SLC10A4 is a protease-activated transporter that transports bile acids.

SLC10A4 belongs to the sodium bile acid cotransporter family, but has no transport activity for bile acids. We performed multiple amino acid alignments and examined the relationships between the SLC10 proteins. The extracellular N-terminus of SLC10A4 was predicted to be relatively longer at the amino acid level than those of SLC10A1, SLC10A2 and SLC10A6. We examined the relationship between the N-terminus and transport activity of SLC10A4. Rat Slc10a4 is predominantly expressed in rat cholinergic neurons; therefore, TE671 cells expressing the acetylcholine receptor and acetylcholinesterase were used. After thrombin treatment, western blotting and immunofluorescence staining demonstrated that the N-terminus of SLC10A4 might be cleaved. Substrates were added to the cells, and their uptake was quantified by liquid chromatography tandem mass spectrometry. Lithocholic acid (LCA) and taurocholic acid (TCA) uptake and cell death effects of LCA were increased by thrombin treatment. After RNA interference treatment for SLC10A4, bile acid uptake was also quantified. In consequence, increases in the LCA and TCA uptake did not occur. Therefore, SLC10A4 may have low activity but becomes activated by proteases, including thrombin, following cleavage. We have demonstrated that SLC10A4 appears to be a protease-activated transporter and transports bile acids.

Repeated treatment with nicotine induces phosphorylation of NMDA receptor NR2B subunit in the brain regions involved in behavioral sensitization.

Protein phosphorylation is an important mechanism for the post-translational modulation of N-methyl-d-aspartate (NMDA) receptor functions. In the present study, we investigated the levels of NR2B phosphorylation at Tyr1472 and Ser1303 in the nucleus accumbens, striatum, frontal cortex, and hippocampus of rats that exhibit behavioral sensitization to nicotine. Repeated treatment of rats with nicotine (0.6mg/kg, s.c., for 7 days) produced locomotor sensitization accompanied by increased NR2B phosphorylation at Tyr1472 in the nucleus accumbens and striatum, brain regions involved in behavioral sensitization. In contrast, no changes in NR2B phosphorylation were observed after a single treatment with nicotine in these brain regions. In addition, no changes in NR2B phosphorylation at Ser1303 were observed after repeated treatment with nicotine in any examined brain regions. These results suggest that repeated treatment with nicotine induces NR2B phosphorylation at Tyr1472 in the nucleus accumbens and striatum, which might contribute to the development of synaptic and behavioral plasticity in response to nicotine.

Simultaneous quantitation of sphingoid bases and their phosphates in biological samples by liquid chromatography/electrospray ionization tandem mass spectrometry.

We developed a liquid chromatography/electrospray ionization tandem mass spectrometry method for the simultaneous quantitative determination of C18 sphingosine (Sph), C18 dihydrosphingosine (dhSph), C18 phytosphingosine (pSph), C18 sphingosine-1-phosphate (S1P), C18 dihydrosphingosine-1-phosphate (dhS1P), and C18 phytosphingosine-1-phosphate (pS1P). Samples were prepared by simple methanol deproteinization and analyzed in selected reaction monitoring modes. No peak tailing was observed on the chromatograms using a Capcell Pak ACR column (1.5 mm i.d. × 250 mm, 3 µm, Shiseido). The calibration curves of the sphingoids showed good linearity (r > 0.996) over the range of 0.050-5.00 pmol per injection. The accuracy and precision of this method were demonstrated using four representative biological samples (serum, brain, liver, and spleen) from mice that contained known amounts of the sphingoids. Samples of mice tissue such as plasma, brain, eye, testis, liver, kidney, lung, spleen, lymph node, and thymus were examined for their Sph, dhSph, pSph, S1P, dhS1P, and pS1P composition. The results confirmed the usefulness of this method for the physiological and pathological analysis of the composition of important sphingoids.

Screening of antibiotics that interact with organic anion-transporting polypeptides 1B1 and 1B3 using fluorescent probes.

