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PubMed1961

Sialic acids (N,7-O-diacetylneuraminic acid and N-acetylneuraminic acid) in Escherichia coli. I. Isolation and identification.

DeWitt, Charles W. (The Upjohn Co., Kalamazoo, Mich.) and Janet A. Rowe. Sialic acids (N,7-O-diacetylneuraminic acid and N-acetylneuraminic acid) in Escherichia coli. I. Isolation and identification. J. Bacteriol. 82:838-848. 1961.-Two sialic acids, N-acetylneuraminic acid and N,7-O-diacetylneuraminic acid, were obtained in crude mixtures from whole cells of Escherichia coli and from its endotoxin by weak acid hydrolysis followed by anion exchange resin chromatography. Yields from whole cells were 0.1 to 0.2% (dry weight) with 50 to 60% purity. Identification of the sialic acids was by comparative paper chromatography and colorimetric assays using the acidic p-dimethylaminobenzaldehyde (direct Ehrlich), resorcinol and thiobarbituric acid reactions. The N-acetyl derivative was also shown to be susceptible to hydrolysis by clostridial N-acetylneuraminic aldolase and the end products identified, N-acetylamannosamine by paper chromatography and pyruvic acid by oxidation of DPNH with lactic acid dehydrogenase. The two sialic acids were separated on paper chromatograms, eluted, and assays for total and ester acyl groups showed the suspected N-acetyl derivative to contain 0.11 O-acyl and 1.16 N-acetyl groups per mole sialic acid and the diacetyl derivative to have 1.10 O-acyl and 0.93 N-acetyl groups per mole. The O-acyl group was identified as acetyl by preparation of the hydroxamate.

Escherichia coli↗

PubMed1997

Effects of medium chain fatty acids (MCFA), myristic acid, and oleic acid on serum lipoproteins in healthy subjects.

In this study we investigated the effects on lipoproteins of medium chain fatty acids (MCFA) and myristic acid relative to those of oleic acid. Thirty-seven women and 23 men consumed a 3-wk run-in diet enriched in oleic acid followed by a 6-wk test diet rich in MCFA (n = 21), myristic (n = 20), or oleic acid (n = 19). Experimental fats were incorporated into solid foods. Total fat intake was 40 En% fat. The dietary compositions were the same except for 10 En%, which was provided by MCFA, myristic, or oleic acids, respectively. With the myristic acid diet, low density lipoprotein (LDL) cholesterol was 0.37 mmol/L higher compared with the oleic acid diet (P = 0.0064 for difference in changes). The MCFA diet increased LDL cholesterol, though not significantly, with 0.23 mmol/L relative to the oleic acid diet (P = 0.0752). Compared with the oleic acid diet, HDL cholesterol concentrations increased with the myristic acid diet by 0.10 mmol/L (P = 0.0273) but not with the MCFA diet. The MCFA diet slightly elevated triacylglycerol concentrations, but responses did not significantly differ between the diets. The MCFA diet significantly decreased the apoA-I to apoB ratio compared with both other diets (P < 0.02). We conclude that MCFA raise LDL cholesterol concentrations slightly and affect the apoA-I to apoB ratio unfavorably compared with oleic acid. Myristic acid is hypercholesterolemic, although less than predicted earlier, and raises both LDL and HDL cholesterol concentrations compared with oleic acid.

Adult↗

PubMed2004

Agonist-induced internalization of leukotriene B(4) receptor 1 requires G-protein-coupled receptor kinase 2 but not arrestins.

