Masamitsu Maekawa

Identification of novel bile acids as biomarkers for the early diagnosis of Niemann-Pick C disease

This article describes a rapid UPLC‐MS/MS method to quantitate novel bile acids in biological fluids and the evaluation of their diagnostic potential in Niemann‐Pick C (NPC). Two new compounds, NPCBA1 (3β‐hydroxy,7β‐N‐acetylglucosaminyl‐5‐cholenoic acid) and NPCBA2 (probably 3β,5α,6β‐trihydroxycholanoyl‐glycine), were observed to accumulate preferentially in NPC patients: median plasma concentrations of NPCBA1 and NPCBA2 were 40‐ and 10‐fold higher in patients than in controls. However, NPCBA1 concentrations were normal in some patients because they carried a common mutation inactivating the GlcNAc transferase required for the synthesis of this bile acid. NPCBA2, not containing a GlcNAc moiety, is thus a better NPC biomarker.

LC/ESI-MS/MS analysis of urinary 3β-sulfooxy-7β-N-acetylglucosaminyl-5-cholen-24-oic acid and its amides: new biomarkers for the detection of Niemann-Pick type C disease.

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) 2pg of nonamidated 3β-sulfooxy-7β-N-acetylglucosaminyl-5-cholen-24-oic acid, 2pg of its glycine-amide, and 0.6pg of its taurine-amide, and is linear up to 300pg. The method was then used to measure the three analytes in the urine of NPC patients (N=2), 3β-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 3month NPC patient and 40times greater in the adult patient than that of healthy volunteers. The markedly elevated urinary concentration of 3β-sulfooxy-7β-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.

Tandem mass spectrometric characterization of bile acids and steroid conjugates based on low-energy collision-induced dissociation.

We examined the characteristics of several bile acids and some steroid conjugates under low-energy-collision-induced dissociation conditions using a triple quadrupole tandem mass spectrometer. According to conjugation types, we observed characteristic product ions and/or neutral losses in the product ion spectra. Amino acid conjugates afforded specific product ions. For example, glycine-conjugated metabolites routinely produced a product ion at m/z 74, and taurine-conjugated metabolites produced product ions at m/z 124, 107, and 80. When a strong peak appeared at m/z 97, the molecule contained a sulfate group. In contrast to amino acid conjugates, carbohydrate conjugates required a combination of product ions and neutral losses for identification. We could discriminate a glucoside from an acyl galactoside according to the presence or absence of a product ion at m/z 161 and a neutral loss of 180 Da. Discrimination among esters, aliphatic ethers, and phenolic ether types of glucuronides was based upon differences in the intensities of a product ion at m/z 175 and a neutral loss of 176 Da. Furthermore, N-acetylglucosamine conjugates showed a characteristic product ion at m/z 202 and a neutral loss of 203 Da, and the appearance of a product ion at m/z 202 revealed the existence of N-acetylglucosamine conjugated to an aliphatic hydroxyl group without a double bond in the immediate vicinity. Together, the data presented here will help to enable the identification of unknown conjugated cholesterol metabolites by using low-energy collision-induced dissociation.

Preference of Conjugated Bile Acids over Unconjugated Bile Acids as Substrates for OATP1B1 and OATP1B3.

Bile acids, the metabolites of cholesterol, are signaling molecules that play critical role in many physiological functions. They undergo enterohepatic circulation through various transporters expressed in intestine and liver. Human organic anion-transporting polypeptides (OATP) 1B1 and OATP1B3 contribute to hepatic uptake of bile acids such as taurocholic acid. However, the transport properties of individual bile acids are not well understood. Therefore, we selected HEK293 cells overexpressing OATP1B1 and OATP1B3 to evaluate the transport of five major human bile acids (cholic acid, chenodeoxycholic acid, deoxycholic acid, ursodeoxycholic acid, lithocholic acid) together withtheir glycine and taurine conjugates via OATP1B1 and OATP1B3. The bile acids were quantified by liquid chromatography-tandem mass spectrometry. The present study revealed that cholic acid, chenodeoxyxcholic acid, and deoxycholic acid were transported by OATP1B1 and OATP1B3, while ursodeoxycholic acid and lithocholic acid were not significantly transported by OATPs. However, all the conjugated bile acids were taken up rapidly by OATP1B1 and OATP1B3. Kinetic analyses revealed the involvement of saturable OATP1B1- and OATP1B3-mediated transport of bile acids. The apparent Km values for OATP1B1 and OATP1B3 of the conjugated bile acids were similar (0.74–14.7 μM for OATP1B1 and 0.47–15.3 μM for OATP1B3). They exhibited higher affinity than cholic acid (47.1 μM for OATP1B1 and 42.2 μM for OATP1B3). Our results suggest that conjugated bile acids (glycine and taurine) are preferred to unconjugated bile acids as substrates for OATP1B1 and OATP1B3.