Hepatic organic anion transporters OATP1B1 and OATP1B3 are expressed at the sinusoidal membrane of hepatocytes and contribute to the hepatic uptake of a wide variety of clinically used drugs. To identify the antibiotics that interact with the human organic anion transporters OATP1B1 and OATP1B3, we applied a screening system using fluorescent probes. Twenty-six antibiotics with a variety of mechanisms of action were examined. The screening demonstrated that four antibiotics inhibited OATP1B1-mediated transport and 11 antibiotics inhibited OATP1B3-mediated transport in a concentration-dependent manner. Antibiotics that inhibited OATP1B3-mediated transport tended to exhibit higher affinity than those that inhibited OATP1B1-mediated transport. To clarify whether the antibiotics that interacted with OATP1B1 and/or OATP1B3 were substrates for these transporters, an uptake study was performed. Rifampicin and penicillin were transported by both OATP1B1 and OATP1B3. Moreover, OATP1B3 was involved in the transport of ceftriaxone, cefmetazole, cefoperazone, and cefotaxime. Macrolides were not significantly transported by either transporter. In conclusion, the results demonstrated that our system is a useful method for the rapid screening of transporter-antibiotic interaction, and we found novel substrates. Our results indicate that OATP1B1 and/or OATP1B3 contribute to the transport process of some antibiotics, and that drug-drug interactions associated with these transporters could occur after the administration of antibiotics.

Epidermal FABP (FABP5) regulates keratinocyte differentiation by 13(S)-HODE-mediated activation of the NF-κB signaling pathway.

Fatty acid-binding proteins (FABPs) are postulated to serve as lipid shuttles that solubilize hydrophobic fatty acids and deliver them to appropriate intracellular sites. Epidermal FABP (E-FABP/FABP5) is predominantly expressed in keratinocytes and is overexpressed in the actively proliferating tissue characteristic of psoriasis and wound healing. In this study, we found decreased expression of the differentiation-specific proteins keratin 1, involucrin, and loricrin in E-FABP(-/-) keratinocytes relative to E-FABP(+/+) keratinocytes. We also determined that incorporation of linoleic acid was significantly reduced in E-FABP(-/-) keratinocytes. Although linoleic acid did not directly affect keratinocyte differentiation, keratin 1 expression was induced by the linoleic acid derivative 13(S)-hydroxyoctadecadienoic acid (13(S)-HODE), and this induction was concomitant with increased NF-κB activity. In E-FABP(-/-) keratinocytes, the expression of 13(S)-HODE and the subsequent induction of NF-κB activity was lower than in wild-type keratinocytes. The reduction of linoleic acid in E-FABP(-/-) keratinocytes led to decreased cellular 13(S)-HODE content, resulting in decreased keratin 1 expression through downregulation of NF-κB activity. The regulation of fatty acid metabolism by E-FABP during keratinocyte differentiation suggests that E-FABP may have a role in the pathogenesis of psoriasis.

Simultaneous determination of guanidinosuccinic acid and guanidinoacetic acid in urine using high performance liquid chromatography/tandem mass spectrometry.

We present a method for the simultaneous determination of guanidinosuccinic acid (GSA) and guanidinoacetic acid (GAA) from urine by protein precipitation and liquid chromatography/tandem mass spectrometry. The chromatographic separation was performed using a cation exchange column with an elution gradient of 0.1 mM and 20 mM ammonium acetate buffers. GSA was detected with the mass spectrometer in negative ion mode monitoring at m/z 174.1, and GAA, creatinine, arginine, and homoarginine were in positive ion mode monitoring at m/z 118.1, 114.1, 175.1, and 189.1, respectively. As an internal standard, L-arginine-(13)C(6) hydrochloride and creatinine-d(3) (methyl-d(3)) were used. The calibration ranges were 0.50-25.0 µg mL(-1), and good linearities were obtained for all compounds (r>0.999). The intra- and inter-assay accuracies (expressed as recoveries) and precisions at three concentration levels (1.00, 5.00 and 25.0 µg mL(-1)) were better than 83.8% and 7.41%, respectively. The analytical performance of the method was evaluated by determination of the compounds in urine from male C57BL/J Iar db/db diabetes mellitus (DM) mice. The values of GSA and GAA corrected by the ratios of the individual compounds to creatinine were significantly increased in DM mice compared with control mice. These results indicated that the newly developed method was useful for determining urinary guanidino compounds and metabolites of arginine.

Transport of estrone 3-sulfate mediated by organic anion transporter OATP4C1: estrone 3-sulfate binds to the different recognition site for digoxin in OATP4C1.

