Application of gas chromatography–triple quadrupole mass spectrometry to the determination of sterol components in biological samples in consideration of the ionization mode

The hyphenation of gas chromatography (GC) and triple quadrupole mass spectrometry is a promising approach to increase sensitivity and selectivity as compared to single quad mass spectrometry. We present in this paper the application of GC–triple quadrupole mass spectrometry for determination of sterol components in biological samples. Due to the fact that sterols are quite small molecules an appropriate ionization mode has to be found for advantageous exploitation of the triple quad function. Electron ionization (EI), positive and negative chemical ionization (PCI, NCI) have been tested regarding sensitivity improvement in oxysterol and bile acid analysis in plasma samples. Target analytes were 24-, 25- and 27-hydroxycholesterol, 7β-hydroxycholesterol, 7-ketocholesterol, 3β,5α,6β-cholestanetriol, cholic acid, chenodeoxycholic acid, deoxycholic acid and lithocholic acid. In contrast to bile acids, oxysterols could be analyzed with the highest degree of sensitivity by application of PCI in multiple reaction monitoring mode whereas 7β-hydroxycholesterol and 7-ketocholesterol showed even better results with NCI.     

Hepatic bile acid metabolism during early development revealed from the analysis of human fetal gallbladder bile

A detailed study of the qualitative and quantitative composition of bile acids in human fetal gallbladder bile is described. Bile was collected during early gestation (weeks 16-19) and analyzed by gas chromatography and mass spectrometry, fast atom bombardment ionization mass spectrometry, and high performance liquid chromatography. Bile acids were separated into different conjugate groups by chromatography on the lipophilic anion exchange gel, diethylaminohydroxypropyl Sephadex LH-20. Quantitatively more than 80% of the bile acids were secreted into bile conjugated to taurine. Unconjugated bile acids and glycine conjugates accounted for 5-10% of the total biliary bile acids. Bile acid sulfates were present only in trace amounts indicating that quantitatively sulfation is not an important pathway in bile acid metabolism during development. Total biliary bile acid concentrations were low (0.1-0.4 mM) when compared to reported values for adult bile (greater than 10 mM). Chenodeoxycholic acid was the major biliary bile acid and exceeded cholic acid concentrations by 1.43-fold indicating either a relative immaturity in 12 alpha-hydroxylase activity during early life or a dominance of alternative pathways for chenodeoxycholic acid synthesis. A relatively large proportion of the biliary bile acids comprised metabolites not found in adult bile. The presence of relatively high proportions of hyocholic acid (often greater than cholic acid) and several 1 beta-hydroxycholanoic acid isomers indicates that C-1 and C-6 hydroxylation are important pathways in bile acid synthesis during development. We describe, for the first time, evidence for the existence of a C-4 hydroxylation pathway in the metabolism of bile acids, which may be unique to early human development. Mass spectrometry was used to confirm the identification of 3 alpha,4 beta,7 alpha-trihydroxy-5 beta-cholanoic and 3 alpha,4 beta-dihydroxy-5 beta-cholanoic acids. Quantitatively, these C-4 hydroxylated bile acids accounted for 5-15% of the total biliary bile acids of the fetus, suggesting that C-4 hydroxylation is quantitatively an important pathway in the bile acid metabolism during early life.     

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.     

Simultaneous determination of biliary bile acids in rat: ELectron impact and ammonia chemical ionization mass spectrometric analyses of bile acids

Bile acids in the rat bile were fractionated into unconjugated, glycine- and taurine-conjugated fractions by employing piperidino-hydroxypropyl Sephadex LH-20 ion-exchange chromatography. Subsequently, these fractions were analyzed by gas-liquid chromatography (GLC) and GLC-mass spectrometry using a Silicone AN-600 column. Not only lithocholic acid, deoxycholic acid, chenodeoxycholic acid, hyodeoxycholic acid, ursodeoxycholic acid and cholic acid, but also αand β-muricholic acids were quantitatively and simultaneously detectable in conjugated and unconjugated fractions, respectively. In the unconjugated and conjugated fractions, varying amounts of the unidentified bile acid were detected upon GLC. The electron impact and ammonia chemical ionization mass spectrometric results and catalytic hydrogenation on the compound indicate that this bile acid seems to be a derivative of β-muricholic acid having a double bond in the side chain. The present method is suitable to the simultaneous and quantitative determination of unconjugated and glycine- and taurine-conjugated bile acids in the rat bile.     

