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Quantitative determination of individual non-sulfated bile acids and sulfated lithocholic acid in serum by mass fragmentography.

Individual non-sulfated bile acids and sulfated lithocholic acid in serum were determined by mass fragmentography. A hexafluoroisopropyl ester-trifluoroacetyl derivative of bile acid was prepared by the method of Imai et al. (J. Chromatogr. 120, 181, 1976). Deuterium labeled deoxycholic acid was used as an internal standard monitoring at m/z 623. Lithocholic acid, deoxycholic acid, chenodeoxycholic acid, ursodeoxycholic acid, and cholic acid were determined by monitoring the intensities of m/z 622, m/z 620, m/z 620, m/z 620, ad m/z 618, respectively. A serum sample of 200 microliters including 500 ng of internal standard was hydrolyzed with strong alkali, then acidified to pH 1 with 2N HCl under cooling on ice, and extracted with diethyl ether immediately. Ether extracts were derivatized without further purification. Besides this assay of non-sulfated bile acids, total serum lithocholic acid including the sulfated form was determined as follows: extraction was performed after mixing the acidified (pH 1 with 2 N HCl) hydrolysate with ether and incubation at 40 degrees C for 2 h. Bile acid peaks in the mass fragmentogram were not affected by other materials in these serum extracts. The average values of individual non-sulfated bile acids in sera from healthy fasting subjects (n=15) were as follows: lithocholic acid, 0.049 micrograms/ml; deoxycholic acid, 0.462 micrograms/ml; chenodeoxycholic acid, 0.671 micrograms/ml; ursodeoxycholic acid, 0.070 icrograms/ml; and cholic acid, 0.217 micrograms/ml. Total lithocholic acid (non-sulfated and sulfated) in sera was 0.166 microgram/ml.

Bile Acids and Salts↗

Stereoselectivity and mode of inhibition of phosphoinositide-coupled excitatory amino acid receptors by 2-amino-3-phosphonopropionic acid.

DL-2-Amino-3-phosphonopropionic acid, a phosphonate-substituted derivative of aspartic acid, has been shown to be an inhibitor of excitatory amino acid-stimulated phosphoinositide hydrolysis in rat brain slices. In this study, the enantiomers of 2-amino-3-phosphonopropionic acid were synthesized and used to further characterize the stereoselectivity and mechanism of interaction of this compound for inhibiting phosphoinositide-coupled (metabotropic) excitatory amino acid receptors. L-2-Amino-3-phosphonopropionic acid was 3-5 times more potent than D-2-amino-3-phosphonopropionic acid as an inhibitor of ibotenate-stimulated [3H]inositol monophosphate formation in slices of the rat hippocampus or quisqualate-stimulated [3H]inositol monophosphate formation in neonatal rat cerebral cortical slices. Carbachol-stimulated phosphoinositide hydrolysis was not inhibited by L-2-amino-3-phosphonopropionic acid, and L-2-amino-3-phosphonopropionic acid had no appreciable affinity for ionotropic excitatory amino acid receptors at concentrations required to inhibit metabotropic excitatory amino acid responses. The inhibitory effects of L-2-amino-3-phosphonopropionic acid or L-2-amino-4-phosphonobutyric acid on phosphoinositide hydrolysis were not competitive, because they could not be surmounted by increasing concentrations of ibotenate or quisqualate. L-2-Amino-3-phosphonopropionic acid inhibition also could not be prevented by washing the tissue before incubation with ibotenate. Thus, L-2-amino-3-phosphonopropionic acid is a stereoselective inhibitor of metabotropic excitatory amino acid receptors with little affinity for ionotropic receptors. However, the inhibitory effects of L-2-amino-3-phosphonopropionic acid or L-2-amino-4-phosphonobutyric acid were not readily reversed, and the site at which they act to inhibit metabotropic excitatory amino acid receptors remains to be determined.

Alanine↗

Ursodeoxycholic acid in the Ursidae: biliary bile acids of bears, pandas, and related carnivores.

