Drug binding sites on chicken albumin: a comparison to human albumin.

Mammalian albumins have two main structurally selective ligand binding sites. Site I binds drugs such as azapropazone, phenylbutazone and warfarin; whereas benzodiazepines, some dansyl amino acids, such as dansylsarcosine, and short chain fatty acids like octanoic acid interact with site II. However, it is not known if non-mammalian albumins have similar binding loci. In this study, drug binding sites on chicken albumin were investigated using site selective fluorescent probes (warfarin and dansylsarcosine) and p-nitrophenyl acetate (NPA); the hydrolysis of which is selectively inhibited by site II ligands. Azapropazone and phenylbutazone decreased the binding of warfarin and dansylsarcosine to a similar extent. Diazepam and octanoic acid also inhibited binding of the two fluorescent probes in a non-selective manner. However, the fluorescence intensity of the warfarin-chicken albumin complex decreased when the pH was increased from 6.0-9.0; but by contrast, the fluorescence of bound dansylsarcosine remained unchanged. Furthermore, the hydrolysis of NPA was selectively inhibited by dansylsarcosine, diazepam and octanoic acid (ligands selective for site II on mammalian albumins), but not by site I selective ligands such as azapropazone and warfarin. Overall, the results suggest that chicken albumin, like mammalian albumins, has discrete binding sites for warfarin and dansylsarcosine.

Binding of sulphamethazine to pig plasma proteins and albumin.

The interaction of sulphamethazine (SMZ) with pig plasma proteins and albumin was studied by ultrafiltration and equilibrium dialysis. Binding to pig plasma proteins was monophasic (affinity approximately 9.0 mol/L x 10(3)) and the main binding protein was albumin. At 37 degrees C and pH 7.4, the affinity of SMZ for albumin was about 8.0 mol/L x 10(3) and the number of binding sites was estimated as 1.4. Increasing the temperature from 4 to 45 degrees C resulted in a seven-fold decrease in affinity, and increasing pH from 6.0 to 8.0 enhanced affinity for pig albumin ten-fold. The free energy of binding (-delta G) and enthalpy change (-delta H) were around 5.5 and 5.1 Kcal/mol, respectively. The total entropy change (delta S) was small and positive, around 2 cal/mol/degree K. Studies with the fluorescent probes warfarin and dansylsarcosine, suggest that these bind to separate sites on porcine albumin. SMZ displaced both probes and inhibited the deacetylation of p-nitrophenyl acetate by pig albumin. We conclude that: (1) binding of SMZ to pig plasma proteins and albumin is weak; (2) the interaction with albumin is exothermic and enthalpy driven, and (3) pig albumin, like other mammalian albumins, appears to possess discrete binding sites for warfarin and dansylsarcosine. SMZ interacts with both these loci.

A cluster of cases of feline dysautonomia (Key-Gaskell syndrome) in a closed colony of cats.

Twenty-five cases of feline dysautonomia (Key-Gaskell syndrome) occurred in a closed cat colony over a period of three weeks. The clinical and pathological signs were sufficiently similar to those reported during the 1982-1986 outbreak to establish a positive diagnosis. The special epidemiological and environmental circumstances of the outbreak provide a new insight into the cause(s) of the syndrome.

Preliminary evidence for the enterohepatic circulation of progesterone in the pig.

In two gilts, following doses of either 47.7 or 64.6 mumol progesterone, a secondary increase in steroid concentration in portal and mesenteric blood plasma occurred 35-40 min after dosing or 10-15 min after an intravenous (i.v.) dose of cholecystokinin. It is suggested that this increase is splanchnic blood progesterone concentration indicates that the steroid undergoes enterohepatic circulation. In five ovariectomized gilts given single i.v. doses of 47.7 mumol progesterone, the steroid was cleared from the circulating plasma bi-exponentially; the mean half-lives (t 1/2) of the two components were 2.47 (+/- 0.36) and 33.5 (+/- 6.63) min. The t 1/2 of the first component was compatible with the liver being a principal organ of clearance.

Responses in the voluntary intake of hay or silage by lactating cows to intraruminal infusions of sodium acetate or sodium propionate, the tonicity of rumen fluid or rumen distension.

