Measurement of bile acid kinetics in human serum using stable isotope labeled chenodeoxycholic acid and capillary gas chromatography electron impact mass spectrometry

A non invasive method for measurement of bile acid kinetics in serum using (24-13C)chenodeoxycholic acid has been developed. After oral administration of 50 mg (24-13C)chenodeoxycholic acid, the exponential decay of the 13C atom percent excess was measured in serum using capillary gas chromatography mass spectrometry. This required that isotope ratios were measured with high accuracy and coefficients of variation less than 1% by means of selected ion monitoring and scan averaging. The clinical applicability was tested by repeated determination of pool size, fractional turnover and synthesis rate of chenodeoxycholic acid in one healthy volunteer. This method permitted the determination of pool size, synthesis and conversion of chenodeoxycholic acid into lithocholic acid in man without the use of radioactive tracers and without repeated duodenal intubation.     

Metabolism of potential precursors of chenodeoxycholic acid in cerebrotendinous xanthomatosis (CTX).

In patients with cerebrotendinous xanthomatosis (CTX), diminished cholic acid production is associated with incomplete oxidation of the cholesterol side chain and the excretion of C(25)-hydroxy bile alcohols. The aims of this investigation were 1) to provide quantitative information on the pool size and production rate of chenodeoxycholic acid by the isotope dilution technique; and 2) to investigate the possible existence of a block in chenodeoxycholic acid synthesis and explain the absence of chenodeoxycholic acid precursors in CTX. After the injection of [24-(14)C]chenodeoxycholic acid, measurements of chenodeoxycholic acid pool size and production rate in a CTX subject were, respectively, 1/20 and 1/6 as great as controls. Further, three potential precursors of chenodeoxycholic acid, namely [G-(3)H]7alpha-hydroxy-4-cholesten-3-one, [G-(3)H]5beta-cholestane-3alpha,7alpha,25-triol, and [G-(3)H]5beta-cholestane-3alpha,7alpha,26-triol, were administered to the CTX and control subjects and the specific activity curves of [G-(3)H]cholic acid and [G-(3)H]chenodeoxycholic acid were constructed and compared. In the control subjects, the two bile acids decayed exponentially, but in the CTX patient maximum specific activities were abnormally delayed, indicating the hindered transformation of precursor into bile acid. These results show that chenodeoxycholic acid synthesis is small in CTX and that the conversion of 7alpha-hydroxy-4-cholesten-3-one, 5beta-cholestane-3alpha,7alpha,25-triol, and 5beta-cholestane-3alpha,7alpha,26-triol to both chenodeoxycholic acid and cholic acid were similarly impaired.     

Gas chromatography-mass spectrometry assay of the (18)o enrichment of water as trimethyl phosphate

We have developed an assay for determining the 18O enrichment of water in biological fluids. Urine, plasma, or whole blood is reacted with phosphorous pentachloride to yield phosphoric acid. Derivatization of phosphoric acid with diazomethane generates trimethyl phosphate. The enrichment of trimethyl phosphate is nearly four times that of water and is assayed using gas chromatography-mass spectrometry (electron impact ionization). Yang et al. (1998, Anal. Biochem. 258, 315-321) assayed the 2H enrichment of body water after exchange with acetone, by gas chromatography-mass spectrometry. The combination of our 18O method and the 2H method of Yang et al. allows one to measure energy expenditure via "doubly labeled" water (2H(2)O + H(2)18O), using small samples of body fluids. These techniques were used to measure energy expenditure in mice, in which the 18O enrichment of body water can be monitored down to 0.025%.     

