The Distribution and Excretion of Phenobarbital William J. Waddell, Thomas C. Butler J Clin Invest. 1957;36(8):1217-1226. https://doi.org/10.1172/JCI103518. Research Article Find the latest version: https://jci.me/103518/pdf THE DISTRIBUTION AND EXCRETION OF PHENOBARBITALI By WILLIAM J. WADDELL2 AND THOMAS C. BUTLER (From the Department of Pharmacology, University of North Carolina School of Medicine, Chapel Hill, N. C.) (Submitted for publication February 25, 1957; accepted April 4, 1957) The 5,5-disubstituted barbituric acids used tion of pK' for the pH value of 7.4. The function medicinally are weak acids with ionization ex- is without a maximum. It is greater the lower ponents in a range such that their dissociation is the value of pK'. influenced by physiological changes of pH. There The pK' of phenobarbital at 370 C. and ionic is much evidence that cellular membranes in gen- strength of 0.16 (approximately the ionic strength eral are permeable to the undissociated forms of of the inorganic ions of plasma) is 7.2. Owing to weak acids and impermeable to the ionized forms the strong electron-attracting force of the phenyl (1). The experiments of Clowes, KeItch, and group in phenobarbital, this drug is a considerably Krahl (2) are interpreted as indicating that a stronger acid than the commonly used barbituric number of barbituric acid derivatives penetrate acid derivatives having only alkyl or alkenyl groups Arbacia eggs only as the undissociated molecules. in the 5-position. The drugs of the latter types The influence of urinary pH on the renal excretion for the most part have pK' values under the same of weak acids has been explained in terms of tubu- conditions in the range of 7.6 to 8.0 (5) .8 lar permeability to the undissociated species and N-methylated barbituric acid derivatives, such as impermeability to the ionic species (3, 4). hexobarbital, mephobarbital, and metharbital, have Changes in the concentration of the undissociated pK' values about 0.4 unit higher than the corre- form of a weakly acidic drug accompanying physi- sponding non-methylated compounds (6). Thio- ological changes in the pH of blood or urine can barbituric acid derivatives are stronger acids than have important effects on the distribution, excre- the analogous oxygen compounds. Thiopental has tion, and pharmacological actions of the drug. a pK' value of 7.3 at 370 C. and ionic strength of The rate of change of the concentration of un- 0.16.' Thus phenobarbital has a lower pK' value dissociated acid with pH is maximal when the pK' than any other commonly used barbituric acid of the acid equals the pH. However, of greater derivative. It is evident from Figure 1 that the interest is the proportion by which the concentra- proportional change in the concentration of undis- tion of undissociated acid changes with a change sociated form resulting from a small change in pH of pH, i.e., the rate at which the concentration of at pH 7.4 is much greater for phenobarbital than undissociated form is changing divided by that for other hypnotic barbituric acids with pK' values concentration. This function is expressed by the of 7.6 to 8.0. For instance, it is 1.8 times as. great following equation, as for barbital or amobarbital, which have pK' val- ues of 7.7. d (HA) 1 Because of the important influence that physio- d(pH) - log. 10 1OP logical shifts in pH might be expected to have on (HA) 1OpH + 10pK"1 the distribution and excretion of phenobarbital, an in which (HA) is the concentration of undissoci- 8By spectrophotometry and by potentiometric titra- ated acid. In Figure 1 is shown the proportional tion we have found higher values of pK' for several rate of change of the concentration of the undis- barbituric acid derivatives at 25° C. than did Krahl and sociated form of a weak acid with pH as a func- also lower temperature coefficients. Our directly meas- ured values at 370 C. and ionic strength of 0.16 are about I This investigation was supported in part by a re- 0.2 unit higher than the values calculated from Krahl's search grant (B-384) from the National Institute of data. Neurological Diseases and Blindness, National Institutes 4This value was determined spectrophotometrically and of Health, Public Health Service. by potentiometric titration. The value of 8.2 reported by 2Public Health Service Research Fellow of the Na- Brodie et al. (7) was obtained by titration in ethanol- tional Institute of Neurological Diseases and Blindness. water solution. 