Kinetic analysis and comparison of uptake, distribution, and excretion of 48V-labeled compounds in rats

Setyawati, I. A., K. H. Thompson, V. G. Yuen, Y. Sun, M. Battell, D. M. Lyster, C. Vo, T. J. Ruth, S. Zeisler, J. H. McNeill, and C. Orvig. Kinetic analysis and comparison of uptake,distribution, and excretion of⁴⁸V-labeled compounds in rats.J. Appl. Physiol. 84(2): 569-575, 1998.Vanadium has been found to be orally active in lowering plasmaglucose levels; thus it provides a potential treatment for diabetesmellitus. Bis(maltolato)oxovanadium(IV) (BMOV) is a well-characterizedorganovanadium compound that has been shown in preliminarystudies to have a potentially useful absorption profile. Tissuedistributions of BMOV compared with those of vanadyl sulfate (VS) werestudied in Wistar rats by using⁴⁸V as a tracer. In this study,the compounds were administered in carrier-added forms by either oralgavage or intraperitoneal injection. Data analyzed by a compartmentalmodel, by using simulation, analysis, and modeling (i.e., SAAM II)software, showed a pattern of increased tissue uptake with use of⁴⁸V-BMOV compared with⁴⁸VS. The highest⁴⁸V concentrations at 24 h aftergavage were in bone, followed by kidney and liver. Most ingested⁴⁸V was eliminated unabsorbed byfecal excretion. On average, ⁴⁸Vconcentrations in bone, kidney, and liver 24 h after oraladministration of ⁴⁸V-BMOV weretwo to three times higher than those of⁴⁸VS, which is consistent with theincreased glucose-lowering potency of BMOV in acute glucose loweringcompared with VS.

     

Estradiol replacement in ovariectomized rats increases the hepatic concentration and biliary secretion of alpha-tocopherol and polyunsaturated fatty acids

Previously, we showed that estradiol replacement in ovariectomized rats produced prominent increases in serum and liver alpha-tocopherol (alphaTP). The present study was conducted to examine whether the estrogen-induced increase in the liver concentrations of alphaTP affects its biliary secretion and the fatty acid compositions of hepatic and biliary lipids. Ten ovariectomized rats were assigned to two groups: five rats were implanted subcutaneously with time-release estradiol pellets (OXE; 25 microg/day/rat) and five with placebo (OXP). Twice daily rats were pair-fed a modified AIN-93G diet containing soybean oil. At 5 weeks, bile was collected via a bile cannula hourly for 8 hours during duodenal infusion of a lipid emulsion (565 micromol triolein and 396 micromol Na-taurocholate/24 mL phosphate buffered saline, pH 6.45) at 3.0 mL/hr. During the 8-hour period, no difference was noted in the hourly rate of bile flow (0.95 mL/hr in OXE rats vs. 0.99 mL/hr in OXP rats). The biliary output of alphaTP for 8 hours was higher in OXE rats (51.6 +/- 3.6 nmol) than OXP rats (31.7 +/- 2.9 nmol). Likewise, the liver concentration of alphaTP was higher in OXE rats (81.9 +/- 3.5 nmol/g liver) than in OXP rats (53.3 +/- 7.4 nmol/g liver). The biliary secretion of phospholipids (PL) for 8 hours was significantly (P < 0.05) higher in OXE rats (55.1 +/- 4.9 micromol) than in OXP rats (42.3 +/- 4.7 micromol). Among the PL fatty acids, the outputs of 20:4 and 22:6n-3 were increased most markedly by estradiol replacement. The total outputs of 22:6n-3 for 8 hours in OXE and OXP rats were 2.95 +/- 0.20 micromol and 1.37 +/- 0.23 micromol, respectively. In the liver, the concentrations of PL 22:5n-3 and 22:6n-3 were elevated significantly in OXE rats. The present results suggest that estradiol may protect hepatic PL and membranes against oxidative damage by improving the liver status of alphaTP.     

The biliary excretion of trypsin in rats

The serum half-life of bovine [³H]acetyltrypsin was estimated to be 9 rain following intravenous administration in rats. This was maintained when six successive doses of 200 g each were given at 1-h intervals. The enzyme was removed from the circulation after complexing with 2-macroglobulin (2-M). The amount of³H label appearing in bile increased with each successive dose and this was associated with breakdown products (<10 000 daltons) of the ₂–M/[³H] acetyltrypsin. Intact –M/[³H] acetyltrypsin was recovered from bile but represented only 0.06% of the administered dose of active enzyme.     

