Gas-liquid chromatographic assay of serum bile acids

A sensitive method has been established for the analysis of serum bile acids by gas-liquid chromatography (glc). Bile acids are extracted from 0.5–2 ml of serum and analysed as methyl ester trifluoroacetates following enzymatic hydrolysis of the taurine and glycine conjugates. The method as described has been used to estimate serum bile acid levels in health and disease although bile acid sulphates are not detected. Inclusion of a solvolysis procedure before enzymatic hydrolysis would allow their measurement.     

Studies on bile acids: The microquantitative separation of cellular bile acids by gas-liquid chromatography

1. A method is described for the quantitative isolation of bile acids from cellular material. Homogenates of rat liver are freeze-dried and extracted exhaustively with 95% (v/v) ethanol containing 0·1% (v/v) of aq. ammonia (sp.gr. 0·88) and purified by anion-exchange chromatography on Amberlyst A-26. 2. The extracted bile acid conjugates are subjected to either of two hydrolytic procedures, one involving chemical and the other enzymic agents. A unique feature in this study is the introduction of an enzyme, a clostridial peptide-bond hydrolase, for the rapid cleavage of bile acid conjugates, replacing the classical drastic chemical hydrolysis with strong alkali. 3. After hydrolysis, free bile acids are methylated and converted into their trifluoroacetates for final determination by gas–liquid chromatography on a triple component column, FS-1265–SE30–NGS. 4. For the purpose of identification of peaks, bile acid methyl esters are converted into their trimethylsilyl ethers by allowing the methyl esters to react with a new and potent silyl donor, bis(trimethylsilyl)acetamide. 5. The technique affords us a means of studying the metabolism of bile acids at the cellular and subcellular levels in tissues.     

Determination of pethidine and its major metabolites in human urine by gas chromatography

Procedures based on gas chromatography were established to determine pethidine and its major metabolites in human urine. The chromatographic system consisted of a glass column packed with 3% (w/w) SP2250 on Chromosorb W (80–100 mesh) linked to a nitrogen—phosphorus detector. Diethyl ether was used as the extraction solvent. Pethidinic and norpethidinic acids, and their conjugated metabolites (after β-glucuronidase treatment) were determined after conversion into pethidine and norpethidine by acid-catalysed esterification. The retention times of pethidine, norpethidine and chlorpheniramine (internal standard) were 3.3, 4.5 and 7.5 min, respectively. The amount of unchanged drugs and metabolites excreted varied considerably among the subjects. The mean 24-h urinary recoveries in eight patients of pethidine, norpethidine, pethidinic acid, norpethidinic acid, and the glucuronides of pethidinic and norpethidinic acids were 6.62 ± 5.05, 4.33 ± 1.19, 18.9 ± 6.29, 9.10 ± 4.26, 15.1 ± 3.02 and 7.57 ± 2.28%, respectively. This indicates that the major metyabolic pathways of pethidine in the eight patients were hydrolysis followed by conjugation. Over 60% of the dose was accounted for in 24 h after intramuscular administration of 1 mg/kg pethidine.     

Analysis of lipoic acid in biological samples by gas chromatography with flame photometric detection

A selective and sensitive gas chromatographic method for the analysis of lipoic acid in biological samples has been developed. After base hydrolysis of the sample, the liberated lipoic acid was converted into its S,S-diethoxycarbonyl methyl ester derivative and measured by gas chromatography using a DB-210 capillary column and a flame photometric detector. The calibration curve was linear in the range 20–500 ng, and the detection limit was ca. 50 pg injected. The best hydrolysis conditions for the biological samples were obtained by using 2 M potassium hydroxide containing 4% bovine serum albumin at 110°C for 3 h. Using this method, lipoic acid in the hydrolysate could be selectively determined without any interference from matrix substances. Analytical results for the determination of lipoic acid in the mouse tissue and bacterial cell samples are presented.     