Total-body creatine pool size and skeletal muscle mass determination by creatine-(methyl-d3) dilution in rats

There is currently no direct, facile method to determine total-body skeletal muscle mass for the diagnosis and treatment of skeletal muscle wasting conditions such as sarcopenia, cachexia, and disuse. We tested in rats the hypothesis that the enrichment of creatinine-(methyl-d(3)) (D(3)-creatinine) in urine after a defined oral tracer dose of D(3)-creatine can be used to determine creatine pool size and skeletal muscle mass. We determined 1) an oral tracer dose of D(3)-creatine that was completely bioavailable with minimal urinary spillage and sufficient enrichment in the body creatine pool for detection of D(3)-creatine in muscle and D(3)-creatinine in urine, and 2) the time to isotopic steady state. We used cross-sectional studies to compare total creatine pool size determined by the D(3)-creatine dilution method to lean body mass determined by independent methods. The tracer dose of D(3)-creatine (<1 mg/rat) was >99% bioavailable with 0.2-1.2% urinary spillage. Isotopic steady state was achieved within 24-48 h. Creatine pool size calculated from urinary D(3)-creatinine enrichment at 72 h significantly increased with muscle accrual in rat growth, significantly decreased with dexamethasone-induced skeletal muscle atrophy, was correlated with lean body mass (r = 0.9590; P < 0.0001), and corresponded to predicted total muscle mass. Total-body creatine pool size and skeletal muscle mass can thus be accurately and precisely determined by an orally delivered dose of D(3)-creatine followed by the measurement of D(3)-creatinine enrichment in a single urine sample and is promising as a noninvasive tool for the clinical determination of skeletal muscle mass.     

Synthesis and structural identification of four dihydroxy acids and 11,12-leukotriene C4 derived from 11,12-leukotriene A4

Using a partially purified 12-lipoxygenase from porcine leukocytes, (5Z,8Z,10E,14Z)-12-hydroperoxy-5,8,10,14-icosate traenoic acid was synthesized from arachidonic acid with a yield of over 35%. The absolute configuration of C-12 was determined as S by chiral-phase column chromatography. It was chemically converted to at least three epoxides with the conjugated triene structure. Two were identified by proton NMR and mass spectrometry to be (5Z,7E,9E,14Z)-(11S,12S)-11,12-oxido-5,7,9,14-ic osatetraenoic acid (11,12-leukotriene A4) and (5Z,7Z,9E,14Z)-(11S,12S)-11,12-oxido-5,7,9,14-ic osatetraenoic acid (7-cis-11,12-leukotriene A4). 11,12-Leukotriene A4 underwent acid hydrolysis to yield two diastereomers of (6E,8E,10E,14Z)-(12S)-5,12-dihydroxy-6,8,10,14-i cosatetraenoic acid and two isomers of (14Z)-(12S)-11,12-dihydroxy-5,7,9,14-icosatetraenoic acid. Upon incubation with rat liver glutathione S-transferase, 11,12-leukotriene A4 was converted to 11,12-leukotriene C4, a spasmogenic compound.     

Solvolysis of chenodeoxycholic acid sulfates

Chemical solvolysls of chenodeoxycholic acid sulfates was studied using 4 published methods. Quantitative recovery of chenodeoxycholic acid from the 3-sulfate was obtained with each method. However, only 2 methods yielded chenodeoxycholic acid after solvolysis of the 7-sulfate. In each instance a compound resembling lithocholic acid by GLC but identifiable as a derivative of chenodeoxycholic acid by mass spectrometry was obtained and represents a product formed during solvolysis. Failure to obtain adequate solvolysis of chenodeoxycholic acid 7-sulfate can lead to false identification of monohydroxy bile acids and apparent absence of the 7-sulfate and disulfate esters.     

Liquid chromatographic-electrospray ionization-mass spectrometric analysis of cytochrome P450 metabolites of arachidonic acid.

