Bile Acid-Induced Increase in Bile Acid-Independent Flow and Plasma Membrane NaK-ATPase Activity in Rat Liver

Previous studies showed that in rats with obstruction of the bile ducts draining the median and left hepatic lobes, and in rats with normal bile ducts in which the bile acid pool size and secretion were augmented by 48-h intraduodenal infusion of taurocholate, bile acid flux through secreting hepatocytes was increased. Under these conditions, taurocholate transport maximum exhibited a time-dependent adaptation to increased secretory load.Unexpectedly, bile acid-independent canalicular flow in these experimental models also was found to be increased when measured at 48 h. Relative to controls, bile acid-independent flow per gram of nonobstructed liver was increased approximately threefold in selectively obstructed rats and 43% in bile acid-loaded rats with normal ducts. In rats infused with bile acids at similar rates for only 16 h, no increase was observed. Studies with [(14)C]erythritol suggested that the increased bile flow under these conditions was of canalicular origin.NaK-ATPase activity in canaliculi-enriched liver plasma membrane preparations from the nonobstructed lobes of selectively obstructed rats and from 48-h bile acid-loaded rats was increased by 47% and 52%, respectively, relative to controls, but was not increased in membranes from 16-h bile acid-loaded rats. Canalicular membrane 5'-nucleotidase and Mg ATPase also were increased.These studies show that augmented bile acid flux through secreting liver causes an adaptive increase in bile acid-"independent" flow and in the activity of canalicular membrane enzymes. The mechanism by which bile acids modulate this and previously reported aspects of bile secretion remains to be elucidated.     

Canalicular Bile Production in Man*

Abstract. Bile flow, erythritol clearance (measured after administration of ¹⁴C-erythritol) and bile acid excretion were studied in 8 non-icteric patients operated upon for gallstone disease without biliary obstruction in whom a T-tube had been inserted. Bile flow increased from the 2nd (0. 27 ± SEM 0. 01 ml/min) to the 7th (0. 50 ± SEM 0. 05 ml/min) postoperative days and remained stable thereafter. Because the values obtained between the 7th and the 15th postoperative days (average: 0. 46 ± SEM 0. 02 ml/min) were not lower and were of the same order of magnitude as those previously reported in the literature for quantitative bile collections, it was assumed that bile collection had been complete. Bile-to-plasma concentration ratio of erythritol was 0. 77 ± SEM 0. 04. The regression line between erythritol clearance and bile flow had a slope of 0. 99 (indicating that increments in clearance were followed by equivalent increments of flow) and an intercept for a zero-clearance of 0. 11, suggesting a ductular/ductal secretion of 0. 11 ml/min. The relationship between erythritol clearance and bile acid excretion suggested that canalicular bile was formed both of a bile acid-dependent flow (11 μl of water being excreted per umol of bile acid secreted) and a bile acid-independent flow of 0. 16 ml/min (or 35 % of the average bile flow). These findings indicate that erythritol clearance provides a valid estimate of canalicular bile flow in man and that the three bile fractions previously postulated in animals (canalicular bile acid-dependent, canalicular bile acid-independent and ductular/ductal) may also be present in man.     

The Effect of Acetazolamide on Cerebral Blood Flow and Oxygen Utilization in the Rhesus Monkey