Hepatic uptake and biliary excretion of manganese in the little skate,Leucoraja erinacea

The liver is a major organ involved in regulating whole body manganese (Mn) homeostasis; however, the mechanisms of Mn transport across the hepatocyte basolateral and canalicular membranes remain poorly defined. To gain insight into these transport steps, the present study measured hepatic uptake and biliary excretion of Mn in an evolutionarily primitive marine vertebrate, the elasmobranch Leucoraja erinacea, the little skate. Mn was rapidly removed from the recirculating perfusate of isolated perfused skate livers in a dose-dependent fashion; however, only a small fraction was released into bile (< 2% in 6 h). Mn was also rapidly taken up by freshly isolated skate hepatocytes in culture. Mn uptake was inhibited by a variety of divalent metals, but not by cesium. Analysis of the concentration-dependence of Mn uptake revealed of two components, with apparent K_m values 1.1 ± 0.1 µM and 112 ± 29 µM. The K_m value for the high-affinity component was similar to the measured skate blood Mn concentration, 1.9 ± 0.5 µM. Mn uptake was reduced by nearly half when bicarbonate was removed from the culture medium, but was unaffected by a change in pH from 6.5 to 8.5, or by substitution of Na with Li or K. Mn efflux from the hepatocytes was also rapid, and was inhibited when cells were treated with 0.5 mM 2,4-dinitrophenol to deplete ATP levels. These data indicate that skate liver has efficient mechanisms for removing Mn from the sinusoidal circulation, whereas overall biliary excretion is low and appears to be mediated in part by an ATP-sensitive mechanism.     

Testosterone represses urinary excretion of the alpha-tocopherol metabolite alpha-carboxymethylhydroxychroman in rats

In rats, plasma and tissue concentrations of α-tocopherol, a predominant form of vitamin E in mammals, are known to differ between the sexes. In order to examine sex differences in α-tocopherol metabolism, we investigated urinary excretion of the α-tocopherol metabolite α-carboxymethylhydroxychroman (α-CEHC) using Wistar rats. First, we measured α-CEHC in urine of 9-week-old male and female rats in basal and α-tocopherol-administered conditions. We observed that female rats excrete significantly more α-CEHC than male rats via urine. This sex difference was observed in matured 9-week-old rats but not in premature 3-week-old rats, suggesting that the difference may relate to sex hormones. In order to confirm this, we examined the effect of ovariectomy and orchiectomy on female and male rats, respectively. The results of castration clearly demonstrated that orchiectomy enhanced urinary excretion of α-CEHC, supporting the hypothesis that testosterone repressed α-tocopherol metabolism. We then administered testosterone propionate to orchiectomized rats and observed down-regulation of α-CEHC excretion. Taken together, these results indicate that testosterone represses the metabolism and urinary excretion of α-tocopherol in rats. This is the first report to show a sex-dependent difference in urinary excretion rate of an α-tocopherol metabolite and contributes to the understanding of vitamin E metabolism.     

Transport and distribution of alpha-tocopherol in lymph, serum and liver cells in rats

Rats were cannulated in the major mesenteric lymph duct and given an intraduodenal bolus of unlabeled and alpha-[3H]tocopherol, and [14C]oleic acid in soybean oil. The appearance of alpha-tocopherol in lymph was negligible during the first 2 h and peaked 4-15 h after feeding, whereas no detectable amount was recovered in the portal vein. Intestinal absorption via the lymphatic pathway was 15.4 +/- 8.9% (n = 10) and 45.9 +/- 10.8% (n = 4) for alpha-tocopherol and [14C]oleic acid, respectively. About 99% of alpha-tocopherol in lymph was associated with the chylomicron fraction (d less than 1.006 g/ml). In non-fasting rats, 51% of serum alpha-tocopherol was associated with chylomicrons/VLDL (very-low-density lipoprotein, d less than 1.006 g/ml) and 47% with HDL (high-density lipoprotein, 1.05 less than d less than 1.21 g/ml). Our study revealed that the liver, skeletal muscle and adipose tissue contain approx. 92% of the total mass of alpha-tocopherol measured in ten different organs. Parenchymal and nonparenchymal liver cells contributed to 75% and 25% of the total mass of alpha-tocopherol in the liver, respectively.     

Suppression of hepatic prostaglandin F2 alpha in rats by dietary alpha-tocopherol acetate is independent of total hepatic alpha-tocopherol.