Identification of (22R)-3 alpha,7 alpha,12 alpha,22- and (23R)-3 alpha,7 alpha,12 alpha,23-tetrahydroxy-5 beta-cholestanoic acids in urine from a patient with Zellweger's syndrome

The nature of two novel C27 bile acids present as the taurine conjugates in urine from a patient with Zellweger's syndrome was studied. Bile acids conjugated with taurine were isolated from unconjugated and glycine-conjugated bile acids by means of ion-exchange chromatography. After alkaline hydrolysis of the taurine conjugates, the hydrolysate was acidified and extracted with ether; the extract was again subjected to ion-exchange chromatography to separate neutral from acidic compounds. The neutral fraction, which consisted mainly of two steroidal lactones, was treated with lithium aluminum hydride, and the reduction products were identified as (22R)-5 beta-cholestane-3 alpha,7 alpha,12 alpha,22,26-pentol and (23R)-5 beta-cholestane-3 alpha,7 alpha,12 alpha,23,26-pentol by direct comparison of their gas-liquid chromatographic behaviors and mass spectral data with those of chemically synthesized authentic samples. Thus, the chemical structure of two native bile acids present in urine from a patient with Zellweger's syndrome should be formulated as (22R)-3 alpha,7 alpha,12 alpha,22-tetrahydroxy-5 beta-cholestanoic acid and (23R)-3 alpha,7 alpha,12 alpha,12 alpha,23-tetrahydroxy-5 beta-cholestanoic acid, respectively.     

A rapid non-chromatographic analysis of individual bile acids in human bile extracts

It is postulated that the six conjugated bile acids of most common occurrence in human bile could be analyzed by three enzymic and one chemical assay without any prior chromatographic separation of the bile acids. In health, all bile acids in liver or gall bladder bile are conjugated with either glycine or taurine and have an a-hydroxyl group at the 3 position. In addition, the trihydroxy bile acid, cholic (C) has a 7α- and a 12α-hydroxy group while the dihydroxy bile acids either have a second hydroxyl group at the 7α-position (chenodeoxycholic acid, CDC) or at the 12α-position (deoxycholic acid, DC). Hydroxysteroid dehydrogenases (HSDH) specific for oxido-reductase activity at the 3α-, 7α- and 12α-positions would directly quantify these 3α-, 7α- and 12α-hydroxyl groups in a sample of bile or bile extract. Subsequent data would be used to solve three simultaneous equations yielding solutions for the overall concentrations of conjugated C, conjugated CDC and conjugated DC on the assumption that the overall concentration of lithocholic acid is negligible (< 2 %). A suitable assay for the sulphonate group containing taurine conjugates, such as that described by Christie, Macdonald & Williams, 1975, along with the total bile acid measurement would readily facilitate the estimation of the glycine/taurine ($\text{G}\text{T}$) ratio. This ratio applied to the enzymatically derived estimates for conjugated DC, CDC and C would approximate the glycodeoxycholate (GDC), glycochenodeoxycholate (GCDC), glycocholate (GC), taurodeoxycholate (TDC), taurochenodeoxycholate (TCDC) and taurocholate (TC) concentrations. Figures for these concentrations would be based on the assumption that the $\text{G}\text{T}$ ratio is approximately the same for each bile acid and that all the bile acids are conjugated.     