Monitoring Serum Levels of Sorafenib and Its N-Oxide Is Essential for Long-Term Sorafenib Treatment of Patients with Hepatocellular Carcinoma

Sorafenib, an oral multi-kinase inhibitor, is the final therapy prior to palliative care for advanced hepatocellular carcinoma (HCC). However, due to its adverse effects, 20% of patients must discontinue sorafenib within 1 month after first administration. To identify ways to predict the adverse effects and administer the drug for longer periods, we explored the relationship between the duration of sorafenib treatment and the pharmacokinetics of sorafenib and its major metabolite, sorafenib N-oxide. Twenty-five subjects enrolled in the study were divided into two groups: patients with dosage reduced or withdrawn due to adverse effects (n = 8), and patients with dosage maintained for 1 month after initial administration (n = 17). We evaluated early sorafenib accumulation as the area under the curve of sorafenib and sorafenib N-oxide concentrations during days 1-7 (AUC(sorafenib) and AUC(N-oxide), respectively). Inter-group comparison revealed that AUC(N-oxide) and AUC ratio (AUC(N-oxide)/AUC(sorafenib)) were significantly higher in the dosage reduction/withdrawal group (P = 0.031 and P = 0.0022, respectively). Receiver operating characteristic analysis indicated that AUC(N-oxide) and AUC ratio were reliable predictors of adverse effects. When patients were classified by cut-off points (AUC(N-oxide:) 2.0 μg ∙ day/mL, AUC ratio: 0.13), progression-free survival was significantly longer in patients with AUC(N-oxide) ≤ 2.0 μg ∙ day/mL (P = 0.0048, log-rank test). In conclusion, we recommend to simultaneously monitor serum levels of sorafenib and its N-oxide during the early stage after the first administration, which enables us to provide safe and long-term therapy for each HCC patient with sorafenib.

Functional characterization of 21 CYP2C19 allelic variants for clopidogrel 2-oxidation

Genetic variations in cytochrome P450 2C19 (CYP2C19) contribute to interindividual variability in the metabolism of therapeutic agents such as clopidogrel. Polymorphisms in CYP2C19 are associated with large interindividual variations in the therapeutic efficacy of clopidogrel. This study evaluated the in vitro oxidation of clopidogrel by 21 CYP2C19 variants harboring amino acid substitutions. These CYP2C19 variants were heterologously expressed in COS-7 cells, and the kinetic parameters of clopidogrel 2-oxidation were estimated. Among the 21 CYP2C19 variants, 12 (that is, CYP2C19.5A, CYP2C19.5B, CYP2C19.6, CYP2C19.8, CYP2C19.9, CYP2C19.10, CYP2C19.14, CYP2C19.16, CYP2C19.19, CYP2C19.22, CYP2C19.24 and CYP2C19.25) showed no or markedly low activity compared with the wild-type protein CYP2C19.1B. This comprehensive in vitro assessment provided insights into the specific metabolic activities of CYP2C19 proteins encoded by variant alleles, and this may to be valuable when interpreting the results of in vivo studies.

