Rats maintained for 8 months on a level of warfarin sufficient to decrease the vitamin K-dependent protein of bone (bone Gla protein) to 2% ofnormal have an excessive mineralization disorder characterized by complete fusion of the proximal tibial growth plate and cessation of longitudinal growth. The general features of this abnormality resemble the fetal warfarin syndrome in humans, a disorder also characterized by excessive mineralization of the growth plate. These excessive mineralization disorders may be caused by the decreased levels of bone Gla protein, a protein that potently inhibits mineralization in vitro.The metabolism of vitamin K presents several intriguing problems in vertebrate physiology and biochemistry. One of the most interesting ofthese is the role ofvitamin K in systems other than blood coagulation. The classical approach to the analysis ofvitamin K physiology has been the study ofdefects in vitamin K-deficient animals. Such studies have established that the first consequence of acute vitamin K deficiency is bleeding and death due to the synthesis of abnormal forms of blood coagulation factors such as prothrombin and factors VII, IX, and X. It is now known that the abnormality in these factors is the absence of 'y-carboxyglutamate (Gla), a Ca2" binding amino acid whose post-translational synthesis from glutamate requires vitamin K (1, 2).Our primary interest over the past several years has been the function of bone Gla protein (BGP), a 49-residue protein of known structure (3, 4) which is numerically one of the 10 most abundant proteins in a typical vertebrate (5,6). To analyze the function ofthis protein in bone metabolism we developed a simple protocol by which rats can be maintained on the vitamin K antagonist warfarin without problems due to bleeding (7). In previous studies we found that animals maintained from birth to 2 months of age on this protocol grow normally and have normal bone structure and mineralization in spite of bone levels of BGP which are only 2% of normal (7).We report here the discovery of excessive mineralization with growth plate closure in the proximal tibia ofrats maintained on warfarin from birth to 8 months of age. MATERIALS AND METHODSMaintenance of Animals on Warfarin. Simonsen albino rats (Sprague-Dawley-derived, Simonsen Laboratories, Gilroy, CA) were maintained from birth to 8 months of age on daily dosages ofwarfarin and vitamin K1 as described (7). To facilitate comparisons, the 10 control rats were selected from litters born at the same time as experimental animals and received daily doses of saline and vitamin K1 (7). Both experimental and control groups had seven male and three female rats. None of the 10 experimental rats chosen at birth for this study showed signs of bleeding or ill health over the 8 months of the experiment. As reported (7), the weight gain ofexperimental and control rats was identical up to 90 days ofage. At8 months ofage the average weights were 402 g for the male and 237 g for the female experimental rats and 455 g for t...

The microscopic distribution pattern of z4sCf and *49Bk in the soft tissues of beagles, one to three weeks following a single intravenous injection of a citrate solution, was found to be very similar to that of a41Am. Relatively high concentrations occurred in the hepatic cells of the liver, the glomeruli of the kidney, the interfollicular region of the thyroid, the cartilaginous tissues of the lung, and the media of the smaller arterioles of most organs. Very intense, but sparsely scattered "hot spots" were also present in the renal papillae and in the submucosa of the bronchioles. Lesser sites of localization were the endocardium of the AV heart valves, the glassy membranes of the larger hairs of the coat, the zona pellucida of the Graafian follicles and the zona arcuata of the adrenal cortex. With the exception of the liver, where the radionuclide was principally within the hepatic cells, most of the deposition sites were extracellular, within or associated with connective tissue which gave a positive periodic acid-Schiff reaction.

This work reports the comparison of a sol-gel and a screen-printed biosensor format using new mediators and a sensitive thin-film (ref. 1: P. Kataky and D. Parker, Analyst, 1996, 121, 1829) to enhance the sensitivity and stability of biosensors. The new mediators were per-alkylated cyclodextrin linked ferrocenes and a control, ferrocene aminocarboxylic acid. The thin film comprised a cocktail with polyurethane, plasticiser, lipophilic anion and a perethylated beta-CD. The analyte targeted was acetylcholine using the established horseradish peroxidase-choline-oxidase-acetylcholine esterase relay. The screen-printed electrode format showed a marked decrease in oxidation potential, the magnitude of the shift depended on the structure of the mediator and the membrane covering. Although lower oxidation-potentials were observed with the sol-gel format sensors, their response was more akin to aqueous solution behaviour. Electrocatalytic currents were observed suggesting a highly efficient electron transfer process.

The detailed neutron-induced autoradiographic process for bone containing zasPu produces autoradiographs with both fission fragment tracks and a corresponding bone image. Seven-microns-thick undecalcified bone sections were affixed on 300-pm-thick Lexan (polycarbonate) film coated with 0.5 % calf skin gelatin and exposed to neutrons in the thermal column of the Material Testing Reactor (MTR) at the National Reactor Testing Station in Idaho. The specimens were immersed in 28% KOH at 5OoC for 1-14 hr to etch pinholes at the location of each fission track and to pit the film at the location of the bone. When the films were etched immediately after irradiation, no bone image was produced, but when the bone sections were allowed to remain on the film for several weeks after irradiation, the irradiated bone caused a pitting of the film surface. Betas from 32P seemed to be the main charged particles available to produce the pitting. This detailed neutron-induced autoradiographic process has advantages over the nuclear emulsion autoradiography of bones because it eliminates long exposure times and fading of latent image. It is a rapid, effective procedure.

The distribution of bone calcium between morphologically identifiable cortical and trabecular bone obtained by dissection and quantitated by neutron activation analysis (NAA) is described. The skeleton of a female beagle dog was dissected into approximately 400 pieces and assayed for 49Ca produced in the University of California, Irvine TRIGA reactor. For each of the skeletal sections, we give the initial weight of the alcohol-fixed tissue, which includes cortical bone, trabecular bone, marrow, and cartilage, and a final tissue weight after the marrow and trabecular bone have been dissected away; total section and cortical section calcium weights are reported. The level of detail is represented, for example, by the vertebrae, which were divided into three parts (body, spine, and transverse processes) and by the long bones, which were divided into 10-12 parts such that characterization of the epiphysis, metaphysis, and diaphysis was accomplished. The median percentage cortical calcium values for cervical, thoracic, and lumbar vertebrae were 82%, 56%, and 66%, respectively; however, variation within these groups and among individual vertebral sections was about a factor of 2. For long bones, the median percentage cortical calcium varied from 90-100% in the midshaft to below 50% in the proximal and distal sections. The final calculated cortical tissue-to-calcium mass ratio (TCR) varied from about 4.5 for midshafts of the long bones to about 9 for thoracic vertebral bodies and indicated that the mineral fraction of cortical bone is not constant throughout the skeleton. The ratio of cortical to trabecular calcium in the skeleton was 79.6:20.4.