The brain is critically dependent for its moment to moment function and survival on an adequate supply of oxygen. The enzyme carbonic anhydrase (EC 4.2.1.1) may play an important role in oxygen delivery to brain tissue by facilitating the hydration of metabolically produced carbon dioxide in erythrocytes in brain capillaries, thus permitting the Bohr effect to occur. We examined the effect of 30 mg/kg i.v. acetazolamide, a potent inhibitor of carbonic anhydrase, upon cerebral blood flow and oxygen consumption in lightly anesthetized, passively ventilated rhesus monkeys. Cerebral blood flow and oxygen consumption were measured with oxygen-15-labeled water and oxygen-15-labeled oxyhemoglobin, respectively, injected into the internal carotid artery and monitored externally. Acetazolamide produced an immediate and significant increase in cerebral blood flow (from a mean of 64.7 to 83.8 ml/100 g per min), an increase in arterial carbon dioxide tension (from a mean of 40.7 to 47.5 torr), and a decrease in cerebral oxygen consumption (from a mean of 4.16 to 2.82 ml/100 g per min). Because the change in cerebral oxygen consumption occurred within minutes of the administration of acetazolamide, we believe that this effect probably was not due to a direct action on brain cells but was achieved by an interference with oxygen unloading in brain capillaries. A resultant tissue hypoxia might well explain part of the observed increase in cerebral blood flow.     

Cholesterol Metabolism and Placental Transfer in the Pregnant Rhesus Monkey

The placental transfer of cholesterol (5-cholesten-3beta-ol) was investigated by giving pregnant rhesus monkeys cholesterol-1alpha-(3)H or cholesterol-4-(14)C and then determining the cholesterol specific activity (SA) in maternal serum and in fetal serum and tissues. An isotopic steady state was established in five pregnant animals by the daily feeding of a tracer dose of cholesterol-4-(14)C. Comparison of maternal and fetal serum cholesterol SA revealed that an average of 42.6% of the serum cholesterol in the term fetus originated by transfer from the maternal blood. The remainder presumably arose by fetal synthesis de novo. Fetal tissues had cholesterol SA equal to or slightly less than that of fetal serum, except for brain which had a SA only 5% that of fetal serum.In other studies a single intravenous dose of radioactive cholesterol was given to either mother or fetus in late pregnancy. The time for detectable passage across the placenta in either direction was between 4 and 24 hr. With maternal administration of the isotope, there was equilibration of maternal and fetal serum cholesterol SA after 10-12 days. With fetal injection of isotopic cholesterol, however, the maternal cholesterol SA never attained a level more than 5% of fetal SA. This indicated that the net cholesterol flux was strongly in the direction of mother to fetus.Serum cholesterol levels were significantly greater in maternal than in fetal serum (80.3+/-18.5 vs. 59.6+/-15.6 mg/100 ml). Maternal serum cholesterol concentration in the monkey was significantly lower in late pregnancy than during the puerperium. Studies of breast milk indicated that approximately two-thirds of milk cholesterol was transferred from the maternal blood.     

Collagen Degradation and the Response to Parathyroid Extract in the Intact Rhesus Monkey

Proline-(14)C was administered to five adult rhesus monkeys, and the degradation of collagen was followed by the excretion of hydroxyproline-(14)C. The results suggested the presence of at least three separate pools of collagen with half-lives of 1 to 2, 2 to 3, and 50 to 70 days. The monkeys were killed after 44 days; at that time the specific activity of the hydroxyproline-(14)C in urine was found to be four to five times that of the hydroxyproline in soluble collagen and 81 to 93% that of hydroxyproline in insoluble collagen. The relationships between urinary hydroxyproline and the degradation of collagen were similar to those previously demonstrated in rats.Parathyroid hormone was administered daily to two of the monkeys from the 27th to the 44th day of the study. The parathyroid hormone increased the amount of hydroxyproline-(14)C excreted, but there was no significant change in the specific activity of the urinary hydroxyproline-(14)C. Since under the conditions of the experiment insoluble collagen was the only possible source of hydroxyproline-(14)C of relatively high specific activity, the results indicated that parathyroid hormone directly or indirectly increased the degradation of insoluble collagen. The results also suggested that parathyroid hormone increased the degradation of soluble collagen, but the relative magnitude of this effect was not clearly established.     

