Bile acid synthesis and excretion following release of total extrahepatic cholestasis by percutaneous transhepatic drainage

Abstract. Urinary, biliary and serum bile acids were studied in three patients before and after percutaneous transhepatic drainage for total bile duct obstruction.
Before drainage high urinary excretion often different bile acids occurred. The percentage distribution was: cholic and chenodeoxycholic acid (66–86%), hyo-cholic (3–16%), 3β 12α-dihydroxy-5-cholenoic (3–6%) and 3β-hydroxy-5-cholenoic acid (2–8%). These acids were regularly found in serum. In addition small amounts (less than 2%) of norcholic, allocholic, 3β, 7α-dihydroxy-5β-cholanoic, 3α, 7α-dihydroxy-5α-cholanoic and lithocholic acid were excreted in urine. Trace amounts of these bile acids were found in serum.
After start of drainage biliary bile acid excretion increased rapidly during the first day, dropped to a minimum during the second or third day and then slowly increased again. In spite of normal volumes of bile produced, the total serum bile acids and the urinary excretion of bile acids remained increased during a drainage period of 19 days. The bile acids were of the same type as observed during cholestasis. In serum the increase was mainly due to high concentrations of chenodeoxycholic and 3β-hydroxy-5-cholenoic acid, as sulphate esters.
Glycine and taurine conjugates of cholic, chenodeoxycholic and hyocholic acid were mainly excreted in bile. Bile acid sulphate esters were only present in trace amounts in bile and were mainly excreted in urine. This, combined with low renal clearance, explains the elevated serum levels of sulphate esters of chenodeoxycholic and 3β-hydroxy-5-cholenoic acid conjugates.     

Effect of long-term starvation on acetate and ketone body metabolism in obese patients

Abstract. The turnover of ketone bodies and acetate was evaluated as well from the disappearance rate of (3-¹⁴C)acetoacetate or (1-¹⁴C)acetate respectively as from the conversion of FFA into these metabolites in normal weight and obese overnight-fasted and in obese long-term starved patients. The disappearance rate of (1-¹⁴C)oleate was the same in all three groups.
Long-term starvation enhanced ketone body turnover almost 10-fold, whereas the disappearance rate for ketone bodies decreased from 0·035 to 0·015 min^-1. Under the same circumstances the turnover of acetate was about 1 μmol g^-1 min^-1 accounting for about 5% of FFA turnover.
Long-term starvation decreased the conversion of (1-¹⁴C)oleate into triglycerides by almost 50% and increased the (2-C)-(4-C)/(1-C) ratio of radioactivity in ketone bodies. The reincorporation of radioactivity from the (1-C)position of (1-¹⁴C)oleate into the ((2-C)-( n -C)) position of FFA, which is a measure of the reutilization of acetyl-CoA for FFA synthesis decreased significantly during long-term starvation.     

ISOLATED FECAL MICROORGANISMS CAPABLE OF 7 α-DEHYDROXYLATING BILE ACIDS

Strains of microorganisms capable of 7α-dehydroxylation of chenodeoxycholate were isolated from rat and human feces. All the strains were strictly anaerobic, non-motile, moderately themioresistant Gram-positive rods. They showed some saccharolytic properties with the production of both acid and gas. They were H₂S-positive but indole-, skatole-, citrate-, catalase-, and oxidase-negative. The isolated strains capable of 7α-dehydroxylation of chenodeoxycholate were also able to oxidize the hydroxyl groups at C-3 and C-7 to keto groups. The following metabolites were isolated: 3-keto-7α-hydroxy-5β-cholanoic acid, 3α-hydroxy-7-keto-5β-cholanoic acid, 3α-hydroxy-5β-cholanoic acid, and 3-keto-5β-cholanoic acid. The isolated strains did not have the enzymes necessary for hydrolyzing conjugated bile acids. In mixed anaerobic cultures of fecal microorganisms, extensive reduction of the 3-keto group to the 3β-hydroxyl group occurred. The microorganism(s) responsible for this reaction have as yet not been isolated.     