Functional Characterization of 34 CYP2A6 Allelic Variants by Assessment of Nicotine C-Oxidation and Coumarin 7-Hydroxylation Activities

CYP2A6, a member of the cytochrome P450 (P450) family, is one of the enzymes responsible for the metabolism of therapeutic drugs and such tobacco components as nicotine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, and N-nitrosodiethylamine. Genetic polymorphisms in CYP2A6 are associated with individual variation in smoking behavior, drug toxicities, and the risk of developing several cancers. In this study, we conducted an in vitro analysis of 34 allelic variants of CYP2A6 using nicotine and coumarin as representative CYP2A6 substrates. These variant CYP2A6 proteins were heterologously expressed in 293FT cells, and their enzymatic activities were assessed on the basis of nicotine C-oxidation and coumarin 7-hydroxylation activities. Among the 34 CYP2A6 variants, CYP2A6.2, CYP2A6.5, CYP2A6.6, CYP2A6.10, CYP2A6.26, CYP2A6.36, and CYP2A6.37 exhibited no enzymatic activity, whereas 14 other variants exhibited markedly reduced activity toward both nicotine and coumarin. These comprehensive in vitro findings may provide useful insight into individual differences in smoking behavior, drug efficacy, and cancer susceptibility.

Focused metabolomics using liquid chromatography/electrospray ionization tandem mass spectrometry for analysis of urinary conjugated cholesterol metabolites from patients with Niemann–Pick disease type C and 3β-hydroxysteroid dehydrogenase deficiency

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.

Unconjugated bile acids in rat brain: Analytical method based on LC/ESI-MS/MS with chemical derivatization and estimation of their origin by comparison to serum levels

Although some studies have revealed the implication of bile acids (BAs) and neurological diseases, the levels and origin of the BAs in the brain are not fully understood. In this study, we first developed and validated a sensitive and specific method for the determination of three unconjugated BAs [cholic acid (CA), chenodeoxycholic acid (CDCA) and deoxycholic acid (DCA)] in the rat brain by liquid chromatography/electrospray ionization-tandem mass spectrometry combined with chemical derivatization. The measured brain concentrations (mean±standard deviation, n=10) of normal rats were 58.7±48.8, 14.2±11.7 and 13.2±8.7ng/g tissue for CA, CDCA and DCA, respectively. For their origin, we developed the hypothesis that they might be mostly derived from the periphery. To test this hypothesis, the brain BA levels were compared with the serum levels. The brain levels had high correlations with the serum levels, and were always lower than the serum levels for the three unconjugated BAs. Furthermore, the higher brain-to-serum concentration ratios were found for the BAs with higher logD values (higher lipophilicity). Moreover, the brains of the rats intraperitoneally administered with deuterium-labeled CA and CDCA were also analyzed; the deuterium-labeled BAs were detected in the brain of the rats administered with these compounds. Based on all the results, we concluded that the BAs found in the brain are mostly derived from the periphery and the major mechanism for the transportation of the unconjugated BAs to the brain is by passive diffusion.

An efficient synthesis of 4α- and 4β-hydroxy- 7-dehydrocholesterol, biomarkers for patients with and animal models of the Smith-Lemli-Opitz syndrome.

A highly efficient and improved method for the preparation of stereoisomeric 4α- and 4β-hydroxy-7-dehydrocholesterol has been developed. These oxysterols are atypical precursors of cholesterol found to be present in increased concentrations in brain, liver, and serum of animals treated with AY9944, an inhibitor of 3β-hydroxysterol-Δ(7)-reductase (Dhcr7). AY9944 -treated rats are considered a model for Smith-Lemli-Opitz syndrome (SLOS). The principal reactions involved were (1) cis-4α,5α-dihydroxylation of the allylic 3β-acetoxy-Δ(4) intermediate with in situ generated RuO4 and subsequent dehydration with SOCl2, (2) direct 4β-hydroxylation of cholesterol with selenium dioxide, and (3) regioselective dehydrogenation at C-7/-8 of the resulting 4α- and 4β-hydroxylated derivatives with 1,3-dibromo-5,5-dimethylhydantoin/azobisisobutyronitrile, followed by tetrabutyl ammonium bromide/tetrabutyl ammonium fluoride. Chemical instability of these 4-hydroxylated 7-dehydrocholesterols when exposed to UV light, heat or in an acidic medium is briefly discussed.