Rumen-fistulated lactating cows were individually fed on hay or silage and intakes were monitored during 3 h treatment periods and for 2 h after. Each experiment used five, six or seven animals and the treatments were applied in a Latin Square design. Sodium acetate infusions of 1.8-11.0 mol in 4.5 litres water caused a dose-related depression in hay intake, the extent being 82 g dry matter (DM)/mol infused (P < 0.01). Sodium acetate infusions of 6.0-15.0 mol in 4.5 litres water caused a dose-related depression in silage intake of 118 g DM/mol infused. Rumen fluid pH for both diets was unaffected by treatment. Acetate and Na concentrations were increased and significantly negatively correlated with intake of both diets. Infusions of 2-8 mol sodium propionate caused a dose-related depression of hay intake which was significant when cow and day effects were accounted for. Sodium propionate infusions of 4-8 mol significantly depressed silage intake by 140 g DM/mol infused (P < 0.001). Rumen fluid pH was unaffected by treatment while propionate and Na concentrations were elevated and significantly negatively correlated with intake for both diets. Inflation of a rubber balloon in the rumen with 12.5-20 litres warm water resulted in a dose-dependent depression in hay intake of 66 g DM/l distension (P < 0.05). There was significant overeating during the 2 h following the 20 litre treatment. With silage, 15-25 litres of balloon distension for 3 h resulted in a dose-dependent depression in intake of 28 g DM/l distension (P < 0.001). There was no significant overeating during the 2 h following distension. When given in physiological amounts, at the lower end of the range used in these experiments, acetate, propionate and distension of the rumen did not significantly affect hay intakes. However, in each case the linear relationship between intake depression and level of treatment suggested that these factors could contribute to the control of feed intake.

The effect of copper and heliotrope on the composition of bile in sheep.

The effects of interrupting the enterohepatic circulation (EHC) of bile salts for seven hours and of feeding copper and heliotrope alone and combined for 13 weeks, on bile flow and excretion of copper, zinc, iron and alpha-mannosidase were studied in sheep. Interruption of EHC reduced bile flow rate and increased the concentration of copper, zinc, iron and bile acids while alpha-mannosidase's activity remained stable. Changes in concentration were related to changes in bile volume for copper and zinc only. Total output per hour was not changed. Biliary concentration of copper correlated with alpha-mannosidase's activity in control sheep and those given copper or heliotrope, supporting the hypothesis that lysosomes are involved in biliary secretion of copper in sheep. Increasing the intake of copper increased the rate of excretion of copper in bile. Copper output was lower when heliotrope was fed alone.

A surgical technique for the long term collection of bile in the pig and measurement of the biliary excretion of copper and zinc.

A method is described for surgically modifying the gastrointestinal tract of the pig which enables the long term collection of bile and measurement of bile flow and allows the pig to be kept unrestrained in a large pen. Three surgically prepared gilts grew at an average of 0.79 kg d-1. Bile flow increased with bodyweight from 1.6 ml min-1 at 45 to 50 kg to 3.5 ml min-1 at 124.5 kg bodyweight. Mean output in bile of copper and zinc by the three gilts during the period 08.00 to 12.30 was 161.0 +/- 11.1 and 3.94 +/- 0.41 nmol min-1, respectively. The calculated output during 24 hours represented approximately 5.8 and 0.13 per cent of the daily intake of copper and zinc, respectively.

Enhancement of tetrathiomolybdate-induced biliary copper excretion in sheep by general anaesthesia and the effect on copper excretion in urine and bile.

To determine whether copper removed from the liver by tetrathiomolybdate (TTM) but not excreted in bile, is excreted in saliva or urine, the effect of an intravenous injection of 100 mg of TTM, as ammonium tetrathiomolybdate, on copper excretion by these routes was measured in conscious sheep and sheep maintained under general anaesthesia. During 4.5 hours the total amount of copper excreted in bile in the untreated, conscious animal was 0.52 +/- 0.04 mumols (mean +/- SE). A dose of 100 mg TTM increased excretion by an average of 11.7-fold to 6.06 +/- 0.29 mumols. Under halothane or barbiturate general anaesthesia copper excretion was increased 3.4-fold to 1.78 +/- 0.30 mumols. In anaesthetised sheep 100 mg TTM produced a 40.7-fold increase to 21.18 +/- 0.95 mumols. Neither anaesthesia nor TTM caused any increase in copper excretion in saliva or urine.

Enhancement of tetrathiomolybdate-induced copper excretion in bile of sheep by the alpha 2-agonistic action of xylazine.

The effect of intravenous doses of cortisol and xylazine on the quantity of copper excreted in response to 100 and 200 mg doses of tetrathiomolybdate (TTM) was studied in seven sheep. Cortisol alone produced a non-significant 1.4-fold increase and had no enhancing action on the response to TTM. Xylazine produced a significant 2.25-fold increase, doubled the quantity of copper excreted in response to both doses of TTM and reduced bile flow by approximately 35 per cent. The alpha 2 antagonist, idazoxan, prevented both the latter effects showing that they were due to xylazine's alpha 2-agonistic action. It is suggested that the combination of an alpha 2-agonist with the intravenous injection of TTM in the treatment of acute copper toxicity in sheep could reduced by 50 per cent the amount of molybdenum needed.

