Thomas Nelson

The extraneural distribution of gamma-hydroxybutyrate.

Abstract— γ‐Hydroxybutyrate has been found to be widely distributed in both neural and extraneural tissues in the rat. The kidney and brown fat have more than 10 times higher concentrations of y‐hydroxybutyrate than does the brain. This observation suggests that γ‐hydroxybutyrate may participate in the metabolism of many organs, and that GABA may not be the precursor in extraneural tissues.

An overview of γ-hydroxybutyrate catabolism: The role of the cytosolic NADP+-dependent oxidoreductase EC 1.1.1.19 and of a mitochondrial hydroxyacid-oxoacid transhydrogenase in the initial, rate-limiting step in this pathway

SummaryTwo enzymes have been found which catalyze the initial step in the catabolism of GHB. The oxidation of GHB to SSA, catalyzed by both of these enzymes, is coupled to the reduction of an oxoacid. In the case of the mitochondrial transhydrogenase, the coupling is obligatory. Although coupling is not obligatory for the GHB dehydrogenase, the stimulation provided by the coupled reaction, and the nature of the kinetics of the uncoupled reaction, may not only allow the reaction to proceed, but may provide a means of regulating the rate of the reaction under in vivo conditions. Since the oxidation of GHB to SSA is the rate limiting step in the overall catabolic pathway (the rate of conversion of GHB to SSA proceeds at approximately one one thousandth of the rate at which SSA is oxidized to succinate by SSA dehydrogenase (30)), factors which regulate the rate of either or both of these enzymes will, in turn, influence tissue levels of endogenous GHB as well as the duration and magnitude of the physiological effect of a dose of GHB.

PURIFICATION AND CHARACTERIZATION OF AN NADP + -LINKED ALCOHOL OXIDO-REDUCTASE WHICH CATALYZES THE INTERCONVERSION OF γ-HYDROXYBUTYRATE and SUCCINIC SEMIALDEHYDE1

Abstract— An NADP+ ‐linked enzyme, capable of interconverting γ‐hydroxybutyrate and succinic semialdehyde, has been isolated from hamster liver and brain. The enzyme which was isolated from liver has been purified 300‐fold and exhibits a single band by polyacrylamide gel electrophoresis. The molecular weight of the enzyme is ‐ 31,000 as estimated from gel filtration and 38,000 as estimated from sodium dodccyl sulfate gel electrophoresis. The enzyme is inhibited by amobarbital, diphenylhy‐dantoin, 2‐propylvalerate, and diethyldithiocarbamate, but not by pyrazole. The enzymes from brain and liver appear to be very similar with regard to their molecular weights and their kinetic constants for γ‐hydroxybutyrate and succinic semialdehyde.

Invalidity of criticisms of the deoxyglucose method based on alleged glucose-6-phosphatase activity in brain.

Abstract: The observations made by Sacks et al. [Neurochem. Rea.8, 661–685 (1983)] on which they based their criticisms of the deoxyglucose method have been examined and found to have no relationship to the conclusions drawn by them. (1) The observations of Sacks et al. (1983) of constant concentrations of [14C]deoxyglucose and [14C]deoxyglucose‐6‐phosphate. predominantly in the form of product, reflects only the postmortem phosphorylation of the precursor during the dissection of the brain in their experiments. When the brains are removed by freeze‐blowing, the time courses of the [14C]deoxyglucose and [14C]deoxyglucose‐6‐phos‐phate concentrations in brain during the 45 min after the intravenous pulse are close to those predicted by the model of the deoxyglucose method. (2) Their observation of a reversal of the cerebral arteriovenous difference from positive to negative for [14C]deoxyglucose and not for [14C]glucose after an intravenous infusion of either tracer is, contrary to their conclusions, not a reflection of glucose‐6‐phosphatase activity in brain but the consequence of the different proportions of the rate constants for efflux and phosphorylation for these two hexoses in brain and is fully predicted by the model of the deoxyglucose method. (3) It is experimentally demonstrated that there is no significant arteriovenous difference for glucose‐6‐phosphate in brain, that infusion of [12P]glucose‐6‐phosphate results in no labeling of brain, and that the blood‐brain barrier is impermeable to glucose‐6‐phosphate. Glucose‐6‐phosphate cannot, therefore, cross the blood‐brain barrier, and the observation by Sacks and coworkers [J. Appl. Physiol.24, 817–827 (1968); Neuro‐chein. Res.8, 661–685 (1983)J of a positive cerebral arteriovenous difference for [14C]glucose‐6‐phosphate and a negative arteriovenous difference for [14C]glucose cannot possibly reflect glucose‐6‐phosphatase activity in brain as concluded by them. Each of the criticisms raised by Sacks et al. has been demonstrated to be devoid of validity.