Human organic anion transporter OATP4C1 is a member of the OATP family predominantly expressed in the kidney, and contributes to the renal secretion of digoxin. However, little is known about the characteristics of OATP4C1-madiated transport. We examined the transport of estrone 3-sulfate, which is known as a substrate for other OATPs, by OATP4C1-expressing cells. Estrone 3-sulfate was efficiently transported by OATP4C1. The Michaelis-Menten constant for estrone 3-sulfate uptake by OATP4C1 was 26.6+/-4.9 microM. Transport of estrone 3-sulfate was significantly inhibited by triiodothyronine, chenodeoxycholic acid, bromosulfophtalein, and cyclosporine, whereas known substrates of OATP4C1, digoxin and ouabain, did not change OATP4C1-mediated transport. We further examined the mutual inhibition study between estrone 3-sulfate and digoxin. Digoxin partially inhibited the estrone 3-sulfate transport, and estrone 3-sulfate did not significantly inhibit digoxin transport. The estimated IC(50) value of digoxin for OATP4C1-mediated estrone 3-sulfate transport was 119 microM. This value is not comparable with the Michaelis-Menten constant for digoxin uptake by OATP4C1 (7.8 microM) reported by Mikkaichi et al.(1)) In conclusion, we found that estrone 3-sulfate is a novel substrate for OATP4C1. Moreover, our results indicate that estrone 3-sulfate does not bind to the recognition site for digoxin in OATP4C1.

[Introducing hazard prediction training 'KYT' to undergraduate pharmacy education on patient safety].

To develop students' sensitivity toword medication hazards, we have introduced a behavioral approach, "Kiken-Yochi Training" (KYT) for hazard prediction training to pharmacy education. KYT was originally implemented in the field of occupational health and safety in Japan. Only recently it has been introduced in the medical arena. The process consists of four steps; identification of hazards, assessing risks, planning countermeasure, and making action plan. One facilitator organizes the KYT class (20 students divided into four or five small groups). Watching a photo or illustration of everyday occurrences, each group follows the above four steps to discuss predictable hazards. Concepts are intensively presented in short time with brainstorming. KYT has been used with five classes thus far. Students learned KYT theory and exhibited desired attitudes and behaviors. Students presented many ideas, then formulated their own action plan within about one hour. More than 95% of KYT-naïve students assessed themselves as capable of applying the methodology in various situations. They also assessed themselves as being more aware of potential hazards and new points of view through the KYT process. Pharmacists must work for safer and more effective pharmacotherapy, predicting hazards as side effect or human error and solving the problems on each patient. KYT is a very useful and effective tool for pro-active safety training for the skill and attitude development. Repeating problem-based learning like KYT at intervals through undergraduate education should improve patient safety.

SLCO4C1 transporter eliminates uremic toxins and attenuates hypertension and renal inflammation.

Hypertension in patients with chronic kidney disease (CKD) strongly associates with cardiovascular events. Among patients with CKD, reducing the accumulation of uremic toxins may protect against the development of hypertension and progression of renal damage, but there are no established therapies to accomplish this. Here, overexpression of human kidney-specific organic anion transporter SLCO4C1 in rat kidney reduced hypertension, cardiomegaly, and inflammation in the setting of renal failure. In addition, SLCO4C1 overexpression decreased plasma levels of the uremic toxins guanidino succinate, asymmetric dimethylarginine, and the newly identified trans-aconitate. We found that xenobiotic responsive element core motifs regulate SLCO4C1 transcription, and various statins, which act as inducers of nuclear aryl hydrocarbon receptors, upregulate SLCO4C1 transcription. Pravastatin, which is cardioprotective, increased the clearance of asymmetric dimethylarginine and trans-aconitate in renal failure. These data suggest that drugs that upregulate SLCO4C1 may have therapeutic potential for patients with CKD.

Neurobehavioral effects of tetrabromobisphenol A, a brominated flame retardant, in mice.