Cluster headache: increased incorporation of (1-14C)arachidonic acid into phosphatidylserine in polymorphonuclear cells

Vasoactive metabolites deriving from arachidonic acid (AA) have been considered as putative mediators in the pathogenesis of various types of headache. In the present study we therefore compare the ability to synthesize AA containing precursor phospholipids in polymorphonuclear cells (PMNs) from healthy controls and cluster headache patients. 3.7% ± 1.4 (mean ± SD) of the (1-¹⁴C)AA incorporated into total PMN glycerophospholipids (GPLs) was recovered in the phosphatidylserine (PS) in a group of cluster headache patients ( n = 12). This was almost twice the value of 1.9% ± 0.8% found in a corresponding group of 24 healthy controls ( p < 0.001). A significant decrease in the incorporation of (1-¹⁴C)AA into phosphatidylcholine (PC) ( p < 0.01) and an increase in the incorporation of (1-¹⁴C)AA into phosphatidyletanolamine (PE) ( p < 0.05) were also found in cluster headache patients when compared to the control group. The increased incorporation of (1-¹⁴C)AA into PS in PMNs from this group of patients is interesting because PS plays an important role in the activation of protein kinase C, an enzyme involved in transmembrane signalling. The clinical implications of the present findings in cluster headache, if any, cannot yet be defined.     

Blood-brain barrier permeability of L-dopa in man

Abstract . The permeability of L-dopa across the blood-brain barrier was studied in twelve patients by means of the indicator dilution method. The extraction of [l-¹⁴C]L-dopa and [3-¹⁴C]L-dopa was 11.7% and 10.4%, respectively. After pretreatment with carbi-dopa the extraction was unchanged (13.5% and 11.6%). The permeability coefficient ( P ) was calculated to 0.9×10^-5 cm s^-1.     

Formation of Inosine Triphosphate and of 14C-labeled 2,6-diaminopurine Ribonucleoside Di- and Triphosphates in Stored Human Erythrocytes

Inosine triphosphate (ITP) and ¹⁴C-labeled 2,6-diaminopurine ribonucleoside di- and triphosphate (DDP-¹⁴C and DTP-¹⁴C, respectively), were formed during incubation of 2,6-diaminopurine-2-¹⁴C and inosine with stored human erythrocytes at 37 C for five hours. Erythrocytes were obtained from acid-citrate-dextrose blood which had been stored in a blood bank for four weeks. DDP-¹⁴C, DTP-¹⁴C, and ITP were isolated and purified by chromatography on Dowex-1 formate resin and on paper, and identified by their chromatographic mobilities, fluorescence in ultraviolet light, derivatives, and absorption spectra. The yields of DDP-¹⁴C, DTP-¹⁴C, and ITP were 0.16, 1.13, and 0.07 per cent, respectively, of the added 2,6-diaminopurine-2-¹⁴C or inosine.     

Synthesis and release of phosphatidylcholine by isolated porcine gastric mucous cells in primary culture

Phosphatidylcholine (PC) is the major phospholipid of the hydrophobic gastric mucosal barrier and is chiefly released from mucous cells into the gastric mucus. Whereas the mucosa contains highly unsaturated PC, gastric mucus predominantly contains palmitoyl-oleoyl-PC and palmitoyl-linoleoyl-PC, indicating a selective release of these PC species into the gastric lumen. In order to understand gastric PC metabolism, we investigated synthesis and release of PC in cultivated porcine gastric mucous cells, using dual labelling with [methyl-³H]-choline and [1-¹⁴C]-palmitate, in the presence of 12- O -tetradecanoylphorbol-13-acetate (TPA), indomethacin and prostaglandin E₂ (PGE₂). Linear incorporation of [methyl-³H]-choline and [1-¹⁴C]-palmitate into PC was achieved for at least 8 h. In contrast to type II pneumocytes TPA increased PC synthesis in gastric mucous cells but not its release. Indomethacin did not influence PC synthesis, but it decreased the release of newly synthesized PC. PGE₂ antagonized the effect of indomethacin on PC release. We conclude that PC release by isolated porcine gastric mucous cells is regulated in a manner different from type II pneumocytes. PC release is impaired by indomethacin and this impairment is restored by PGE₂.     

