Calciferol 25-hydroxylase activity in smooth and rough endoplasmic reticulum of rat liver.

Calciferol 25-hydroxylase activity of vitamin D-deficient rats was measured in both liver microsomes and submicrosomal fractions. The smooth and rough-surfaced microsomes were prepared by a density gradient centrifugation technique in the presence of cesium chloride. Purity of the isolated microsomal membranes was ascertained by electron microscopy, RNA determination, measurement of enzyme markers, and by labeling of the cytoplasmic RNA with [5-³H]orotic acid. Calciferol 25-hydroxylase activity was present in both smooth and rough-surfaced microsomes. The specific activity of the enzyme was greater in the rough fraction. There was a linear relation between enzymic activity and the concentration of enzyme for both total and submicrosomal fractions. These data show the presence of calciferol 25-hydroxylase activity in both smooth and rough-surfaced microsomes isolated from livers of vitamin D-deficient rats.     

The metabolic fate of 13N-labeled ammonia in rat brain.

13N-labeled ammonia was used to study the cerebral uptake and metabolism of ammonia in conscious rats. After infusion of physiological concentrations of [13N]ammonia for 10 min via one internal carotid artery, the relative specific activities of glutamate, glutamine (alpha-amino), and glutamine (amide) in brain were approximately 1:5:400, respectively. The data are consistent with the concept that ammonia, entering the brain from the blood, is metabolized in a small pool of glutamate that is both rapidly turning over and distinct from a larger tissue glutamate pool (Berl, S., Takagaki, G., Clarke, D.D., and Waelsch, H. (1962) J. Biol. Chem. 237, 2562-2569). Analysis of 13N-metabolites, after infusion of [13N]ammonia into one lateral cerebral ventricle, indicated that ammonia entering the brain from the cerebrospinal fluid is also metabolized in a small glutamate pool. Pretreatment of rats with methionine sulfoximine led to a decrease in the label present in brain glutamine (amide) following carotid artery infusion of [13N]ammonia. On the other hand, 13N activity in brain glutamate was greater than that in the alpha-amino group of glutamine, i.e. following methionine sulfoximine treatment the expected precursor-product relationship was observed, indicating that the two pools of glutamate in the brain were no longer metabolically distinct. The amount of label recovered in the right cerebral hemisphere, 5 s after a rapid bolus injection of [13N]ammonia via the right common carotid artery, was found to be independent of ammonia concentration within the bolus over a 1000-fold range. This finding indicates that ammonia enters the brain from the blood largely by diffusion. In normal rats that were killed by a freeze-blowing technique 5 s after injection of an [13N]ammonia bolus, approximately 60% of the label recovered in brain had already been incorporated into glutamine, indicating that the t1/2 for conversion of ammonia to glutamine in the small pool is in the range of 1 to 3 s or less. The data emphasize the importance of the small pool glutamine synthetase as a metabolic trap for the detoxification of blood-borne and endogenously produced brain ammonia. The possibility that the astrocytes represent the anatomical site of the small pool is considered.     