Groups of eight weanling female F344/N rats were fed semipurified diets that supplied 0, 50, 500, 5000, or 15,000 mg alpha-tocopherol acetate/kg diet, with and without 0.05% phenobarbital (PB) for 9 weeks. Both plasma and hepatic alpha-tocopherol levels, measured by HPLC, strongly correlated with alpha-tocopherol intake (r greater than 0.73, p less than 0.0001). Phenobarbital both depleted hepatic alpha-tocopherol and increased plasma alpha-tocopherol significantly. Although treatment with PB for 9 weeks significantly increased GST activity, PB did not affect hepatic prostaglandin (PG)F2 alpha status, as determined by radioimmunoassay. PGF2 alpha was significantly greater (by 52%) in rats fed no alpha-tocopherol than in rats fed 15,000 mg alpha-tocopherol acetate/kg diet. Hepatic PGF2 alpha status was correlated inversely but weakly with dietary alpha-tocopherol (r = -0.24, p less than 0.05). Hepatic PGF2 alpha status was not correlated with hepatic or plasma alpha-tocopherol status. This finding suggests either that there is a small depletion-resistant subcellular alpha-tocopherol pool which regulates PGF2 alpha production or that alpha-tocopherol alters PGF2 alpha production in vivo by an indirect mechanism.     

Dosing-time-dependent variation in biliary excretion of flomoxef in rats

We previously reported that the biliary excretion of flomoxef, an oxacephem antibiotic, was greater after dosing at 21:00 than at 09:00h in diurnally active human subjects. The present study was undertaken to examine whether the biliary excretion of flomoxef is also dependent on its dosing time in rats. Adult male Wistar rats were housed under light on at 07:00h and off at 19:00h. Bile fluid was completely drained through a polyethylene catheter from conscious animals. Flomoxef (20 mg/kg) was injected into the tail vein at 09:00 or 21:00h by a cross-over design, and drained bile fluid was collected for 8h after each dosing. The maximum concentration of biliary flomoxef was significantly greater and its total excretion tended to be greater after dosing at 09:00 than 21:00h. These results suggest the biliary excretion of flomoxef is enhanced after dosing at the beginning of the rest period in rats, as it is in humans.     

Stereoselectivity of biliary excretion of 2-arylpropionates in rats

To examine the stereoselectivity of biliary excretion, the optically pure enantiomers of ketoprofen (KT), ibuprofen (IBU), and flurbiprofen (FLU) were intravenously administered to normal and bile duct-cannulated rats at 10 mg/kg. The recovery of total KT in bile was significantly higher after administration of (S)-KT than after (R)-KT [90.1 ± 3.5% vs 68.8 ± 8.2%, n =3, P < 0.05]. In normal rats the terminal half-life of (R)-KT was significantly shorter than that of (S)-KT after administration of (R)-KT (2.2 ± 0.6 h vs 14.3 ± 4.9 h, n = 3, P < 0.05). The terminal half-life of both enantiomers was significantly shorter in rats with continuous bile drainage as compared to normal rats. No significant differences in pharmacokinetic parameters could be found between both enantiomers in bile duct-cannulated animals. The total amount of IBU in bile was slightly higher after administration of (S)-IBU than after (R)-IBU administration. The percentage of (R)-IBU after (R)-IBU administration, however, was very low [(R)-IBU: 1.5 ± 0.9%, (S)-IBU: 23.4 ± 5.8%]. In normal rats the clearance of (R)-IBU was significantly higher as compared to (S)-IBU. Differences in pharmacokinetic parameters between normal and bile duct-cannulated rats were not statistically significant due to high interindividual variability. The total recovery of FLU, which was excreted in bile to a lower extent than either KT or IBU, also tended to be greater after S-enantiomer administration. Only small amounts of (S)-FLU could be recovered in bile after (R)-FLU administration. The pharmacokinetic parameters did not differ significantly between (R)- and (S)-FLU or between normal and bile duct-cannulated rats due to its low inversion rate and low excretion via bile. © 1993 Wiley-Liss, Inc.     

Azelastine and suplatast shorten the distribution half-life of IgE in rats

We aim to clarify whether suplatast and azelastine (anti-allergic drugs) can shorten the half-life of imnunoglobulin E (IgE) in the circulating blood. Thirty Wistar rats were divided into six groups. Distilled water or anti-allergic drugs were given orally for 6 days after the first sensitization. Two milligrams of monoclonal dinitrophenyl (DNP)-specific rat IgE was administered to the rats, which had been given suplatast or azelastine orally. The level of DNP-specific rat IgE in the serum was estimated by IgE-capture enzyme-linked immunosorbent assay, and the turnover of IgE was analyzed from its pharmacokinetic parameters. The elimination half-life of rat IgE was about 12 h irrespective of the sensitized state. The intercompartmental rate constants (Kct and Ktc) in the suplatast-administered or azelastine-administered group were larger than those of the distilled water-administered group under non-sensitized conditions. These findings suggested that the anti-allergic drugs used in the present study facilitated the excretion of IgE from the circulation in rats.     