Arachidonic acid (AA) can be metabolized by cytochrome P450 (CYP) enzymes to many biologically active compounds including 5,6-, 8,9-, 11,12-, and 14,15-epoxyeicosatrienoic acids (EETs), their corresponding dihydroxyeicosatrienoic acids (DHETs), and 20-hydroxyeicosatetraenoic acid (20-HETE). These eicosanoids are potent regulators of vascular tone. We developed a liquid chromatography-electrospray ionization-mass spectrometry method to simultaneously determine 5,6-, 8,9-, 11,12-, and 14,15-EETs; 5,6-, 8,9-, 11,12-, and 14,15-DHETs; and 20-HETE. [2H8]EETs, [2H8]DHETs, and [2H2]20-HETE were used as internal standards. These compounds are readily separated on a C18 reverse-phase column using water:acetonitrile with 0.005% acetic acid as a mobile phase. The internal standards, [2H8]EETs, [2H8]DHETs, and [2H2]20-HETE, eluted slightly faster than the natural eicosanoids. The samples were ionized by electrospray with fragmentor voltage of 120 V and detected in a negative mode. The negative ion detection gave a lower background than the positive ion detection for these compounds. These eicosanoids exhibited high abundance of the ions corresponding to [M - 1]-. The m/z = 319, 337, and 319 ions were used for quantitation of EETs, DHETs, and 20-HETE, respectively. The detection limits using selected ion monitoring of these compounds are about 1 pg per injection. The position of functional groups and water content of mobile phase had a significant effect on the sensitivity of detection. Water content of 40% was found to give maximal sensitivity. The method was used to determine EETs, DHETs, and 20-HETE in bovine coronary artery endothelial cells, dog plasma, rat astrocytes, and rat kidney microsome samples.     

Difference between cholic acid and chenodeoxycholic acid in dependence upon cholesterol of hepatic and plasmatic sources as the precursor in rats

Some difference in functional pool of cholesterol acting as the precursor of bile acids is pointed out between cholic acid and chenodeoxycholic acid. In order to elucidate this problem further, some experiments were performed with rats equilibrated with [7(n)-3H, 4-(14)C] cholesterol by subcutaneous implantation. The bile duct was cannulated in one series of experiments and ligated in another. After the operation 14C-specific radioactivity of serum cholesterol fell, but reached practically a new equilibrium within three days. 14C-Specific radioactivity of serum cholesterol as well as of biliary bile acids in bile-fistula rats and urinary bile acids in bile duct-ligated rats was determined during a three days-period in the new equilibrated state. The results were as follows: (1) 14C-Specific radioactivity of cholic acid and chenodeoxycholic acid in bile was lower than that of serum cholesterol, and 14C-specific radioactivity of cholic acid was clearly lower than that of chenodeoxycholic acid. (2) 14C-Specific radioactivity of cholic acid and beta-muricholic acid in urine was lower than that of serum cholesterol, and 14C-specific radioactivity of cholic acid was lower than that of beta-muricholic acid. (3) Biliary as well as urinary beta-muricholic acid lost tritium label at 7-position entirely during the course of formation from [7(n)-3H, 4-(14)C]cholesterol.     

Measurement of parameters of cholic acid kinetics in plasma using a microscale stable isotope dilution technique: application to rodents and humans.

A stable isotope dilution method is described that allows measurement of cholic acid (CA) kinetics, that is, pool size, fractional turnover rate (FTR), and synthesis rate in mice, rats, and humans. Decay of administered [2,2,4,4-2H4]CA enrichment was measured in time in 50-microl plasma samples by gas-liquid chromatography/electron capture negative chemical ionization-mass spectrometry, applying the pentafluorobenzyl-trimethylsilyl derivative. The kinetic data expressed species-dependent differences. The CA pool sizes were 16.8 +/- 2.1, 10.6 +/- 1.2, and 2.4 +/- 0.7 micromol/100 g body weight for mice, rats, and humans, respectively. The FTR values were 0.44 +/- 0.03, 0.88 +/- 0.10, and 0.46 +/- 0.14 per day for mice, rats, and humans. The corresponding synthesis rates were 7.3 +/- 1.6, 9.3 +/- 0.1, and 1.0 +/- 0.2 micromol/100 g body weight per day. The human data agreed well with literature data obtained by conventional isotope dilution techniques. For rats and mice these are the first reported isotope dilution data. The method was validated by confirmation of isotopic equilibrium between biliary CA and plasma CA in the rat. Its applicability was demonstrated by the observation of increased CA FTR and CA synthesis rate in rats fed cholestyramine, which is known to increase fecal bile acid excretion. The presented stable isotope dilution method enables the determination of CA kinetic parameters in small plasma samples. The method can be applied in unanesthetized rodents with an intact enterohepatic circulation and may also be valuable when studying the development of human neonatal bile acid kinetics.     