The biliary bile acid composition of gallbladder bile obtained from six species of bears (Ursidae), the Giant panda, the Red panda, and 11 related carnivores were determined by reversed phase liquid chromatography and gas chromatography-mass spectrometry. Bile acids were conjugated solely with taurine (in N-acyl linkage) in all species. Ursodeoxycholic acid (3 alpha, 7 beta-dihydroxy-5 beta-cholan-24-oic acid) was present in all Ursidae, averaging 1-39% of biliary bile acids depending on the species; it was not detected or present as a trace constituent (< 0.5%) in all other species, including the Giant panda. Ursodeoxycholic acid was present in 73 of 75 American Black bears, and its proportion averaged 34% (range 0-62%). Ursodeoxycholic acid averaged 17% of biliary bile acids in the Polar bear (n = 4) and 18% in the Brown bear (n = 6). Lower proportions (1-8%) were present in the Sun bear (n = 2), Ceylon Sloth bear (n = 1), and the Spectacled bear (n = 1). Bile of all species contained taurine-conjugated chenodeoxycholic acid and cholic acid. In some related carnivores, deoxycholic acid, the 7-dehydroxylation product of cholic acid, was also present. To determine whether the 7 beta hydroxy group of ursodeoxycholic acid was formed by hepatic or bacterial enzymes, bile acids were determined in hepatic bile obtained from bears with chronic biliary fistulae. Fistula bile samples contained ursodeoxycholic acid, chenodeoxycholic acid, and a trace amount of cholic acid, all as taurine conjugates, indicating that ursodeoxycholic acid is a primary bile acid formed in the liver in Ursidae.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Renal underexcretion of uric acid is present in patients with apparent high urinary uric acid output.

OBJECTIVE: To compare renal handling of uric acid in patients with primary gout with that of a control group. METHODS: A case-control study of 100 patients with primary gout and 72 healthy controls was undertaken. Creatinine clearance, uric acid clearance, 24-hour uric acid urinary excretion, fractional excretion of uric acid, excretion of uric acid per volume of glomerular filtration, urinary uric acid to creatinine ratio, and glomerular uric acid filtered load were calculated using 24-hour urine samples. After treatment with allopurinol to achieve similar glomerular filtered load of uric acid, patients were again compared with controls. RESULTS: Patients with gout showed lower uric acid clearance, fractional excretion of uric acid, excretion of uric acid per volume of glomerular filtration, and urinary uric acid to creatinine ratio than controls at baseline, when patients showed hyperuricemia. Although the glomerular uric acid filtered load was much higher in patients with gout than controls, 24-hour uric acid excretion was not statistically different. After treatment with allopurinol, and achieving similar uric acid filtered loads, patients still showed lower figures than controls. When patients with 24-hour urinary uric acids levels >700 mg/day were compared with controls, they had lower uric acid clearance and fractional excretion of uric acid than controls, both at baseline and after achieving similar filtered loads with allopurinol therapy. CONCLUSIONS: Renal underexcretion is the main mechanism for the development of primary hyperuricemia in gout, but even patients showing apparent high 24-hour uric acid output show lower uric acid clearance than controls, indicating that relative, low-grade underexcretion of uric acid is at work.

Adult↗

Acidic amino acid transport in animal cells and tissues.

1. The occurrence and characterization of acidic amino acid transport in the plasma membrane of a variety of cells and tissues of a number of organisms is reviewed. 2. Several cell types, especially in brain, possess both high- and low-affinity transport systems for acidic amino acids. 3. High-affinity systems in brain may function to remove neurotransmitter amino acid from the extracellular environment. 4. Many cell systems for acidic amino acid transport are energized by an inwardly directed Na+ gradient. Moreover, certain cell types, such as rat brain neurons, human placental trophoblast and rabbit and rat kidney cortex epithelium, respond to an outwardly directed K+ gradient as an additional source of energization. This simultaneous action may account for the high accumulation ratios seen with acidic amino acids. 5. Rabbit kidney has been found to have a glutamate-H+ co-transport system which is subject to stimulation by protons in the medium. 6. Acidic amino acid transport in rat brain neurons occurs with a stoichiometric coupling of 1 mol of amino acid to 2 mol of Na+. For rabbit intestine, one Na+ is predicted to migrate for each mol of amino acid. 7. Uptake in rat kidney cortex and in high-K+ dog erythrocytes is electrogenic. However, uptake in rabbit and newt kidney and in rat and rabbit intestine is electroneutral. 8. Na+-independent acidic amino acid transport systems have been described in the mouse lymphocyte, the human fibroblast, the mouse Ehrlich cell and in rat hepatoma cells. 9. In a number of cell systems, D-acidic amino acids have substantial affinity for transport; D-glutamate, in a number of systems, however, appears to have little reactivity. 10. Acidic amino acid transport in some cell systems appears to occur via the "classical" routes (Christensen, Adv. Enzymol. Relat. Areas Mol. Biol. 49, 41-101, 1979). For example, uptake in the Ehrlich cell is partitioned between the Na+-dependent A system (which transports a wide spectrum of neutral amino acids), the Na+-dependent ASC system (which transports alanine, serine, threonine, homoserine, etc.), and the Na+-independent L system (which shows reactivity centering around neutral amino acids such as leucine and phenylalanine). Also, a minor component of uptake in mouse lymphocytes occurs by a route resembling the A system. 11. Human fibroblasts possess a Na+-independent adaptive transport system for cystine and glutamate that is enhanced in activity by cystine starvation.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acids↗

Effects of acid adaptation of Escherichia coli O157:H7 on efficacy of acetic acid spray washes to decontaminate beef carcass tissue.

