Biological potency and radioimmunoassay of canine calcitonin.

Calcitonin (CT) is a major calcitropic hormone. Because of low cross reactivity of canine CT (cCT) in radioimmunoassays (RIA) developed for other species, a homologous RIA is needed. Synthesis of cCT allowed study of its biologic potency using a rat bioassay and its plasma half-life in dogs. The availability of cCT also made possible the development of a homologous RIA for measurement of basal and stimulated plasma CT concentrations in dogs. The biologic potency of the synthesized cCT in rats is 24 IU/mg of peptide, which is low in comparison with the 4,000 IU/mg of the salmon CT standard. In the dog, an even lower potency of 4.4 IU/mg of cCT was found. Measurement of the disappearance of iv-injected radioiodinated or nonradioiodinated cCT revealed a short biologic half-life of less than 3 min, followed by a long half-life of 20 min. A polyclonal antiserum against synthetic cCT was raised in a goat. Using a final antiserum dilution of 1:12,000 and 125I-labeled synthetic cCT, the RIA had a detection limit of 6.5 ng/l. The antibody did not crossreact with standard human CT and had <0.1% cross reactivity with porcine CT. For measurement of plasma cCT concentrations, an extraction procedure was developed using ethanol. Dilutions of synthetic cCT and canine plasma extracts revealed parallelism over a wide range of concentrations. Size exclusion chromatography of canine plasma extracts on Biogel P-10 revealed a single cCT peak at the same position as [125I]-cCT, showing that there was little interference by other proteins or cCT prohormone. Basal plasma CT concentrations were 12-80 ng/l, and there was an 8- and 20-fold increase after calcium (1 and 2.5 mg/kg body weight) bolus infusion.

Absorption of hydrophilic and lipophilic compounds through epidermal and subepidermal strata of rat skin in vitro.

The absorption of a model hydrophilic compound (benzoic acid) and a model lipophilic compound [di(2-ethylhexyl)phthalate; DEHP] was measured through separated epidermal membranes and through the residual stripped dermis of rat skin. Absorption was determined using both phosphate buffered saline (PBS) and 50% ethanol/water as the receptor fluid. The rate and extent of DEHP absorption through epidermis was greatly increased (40- to 80-fold) using 50% ethanol/water as receptor fluid compared with using PBS, but with the stripped dermis only a small difference (approximately two- to threefold) was detected. The use of 50% ethanol/water as receptor fluid also promoted the penetration of benzoic acid through the epidermis, but only to a limited extent (two- to threefold increase). As with DEHP, the penetration of benzoic acid through the dermis was also increased. The permeability to (3)H(2)O of epidermal, but not dermal membranes was also increased by the use of a 50% ethanol/water receptor fluid. It was concluded that 50% ethanol/water can increase the permeability of rat skin strata to both lipophilic and hydrophilic compounds. Further work is needed to provide a basis for selecting suitable receptor fluid formulations for in vitro dermal absorption studies.

The international validation of a fixed-dose procedure as an alternative to the classical LD50 test.

In an international study involving 33 laboratories in 11 countries, the acute oral toxicity to the rat of 20 substances and preparations was evaluated using a fixed-dose procedure and the results compared with those obtained for the test materials using the classical LD50 test. The study has shown that the fixed-dose approach to acute oral toxicity testing: (1) produces consistent results that are not substantially affected by inter-laboratory variations; (2) provides adequate information for risk assessment purposes on signs of toxicity, including their nature, time to onset, duration and outcome; (3) uses fewer animals than the current internationally agreed OECD procedure (Guideline 401-revised); (4) subjects animals to less pain and distress than the classical LD50 test and causes less compound-related mortality; and (5) enables substances and preparations to be ranked according to the EEC classification system on the basis of their acute oral toxicity, such ranking being compatible with that allocated by the results of classical LD50 studies.

The effect of chronic low level lead exposure on blood-brain barrier function in the developing rat.