In vivo regulation of phenylalanine hydroxylation to tyrosine, studied using enrichment in apoB-100

Phenylalanine hydroxylation is necessary for the conversion of phenylalanine to tyrosine and disposal of excess phenylalanine. Studies of in vivo regulation of phenylalanine hydroxylation suffer from the lack of a method to determine intrahepatocyte enrichment of phenylalanine and tyrosine. apoB-100, a hepatic export protein, is synthesized from intrahepatocyte amino acids. We designed an in vivo multi-isotope study, [(15)N]phenylalanine and [2H2]tyrosine to determine rates of phenylalanine hydroxylation from plasma enrichments in free amino acids and apoB-100. For independent verification of apoB-100 as a reflection of enrichment in the intrahepatocyte pool, [1-(13)C]lysine was used as an indicator amino acid (IAA) to measure in vivo changes in protein synthesis in response to tyrosine supplementation. Adult men (n = 6) were fed an amino acid-based diet with low phenylalanine (9 mg.kg(-1).day(-1), 4.54 mumol.kg(-1).,h(-1)) and seven graded intakes of tyrosine from 2.5 (deficient) to 12.5 (excess) mg.kg(-1).day(-1). Gas chromatography-quadrupole mass spectrometry did not detect any tracer in apoB-100 tyrosine. A new and more sensitive method to measure label enrichment in proteins using isotope ratio mass spectrometry demonstrated that phenylalanine hydroxylation measured in apoB-100 decreased linearly in response to increasing tyrosine intake and reached a break point at 6.8 mg.kg(-1).day(-1). IAA oxidation decreased with increased tyrosine intake and reached a break point at 6.0 mg.kg(-1).day(-1). We conclude: apoB-100 is an accurate and useful measure of changes in phenylalanine hydroxylation; the synthesis of tyrosine via phenylalanine hydroxylation is regulated to meet the needs for protein synthesis; and that plasma phenylalanine does not reflect changes in protein synthesis.     

The determination of amino acid pool sizes and turnover rates by gas chromatographic mass spectrometric analysis of stable isotope enrichment

Gas chromatography mass spectrometry has been used to determine the 15N enrichment of plasma glycine of rabbits at various times following the intravenous administration of 15N-glycine. These data were used to prepare isotope enrichment time decay curves for eleven individual animals. The slopes and intercepts of least squares lines that describe the decay curves were considerably more accurately than those reported in similar studies employing radioactive tracers. Individual glycine pool sizes (13.8-37.4 micronmoles per 100 g body wt), turnovers rates (2.66-3.36 pools h-1) and flux (50.4-99.7 micronmoles h-1 per 100 g body wt) were estimated from these parameters in a group of animals and compared with the literature values. These results demonstrate that low risk non-radioactive stable isotopes can be substituted for radioactive tracers in studies of human amino acid metabolism, with considerable saving in time and without loss in accuracy, when gas chromatography mass spectrometry is used to determine plasma amino acid and stable isotope enrichment.     