Alteration of Bile Canalicular Enzymes in Cholestasis. A POSSIBLE CAUSE OF BILE SECRETORY FAILURE

Bile secretory failure (cholestasis) may result from several possible mechanisms involved in bile secretion. We have examined the possibility that abnormalities in enzyme content, composition, and turnover of liver plasma membrane constituents are altered in cholestasis.Severe and mild cholestasis were produced by 5 days of bile duct ligation and ethinyl estradiol administration, respectively. Bile duct ligation but not ethinyl estradiol treatments was associated with elevations of the serum bilirubin level and 5'-nucleotidase activity. However, basal bile flow and bilirubin transport maximum (T(m)) were significantly reduced after ethinyl estradiol treatment. Liver plasma membrane fractions rich in canalicular membranes were prepared from groups of rats in each of three categories; normal, after bile duct ligation, or ethinyl estradiol administration, and their respective controls. Electron microscopy and enzyme marker studies demonstrated plasma membrane fractions free of significant contamination.Plasma membrane fractions prepared from mild as well as severe cholestasis had increased alkaline phosphatase activity, and reduced 5'-nucleotidase and Mg(2+)-ATPase activities. Co(2+)-CMPase activity was unchanged. Kinetic analysis of 5'-nucleotidase and Mg(2+)-ATPase activities in plasma membrane fractions demonstrated reduced V(maz) (but unaltered K(m)). Reducted V(maz) was unrelated to addition in vitro of di-or trihydroxy bile salts or ethinyl estradiol and, therefore, suggests that reduced activities in cholestasis are due to decreased enzyme content. Cholestasis was not associated with changes in the synthesis or degradation rate of pulse-labeled plasma membrane proteins or alterations in the major protein bands separated on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis.Plasma membrane cholesterol, phospholipid, and neutral sugar content was unaltered, but sialic acid content was significantly increased in both forms of cholestasis. Alterations in specific canalicular enzymes in two forms of cholestasis suggest that these changes may be involved in the pathogenesis of bile secretory failure, or may result from cholestasis.     

Canalicular Bile Secretion in Man STUDIES UTILIZING THE BILIARY CLEARANCE OF [14C]MANNITOL

[¹⁴C]Mannitol was administered i.v. as a bolus injection to five postcholecystectomy patients with indwelling T-tubes and re-established enterohepatic circulations to evaluate the biliary clearance of [¹⁴C]mannitol as a means of estimating canalicular bile flow in man. [¹⁴C]Mannitol appeared in collections of bile 9-22.5 min after intravenous injection, rose to a peak, and thereafter paralleled the plasma [¹⁴C]mannitol disappearance curve. Bile-plasma [¹⁴C]mannitol ratios and [¹⁴C]mannitol clearances were determined during control and choleretic periods after correction of the bile [¹⁴C]mannitol points for the transit time of a given sample. After i.v. injection of sodium dehydrocholate in five studies, bile flow and mannitol clearance increased proportionately. However, when ductular secretion was stimulated with an i.v. bolus of secretin in three other studies, [¹⁴C]mannitol clearance remained essentially unchanged, indicating that [¹⁴C]mannitol entered bile at the level of the hepatocyte and could be utilized as a marker of canalicular flow in man.     

The Rhesus Monkey as a Model for Evaluation of the Preservation of Stored Whole Blood

Blood was collected into Acid Citrate Dextrose solution (ACD) from a group of 20 large, adult rhesus monkeys. In ten of the experiments, the blood was labeled immediately with radioisotopes and reinfused into the donor monkeys. The other ten blood units were stored at 4 C for 21 days, then tabled and infused into the donor monkeys. In vitro determinations were made immediately after collection and at the end of the storage period and compared with the results obtained in stored human blood.
The ten fresh blood infusions had a mean survival of 101.3 per cent with an average half time of 10.4 days. The 21 (lay‐old bloods had a mean survival of 73.7 per cent (S.D. = 5.1%) with an average half time of 11.3 days. The average red blood cell survival of 123 human blood units stored 21 days and reinfused into their donors had an average red cell survival of 81.0 per cent (S.D. = 8.6%). The determinations in rhesus blood and human blood were comparable.
These data strongly suggest that in vivo studies done in the rhesus monkey may be substituted for those performed in man to evaluate red cell preservation.     