Tetrabromobisphenol A (TBBPA) is widely used as a flame retardant and is suspected to be stable in the environment with possible widespread human exposures. In the present study, we investigated the behavioral effects of TBBPA and measured the levels of TBBPA in the brain after oral administration in mice. Acute treatment with TBBPA (5mg/kg body weight) 3h before the open-field test induced an increase in the horizontal movement activities. In contextual fear conditioning paradigm, mice treated with TBBPA (0.1mg/kg or 5mg/kg body weight) showed more freezing behavior than vehicle-treated mice. In addition, TBBPA (0.1mg/kg body weight) significantly increased the spontaneous alternation behavior in the Y-maze test. The levels of TBBPA in the brain following TBBPA treatment were determined by using LC/ESI-MS/MS system. In the brain regions examined, high amounts of TBBPA were detected in the striatum after treatment with 0.1mg/kg or 5mg/kg body weight TBBPA, whereas non-specific accumulation of TBBPA in the brain was found after treatment with 250 mg/kg body weight TBBPA. These results suggest that TBBPA accumulates in brain regions including the striatum and induces the behavioral alterations. Together, the possibility of widespread human exposure to TBBPA warrants further studies to characterize its neurotoxicity.

Cysteinyl leukotrienes enhance the degranulation of bone marrow-derived mast cells through the autocrine mechanism.

The cysteinyl leukotrienes (LTs), LTC(4), LTD(4), and LTE(4), are potent inflammatory mediators and are involved in allergic reactions, such as bronchoconstriction, eosinophilic inflammation, and allergic cell proliferation. The present study aimed to elucidate the role of constitutively produced cysteinyl LTs in mast cell activation. We used a newly developed quantification method based on mass spectrometry to detect cysteinyl LTs in the cultured medium of mouse bone marrow-derived mast cells (BMMCs), which were obtained by interleukin (IL)-3-conditioned culture of mouse bone marrow. BMMCs were stimulated with immunoglobulin (Ig) E and antigen (IgE/Ag) or lipopolysaccharide for 1 or 24 h. This new quantification method revealed that unstimulated BMMCs produced and secreted LTB4 and LTE4 after 24 h of incubation. The treatment of unstimulated BMMCs for 2 h with montelukast, an antagonist of a cysteinyl LT receptor, CysLT1, resulted in the suppression of a downstream signaling event of this receptor, i.e., the decrease in phosphorylation of extracellular responsive kinases. Thus, cysteinyl LTs constitutively simulate BMMCs through the CysLT1 receptor in an autocrine manner. Treatment of BMMCs for 3 weeks with montelukast, which caused long-term inhibition of the autocrine cyteinyl LT-derived signal, significantly attenuated the IgE/Ag-dependent degranulation, as judged by the decrease in the release of beta-hexosaminidase, an enzyme contained in the granules, whereas the production of cytokines, such as IL-6 and tumor necrosis factor-alpha, were largely unaffected. In conclusion, an autocrine signal derived from constitutively produced cysteinyl LTs predisposes mast cells to the degranulation upon allergic stimulation.

The nutrient formula containing eicosapentaenoic acid and docosahexaenoic acid benefits the fatty acid status of patients receiving long-term enteral nutrition.

Currently, various formulas with different fatty acid compositions are used for enteral nutrition (EN). All formulas contain various concentrations of essential fatty acids: linoleic acid (LA) and alpha-linolenic acid (ALA); LA is biotransformed into arachidonic acid (AA) and ALA into eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in vivo. Some formulas contain preformed EPA and DHA. However, the effects of the differences in the fatty acid composition on the fatty acid status of patients receiving long-term EN is not clear. We measured serum fatty acid concentrations in 50 patients with neurological diseases receiving long-term EN. The data were then compared retrospectively with reference to the fatty acid compositions of the formulas used. All of the patients received almost their entire nutritional intake via EN for at least 1 year. Blood samples were obtained just before injecting the EN solution. Among the formulas that did not include EPA or DHA, formulas with low ALA concentrations were associated with low serum EPA and DHA. Conversely, the ALA-enriched formulas with reduced LA concentrations significantly increased EPA and DHA levels, although the levels remained lower than the control values. With the formula containing EPA and DHA, the EPA and DHA levels reached control values. Therefore, the fatty acid composition of the EN formulas affected the fatty acid status of patients receiving long-term EN. Formulas containing preformed EPA and DHA with suitable amounts of essential fatty acids may benefit these patients.

Simple quantification of lansoprazole and rabeprazole concentrations in human serum by liquid chromatography/tandem mass spectrometry.

