Thomas C. Butler

Metabolic Conversion of Primidone (Mysoline) to Phenobarbital.

Summary Primidone (Mysoline) is in part converted to phenobarbital by dog and by man. Concentrations of phenobarbital found in plasma of patients receiving therapeutic doses of primidone are high enough to exert a significant antiepileptic effect.

The effects of nigericin, valinomycin, and 2,4-dinitrophenol on intracellular pH, glycolysis, and K+ concentration of ehrlich ascites tumor cells

Abstract The effects of nigericin, valinomycin, 2,4-dinitrophenol, and combinations of these drugs on intracellular pH (pH i ), glycolysis, and K + concentrations of Ehrlich ascites tumor cells have been studied. All of the drugs and combinations raised extracellular pH (pH e ) and lowered pH i in non-glycolyzing cells. All drugs administered with glucose except nigericin increased the rate of glycolysis and produced lower values of pH e and pH i than did glucose alone. Nigericin caused some increase in the rate of lactate production with little if any effect on glucose utilization. The values of pH e and pH i obtained with glycolyzing cells treated with nigericin alone and in combination with valinomycin indicate effective transfer of H + from the external medium into the cells, presumably in exchange for K + . All drugs and combinations caused loss of cellular K + that was partially inhibited by glucose. The effects of nigericin and valinomycin on K + loss were additive. Those of dinitrophenol and valinomycin were not. The effects of dinitrophenol and valinomycin are attributed to depletion of ATP required for Na + -K + membrane transport. An additional effect of nigericin may be promotion of K + -H + exchange across the plasma membrane. The combination of valinomycin plus dinitrophenol does not have an effect equivalent to that of nigericin on the plasma membrane.

Effects of valinomycin, ouabain, and potassium on glycolysis and intracellular pH of Ehrlich ascites tumor cells.

SummaryBoth valinomycin and ouabain block reaccumulation of K+ by Ehrlich ascites tumor cells depleted of K+ and cause loss of K+ from high-K+ cells. Glucose largely reverses the effect of valinomycin and to a lesser extent that of ouabain.In cells depleted of K+, glucose utilization and lactate production are impaired. Neither extracellular pH (pHe) nor intracellular pH (pHi) falls to the extent seen in non-depleted glycolyzing cells. Addition of K+ to depleted cells reverses these effects. Valinomycin increases glycolysis in K+-depleted cells but to a greater extent in nondepleted or K+-repleted cells. The increase in lactate production caused by valinomycin is accompanied by a correspondingly greater fall in pHe and pHi. Valinomycin, unlike other uncoupling agents, does not abolish the pH gradient across the plasma membrane. Increased utilization of glucose resulting from addition of K+ to K+-depleted cells or addition of valinomycin either to depleted or non-depleted cells can be entirely accounted for by increased lactate production. Ouabain blocks the stimulatory effect of added K+ on K+-depleted cells and has an inhibitory effect on glycolysis in non-depleted cells. It does not obliterate the difference in glycolytic activity between K+-depleted and nondepleted cells. Ouabain does not completely block the effect of valinomycin in augmenting glycolysis in depleted or non-depleted cells. Increased accumulation of glycolytic intermediates, particularly dihydroxyacetone phosphate, is found in glycolyzing K+-depleted cells. The most marked accumulation was found in ouabain-treated K+-deficient cells.

Renal excretion of 5,5-dimethyl-2-4-oxazolidinedione (product of demethylation of trimethadione.

Summary A method is described for determination of 5,5-dimethyl-2,4-oxazolidinedione (DMO) in urine. DMO is not bound to serum albumin. In the dog DMO is excreted completely unchanged in the urine. In the dog and in man the renal clearance of DMO is much higher in alkaline urine than in acid urine. This is explained on the assumption that the compound is reabsorbed in the renal tubule by a process of passive diffusion, the tubular epithelium being permeable to the un-dissociated form and impermeable to the ionic form.

Metabolic Demethylation of 3,5,5-Trimethyl-2,4-Oxazolidinedione (Trimethadione, Tridione).∗

Summary 3,5,5-Trimethyl-2.4-oxazolidine-dione (trimethadione, Tridione) is demethylated by the dog to yield 5,5-dimethyl-2,4-oxazolidinedione. This metabolic product has been isolated from urine and identified.

Demethylation of N-methyl derivatives of barbituric acid, hydantoin, and 2,4-oxazolidinedione by rat liver microsomes

Abstract Several N-methyl derivatives of barbituric acid, hydantoin, and 2,4-oxazolidinedione were demethylated by a preparation of the microsomal and soluble fractions of a rat liver homogenate with added NADP, glucose-6-phosphate, and nicotinamide and under an atmosphere of pure oxygen. Demethylation was accompanied by production of formaldehyde. There were large differences among the substrates in their rates of demethylation. Livers of rats pretreated with phenobarbital or nikethamide had higher demethylating activity toward all the substrates than did livers of untreated rats, but the degree of augmentation of activity was quite different for the different substrates.