Determination of plasma bile acids by capillary gas-liquid chromatography-electron capture negative chemical ionization mass fragmentography

Combined capillary gas-liquid chromatography-electron capture negative chemical ionization mass spectrometry of pentafluorobenzyl ester-TMSi ether derivatives of bile acids and isotope dilution using deuterated internal standards are introduced as a sensitive and selective analysis technique for plasma bile acids. As a result of the high ionization efficiency of pentafluorobenzyl derivatives under electron capturing conditions and minimal fragmentation, the detection limit of this technique is low: 1 pg for each bile acid. The high sensitivity enabled the detection and quantitation of atypical bile acids in 200-microliters aliquots of plasma from fasting healthy adults as exemplified by trihydroxycoprostanic acid (0.002 +/- 0.001 mumol/l) and dihydroxycoprostanic acid (0.013 +/- 0.002 mumol/l).     

Profiling circulating and urinary bile acids in patients with biliary obstruction before and after biliary stenting

Bile acids are considered as extremely toxic at the high concentrations reached during bile duct obstruction, but each acid displays variable cytotoxic properties. This study investigates how biliary obstruction and restoration of bile flow interferes with urinary and circulating levels of 17 common bile acids. Bile acids (conjugated and unconjugated) were quantified by liquid chromatography coupled with tandem mass spectrometry in serum and urine samples from 17 patients (8 men and 9 women) with biliary obstruction, before and after biliary stenting. Results were compared with serum concentrations measured in 40 age- and sex-paired control donors (20 men and 20 women). The total circulating bile acid concentration increases from 2.7 μM in control donors to 156.9 μM in untreated patients with biliary stenosis. Serum taurocholic and glycocholic acids exhibit 304- and 241-fold accumulations in patients with biliary obstruction compared to controls. The enrichment in chenodeoxycholic acid species reached a maximum of only 39-fold, while all secondary and 6α-hydroxylated species--except taurolithocholic acids--were either unchanged or significantly reduced. Stenting was efficient in restoring an almost normal circulating profile and in reducing urinary bile acids. Conclusion: These results demonstrate that biliary obstruction affects differentially the circulating and/or urinary levels of the various bile acids. The observation that the most drastically affected acids correspond to the less toxic species supports the activation of self-protecting mechanisms aimed at limiting the inherent toxicity of bile acids in face of biliary obstruction.     

Metabolism of (24R and 24S)-27-nor-24-methyl-3α,7α-dihydroxy-5β-cholestan-26-oic acids and 27-nor-3α,7α-dihydroxy-5β-cholestan-26-oic acid in guinea pigs

[7β-³H]-(24R and 24S)-27-nor-24-methyl-3α,7α-dihydroxy-5β-cholestan-26-oic acids and [7β-³H]-27-nor-3α,7α-dihydroxy-5β-cholestan-26-oic acid (C₂₇ and C₂₆ bile acids having the same nuclear configuration as cheno-deoxycholic acid and its precursor, 3α,7α-dihydroxy-5β-cholestan-26-oic-acid) were synthesized and administered intraperitoneally to bile fistula guinea pigs. The biliary bile acids formed were hydrolyzed and analyzed by thin layer chromatography, and the metabolites were identified by the inverse isotope dilution method. The results showed that both (24R and 24S)-27-nor-24-methyl-3α,7α-dihydroxy-5β-cholestan-26-oic acids were not metabolized by the liver and were excreted unchanged as their taurine and glycine conjugates whereas 27-nor-3α,7α-dihydroxy-5β-cholestan-26-oic acid was converted to chenodeoxycholic acid.     

BILIARY BILE ACID COMPOSITION OF THE PHYSETERIDAE (SPERM WHALES)

