Radioimmunoassay of the anabolic agent zeranol. V. Residues of zeranol in the edible tissues, urine, faeces and bile of steers treated with Ralgro.

Two trials were conducted on steers implanted with zeranol (Ralgro) to determine the edible tissue residues and the secretion pattern in faeces, urine and bile of zeranol residues throughout and beyond the recommended withdrawal period (70 days) for this drug. In the first trial there was considerable variation in the zeranol residue concentration in all edible tissues, the highest concentrations found in the liver being significantly above the control values (P less than 0.05). In the other tissues, only fat sampled 14 days after implanting was significantly above the control value (P less than 0.05). The zeranol concentration in bile samples obtained at slaughter [70 days (18), 90 days (5) and 120 days (2)] were all higher than the apparent concentration in the bile of untreated steers. The mean concentration of zeranol in the faeces and urine varied from day to day and between animals sampled on the same day following implantation. The highest mean concentrations were observed during the first 40 days following implanting, declining steadily to approach the control values 70 days after implantation. The second trial using steers prepared with bile duct re-entrant cannulae resulted in a similar pattern of zeranol excretion in bile, faeces and urine. The highest concentrations of zeranol were observed in bile and ranged from 24 to 34 micrograms/l; there was considerable variation between animals and within animals sampled on successive days. Although the concentration declined steadily, zeranol was still readily detectable 120 days after implanting.(ABSTRACT TRUNCATED AT 250 WORDS)

An improved radioimmunoassay for the anabolic agent hexoestrol, using a monoclonal antibody.

A monoclonal antibody was raised against hexoestrol coupled to bovine serum albumin. The antibody cross-reacted with the stilbenes, diethylstilboestrol (10%) and dienoestrol (4%), but had no cross-reaction (less than 0.01%) with other anabolic agents. A radioimmunoassay method using the monoclonal antibody has been validated and used to measure residues of hexoestrol in the urine of treated cattle. The limit of detection was 0.6 pg/ml urine at the 95% confidence limit. The results were compared with those obtained using polyclonal antibodies. Although there was a good correlation between the results, the use of monoclonal antibody gave more reliable results than those obtained with available polyclonal antibodies. The monoclonal antibody, because of its quality and theoretically limitless supply, is very suitable for use in large scale screening or monitoring programmes for regulating the use of hexoestrol.

The absorption of anabolic agents from pellets implanted at the base of the ear in sheep.

Twenty wether sheep were allocated to seven groups and received implants near the base of one ear with pellets containing: for group 1, (OE) 20 mg oestradiol-17 beta alone; for group 2, (TBA/OE) 20 mg oestradiol-17 beta intimately mixed with 140 mg trenbolone acetate; for group 3, (T/OE) 20 mg oestradiol-17 beta mixed with 200 mg testosterone; for group 4, (P/OE) 20 mg oestradiol-17 beta mixed with 200 mg progesterone; for group 5, (TBA/OE2) 20 mg oestradiol-17 beta in one ear and 140 mg trenbolone in the other ear; for group 6, (TBA/OE1) 20 mg oestradiol-17 beta and 140 mg trenbolone as separate pellets in one ear; group 7 sheep received implants of carrier material and served as controls. The concentrations of steroids were measured in plasma samples collected from both jugular veins during the 16-week period after implantation. The absorption of oestradiol-17 beta was slower and more sustained from the pellets in which it was mixed with other steroids (groups 2, 3 and 4) than from the pellets containing oestradiol-17 beta alone (groups 1, 5 and 6). The concentration of each steroid in plasma was higher in the jugular vein ipsilateral to the implant than in the vein on the opposite side. The difference between the concentrations in the two veins was used to calculate the biological half-lives of the steroids; for oestradiol-17 beta and trenbolone the mean values ranged from 1.8 to 6.8 min and from 3 to 4 min, respectively, and for testosterone and progesterone the mean values were 4.7 and 3.5 min, respectively.

The absorption, distribution and excretion of anabolic agents.