2-Deoxyglucose Incorporation into Rat Brain Glycogen During Measurement of Local Cerebral Glucose Utilization by the 2-Deoxyglucose Method

Abstract: The incorporation of 14C into glycogen in rat brain has been measured under the same conditions that exist during the measurement of local cerebral glucose utilization by the autoradiographic 2‐[14C]deoxyglucose method. The results demonstrate that approximately 2% of the total 14C in brain 45 min after the pulse of 2‐[14C]deoxyglucose is contained in the glycogen portion, and, in fact, incorporated into α‐1‐4 and α‐1‐6 deoxyglucosyl linkages. When the brain is removed by dissection, as is routinely done in the course of the procedure of the 2‐[14C]deoxyglucose method to preserve the structure of the brain for autoradiography, the portion of total brain 14C contained in glycogen falls to less than 1%, presumably because of postmortem glycogenolysis which restores much of the label to deoxyglucose‐phosphates. In any case, the incorporation of the 14C into glycogen is of no consequence to the validity of the autoradiographic deoxyglucose method, not because of its small magnitude, but because 2‐[14C]deoxyglucose is incorporated into glycogen via [14C]deoxyglucose‐6‐phosphate, and the label in glycogen represents, therefore, an additional “trapped” product of deoxyglucose phosphorylation by hexokinase. With the autoradiographic 2‐[14C]deoxyglucose method, in which only total 14C concentration in the brain tissue is measured by quantitative autoradiography, it is essential that all the labeled products derived directly or indirectly from [14C]deoxyglucose phosphorylation by hexokinase be retained in the tissue; their chemical identity is of no significance.

Pyretic action of low doses of γ-hydroxybutyrate in rats

Abstract γ-Hydroxybutyrate (GHB) has been found to have a biphasic effect on body temperature with increased body temperature after low doses (5–10 mg/kg) and decreased body temperature after high doses (300–500 mg/kg). Brain levels of GHB between 30 and 60 min post-injection of GHB were not altered by the low doses (5–10 mg/kg), although a dose of 200 mg/kg produced a large increase in the brain concentration.

Direct chemical measurement of the λ of the lumped constant of the [14C]deoxyglucose method in rat brain: effects of arterial plasma glucose level on the distribution spaces of [14C]deoxyglucose and glucose and on λ

The lumped constant in the operational equation of the 2-[14C]deoxyglucose (DG) method contains the factor λ that represents the ratio of the steady-state tissue distribution spaces for [14C]DG and glucose. The lumped constant has been shown to vary with arterial plasma glucose concentration. Predictions based mainly on theoretical grounds have suggested that disproportionate changes in the distribution spaces for [14C]DG and glucose and in the value of λ are responsible for these variations in the lumped constant. The influence of arterial plasma glucose concentration on the distribution spaces for DG and glucose and on λ were, therefore, determined in the present studies by direct chemical measurements. The brain was maintained in steady states of delivery and metabolism of DG and glucose by programmed intravenous infusions of both hexoses designed to produce and maintain constant arterial concentrations. Hexose concentrations were assayed in acid extracts of arterial plasma and freeze-blown brain. Graded hyperglycemia up to 28 mM produced progressive decreases in the distribution spaces of both hexoses from their normoglycemic values (e.g., ∼ – 20% for glucose and – 50% for DG at 28 mM). In contrast, graded hypoglycemia progressively reduced the distribution space for glucose and increased the space for [14C]DG. The values for λ were comparatively stable in normoglycemic and hyperglycemic conditions but rose sharply (e.g., as much as 9–10-fold at 2 mM) in severe hypoglycemia.