In vitro and in vivo vasodilating effects of KRN4884, Ki1769 and Ki3005, pyridinecarboxamidine derivatives

Summary— The vasodilating potencies and mechanism of action of a novel pyridinecarboxamidine derivative, KRN4884 (5-amino-N-(2-(2-chlorophenyl)ethyl]-N'-cyano-3-pyridinecarboxamidine] were compared with those of Ki 1769 [N-cyano-N'-(2-phenylethyl)-3-pyridinecarboxamidine] and Ki3005 [N-[2-(2-chlorophenyl)ethyl]-N'-cyano-3-pyridinecarboxamidine] in rat isolated aortas and in anesthetized normotensive rats. In vitro, KRN4884 (10-¹⁰-10-⁶ M), Ki3005 (10-¹⁰-10-⁶ M) and Ki30O5 (10-¹⁰-10-⁶ M) produced concentration-dependent relaxations. KRN4884 was about 100- and 10-fold more potent than KU769 and Ki3005, respectively. The relaxant effects of these compounds were antagonized by glibenclamide. In vivo, KRN4884 (1–10 μg/kg, intravenously [iv]), KM769 (10–100 μg/kg, iv) and Ki3005 (3–30 μg/kg, iv) produced dose-dependent decreases in mean blood pressure with slight increases in heart rate. At 10 μg/kg, iv, the hypotensive effect of KRN4884 was about the same as that of Ki3005 and about 5-fold more pronounced than that of Ki 1769.
The hypotensive action remained for a longer period after KRN4884 administration. In rats pre-treated with glibenclamide (20 mg/kg, iv), the hypotensive effect of KRN4884 was abolished. These results suggest that the effect of KRN4884 in vitro and in vivo is based on its K channel opening action and that the in vitro vasorelaxant effect of these compounds in aortic rings does not predict their relative hypotensive effect in vivo.     

Formation of bile acids in man: conversion of cholesterol into 5β-cholestane-3α,7α,12α-triol in liver homogenates

The mechanisms of the conversion of cholesterol into bile acids in man were studied by examining the metabolism of cholesterol-1,2-(3)H, cholest-5-ene-3beta,7alpha-diol-7beta-(3)H, tritiumlabeled 7alpha-hydroxycholest-4-en-3-one, 7alpha,12alpha-dihydroxycholest-4-en-3-one, and cholest-5-ene-3beta,7alpha,12alpha-triol in fractions of liver homogenates. The 20,000 g supernatant fluid catalyzed the conversion of cholesterol into cholest-5-ene-3beta,7alpha-diol, 7alpha-hydroxycholest-4-en-3-one, 7alpha-12alpha-dihydroxycholest-4-en-3-one, and 5beta-cholestane-3alpha,7alpha,12alpha-triol. In the presence of microsomal fraction fortified with NAD(+), cholest-5-ene-3beta,7alpha-diol was converted into 7alpha-hydroxycholest-4-en-3-one, and when this fraction was fortified with NADPH small amounts of cholest-5-ene-3beta-7alpha,12alpha-triol were formed. 7alpha-Hydroxycholest-4-en-3-one was metabolized into 7alpha-12alpha-dihydroxycholest-4-en-3-one in the presence of microsomal fraction fortified with NADPH and into 5beta-cholestane-3alpha,7alpha-diol in the presence of 100,000 g supernatant fluid. Cholest-5-ene-3beta,7alpha,12alpha-triol was converted into 7alpha,12alpha-dihydroxycholest-4-en-3-one in the presence of microsomal fraction fortified with NAD(+). The 100,000 g supernatant fluid catalyzed the conversion of 7alpha,12alpha-dihydroxycholest-4-en-3-one into 5beta-cholestane-3alpha,7alpha,12alpha-triol. The sequence of reactions in the conversion of cholesterol into 5beta-cholestane-3alpha,7alpha-diol and 5beta-cholestane-3alpha,7alpha,12alpha-triol, the subcellular localization of the enzymes, and the cofactor requirements were found to be the same as those described for rat liver.     