The metabolic fate of the anabolic agents, diethylstilbestrol, hexestrol, trenbolone acetate, zeranol and the endogenous steroids are discussed under the headings absorption, distribution and excretion. There is an optimum concentration of anabolic agent in the systemic circulation that results in a maximum increase in growth rate of farm animals. This optimum blood concentration should be maintained over a long period. However, there is rapid metabolism and excretion of anabolic agents with short half-lives in blood, and metabolic clearance rate equals entry rate. The rate of absorption of the agent, which is determined by formulation and site of administration, is most important and is best achieved by the use of slow release implants. The pattern of exponential absorption from compressed pellets of single anabolic agents is not ideal, and a more constant payout of drug, in particular estradiol-17 beta, is best achieved in combined preparations of agents or from silicone rubber implants impregnated with the agent. The high metabolic clearance rate and rapid excretion of anabolic substances influences the distribution of residues. Outside the site of administration, less than 1% of the administered dose is present in the animal. The lowest concentrations of residues are found in muscle and fat, higher concentrations are present in liver and kidney and the highest concentrations are in the bile, urine and feces.

Radioimmunoassay of hexoestrol residues in faeces, tissues and body fluids of bulls and steers.

A fast, reliable and sensitive radioimmunoassay method using either 3H-hexoestrol or a 125I-hexoestrol derivative as the competitive radioligand has been developed for the measurement of residues of hexoestrol in faeces. The blank value for untreated cattle was 113 +/- 98 pg/g faeces, the intra-assay coefficient of variation 10.8 per cent, interassay coefficient of variation 11.1 per cent and recovery of 3H-hexoestrol added to faeces was 58.7 per cent. The concentration of hexoestrol in faeces was measured in 18 steers and 14 bulls at regular intervals after implantation of 36, 45 and 60 mg of hexoestrol. Residues were detected in all samples up to 104 days after implantation and in nine out of 13 samples taken 111 to 153 days after implantation. Residues of hexoestrol were also measured in edible tissues and body fluids of 14 bulls and eight steers slaughtered between 90 and 153 days after implantation of 45 mg hexoestrol. The percentages of samples containing residues were 82, 73, 64, 82 and 27 per cent for bile, urine, liver, kidney and muscle, respectively.

Differences in the biotransformation of a 17 beta-hydroxylated steroid, trenbolone acetate, in rat and cow.

1. The metabolism of trenbolone acetate, 17 beta-acetoxyestra-4,9,11-trien-3-one (TBA), an anabolic compound used as a growth promoter, was compared in rat and cow. 2. [6,7-3H] TBA was injected i.v. into rats and a heifer, and bile was collected for 24 h. In both species, the bile was the major route of excretion. TBA undergoes an extensive hydrolysis to 17 beta-hydroxyestra-4,9,11-trien-3-one and the unchanged compound was not detected, but subsequent major metabolic pathways are different in the two species. 3. In the rat, oxidation of the 17 beta-hydroxyl to the 17-oxo group and hydroxylation in the 16 alpha-position are the major routes. The three major metabolites are 17 beta-hydroxyestra-4,9,11-trien-3-one, 16 alpha, 17 beta-dihydroxyestra-4,9,11-trien-3-one and 16 alpha-hydroxyestra-4,9,11-trien-3, 17-dione. 4. In the heifer, 17 alpha-epimerization is the major pathway and the main metabolite is the 17 alpha-hydroxyestra-4,9,11-trien-3-one. 5. In both species, estra-4,9,11-trien-3,17-dione and the other metabolites, resulting either from hydroxylation in 1, 2, 6 beta, 16 alpha or 16 beta positions, or from aromatization of the A ring, were minor products. 6. Overall, 60% of the 3-oxotriene structures identified in the rat bile were 17 beta-hydroxylated and the remainder were 17-keto metabolites, whereas in the heifer bile 90% were 17 alpha-hydroxylated compounds. 7. Thus, in bovine species, the major pathway is similar to those of testosterone or 17 beta-estradiol which are mainly excreted as their 17 alpha-epimers. The epimerization strongly decreases the biological potency, as with the natural 17 beta-hormones, and leads to detoxication of tissue residues.

Growth hormone, insulin, prolactin and total thyroxine in the plasma of sheep implanted with the anabolic steroid trenbolone acetate alone or with oestradiol.