Abstract: The bile acid composition of bile obtained from the hepatopancreatic ducts of three species of sperm whales (Cetacea: Physeteridae) was investigated. Bile acids were isolated by adsorption chromatography and analyzed by sequential HPLC, SIMS, and GLC-MS. In each species the dominant bile acids were deoxycholic acid (a secondary bile acid formed by bacterial 7α-dehydroxylation of cholic acid), and chenodeoxycholic acid (a primary bile acid) which together composed more than 86% of biliary bile acids in all three species. In Physeter catodon (sperm whale) deoxycholic acid constituted 79%, and in Kogia breviceps (pygmy sperm whale) it was 61% of biliary bile acids. The sperm whale, which differs from other whales in having a remnant of a large intestine, is the second mammal identified to date in which deoxycholic acid is the predominant bile acid. The high proportion of deoxycholic acid indicates that in the Physeteridae, anaerobic fermentation occurs in its cecum, and that bile acids undergo enterohepatic cycling. Also found were minor proportions of cholic acid, as well as bacterial derivatives of chenodeoxycholic acid (ursodeoxycholic acid, lithocholic acid, and the 12β-epimer of allo-deoxycholic acid). Bile acids were conjugated with taurine in all species; however, in the sperm whale ( Physeter ) glycine conjugates were present in trace proportions. The bile acid hydroxylation pattern (12α- but not 6α-hydroxylation), lack of primary 5α- (allo) bile acids, and presence of glycine conjugated bile acids suggests the possibility that sperm whales originated from ancient artiodactyls.     

Cholesterol 7α-hydroxylase activity and bile acid synthesis in hepatocytes of unwearied and weaned pigs in monolayer culture

Activity of cholesterol 7α-hydroxylase (EC 1.14.13.17) in freshly isolated hepatocytes from unweaned piglets (2 to 3 weeks old) was 16-times lower as compared to hepatocytes from weaned piglets (7 to 8 weeks old). The monolayer culture activity of the enzyme remained low in unweaned piglet hepatocytes. In contrast, in cultured hepatocytes from weaned piglets, cholesterol 7α-hydroxylase activity declined during the first day of culture, but was restored during the next 2 culture days, provided that fetal bovine serum (10%) was added to the culture medium. Addition of dexamethasone (50 nM) and insulin (135 nM) to the medium, further enhanced cholesterol 7α-hydroxyease activity to values similar to those in freshly isolated hepatocytes and retarded the decline of enzyme activity after the 3rd culture day. Cultured hepatocytes from weaned and unweaned piglets synthesized similar types of bile acids from [¹⁴C]cholesterol. among which hyocholic acid (the most prominent), hyodeoxycholic acid, chenodeoxycholic acid, murocholic acid and lithocholic acid could be identified. 95% of radiolabelled bile acids synthesized was conjugated, mainly with glycine, but also with taurine, sulfate and glucuronic acid. The rate of mass production of bile acids by cultured hepatocytes of weaned piglets (as measured by gas-chromatography) parallelled cholesterol 7α-hydroxylase activity, and was low in the absence of serum, but increased in medium containing fetal bovine serum, dexamethasone and insulin to a rate lying in the range of 75% of the in vivo bile acid production during the 3rd culture day. Bile acid production by unweaned piglet hepatocytes was 3-times lower under these conditions. It is concluded that hepatocytes from young weaned pigs cultured in medium containing 10% fetal bovine serum, offer a suitable in vitro model for the study of bile acid synthesis, in view of the high cholesterol 7α-hydroxylase activities and bile acid production rates.     

Presence of protein-bound unconjugated bile acids in the cytoplasmic fraction of rat brain

Using liquid chromatography/electrospray ionization mass spectrometry, we have found three unconjugated bile acids [cholic acid (CA), chenodeoxycholic acid (CDCA), and deoxycholic acid (DCA)] in the rat brain cytoplasmic fraction. CDCA was detected only upon extraction with high concentrations of guanidine, indicating that it is bound noncovalently to protein in the brain. The most abundant of the three, it was present at a concentration of 1.6 nmol/g wet weight (approximately 15 mg of protein) of brain, corresponding to almost 30 times its serum concentration. CA and DCA were present at 1/30th the concentration of CDCA. Bile acids conjugated with amino acids, sulfuric acid, and glucuronic acid were not detected. These data clearly demonstrate that unconjugated CDCA and, to a lesser extent, CA and DCA, exists in the rat brain.     

