In the past few years, considerable evidence has been presented indicating that sulfobromophthalein sodium (BSP) is metabolized in the liver (1)(2)(3)(4)(5)(6). Recently, we have demonstrated that the major pathway of BSP metabolism in man and in the rat involves conjugation of BSP with.the tripeptide glutathione (5). Similar results have been obtained by Javitt and his associates in the dog (6). Grodsky, Carbone and Fanska concluded "that BSP is excreted at least in part as a mercaptide with cysteine or the peptide glutathione," in man (4). These latter authors are not certain about the presence of glutathione, however, since they feel that glycine and glutamic acid are possible contaminants of the BSP metabolites.The results of the present investigation disclosed three critical features of hepatic BSP metabolism; first, an enzyme is described, identified in liver, which catalyzes the conjugation of BSP and glutathione; second, glutathione is shown to be the preferred substrate for the enzyme; finally, it is demonstrated that 1 mole of bromide ion is released from BSP for each mole of BSP-glutathione formed. METHODS1. Preparation of liver homogenates and subcellular fractions. Adult, male, Sprague-Dawley rats were stunned by a blow on the head, their throats cut, and their livers perfused through the portal vein with 20 ml of ice-cold phosphate buffer, 0.1 M, pH 7.8. The livers were then excised and placed in cold buffer to cool. After weighing, the livers were homogenized in a Dounce homogenizer (7) with a volume of phosphate buffer equal to the weight of the liver. The resulting homogenate *This work was supported by research grants from the United States Public Health Service [H-3439], and the American Heart Association. Part of this work has appeared in abstract form (J. clin. Invest. 1960, 39, 978) and was presented at the annual meeting of the American Association for the Study of Liver Diseases, Chicago, Ill., November, 1960. tEstablished Investigator of the American Heart Association.was centrifuged at 800 G for 10 minutes at -10 C.The supernatant containing the broken liver cells minus nuclei and strands of connective tissue was called homogenate and was used as follows. For preparation of subcellular fractions, mitochondria were spun at 12,800 G for 10 minutes at -10 C in a Servall refrigerated centrifuge. Microsomes were then separated from the resulting supernatant by centrifuging at 144,000 G for 30 minutes at 00 C in a Spinco model L ultracentrifuge.The mitochondria and microsomes were washed once in ice-cold phosphate buffer, recentrifuged at 12,800 G for 10 minutes, and at 144,000 G for 30 minutes, respectively, and after discarding the wash, were resuspended in a volume of phosphate buffer equivalent to the volume of the homogenate from which they were obtained. The fraction remaining after removal of microsomes was called the supernatant. A portion of the original homogenate was boiled for 5 minutes at 1000 C. It was then centrifuged at 144,000 G for 30 minutes. The resulting supernatant ...

Most of the sulfobromophthalein sodium (BSP) appearing in the bile of man, rat, dog, sheep and cat differs chromatographically from the free BSP originally administered (1-4). The altered BSP compounds in man, rat and dog appear to be anmino acid conjugates (2-4). Thus, after hydrolysis of the major BSP compounds with 6 N HC1, several amino acids have been detected by filter paper chromatography. Glycine, glutamic acid and alanine were identified in man (2, 3), glycine and glutamic acid in the rat (2) and glycine in the dog (3).Recently, in a preliminary report, Javitt. Wheeler, Baker and Ramiios (5) suggested that BSP appears in bile of the dog as a conjugate of glutathione. These authors found glycine, glutamic acid and evidence of free sulfhydryl groups, as miianifested by a positive sodium azide-iodine reaction, after alkaline hydrolysis of the major BSP conjugates. Occasionally, cysteine was detectable. BSP conjugates formed in vitro by admixture of BSP and glutathione were identical electrophoretically and chromatographically with conjugates found in bile. Cysteine has also been identified in human BSP conjugates by Grodsky, Carbone and Fanska (4 acid as possible contaminants of the BSP metabolites.In the present report, evidence will be presented that BSP is conjugated with glutathione in the rat. Furthermore, it will be demonstrated that the linkage of glutathione to BSP occurs through the sulfhydryl group of cysteine. MATERIALS AND METHODSFour to 5 mg of S35-labeled reduced glutathione 1 which contained 