Dietary Factors Affecting Biliary Lipid Secretion in the Rhesus Monkey A Mechanism for the Hypocholesterolaemic Action of Polyunsaturated Fat?*

Abstract Using an experimental model which permits representative bile sampling whilst maintaining an almost “intact” enterohepatic circulation, it was found that different diets markedly influenced biliary lipid secretion in the rhesus monkey. In different studies, we have previously shown that bile volume, bile salt, phospholipid and cholesterol secretion rates were significantly less in animals given a low fat commercial diet then in monkeys given a second commerical diet with higher fat content. The present paired studies confirmed that in the same animals, the higher fat diet stimulated bile volume and biliary lipid secretion. On the higher fat diet there was no change in bile salt concentration or in pool size, the greater secretion rate being due to a greater bile volume and to more frequent circulations of the bile salt pool. Since the major differences between the two commercial diets were in fat and calories, the effect of supplementing the low fat diet with individual additives of olive, corn and medium chain triglyceride oil and with 40% dextrose was examined in further paired studies. In six monkeys, supplements of mono-unsaturated olive oil had no effect on biliary lipid secretion, but in seven animals given comparable doses of polyunsaturated corn oil, there was a significant increase in biliary bile salt, phospholipid and cholesterol output. The addition of medium chain triglyceride oil or of 250 ml of 40% dextrose did not change biliary lipid secretion rates. None of the dietary manipulations significantly altered cholesterol solubility in bile. In monkeys where hepatic bile salt synthesis had been maximally induced by partial biliary diversion (33.3% interruption of the EHC), corn oil no longer increased bile salt secretion suggesting that the stimulatory effect of corn oil was due to stimulation of bile salt synthesis. It is postulated that the increased turnover of bile salts seen after feeding corn oil, with a resultant acceleration in cholesterol catabolism, may explain, at least in part, the hypocholesterolaemic action of polyunsaturated fat.     

Introduction of a Xenogeneic Gene via Hematopoietic Stem Cells Leads to Specific Tolerance in a Rhesus Monkey Model

Host immune responses against foreign transgenes may be a major obstacle to successful gene therapy. To clarify the impact of an immune response to foreign transgene products on the survival of genetically modified cells, we studied the in vivo persistence of cells transduced with a vector expressing a foreign transgene compared to cells transduced with a nonexpressing vector in the clinically predictive rhesus macaque model. We constructed retroviral vectors containing the neomycin phosphotransferase gene (neo) sequences modified to prevent protein expression (nonexpressing vectors). Rhesus monkey lymphocytes or hematopoietic stem cells (HSCs) were transduced with nonexpressing and neo-expressing vectors followed by reinfusion, and their in vivo persistence was studied. While lymphocytes transduced with a nonexpressing vector could be detected for more than 1 year, lymphocytes transduced with a neo-expressing vector were no longer detectable within several weeks of infusion. However, five of six animals transplanted with HSCs transduced with nonexpression or neo-expression vectors, and progeny lymphocytes marked with either vector persisted for more than 2 years. Furthermore, in recipients of transduced HSCs, infusion of mature lymphocytes transduced with a second neo-expressing vector did not result in elimination of the transduced lymphocytes. Our data show that introduction of a xenogeneic gene via HSCs induces tolerance to the foreign gene products. HSC gene therapy is therefore suitable for clinical applications where long-term expression of a therapeutic or foreign gene is required.     

