Radiological protection of the patient in veterinary medicine and the role of ICRP.

At the request of the Main Commission of the International Commission on Radiological Protection (ICRP), Task Group 107 (TG107) was set up to consider the issue of radiological protection of the patient in veterinary medicine. TG107, who authored this article, brought together information relating to the use of diagnostic imaging and radiation oncology in veterinary medicine. A number of specific areas were identified that appeared to be appropriate for attention by ICRP. These included the use of dose quantities and units, the need for re-evaluation of stochastic and deterministic risks from ionising radiation in animals, and the growing use of imaging and therapeutic equipment for animals that is little different from that available to humans. TG107 unanimously recommended that it was both appropriate and timely for ICRP to consider and advise on these issues, and the Main Commission agreed. This paper summarises the findings of TG107.

Quantitative analysis of the effect of probenecid on pharmacokinetics of 99mTc-mercaptoacetyltriglycine in dogs.

Effect of probenecid on pharmacokinetics of 99mTc-mercaptoacetylytriglycine (99mTc-MAG3) in dogs was investigated before (control), and after 15 min and 24 h of i.v. injection of probenecid (20 mg/kg). Plasma concentration-time profiles of 99mTc-MAG3 were described with a two-compartment open model. Plasma 99mTc-MAG3 clearances (Clp, ml/min/kg) were 7.9 +/- 0.5, 3.3 +/- 0.5 and 4.8 +/- 1.3 in control, 15 min and 24 h after probenecid administration respectively. Similarly, the biological half-lives at elimination phase (t(1/2), h) were 0.61 +/- 0.09, 0.79 +/- 0.11 and 0.74 +/- 0.12, and volumes of distribution at steady state (Vdss, L/kg) were 0.29 +/- 0.04, 0.20 +/- 0.05 and 0.25 +/- 0.06 respectively. The prolonged biological half-life and decreased Vdss decreased Clp significantly. Clp was a function of plasma probenecid concentration based on Michaelis-Menten kinetics. The maximum Clp inhibition (Imax) by probenecid and the plasma probenecid concentration that induced 50% of Imax (I50) were estimated to be 72 +/- 12% and 13 +/- 8 microg/ml respectively. This means that the rest (about 28%) of the Clp is not blocked by probenecid alone, suggesting the possibility of another route(s) of elimination or renal transporters which are independent from probenecid. Moreover, inter-species correlation between Clp of 99mTc-MAG3 and body weight are discussed.

Binding characteristics of folate to high affinity folate binding protein purified from porcine serum.

High affinity folate binding protein (HFBP) in porcine serum was purified 2,000-fold to a specific activity of 1.4 nmol of tetrahydrofolic acid (THF) bound per mg of protein, using folic acid (FA) coupled EAH-Sepharose gel affinity chromatography. Binding activity of purified HFBP to folate was examined by ultrafiltration method linked to high-performance liquid chromatography with electrochemical detection or to liquid scintillation counting. Binding affinity of HFBP to folate was characterized by dissociation constants (Kd): 13, 17, and 31 pM for tritiated FA (3HFA), THF, and N5-methyltetrahydrofolic acid (5MF), respectively. FA, THF, and 5MF significantly inhibited binding of HFBP to 3HFA, and according to the magnitude of intensity of the binding inhibition, the order of affinity of each folate was confirmed to be FA > THF > 5MF. Binding activity was rather high and stable for THF and 5MF at pH ranging from 6.0 to 10.0. The binding activity, however, rapidly decreased at pH below 6.0 and over 10.0. No binding activity was observed pH below 3.0 and over 12. Gel filtration analysis showed that the prepared HFBP solution had specific binding activity at around 77 kDa of apparent molecular weight, which was 82 kDa by SDS-PAGE. It is considered that this specific and stable binding enables THF to distribute in porcine plasma.

Isolation of a bovine full length cytochrome P450 (CYP3A) cDNA sequence and its functional expression in V79 cells.

