Excretion of foetal bilirubin by the rat placenta-maternal liver tandem.

Using plasma membrane vesicles from human trophoblast, carrier-mediated transport of unconjugated bilirubin (UCBR) has been reported. In the present work, using the in situ perfused rat placenta-maternal liver tandem, the relevance of this pathway in vivo was investigated. After single-pass perfusion of rat placenta through the umbilical artery with 0.25 micromol [(3)H]-UCBR, approximately 15 per cent of it was taken up by the placenta, detected in maternal serum (>96 per cent was unconjugated) and subsequently secreted into maternal bile (approximately 15 per cent of administered dose; >88 per cent was glucuronidated bilirubin). Co-administration through the umbilical artery of 0.25 micromol [(3)H]-UCBR and 2.5 micromol unlabelled UCBR, bromosulfophthalein, cholic acid or biliverdin IXalpha, reduced [(3)H]-UCBR placenta uptake, and the amount of radioactivity found in the maternal serum and bile. Co-administration into maternal jugular vein of 0.1 micromol [(3)H]-UCBR-a dose 3-fold higher than that reaching the maternal compartment in placenta perfusion experiments-and 1.0 micromol bromosulfophthalein, cholic acid or biliverdin IXalpha, resulted in no marked inhibition of the amount of radioactivity bile output. When antipyrine and [(3)H]-UCBR were continuously co-infused to the mother, similar antipyrine concentrations in maternal and foetal serum were reached in approximately 15 min, while progressive increase in [(3)H]-bilirubin concentrations in maternal serum above 70 microM was accompanied by a very low transfer of this compound into foetal compartment where [(3)H]-bilirubin concentrations were always <10 microM. These results suggest that the transfer of UCBR across the rat placenta occurs, without biotransformation, via a foetal-to-maternal mainly unidirectional pathway that can be cis-inhibited by UCBR and other cholephilic organic anions.

Evidence for carrier-mediated transport of unconjugated bilirubin across plasma membrane vesicles from human placental trophoblast.

Unconjugated bilirubin (UCB) is currently believed to cross the placenta only by passive diffusion. To assess whether carrier-mediated transport might be involved, the uptake of [(3)H]-UCB by basal (bTPM) and apical (aTPM) plasma membrane vesicles from human placental trophoblast at term was investigated. In both types of vesicles, the uptake of [(3)H]-UCB into an osmotically sensitive space was temperature-dependent, independent of the presence of Na(+), and not affected by changes in membrane potential. The uptake of [(3)H]-UCB by aTPM, but not bTPM, was activated by ATP hydrolysis and inhibited by vanadate. Thus, the exact contribution of both inside out and right-side out bTPM to UCB uptake could not be distinguished, while only inverted aTPM were expected to carry out ATP-dependent UCB uptake. In bTPM and aTPM, uptake of free (unbound) [(3)H]-UCB (B(f)) consisted of a dominant, saturable, presumably carrier-mediated process and a diffusional component that became predominant only at B(f) near or above aqueous solubility limit for UCB (70 nM ). For bTPM, K(m)=7.2 nM; V(max)=9.8 pmol/20s/mg protein; and diffusion coefficient (K(D))=0.14 ml/20s/mg protein. For aTPM in the presence of 9.5m M ATP, K(m)=18 n M; V(max)=131 pmol/20s/mg protein; and K(D)=0.47 ml/20s/mg protein. The uptake of [(3)H]-UCB by bTPM was cis-inhibited by estrone-3-sulfate and estradiol-17 beta-glucuronide and trans-stimulated by unlabelled UCB and bromosulphopthalein. ATP-dependent UCB uptake by aTPM was cis-inhibited by doxorubicin, cholic acid, methotrexate and pronenecid. These findings suggest the presence of distinct transporters in the two domains of human placental trophoblast that could cooperate to transfer UCB from the foetus to the maternal circulation.

Pitfalls in preparation of (3)H-unconjugated bilirubin by biosynthetic labeling from precursor (3)H-5-aminolevulinic acid in the dog.