Synthesis and biliary excretion of tyrosine-conjugated bile salts in Wistar rats

Tyrosine-labelled free and glycine-conjugated bile acids were synthesized and radiolabelled with 125I to high purity. The synthetic method utilized excess tyrosine methyl ester hydrochloride (1.4 equiv.) and bile acid (one equiv.) via dicyclohexylcarbodiimide (1.4 equiv.) with yields of 90-93% for tyrosine bile acid conjugates and glycyltyrosine conjugates and 56-60% yields for the glycylglycyltyrosine conjugates. All of the eight iodinated tyrosine bile acids tested were rapidly excreted into bile following intravenous injection. In bile duct-cannulated rats with ligated renal pedicles under pentobarbital anaesthesia the percentages of injected dose recovered from bile within 20 min were as follows: cholylglycine ([14C]cholylGly), 81.2 +/- 1.3%; taurocholate ([14C]taurocholate), 94.3 +/- 1.0%; cholyltyrosine (125I-cholylTyr), 85.5 +/- 3.3%; deoxycholyltyrosine (125I-deoxycholylTyr), 87.9 +/- 6.3%; chenodeoxycholyltyrosine (125I-chenodeoxycholylTyr), 93.4 +/- 2.9; cholylglycyltyrosine (125I-cholylGlyTyr), 95.7 +/- 6.7%; deoxycholylglycyltyrosine (125I-deoxylcholylGlyTyr), 92.5 +/- 3.2%; chenodeoxycholylglycyltyrosine (125I-chenodeoxycholylGlyTyr), 94.1 +/- 3.1%; cholyldiglycyltyrosine (125I-cholylGlyGlyTyr), 85.2 +/- 3.6%, and deoxycholyldiglycyltyrosine (125I-deoxycholylGlyGlyTyr), 85.5 +/- 2.7%. Values are means +/- SD. Thus the biliary excretion of 125I-chenodeoxycholylGlyTyr, 125I-chenodeoxycholylTyr, 125I-deoxycholylGlyTyr and 125I-cholylGlyTyr was similar to that of [14C]taurocholate, the major naturally occurring bile acid in the rat, and the biliary excretion of all the tyrosine conjugates was similar to or exceeded that of [14C]cholylglycine. Conjugation with tyrosine enhanced the efficiency of plasma-to-bile transport of most naturally occurring bile acids. Comparison of glycyltyrosine conjugates with glycylglycyltyrosine conjugates suggests that any additional benefit derived by elongation of the side-chain is probably negated by obscuring the 12 alpha-hydroxyl function on the steroid nucleus in the bile acid glycylglycyltyrosine conjugates.     

Biliary copper excretion capacity in intact animals: correlation between ATP7B function, hepatic mass, and biliary copper excretion

Copper toxicosis can occur in the absence of biliary copper excretion. To demonstrate whether biliary copper excretion capacity is correlated with hepatic mass and ATP7B function, we undertook studies in intact animals. Copper-histidine was injected intrasplenically after baseline bile collection, followed by measurement of copper excretion in Long-Evans Cinnamon (LEC) rats lacking atp7b function and in normal, syngeneic Long-Evans Agouti (LEA) rats. The basal biliary copper excretion was very low in LEC rats compared with LEA rats, 8+/-4 and 37+/-18 ng copper/min, respectively; p<0.05. After addition of copper, copper excretion increased significantly (by two- to five-fold) in LEA rats during the 30 minute study period, whereas LEC rats showed only a slight and transient increase in copper excretion. After one-third and two-thirds partial hepatectomy immediately before copper loading, copper excretion decreased progressively. The studies indicate that biliary copper excretion depends on hepatocyte mass and ATP7B gene function. Analysis of copper excretion with our non-radioactive method will facilitate testing of novel therapies and pathophysiological mechanisms in copper toxicity.     