Synthesis of 11,12-leukotriene A4, 11S,12S-oxido-5Z,7E,9E,14Z-eicosatetraenoic acid, a novel leukotriene of the 12-lipoxygenase pathway

A simple and efficient method for preparing 11,12-leukotriene A4 has been established by the stereospecific biomimetic route from arachidonic acid. 12S-Hydroperoxy-5Z,8Z,10E,14Z-eicosatetraenoic acid was synthesized using a partially purified 12-lipoxygenase of porcine leukocytes. The methyl ester of the compound was then chemically converted to two labile epoxides with a conjugated triene structure. These compounds were identified by proton NMR and mass spectrometry to be 11S,12S-oxido-5Z,7E,9E,14Z-eicosatetraenoic acid (11,12-leukotriene A4) and its geometric isomer.     

Chemical synthesis of deuterated folate monoglutamate and in vivo assessment of urinary excretion of deuterated folates in man

the synthesis and in vivo application of stable-isotopically labeled folic acid was investigated to devise methods suitable for studies of folate metabolism in human subjects. Glutamate-labeled tetradeutero-pteroylglutamic acid (d4-folic acid) was prepared by mixed anhydride coupling of N10-trifluoroacetylpteroic acid and dimethyl L-[3,3,4,4-2H4]glutamic acid, saponification in sodium deuteroxide, and chromatographic purification. Retention of the isotopic label was verified by proton NMR and mass spectrometry of the para-aminobenzoylglutamic acid product of C9-N10 bond cleavage. A method was devised for determination of of isotopic enrichment of urinary d4-folates derived from orally administered d4-folic acid using affinity chromatographic purification, chemical cleavage of the C9-N10 bond, HPLC isolation of the p-[2H4]aminobenzoylglutamate product, followed by negative-ion chemical-ionization gas chromatography/mass spectrometry. Data concerning the urinary excretion of d4-folates derived from an oral dose of d4-folic acid in an adult human are presented.     

Characterization and in vivo production of three glycolipids from Candida Bogoriensis: 13-glucopyranosylglucopyranosyloxydocosanoic acid and its mono- and diacetylated derivatives

Three glycosides of 13-hydroxydocosanoic acid isolated from Candida bogoriensis were characterized by quantitating the amount of carbohydrate, acetate, and hydroxy acid in each, and by gas-liquid chromatography and mass spectrometry of their methyl ester, trimethylsilyl ether derivatives. One of the glycosides was the diacetylated derivative of 13-glucosylglucosyloxydocosanoic acid previously characterized by Tulloch, Spencer, and Deinema (Can. J. Chem., 46: 345 [1968]), in which the disaccharide had the beta(1 --> 2) sophorose linkage and the acetyl groups were attached to the 6' and 6" positions of the glucose residues. The other two glycosides were 13-glucosylglucosyloxydocosanoic acid and its monoacetylated derivative. A comparison of the mass spectra of derivatives indicates that the acetyl group of the monoacetyl lipid is on the internal glucose. Methyl 13-glucosyloxydocosanoate was produced by acid hydrolysis of the methyl ester of the unacetylated glycolipid and was characterized by the same techniques as the other glycolipids. Time course of production of the three glycolipids is consistent with the diacetylated derivative being the first extra-cellular product and the other two glycolipids being formed by deacetylation. 13-Hydroxy[13-(3)H]docosanoic acid, methyl 13-hydroxy[13-(3)H]docosanoate, and 9-hydroxy[11,12-(3)H]-stearic acid were each incorporated into the glycolipid fraction.     

Rabbit renal cortical microsomes metabolize arachidonic acid to trihydroxyeicosatrienoic acids

(1-¹⁴C) Eicosatetraenoic (Arachidonic) acid was incubated wiht microsomes from rabbit renal cortex and NADPH (1 mM) for 15 min at 37°C. The products were extracted and purified by high pressure liquid chromatography. Some of the most polar metabolites were identified by gas chromatography mass spectrometry. They were 11, 12, 19- and 11, 12,20-trihydroxy-5,8-14-eicosatrienoic acid, 14,15,19- and 14,15,20- trihydroxy-5,8,11-eicosatrienoic acid, and 11,12-dihydroxy-19-oxo- 5,8,14-eicosatrienoic acid. These products were likely formed by ω- and (ω−1)-hydroxylation of 11,12-dihydroxy-5,8,14-eicosatrienoic aic and 14,15-dihydroxy-5,8,11-eicosatrienoic acid, two recently identified metabolites of arachidonic acid in fortified rabbit kidney microsomes.     