A Flow Cytometric Method for Determination of the Blood Neutrophil Fraction in Rats

Determination of the proportion of neutrophils in the peripheral blood is important for diagnostic purposes in medicine and for evaluating new drugs in the pharmaceutical industry. To measure the neutrophil concentration in rat blood, a fast and accurate flow cytometric method was developed. Rat neutrophils were quantified by using primary antibodies that recognize the RP1 antigen and secondary antibodies conjugated with fluorescein isothiocyanate. The flow cytometric method was calibrated by comparing cytometric results with data from a manual differential count. The results obtained by these 2 methods correlated with a Pearson correlation coefficient of 0.91 and were in agreement according to subsequent statistical analysis. To confirm the usefulness of the method in preclinical applications, the production of neutrophils in rats was stimulated by pegfilgrastim. Blood samples were taken at predetermined time points, and the pharmacodynamic profile was determined. These results confirmed that the flow cytometric method for neutrophil quantification is accurate and much faster than the manual microscopic method. Moreover, the flow cytometric method is easy to use, suggesting that it could become the method of choice for preclinical applications.Abbreviations: ANC, absolute neutrophil count; FITC, fluorescein isothiocyanate; WBC, white blood cell countDetermination of neutrophil concentration is a widespread practice in human medicine, in veterinary medicine, and in the pharmaceutical industry.^10,13,16,19 In particular, neutrophil concentration is an important parameter for preclinical tests on laboratory animals.^14,24 Neutrophil granulocytes are myeloid blood cells with diverse functions in vertebrate organisms, including defense against bacterial, fungal, and other infections, with specialized cellular functions such as phagocytosis, adherence, chemotaxis, and secretion.² The number of neutrophils can change dramatically due to many diverse pathologic conditions and drugs. Pegfilgrastim is pegylated granulocyte-colony stimulating factor, a drug used to accelerate neutrophil recovery and reduce the incidence of infections after chemotherapy.⁴ Bacterial infections, acute inflammation, and some malignancies typically lead to neutrophilia, whereas leukemias, autoimmune diseases, and chemotherapy frequently cause neutropenia.¹⁹Microscopic determination of the neutrophil percentage in blood is a common method used in human and veterinary clinics as well as in the pharmaceutical industry. The microscopic assessment of neutrophil concentration suffers from low reliability, owing to the quality of the blood smear and skill of the analyst. Furthermore, microscopy is time-consuming. Manual differentiation of white blood cells is still considered to be the routine method for validation of automated instrumentation.^10,22Parallel with the classic manual method, flow cytometry has emerged as a key technology in clinical laboratories. Flow cytometry offers the possibility of analyzing a high number of samples in a short time. In comparison to classic microscopic counting techniques, flow cytometry offers less biased, more accurate, and more precise sample assessment. Flow cytometers in current use include stand-alone instruments and those incorporated in hematology analyzers. Automated hematology analyzers using optical measurements typically are used for testing a restricted set of parameters. Automated analyzers have not replaced the manual method due to the inaccuracy of assignment of monocytes, eosinophils, and basophils from many animal species,²⁷ and they fail to resolve band neutrophils adequately.³ In contrast, flow cytometers are highly flexible instruments that can measure various properties of cells and their relative counts in different cell systems. Therefore flow cytometers may be more desirable than automatic hematology machines in many settings, especially in the research area. Flow cytometry has been developing in parallel with the development of new fluorescent dyes conjugated with antibodies. Because most antibodies are made for human clinical applications, the main obstacle to maximal application of flow cytometry in veterinary medicine is the lack of species-specific antibodies. Although flow cytometry can rapidly evaluate many cells,^27,30 the accuracy of flow-based cell quantification compared with the standard manual method is uncertain until direct comparison studies are reported.In the current study, a flow cytometric method for the determination of rat neutrophil concentration in peripheral blood was developed. Such a technique would be useful for evaluating new drugs in preclinical studies, particularly as a screening method for selection of the best analog of a drug. The flow cytometric method was compared with traditional manual counting under light microscopy. Various characteristics of the new method, such as accuracy and ease of use, were examined.     

