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A B S T R A C T The mechanism of drug-induced displacement of bilirubin from the blood into tissues was studied.

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The following agents were found to oxidize bilirubin in vitro : hemoglobin and horse-radish peroxidase (both with hydrogen peroxide), cytochrome c, xanthine oxidase, and an insoluble oxidase, present in brain and other tissues. Kinetic constants were determined. The process with hemoglobin was inhibited competitively by two product molecules. The insoluble oxidase from brain was present in mitochondria. The supernatant fraction contained an inhibitor. The oxidase was inactive in the absence of salt and was unspecifically activated by a number of salts, the activity depending upon ionic strength, irrespective of which ions were present. Reaction products included biliverdin and a yellow, diazo-negative, polar pigment with the same oxidation level as bilirubin. Schmid and Hammaker [l]in 1963 demonstrated the existence of an alternative route of bilirubin metabolism, operating in the absence of a functioning conjugating apparatus. After administration of [14C]bilirubin to a child with congenital unconjugated hyperbilirubinemia the label was recovered mainly as polar, diazo-negative substances in the bile. The present paper deals with enzymes, obtained from brain and other tissues, catalysing a similar conversion of bilirubin.The work was initiated by an incidental observation. The yellow colour of bilirubin-stained brain specimens, obtained by autopsy from a child who died with kernicterus, faded in the course of a few hours. After homogenization in saline practically no bilirubin could be found. Added bilirubin (10 p.M) was metabolized by the homogenates at 37" with velocities up to 3 nmoles/min per gram tissue. METHODS Crude Preparation of Bilirubin Oxidase from BrainGuinea pigs were killed by luxation of the neck and were bled from the carotides. The brains were removed, chilled on ice and homogenized in 20volumes of water in a Virtis "45" homogenizer for 1 min a t medium speed. The homogenate was centrifuged a t 100OOxg for 20 min at 2". The supernatant showed a bilirubin oxidizing activity due to its content of hemoglobin and cytochrome c and was discarded. The sediment was freeze-dried and showed constant activity during storage a t -20" for several weeks.

Binding equilibria of long-chain fatty acids to human serum albumin, in serum or plasma, were studied by a dialysis exchange rate technique. Palmitate was added to citrated plasma in vitro and it was observed that between six and ten palmitate molecules were bound to albumin with nearly equal affinity. Observations in vivo gave similar results in the following series: (a) in two volunteers with increased fatty acid concentrations after fasting, exercise, and a cold shower: (b) in three male volunteers in whom high concentrations of non-esterified fatty acids, up to 4.6 mM, were induced by intravenous administration of a preparation of lecithin/glycocholate mixed micelles, and (c) in 81 patients with diabetes mellitus, type I.The binding pattern of palmitate in serum or plasma is essentially different from that observed with palmitate added to buffered solutions of pure albumin when two molecules are tightly bound and about four additional molecules with lower affinity. The differences may partly be explained by the presence of chloride ions in blood plasma, reducing the affinity for binding of the first two fatty acid molecules, and partly by facilitated binding of several molecules of mixed fatty acids, as found in plasma.Binding equilibrium studies for long-chain fatty acids to serum albumin are complicated by very low ligand solubility. We have in previous work failed to demonstrate any solubility of palmitic, stearic and oleic acid at neutral pH and 37°C [l]. It is, in consequence, not possible to describe binding of these acids in terms of equilibria between free and bound ligand, as in usual binding studies. On the other hand, equilibrium with respect to transfer of an insoluble ligand from one carrier to another can be assessed quantitatively, utilizing the reserve carrier concept. The concentration of reserve albumin for binding of a ligand has previously been defined as the concentration of a purified standard albumin preparation which in buffered solution binds a trace amount of the radiolabeled ligand as tightly as it is bound in the sample [2]. The concentration of reserve albumin for binding of palmitate or stearate is thus an inverse measure of how tight the fatty acids are bound and this parameter can be measured in albumin solutions and in serum or plasma even if the fatty acids are insoluble.The aim of the present work is to investigate binding equilibria of long-chain fatty acids in human blood plasma (serum) under varying conditions and to compare the results with previous observations on binding to serum albumin in buffered solutions. MATERIALS AND METHODS Human serum albuminHuman serum albumin was obtained from AB Kabi Vitrum (Stockholm, Sweden; lot no. RFM 57). More than 95% of the total protein of this preparation is serum albumin. Fatty acidsPalmitic, oleic, linoleic and myristic acids were obtained from Fluka AG, Buchs, Switzerland. The purity of all four acids was determined as >99% by gas-liquid chromatography.[l-'4C]Palmitic acid (specific activity 58 Ci/mol) was from Amersham Intern...