Calculation of stable isotope enrichment tracer kinetic procedures

The choice of method of expressing isotopic enrichment in tracer kinetic experiments utilizing stable isotopes was found to affect the calculation of tracee pool size and half-life. The most commonly used definition, the difference between enriched and natural abundance, i.e. atom percent excess, was found to result in significant error in model systems when the dose of tracer was 10% of the pool size. Errors in determining first-order rate constants of efflux and in pool sizes decreased with decreasing ratio of tracer to tracee. Error in determining pool size increased with longer 'sampling' periods, while error in determining the rate constant increased with shorter sampling periods. Of three less frequently used expressions of isotopic enrichment two were found to yield the exact answers in model systems. The correct expressions of isotopic enrichment were linear functions of the quantity of tracer in the system. A practical example demonstrated the effect of choice of expression of enrichment on estimates of whole body copper pool size and turnover in dairy cattle.     

Quantitative aspects of the conversion of 5 beta-cholestane intermediates to bile acids in man.

The in vivo conversion of several 5 beta-cholestane intermediates to primary bile acids was investigated in three patients with total biliary diversion. The following compounds were administered intravenously: 5 beta-[G-3H]-cholestane-3 alpha, 7 alpha-diol, 5 beta-[G-3H]cholestane-3 alpha, 7alpha, 26-triol, and 5 beta-[24-14C]cholestane-3 alpha, 7 alpha-25-triol. Bile was then collected quantitatively at frequent intervals for the next 21 to 28 h. The administered 5 beta-[G-3H]cholestane-3alpha, 7alpha, 26-triol was found to be efficiently converted to cholic and chenodeoxycholic acids in two patients; 61 and 75% of the administered label was found in primary bile acids. The proportion of labeled cholic to chenodeoxycholic acid was 1.20 and 1.02 in the bile of these patients, indicating that the C-26 triol was efficiently converted to cholic acid. The ratio of cholic to chenodeoxycholic acid (mass) in the bile of these patients was 1.23 and 2.32. The 5 beta-cholestane-3alpha, 7alpha-diol intermediate was also efficiently converted (71%) to both primary bile acids. The cholic to chenodeoxycholic acid ratios by mass and label were similar (2.97 versus 2.23). By contrast, the 5beta-cholestane-3alpha, 7alpha, 25-triol was poorly converted to bile acids in three patients. Following the administration of this compound almost all of the administered radioactivity found in the bile acid fraction was in cholic acid (5 to 19%) and very little (less than 5%) was found in chenodeoxycholic acid. These findings indicate that ring hydroxylation at position 12 is not materially hindered by the presence of a hydroxyl group on the side chain at C-26 in patients with biliary diversion. The labeled C-26-triol which was efficiently converted to both primary bile acids in a proportion similar to that which was observed for the bile acids synthesized by the liver suggests that this 5beta-cholestane derivative may be a major intermediate in the synthesis of both cholic and chenodeoxycholic acids.     

Arachidonic acid inhibits epithelial Na channel via cytochrome P450 (CYP) epoxygenase-dependent metabolic pathways