20 to 30 /Ac of radioactive sulfur was injected intraperitoneally into 7 rats. Two rats received approximately 5 mg of S35-labeled L-cystine 1 containing 25 tuc of radioactivity. Ten uc of inorganic S'604 in normal saline wxas administered to an additional 2 rats. Two to 4 hours later, polyethylene tubing was inserted into the commoni bile duct and a control bile sample was collected for 45 minutes. BSP in amounts of 4 to 5 mg per 100 g body weight was then in;jected intravenously and additional bile was collected over the ensuing 45 minutes. In four experiments, S35-labeled BSP 2 was given intravenously and bile was collected for 3 hours after the common bile duct was intubated.Descending chromatograms of bile were made onl Whatman no. 1 filter paper strips. The distribution of radioactivity and of BSP oni the chromatograms was then determined. The chromatographic techniques and the methods of quantitatinig radioactivity and BSP color on chromatograms have been described in detail in a previous publication (2). Briefly, two adjacent strips 1 to 1.5 cm wide were cut longitudinally from the chromatograms. After sectioning at 1 cm intervals, radioactivity was measured on one strip by placing each segment of paper in a counting bottle containing a phosphor and counting in a liquid scintillation counter. The distribution of BSP on the adjacent strip was determined by eluting the BSP contained in each segment of paper into 0.1 N potassium hydroxide and measuring the optical density of the eluate in a Beckman ...

Sulfobromophthalein sodium, hereafter referred to as BSP, has frequently been employed to appraise hepatic function in the newborn infant (1-7). With but one exception (2), many studies have demonstrated delayed disappearance of BSP from blood and increased BSP retention in both the full-term and premature infant in the immediate postpartum period (1, 3-7). Subsequently, the rate of removal of BSP from blood gradually increased, and within a few weeks after birth, approached values found in older children and adults. In general, the impairment of BSP clearance in the first few days after delivery has been attributed to immaturity, and the gradual improvement to maturation of the factors involved in the hepatic removal of BSP from blood and the subsequent excretion of BSP into bile.Based on a considerable body of evidence which has been accumulated within the past few years (8-15), it is now known that a major fraction of the BSP which is removed from blood and excreted into bile undergoes metabolic transformation within the liver. There appears to be general agreement that the major pathway of BSP metabolism in man, rat, dog, and other species involves conjugation with the tripeptide glutathione in thioether linkage (12)(13)(14)(15). Recently, we identified an enzyme in the soluble supernatant fraction of liver that catalyzes BSP-glutathione conjugation (15). During conjugation, whether enzymecatalyzed or not, bromine is released from BSP, and the sulfur group of glutathione attaches to BSP at the site of bromine removal (13, 15). * This work was supported by research grants from the United States Public Health Service (H-3439), and the American Heart Association. Part of this work has appeared in abstract form (J. clin. Invest. 1961(J. clin. Invest. , 40, 1030 and was presented at the annual meeting of the American Association for the Study of Liver Diseases, Chicago, Ill., November, 1960. t Established Investigator of the American Heart Association.Glutathione appears to be the only necessary substrate for BSP conjugation. No requirement for cofactor has been demonstrated (15).The objective of the present study was to determine if the initial delay, then improvement in BSP clearance observed in the neonatal period, might be related to alterations in BSP metabolism. To achieve this, the BSP-glutathione conjugating system was appraised by assaying conjugating enzyme activity and measuring glutathione content of livers obtained from rats at various stages of development. METHODSESP-glutathione conjugating enzyme activity and glutathione content were measured in livers of SpragueDawley rats of various ages. In assigning an age to fetuses in utero, it was assumed that the gestation period of the rat was 21 days from the date of fertilization. The animals were killed by a blow on the head, their throats cut, the livers removed rapidly, chilled, and weighed. Livers obtained from several litter mates of the younger rats were pooled in order to provide enough tissue for duplicate measurements.1. Incubation procedur...