Blood-brain barrier (BBB) function was assessed in 19-21-day-old rats exposed to low level lead from birth. Newborn rats received lead via milk from lactating dams given drinking water containing 0.1% lead acetate [Pb(Ac)2]. The treatment regime produced lead levels in the neonates within the range 20-80 micrograms dl-1 blood, without affecting growth. Cerebrovascular permeability (PS-product) to the diffusion-limited solute mannitol was unchanged in six regions of the cerebral hemisphere, the cerebellum and the brainstem, suggesting that barrier integrity was not affected by the low dose lead treatment. Regional cerebrovascular permeability to nutrient tracers representing seven BBB transport classes was not impaired by lead treatment. However, the PS estimates for the amino acids lysine and histidine and for thiamine were greater than control in some regions of the cerebral hemisphere. These alterations in nutrient supply to the brain may reflect altered substrate utilization associated with repair processes or delayed maturation of the CNS.

Regional cerebral glucose metabolism and blood flow during the silent phase of methylmercury neurotoxicity in rats.

Methylmercuric chloride was given to rats in a neurotoxic dose regimen (six daily doses of 8 mg kg-1 p.o.). During the silent (asymptomatic) phase of intoxication, the rates of cerebral glucose influx and cerebral glucose phosphorylation were measured simultaneously using 2-deoxyglucose. Regional cerebral blood flow was also measured using iodoantipyrine. The unidirectional flux of glucose into brain was not affected by methylmercury, and differences in the rates of glucose phosphorylation from region to region remained coupled to the regional cerebral blood flow. However, the blood flow was reduced throughout the brain, an observation suggesting that the operational level of metabolically regulated blood flow had been reset. Thus, in spite of a generalised reduction in blood flow, there was no indication of impaired cerebral glucose supply or utilization during the silent phase of methylmercury intoxication.

Regional blood-brain glucose transfer in the rat: a novel double-membrane kinetic analysis.

Regional blood-brain glucose transfer was studied in pentobarbitone-anaesthetized rats using a programmed intravenous infusion technique that maintained steady levels of unlabeled (up to 55 mM) and tracer D-glucose in the circulating plasma. Regional cerebral blood flow, glucose phosphorylation rate, and tissue glucose content were also measured under comparable conditions. Data were analysed in terms of irreversible Michaelis-Menten kinetics assuming independent influx and efflux (Type I) and reversible Michaelis-Menten kinetics (Type II) across both the luminal and the abluminal membranes of the endothelial cell. The latter analysis corresponds to simple stereospecific membrane pores. The mathematical model allowed for changes in tissue glucose content and back-diffusion of tracer during the experiments. Type I analyses gave Kt values of approximately 6.6 mM, whereas those by Type II were consistently lower. Interregional differences were not significant using either scheme. Comparison of Type II with Type I analyses revealed a possible explanation for discrepancies in the estimates of nonsaturable glucose transfer by different methods and highlighted the importance of tissue glucose measurements in studies of unidirectional glucose influx. Since the experimental data may be described equally well by either scheme and some interaction between influx and efflux across the endothelial cell might be expected, consideration of this alternative approach is suggested.

Long-term peritoneal tissue response in rats to mould-release agents and lubricant powder used on surgeons' gloves.

Following implantation of 50-mg samples of a starch powder used for glove lubrication, or of talc or calcium carbonate mould-release agents for gloves, in the peritoneal cavity of rats, the treated animals were killed in groups of ten after 2, 4, 8, 13, 26 and 52 wk and were examined. Groups of sham-operated animals were used as controls. Within each treatment group the frequency of peritoneal adhesions remained constant throughout the study. Talc produced significantly more adhesions than the other treatments and caused a more severe granulomatous reaction, which persisted. Calcium carbonate and starch powder produced similar numbers of adhesions and in both cases the residues became invested by a thin fibrous capsule. Calcium carbonate appeared a safer material than talc.

Changes in the distribution of histochemically localized mercury in the CNS and in tissue levels of organic and inorganic mercury during the development of intoxication in methylmercury treated rats.

The development of neurotoxicity in rats after exposure to methylmercuric chloride was monitored using behavioural indices. At selected time points the cellular localization of mercury and the relative amounts of organic and inorganic mercury were determined in several regions of the CNS, and in some non-neural tissues. The CNS showed an affinity for organic mercury, the levels of inorganic mercury remaining low throughout symptomatic intoxication. Histopathological changes were not closely related to the regional tissue content of the organic or inorganic forms, nor to mercury localized histochemically at the cellular level. The stained deposits, which had focal cytoplasmic distribution, appeared in glial cells initially then in larger neurones as the intoxication progressed. These observations may represent changes in the mercury content of different cell types or reflect differences in the way that they handle a similar burden of mercury. A transitory accumulation of mercury in glial cells may be a factor contributing to the occurrence of a latent period and sequestration of mercury in cytoplasmic organelles may serve to protect some cell types from injury.