CEREBRAL BLOOD FLOW AND CEREBRAL METABOLIC RATES FOR OXYGEN, GLUCOSE, AND KETONE BODIES IN NEWBORN DOGS

Cerebral blood flow (CBF) and the cerebral metabolic rates for oxygen, glucose, acetoacetate, β-hydroxybutyrate and lactate were measured in 1- to 5-day old Beagle dogs under nitrous oxide anesthesia. CBF was determined by ¹³³Xe washout with mechanically integrated blood samples withdrawn simultaneously from a femoral artery and from the posterior one-third of the superior sagittal sinus. CBF and CMRO₂ in normocapnia (PaCO₂ 40 × 1 mm Hg) were 48 × 5 ml/100 g/min and 2.15 ml/100 g/min, respectively. There was a positive, linear relationship between CBF and PaCO₂, calculated for PaCO₂ values ranging from 26 to 70 mm Hg. Induced hypocapnia (PaCO₂ 31 × 1 mm Hg) or hypercapnia (PaCO₂ 58 × 2 mm Hg) did not alter the CMRO₂. Glucose and acetoacetate were taken up by the brain at all PaCO₂ levels examined; however, the cerebral uptake of glucose always exceeded the combined uptake of ketone bodies by more than a factor of ten. The cerebral metabolic rate for glucose (94.6 × 3.6 μmol/100 g/min) more than accounted for overall cerebral oxygen consumption, and yielded an oxygen:glucose ratio (mol:mol) of 5.1. Thus, as in adult animals, PaCO₂ is an important regulator of cerebral blood flow in puppies, and glucose is the major substrate for oxidative energy production in the immature brain. The oxidation of ketone bodies by the newborn dog brain accounts for not more than 6% of the in vivo cerebral oxygen consumption.     

Toxicological evaluation of methyl tertiary butyl ether (MTBE): Testing performed under TSCA consent agreement

MTBE is a colorless, relatively volatile liquid that has found widespread use as an octane‐enhancing gasoline additive. In 1987, the Environmental Protection Agency's (EPA) Interagency Testing Committee identified MTBE for priority testing consideration based on large production volume, potential widespread exposure, and limited data on chronic health effects. In response, the industry formed the MTBE Health Effects Testing Task Force, which in 1988 signed a Consent Agreement with the EPA requiring the task force member companies to perform toxicological testing on MTBE.

The testing program, which began in the second quarter of 1988, consists of a full complement of short‐ and long‐term tests. The testing completed to date includes genotoxicity (in vivo bone marrow cytogenetics and Drosophila sex‐linked recessive lethal assays), developmental toxicity, acute and subchronic neurotoxicity (motor activity, functional observation battery, and neuropathology), subchronic toxicity, reproductive/fertility effects, and pharmacokinetic studies. There is also an ongoing oncogenicity study in rats and mice. The final report for this chronic study is expected at the end of 1992. The total cost for the program is approximately $3.75 million, which is funded by the 11 Task Force member companies based on market share.

These studies were sponsored by the MTBE Health Effects Testing Task Force, Oxygenated Fuels Association, Washington, D.C.     

Glutathione and Ascorbate During Ischemia and Postischemic Reperfusion in Rat Brain

Thirty minutes of total cerebral ischemia (decapitation) decreased total glutathione (GSH + GSSG) by 7% but had no detectable effect on the concentration of oxidized glutathione (GSSG), reduced ascorbate, or total ascorbate. In a model of reversible, bilateral hemispheric ischemia (four-vessel occlusion) no changes in glutathione or ascorbate were detected after 30 min of ischemia. During 24 h of reperfusion following such an insult no detectable change in total ascorbate, reduced ascorbate, or oxidized glutathione was noted; however, total brain glutathione declined by 25%. The findings are discussed in relation to the hypothesis that the deleterious effects of ischemia are due to an increase in free radical production which in turn leads to increased lipid peroxidation.     

Amino acid differences between the α-chains from two hemoglobins of the yellow-cheeked vole (family cricetidae)

The yellow-cheeked vole (Microtus xanthognathus) shows two electrophoretic hemoglobin components. Electrophoresis of the polypeptide chains from the separated hemoglobin components shows identical β-chains but two α-chains of different mobility, α ^f and α ^s . The composition of soluble tryptic peptides was determined for each α-chain. Amino acid differences were found in peptides αT1 and αT9; the compositions of the remainder of the homologous peptides were identical. Differences in αT1, found at α4 (α ^s -Gly-α ^f -Val) and α5 (α ^s -Thr-α ^f -Asp), were confirmed after a run to residue 20 of the fast component in an automatic sequencer. The differences in charge between αT1 peptides can account for the electrophoretic pattern of two hemoglobins. This is the first time that it has been possible to identity the residues which can account for the charge difference between the two hemoglobins observed in a Microtus species.