Effect on feed intake of infusing sodium propionate or sodium acetate into a mesenteric vein of cattle.

Solutions containing sodium propionate or sodium acetate were infused into a mesenteric vein of eight steers in order to examine the effect of increasing the entry rate of these metabolites on feed intake. Infusion of propionate inhibited feed intake to varying degrees, but acetate infused at equivalent rates had no effect. Rate of entry of propionate into the visceral circulation may be a physiological mechanism for controlling feed intake in cattle, but it is mainly effective when the animal has eaten close to its voluntary maximum intake.

A novel form of dependency of hepatic extraction ratio of opioids in vivo upon the portal vein concentration of drug: comparison of morphine, diamorphine, fentanyl, methadone and buprenorphine in the chronically cannulated cow.

In the chronically cannulated cow, the hepatic extraction ratio for intravenous boluses of morphine, diamorphine, fentanyl, methadone and buprenorphine increased towards a plateau value as portal vein drug concentration increased. An extraction ratio close to zero for morphine was observed at a portal vein plasma drug concentration of about 200 nanomol per litre, which is within the range for significant pharmacodynamic effects. The similar concentrations extrapolated for the other narcotics would be of less pharmacodynamic importance. The phenomenon did not depend with morphine on the history of drug delivery to the liver; measurement of hepatic blood flow showed the effect was not an artifact of unrepresentative blood sampling, and was not related to any action of the narcotics on hepatic blood flow. The existence of this novel type of concentration dependent hepatic extraction ratio in vivo can explain a number of anomalous observations on narcotic pharmacokinetics, especially for morphine. Furthermore, similar behaviour may be expected for non-opioid drugs having similar pharmacokinetic properties.

Bile flow, bile salt secretion and the excretion of iron, copper, zinc and manganese in the bile of calves infected with Fasciola hepatica.

The rate of flow of bile and the concentrations of iron, copper, zinc and manganese in bile were measured in four bull calves, before and for at least 23 weeks after infection with 1000 metacercariae of Fasciola hepatica. Bile flow rate began to increase about 10 weeks after infection and had increased nearly three-fold by 23 weeks but bile salt (total-cholate) secretion rate remained similar to that of two control calves. The excretion of iron in bile increased rapidly eight weeks after infection from a mean of 21.5 nmol per minute to reach 469 nmol per minute at 14 weeks and this increase was accompanied by an increase in gamma-glutamyl transpeptidase activity in plasma. 59Fe studies showed that most of the iron in the bile of infected calves was derived from red blood cells. There were no changes in the excretion of zinc, copper or manganese in bile which could be associated with the infection.

Acute manganese toxicity and the absorption and biliary excretion of manganese in cattle.

The concentration of manganese in the duodenum of three steers was increased by infusing manganese chloride intraduodenally at approximately 100, 515 and 840 mumol per minute for 30 hours and the rate at which manganese was excreted in bile was measured. In one steer an infusion of manganese chloride at 529 mumol per minute was toxic and in two steers infusions at approximately 840 mumol per minute were also toxic, causing inappetence, a reduced bile flow and abdominal discomfort. The maximum rates at which manganese was excreted in bile during the first three hours of the infusions at 515 and 840 mumol per minute were between 2.1 and 3.6 per cent of the rate of infusion. During the last three hours of all nontoxic infusions the rate of excretion of manganese in bile was 0.82 +/- 0.08 per cent of its rate of infusion. Systemic plasma manganese concentrations did not increase during these infusions. In a fourth steer the longer term capacity of the liver to excrete manganese in bile when the manganese chloride was given intravenously was measured, the concentration of manganese in the portal vein being increased by infusions of manganese chloride solution into a mesenteric vein. The rate of infusion was increased at hourly intervals. The liver's capacity to remove all manganese during first pass was only exceeded at an infusion rate of 84 mumol per minute. Infusions could be made into the mesenteric vein at 24 and 35.8 mumol per minute for 53 and 26 hours respectively before signs of toxicity were observed. Following signs of toxicity the excretion of copper in bile was decreased for two days.

Maximum capacity of the bovine liver to remove manganese from portal plasma and the effect of the route of entry of manganese on its rate of removal.

The rate of uptake of manganese by the liver was measured during infusions of manganese into either the visceral or the systemic circulation of four cows surgically prepared with cannulae in a mesenteric vein, the portal vein and a hepatic vein and in one carotid artery. In three experiments the maximum capacity of the liver to remove manganese from plasma was measured by infusing manganese chloride solution into a mesenteric vein at increasing rates for six hours. In two experiments the rate of uptake of manganese by the liver was determined during infusions of manganese chloride solution into a jugular vein for six hours at a constant rate. Virtually all the manganese infused into a mesenteric vein was cleared during its first pass through the liver, up to a maximum rate (mean +/- SEM) of 97.1 +/- 14.1 mumol per minute or 12.7 +/- 2.3 mumol per minute per kg of liver weight. However, when manganese was infused into a jugular vein at rates less than this maximum rate of uptake only approximately 50 per cent of the manganese in portal plasma was removed during a single passage through the liver.