A rapid, simple and highly sensitive method was developed for the quantitative determination of lansoprazole and rabeprazole concentrations in 20 microL of human serum using high-performance liquid chromatography/tandem mass spectrometry (LC/MS/MS). Analytes, along with an internal standard (lansoprazole deuterium derivatives), were separated using a mobile phase of acetonitrile/1mM ammonium formate (140/60, v/v) on a C18 analytical column and analyzed in the selected reaction-monitoring (SRM) mode. The lower limit of quantification was 0.25 ng/mL. A good linear response was observed for each analyte (from 0.25 ng to 2.5 microg/mL). This method was useful for therapeutic drug monitoring and pharmacokinetic studies.

Stronger inhibition of gastric acid secretion by lafutidine, a novel H2 receptor antagonist, than by the proton pump inhibitor lansoprazole.

AIM: To compare the antisecretory activity and plasma drug concentrations of a single oral dose of 10 mg lafutidine, a novel H2 receptor antagonist, with those of the proton pump inhibitor lansoprazole (LPZ) 30 mg. METHODS: Ten volunteers without H pylori infection participated in this crossover study comparing lafutidine 10 mg with LPZ 30 mg. Intragastric pH was monitored for 6 h in all participants, and blood samples were collected from four randomly selected individuals after single-dose administration of each drug. RESULTS: The median intragastric pH was significantly higher in individuals who received lafutidine 10 mg than in those who received LPZ 30 mg 2, 3, 4, 5, and 6 h after administration. Maximal plasma drug concentration was reached more promptly with lafutidine 10 mg than with LPZ 30 mg. CONCLUSION: In H pylori-negative individuals, gastric acid secretion is more markedly inhibited by lafutidine than by LPZ.

Early effects of Lansoprazole orally disintegrating tablets on intragastric pH in CYP2C19 extensive metabolizers.

AIM: To compare rabeprazole (RPZ; 10 mg) with Lansoprazole orally disintegrating tablets (LPZ; 30 mg OD) in terms of antisecretory activity and blood drug concentration after a single dose. METHODS: Eight H pylori -negative cytochrome P450 (CYP) 2C19 extensive metabolizers were assigned to receive a single oral dose of RPZ 10 mg or LPZ 30 mg OD. Twelve hour intragastric pH monitoring was performed on the day of treatment. Blood samples were also collected after the administration of each drug. RESULTS: LPZ 30 mg OD induced a significantly earlier rise in blood drug concentration than RPZ 10 mg; consequently, LPZ 30 mg OD induced a significantly earlier rise in median pH in the third and fourth hours of the study. CONCLUSION: In H pylori-negative CYP2C19 extensive metabolizers, LPZ 30 mg OD induced a significantly faster inhibition of gastric acid secretion than RPZ 10 mg.

Prostaglandin F(2alpha) regulates cytokine responses of mast cells through the receptors for prostaglandin E.

There is an increasing body of evidence that prostanoids modulate mast cell functions and contribute to the development of allergic inflammation. The present study aimed to identify an undetermined function of prostaglandin (PG) F(2alpha) in mast cell activation and the signaling mechanism involved in it. Simultaneous quantification of prostanoids by liquid chromatography/tandem mass spectrometry revealed the constitutive release of PGF(2alpha), thromboxane B(2), and 6-keto-PGF(1alpha) from bone marrow-derived mast cells (BMMCs). Upon activation of BMMCs by lipopolysaccharide, the cytokine production in BMMCs was enhanced when the culture was supplemented with PGF(2alpha). However, F prostanoid receptor-a selective receptor for PGF(2alpha)-was not detected in BMMCs. Further investigations performed using prostanoid receptor antagonists revealed an alternative mechanism wherein the receptors for PGE species-E prostanoid receptors-mediated the PGF(2alpha) signal in BMMCs. The present study provides an insight into a novel function of PGF(2alpha), i.e., an autocrine accelerator for mast cell activation.

Rapid screening of antineoplastic candidates for the human organic anion transporter OATP1B3 substrates using fluorescent probes.

A rapid screening system has been established to extract novel candidates that exhibit potent inhibition of the transport of fluorescent substrate by organic anion transporting polypeptide (OATP) 1B3. OATP1B3 is abundantly expressed in solid digestive organ cancers. Thus, the identification of new substrates leads to novel strategies for effective cancer chemotherapy with minimal adverse effects. We used an automated image acquisition and analysis system (IN Cell Analyzer 1000) to visualize the transport and subsequent accumulation of the fluorescent substrate chenodeoxycholyl-(Nepsilon-NBD)-lysine (CDCA-NBD). Antineoplastic screening demonstrated that five candidates agents, docetaxel, actinomycin D, mitoxantrone, paclitaxel, and SN-38, exhibited potent inhibitory effects on OATP1B3-mediated transport of CDCA-NBD. To clarify if these antineoplastic drugs are substrates for OATP1B3, we performed transport assays in OATP1B3-expressing cells. We determined that SN-38 is a novel substrate for OATP1B3. In conclusion, our results demonstrate that the screening system established in this study is a useful method for the rapid extraction of candidate therapeutic agents from the large numbers of compounds.

