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Maekawa M.,Tohoku University | Shimada M.,Tohoku University | Ohno K.,Tottori University | Togawa M.,Tottori University | And 6 more authors.
Annals of Clinical Biochemistry | Year: 2015

Background Various conjugated cholesterol metabolites are excreted in urine of the patients with metabolic abnormalities and hepatobiliary diseases. We aimed to examine the usefulness of precursor ion scan and neutral loss scan for the characterization of conjugated cholesterol metabolites in urine. Methods A mixture of authentic standards of conjugated cholesterol metabolites was used for investigating the performance of the present method. The urine of patients with Niemann–Pick diseases type C and 3β-hydroxysteroid dehydrogenase deficiency were analysed by precursor ion scan of m/z 97, 74, and 124. Results A precursor ion scan of m/z 97 was effective for identifying conjugates with ester sulphates on hydroxyl groups whereas ester sulphates on phenolic alcohols were signalled by a neutral loss scan of 80 Da. Monosaccharide-conjugated cholesterol metabolites were signalled by a precursor ion scan of m/z 113. Although precursor ion scan of m/z 74 and 124 was effective for finding glycine- and taurine-conjugated metabolites, high intensity of product ions (m/z 74 and 124) disturbed measurement of other multiply conjugated metabolites. The urine samples contained many conjugated cholesterol metabolites, and there were several disease-specific intense peaks. We found several unknown intense peaks with three known peaks in urine of the Niemann–Pick type C patient. In the patient with 3β-hydroxysteroid dehydrogenase deficiency, intense peaks that were tentatively identified as 5-cholenoic acid sulphates and their glycine and taurine conjugates were present. Conclusion The method should lead to the discovery of new urinary biomarkers for these disturbances of cholesterol catabolism and transport. © 2015, © The Author(s) 2015. Source


Naritaka N.,Juntendo University | Suzuki M.,Juntendo University | Sato H.,Jichi Medical University | Takei H.,Junshin Clinic Bile Acid Institute | And 5 more authors.
Clinica Chimica Acta | Year: 2015

Background: The primary bile acids found in meconium vary with the gestational age of the fetus and the intestinal location of the meconium. We determined the composition of bile acids in samples that were collected from the gallbladder and intestine. Methods: The bile-acid profiles of intestinal contents and the gallbladder were obtained from nine fetuses who died from abortion or respiratory failure within 72. h after birth. Intestinal content samples were collected from seven intestinal locations. The bile-acid profiles of meconium were also obtained from seven full-term live births for comparison. The profiles were analyzed using liquid chromatography-tandem mass spectrometry. Results: The bile acids in meconium collected from stillborn and live births were mainly chenodeoxycholic acid and cholic acid, conjugated with taurine, glycine, and sulfate. The same bile acids were found in the gallbladder, except that sulfate was not found. Conclusions: Sulfate-conjugated bile acid is found in urine, but rarely in stool. In this study, the gallbladder bile acid contained no sulfate conjugates, but these were present in intestinal contents and meconium. These results indicate that sulfate-conjugated bile acids are not excreted into the intestine through the biliary tract but originate from swallowed amniotic fluid that contains fetal urine. © 2015 Elsevier B.V. Source


Muto A.,Junshin Clinic Bile Acid Institute | Takei H.,Junshin Clinic Bile Acid Institute | Unno A.,Junshin Clinic Bile Acid Institute | Murai T.,Health Sciences University of Hokkaido | And 12 more authors.
Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences | Year: 2012

The synthesis of bile salts from cholesterol is a complex biochemical pathway involving at least 16 enzymes. Most inborn errors of bile acid biosynthesis result in excessive formation of intermediates and/or their metabolites that accumulate in blood and are excreted in part in urine. Early detection is important as oral therapy with bile acids results in improvement. In the past, these intermediates in bile acid biosynthesis have been detected in neonatal blood or urine by screening with FAB-MS followed by detailed characterization using GC-MS. Both methods have proved difficult to automate, and currently most laboratories screen candidate samples using LC-MS/MS. Here, we describe a new, simple and sensitive analytical method for the identification and characterization of 39 conjugated and unconjugated bile acids, including Δ 4-3-oxo- and Δ 4,6-3-oxo-bile acids (markers for Δ 4-3-oxo-steroid 5β-reductase deficiency), using liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). In this procedure a concentrated, desalted urinary sample (diluted with ethanol) is injected directly into the LC-ESI-MS/MS, operated with ESI and in the negative ion mode; quantification is obtained by selected reaction monitoring (SRM). To evaluate the performance of our new method, we compared it to a validated method using GC-MS, in the analysis of urine from two patients with genetically confirmed Δ 4-3-oxo-steroid 5β-reductase deficiency as well as a third patient with an elevated concentration of abnormal conjugated and unconjugated Δ 4-3-oxo-bile acids. The Δ 4-3-oxo-bile acids concentration recovered in three patients with 5β-reductase deficiency were 48.8, 58.9, and 49.4μmol/mmol creatinine, respectively by LC-ESI-MS/MS. © 2012 Elsevier B.V.. Source