The leukotriene B(4) (LTB(4)) receptor (BLT1) becomes desensitized upon repeated agonist stimulation. Little is known, however, about BLT1 internalization, which follows desensitization in most G-protein-coupled receptors (GPCR). In the current study, transiently expressed BLT1 readily internalized, after LTB(4) stimulation, in RBL-2H3 cells that express high levels of endogenous GPCR kinase 2 (GRK2) but did not in COS-7 or human embryonic kidney (HEK) 293 cells, which do not overexpress GRK. The internalization of BLT1 could be blocked in RBL-2H3 cells by coexpressing dominant-negative (DN) GRK2 K220R and could be promoted in HEK293 cells by coexpressing wild-type (WT) GRK2. Coexpression of WT or DN nonvisual arrestins had no effect on BLT1 internalization. Moreover, upon stimulation with LTB(4), BLT1 did not induce arrestin-green fluorescence protein redistribution in either cell type, even in the presence of overexpressed GRK2. Coimmunoprecipitation experiments confirmed that BLT1 could associate with GRK2 but not with arrestins. A C-tail-truncated mutant of BLT1 lost the capacity to internalize and associate with GRK2 upon exposure to LTB(4), suggesting that the C-tail was required for receptor internalization and association with GRK2. Taken together, our results indicate that the C terminus of BLT1 plays a pivotal role in receptor internalization and GRK2 association. Moreover, ligand-induced BLT1 internalization is dependent on GRK2 but independent of arrestins. This may allow differential, cell-type-specific signaling in response to LTB(4), depending on GRK expression levels.

Animals↗

PubMed1992

Formula alpha-linolenic (18:3(n - 3)) and linoleic (18:2(n - 6)) acid influence neonatal piglet liver and brain saturated fatty acids, as well as docosahexaenoic acid (22:6(n - 3)).

Saturated fatty acids can be synthesized de novo and play a role in determining properties of structural membranes. The effect of dietary essential fatty acids, linoleic acid (18:2(n - 6)) and alpha-linolenic acid (18:3(n - 3)), on the saturated fatty acid content of membrane phospholipid has not previously been considered in newborn nutrition. The studies report the effect of low (1% fatty acids) or high (4%) formula 18:3(n - 3) with low (16%) or high (30-35%) formula 18:2(n - 6) on the saturated and unsaturated fatty acid composition of liver and brain structural lipid of piglets fed formula from birth for 15 days. A significant inverse relationship between the formula % 18:3(n - 3), but not 18:2(n - 6), and the liver phospholipid palmitic acid (16:0) was found. This may indicate a possible effect of dietary 18:3(n - 3) on de novo synthesis of 16:0 and requires further investigation. Monounsaturated fatty acids in both liver and brain were significantly lower in response to high 18:3(n - 3) and to high 18:2(n - 6) plus low 18:1(n - 9) in the formula. Liver phospholipid and brain total lipid % docosahexaenoic acid (22:6(n - 3)) were significantly higher when formula containing 4% rather than 1% 18:3(n - 3) was fed, suggesting that 1% 18:3(n - 3) may limit tissue (n - 3) fatty acid accretion. These results suggest that future studies of essential fatty acid requirements, specifically 18:3(n - 3), should consider possible influences on the saturated fatty acids which also play a functional role in tissue structural lipids.

Animals↗

PubMed1986

Incorporation and distribution of dihomo-gamma-linolenic acid, arachidonic acid, and eicosapentaenoic acid in cultured human keratinocytes.

Human keratinocytes in culture were labelled with 14C-dihomo-gamma-linolenic acid, 14C-arachidonic acid or 14C-eicosapentaenoic acid. All three eicosanoid precursor fatty acids were effectively incorporated into the cells. In phospholipids most of the radioactivity was recovered, in neutral lipids a substantial amount, and as free unesterified fatty acids only a minor amount. The most of the radioactivity was found in phosphatidylethanolamine which was also the major phospholipid as measured by phosphorous assay. The incorporation of dihomo-gamma-linolenic acid and arachidonic acid into lipid subfractions was essentially similar. Eicosapentaenoic acid was, however, much less effectively incorporated into phosphatidylinositol + phosphatidylserine and, correspondingly, more effectively into triacylglycerols as compared to the two other precursor fatty acids. Once incorporated, the distribution of all three precursor fatty acids was relatively stable, and only minor amounts of fatty acids were released into the culture medium during short term culture (two days). Our study demonstrates that eicosanoid precursor fatty acids are avidly taken up by human keratinocytes and esterified into membrane lipids. The clinical implication of this finding is that dietary manipulations might be employed to cause changes in the fatty acid composition of keratinocytes.

8,11,14-Eicosatrienoic Acid↗

PubMed1992

Comparative hepatotoxicity of cholic acid, deoxycholic acid and lithocholic acid in the rat: in vivo and in vitro studies.