Chemical lonization-mass spectrometric approach to structure determination of an intermediate in bile acid biosynthesis

In the course of a study on the details of the biosynthesis of cholic acid from cholesterol, 5β-[26,27-¹⁴C]cholestan-3α,7α,12α,24S,25-pentol, an intermediate in the 25-hydroxylation pathway of cholic acid, was incubated for 2 min with the cytosolic fraction of rat liver homogenate in the presence of NAD. A precursor to cholic acid which appeared to be a ketone was isolate from the reaction mixture by thin-layer chromatography. This material proved to be of inadequate volatility for electron impact mass spectrometry and was therefore studied, without further purification, by techniques of chemical ionization mass spectrometry using ammonia as the reagent gas. The spectrum was rerecorded using argon mixed with ammonia to induce additional fragmentation. One of these fragments corresponded to a McLafferty rearrangement of a 24-keto-25-hydroxycholestane derivative. To obtain additional evidence for this structure the following sequence of reactions was conducted on about 20 μg of the intermediate: (1) periodic acid oxidation, (2) diazomethane treatment, and (3) chromic acid oxidation. The change in molecular weight after each reaction agreed with the presence of a 25-hydroxy-24-keto side chain and three secondary hydroxyl groups in the molecule. Therefore, it could be deduced that the intermediate was 3α,7α,12α,25-tetrahydroxy-5β-cholestan-24-one. This work demonstrates that chemical ionization-mass spectrometic techniques can be a labor-saving alternative to other methods of structure determination and that 3α,7α,12α,25-tetrahydroxy-5β-cholestan-24-one is probably an intermediate in the 25-hydroxylation pathway of cholic acid from cholesterol.     

Sequence analysis of the sulfated rhamno-oligosaccharides derived from a sulfated rhamnan

Three sulfated rhamno-oligosaccharides, designated O1, O2 and O3, were obtained by mild acid hydrolysis of the sulfated rhamnan and purified by gel-permeation chromatography. On the basis of one- and two-dimensional nuclear magnetic resonance (1D, 2D NMR) spectroscopic analyses, the oligosaccharide O1 was characterized to be α-l-Rhap-(2SO4)-(1→3)-α-l-Rhap. The fragmentation pattern of the homogeneous disaccharide in the product ion spectra was recognized by negative-ion electrospray tandem mass spectrometry with collision-induced dissociation (ES-CID MS/MS). With the principles established, the sequences of the oligosaccharides O2 and O3 were deduced to be α-l-Rhap-(2SO4)-(1→3)-α-l-Rhap-(1→3)-α-l-Rhap, and α-l-Rhap-(2SO4)-(1→3)-α-l-Rhap-(1→3)-α-l-Rhap-(1→3)-α-l-Rhap (2SO(4)), respectively. The investigation demonstrated that the sulfated rhamnan-derived oligosaccharides were novel sulfated oligosaccharides different from those of other polysaccharides-degraded from algae, and it could be possible to determine the sequence of the sulfated rhamno-oligosaccharides directly from the glycosidic cleavage fragmentation in the product ion spectra.     

Bile acids XLII. Preparation of 5-alpha-cholestan-26-oic acids from kryptogenin

3β, 7α, 26-Triacetoxy-5α-cholestane was prepared from 25R-3β, 26-diacetoxy-5α-cholestan-7α-ol, and partially hydrolyzed with potassium carbonate in methanol-benzene. The three acetylated products thus obtained were characterized by thin layer and gas liquid chromatography, and mass spectrometry. By oxidation and alkaline hydrolysis, 3β, 7α-diacetoxy-5α-cholestan-26-ol was converted to 3β, 7α-dihydroxy-5α-cholestanoic acid. 7α, 26-Diacetoxy-5α-cholestan-3β-ol was characterized as indicated. The third product, 7α-acetoxy-5α-cholestane-3β, 26-diol was oxidized to 3-oxo-7α-acetoxy-5α-cholestanoic acid which was reduced catalytically and hydrolyzed to provide 3α, 7α-dihydroxy-5α-cholestanoic acids and its 3β-isomer. By comparison of the specific rotation of this sample of 3α, 7α-dihydroxy-5α-cholestanoic acid derived from 25R-kryptogenin with a similar product derived from arihydro-5α-cyprinol obtained from carp bile, the latter derivative appears to be primarily the 25S material.     