Identification of 3 beta, 7 beta-dihydroxy-5 beta-cholan-24-oic acid in serum from patients treated with ursodeoxycholic acid

An unknown bile acid was found by gas-liquid chromatography in the serum of patients who were administered ursodeoxycholic acid for the treatment of cholesterol gallstones. Identification of the chemical structure of the unknown bile acid was performed by the use of gas-liquid chromatography-mass spectrometry. Mass spectrum analysis of the methyl ester trimethylsilyl ether of the bile acid showed explicitly that this is dihydroxy-5 beta-cholanoic acid, since peaks at m/e 460 and 370 characteristic of methyl ester trimethylsilyl ether of dihydroxy bile acid were clearly exhibited. Sites of the two hydroxyl groups on the steroid nucleus were determined to be at the 3- and 7-positions by conversion of the bile acid to the corresponding dioxo-cholanoic acid and by comparison of the gas-liquid chromatographic behavior with those of authentic dioxo bile acids. Four authentic 3,7-dihydroxy-5 beta-cholan-24-oic acids were chemically synthesized and retention times and mass spectra of their methyl ester trimethylsilyl ether derivatives compared precisely with that of the unknown bile acid. The results indicate that the unknown bile acid is 3 beta, 7 beta-dihydroxy-5 beta-cholan-24-oic acid. Preliminary experiments suggest that 3 beta, 7 beta-dihydroxy-5 beta-cholan-24-oic acid is absent as amino acid-conjugated forms in serum. It is also suggested that the bile acid is excreted into urine but not into bile.     

Method for combined analysis of profiles of conjugated progesterone metabolites and bile acids in serum and urine of pregnant women

A method for analysis of profiles of conjugated progesterone metabolites and bile acids in 10 ml of urine and 1–4 ml of serum from pregnant women is described. Total bile acids and neutral steroids from serum and urine were extracted with octadecylsilane-bonded silica. Groups of conjugates were separated on the lipophilic ion-exchanger triethylaminohydroxypropyl Sephadex LH-20 (TEAP-LH-20). Fractions were divided for steroid or bile acid analyses. Sequences of hydrolysis/ solvolysis and separations on TEAP-LH-20 permitted separate analyses of steroid glucuronides, monosulfates and disulfates and bile acid aminoacyl amidates, sulfates, glucuronides and sulfate-glucuronides. Radiolabelled compounds were added at different steps to monitor recoveries and completeness of separation, and hydrolysis/solvolysis of conjugates was monitored by fast-atom bombardment mass spectrometry. The extraction and solvolysis of steroid disulfates in urine were studied in detail, and extraction recoveries were found to be pH-dependent. Following methylation of bile acids, all compounds were analysed by capillary gas chromatography and gas chromatography—mass spectrometry of their trimethylsilyl ether derivatives. Semiquantification of individual compounds in each profile by gas—liquid chromatography had a coefficient of variation of less than 30%. The total analysis required 3 days for serum and 4 days for urine.     

Rapid determination of trans-fatty acids in human adipose tissue: Comparison of attenuated total reflection infrared spectroscopy and gas chromatography

A rapid attenuated total reflection (ATR) infrared (IR) spectroscopy procedure was used for quantitating the levels of total trans-fatty acid methyl ester (FAME) derivatives in neat (without solvent) test samples isolated from human adipose tissue. This procedure requires no weighing of the laboratory sample. The single-beam spectrum of the trans-containing FAMEs was ‘ratioed' against that of a reference material having only cis double bonds in order to obtain a symmetric absorption band at 966 cm⁻¹ on a horizontal background. A single-reflection ATR diamond cell that requires only about 1 μl of neat FAMEs was used. The average level of trans-fatty acids in human adipose tissue found by ATR (3.07±0.27%) was generally higher than that obtained by gas chromatography (2.59±0.20%). Reasons for such a difference are discussed.     