Characteristics of sinusoidal uptake and biliary excretion of cysteinyl leukotrienes in perfused rat liver

In single-pass perfused rat liver, the sinusoidal uptake of infused 3H-labelled leukotriene (LT) C4 (10 nmol.l-1) was inhibited by sulfobromophthalein. Inhibition was half-maximal at sulfobromophthalein concentrations of approximately 1.2 mumol.l-1 in the influent perfusate and leukotriene uptake was inhibited by maximally 34%. Sulfobromophthalein (20 mumol.l-1) also decreased the uptake of infused [3H]LTE4 (10 nmol.l-1) by 31%. Indocyanine green (10 mumol.l-1) inhibited the sinusoidal [3H]LTC4 uptake by 19%. Replacement of sodium in the perfusion medium by choline decreased the uptake of infused [3H]LTC4 (10 nmol.l-1) by 56%, but was without effect on the uptake of sulfobromophthalein. The canalicular excretion of LTC4, LTD4 and N-acetyl-LTE4 was inhibited by sulfobromophthalein. In contrast, the proportion of polar omega-oxidation metabolites recovered in bile following the infusion of [3H]LTC4 was increased. Taurocholate, which had no effect on the sinusoidal leukotriene uptake, increased bile flow and also the biliary elimination of the radioactivity taken up. With increasing taurocholate additions, the amount of LTD4 recovered in bile increased at the expense of LTC4. Following the infusion of [3H]LTD4 (10 nmol.l-1), a major biliary metabolite was LTC4 indicating a reconversion of LTD4 to LTC4. In the presence of taurocholate (40 mumol.l-1), however, this reconversion was completely inhibited. The findings suggest the involvement of different transport systems in the sinusoidal uptake of cysteinyl leukotrienes. LTC4 uptake is not affected by bile acids and has a sodium-dependent and a sodium-independent component, the latter probably being shared with organic dyes. Sulfobromophthalein also interferes with the canalicular transport of LTC4, LTD4 and N-acetyl-LTE4, but not with the excretion of omega-oxidized cysteinyl leukotrienes. The data may be relevant for the understanding of hepatic leukotriene processing in conditions like hyperbilirubinemia or cholestasis.     

The biliary excretion of enterokinase in rats. Studies in normal, chronic ethanol-maintained and cirrhotic rats.

The excretion of catalytically active human or pig enterokinase in hepatic bile after intravenous administration to normal rats or rats that had been maintained on 20% (v/v) ethanol for 1 year showed similar kinetics to that described for other serum-derived bile proteins. The half-life in serum was 2.5 min or less, and most of the enzyme was excreted within 45 min of administration. This was maintained when up to six successive doses were given at 90 min intervals. The mean amount excreted per dose was independent of the dose number and varied from 0.8% to 2.1% in the normal animals and 1.2% to 2.0% in the chronic ethanol-maintained animals. When three doses of enzyme were given at 30 min intervals, the total amount of active enterokinase recovered in bile was dose-dependent and was consistently higher in the rats drinking 20% (v/v) ethanol. The serum half-life of enterokinase in rats made cirrhotic by inhalation of carbon tetrachloride vapour was extended to 6 min or more. The amount of active enzyme recovered in bile was at least 50% less than in weight-matched normal rats, and excretion was not complete 2h after intravenous administration. The possible significance of these findings in liver and pancreatic disease is discussed.     

High biliary and systemic excretion of a glucuronide during its hepatic synthesis

Using isolated perfused rat liver and ¹⁴C-p-nitrophenol as a model substrate for glucuronidation, evidence was obtained that during synthesis of p-nitrophenyl-glucuronide biliary excretion of the metabolite is much higher than would be expected from concentrations of the metabolite in the circulation. Isotopic dilution experiments indicate that newly synthesized glucuronide is in a pool which is readily available for biliary excretion but is not easily miscible, or diluted, by the glucuronide in the circulation.     

Fecal excretion, uptake and metabolism by colon mucosa of diacylglycerol in rats.

In a previous paper we demonstrated that human fecal bacteria can convert phosphatidylcholine to diacylglycerol (DAG), an activator of protein kinase C. The present study demonstrates that several foods contain appreciable levels of DAG, especially certain vegetable oils. On the other hand, when rats were administered [14C]-labeled DAG by intragastric intubation less than 0.1% of the administered radioactivity was recovered as DAG in the feces. Thus only negligible amounts of dietary DAG actually reach the colon. When [14C]DAG was injected directly into ligated segments of rat colon we found appreciable uptake of the intact DAG by the mucosal cells. The major metabolite was arachidonic acid, suggesting that the DAG lipase pathway is more active than the DAG kinase pathway in these cells. Taken together, these results are consistent with our hypothesis that much of the DAG present in the colonic lumen is produced by the intestinal bacteria and that this DAG can actually enter the colonic mucosal cells, where it might influence their function.