The Clara cell and pulmonary surfactant: a study using selective chemical ablation.

Administration of 3-hydroxymethylfuran-N-ethylcarbamate (HFC) to female hamsters via the jugular vein under pentobarbitone anaesthetic at 20 mg per kg body weight produced pronounced necrosis of the Clara cells without apparent morphological effect on other cell types as judged by transmission electron microscope examination. The surfactant material recoverable by minimal lavage followed by purification by sucrose gradient ultracentrifugation increased, reaching a maximum around 48 h after treatment. At this time static pressure/volume measurements on isolated lungs indicated an increase in airway surface compliance. Lavageable surfactant phospholipid composition was examined by 31P nuclear magnetic resonance (n.m.r.). The distribution of phospholipids between the various classes was unchanged by HFC treatment. No change in the total lung surfactant pool size was seen. These results are discussed in relation to the possible roles of the Clara cell in influencing airway surfactant levels.

Influence of the sterilization method and of magnesium oxide on the tissue responses in the rat to modified starch glove powders.

The tissue response to samples of a surgical glove powder was assessed from counts of the adhesions and granulomas found 7 and 14 days after introduction into the peritoneal cavity of rats, and by a histological study of affected tissues. Negative control groups treated with no powder and positive controls treated with talc were included. Various sample treatments were implemented to examine the effect of the sterilization method on the tissue response and the influence of magnesium oxide, normally added as a dispersing agent. The glove powder produced less reaction when steam-sterilized than when gamma-irradiated and the presence of magnesium oxide at 2% concentration made no detectable difference.

Amino acid concentrations in portal venous plasma during absorption from the small intestine of the guinea pig of an amino acid mixture simulating casein and a partial enzymic hydrolysate of casein.

1. The characteristics of absorption of individual amino acids from amino acid mixtures simulating casein and from enzymic hydrolysates of casein containing oligopeptides as well as free amino acids are known to be different. The differences, which are attributable to mucosal uptake of small peptides, involve more rapid absorption from the enzymic hydrolysates of certain amino acids which are relatively slowly absorbed from the amino acid mixtures. This could lead to more effective utilization of amino acids from the enzymic hydrolysates than from the amino acid mixtures. 2. To obtain further information bearing on this hypothesis, we have used a recently developed technique for portal cannulation in the guinea pig to make a preliminary investigation of amino acid concentrations in the portal venous plasma at intervals after the infusion into the duodenum of equivalent amounts of (a) an amino acid mixture simulating casein and (b) a partial enzymic (papain followed by kidney peptidases) hydrolysate of casein, the two preparations being infused in separate experiments. 3. For some amino acids, such as leucine, isoleucine, valine, phenylalanine and lysine, the curves after the enzymic hydrolysate were fairly similar to the corresponding curves after the amino acid mixture, though usually slightly lower. With other amino acids, the curves after the enzymic hydrolysate were very much lower than the corresponding curves after the amino acid mixture. With serine, glutamine, proline and glycine this discrepancy was particularly great. 4. The results cannot yet be fully explained, but their main features are explicable by the hypothesis that the lower amino acid concentrations in portal plasma after the enzymic hydrolysate are the result of entry of amino acids into the portal blood in peptide form, in which they would not be detectable by the analytical technique employed, and possibly also of more rapid clearance of amino acids from the blood during absorption of this preparation.

Cardiovascular effects of acrylic bone cement in rabbits and cats.

The cardiovascular responses to forcing acrylic bone cement, Plasticine, or soft paraffin wax into the medullary cavity of the femur have been studied in rabbits and cats. An acute fall in blood pressure, occurring within a few seconds of insertion, was demonstrated with each substance. In a few of the animals the blood pressure response had a second more protracted component and it is suggested that more than one mechanism is involved. The cardiovascular effects that have been observed in man when acrylic cement is used in prosthetic hip surgery also may be due to more than one mechanism.