Synthesis of potential cholelitholytic agents: 3 alpha,7 alpha,12 alpha-trihydroxy-7 beta-methyl-5 beta-cholanoic acid, 3 alpha,7 beta,12 alpha-trihydroxy-7 alpha-methyl-5 beta-cholanoic acid, and 3 alpha,12 alpha-dihydroxy-7 xi-methyl-5 beta-cholanoic acid

This report describes the chemical synthesis of six new bile acid analogs, namely, 3 alpha,7 alpha,12 alpha-trihydroxy-7 beta-methyl-5 beta-cholanoic acid (7 beta-methyl-cholic acid), 3 alpha,7 beta,12 alpha-trihydroxy-7 alpha-methyl-5 beta-cholanoic acid (7 alpha-methyl-ursocholic acid), 3 alpha,12 alpha-dihydroxy-7 xi-methyl-5 beta-cholanoic acid (7 xi-methyl-deoxycholic acid), 3 alpha,12 alpha-dihydroxy-7-methyl-5 beta-chol-7-en-24-oic acid, 3 alpha,12 alpha-dihydroxy-7-methyl-5 beta-chol-6-en-24-oic acid, and 3 alpha,12 alpha-dihydroxy-7-methylene-5 beta-cholan-24-oic acid. The carboxyl group of the starting material 3 alpha,12 alpha-dihydroxy-7-oxo-5 beta-cholanoic acid was protected by conversion to its oxazoline derivative. A Grignard reaction of the bile acid oxazoline with CH3MgI followed by acid hydrolysis gave two epimeric trihydroxy-7-methyl-cholanoic acids and three dehydration products. The latter were purified by silica gel column chromatography and silica gel-AgNO3 column chromatography of their methyl ester derivatives. Catalytic hydrogenation of 3 alpha,12 alpha-dihydroxy-7-methyl-5 beta-chol-6-en-24-oic acid and 3 alpha,12 alpha-dihydroxy-7-methylene-5 beta-cholan-24-oic acid gave 3 alpha,12 alpha-dihydroxy-7 xi-methyl-5 beta-cholanoic acid. The configuration of the 7-methyl groups and the position of the double bonds were assigned by proton nuclear magnetic resonance spectroscopy and the chromatographic and mass spectrometric properties of the new compounds. These compounds were synthesized for the purpose of exploring new and potentially more effective cholelitholytic agents. The hydrophilic bile acids 7 beta-methyl-cholic acid and 7 alpha-methyl-ursocholic acid are of particular interest because they should be resistant to bacterial 7-dehydroxylation.     

Synthesis and characteristics of the specific monosulfates of chenodeoxycholate,deoxycholate and their taurine or glycine conjugates

The isomeric monosulfates of chenodeoxycholate, deoxycholate, and their taurine or glycine conjugates, were synthesized and characterized. Reaction with chlorosulfonic acid in pyridine for 2 minutes mainly afforded the 3-monosulfates. To prepare the 7- or the 12-monosulfates, the 3-hydroxyl group was protected by carbethoxylation prior to sulfation of the 7- or 12-hydroxyl group for 24 h to 5 days; after sulfation, the protecting 3-carbethoxy function was removed by mild alkaline hydrolysis. The crude bile salt monosulfates were purified by chromatography on silica gel and on Sephadex LH-20 and were crystallized from methanolethanol-ethyl acetate. The results of elemental analysis demonstrated that the compounds were disodium dihydroxy bile salt monosulfates. Thin layer chromatography of the sulfates, and gas-liquid chromatography after oxidation and solvolysis, showed that the substances were pure and that the sulfate group was at the expected position.     