Kinetics of primary bile acids in patients after orthotopic liver transplantation

Administration of cyclosporin A (CsA) may induce cholestasis, and this effect has been attributed to impaired hepatocellular uptake, transport, secretion and intestinal absorption of bile acids. Disturbances of the enterohepatic circulation may affect metabolism of bile acids. To test whether liver transplantation and treatment with CsA alters pool sizes or synthesis and turnover rates, we determined kinetics of primary bile acids in patients after orthotopic liver transplantation on CsA. Two male and four female patients were studied 6–20 months after transplantation. They had no overt signs of cholestasis, graft dysfunction or rejection. Kinetics of cholic acid (CA) and chenodeoxycholic acid (CDCA) were simultaneously determined after oral administration of [24-¹³C]-CA and [24-¹³C]-CDCA on the basis of isotope dilution in a single pool of bile acids. Ten healthy volunteers served as controls. After orthotopic liver transplantation, pool sizes, fractional turnover rates and synthesis rates of both primary bile acids, CA and CDCA were not significantly different from control subjects. In spite of the known interference of CsA with the enterohepatic circulation of bile acids, in the majority of patients after orthotopic liver transplantation without cholestasis, graft dysfunction or rejection, treatment with CsA does not disturb kinetics of primary bile acids.     

Utilization of myristic and palmitic acid in humans fed different dietary fats

To assess the influence of dietary fat composition on the contribution of dietary myristic and palmitic acid to total fat oxidation and energy production, eight healthy men consumed diets containing 40% of total energy as fat, largely as either butter, tallow or corn oil, for 11 days. On days 8 and 11 of each diet, [1-¹³C]-myristic or [1-¹³C]-palmitic acid (20 mg kg^–1 body weight) was ingested mixed with the test breakfast meal. Respiratory gas exchange was measured before, and for 9 h after, consumption of the meal. Breath ¹³CO₂ enrichments were determined hourly by isotope ratio mass spectrometry. Cumulative 9-h percentage oxidation of dietary myristic acid exceeded that of palmitic acid ( P  < 0.01), but neither was influenced by fat treatment [ n  = 8, 7.1% (1.0) (SEM), 8.6% (0.9) and 8.9% (0.6) of dietary myristic acid and 3.3% (0.7), 3.0% (0.9), and 2.5% (0.6) of dietary palmitic acid from butter, tallow and corn oil meals respectively]. Net dietary myristic acid oxidation was greater ( P  <0.05) after consumption of the meal high in butter than after consumption of other fats. Net dietary palmitic acid oxidation was similar after consumption of all test meals. Precedent fat treatment had no measurable effect on net fat or carbohydrate oxidation or energy expenditure. The overall contribution of dietary myristic or palmitic acid to total fat oxidation did not exceed 1% over 9 h for any dietary fat. These results suggest that, although dietary fatty acid content is the principal determinant of net dietary fatty acid oxidation, dietary fat sources with moderate differences in fat composition do not measurably alter total energy or substrate utilization after a meal.     

Tritium and 14C isotope effects using tracers of leucine and alpha-ketoisocaproate

Abstract. To test if different leucine tracers behave in an indistinguishable manner and, by implication, that their metabolism is identical to that of natural leucine, we measured whole body leucine turnover in dogs and humans and fibrinogen synthesis in dogs by simultaneously infusing either [1–¹⁴C]leucine or [4,5–³H]leucine or [I-¹⁴C]α-ketoisocaproate (KIC) and [4,5–³H]KIC. Whole body leucine fluxes calculated from the plasma specific activity of the transaminated product of the infused tracer (reciprocal pool model) were lower (dogs by 5.7%; humans by 6.4%, both P<0.02) when the plasma 'H specific activity compared to ¹⁴C specific activity were used with leucine tracers and were also lower (dogs by 4.4%, P<0.02; humans by 86%, P<0.06 ) using the KIC tracers. Using leucine or KIC tracers in dogs, the fractional rate of fibrinogen synthesis was 6.7% or 9.4% lower, respectively, (P<0.02) using the ³H versus the ¹⁴C tracer. The apparently lower incorporation of ³H into protein was only in part accounted for by detritiation (2.1%, P = 0.05) of [³H]leucine during acid hydrolysis of proteins. These results suggest that in vivo and/or in vitro differential isotope effects are small (˜5%), but should be considered when dual isotopes infusions are employed to partition amino acid metabolism.     