From a bovine liver cDNA library in λMaxl a 1870 bp cDNA was isolated using the human CYP3A4 cDNA as a probe. The cDNA-deduced amino acid sequence encoded a protein of 507 amino acids and exhibited homologies of 76, 72 and 64% with canine CYP3A12, human CYP3A4 and rat CYP3A1, respectively. Furthermore, a very high homology of 91.7% was observed with the deduced amino acid sequence of a partial CYP3A cDNA from dwarf goat. A striking observation was that both the bovine and the goat cDNA exhibit a 4 amino acid extension at the C-terminus, which is due to a frame-shifting insertion of 2 nt. The bovine CYP3A cDNA was cloned in a retroviral vector, transfected to V79 cells and cells were selected for cytochrome P450 expression. The expressed enzyme was shown to catalyze the 6ß-hydroxylation of testosterone, which could also be observed in a V79 cell line expressing human CYP3A4. In the bovine CYP3A cell line, however, 6ß-hydroxytestosterone was not found to be the major metabolite. This cell line additionally showed high levels of hydroxylase activity at the 2ß and 12ß position of testosterone. The cDNA-expressed testosterone hydroxylase activity could be inhibited with the specific CYP3A inhibitors, tiamulin and ketoconazole.

Alteration of plasma folates in gestating sows and newborn piglets.

Plasma folates tetrahydrofolate (THF) and N5-methyltetrahydrofolate (5MF) were determined in nongestating, gestating, and lactating sows (2 to 4 yr old, 2-6 parities, n = 88) and in newborn, growing, and finishing pigs (0 to 158 days old, n = 191) with the use of high-performance liquid chromatography with an electrochemical detector. Plasma folates after folic acid injection (1 mg/kg iv) were also monitored in 2-day-, 2-wk-, and 2-mo-old pigs. In sows, plasma folates decreased significantly as pregnancy advanced. This was mainly due to the decrease of THF, which must be caused by the folate supply from the maternal body to the fetuses. In newborn piglets, levels of "total" plasma folates were much higher than those in adults (nongestating sows), and 5MF was the major folate content. This result may be related to the observation that, after folic acid injection, newborn piglets showed much higher metabolic activity of folic acid to 5MF than adults. Rapid and drastic decrease of plasma folate was observed during nursing in piglets, which is considered to be associated with the decrease of the metabolic activity of folic acid and rapid expansion of body volume together with negative folate intake during the nursing period.

Role of high-affinity folate-binding protein in the plasma distribution of tetrahydrofolate in pigs.

Stability and protein-binding properties of tetrahydrofolate (THF) in pig plasma were studied in vitro. THF in plasma was stable for more than 120 min when it existed in a bound form, whereas THF both in plasma ultrafiltrate and in plasma ultrafiltrate plus porcine albumin was degraded rapidly and disappeared soon after its addition. These results suggest that high-affinity folate-binding protein (HFBP) is related to the stability of THF. THF-protein binding kinetic analysis showed that porcine plasma had HFBP and low-affinity binding protein (albumin) for THF. Dissociation constant and maximal binding capacity of HFBP were calculated to be 0.4 and 70 nM, respectively, indicating that > 98% of endogenous plasma THF existed in bound form with HFBP. Porcine albumin was not essentially a protein that binds and protects endogenous THF from degradation. We conclude that most endogenous THF binds to HFBP and only the unbound form of THF is rapidly degraded in pig plasma. HFBP protects THF from degradation and allows THF to exist stably in pig plasma. In addition, HFBP may govern the species specificity of plasma folate distribution in pigs.

Pharmacokinetics and bioavailability of folic acid and plasma levels of bioactive folates after folic acid administration to pigs.

After intravenous (1 mg/kg body weight), intramuscular (1 mg/kg body weight) and oral (1 and 50 mg/kg body weight) administration of folic acid (FA) to pigs, plasma levels of FA, tetrahydrofolic acid (THF), 5-methyltetrahydrofolic acid (5MF) were determined by using high-performance liquid chromatography. The pharmacokinetic profile of plasma FA after oral administration indicated an absorption rate-limited elimination, i.e., so called 'flip-flop' phenomenon. The bioavailability of FA was quite low after a high oral dose (F = 0.01), in contrast to a high value after intramuscular administration (F = 0.95). The plasma levels of biologically active, reduced forms of folates (THF and 5MF) were significantly increased over their basal levels after IV and IM administration to FA. The levels of these active folates were not increased after oral administration of a similar dose of FA. A 50 times higher dose was required to increase the active folates to the levels observed after IV and IM administration.