We report problems encountered during preparation of tritium-labeled unconjugated bilirubin ((3)H-UCB) from precursor (3)H-5-aminolevulinic acid ((3)H-ALA) in 2 dogs with external biliary drainage installed into the animals under general anesthesia. Under prolonged sedation, 12.9 or 14.0 mCi of (3)H-ALA was administered intravenously in two divided doses, and bile was collected for 9 hours. In one animal, taurocholate (TC) infusion was needed to maintain bile flow. (3)H-UCB was isolated from the bile and recrystallized with the improved method of Webster et al (Webster CC, Tiribelli C, Ostrow JD. J Lab Clin Med 2001;137:370-3). Based on radioactivity and pigment content, hourly bile collections were pooled to optimize specific activities. Surprisingly, in the first dog, only 2.9% of injected radioactivity was recovered in bile and only 14.1% in urine, and the specific activities of the crystalline (3)H-UCB from the two pools were only 39.5 and 30.0 x 10(3) dpm/microg. High-performance liquid chromatography analysis revealed that only 4% of ALA degraded during 5 minutes in injection solution at pH 6.8. The low incorporation of (3)H-ALA and low specific activity of (3)H-UCB was apparently caused mainly by prior degradation and exchange of labile tritium of the (3)H-ALA and probably by enhanced endogenous ALA synthesis caused by the anesthetic/sedative agents. Revised procedures in the second dog improved the incorporation of (3)H-ALA to 11.9% excreted in bile and the specific activity of the crystalline (3)H-UCB to 122.0 and 50.8 x 10(3) dpm/microg, while urinary excretion of tritium increased to 28.5%. These experiences emphasize possible pitfalls in preparing (3)H-UCB by biosynthetic labeling from (3)H-ALA administered to dogs.

Effect of experimental fasciolosis on antipyrine metabolism and clearance in water buffaloes.

The effect of chronic Fasciola hepatica infection on the metabolism of antipyrine, a marker of microsomal oxidative metabolism, was investigated in male water buffaloes dosed daily with 60 F. hepatica metacercariae over 20 days. The plasma elimination half-life of antipyrine was significantly elevated by 23% at 11 weeks postinfection (p.i.) but did not significantly differ from the control period at 20 weeks p.i. The systemic clearance of antipyrine decreased by 48% at 11 weeks p.i. and then returned to normal. The renal clearance for each of the main antipyrine metabolites decreased at 11 weeks p.i. (hydroxymethylantipyrine (HMA), -42%; norantipyrine (NORA), -58%; and 4-hydroxyantipyrine (OHA), -70%) and did not significantly differ from the control period at 20 weeks p.i. These findings indicate that experimental subclinical fasciolosis leads to altered antipyrine kinetics and to an inhibition of the different antipyrine metabolic pathways in water buffaloes.

Changes in metabolism and urinary excretion of antipyrine induced by aerobic conditioning.

BACKGROUND: Physical conditioning has been reported to increase liver oxidative metabolism determined by antipyrine clearance. The purpose of this investigation was to study effects of aerobic conditioning on the different metabolic pathways of antipyrine by comparing the production clearances of antipyrine metabolites. PARTICIPANT: volunteers not engaged in the systematic practice of any sport (n = 14) were compared with aerobically-conditioned subjects (n = 14) (long distance runners, defined as men running > 80 km/week). INTERVENTIONS: antipyrine was administered orally. Saliva samples were collected under basal conditions and at 8, 16, 24, 32 and 40 hrs following antipyrine administration. Urine was collected for 24 hrs after antipyrine ingestion. MEASURES: endurance performance was expressed by the maximal oxygen uptake (VO2max), the ventilatory threshold and the 4 mM.l-1 lactate threshold (OBLA). Antipyrine pharmacokinetic parameters (antipyrine clearance and half-live) were obtained from saliva samples by the standard multiple-sample method. RESULTS: VO2max, ventilatory threshold and OBLA were higher in trained than in control subjects (+32%, +16% and +74%, respectively). Salivary antipyrine clearance was higher, whether or not this variable was corrected for weight (+26% and +38%, respectively), and antipyrine half-life was significantly reduced (-31%) in runners. There was no significant change with training in the renal clearance of antipyrine or in the norantipyrine (NORA) formation clearance but significant increases were observed in hydroxymethylantipyrine (HMA) and 4-hydroxyantipyrine (OHA) formation clearances (+42 and +37%, respectively). CONCLUSIONS: The findings indicate that aerobic conditioning leads to increased disposition of antipyrine and that the main metabolic pathways of the compound are changed differently.

Plasma aspartate aminotransferase (AST), glutamate dehydrogenase (GLDH) and gamma-glutamyl transpeptidase (GGT) activities in water buffaloes with experimental subclinical fasciolosis.

The effect of chronic Fasciola hepatica infection on the activity of plasma aspartate aminotransferase (AST), glutamate dehydrogenase (GLDH) and gamma-glutamyl transpeptidase (GGT) was investigated in water buffaloes dosed daily with 60 F. hepatica metacercariae over 20 days. Experimental fluke infection caused no clinical signs but provoked an increase in plasma level of IgG directed against F. hepatica from 4 weeks after infection. There was a significant increase in plasma AST from 6 weeks post-infection. Maximal values were reached at 14 weeks and remained significantly elevated by 23 weeks. Plasma GLDH was significantly elevated from 6 to 21 weeks post-infection. Significant increases in plasma GGT occurred from 8 to 26 weeks post-infection, reaching maximal values at 15 weeks. This study shows that plasma enzyme activities may be useful in studies of fluke-induced liver damage in water buffaloes.