Until now, the cytotoxicity of the bile acids was mostly seen as being inversely associated with their degree of lipophilicity. The present study aimed at comparing the hepatotoxicity of cholic acid (CA), deoxycholic acid (DCA) and lithocholic acid (LCA), which are respectively, tri-, di- and monohydroxylated bile acids. For in vivo studies, the bile acids have been given at the dose of 0.5% or 1% in the diet of male Wistar rats for 2 weeks. The histological analysis of the liver, and the measurement of serum parameters of cytotoxicity and cholestasis (aminotransferases activity, bilirubin and total bile acids concentration), indicate that, among the bile acids tested, DCA is the most hepatotoxic, at both doses, while CA is the least hepatotoxic and cholestatic compound. Moreover, DCA is the only bile acid which, when given at the dose of 0.5%, induces lipid peroxidation in the liver, as evidenced by the measurement of thiobarbituric reactive substances in liver homogenates. The analysis of bile acids in liver homogenates by gas liquid chromatography revealed that feeding the animals with DCA results in its hepatic accumulation. Feeding rats with LCA or CA only slightly modifies the proportion of tri-, di- and monohydroxylated bile acids in the liver, as compared to controls. An in vitro experiment aimed at studying the hepatocellular lysis induced in vitro by the three bile acids by measuring the release of lactate dehydrogenase in the incubation medium of surviving hepatocytes in suspension. At a concentration of 1 mM, only DCA induces a significant cellular lysis, while at this concentration the lytic effects of CA and LCA are progressive and time-dependent. From this study, we gather that the hepatotoxicity of bile acids does not necessarily depend on their degree of hydroxylation. Our results are in accordance with some studies in rat hepatocarcinogenesis, showing a predominant initiating and promoting effects of DCA, as compared to LCA.

Administration, Oral↗

PubMed1984

Fatty-acid-binding proteins. Occurrence of two fatty-acid-binding proteins in bovine liver cytosol and their binding of fatty acids, cholesterol, and other lipophilic ligands.

Fatty-acid-binding proteins (FABPs) are known as cytosolic binding sites for fatty acids and their CoA esters. Radioactively labeled and fluorescent fatty acids were used to locate and identify these proteins in bovine liver cytosol. The occurrence of two species of FABPs was demonstrated and these were designated pI6.0-FABP and pI7.0-FABP according to their isoelectric points in the delipidated state. Oleic acid/FABP binding ratios were 1 with pI6.0-FABP and 2 with pI7.0-FABP. Upon binding of oleic acid the isoelectric points of liganded FABPs shifted to pH 5.0-5.1 in each case. Both proteins were purified by removing nonbinding proteins by acid and heat denaturation and subsequent gel filtration. By making use of the pI shifts observed upon lipidation and delipidation of the binding proteins with ligand fatty acids, final purification was achieved in two fractionations by isoelectric focusing. The binding proteins (Mr 11 800 +/- 1 000) had similar amino-acid compositions (no Trp) and were not covalently modified by carbohydrate and fatty acid. Fatty acids and their CoA esters were complexed by either FABP, cholesterol only by pI-7.0-FABP, though non-stoichiometrically. 16-(9-Anthroyloxy)palmitic acid was bound by pI-7.0-FABP in a 1:1 ratio and precluded the additional binding of a straight-chain fatty acid. Electrophoretic titration curves indicated dissociation of the oleic acid/pI7.0-FABP complex below pH 5.0. It appears that fatty acids and their CoA esters are the foremost binding partners of FABPs in vivo. The results are discussed in terms of a single binding site for fatty acids per molecule FABP.

Amino Acids↗

PubMed1985

Digestion and absorption of lipids and bile acids in sheep fed stearic acid, oleic acid, or tristearin.

