It has been shown recently by several research groups (1-5) that part of sulfobromophthalein (BSP), after intravenous injection into humans and animals, is excreted in the bile in a conjugated form. BSP conjugates also have been demonstrated in serum and urine (6,7). Evidence has been presented that the amino acids, glycine, glutamic acid, and cysteine, are present in the conjugates (4, 8, 9). The peptide, glutathione, appears to be conjugated with BSP (8,10,11). The question as to how important conjugation is for BSP excretion has not been settled so far. It is clear, however, that conjugation is not essential for BSP secretion and that at least part of the BSP can be secreted in the free form. In liver disease the relative percentage of free BSP and various conjugates in bile, serum, and urine differs from the normal (6,7,12).Because the elimination of BSP is retarded in both full term (13-15) and premature infants (16-19) it seemed of interest to investigate the extent of BSP conjugation in these age groups. Because of the insufficient conjugation that leads to the diminished bilirubin excretion after birth (20, 21), a similar mechanism for the inadequate BSP clearance was considered possible.
METHODSThe appearance of BSP conjugates in the serum was studied by chromatograms from 10 newborn infants, 2 to 9 days old, with body weights between 2.5 and 4 kg; and from 13 children, aged 2.5 to 15.5 years. After intravenous injection of 5 mg of BSP per kg body weight, blood samples were drawn at designated intervals, generally 3, 15, 30, and 45 minutes and sometimes later. In 7 newborn infants and 7 children duodenal fluid also was obtained and chromatographed. Starting with the BSP injection, the collecting tubes were changed generally every 15 minutes in the older children and every 30 minutes in the newborn infants, in an attempt to follow the sequence of BSP conjugation in the bile. When feasible, bile collection was continued for about 2 to 3 hours until 5 or 6 consecutive samples had been obtained.Before proceeding with the chromatographic separation, the concentration of total BSP was measured in samples of serum and suitably diluted bile by the method of Hofmann and Oettel (22). In preparing the plasma and bile samples for BSP chromatography the procedure of Carbone, Grodsky and Hjelte (6) was followed. One to 2 ml of serum or a sufficient amount of duodenal juice was extracted with 3 times the volume of acetone. More than 90 per cent of the BSP present is recovered in this way. The acetone extract was taken to dryness by a stream of air in a water bath at 40'C, redissolved in water (0.2 ml for each ml of the initial sample), and chromatographed on Whatman 3MM paper in an ascending system using tert-butanol: water (1.73: 1, vol/vol) for about 15 to 20 hours. Occasionally a system of n-butanol: glacial acetic acid: water (8: 2: 2, vol/vol) was used with Whatman no. 1 paper.The different bands on the chromatograms were identified by a purple color upon exposure to ammonia fumes. Of the 23 serum chromatograms, ...