The effect of ketosis induced by medium chain triglycerides on intracellular pH of mouse brain.

Ketosis was induced in mice in acute and chronic regimens with the use of medium chain triglycerides (MCT). Plasma levels of y3‐hydroxybutyrate and acetoace‐tate were measured. Intracellular pH (pHj) of brain was calculated from the distribution of 5,5‐dimethyl‐2,4‐oxazolidinedione‐,4C (DMO‐,4C). When mice fed a normal diet and starved overnight were given a large dose of MCT by stomach tube, plasma levels of the ketone bodies rose and blood pH and brain pHs fell significantly. Chronic ketosis was produced by feeding ad libitum a diet high in MCT and supplementing it with two daily intragastric doses of MCT. Although elevated levels of ketone bodies were maintained throughout the day, only for short periods after the supplemental doses of MCT were blood pH and brain pHj significantly lowered. This gives evidence, consonant with the work of others, of a compensatory mechanism opposing change of brain pHj that can in effect pump hydrogen ions out of the cell when acid has entered. It is suggested that the antiepileptic effect of ketosis may depend not so much on an actual lowering of brain pHi as on the imposition of an additional demand on the proton‐pumping mechanism maintaining pHj.

Demethylation of trimethadione and metharbital by rat liver microsomal enzymes: Substrate concentration-yield relationships and competition between substrates☆

Abstract A study has been made of the relationship between substrate concentration and yield and of competition between substances in the demethylation of trimethadione (3,5,5-trimethyl-2,4-oxazolidinedione) and metharbital (5,5-diethyl-1-methyl barbituric acid) by microsomal enzymes of rat liver. The syntheses of trimethadione-14C and metharbital-14C for use as substrates are described. Trimethadione has a much lower affinity for the demethylating enzyme or enzymes than has metharbital. Each of the two substrates has an inhibitory effect on the demethylation of the other. 5,5-Dimethyl-2,4-oxazolidinedione (DMO), the product of dimethylation of trimethadione, has an inhibitory effect about equal to that of trimethadione on the demethylation of metharbital. DMO also inhibits the demethylation of trimethadione.

Acid-Labile Carbon Dioxide in Muscle Its Nature and Relationship to Intracellular pH.

Summary A portion of the acid-labile CO2 in alkaline extracts of rat muscle is not precipitable by BaCl2. Carbonate added to an extract that has been acidified, freed of CO2 by evacuation, and realkalinized is only partially precipitated. It is concluded that barium solubility is the effect of some substance in the muscle extract in inhibiting crystallization of a limited amount of BaCO3. Calculation of intracellular pH from total acid-labile CO2 appears valid.

The pH of intracellular water.

The water contained within cellular membranes comprises nearly half of the total body weight of a mammal, and it is approximately double the amount of the water situated outside cells. Of the various fluid compartments of the body, only plasma and a few minor compartments are accessible for direct and unequivocal chemical analysis. There is a striking contrast between the rather adequate knowledge of the chemistry of plasma and the much less adequate knowledge of the chemistry of the larger intracellular compartment. One of the chemical characteristics of the intracellular water that is of the greatest interest is its pH value. It is to be emphasized that pH is a practical scale of acidity defined in terms of standards, and that it does not furnish information necessary for the calculation of absolute hydrogen ion activity. Furthermore, a t physiological values of p H , the concentration of the free hydrogen ion is too low to play any significant role in physical processes. Failure to appreciate these facts has beguiled some workers, through a misguided obsession with the hydrogen ion, to the deplorable and unjustifiable practice of calculating hydrogen ion concentrations from measured pH values. The significance of pH is that it is an index of the chemical potential of protons, not only the protons existing free but also the dissociable protons incorporated in proton donor molecules. Even when free protons are present in negligible amounts, those in proton donor molecules may be in abundance. pH can be considered an expression of the ease with which protons may be released from proton donors and as an index to the ratios of proton donors to proton acceptors. Since numerous biochemical reactions involve the transfer of protons or are influenced by the availability of protons it is evident that no adequate study of such reactions can be made without knowledge of the chemical potential of the proton. Many assumptions have been made concerning the pH of intracellular water and many attempts have been made to measure it, but actual knowledge of the intracellular pH of mammalian cells has been meager and unsatisfactory. The various methods that have been used to measure intracellular pH have been reviewed by Caldwell (1956). These methods include measurements on preparations of broken cells, measurements on fluid withdrawn from cells, observations with visible indicators, measurements with micro-