The Biliary Excretion of Sulfobromophthalein Sodium (Bsp) in the Rat as a Conjugate of Glycine and Glutamic Acid *†

Abstract

Sulfobromophthalein sodium (BSP) is a phthalein dye that is removed from blood predominantly by the liver and excreted into the bile (2). Removal is impaired in the presence of hepatocellular damage, and BSP retention in blood has proved to be a sensitive index of hepatic dysfunction (3,4). Although BSP has been used in the clinical evaluation of hepatic disease for over 30 years, the precise mechanisms by which it is handled by the liver remain poorly understood.Based on a considerable body of evidence (5-14), it is currently believed that hepatic removal of BSP depends upon the simultaneous operation of at least two processes: 1) uptake of BSP by liver cells in an amount proportional to a given blood level, and 2) transfer from blood to bile by a rate-limited transfer mechanism. Recently, a chromatographic and chemical analysis of BSP as it appeared in bile was undertaken in our laboratory in order to examine the biochemical mechanisms involved in its excretion. In a preliminary communication (1), it was reported that BSP is excreted as four distinct compounds in the bile of the rat. The major excretory product was demonstrated to be a conjugate of BSP with the amino acids, glycine and glutamic acid. A more complete presentation of these and additional observations forms the basis of the present report. MATERIALS AND METHODSThe standard BSP used in these studies was the 5 per cent solution sold commercially by Hynan, Westcott and Dunning, Inc., of Baltimore, Md. Chromatographically * A preliminary report of these studies has been previously published (1). The common bile ducts of rats (Long Evans, Wistar) were cannulated with fine polyethylene tubing under ether anesthesia. Bile was allowed to drain into small bottles while the rats were gently restrained in special cages. After collection of a control sample of bile, varying doses of BSP were injected and additional bile samples were obtained. The bile samples were treated as follows:1) Quantitation of BSP in bile. Colorimetric determinations were made on a Beckman DU spectrophotometer, set at 575 mu, on appropriately diluted bile specimens to which alkali had been added. Complete recovery of BSP was obtained over the range of concentrations, 30 to 350 mg. per cent, encountered in bile in these studies.2) Chromatography of BSP compounds. Bile samples were applied 25 Iul. at a time as a band 5 inches wide on Whatman No. 1 filter paper strips (6 X 21 inches). Each 25 ,IA. volume was dried with cold air from a hair dryer before the next aliquot was added to the paper. A total of 100 to 200 ul. of bile containing 0.045 to 0.35 mg. of BSP was applied at the origin. The chromatograms were then developed for 16 to 20 hours at 22°C. in a descending system consisting of glacial acetic acid: water: n-propyl alcohol (1: 5: 10 v/v). After the papers were dried, BSP bands were identified by: 1) the development of a purplish color when exposed to ammonia vapors, and 2) their absorption in the ultraviolet range (X max = 232 mM) .3) Quantitation of BSP on chromatograms. In s...

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.…”

Biliary Transport and Hepatic Storage of Sulfo-Bromophthalein Sodium in the Unanesthetized Dog, in Normal Man, and in Patients With Hepatic Disease *

“…Although it has been shown that a major proportion of BSP appears in the bile in the form of chromotographically distinct conjugates (21)(22)(23)(24), it is apparent that conjugation is not essential for BSP secretion since a considerable amount of unconjugated BSP is also found in all species. However, in view of the constant ratio between the amount of BSP and its conjugates., which was observed during concturrent studies (22), one cannot be certain whether the BSP Tm refers to a single transport mechanism or to the sum of separate transport mechanisms for BSP and each of its conjugates.…”

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

“…BSP conjugates and free BSP are then excreted into bile. In man (5-7), the rat (6,8,9), and dog (2,5), BSP conjugates account for most of the BSP appearing in bile, approximately 75 to 85%ob in some studies. The preponderance of conjugated BSP in bile suggests that metabolized BSP is preferentially excreted.…”

“…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.…”