Sheep were fed diets containing 7.5% added stearic acid, oleic acid, or tristearin for 21 days. In addition, 50 microCi/kg cerium-141 was included for the last 10 days on experimental diets as an unabsorbed reference substance. In the rumen dietary triglycerides were approximately 50% hydrolyzed, and hydrogenation resulted in saturation of the free fatty acid fraction. Some net synthesis of phospholipids, presumably microbial phospholipids, occurred in the rumen. In the intestine immediately distal to the pylorus, extensive secretion of bile acids, cholesterol, phospholipids, triglycerides, free fatty acids, and lipase occurred. This resulted in doubling of fatty acid fluxes through the duodenum. These endogenous secretions were reabsorbed rapidly however, with the major site of lipid and bile acid absorption in the region .6 to 4 m distal to the pylorus. Additional but less absorption occurred in the more distal segments of the small intestine. Overall absorption of stearic acid, oleic acid, and tristearin supplements was in the range 60 to 70%, and no differences were apparent between fats. Unsaturated fatty acids were over 90% absorbed as compared with 55 to 65% for saturated fatty acids. No significant effect of any of the supplements was observed on ruminal total volatile fatty acids, ratios of volatile fatty acids, or on overall cellulose or caloric digestion.

Abomasum↗

PubMed1993

Effects of stearic acid and trans fatty acids versus linoleic acid on blood pressure in normotensive women and men.

The objective of this study was to compare the effect of linoleic acid (cis,cis-C18:2) with that of its hydrogenation products stearic acid (C18:0) and elaidic acid (trans-C18:1) on blood pressure levels in normotensive humans. We therefore measured the effects of these fatty acids on systolic and diastolic blood pressure in 30 women and 25 men. Three strictly controlled experimental diets were supplied to every subject for 3 weeks each, in different order (multiple cross-over). The composition of the three diets was constant, except for 8% of daily energy, which was provided by either linoleic acid, stearic acid, or monounsaturated trans fatty acids. The statistical power for detecting a true difference between two diets of 3 mmHg in systolic and diastolic blood pressure was over 90%. Mean systolic/diastolic blood pressure at the end of the dietary periods was 114/69 mmHg on the linoleic acid diet, 113/70 on the stearic acid diet, and 113/69 on the trans fatty acid diet. No significant differences were observed in blood pressure levels after 3 weeks on each diet. We conclude that a major increase in the intake of linoleic acid at the expense of stearic acid or trans fatty acids has no effect on blood pressure in normotensive young women and men.

Adult↗

PubMed1988

Further studies on the hydrolysis of salicyluric acid in intestinal microorganisms and prolonged blood concentration of salicylic acid following rectal administration of salicyluric acid in rabbits.

The blood concentrations of salicyluric acid and salicylic acid following intracecal and rectal administration of salicyluric acid were determined in rabbits. Immediate and very extensive salicylic acid formation in the cecum was found following intracecal administration. After rectal administration, a small amount of salicyluric acid was absorbed in intact form. The rest was rapidly hydrolyzed to salicylic acid, which was subsequently absorbed. The blood concentration of salicylic acid was maintained at 1.3-1.8 micrograms/ml from 2 to 12 h. Three doses of salicyluric acid were administered rectally. The peak level of salicyluric acid increased with dose. However, salicylic acid concentration in the blood following administration of salicyluric acid at 10.0 mg/kg (salicylic acid equivalent) was not double that observed following administration of salicyluric acid at 5.0 mg/kg (salicylic acid equivalent). It appears that a larger amount of salicyluric acid in the rectal lumen may have saturated the glycine deconjugation system.

Administration, Rectal↗

PubMed1980

Simultaneous liquid-chromatographic quantitation of salicylic acid, salicyluric acid, and gentisic acid in urine.

We have developed a specific and sensitive method for the determination of salicylic acid, salicyluric acid, and gentisic acid in urine. Any proteins present are precipitated with methyl cyanide. After centrifugation, an aliquot of the supernate is directly injected into an octadecyl silane reversed-phase chromatographic column, then eluted with a mixture of water, butanol, acetic acid, and sodium sulfate, and quantitated at 313 nm by ultraviolet detection according to peak-height ratios (with internal standard, o-methoxybenzoic acid) or peak heights (no internal standard). The method allows estimates within 25 min. Sensitivity was 0.2 mg/L for gentisic acid, and 0.5 mg/L for both salicyluric and salicylic acid (20-micro L injection volume); response was linear with concentration to at least 2.000 g/L for salicylic acid and metabolites. Analytical recovery of salicylic acid and metabolites from urine is complete. Intra-assay precision (coefficient of variation) is 5.52% at 7.5 mg/L for salicylic acid, 5.01% at 9.33 mg/L for salicyluric acid, and 3.07% at 7.96 mg/L for gentisic acid. Interassay precision is 7.32% at 7.51 mg/L for salicylic acid, 5.52% at 8.58 mg/L for salicyluric acid, and 3.97% at 8.32 mg/L for gentisic acid. We saw no significant interference in urine from patients being treated with various drugs other than aspirin.