We used the patch-clamp technique to study the effect of arachidonic acid (AA) on epithelial Na channels (ENaC) in the rat cortical collecting duct (CCD). Application of 10 microM AA decreased the ENaC activity defined by NPo from 1.0 to 0.1. The dose-response curve of the AA effect on ENaC shows that 2 microM AA inhibited the ENaC activity by 50%. The effect of AA on ENaC is specific because neither 5,8,11,14-eicosatetraynoic acid (ETYA), a nonmetabolized analogue of AA, nor 11,14,17-eicosatrienoic acid mimicked the inhibitory effect of AA on ENaC. Moreover, inhibition of either cyclooxygenase (COX) with indomethacin or cytochrome P450 (CYP) omega-hydroxylation with N-methylsulfonyl-12,12-dibromododec-11-enamide (DDMS) failed to abolish the effect of AA on ENaC. In contrast, the inhibitory effect of AA on ENaC was absent in the presence of N-methylsulfonyl-6-(propargyloxyphenyl)hexanamide (MS-PPOH), an agent that inhibits CYP-epoxygenase activity. The notion that the inhibitory effect of AA is mediated by CYP-epoxygenase-dependent metabolites is also supported by the observation that application of 200 nM 11,12-epoxyeicosatrienoic acid (EET) inhibited ENaC in the CCD. In contrast, addition of 5,6-, 8,9-, or 14,15-EET failed to decrease ENaC activity. Also, application of 11,12-EET can still reduce ENaC activity in the presence of MS-PPOH, suggesting that 11,12-EET is a mediator for the AA-induced inhibition of ENaC. Furthermore, gas chromatography mass spectrometry analysis detected the presence of 11,12-EET in the CCD and CYP2C23 is expressed in the principal cells of the CCD. We conclude that AA inhibits ENaC activity in the CCD and that the effect of AA is mediated by a CYP-epoxygenase-dependent metabolite, 11,12-EET.     

Formation of ursodeoxycholic acid from chenodeoxycholic acid in the human colon: studies of the role of 7-ketolithocholic acid as an intermediate

The formation of ursodeoxycholic acid from chenodeoxycholic acid and the role of 7-ketolithocholic acid as an intermediate in this biotransformation were studied in vitro in fecal incubations as well as in vivo in the human colon. [24-14C]-Labeled 7-ketolithocholic and chenodeoxycholic acids were studied at various concentrations, and the biotransformation products were analyzed by thin-layer chromatography, gas-liquid chromatography, and mass spectrometry. There was rapid colonic conversion of 7-ketolithocholic acid to ursodeoxycholic acid and, to a lesser extent, to chenodeoxycholic acid. The reduction of 7-ketolithocholic to ursodeoxycholic acid proceeded significantly faster anaerobically and at acid pH than under aerobic and alkaline conditions. When chenodeoxycholic acid was incubated in vitro or instilled into the colon, various amounts of 7-ketolithocholic and ursodeoxycholic acids were formed. The formation of 7-ketolithocholic acid was favored by alkaline conditions. Isotope dilution studies, in which trace amounts of labeled 7-ketolithocholic acid were incubated with unlabeled chenodeoxycholic acid, indicate 7-ketolithocholic acid to be the major intermediate in the intestinal bacterial conversion of chenodeoxycholic to ursodeoxycholic acid.     

Simultaneous evaluation of six human glucuronidation activities in liver microsomes using liquid chromatography-tandem mass spectrometry

This article describes the development of a procedure for the simultaneous evaluation of the activity of six different uridine diphosphate (UDP)-glucuronyltransferases (UGTs) in human liver microsomes (HLMs). The method consists of incubations of probe substrates for UGT1A1 (etoposide), UGT1A3 (chenodeoxycholic acid), UGT1A4 (trifluoperazine), UGT1A6 (serotonin), UGT1A9 (mefenamic acid), and UGT2B7 (azidothymidine) with HLMs. The six substrates were divided into three different incubations (etoposide + mefenamic acid; chenodeoxycholic acid + serotonin + azidothymidine; and trifluoperazine alone), the media of which were pooled before analysis. Glucuronide formation rates were determined in a single run of 20 min using a validated liquid chromatography-tandem mass spectrometry method. No significant difference was observed between glucuronidation activities measured using the current procedure and individual incubations of the probes. The method was used successfully for the determination of UGT activities in 44 individual HLM preparations and for the phenotyping of preparations predicted to have altered UGT1A1 and UGT2B7 activities because of known genetic polymorphisms.