Synthesis and chiroptical characterization of prostacyclin diastereomers

In the mercuri- and halo-cyclizations of PGF₂α methyl ester and its 11,15-bis(α-ethoxyethyl)-ether (or other protected forms) the exo-PGI₁ derivative predominates independent of reagent and degree of protective of the PGF₂α sample used. Diastereomerically pure samples of exo- and endo-PGI₁ and prostacyclin (PGI₂) were prepared. PGI₀ epimers were prepared: catalytic hydrogenation of PGI₂ Me ester provides exclusively the endo isomer. PGI₂ methyl ester was found to be stable to extensive chromatography on silica, and to storage for at least a year in anhydrous ethanol at −20°C. At pH 7.4 in 2:1 H₂O:EtOH, the ester has a half-life in excess of 5 hr at 25°C. A reproducible small scale (0.4–3 mg) synthesis of prostacyclin uses a modification of Whittaker's iodocyclization followed by DBN treatment. This procedure, developed with 15-³H-PGF₂α, proved widely applicable to PGF₂α analogs and diastereomers. The following prostacyclins (in the Me ester and Na salt forms) bearing the 5-en-6-yl ether unit were prepared in this way: ent-PGI₂, rac-PGI₂, 15-epi-PGI₂, ent-15-epi-PGI₂, 11-epi-PGI₂, 8,9,12-epi-PGI₂, -PGI₂, 13,14-dihydro-PGI₂, and 13,14-dihydro-15-epi-PGI₂. NMR comparisons for the methyl esters reveal that of the resonances (H-5,9,11,15) that appear at δ4.0±0.6 ppm, the most deshielded is H-9 so long as the 5,6-olefin is . The 8 ,9 -6,9-oxido- -5,6-ene unit is most readily characterized by its strong positive dichroic absorption at 210–230 nm. CD spectroscopy not only serves to confirm the presence of this unit in analogs, but also can be used for quantitative analysis of PGI₂ solutions and for monitoring the rate of hydrolytic cleavage of these enol ethers.     

Determination of sulfated and nonsulfated bile acids in serum by mass fragmentography

A Sep-Pak C18 cartridge was used for purification of bile acids from serum. Three kinds of deuterium labeled internal standards were required for accurate measurement of individual sulfated and nonsulfated bile acids. These internal standards were added to the serum before its application to the cartridge. Separation of sulfated and nonsulfated bile acids was performed on piperidinohydroxypropyl Sephadex LH-20 column chromatography. The nonsulfate fraction was submitted to alkaline hydrolysis, and the sulfate fraction to solvolysis followed by alkaline hydrolysis. Each fraction was converted to the hexafluoroisopropyl-trifluoroacetyl derivatives and quantitated by mass fragmentography. The recovery of each bile acid sulfate was quite satisfactory. In fasting healthy subjects the mean of total nonsulfated bile acids in serum was 1.324 micrograms/ml, and that of total sulfated bile acids was 0.450 micrograms/ml. Sulfated lithocholic acid comprised a large part of sulfated bile acids in healthy subjects.     

Does dietary DHA improve neural function in children? Observations in phenylketonuria

Children with phenylketonuria (PKU) have a restricted protein intake and thus low dietary intakes of long-chain polyunsaturated fatty acids (LC-PUFA), which may cause subtle neurological deficits. We measured plasma phospholipid fatty acids and visual evoked potential (VEP) in 36 children with well-controlled PKU (6.3±0.6 years, 19 girls), before and after 3 months of supplementing fish oil capsules providing 15 mg docosahexaenoic acid (DHA)/kg daily. The motometric Rostock-Oseretzky Scale (ROS) was performed before and after supplementation in the 24 PKU children aged >4 years. VEP latencies and ROS were also assessed in omnivorous, age-matched controls without fish oil supply at baseline and after 3 months. Fish oil supply increased plasma phospholipid eicosapentaenoic acid (EPA) (0.40±0.03 vs 3.31±0.19%, p<0.001) and DHA (2.37±0.10 vs 7.05±0.24%, p<0.001), but decreased arachidonic acid (AA) (9.26±0.23 vs 6.76±0.16%, p<0.001). Plasma phenylalanine was unchanged. VEP latencies and ROS results significantly improved after fish oil in PKU children, but remained unchanged in controls. The improvements of VEP latencies, fine motor and coordination skills indicate that preformed n-3 LC-PUFA are needed for neural normalcy in PKU children. The optimal type and dose of supply still needs to be determined. Since PKU children are generally healthy and have normal energy and fatty acid metabolism, these data lead us to conclude that childhood populations in general require preformed n-3 LC-PUFA to achieve optimal neurological function.     