Aspirin↗

PubMed1998

Spontaneous conversion of L-dehydroascorbic acid to L-ascorbic acid and L-erythroascorbic acid.

Dehydroascorbic acid, an oxidation product of ascorbic acid (vitamin C), spontaneously decomposed at neutral and higher pH levels to form three products that could be quantitated by HPLC-electrochemical analysis. One of the products was ascorbic acid, suggesting that dehydroascorbic acid was reduced to ascorbic acid without adding an exogenous reductant. The major newly produced compound was almost identical to ascorbic acid by UV spectroscopy, which therefore potentially interfered in the study of ascorbic acid metabolism. The ascorbic acid-like compound was isolated by reversed-phase HPLC and identified as L-erythroascorbic acid by mass spectrometry. Fe(II) and Cu(I) increased, whereas desferrioxamine, a potent iron chelator, inhibited L-erythroascorbic acid production. Phosphate, used as buffer, and cyanide greatly enhanced dehydroascorbic acid conversion to L-erythroascorbic acid. The identification of L-erythroascorbic acid and its quantitation by an electrochemical method provides a useful tool for future study of dehydroascorbic acid metabolism.

Ascorbic Acid↗

PubMed1990

Evaluation of clastogenicity of formic acid, acetic acid and lactic acid on cultured mammalian cells.

Using Chinese hamster ovary K1 cells, chromosomal aberration tests were carried out with formic acid, acetic acid and lactic acid, and the relationship between the pH of the medium and the clastogenic activity was examined. The medium used was Ham's F12 supplemented with 17 mM NaHCO3 and 10% fetal calf serum. All of these acids induced chromosomal aberrations at the initial pH of ca. 6.0 or below (about 10-14 mM of each acid) both with and without S9 mix. Exposure of cells to about pH 5.7 or below (about 12-16 mM of each acid) was found to be toxic. When the culture medium was first acidified with each of these acids and then neutralized to pH 6.4 or pH 7.2 with NaOH, no clastogenic activity was observed. Using F12 medium supplemented with 34 mM NaHCO3 as a buffer, no clastogenic activity was observed at doses up to 25 mM of these acids (initial pH 5.8-6.0). However, it was found that about 10% of the cells had aberrations at pH 5.7 or below (27.5-32.5 mM of each acid). Furthermore, when 30 mM HEPES was used as a buffer, chromosomal aberrations were not induced at doses up to 20 mM formic acid and acetic acid (initial pH 7.0-7.1), and at doses up to 30 mM lactic acid (initial pH 6.6). In the initial pH range of 6.4-6.7 (25-32.5 mM of each acid), chromosomal aberrations were observed. The above results show that these acids themselves are non-clastogenic, and the pseudo-positive reactions attributable to non-physiological pH could be eliminated by either neutralization of the treatment medium or enhancement of the buffering ability.

Acetates↗

PubMed1991

Radical adducts of nitrosobenzene and 2-methyl-2-nitrosopropane with 12,13-epoxylinoleic acid radical, 12,13-epoxylinolenic acid radical and 14,15-epoxyarachidonic acid radical. Identification by h.p.l.c.-e.p.r. and liquid chromatography-thermospray-m.s.