PET Imaging Studies in Rhesus Monkey with the Cannabinoid-1 (CB1) Receptor Ligand [11C]CB-119

Purpose  The in vitro and in vivo evaluation of the selective, high affinity (human CB1 IC₅₀ 0.49 nM) inverse agonist CB1R tracer [¹¹C]CB-119, a close analog of the previously disclosed [¹⁸F]MK-9470, was undertaken. Procedures  [¹¹C]CB-119 was synthesized with high specific activity by alkylation of a phenolic precursor with [¹¹C]methyl iodide. In vitro autoradiographic studies using rhesus brain slices were carried out using [³H]CB-119, and in vivo imaging studies were carried out using [¹¹C]CB-119 in rhesus monkeys under baseline and blocked conditions. Results  Autoradiographic studies in rhesus brain showed the expected distribution pattern for CB1R with highest binding in the cerebral cortex, cerebellum, caudate/putamen, globus pallidus, substantia nigra, and hippocampus. Lower binding was seen in the posterior hypothalamus, ventral tegmental area, and periventricular gray area, and the lowest binding was in the thalamic nuclei. The binding of [³H]CB-119 was fully blocked by the addition of 10 μM CB-119. Rhesus positron emission tomography imaging studies showed very good brain uptake and a distribution pattern consistent with that seen in the autoradiographic studies. The kinetics of tracer uptake was slow. The brain uptake was blocked by pretreatment with taranabant, a CB1R inverse agonist. The specific signal (total/nonspecific) in rhesus putamen at 90 min was ~6:1. Conclusions  [¹¹C]CB-119 is a suitable tracer for imaging central CB1 receptors.     

Taurocholate pool size and distribution in the fetal rat.

Taurocholate concentrations in fetal and neonatal rats were determined by radioimmunoassay. Total body taurocholate pool size varied from 0.0049 +/- 0.0008 to 203 +/- 8 nmol/g body weight from day 5 of gestation to 5 d after birth. A 50-fold increase in taurocholate pool size was observed between days 15 and 19 of gestation. The distribution of taurocholate between liver, intestine, and the remainder of the carcass was determined for rats of gestational age 19 d to 5 d after birth. The major fraction of total body taurocholate was in the liver and intestine, with less than 15% in the remainder of the carcass. The ratio of taurocholate in intestine to taurocholate in liver, which was 1:17 at 19 d of gestation, had altered substantially to a ratio of 6:1 by 5 d after birth. Treatment of pregnant rats with 60 microgram/d of dexamethasone from gestational day 9 until sacrifice increased fetal taurocholate pool size by 80% at 15 d, 40% at 19 d, and 16% at 1 d after birth. Administration of dexamethasone to the mother also changed the ratio of taurocholate in intestine to taurocholate in liver. At 19 d of gestation, dexamethasone-treated mothers had fetuses with approximately equal amounts of taurocholate in intestine and liver. This suggested that adrenocorticosteroids stimulate the early maturation of factors controlling taurocholate pool size and tissue distribution in the rat fetus.     

Role of Sodium Taurocholate Cotransporting Polypeptide as a New Reporter and Drug-Screening Platform: Implications for Preventing Hepatitis B Virus Infections

Molecular Imaging and Biology - Sodium taurocholate cotransporting polypeptide (NTCP) is a transmembrane protein responsible for delivering indocyanine green (ICG), an ideal infrared fluorescent...     

Pharmacokinetics of Cefovecin in Squirrel Monkey (Saimiri sciureus), Rhesus Macaques (Macaca mulatta), and Cynomolgus Macaques (Macaca fascicularis)