The mode of action of the anabolic steroid trenbolone acetate (19-norandrost-4,9,11-trien-3-one-17-acetate) was studied through the endogenous hormonal response of castrated male sheep to subcutaneous implantation of 140 mg of trenbolone acetate and 20 mg of oestradiol both separately and in combination. Radioimmunoassay of delta-4,9,11-trienic steroids and oestradiol-17 beta in plasma confirmed that simultaneous administration of trenbolone acetate with oestradiol led to a significantly greater persistence of oestradiol-17 beta residues in plasma (P less than 0.05) than with implantation of oestradiol alone. Oestradiol treatment increased concentrations of growth hormone and insulin (P less than 0.05; P less than 0.001 respectively) in plasma samples collected weekly. Trenbolone acetate by itself had no significant effect and the oestrogenic response was blocked on the simultaneous implantation of trenbolone acetate and oestradiol (despite higher plasma levels of oestradiol-17 beta with this treatment). Plasma total thyroxine was markedly depressed to 45 per cent of its basal level by trenbolone acetate, alone or with oestradiol (P less than 0.001) and depressed to 80 per cent of basal by oestradiol treatment alone (P less than 0.001). Plasma prolactin was unaltered by the above treatments.

Plasma concentrations of ovarian hormones during the oestrous cycle of the sheep and cow.

Measurements were made of the plasma concentrations of testosterone, progesterone and oestradiol-17 beta during the oestrous cycle of the ewe, and of the plasma concentrations of testosterone and progesterone during the oestrous cycle of the heifer. The maximum concentration of progesterone in the heifer was nearly twice that in the ewe. The maximum concentration of testosterone was similar in both species and occurred at the onset of luteolysis; in the ewe the peak concentration of 19.5 ng/ml plasma was significantly greater (P less than 0.05) than the concentrations of testosterone measured during the remainder of the oestrous cycle.

Effects of food deprivation on ketonaemia, ketogenesis and hepatic intermediary metabolism in the non-lactating dairy cow.

1. The aim of this work was to investigate why non-lactating dairy cows are less susceptible to the development of ketonaemia during food deprivation than are dairy cows in early lactation. 2. The first experiment (Expt. A) consisted of determining the effect of 6 days of food deprivation on the concentrations of ketone bodies, and of metabolites related to the regulation of ketogenesis, in jugular blood and liver of non-lactating cows. 3. During the food deprivation, blood ketone-body concentrations rose significantly, but to a value that was only 16% of that achieved in lactating cows deprived of food for 6 days [Baird, Heitzman & Hibbitt (1972) Biochem. J. 128, 1311--1318]. 4. In the liver, food deprivation caused: a rise in ketone-body concentrations; a fall in the concentration of glycogen and of various intermediates of the Embden-Meyerhof pathway and the tricarboxylic acid cycle; an increase in cytoplasmic reduction; a decrease in the [total NAD+]/[total NADH] ratio; a decrease in energy charge. These changes were all qualitatively similar to those previously observed in the livers of the food-deprived lactating cows. 5. There appeared therefore to be a discrepancy in the food-deprived non-lactating cows between the absence of marked ketonaemia and the occurrence of metabolic changes within the liver suggesting increased hepatic ketogenesis. This discrepancy was partially resolved in Expt. B by the observation in two catheterized non-lactating cows that, although there was a 2-fold increase in hepatic ketogenesis during 6 days of food deprivation, ketogenesis from the splanchnic bed as a whole (i.e. gut and liver combined) declined slightly owing to cessation of gut ketogenesis.

Effects of anabolic steroids on liver cell ultrastructure in sheep.

Castrated male sheep were sham inplanted or implanted with estradiol, trenbolone acetate or trenbolone acetate in combination with estradiol, and slaughtered after 56 days. Ultrastructural and morphometric examination of the livers of steroid-treated sheep showed an increase in the volume density of the cell occupied by rough endoplasmic reticulum and microbodies. In four of the eight sheep treated with trenbolone acetate, either alone or in combination with estradiol, enlarged mitochondria with crystalline inclusions were seen. The increase in hepatic rough endoplasmic reticulum may be of benefit to the sheep whereas the mitochondrial changes after anabolic steroid use indicate a cellular lesion of unknown significance for health.