Maturation of the Sulfobromophthalein Sodium-Glutathione Conjugating System in Rat Liver*

Abstract

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...

Alterations in Sulfobromophthalein Sodium-Removal Mechanisms From Blood During Normal Pregnancy*

“…BSP bands were identified by exposing the dried papers to ammonia vapors. BSP was eluted from each band and the concentration determined by methods described in detail in previous publications (13,14). One or two bands corresponding to BSP conjugates and a band containing free BSP were identified on the chromatograms.…”

“…The components of the BSP-glutathione conjugating system have not been examined in human liver during pregnancy. In the rat, a decrease in BSP conjugating enzyme activity with normal levels of glutathione has been found during the last 5 days of gestation (14). When liver glutathione levels have been maintained, however, a moderate decrease in enzyme activity has not resulted in a decrease in BSP Tm in the rat (32).…”

The Importance of Conjugation with Glutathione for Sulfobromophthalein Sodium (BSP) Transfer from Blood to Bile*

“…The product of BSP concentration and bile volume yields a value for total BSP excretion in bile. Distribution of BSP compounds appearing in bile was determined chromatographically by methods described in detail in previous publications (6,12). The quantity of BSP excreted as conjugated (chromatographic bands A, B, C) and free (band D) BSP was calculated as the product of total BSP excreted in bile and the per cent of total BSP appearing in each of the chromatographic bands.…”

Multiple Halothane Exposure and Hepatic Bromsulphthalein Clearance

“…al., 1958;Burnap et al, 1958;Carson et al, 1959;Griffiths and Ozguc, 1964;Joseph, 1964;Keeri-SzAt6, 1965), Retention (± S.D.) after in dogs (Brindle et al, 1957), in sheep (Hull and Reilly, 1968), res and in calves (Middleton et al, 1966 (Combes and Stakelum, 1962) to support the view that the 5 ±3 6±3 removal of B.S.P. from plasma is dependent on the simultaneous operation of (two processes: (a) uptake into a hepatic Retention (± storage compartment, and (b) excretion into bile.…”

“…It is now generally agreed (Combes and Stakelum, 1962 Two further mechanisms of interference with B.S.P. clearance merit attention.…”