Cefovecin sodium is a third-generation broad-spectrum cephalosporin antibiotic licensed for the treatment of skin infections in cats and dogs. The objective of our study was to assess whether its pharmacokinetic profile in squirrel monkey, rhesus macaques, and cynomolgus macaques was similar to that of dogs. Plasma levels were determined by using protein precipitation followed by liquid chromatography tandem mass spectrometry. After subcutaneous dosing at 8 mg/kg, the plasma terminal half-life of cefovecin was substantially shorter in the nonhuman primates (2.6 to 8.0 h) than in dogs (102 h). The total plasma exposure (AUC_0-96h) was 10- to 40-fold lower in nonhuman primate species. In cynomolgus macaques, cefovecin showed a similar subcutaneous bioavailability (82% compared with 100%) and volume of distribution (0.16 compared with 0.12 L/kg) as compared to dogs; however, the plasma clearance of cefovecin was 20-fold higher. Cefovecin susceptibility testing and minimum inhibitory concentrations were not established for clinical isolates in nonhuman primates. However, if the minimum inhibitory concentrations of cefovecin for various nonhuman primates pathogens are in the same range as those observed for canine pathogens, our results suggest that cefovecin used at the same dosing regimen and frequency prescribed for the dogs will be ineffective and that increases in dose or frequency (or both) may be required.Abbreviation: AUC, area under the plasma concentration–time curveCefovecin sodium (CAS no. 141195-77-9) is a recently approved, third-generation cephalosporin antibiotic licensed for use in dogs and cats in Europe and North America.⁷ It has a broad-spectrum of antimicrobial activity against gram-positive and gram-negative bacteria and is active against clinically relevant canine and feline pathogens associated with skin, soft tissue, and urinary tract infections.¹¹ Cefovecin is administered as a single subcutaneous injection at 8 mg/kg once every 14 d.¹¹ Published canine data indicate that the drug is fully bioavailable after subcutaneous administration, with a mean maximum concentration of 121 µg/mL, a mean elimination half-life of 133 h, and a clearance of 0.76 mL/h/kg after intravenous dosing.¹² Cefovecin is highly bound to plasma proteins (96.0% to 98.7%).¹² In dogs, it is not metabolized by the liver; the majority of the dose is eliminated in the urine, with some fraction excreted as unchanged drug in the bile.⁷Cephalosporins are used widely as antibiotics and have well-established safety and efficacy profiles. The use of an injectable antibiotic with an extended duration of action would greatly benefit zoo and laboratory animal veterinarians working with nonhuman primates, because it would reduce the frequency of capture and restraint of animals for treatment, decrease stress to animals, and lessen the risk of injuries of animal handlers. Current antibiotic therapies available for nonhuman primates have impractical dosing regimens and can be difficult to administer. These challenges may contribute to treatment failure and potential development of drug resistance.¹ A review of listservs and online correspondence (for example, Compmed, Industry Veterinarians) suggested the off-label use of cefovecin by primate veterinarians. Therefore, to assess the potential use of cefovecin in nonhuman primates, we determined its pharmacokinetic parameters in squirrel monkeys, rhesus macaques, and cynomolgus macaques and compared them with those in dogs. Investigation of antibiotic efficacy and the safety profile of cefovecin in nonhuman primates was outside the scope of this evaluation.     

Hepatic Storage and Biliary Transport Maximum of Taurocholate and Taurochenodeoxycholate in the Dog

The purpose of this work was to validate for taurocholate and taurochenodeoxycholate the multiple infusion method of Wheeler et al. previously used for the study of hepatic sulphobromophthalein transport and to obtain numerical estimates of the relative storage capacity and secretory transport maximum of both bile acids. Experiments were performed in anaesthetized dogs after depletion of the endogenous bile acid pool. Bile was collected continuously to prevent recirculation of bile acids and to measure their secretion rates. Taurocholate or taurochenodeoxycholate were infused intravenously at 3 different rates and blood samples were collected every ten min to measure serum bile acid concentrations. Extrahepatic distribution spaces of taurocholate and taurochenodeoxycholate were measured by an isotope dilution method. Serum bile acid concentrations varied linearly with time during the last 30 min of each infusion period. A linear relationship was found between the calculated hepatic removal rate and the rate of change of serum bile acid concentration. The mean values of relative storage capacity were (in μmol. μmol^-1 1^-1. kg body weight^-1) 0.16 ± SD 0.07 for taurocholate and 0.08 ± SD 0.03 for taurochenodeoxycholate. The mean values for secretory transport maximum were (in μmo 1.min^-1. kg body weight^-1) 4.81 ± SD 1.24 for taurocholate and 3.56 ± SD 0.60 for taurochenodeoxycholate. The values of secretory transport maximum with the multiple infusion method were only slightly higher than those obtained by direct measurement of biliary secretion. The values of relative storage capacity obtained during infusions resulting in decreasing plasma concentrations were usually lower than those obtained when the plasma concentration increased. This suggests that the limitations of the method previously noted for sulphobromophthalein may apply to bile acids.     