1217 1218 WILLIAM J. WADDELL AND THOMAS C. BUTLER residue was taken up in the 1 M phosphate buffer of pH 8 used in the original method (8), and the subsequent -2.0 steps of that method carried out as usual. When the specificity of this method was investigated by the pro- cedure described by Butler, Mahaffee, and Waddell (8), v -1.5- the ether/buffer partition coefficients were found to con- form closely to the values expected for phenobarbital. The methods used for the determination of phenobarbi- x v-0. tal in dog and human urine when applied to urine free of drug gave calculated values that did not exceed 5 per cent of the phenobarbital concentrations that were to be 0.5- experimentally measured in urine. The various drugs that were administered to dogs did not interfere with the determination of phenobarbital in plasma or urine. Weighed samples of 1 to 5 Gm. of tissues were ho- 10o 62 64 " i0 i72 74 f. I0 SI 14 mogenized with weighed 5-ml. portions of 1 M phos- pK phate buffer of pH 8. The homogenizer was a device FIG. 1. THE PROPORTIONAL RATE OF CHANGE OF THE consisting of sharp blades mounted on a shaft driven at CONCENTRATION OF THE UNDISsocIATED FoRM OF A WEAK high speed by an electric motor. A 2-ml. sample of the ACD WITH PH AT PH 7.4 AS IT Is RELATED TO THE PK' homogenate was weighed, 1 ml. of the buffer of pH 8 OF THE AcID was added, and the subsequent steps of the analytical pro- The dotted line is the asymptote approached by the cedure were carried out in the same way as previously function as pK' decreases. described for plasma (8). When this method was ap- plied to tissues free of drug, the calculated phenobarbital investigation has been made of these effects. It concentrations did not exceed 1 #g. per Gm. Recovery of added to tissues was complete. was anticipated that the information to be gained phenobarbital Because higher concentrations of phenobarbital were might be of value in the treatment of phenobarbital found in liver than in other tissues, the specificity of the poisoning. An investigation has also been made method as applied to liver was investigated. A homoge- of other factors influencing the renal excretion of nate of a liver sample from a dog that had received phenobarbital. phenobarbital was extracted with ether in the same man- ner as used in the first extraction step of the regular METHODS analytical procedure. The ether extract was evaporated and the residue distributed between ether and 1 M phos- Determination of phenobarbital. For dog and human phate buffer of pH 8.2 in a 30-cell countercurrent distri- plasma and for dog urine the method of Butler, Mahaffee, bution train of the type described by Craig, Hausmann, and Waddell (8) was used. By measurements of parti- Ahrens, and Harfenist (10). Twenty-two effluent frac- tion coefficients between ether and buffers, the specificity tions of ether were withdrawn from the train. Frac- of this method for plasma had previously been demon- tions 11 to 22, which would contain p-hydroxy pheno- strated. By the same procedure it has been shown to barbital. if any were present, were analyzed for that have adequate specificity when applied to the determina- compound by the method described by Butler (9). No evi- tion of phenobarbital in dog urine. This method deter- dence was found of the presence of any p-hydroxy pheno- mines bound as well as unbound phenobarbital in plasma. barbital. Fractions 1 to 10 were combined and evaporated Human urine, unlike dog urine, contains unconjugated and the residue was put through the countercurrent dis- p-hydroxy phenobarbital (9), which interferes with the tribution train, the light solvent being a mixture of 30 per determination of phenobarbital. The interfering meta- cent (v/v) ether and 70 per cent 2,2,4-trimethylpentane bolic product was separated by partitioning between ben- and the heavy solvent a 1 M phosphate buffer of pH 7.2. zene and water. To 1 ml. of urine in a glass stoppered Thirty effluent fractions of light solvent were collected. tube was added 1 ml. of 1 N HCl and 12 ml. of benzene. These were individually extracted with 4-ml. portions of The tube was shaken and centrifuged. The aqueous phase the buffer of pH 11 used in the regular analytical pro- was then transferred by pipette to a second tube, where cedure. Each buffer extract was washed with 2 ml. of it was shaken with a fresh 12-ml. portion of benzene. purified ether and the phenobarbital concentration calcu- After centrifugation, the aqueous phase was removed and lated from the difference between the absorbency of the discarded. The first benzene extract was shaken with buffer at 240 m/ and that at 260 my. In Fractions 11 to 1 ml. of 0.1 N HCI. After centrifugation, the aqueous 30 the amounts of phenobarbital measured in this way con- phase was transferred to the second benzene extract.