Reduced in vitro binding of tryptophan by plasma in uremia.

Reduced in vitro binding of tryptophan by plasma in uremia. Since markedly depressed binding of the essential amino acid tryptophan to plasma proteins of fasting chronically uremic patients was previously shown, we have now determined the association constants (k) and binding sites (n) for tryptophan for whole plasma and purified albumin from normal subjects and patients on intermittent hemodialysis. We used the in vitro equilibrium dialysis technique (plasma:buffer ratio=1:25) and analyzed the data by computer with a one ligand-two binding sites model. Over a wide range of free tryptophan concentrations (0.26 to 10 mg/100 ml), binding was markedly depressed in the patients. The diminution was mainly due to a decrease of n 1 (binding capacity of the strong, first site on albumin) from the normal value of 0.74 ± 0.14 (sd) to 0.52 ± 0.15 (P 1 of 4.1 ± 1.1 for the patients and 4.6 ± 0.7 × 10 3 L/M for normal subjects were not different. In four of eight patients, binding of tryptophan at the second site was 20% of the total at low tryptophan concentrations. In contrast, binding indexes of albumin isolated from four patients by (NH 4 ) 2 SO 4 fractionation plus DEAE-Sephadex column chromatography were identical to those of albumin similarly isolated from three normal controls. We conclude that abnormal binding of tryptophan in uremia is probably not due to an intrinsic defect of albumin but to the presence of a competing solute, which is either a nondialyzable large molecule or a small solute which binds extremely tightly to albumin. On the other hand, the possibility was not excluded that abnormal albumin was present in vivo but was either lost or "normalized" during the purification procedure. This abnormality may also be the basis for the observed reduced binding of many drugs, particularly of the acidic type, to plasma proteins in chronically uremic patients. Diminution in vitro de la liaison du tryptophane au plasma au cours de l'uremie. Il a ete montre anterieurement une diminution importante de la liaison du tryptophane aux proteines plasmatiques des sujets a jeun atteints d'uremie chronique. Nous avons determine les constantes d'association (k) et les sites de liaison (n) du tryptophane au plasma total et a l'albumine purifiee de sujets normaux et de sujets soumis a l'hemodialyseiterative. Nous avons utilise la technique de la dialyse a l'equilibre (rapport plasma/tampon = 1,25) et analyse mathematiquement les resultats pour un modele a un ligand et deux sites de liaison. Pour un large eventail de concentration de tryptophane libre (0,26 a 10 mg/100 ml), la liaison est nettement diminuee chez les malades. La diminution est due essentiellement a la baisse de n 1 (capacite de liaison du premier site fort sur l'albumine) d'une valeur normale de 0,74 ± 0,14 (SD) a 0,52 ± 0,15 (P 1 de 4,1 ± 1,1 des malades et de 4,6 ± 0,7 × 10 3 L/M des normaux ne sont pas differents. Chez quatre des huite malades la liaison du tryptophane au deuxieme site est de 20% du total aux concentrations faibles de tryptophane. A l'oppose, les parametres de liaison de l'albumine isolee chez quatre malades par fractionnement avec (NH 4 ) 2 SO 4 et chromatographie sur colonne de DEAE-Sephadex sont identiques a ceux de l'albumine isolee de la meme facon chez trois sujets temoins. Nous concluons que la liaison anormale du tryptophane au cours de l'uremie n'est probablement pas due a un deficit intrinseque de l'albumine mais a la presence d'un solute competiteur qui est soit une grosse molecule non dialysable soit une petite molecule qui se lie fortement a l'albumine. D'autre part, la possibilite n'est pas exclue d'une albumine anormale presente in vivo et perdue ou "normalisee" durant la purification. Cette anomalie peut aussi etre la base de la diminution observee de la liaison de divers medicaments, particulierement du type acide, aux proteines plasmatiques chez des malades atteints d'uremie chronique.