Linoleic acid-derived radicals, which are formed in the reaction of linoleic acid with soybean lipoxygenase, were trapped with nitrosobenzene and the resulting radical adducts were analysed by h.p.l.c.-e.p.r. and liquid chromatography-thermospray-m.s. Three nitrosobenzene radical adducts (peaks I, II and III) were detected; these gave the following parent ion masses: 402 for peak I, 402 for peak II, and 386 for peak III. The masses of peaks I and II correspond to the linoleic acid radicals with one more oxygen atom [L(O).]. The radicals are probably carbon-centred, because the use of 17O2 did not result in an additional hyperfine splitting. Computer simulation of the peak I radical adduct e.p.r. spectrum also suggested that the radical is carbon-centred. The peak I radical was also detected in the reaction of 13-hydroperoxylinoleic acid with FeSO4. From the above results, peak I is probably the 12,13-epoxylinoleic acid radical. An h.p.l.c.-e.p.r. experiment using [9,10,12,13-2H4]linoleic acid suggested that the 12,13-epoxylinoleic acid radical is a C-9-centred radical. Peak II is possibly an isomer of peak I. Peak III, which was observed in the reaction mixture without soybean lipoxygenase, corresponds to a linoleic acid radical (L.). The 12,13-epoxylinoleic acid radical, 12,13-epoxylinolenic acid radical and 14,15-epoxyarachidonic acid radical were also detected in the reactions of linoleic acid, linolenic acid and arachidonic acid respectively, with soybean lipoxygenase using nitrosobenzene and 2-methyl-2-nitrosopropane as spin-trapping agents.

Chromatography, High Pressure Liquid↗

PubMed2000

Inactivation of the peroxisomal multifunctional protein-2 in mice impedes the degradation of not only 2-methyl-branched fatty acids and bile acid intermediates but also of very long chain fatty acids.

According to current views, peroxisomal beta-oxidation is organized as two parallel pathways: the classical pathway that is responsible for the degradation of straight chain fatty acids and a more recently identified pathway that degrades branched chain fatty acids and bile acid intermediates. Multifunctional protein-2 (MFP-2), also called d-bifunctional protein, catalyzes the second (hydration) and third (dehydrogenation) reactions of the latter pathway. In order to further clarify the physiological role of this enzyme in the degradation of fatty carboxylates, MFP-2 knockout mice were generated. MFP-2 deficiency caused a severe growth retardation during the first weeks of life, resulting in the premature death of one-third of the MFP-2(-/-) mice. Furthermore, MFP-2-deficient mice accumulated VLCFA in brain and liver phospholipids, immature C(27) bile acids in bile, and, after supplementation with phytol, pristanic and phytanic acid in liver triacylglycerols. These changes correlated with a severe impairment of peroxisomal beta-oxidation of very long straight chain fatty acids (C(24)), 2-methyl-branched chain fatty acids, and the bile acid intermediate trihydroxycoprostanic acid in fibroblast cultures or liver homogenates derived from the MFP-2 knockout mice. In contrast, peroxisomal beta-oxidation of long straight chain fatty acids (C(16)) was enhanced in liver tissue from MFP-2(-/-) mice, due to the up-regulation of the enzymes of the classical peroxisomal beta-oxidation pathway. The present data indicate that MFP-2 is not only essential for the degradation of 2-methyl-branched fatty acids and the bile acid intermediates di- and trihydroxycoprostanic acid but also for the breakdown of very long chain fatty acids.

3-Hydroxyacyl CoA Dehydrogenases↗

PubMed1977

Effects of branched-chain amino acid antagonism in the rat on tissue amino acid and keto acid concentrations.

Growth rate, plasma amino acid, and alpha-keto acid concentrations and activities of the branched-chain amino acid degradative enzymes of rats were measured. Effects of ingestion of excessive amounts of branched-chain amino acids on these variables were determined. Excessive intake of a single branched-chain amino acid led rapidly to elevated plasma concentration of both the amino acid administered and its corresponding alpha-keto acid and, if the rats had previously been fed a low protein diet, to an increase in liver branched-chain alpha-keto acid dehydrogenase activity. Only leucine caused, in addition, marked growth and food intake depression and decreased plasma isoleucine, valine, alpha-keto-beta-methylvaleric acid and alpha-keto isovaleric acid concentrations. The growth depression was associated food intake depression and could be moderated by addition of isoleucine and valine to the diet. The decreases in plasma isoleucine, valine, alpha-keto-beta-methylvaleric acid and alpha-keto isovaleric acid were not caused by increased degradation of these metabolites to carbon dioxide as branched-chain amino acid oxidation rates in vivo were unchanged by leucine loading and the degradative enzymes were unchanged in adequately fed rats. The decreased concentrations of these amino and keto acids may be the result of decreased protein degradation or increased protein synthesis, possibly mediated by insulin.