The Formation of Bile Acids in Patients with Three Types of Hyperlipoproteinemia

Abstract. ¹⁴C-cholic and ³H-chenodeoxycholic acid were administered orally to patients who were given a standardized diet of the regular type. The total formation of bile acids and the combined cholic and chenodeoxycholic pool in 8 patients with hyper-β-lipoproteinaemia (hyperlipoproteinaemia type II) were significantly lower than those recorded for 10 patients with hyperpre-β-lipoproteinaemia (hyperlipoproteinaemia type IV) and for 3 patients with combined hyperpre-β-lipoproteineamia and hyperchylemicronaemia (hyperlipoproteinaemia type V). These differences were exclusively related to variations of the pool size and turnover of cholic acid. — When compared to a younger group of healthy subjects with a somewhat higher caloric intake, the formation of cholic acid was subnormal in type II and markedly elevated in type TV and V.     

Role of primary and secondary bile acids as feedback inhibitors of bile acid synthesis in the rat in vivo.

The effect of various primary and secondary bile acids on the rates of synthesis of all major bile acids was studied in the live rat with an extracorporal bile duct. Bile acid synthesis was determined using HPLC based on mass or by isotope dilution. Derepressed rates of bile acid synthesis (30-54 h) were inhibited by an infusion of taurocholic acid only at a supraphysiological dose of 500 mumol/kg per h, but not at 300 mumol/kg per h, which approximates the initial bile acid secretion (250 mumol/kg per h). When administered together with taurocholic acid (200 mumol/kg per h) only a high dose of taurochenodeoxycholic acid (100 mumol/kg per h) decreased taurocholic but not tauromuricholic or taurochenodeoxycholic acid synthesis. The only bile acid suppressing taurocholic acid (36-71%) and taurochenodeoxycholic acid (up to 33%) formation at an infusion rate close to the normal portal flux was deoxy- or taurodeoxycholic acid at 15-50 mumol/kg per h. It may be concluded that deoxycholic acid and possibly other secondary bile acids are much more potent inhibitors than primary bile acids.     

Excretion of bile acids in feces in pathology of the liver and intestines]

Intestinal excretion of free bile acids (BA), i.e. of cholic, chenodeoxycholic, deoxycholic, lithocholic, and of conjugated BA, i.e. of glycocholic, glycodeoxycholic together with glycochenodeoxycholic, taurocholic, taurodeoxycholic together with taurochenodeoxycholic acids, was examined in patients with viral hepatitides (VH), chronic hepatitis (CH), biliary cirrhosis of the liver (BCL), and acute dysentery (AD), as were the effects of therapy on these acid levels. The findings evidence that fecal levels of free BA are significantly reduced during the acute period of VH, CH, AD, and BCL, whereas the levels of conjugated acids are elevated in all the examinees except the BCL patients, in whom these acids are unchanged. Study of BA excretion in VH patients treated with prednisolone has demonstrated a normalizing effect of this therapy on the spectrum of excreted free BA. Furazolidone and erythromycin therapy resulted in disordered transformation of 'primary' BA into 'secondary' ones and to deconjugation of BA, this being possibly related to these drugs effects on intestinal microflora.     

Activation of GPVI by collagen is regulated by α2β1 and secondary mediators

Summary.  We have compared the roles of adenosine diphosphate (ADP), thromboxanes and the integrin α₂β₁ in the activation of washed platelets by collagen in the presence of the α_IIbβ₃ antagonist lotrafiban. The stimulation of protein tyrosine phosphorylation by a collagen suspension is markedly delayed in the presence of the above inhibitors but shows substantial recovery with time. In comparison, activation of phospholipase C (PLC), Ca²⁺ elevation and dense granule secretion are more severely suppressed by the above inhibitors. α₂β₁ blockade has a slightly greater inhibitory effect on all of the above responses than a combination of ADP receptor antagonists and cyclooxygenase inhibitor. Platelets exposed to a collagen monolayer show robust elevation of Ca²⁺ that is delayed in the presence of the above inhibitors and which is accompanied by α-granule secretion. These results demonstrate that secondary mediators and α₂β₁ modulate collagen-induced intracellular signaling but have negligible effect on GPVI signaling induced by the specific agonist convulxin. This work supports the postulate that the major role of α₂β₁ is to increase the avidity of collagen for the platelet surface and by doing so enhance activation of GPVI. Therefore we propose an important role of secondary mediators in collagen-induced signaling is the indirect regulation of GPVI signaling via activation of α₂β₁.     

