C) determination by the hemoglobin variant Shelby.

Hemoglobin variant carrier status was found in a 46-year-old African American man following detection of a falsely elevated hemoglobin A1c (HbA1c) by ionexchange high-performance liquid chromatography (HPLC, VARIANT A1c, Bio-Rad Laboratories, Hercules, CA). Additional analysis of the hemoglobin variant using the Beta Thal Short program (Bio-Rad) revealed an unknown peak with a retention time of 4.84 minutes and a proportion of 26.3%. No mass shift in alpha-globin or beta-globin proteins was observed by mass spectrometry. DNA sequencing revealed a missense mutation in 1 beta-globin allele corresponding to the hemoglobin Shelby trait. The patient was asymptomatic with a normal hemoglobin value of 13.6 g/dL (136 g/L) but had increased target cells on a peripheral blood smear. An alternative method for HbA1c determination using boronate-affinity HPLC provided a value of 3.9% (0.04; reference range, 4.0%-6.9% [0.04-0.07]), more consistent with the patient's recent blood glucose values in the normal range.

Dilute and shoot: analysis of drugs of abuse using selected reaction monitoring for quantification and full scan product ion spectra for identification.

Our objective was to develop a "dilute and shoot" liquid chromatography-tandem mass spectrometry confirmatory procedure that uses full scan product ion spectra to identify drugs that are present above cutoff values as determined by isotope dilution relative to a deuterium-labeled internal standard. Deuterium-labeled internal standards are added to urine which is then diluted prior to analysis. Full scan product ion spectra were obtained in the data-dependent mode using a linear ion trap (ABI 4000 Qtrap). Identification was based on a purity fit of greater than 70. Ninety-seven urine specimens were analyzed by the method described, and results were compared to values obtained from a reference laboratory using selected reaction monitoring (SRM). The ion trap provided about 30-fold increase in signal-to-noise ratio as compared with the same instrument operated in a traditional full scan product ion mode. The assays were linear to at least 10 times the cutoff. Selecting appropriate triggers for obtaining full scan product ion spectra minimized space charging for specimens that contained high concentrations of drugs. There was 100% concordance between the full scan identification and the SRM results for identification of amphetamine, methamphetamine, benzoylecgonine, morphine, codeine, hydrocodone, and hydromorphone. The ability to "dilute and shoot" reduces the turnaround time for results. The data acquired with SRM and full scan product ion spectra provide accurate quantification and a high degree of specificity.

Analysis of testosterone in serum using mass spectrometry.

For either gas chromatography mass spectrometry (GC-MS) or liquid chromatography tandem mass spectrometry (LC-MS-MS) methods, the first step in the analysis is to add a deuterium-labeled internal standard such as testosterone-16,16,17-d(3). Testosterone in the sample is then isolated by liquid-liquid extraction and the extract is dried under a stream of nitrogen. For the GC-MS method we describe; the residue is transformed to the pentafluorobenzyl/trimethylsilyl derivative and is injected into the GC-MS, separated on a dimethypolysiloxane column, and ionized using electron capture negative chemical ionization (ECNCI). Quantification of testosterone in the samples is by selected ion monitoring, measuring peak ratios of testosterone relative to the deuterium-labeled internal standard. For the LC-MS-MS analysis of testosterone, the sample extract is reconstituted in mobile phase, injected on a C18 column, and quantified using multiple reaction monitoring of testosterone relative to the internal standard. There are no interferences from common steroids found in human serum. For both methods the run-to-run precision and accuracy is generally less than 6% and the methods are linear from 5 to 2000 ng/dL.

Human cecal bile acids: concentration and spectrum.

To obtain information on the concentration and spectrum of bile acids in human cecal content, samples were obtained from 19 persons who had died an unnatural death from causes such as trauma, homicide, suicide, or drug overdose. Bile acid concentration was measured via an enzymatic assay for 3alpha-hydroxy bile acids; bile acid classes were determined by electrospray ionization mass spectrometry and individual bile acids by gas chromatography mass spectrometry and liquid chromatography mass spectrometry. The 3alpha-hydroxy bile acid concentration (mumol bile acid/ml cecal content) was 0.4 +/- 0.2 mM (mean +/- SD); the total 3-hydroxy bile acid concentration was 0.6 +/- 0.3 mM. The aqueous concentration of bile acids (supernatant after centrifugation) was identical, indicating that most bile acids were in solution. By liquid chromatography mass spectrometry, bile acids were mostly in unconjugated form (90 +/- 9%, mean +/- SD); sulfated, nonamidated bile acids were 7 +/- 5%, and nonsulfated amidated bile acids (glycine or taurine conjugates) were 3 +/- 7%. By gas chromatography mass spectrometry, 10 bile acids were identified: deoxycholic (34 +/- 16%), lithocholic (26 +/- 10%), and ursodeoxycholic (6 +/- 9), as well as their primary bile acid precursors cholic (6 +/- 9%) and chenodeoxycholic acid (7 +/- 8%). In addition, 3beta-hydroxy derivatives of some or all of these bile acids were present and averaged 27 +/- 18% of total bile acids, indicating that 3beta-hydroxy bile acids are normal constituents of cecal content. In the human cecum, deconjugation and dehydroxylation of bile acids are nearly complete, resulting in most bile acids being in unconjugated form at submicellar and subsecretory concentrations.

41Ca and accelerator mass spectrometry to monitor calcium metabolism in end stage renal disease patients.

BACKGROUND: Monitoring bone resorption with measurements of bone density and biochemical markers is indirect. We hypothesized that bone resorption can be studied directly by serial measurements of the ratio (41)Ca/Ca in serum after in vivo labeling of calcium pools with (41)Ca. We report the preparation of an intravenous (41)Ca dose suitable for humans, an analytical method for determining (41)Ca/Ca isotope ratios in biological samples, and studies in human volunteers. METHODS: (41)Ca was formulated and aliquoted into individual vials, and to the extent possible, the (41)Ca doses were tested according to US Pharmacopeia (USP) guidelines. A 10 nCi dose of (41)Ca was administered intravenously to 4 end stage renal disease (ESRD) patients on hemodialysis and 4 healthy control individuals. Distribution kinetics were determined over 168 days. Calcium was isolated with 3 precipitation steps and a cation-exchange column, and (41)Ca/Ca ratios in serum were then measured by accelerator mass spectrometry. RESULTS: The dosing solution was chemically and radiologically pure, contained <0.1 endotoxin unit/mL, and passed USP sterility tests. Quantification of (41)Ca/Ca ratios was linear from 6 x 10(-14) to 9.1 x 10(-10). The run-to-run imprecision (as CV) of the method was 4% at 4.6 x 10(-11) and 6% at 9.1 x 10(-10). The area under the curve of (41)Ca in the central compartment vs time was significantly less for ESRD patients than for controls (P < 0.005). CONCLUSIONS: Isotope ratios spanning 5 orders of magnitude can be measured by accelerator mass spectrometry with excellent precision in the range observed in samples collected from patients who have received 10 nCi of (41)Ca. The (41)Ca at this dose caused no adverse effects in 8 volunteers. This is the first report of the use of (41)Ca to monitor differences in bone turnover between healthy individuals and ESRD patients.