16例下泪小管断裂吻合术的治疗体会

16例下泪小管断裂均于手术显微镜下 ,直视注水、注气或经上泪点注入稀释美蓝寻找出泪小管鼻侧断端 ,以 1mm宽的硬膜外导管为支撑物 ,再用 8 0可吸收缝线间断缝合两侧断端泪小管壁及周围组织 3针。术后每周冲洗泪道 1次 ,术后 3个月拔管。结果 16例患者随访 6～ 12个月 ,全部治愈。应用硬膜外导管作支撑物 ,在显微镜下行泪小管吻合术 ,操作简单 ,取材方便 ,疗效满意。     

Studies of Relationships among Bile Flow, Liver Plasma Membrane NaK-ATPase, and Membrane Microviscosity in the Rat

Liver plasma membrane (LPM) NaK-ATPase activity, LPM fluidity, and bile acid-independent flow (BAIF) were studied in rats pretreated with one of five experimental agents. Compared with controls, BAIF was increased 24.6% by thyroid hormone and 34.4% by phenobarbital, decreased by ethinyl estradiol, but unchanged by propylene glycol and cortisone acetate. Parallel to the observed changes in BAIF, NaK-ATPase activity also was increased by thyroid hormone (40.8%) and decreased by ethinyl estradiol (26.2%). In contrast, NaK-ATPase activity failed to increase after phenobarbital but did increase 36% after propylene glycol and 34.8% after cortisone acetate. Thus BAIF and NaK-ATPase activity did not always change in parallel. The NaK-ATPase K(m) for ATP was not affected by any of these agents.LPM fluidity, measured by fluorescence polarization using the probe 1,6-diphenyl-1,3,5-hexatriene, was found to be increased by propylene glycol, thyroid hormone, and cortisone acetate, decreased by ethinyl estradiol, and unaffected by phenobarbital. Thus in these cases, induced changes in LPM fluidity paralleled those in NaK-ATPase activity. In no case did Mg-ATPase or 5'-nucleotidase activities change in the same direction as NaK-ATPase, and the activity of neither of these enzymes correlated with LPM fluidity, thus indicating the selective nature of the changes in LPM enzyme activity caused by the agents.These findings indicate that LPM fluidity correlates with NaK-ATPase activity and may influence the activity of this enzyme. However, the nature of the role of LPM NaK-ATPase in bile secretion is uncertain and needs further study.     

Sodium-coupled Taurocholate Transport in the Proximal Convolution of the Rat Kidney In Vivo and In Vitro

Using the standing droplet technique in the renal proximal convolution and simultaneous microperfusion of the peritubular capillaries, the zero net flux transtubular concentration difference of taurocholate (ΔC_TC⁻) at 45 s was determined as a measure of active bile acid reabsorption in vivo. Starting with 0.1 mmol/liter taurocholate in both perfusates the control ΔC_TC⁻ of 0.042 mmol/liter fell to 0.006 mmol/liter (P < 0.001) when the Na⁺ concentration in the perfusates was reduced to zero. Removal of bicarbonate from the perfusates to alter pH had no influence on ΔC_TC⁻. When glycocholate was added to the perfusates ΔC_TC⁻ was decreased, while probenecid increased ΔC_TC⁻.