Beta3-adrenoceptors in the cardiovascular system

Summary— Behind the classic beta₁, and beta₂-adrenoceptors, recent molecular and pharmacological studies have described a new receptor, called the beta₃-adrenoceptor, in various mammalian tissues (brown and white adipose tissue, digestive smooth muscle). Few authors have investigated the putative existence of the beta₃-adrenoceptor in the cardiovascular system. This paper reviews the available data. In vitro studies show that beta₃-adrenoceptor agonists (BRL 37344, CGP 12177) induce a relaxation of fragments of rat carotid artery which is not antagonized by propranolol. In dogs, these drugs elicit a decrease in blood pressure due to peripheral vasodilation and an increase in heart rate which is of baroreflex origin. The vasodilating effects are mainly observed in cutaneous and adipose tissue vessels and cannot be explained by any known transductional mechanism. Activation of this vascular β₃-adrenoceptor requires higher doses of catecholamines than for β₁- or β₂-adrenoceptors. In humans, the cardiovascular effects of beta₃-adrenoceptor agonists are explained by the activation of beta₁- or beta₂ (and not beta₃-)-adrenoceptors. These studies suggest the presence of vascular (but not cardiac) beta₃-adrenoceptors in dogs. In other species, including man, the presence of such cardiac β₃-adrenoceptors remains to be resolved. Their physiological relevance remains unknown.     

Characterization of the collagen types synthesized by human and rat adipocyte precursors in vitro

Abstract. The types of collagen molecules synthesized by newly confluent rat and human adipocyte precursor cells were studied and compared with those synthesized by fibroblasts in culture. For this the cells were incubated in the presence of [2-³H]L-glycine and [5-³H]L-proline and the newly synthesized radiolabelled collagen produced was purified and analysed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. This analysis revealed that the ratio of Types I and III collagen produced by four adipocyte precursor cell strains, each of distinct origin, was identical to that found with two different strains of lung fibroblasts. The mean ratio of Type I: Type III collagen in all cases was approximately 3·5:1. Because mature fat cells do not synthesize collagen and the adipocyte precursor cells lose their capacity for collagen synthesis during their post-confluent differentiation in vitro these data provide a biochemical basis to substantiate previous suggestions, based on morphology only, that the adipocyte stem cell is fibroblastic in nature.     

25-Hydroxylase activity in subcellular fractions from human liver. Evidence for different rates of mitochondrial hydroxylation of vitamin D2 and D3

25-Hydroxylation of vitamin D2 and D3 was studied in subcellular fractions from human liver, using a technique based on isotope dilution-mass spectrometry. The mitochondrial fraction fortified with isocitrate catalysed 25-hydroxylation of vitamin D3 at a rate of about 10 pmol/mg protein X min. Under the same conditions, the rate of 25-hydroxylation of vitamin D2 was less than 2 pmol/mg protein X min. Crude microsomes fortified with NADPH catalysed 25-hydroxylation of vitamin D3 to a very low extent, and this activity was not linear with the amount of microsomal protein. A higher rate of conversion was obtained with a partially purified cytochrome P-450 fraction in the presence of NADPH-cytochrome P-450 reductase and NADPH. This fraction also catalysed 25-hydroxylation of 1 alpha-hydroxyvitamin D3 and 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol. 25-Hydroxylation of vitamin D2 could not be detected, neither with crude microsomes, nor with the microsomal cytochrome P-450 fraction. Since the assay for 25-hydroxyvitamin D2 was less sensitive than that for 25-hydroxyvitamin D3, these experiments do not rule out the presence of some 25-hydroxylase activity towards vitamin D2 in the microsomes. The results are discussed in relation to previous work in which a lower toxicity has been reported for vitamin D2 than for vitamin D3 in some mammalian species.