Comparative studies of bile salts. Bile salts of the lamprey Petromyzon marinus L

1. Bile salts of Petromyzon marinus L. ammocoetes appeared to consist solely or chiefly of a crystalline substance, whose chromatographic and i.r.-spectral characteristics suggested that it was a monosulphate ester of a bile alcohol having the 3α,7α,12α-trihydroxy pattern of substitution in a 5α-steroid nucleus. 2. This substance on cleavage with dioxan–trichloroacetic acid gave petromyzonol, n.m.r. and mass-spectral examination of which suggested the structure 5α-cholane-3α,7α,12α,24-tetrol. 3. 3α,7α,12α-Trihydroxy-5α-cholanoic acid (allocholic acid) from the lizards Anolis lineatopus lineatopus Gray and Cyclura carinata Harlan (family Iguanidae) was esterified with propan-1-ol and reduced by lithium aluminium hydride to 5α-cholane-3α,7α,12α,24-tetrol, identical with petromyzonol. 4. Chromic acid oxidation of petromyzonol sulphate from lamprey bile, followed by acid hydrolysis, gave 24-hydroxy-5α-cholane-3,7,12-trione; hence the sulphate ester group is at C-24. 5. Petromyzonol sulphate is both primitive and unique: a study of its biogenesis might improve our understanding of evolution at the molecular level.     

Vulpecholic acid (1 alpha, 3 alpha, 7 alpha-trihydroxy-5 beta-cholan-24-oic acid): a novel bile acid of a marsupial, Trichosurus vulpecula (Lesson)

A novel trihydroxylated C24 bile acid was isolated from the gallbladder bile of the Australian opossum, Trichosurus vulpecula (Lesson). This acid, for which the name vulpecholic acid is proposed, was identified as 1 alpha, 3 alpha, 7 alpha-trihydroxy-5 beta-cholan-24-oic. The structure proof included mass spectral and 1H and 13C nuclear magnetic resonance characterization of all crucial derivatives obtained by: oxidation of the methyl ester to a triketone with the enolizable 1,3-diketone function; methylation of this triketone to two isomeric methyl enol ethers; and reductive removal of oxygen functions from this triketone to give 5 beta-cholan-24-oic and 7-oxo-5 beta-cholan-24-oic acids. Vulpecholic acid was found in the bile in the unconjugated form; it accounted for more than 60% of the solid bile material. The marsupial T. vulpecula is the first example of a mammal secreting a 1 alpha-hydroxylated bile acid as well as the first example of a mammal secreting the major bile acid in a free form.     

Importance of high-performance liquid chromatographic analysis of serum N-acylneuraminic acids in evaluating surgical treatment in patients with early endometrial cancer

The objectives of this study were the quantification of the two major sialic acid (Sia) forms – N-acetylneuraminic (Neu5Ac) and N-glycolylneuraminic acids (Neu5Gc) – in serum before and after surgical treatment of early endometrial cancer and the relation of their levels with the progress of surgical therapy. The major Sia forms were liberated from sera glycoconjugates by mild acid hydrolysis, separated as per-O-benzoylated derivatives by a highly sensitive reversed-phase HPLC method and detected at 231 nm. Total Sia content in sera of healthy women was not related to age and body weight. Neu5Ac was identified as the major Sia in sera from both cancer patients, healthy individuals as well as in tissue specimens (≥94% of total Sia). In patients with endometrial cancer the total Sia level before surgical treatment (709.5±306.5 mg/l) was significantly higher (p≤0.0001) than that of the control group (213.5±88.7 mg/l). The elevation in Sia level was exclusively due to Neu5Ac. Following surgical therapy, serum Neu5Ac levels (699.4±305.6 mg/l) were significantly decreased (305.9±114.5 mg/l). In one case, where Neu5Ac level was increased 15 days and eight months after surgery (1.8 and 2.5 times as compared to control, respectively), a metastasis not detected during surgery was recorded. The obtained results suggest that Neu5Ac level in serum may be used as a tumor marker in evaluating the suitability of surgical treatment in early endometrial cancer.     