Behavior of 3α- and 7α-hydroxysteroid dehydrogenases on chenodeoxycholate substituted sepharose

Chenodeoxycholate (3α-, 7α-dihydroxy-5β-cholanoate) was linked to Sepharose 4B by an ethylenediamine bridge. When 3α-hydroxysteroid dehydrogenase and 7α-hydroxysteroid dehydrogenase preparations were applied to a column of covalently linked Chenodeoxycholate, both enzymes were retarded at pH 6.7; the 7α-OH oriented enzyme more than the 3α-OH enzyme. Approximately forty-fold purification of 7a-hydroxysteroid dehydrogenase was achieved in one step. Although no significant purification of 3α-hydroxysteroid dehydrogenase occurred, the background value in the fluorometric enzymatic estimation of bile acids by eluted 3α-hydroxysteroid dehydrogenase was markedly reduced. Molecular weight estimation by Sephadex G-200 gave the values of 47,000 for 3α-hydroxysteroid dehydrogenase and 105,000 for 7α-hydroxy-steroid dehydrogenase.     

Determination of bile acids in biological fluids by liquid chromatography-electrospray tandem mass spectrometry

A simple, sensitive, and specific liquid chromatography-electrospray tandem mass spectrometry (LC-MS/MS) method for the determination of bile acids in human bile has been developed. The bile acids were extracted with a C(18) (octadecyl) reversed-phase column and identified and quantified by simultaneous monitoring of their parent and daughter ions, using the multiple reaction monitoring mode. Identification and quantification of conjugated bile acids in bile was achieved in 5 min. The detection limit was 1 ng, and the determination was linear for concentrations up to 100 ng. The percent recovery of standards made of single conjugated (glycine and taurine) bile acid or of mixture of glycine- or taurine-conjugated cholic acid, chenodeoxycholic acid, deoxycholic acid, ursodeoxycholic acid, and lithocholic acid averaged 71.73% to 95.92%. The percent recovery of the same standard bile acids was also determined by gas chromatography-mass spectrometry (GC-MS), using the selected ion monitoring mode, and averaged 66% to 96%. A biliary bile acid profile of human gallbladder bile was obtained by LC-MS/MS and GC-MS.The results showed a good correlation between the two techniques and no significant differences between the two methods were observed. The LC-MS/MS method was also used for the analysis of serum, urine, and fecal bile acids. In conclusion, LC-MS/MS is a simple, sensitive, and rapid technique for the analysis of conjugated bile acids in bile and other biological samples. - Perwaiz, S., B. Tuchweber, D. Mignault, T. Gilat, and I. M. Yousef. Determination of bile acids in biological fluids by liquid chromatography-electrospray tandem mass spectrometry. J. Lipid Res. 2001. 42: 114;-119.     

Chemical synthesis of the 3-sulfooxy-7-N-acetylglucosaminyl-24-amidated conjugates of 3beta,7beta-dihydroxy-5-cholen-24-oic acid, and related compounds: unusual, major metabolites of bile acid in a patient with Niemann-Pick disease type C1

The chemical synthesis of 3beta,7beta-dihydroxy-5-cholen-24-oic acid, triply conjugated by sulfuric acid at C-3, by N-acetylglucosamine (GlcNAc) at C-7, and by glycine or taurine at C-24, is described. These are unusual, major metabolites of bile acid found to be excreted in the urine of a patient with Niemann-Pick disease type C1. Analogous double-conjugates of 3beta-hydroxy-7-oxo-5-cholen-24-oic acid were also prepared. The principal reactions involved were: (1) beta-d-N-acetylglucosaminidation at C-7 of methyl 3beta-tert-butyldimethylsilyloxy (TBDMSi)-7beta-hydroxy-5-cholen-24-oate with 2-acetamido-1alpha-chloro-1,2-dideoxy-3,4,6-tri-O-acetyl-d-glucopyranose in the presence of CdCO(3) in boiling toluene; (2) sulfation at C-3 of the resulting 3beta-TBDMSi-7beta-GlcNAc with sulfur trioxide-trimethylamine complex in pyridine; and (3) direct amidation at C-24 of the 3beta-sulfooxy-7beta-GlcNAc conjugate with glycine methyl ester hydrochloride (or taurine) using 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride as a coupling agent in DMF. The structures of the multi-conjugated bile acids were characterized by liquid chromatography-mass spectrometry with an electrospray ionization probe under the positive and negative ionization modes.     