Hepatic cytochrome P450 induction in goats. Effects of model inducers on the metabolism of alkoxyresorufins, testosterone and ethylmorphine, and on apoprotein and mRNA levels.

Male and female African dwarf goats were treated orally with phenobarbital (PB) or triacetyloleandomycin (TAO), or subcutaneously with beta-naphthoflavone (BNF). Hepatic microsomal cytochrome P450 content was increased by PB and TAO, but not by BNF. PB effects on P450 activities were non-selective: ethoxyresorufin deethylase (EROD) and pentoxyresorufin depentylase (PROD), hydroxylation of testosterone (TST) and demethylation of ethylmorphine (ETM) were all induced by a factor of 2-3. A similar non-selective induction was observed with TAO, except for EROD and PROD (no effects). After PB and TAO treatment, increased levels of a protein cross-reactive with anti-sheep P450 3A and 2B were found. Thus, in dwarf goats, both PB and TAO appeared to be P450 3A inducers. Selective PB effects related to a P450 2B form on PROD are lacking but 16 alpha-hydroxylation of TST was induced markedly. At the mRNA level, PB induced an mRNA that showed good sequence homology with a human P450 3A4 cDNA probe, rather than with a rat 3A1 probe. BNF selectively induced EROD, whereas TST hydroxylation and ETM dealkylation were inhibited. With BNF-treated animals, increased concentrations of a protein cross-reactive with anti-rat P450 1A1/1A2 and of an mRNA that showed homology with a human 1A1 cDNA probe, but not with a mouse 1A1/1A2 probe, were observed.

Metabolism of antipyrine and sulphadimidine in dwarf goats: effects of the enzyme-inducing agents phenobarbital, troleandomycin and rifampicin.

1. Antipyrine (AP) and sulphadimidine (SDD) plasma elimination and metabolite formation were studied in dwarf goats before and after treatment with phenobarbital (PB), triacetyloleandomycin (TAO), and rifampicin (RIF). 2. PB treatment significantly increased AP plasma clearance in both male and female goats. With SDD, only male goats were studied, which showed a significant increase of SDD plasma clearance following PB treatment. 3. After PB treatment, partial clearance values of four AP metabolites, 3-hydroxymethylantipyrine (HMA), norantipyrine (NORA), 4-hydroxyantipyrine (OHA) and 4,4'-dihydroxyantipyrine (DOHA), were significantly increased. This induction effect was different for the individual metabolites and also showed sex-dependency. 4. In PB-induced male goats the formation of the hydroxylated SDD metabolites, 6-hydroxymethyl-SDD and 5-hydroxy-SDD, was significantly increased. 5. After TAO treatment, female goats showed a slightly reduced AP plasma clearance and a decreased partial clearance of two AP metabolites, HMA and DOHA. There was no effect on SDD plasma elimination or metabolite excretion. 6. In male goats, RIF had no effect on plasma elimination of AP and SDD. With SDD, it decreased the urinary excretion of the unchanged drug and its N4-acetylated metabolite. 7. Induction/inhibition studies of drug metabolism in food-producing animal species are desirable to gain more insight into the regulation of enzymes involved in the metabolism of xenobiotics.

Tetrahydrofolic acid as the principal congener of plasma folates in pigs.

Concentrations of pig plasma folates were determined by a high-performance liquid chromatography with an electrochemical detector (HPLC-ECD). Tetrahydrofolic acid (THF) and 5-methyltetrahydrofolic acid (5-MF) were found to be the plasma folates. The concentration of THF (10.3 +/- 4.3 ng/ml) was significantly higher than that of 5-MF (3.5 +/- 1.0 ng/ml). The sum of the THF and 5-MF concentrations determined by the HPLC-ECD analysis in Göttingen miniature pigs was comparable to the total folate concentration of the same plasma sample determined by a radioligand assay. No or only trace amounts of THF were found in the plasma of the rats, mice, rabbits, dogs, cows, horses, or humans. Although 5-MF is generally recognized as the only principal plasma folate in many species, THF may be the principal congener with 5-MF as a secondary congener in the pig.