Alanine↗

PubMed1997

Differential interaction of bile acids from patients with inborn errors of bile acid synthesis with hepatocellular bile acid transporters.

People with genetic or acquired defects in the biosynthesis of bile acids may suffer from cholestasis. Patients with a deficiency of 3 beta-hydroxy-delta 5-C27-steroid dehydrogenase/isomerase from 3 beta, 7 alpha-dihydroxy- and 3 beta, 7 alpha, 12 alpha-trihydroxy-5-cholenoic acids, the sulfated and partially glycine-conjugated forms of which are found in their urine and bile. 3-Oxo-delta 4 bile acids are detected in the urine of patients with a deficiency of 5 beta-reductase. It has been postulated that these unusual bile acids might act as cholestatic agents in these patients. The aim of the present study was to test this hypothesis in an in vitro system, since the abnormal bile acids would be metabolized in in vivo experiments. Basolateral (sinusoidal) and canalicular plasma membrane vesicles were isolated from rat liver. A rapid filtration method was used to determine transport of cholyltaurine in the presence of model bile acids into the isolated vesicles. It was found that 3 beta, 7 alpha-dihydroxy-5-cholenoic acid and 7 alpha-hydroxy-3-oxo-4-cholenoic acid both inhibited the apical, ATP-dependent transport system for cholyltaurine in a competitive manner with K(m) values of 15 microM and 16 microM, respectively. Radioactively labeled 3 beta, 7 alpha-dihydroxy-5-cholenoyltaurine and 7 alpha-hydroxy-3-oxo-4-cholenoyltaurine were not transported by the same transport system. The same types of experiments were performed with basolateral plasma membrane vesicles. It was found that, in contrast to the canalicular ATP-dependent bile acid transport system, only 7 alpha-hydroxy-3-oxo-4-cholenoyltaurine was a competitive inhibitor of the sodium-dependent transport system for cholyltaurine with a K(m) of 16 microM. Studies with radioactively labeled 7 alpha-hydroxy-3-oxo-4-cholenoyltaurine and 3 beta, 7 alpha-dihydroxy-5-cholenoyltaurine revealed that 7 alpha-hydroxy-3-oxo-4-cholenoyltaurine was transported in a sodium-dependent manner into basolateral rat liver plasma membrane vesicles, whereas 3 beta, 7 alpha-dihydroxy-5-cholenoyltaurine was not transported in a sodium-dependent way. These results support the hypothesis that the unusual bile acids found in patients with defects in bile acid biosynthesis might act as cholestatic agents by inhibiting the canalicular ATP-dependent transport system for bile acids which constitutes the rate-limiting step in the overall process of bile acid transport across hepatocytes. Furthermore, the experiments demonstrated that, despite similar substrate specificities, the basolateral sodium-dependent and the apical ATP-dependent transport system for cholyltaurine might have different recognition sites for bile acids.

Animals↗

PubMed1981

Turnover of free sialic acid, CMP-sialic acid, and bound sialic acid in rat brain.

Adult male rats were injected intraventricularly with N-[3H]acetylmannosamine. After different time intervals the rats were killed and free sialic acid, CMP-sialic acid, lipid- and protein-bound sialic acid were isolated from brain and the specific radioactivities determined. Maximal specific radioactivity was reached after approximately 4 h for CMP-sialic acid, after 10-12 h for free sialic acid and after approximately 42 h for lipid- and protein-bound sialic acid. After some days the specific radioactivities of all four pools were the same and decreased equally, with a calculated turnover rate of approximately 3.5 weeks. The conclusion was that this phenomenon was the result of reutilisation of sialic acid and/or precursors. Therefore, the calculated turnover is not the turnover of bound sialic acid, but merely the rate of leakage of sialic acid and/or precursors out of the brain, so that no real turnover can be measured by this method. The first few hours after injection the specific radioactivity of CMP-sialic acid rose above that of free sialic acid. It is supposed that a compartmentalization exists of free sialic acid. The newly synthesised sialic acid molecules are not secreted into the cytoplasmic pool but are preferentially used for the synthesis of CMP-sialic acid. The results and conclusions are discussed in view of the general problems concerning turnover measurements of glycoconjugates.

Animals↗