Preparation of I-131-labeled human serum prealbumin and its metabolism in normal and sick patients.

Abstract

Despite some early doubts concerning its presence in plasma and its physiological function (1-4), the thyroxine-binding prealbumin of human serum (TBPA) is now known to play a significant role in the transport of thyroxine (T4) (5-8). Normally, as assessed by in vitro techniques, TBPA appears to bind at least 25 to 35%o of T4 in serum (5,6). In the serum of many patients with severe acute or chronic illness, however, the proportion of endogenous T4 in serum bound by TBPA and the T4-binding capacity of TBPA decline (8,9). The recent availability of substantial quantities of a highly purified preparation of TBPA has made possible an investigation of the in vivo metabolism of this protein in normal patients and in patients with a variety of disorders that lead to decreased binding of T4 by TBPA in serum. In addition, the cause of this decrease in T4 binding in the serum of such "sick" patients has been evaluated. A portion of the findings has been presented in abstract form (10). While these studies were in progress, Oppenheimer, Surks, Bernstein, and Smith reported similar studies in abstract form (11,12). MethodsHighly purified TBPA was prepared from plasma Fraction IV-6 (method 6) of Cohn and colleagues (13).* Submitted for publication April 9, 1965; accepted June 16, 1965. This The method of purification of the protein and details of its characterization will be described in detail elsewhere (14). Additional characterization of the specific batch of protein employed in the present studies was carried out. Solutions of TBPA (2.0 g per 100 ml), enriched with I'-labeled T4,1 were subjected to electrophoresis in acrylamide gel in a Tris-maleate buffer system,2 pH 9.2. Gel concentrations of 5.0, 7.0, and 8.0 g per 100 ml were employed, and, in some instances, two dimensional gel electrophoretic studies were conducted (15). After electrophoresis, gels were radioautographed and then stained with amido black 10 B.Iodination of TBPA. To minimize the possibility of denaturing the TBPA during the iodination process, a technique was developed for iodinating protein by a microdiffusion process. The technique, an adaptation of that described by Banerjee and Ekins (16), was first employed in tests with human serum albumin (HSA) or with purified TBPA for relatively low specific activity labeling. When the method had been standardized, it was employed for higher specific activity labeling of the TBPA used in the present studies. This was performed as follows. All glassware and buffer media employed were autoclaved before use. A 2.0 g per 100 ml solution of TBPA was prepared in 0.01 M phosphate buffer, pH 7.5, and 200 Al was pipetted into the center well of a single side-armed Warburg flask of 5.0 ml capacity. NaI' (10 to 15 mc, free of carrier iodide and reducing agent),8 together with 60 Ag of unlabeled NaI, was introduced into the main compartment of the vessel in approximately 1 ml of phosphate buffer. Five hundred Al of fresh 3% H202 4 was added to the side arm, and the vessel was closed with a greased ground-glass stopp...

Estimation of Rapidly Exchangeable Cellular Thyroxine from the Plasma Disappearance Curves of Simultaneously Administered Thyroxine-131I and Albumin-125I*

“…Plasma levels of the two iodinated proteins do not differ appreciably during the period of observation. The data indicate that the acute distribution kinetics of TBPA and albumin are similar and consonant with previous demonstrations that the space of TBPA distribution is the same as that of serum albumin (16,17 were, therefore, applied to obtain information about the distribution of this protein. As will be shown subsequently, a large fraction of administered thyroxine-'31I leaving the plasma compartment enters the liver.…”

“…The function X can be determined graphically from a plot of log (Imax-It) against t (Equation 5). From Equations 10 and 11, it follows that [17] where Z, and ZE are the pool sizes of thyroxine in compartments I and E, respectively, and r is the concentration of circulating thyroxine in plasma. It is apparent that if thyroxine-'I accumulation in the intracellular compartment were followed for a longer period and plotted on a time scale of days rather than hours, the apparent plateau level of It would be reduced to a maximal point with a subsequent downward deflection of the curve representing the net effects of thyroxine metabolism and the further distribution of thyroxine into the extracellular compartment.…”

The kinetics of distribution between plasma and liver of 131-I-labeled L-thyroxine in man: observations of subjects with normal and decreased serum thyroxine-binding globulin.

“…Studies of the distribution of labeled serum albumin in humans, however, indicate that the liver contains only a small pool of exchangeable albumin (29). The results of similar investigations have recently been reported on 131I-labeled prealbumin in which there was no evidence for selective hepatic accumulation of TBPA (30,31 (Table II). These results would indicate that TBG does not play an important role in the binding of T4 within the liver.…”

Thyroxine Turnover and Transport in Laennec's Cirrhosis of the Liver*

“…Diminished TBPA capacity, found in all of the present sera, has been shown to be associated with reduced concentration of this protein due to impaired synthesis in acute injury and in chronic illness including hepatic cirrhosis (17,18). While such evidence is not available regarding TBG, similar phenomena would seem reasonable.…”