Maekawa M.,Tohoku University | Misawa Y.,Tohoku University | Sotoura A.,Tohoku University | Yamaguchi H.,Tohoku University | And 11 more authors.
Steroids | Year: 2013

We developed a sensitive, reliable, and accurate LC/ESI-MS/MS method for measurement of 3β-sulfooxy- 7β-N-acetylglucosaminyl-5-cholen-24-oic acid and its glycine and taurine amides in urine. This atypical C24 bile acid has been reported previously to be present in the urine of patients with Niemann-Pick Type C (NPC) disease. In the method, targeted analytes are concentrated at the front edge of a trapping column, Shim-pack MAYI-C8, which permits elimination of contaminating molecules in the urinary matrix. The trapped analytes are then eluted, separated on a YMC-Pack Pro C18, and quantified with MS/MS using selected reaction monitoring. The method could detect (as amount injected) 2 pg of nonamidated 3b-sulfooxy- 7b-N-acetylglucosaminyl-5-cholen-24-oic acid, 2 pg of its glycine-amide, and 0.6 pg of its taurineamide, and is linear up to 300 pg. The method was then used to measure the three analytes in the urine of NPC patients (N = 2), 3b-hydroxysteroid dehydrogenase deficiency patients (N = 2), and healthy volunteers (N = 8). Measurable concentrations of all three analytes were present in all subjects. The urinary concentration of the sum of all three analytes was four hundred times greater in the 3 month NPC patient and 40 times greater in the adult patient than that of healthy volunteers. The markedly elevated urinary concentration of 3b-sulfooxy-7b-N-acetylglucosaminyl-5-cholen-24-oic acid and its amides in NPC patients suggests that these compounds may be valuable biomarkers for detection of NPC disease. © 2013 Elsevier Ltd. All rights reserved. Source


Kakiyama G.,Virginia Commonwealth University | Muto A.,Junshin Clinic Bile Acid Institute | Takei H.,Junshin Clinic Bile Acid Institute | Nittono H.,Junshin Clinic Bile Acid Institute | And 5 more authors.
Journal of Lipid Research | Year: 2014

We have developed a simple and accurate HPLC method for measurement of fecal bile acids using phenacyl derivatives of unconjugated bile acids, and applied it to the measurement of fecal bile acids in cirrhotic patients. The HPLC method has the following steps: 1 ) lyophilization of the stool sample; 2 ) reconstitution in buffer and enzymatic deconjugation using cholylglycine hydrolase/sulfatase; 3 ) incubation with 0.1 N NaOH in 50% isopropanol at 60°C to hydrolyze esterified bile acids; 4 ) extraction of bile acids from particulate material using 0.1 N NaOH; 5 ) isolation of deconjugated bile acids by solid phase extraction; 6 ) formation of phenacyl esters by derivatization using phenacyl bromide; and 7 ) HPLC separation measuring eluted peaks at 254 nm. The method was validated by showing that results obtained by HPLC agreed with those obtained by LC-MS/ MS and GC-MS. We then applied the method to measuring total fecal bile acid (concentration) and bile acid profile in samples from 38 patients with cirrhosis (17 early, 21 advanced) and 10 healthy subjects. Bile acid concentrations were significantly lower in patients with advanced cirrhosis, suggesting impaired bile acid synthesis.-Kakiyama, G., A. Muto, H. Takei, H. Nittono, T. Murai, T. Kurosawa, A. F. Hofmann, W. M. Pandak, and J. S. Bajaj. A simple and accurate HPLC method for fecal bile acid profile in healthy and cirrhotic subjects: validation by GC-MS and LC-MS. J. Lipid Res. 2014. 55: 978-990. Copyright © 2014 by the American Society for Biochemistry and Molecular Biology, Inc. Source

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