Angiopoietin-related growth factor suppresses gluconeogenesis through the Akt/forkhead box class O1-dependent pathway in hepatocytes.

Angiopoietin-related growth factor (AGF; or Angptl6) is a liver-derived, circulating factor and is considered to be a regulator of metabolic homeostasis. AGF is capable of counteracting both obesity and obesity-related insulin resistance. However, the target tissues and the molecular mechanisms underlying the antiobesity and antidiabetic actions of AGF have not been completely defined. Using rat hepatoma H4IIEc3 cells or primary hepatocytes, we demonstrate that AGF suppresses glucose production in a concentration-dependent manner through reduced expression of a key gluconeogenic enzyme, glucose-6-phosphatase (G6Pase), at both transcriptional and translational levels. The action of AGF on glucose production was inhibited by pretreatment of the cells with LY294002 [2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one], a phosphoinositide 3-kinase (PI3K) inhibitor, and Akt (protein kinase B) inhibitors. AGF increased the phosphorylation of Akt and its substrates, glycogen synthase kinase 3beta and forkhead box class O1 (FoxO1), a key transcription factor for G6Pase expression. Furthermore, an immunohistochemical approach with anti-FoxO1 antibody demonstrated that AGF stimulation promoted translocation of FoxO1 from the nucleus to the cytoplasm in the cells. These results suggest that in hepatocytes, AGF suppresses gluconeogenesis via reduced transcriptional activity of FoxO1 resulting from the activation of PI3K/Akt signaling cascades.

Simultaneous quantification of seven prostanoids using liquid chromatography/tandem mass spectrometry: the effects of arachidonic acid on prostanoid production in mouse bone marrow-derived mast cells.

We have developed a method for the simultaneous estimation of the levels of the prostanoids 6-keto prostaglandin (PG) Flalpha, PGB2, PGD2, PGE2, PGF2(alpha), PGJ2, and thromboxane (TX) B2 in blood- or serum-containing medium using liquid chromatography-tandem mass spectrometry. These prostanoids and their deuterium derivatives, which were used as internal standards, were subjected to solid-phase extraction using Empore C18 HD disk cartridges and analyzed in the selected reaction-monitoring mode. A linear response curve starting at 10 pg of prostanoid/tube was observed for each prostanoid. The accuracy of the method was demonstrated with samples containing known amounts of the prostanoids. Furthermore, we used this method to analyze the prostanoids produced in mouse bone marrow-derived mast cells stimulated with arachidonic acid, which resulted in the production of PGD2, PGE2, PGF2alpha, and TXB2. The results suggest that this simultaneous quantification method is useful for the analysis of the production of biomedically important prostanoids.

LC/ESI-tandem mass spectrometric determination of bile acid 3-sulfates in human urine 3beta-Sulfooxy-12alpha-hydroxy-5beta-cholanoic acid is an abundant nonamidated sulfate.

We developed a highly sensitive and quantitative method to detect bile acid 3-sulfates in human urine employing liquid chromatography/electrospray ionization-tandem mass spectrometry. This method allows simultaneous analysis of bile acid 3-sulfates, including nonamidated, glycine-, and taurine-conjugated bile acids, cholic acid (CA), chenodeoxycholic acid (CDCA), deoxycholic acid (DCA), ursodeoxycholic acid (UDCA), and lithocholic acid (LCA), using selected reaction monitoring (SRM) analysis. The method was applied to analyze bile acid 3-sulfates in human urine from healthy volunteers. The results indicated an unknown compound with the nonamidated common bile acid 3-sulfates on the chromatogram obtained by the selected reaction monitoring analysis. By comparison of the retention behavior and MS/MS spectrum of the unknown peak with the authentic specimen, the unknown compound was identified as 3beta,12alpha-dihydroxy-5beta-cholanoic acid 3-sulfate.