Metabolic Stability of 3-O-Methyl-d-Glucose in Brain and Other Tissues

Abstract: 3‐O‐Methyl‐d‐glucose (methylglucose) is often used to study blood‐brain barrier transport and the distribution spaces of hexoses in brain. A critical requirement of this application is that it not be chemically converted in the tissues. Recent reports of phosphorylation of methylglucose by yeast and heart hexokinase have raised questions about its metabolic stability in brain. Therefore, we have re‐examined this question by studying the metabolism of methylglucose by yeast hexokinase and rat brain homogenates in vitro and rat brain, heart, and liver in vivo. Commercial preparations of yeast hexokinase did convert methylglucose to acidic products, but only when the enzyme was present in very large amounts. Methylglucose was not phosphorylated by brain homogenates under conditions that converted 97% of [U‐14C]glucose to ionic derivatives. When [14C]methylglucose, labeled in either the methyl or glucose moiety, was administered to rats by an intravenous pulse or a programmed infusion that maintained the arterial concentration constant and total 14C was extracted from the tissues 60 min later, 97–100% of the 14C in brain, >99% of the 14C in plasma, and >90% of that in heart and liver were recovered as unmetabolized [14C]methylglucose. Small amounts of 14C in brain (1–3%), heart (3–6%), and liver (4–7%) were recovered in acidic products. Plasma glucose levels ranging from hypoglycemia to hyperglycemia had little influence on the degree of this conversion. The distribution spaces for methylglucose were found to be 0.52 in brain and heart and 0.75 in liver.

Refinement of the Kinetic Model of the 2-[14C]Deoxyglucose Method to Incorporate Effects of Intracellular Compartmentation in Brain

A translocase to transport hexose phosphate formed in the cytosol into the cisterns of the endoplasmic reticulum, where the phosphatase resides, is absent in brain (Fishman and Karnovsky, 1986). 2-Deoxyglucose-6-phosphate (DG-6-P) may therefore have limited access to glucose-6-phosphatase (G-6-Pase), and transport of the DG-6-P across the endoplasmic reticular membrane may be rate limiting to its dephosphorylation. To take this compartmentation into account, a five-rate constant (5K) model was developed to describe the kinetic behavior of 2-deoxyglucose (DG) and its phosphorylated product in brain. Loss of DG-6-P was modeled as a two-step process: (a) transfer of DG-6-P from the cytosol into the cisterns of the endoplasmic reticulum; (b) hydrolysis of DG-6-P by G-6-Pase and subsequent return of the free DG to the precursor pool. Local CMRglc (LCMRglc) was calculated in the rat on the basis of this model and compared with values calculated on the basis of the three-rate constant (3K) and the four–rate constant (4K) models of the DG method. The results show that under normal physiological conditions all three models yield values of LCMRglc that are essentially equivalent for experimental periods between 25 and 45 min. Therefore, the simplest model, the 3K model, is sufficient. For experimental periods from 60 to 120 min, the 4K and 5K models do not correct completely for loss of product, but the 5K model does yield estimates of LCMRglc that are closer to the values at 45 min than those obtained with the 3K and 4K models.

Evidence for the Participation of a Cytosolic NADP+‐Dependent Oxidoreductase in the Catabolism of γ‐Hydroxybutyrate In Vivo

Abstract: The concentration of γ‐hydroxybutyrate (GHB) in brain, kidney, and muscle as well as the clearance of [1‐14C]GHB in plasma have been found to be altered by the administration of a number of metabolic intermediates and drugs that inhibit the NADP+‐dependent oxidoreductase, “GHB dehydrogenase,” an enzyme that catalyzes the oxidation of GHB to succinic semialdehyde. Administration of valproate, salicylate, and phenylacetate, all inhibitors of GHB dehydrogenase, significantly increased the concentration of GHB in brain; salicylate increased GHB concentration in kidney, and α‐ketoisocaproate increased GHB levels in kidney and muscle. The half‐life of [1‐14C]GHB in plasma was decreased by D‐glucuronate, a compound that stimulates the oxidation of GHB by this enzyme and was increased by a competitive substrate of the enzyme, L‐gulonate. The results of these experiments suggest a role for GHB dehydrogenase in the regulation of tissue levels of endogenous GHB.