The effect of physical conditioning on antipyrine clearance.

The effects of physical conditioning on antipyrine clearance were studied in two groups of subjects. Healthy men not engaged in the systematic practice of any sport were compared with endurance runners (defined as men running > 80 km/week). Studies were carried out at three different periods of the annual plan training at 4-month intervals. Antipyrine was administered orally and pharmacokinetic parameters were obtained from saliva samples by the multiple-sample method. Endurance performance, expressed in terms of the maximal oxygen uptake (VO2max), the ventilatory threshold and the 4-mM x l(-1) lactate threshold (OBLA), was higher in trained than in control subjects at each of the three periods. Antipyrine clearance was also significantly elevated and antipyrine half-life reduced in runners during all periods. No significant difference in VO2max or antipyrine clearance was found between the various periods in either trained or control subjects. Both ventilatory threshold and OBLA increased significantly along the training period in conditioned subjects. Significant correlations were found between antipyrine clearance and VO2max, ventilatory threshold and OBLA. In summary, these results indicate an association between aerobic conditioning and increased hepatic oxidative metabolism of low-clearance drugs.

Pectin feeding influences fecal bile acid excretion, hepatic bile acid and cholesterol synthesis and serum cholesterol in rats.

This study was designed to investigate the effects of pectin on cholesterol and bile acid metabolism and to elucidate the mechanisms involved in its hypolipidemic effect in rats. The key regulatory enzymes in cholesterol and bile acid metabolism, 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase) and cholesterol 7alpha-hydroxylase, were determined. Circulating, hepatic, and biliary lipid concentrations and fecal bile acid excretion were also measured. Male Wistar rats were fed a fiber-free or a pectin-supplemented (7 g/100 g) diet for 4 wk. Bile flow and the biliary secretion of both bile acids and cholesterol were not significantly different than controls in pectin-fed rats. The addition of pectin to the diet resulted in lower serum and liver cholesterol concentrations (-27 and -17%, respectively). Fecal bile acid excretion (+168%) and the hepatic activity of cholesterol 7alpha-hydroxylase (+70%) were significantly higher in pectin-fed animals. HMG-CoA reductase activity was also significantly greater (+11%) in the presence of dietary pectin. Results of our study indicate that pectin, by enhancing fecal bile acid excretion, may cause increased hepatic synthesis of bile acids and liver depletion of cholesterol in rats, which results in a higher rate of cholesterol synthesis and reduced serum cholesterol concentrations.

Albumin binding of unconjugated [3H]bilirubin and its uptake by rat liver basolateral plasma membrane vesicles.

Using highly purified unconjugated [3H]bilirubin (UCB), we measured UCB binding to delipidated human serum albumin (HSA) and its uptake by basolateral rat liver plasma membrane vesicles, in both the absence and presence of an inside-positive membrane potential. Free UCB concentrations ([Bf]) were calculated from UCB-HSA affinity constants (K'f), determined by five cycles of ultrafiltration through a Centricon-10 device (Amicon) of the same solutions used in the uptake studies. At HSA concentrations from 12 to 380 microM, K'f (litre/mol) was inversely related to [HSA], irrespective of the [Bf]/[HSA] ratio. K'f was 2.066 x 10(6) + (3.258 x 10(8)/[HSA]). When 50 mM KC1 was isoosmotically substituted for sucrose, the K'f value was significantly lower {2.077 x 10(6) + (1.099 x 10(8)/[HSA])}. The transport occurred into an osmotic-sensitive space. Below saturation ([Bf] < or = 65 nM), both electroneutral and electrogenic components followed saturation kinetics with respect to [Bf], with K(m) values of 28 +/- 7 and 57 +/- 8 nM respectively (mean +/- S.D., n = 3, P < 0.001). The Vmax was greater for the electrogenic than for the electroneutral component (112 +/- 12 versus 45 +/- 4 pmol of UCB. mg-1 of protein. 15 s-1, P < 0.001). Sulphobromophthalein trans-stimulated both electrogenic (61%) and electroneutral (72%) UCB uptake. These data indicate that: (a) as [HSA] increases, K'f decreases, thus increasing the concentration of free UCB. This may account for much of the enhanced hepatocytic uptake of organic anions observed with increasing [HSA]. (b) UCB is taken up at the basolateral membrane of the hepatocyte by two systems with K(m) values within the range of physiological free UCB levels in plasma. The electrogenic component shows a lower affinity and a higher capacity than the electroneutral component. (c) It is important to calculate the actual [Bf] using a K'f value determined under the same experimental conditions (medium and [HSA]) used for the uptake studies.