Exposure to low pH and organic acids in the bovine gastrointestinal tract may result in the induced acid resistance of Escherichia coli O157:H7 and other pathogens that may subsequently contaminate beef carcasses. The effect of acid adaptation of E. coli O157:H7 on the ability of acetic acid spray washing to reduce populations of this organism on beef carcass tissue was examined. Stationary-phase acid resistance and the ability to induce acid tolerance were determined for a collection of E. coli O157:H7 strains by testing the survival of acid-adapted and unadapted cells in HCl-acidified tryptic soy broth (pH 2.5). Three E. coli O157:H7 strains that were categorized as acid resistant (ATCC 43895) or acid sensitive (ATCC 43890) or that demonstrated inducible acid tolerance (ATCC 43889) were used in spray wash studies. Prerigor beef carcass surface tissue was inoculated with bovine feces containing either acid-adapted or unadapted E. coli O157:H7. The beef tissue was subjected to spray washing treatments with water or 2% acetic acid or left untreated. For strains ATCC 43895 and 43889, larger populations of acid-adapted cells than of unadapted cells remained on beef tissue following 2% acetic acid treatments and these differences remained throughout 14 days of 4 degrees C storage. For both strains, numbers of acid-adapted cells remaining on tissue following 2% acetic acid treatments were similar to numbers of both acid-adapted and unadapted cells remaining on tissue following water treatments. For strain ATCC 43890, there was no difference between populations of acid-adapted and unadapted cells remaining on beef tissue immediately following 2% acetic acid treatments. These data indicate that adaptation to acidic conditions by E. coli O157:H7 can negatively influence the effectiveness of 2% acetic acid spray washing in reducing the numbers of this organism on carcasses.

Abattoirs↗

Fatty acids of pulmonary surfactant phosphatidylcholine from fetal rabbit lung tissue in culture. Biosynthesis of n-10 monoenoic fatty acids.

We have previously reported that fetal rabbit lung tissue in organ culture produces a lamellar body material (pulmonary surfactant) with a lower percentage of disaturated phosphatidylcholine than is typically found in rabbit lung in vivo (Longmuir, K.J., C. Resele-Tiden, and L. Sykes. 1985. Biochim. Biophys. Acta. 833: 135-143). This investigation was conducted to identify all fatty acids present in the lamellar body phosphatidylcholine, and to determine whether the low level of disaturated phosphatidylcholine is due to excessive unsaturated fatty acid at position sn-1, sn-2, or both. Fetal rabbit lung tissue, 23 days gestation, was maintained in culture for 7 days in defined (serum-free) medium. Phospholipids were labeled in culture with [1-14C]acetate or [U-14C]glycerol (to follow de novo fatty acid biosynthesis), or with [1-14C]palmitic acid (to follow incorporation of exogenously supplied fatty acid). Radiolabeled fatty acid methyl esters obtained from lamellar body phosphatidylcholine were first separated by reverse-phase thin-layer chromatography (TLC) into two fractions of 1) 14:0 + 16:1 and 2) 16:0 + 18:1. Complete separation of the individual saturated and monoenoic fatty acids was achieved by silver nitrate TLC of the two fractions. Monoenoic fatty acid double bond position was determined by permanganate-periodate oxidation followed by HPLC of the carboxylic acid phenacyl esters. Lamellar body phosphatidylcholine contained four monoenoic fatty acids: 1) palmitoleic acid, 16:1 cis-9; 2) oleic acid, 18:1 cis-9; 3) cis-vaccenic acid, 18:1 cis-11; and 4) 6-hexadecenoic acid, 16:1 cis-6. In addition, 8-octadecenoic acid, 18:1 cis-8, was found in the fatty acids of the tissue homogenate. The abnormally low disaturated phosphatidylcholine content in lamellar body material was the result of abnormally high levels of monoenoic fatty acid (principally 16:1 cis-9) found at position sn-2. Position sn-1 contained normal levels of saturated fatty acid. The biosynthesis of the unusual n-10 fatty acids was observed from the start of culture throughout the entire 7-day culture period, and was observed in incubations of tissue slices of day 23 fetal rabbit lung. This is the first report of the biosynthesis of n-10 fatty acids (16:1 cis-6 and 18:1 cis-8) in a mammalian tissue other than skin, where these fatty acids are found in the secretory product (sebum) of sebaceous glands.

Animals↗