Synthesis of the specific monosulfates of cholic acid.

The three isomeric cholic acid-monosulfates were synthetized and characterized. Cholic acid-3-sulfate was obtained by reacting cholic acid for 2 min with chlorosulfonic acid in pyridine and chromatography of the resulting bile salt mixture on Sephadex LH-20. The 7- and the 12-monosulfate were prepared by sulfation of the corresponding monohydroxy-diacetates followed by removal of the acetyl groups by alkaline hydrolysis and purification by chromatography on Sephadex LH-20. On TLC in n-butanol-acetic acid-water (10:1:1, v/v) the Rf values were 0.59 for cholic acid-3-sulfate, 0.52 for cholic acid-7-sulfate and 0.48 for cholic acid-12-sulfate. The time required for complete solvolysis at 37 degrees C in acid methanol-acetone (1:9) was 3 h for cholic acid-3-sulfate, 12 h for the 12-monosulfate and 18 h for the 7-monosulfate.     

In vitro biohydrogenation of four dietary fats

This study was designed to determine in vitro rates of biohydrogenation of dietary unsaturated fatty acids by a mixed population of rumen microbes. The four dietary fats [Alifet High-Energy^® (AHE), Alifet-Repro^® (AR), Megalac^® (MG), and Energy Booster^® (EB)] differ in method of preparation, fatty acid composition, or both of these factors. Dietary fats (20 mg) were incubated with 4 mL strained rumen fluid diluted with 16 mL of medium, 0.8 mL of reducing solution buffer, and 200 mg of a synthetic diet (370 g cellulose, 370 g starch, and 160 g casein per kg DM) at 37 °C. Total contents were collected after 0, 6, 12, 24, or 36 h and change in fatty acid content determined. Disappearance of oleic acid was minimal (0.05–0.20) in AR and MG but moderate (about 0.60) in AHE and EB after 36 h of incubation. Rate of biohydrogenation of linoleic and linolenic acids from AR were similar (0.025 ± 0.009 h⁻¹) and 0.65 of these fatty acids remained intact after 36 h. Rate of biohydrogenation of linoleic acid was four times greater than for oleic acid (0.040 ± 0.013 h⁻¹ versus 0.009 ± 0.002 h⁻¹) in MG. Thus, 0.65 of the linoleic acid but only 0.20 of the oleic acid had disappeared from MG after 36 h. Trans-11 and trans-12 were the predominant trans-isomers in AHE and AR cultures whereas trans-9 and trans-10 were the predominant trans-isomers in EB and MG cultures. None of the dietary fats contained conjugated linoleic acid (CLA) but CLA was present in the incubation inoculum. The amount of CLA decreased with time but this was not affected by source of dietary fat. Most (0.90–0.95) of the long-chain fatty acids eicosapentaenoic (EPA) and docosahexaenoic (DHA) in AR remained after 36 h of incubation. Results demonstrate that biohydrogenation varied among fatty acids and among source of dietary fat and indicate that AR can be used to increase post-ruminal supply of linolenic, EPA and DHA.     

Quality of the Entomopathogenic NematodeSteinernema carpocapsaeProduced on Different Media