The choleretic effect of menbutone and clanobutin sodium in steers.

Adult steers were given either clanobutin or menbutone intravenously and the effects on bile flow measured. Doses of 4.3 g of clanobutin or 3.0 g of menbutone had no effect on bile flow. However, when bile flow was previously reduced by reducing the total bile salts in the enterohepatic circulation both compounds were potent choleretics, increasing the volume of bile flow up to four-fold.

Surgical procedure for modifying the duodenum in cattle to measure bile flow and the diurnal variation in biliary manganese, iron, copper and zinc excretion.

A surgical procedure is described for modifying the duodenum of cattle so that bile can be sampled and its rate of flow measured for long periods. In 12 steers, aged three months to three years and weighing between 50 and 650 kg, bile flow ranged from 2 to 12 ml per minute, the rate of flow increasing with bodyweight. The rate of flow expressed as ml per minute per 100 kg bodyweight decreased as bodyweight increased, the regression equation being in (ml per minute per 10 kg) = 1.65-0.0022 x where x = bodyweight (kg), r = 0.871. The mean concentrations of copper, iron, manganese and zinc in bile were 8.2, 6.2, 17.1 and 3.3 mumol per litre respectively. The concentration of iron was the least variable between the steers. The average total cholate concentration was 100 mmol per litre and total solids ranged from 5.4 to 7.2 per cent. Diurnal patterns in bile flow and trace element excretion were measured in four adult steers during a period of 38.5 hours. Although the rate of excretion of iron, zinc and copper and bile flow varied throughout the period, changes could not be associated with feeding or time of day. The concentration of manganese in bile and its excretion rate varied in a reproducible manner which may be related to feeding activity. The mean output of the four trace elements in bile from the four steers during 24 hours was, copper 37.7 mumol, iron 68.0 mumol, manganese 80.3 mumol and zinc 59.6 mumol.

The maximum capacity of the liver of the adult dairy cow to metabolize ammonia.

1. Three adult dairy cows were fitted with cannulas in a mesenteric, portal, hepatic and jugular vein and a carotid artery. They received infusions of step-wise increasing amounts of ammonia as ammonium acetate via a mesenteric vein until NH 3 intoxication occurred. Sodium acetate was used in control infusions. The maximum rate of uptake of NH3 by the liver and the concentrations of glucose, urea, lactate, acetate and bilirubin in blood were measured. 2. During the infusions of ammonium acetate the liver extracted almost all the NH3 present in the portal vein until an infusion rate of approximately 15.0 mmol/min was reached. The maximum capacity of the liver to remove NH3 during its first pass was on average 1.84 mmol/min per kg wet weight. The cows became intoxicated when arterial plasma ammonia concentrations reached 0.8 mmol/l. Concentrations of NH3 in jugular venous blood were between 66 and 74% of those in the carotid.

The excretion of selenium in bile and urine of steers: the influence of form and amount of Se salt.

1. The excretion of 75Se and stable Se in bile and urine was measured in four steers during 6 h after intravenous injections of 75Se as either selenite or selenate containing either 5 or 5000 microgram carrier Se. 2. When 5000 microgram Se were given, the rate of urinary excretion and plasma clearance of 75SE was similar for both salts. Approximately 23% was excreted in urine and plasma clearance was triexponential, the mean half-life (t 1/2) of the successive components, alpha, beta, and gamma, being 2.3, 15-2 and 465 min respectively. The amount of 75Se excreted in bile was small; 1.94% of the 75SeO3(2-) and 0.86% of the 75SeO4(2-) dose. 3. When 5 microgram Se were given the plasma clearance of 75Se was initially biexponential but the entry of 75Se-labelled protein from the liver caused an increase in plasma radioactivity after 30-40 min. The effect was most marked after 5 microgram 75SeO3(2-) when plasma 75Se radioactivity returned to 69% of the activity present at 2 min. Values for t 1/2 of the two components of clearance for 75SEO4(2-) were respectively alpha 2.6 and 2.5 min, and beta 15.9 and 36.6 min. Similar amounts of 75Se appeared in bile (0.2% of the dose) after injection of either salt but much less 75Se was excreted in urine after 75SeO3(2-) (6%) than after 75SeO4(2-) (17%). 4. At low dosage rates (5 microgram) Se is more readily incorporated into tissues from SeO3(2-) than from SeO4(2-).