Collisionally induced dissociation of epoxyeicosatrienoic acids and epoxyeicosatrienoic acid-phospholipid molecular species.

Four isomers of epoxyeicosatrienoic acid (EET) can be formed by cytochrome P-450 oxidation of arachidonic acid: 5,6-, 8,9-, 11,12-, and 14,15-epoxyeicosatrienoic acid. The collision-induced dissociation of the [M-H]- anion at m/z 319 from each of these isomers, using negative-ion fast atom bombardment ionization and a triple quadrupole mass spectrometer, resulted in a series of common ions as well as ions characteristic of each isomer. The common ions were m/z 301 [M-H2O]- and 257 [M-(H2O + CO2)]-. Unique ions resulted from cleavages alpha to the epoxide moiety to form either conjugated carbanions or aldehydes. Mechanisms involving charge site transfer are suggested for the origin of these ions. A distonic ion series that may involve a charge-remote fragmentation mechanism was also observed. The epoxyeicosatrienoic acids were also incorporated into cellular phospholipids following incubation of the free acid with murine mast cells in culture. Negative fast atom bombardment mass spectrometry of purified glycerophosphoethanolamine-EET species and glycerophosphocholine-EET species yielded abundant [M-H]- and [M-CH3]- ions, respectively. The collision-induced dissociation of these specific high-mass ions revealed fragment ions characteristic of the epoxyeicosatrienoic acids incorporated (m/z 319, 301, and 257) and the same unique ions as those seen with each isomeric epoxyeicosatrienoic acid. With this direct method of analysis, phospholipids containing the four positional isomers of EET, including the highly labile (5,6-EET), could be identified as unique molecular species in mast cells incubated with EET.     

Biliary bile acids in birds of the Cotingidae family: taurine-conjugated (24R,25R)-3α,7α,24-trihydroxy-5β-cholestan-27-oic acid and two epimers (25R and 25S) of 3α,7α-dihydroxy-5β-cholestan-27-oic acid

Three C(27) bile acids were found to be major biliary bile acids in the capuchinbird (Perissocephalus tricolor) and bare-throated bellbird (Procnias nudicollis), both members of the Cotingidae family of the order Passeriformes. The individual bile acids were isolated by preparative RP-HPLC, and their structures were established by RP-HPLC, LC/ESI-MS/MS and NMR as well as by a comparison of their chromatographic properties with those of authentic reference standards of their 12α-hydroxy derivatives. The most abundant bile acid present in the capuchinbird bile was the taurine conjugate of C(27) (24R,25R)-3α,7α,24-trihydroxy-5β-cholestan-27-oic acid, a diastereomer not previously identified as a natural bile acid. The four diastereomers of taurine-conjugated (24ξ,25ξ)-3α,7α,24-trihydroxy-5β-cholestan-27-oic acid could be distinguished by NMR and were resolved by RP-HPLC. The RRT of the diastereomers (with taurocholic acid as 1.0) were found to be increased in the following order: (24R,25R)<(24S,25R)<(24S,25S)<(24R,25S). Two epimers (25R and 25S) of C(27) 3α,7α-dihydroxy-5β-cholestan-27-oic acid were also present (as the taurine conjugates) in both bird species. Epimers of the two compounds could be distinguished by their NMR spectra and resolved by RP-HPLC with the (25S)-epimer eluting before the (25R)-epimer. Characterization of the taurine-conjugated (24R,25R)-3α,7α,24-trihydroxy-5β-cholestan-27-oic acid and two epimers (25R and 25S) of 3α,7α-dihydroxy-5β-cholestan-27-oic acid should facilitate their detection in peroxisomal disease and inborn errors of bile acid biosynthesis.