The characteristics of morphometric (body length and width), biochemical (fatty acid content), motility, and penetration rate of infective juveniles ofSteinernema carpocapsaeBeijing strain reared on four different culture media [e.g., plant protein medium (I), animal protein medium (II), plant and animal protein medium (III), andin vivoculture (IV) were systematically compared in this research. The results showed that the average lengths of infective juveniles were 497.4 ± 0.09, 514.3 ± 0.08, 525.7 ± 0.09, and 556.6 ± 0.09 μm, the average widths were 24.9 ± 0.006, 25.6 ± 0.005, 26.1 ± 0.006, and 27.9 ± 0.004 μm, and the average dry weights per million infective juveniles were 49.2 ± 2.2, 58.6 ± 2.4, 59.6 ± 1.8, and 80.7 ± 1.7 mg cultured by media I, II, III, and IV, respectively. The highest relative content of fatty acid of infective juveniles was obtained from medium IV at 15.4 ± 1.2 × 10⁵μV/s, and the lowest one was 6.76 ± 0.3 × 10⁵μV/s from medium I and 11.8 ± 0.2 × 10⁵and 13.7 ± 0.3 × 10⁵μV/s from media II and III, respectively. The numbers of nematodes that moved a vertical distance of 5 cm in sand column within 48 h were 24 ± 3.6, 75 ± 11.6, 69 ± 9.7, and 92 ± 13.2 and the penetration rates into theGallerialarva within 24 h were 2.8 ± 0.45, 6.0 ± 1.14, 6.4 ± 0.74, and 6.0 ± 0.7% from media I to IV, respectively. The results indicated that the quality of entomopathogenic nematode was influenced by the cultural medium component. The animal protein, especially from insects which were presented in media II, III, and IV, has a strong positive effect on nematode quality.     

Application of high-performance liquid chromatography of plasma fatty acids as their phenacyl esters to evaluate splanchnic and renal fatty acid balance in vivo

Plasma fatty acids from renal and hepatic veins, and arterialized hand vein obtained in 20 subjects before and after insulin infusion were separated by reversed-phase high-performance liquid chromatography following phenacyl esterification. Separation and quantification over the range 1.0–100 nmol per injection of nine fatty acids was achieved within 60 min using [²H₃₁]palmitic acid as internal standard. Analytical recoveries were greater than 90% and the intra- and inter-assay coefficients of variation were less than 2.5 and 4.0%, respectively. Following insulin infusion, net splanchnic uptake of total fatty acids decreased from 3.0±0.3 to 1.0±0.1 μmol/kg min (p<0.01), whereas net renal balance remained neutral (−0.04±0.04 vs. −0.06±0.03 μmol/kg min, p=N.S.). Individual fatty acid balance varied from a low of 0.012±0.005 (myristic acid) to a high of 0.95±0.08 (oleic acid) μmol/kg min across the splanchnic tissues and from 0.005±0.002 (stearic acid) to 0.21±0.1 (oleic acid) μmol/kg min across the kidney. There is a substantial diversity in changes in plasma concentration and regional balance of individual fatty acid during short-term fasting and hyperinsulinemia. This method is simple, accurate, and can be applied to assess individual fatty acid metabolism in vivo.     

Bile acid metabolism in early life: studies of amniotic fluid

Bile acid metabolism of the human fetus was examined in early gestation (weeks 13-19) and compared with the full-term fetus from the analysis of amniotic fluid collected from healthy pregnant women. Total individual bile acids were determined by gas-liquid chromatography-mass spectrometry after solvolysis and hydrolysis of bile acid conjugates. Additionally, bile acids were separated according to their mode of conjugation by lipophilic anion exchange chromatography. Qualitatively the bile acid profiles of amniotic fluid in early gestation were similar and markedly different from those of full-term fetuses. Chenodeoxycholic acid was the major bile acid identified in early gestation and concentrations exceeded those of cholic acid, but by full term this relationship was reversed. Over 50 bile acids were identified in the amniotic fluids, these included C-1, C-4, and C-6 hydroxylated species and reflected primary hepatic synthesis by the fetus. At full term, 7 alpha,12 alpha-dihydroxy-3-oxo-4-cholenoic acid was one of the major bile acids identified in amniotic fluid. The monohydroxy bile acids lithocholic and 3 beta-hydroxy-5-cholenoic acids were present in significant proportions during early gestation, but by full term these accounted for only a few percent of the total bile acids. Quantitatively the total bile acid concentration of amniotic fluid was less than 4 mumol/l. The majority of bile acids were found to be glyco-, tauro-, and sulfate-conjugates. The more hydrophobic bile acids tended to be preferentially sulfated. These data indicate that significant and major changes in bile acid metabolism take place between early and late gestation in the human fetus.