binding of radio-hippuran to serum protein and red blood cells

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  • Thus, five patients with normal kidney dification of the technique of Taplin et al. function (group I), three with unilateral- (1956). ly damaged kidney function (group II), Naturally, other problems too, have and two with bilaterally severely damaged been investigated in the clinical part, for kidney function (group 111) have been instance, the influence which various investigated with combined external ra- amounts of free radioactive iodide might diorenography which is the author's mo- have on the kidney function test.

    Part I

    Binding of Radio-Hippuran to serum protein and red blood cells

    The affinity of Radio-Hippuran to different blood components was investi- gated with the same technique as in the animal experiments.

    Investigation on serum protein in vitro

    Serum with normal electrophoretical picture was mixed with Radio-Hippuran in a tracer and a carrier dose. The carrier mixture contained 10 mg ortho-iodohip- purate per 100 ml serum. Both mixtures were allowcd to stand at room tempera- ture for 30-60 minutes and were then examined in a way similar to that de- scribed in Chapter V. Dialysis was per- formed for 48 hours, continued for an- other 24 hours after change of the buffer solution. Ultrafiltration was carried out for about 15 hours. In electrophoretic examinations two of the buffer solutions described in Chapter V were used: a bar- bital buffer with pH of 8.6, and a Soren- sen's phosphate buffer with pH of 7.4.

    Migration of Radio-Hippuran into the red blood cells in uitro

    A tracer dose of Radio-Hippuran was added to a freshly drawn whole blood sample (hematocrit 40 per cent) and the

    mixture gently shaken and stored a t a temperature of + 37" C. Samples were removed during the first two hours and treated as described in Chapter V.

    The influence of the in uiuo milieu

    In one patient (case 10, hematocrit 36 per cent), after the injection of Radio- Hippuran, blood samples were withdrawn at intervals, and centrifuged in hepariniz- ed glass tubes as soon as possible after collection, and the radioactivity measur- ed separately in plasma and in the red blood cells. A blood sample taken a t 3 minutes after injection of Radio-Hippuran was allowed to coagulate spontaneously, and the serum subjected to electrophoret- ic examination. The electrophoretic strips were then scanned for beta ray activity.


    Binding of Radio-Hippuran to serum pro- teins i n v i t r o and i n v i u o

    1. Protein precipitation. The radioac- tivity of the precipitate amounted respec- tively to 1.1 and 0.5 per cent of the total radioactivity in the tracer and carrier ex- periments in vitro.


  • 2. Dialysis. The ratios of serum to buf- Discussion fer Radio-Hippuran at 24 hours were 3.9 and 2.4, and at 48 hours 1.6 and 1.5 in the tracer and carrier experiments in uitro, respectively. At 48 hours, 2.2 and 1.2 per cent, and at 72 hours, 0.3 and 0.4 per cent of the radioactivity remained in the bags containing the tracer and the carrier doses of ortho-iodohippurate, re- spectively.

    3. Ultrafiltration. After 15 hours SUC- tion, 6.5 and 4.3 per cent of the radioac- tivity, respectively, remained in the tubings in the tracer and carrier experiments in

    The results of the investigations on the binding of Radio-Hippuran to human se- rum proteins in uitro and in viuo are in accordance with those found in the ani- mal experiments (Chap. V). They also agree with the findings of Bennhold et al. (1950), who observed that test agents used in examination of the renal excretory power usually moved as unbound sub- stances in free electrophoresis with barbi- tal buffer, and with the suggestions by Burbank et al. (1961). From the results in vitro and in uiuo, the conclusion can

    uitro. be drawn that Radio-Hippuran has the 4. Electrophoresis. Radio-Hippuran possibility to diffuse from blood into other

    body spaces. This fact is of importance in the interpretation of the results of the external measurements over various re- gions of the body.

    moved as a free substance, unattached to any protein, in both of the buffer solu- tions used, in the tracer and carrier ex- periments in uitro as well as in the exa- mination of serum from the patient inject- ed with Radio-Hippuran.

    Migration of Radio-Hippuran into the red blood cells i n u i t r o and i n u i u o

    At about 30 minutes after adding Ra- dio-Hippuran to whole blood in vitro, an equilibrium seemed to exist between the radioactivity in red blood cells and plas- ma. From this time on, about 22 per cent of the radioactivity in whole blood was recovered in the erythrocytes. The migra- tion rate of Radio-Hippuran to reach this equilibrium amounted to about 8 per cent per minute.

    The relation of the radioactivity in the red blood cells to that of the total blood sample in uiuo increased during the first 25 minutes and remained then at a rather constant level with a mean value of 24

    The result of the investigation on the migration of Radio-Hippuran into red blood cells in uitro and in uiuo is in agreement with that found in the animal experiments. The findings in the experi- ments in uitro did not quite agree with those of Smith et al. (1945), who tested non-radioactive ortho-iodohippurate, but agree rather well with those of Burbank et al. (1961), who used the l3lI-labe1led compound. The former and latter investi- gators found ratios, content of ortho-iodo- hippurate in red blood cells to that in plasma, of 0.49 and 0.29, respectively. The examination of the uptake of Radio- Hippuran in red blood cells in uiuo, in blood samples drawn at 25-62 minutes, showed a higher ratio than was found in vitro, but the fact that this ratio re-

    per cent, corresponding to a ratio, red blood cells to plasma, of 0.43 a t a hema- tocrit value of 45 per cent.

    mained a t a rather constant level indi- cates that no firm binding of Radio- Hippuran to the red blood cells existed.


  • Summary

    1. The binding of Radio-Hippuran to human serum proteins in uitro has been examined by protein precipitation, dialy- sis, ultrafiltration and electrophoresis, and in viuo, by electrophoresis. The investiga- tions revealed that none a t all or only very small amounts of Radio-Hippuran seem to be bound to the serum proteins.

    2 The uptake of Radio-Hippuran into human red blood cells in vitro and in viuo was investigated. The experiments in uitro revealed an uptake of Radio-Hip- puran in the red blood cells with a mean ratio, red blood cells to plasma, of 0.33. In the examination in uiuo, a rather con- stant value of this ratio, on the average, 0.43, was obtained.

    Part 2

    Clinical applications of combined radiorenography


    Solutions All solutions were kept in the dark in

    a refrigerator a t +2 C. 1. The stock solutions of Radio-Hippu-

    ran were diluted with physiologic saline so as to obtain a solution with 15-20 pC la*I per 2 ml. The concentration of ortho- iodohippurate amounted to 15-70 pg per 2 ml. The content of fraction A was be- tween 90 and 96 per cent.

    2. The stock Solutions of RISA were di- luted with physiologic saline so that they contained 5-10 pC 1311 per 2 ml.

    Apparatus for external scintillation meas- urements

    The two apparatuses for external meas- urements described in Chapter I11 were used. The collimator inserts in the animal experiment were replaced by two others: a 12 wide angle insert for radiorenogram and radiohepatogram curves and meas- urements over the heart, and a 34 wide angle insert for measurements over the urinary bladder, one foot, and the head. The outer opening of the first insert was 110 mm from the surface of the crystal,

    and had a diameter of 65 mm. The col- limating effect was checked with a point source of lalI at various distances. Thus, plateau-shaped scan-curves were obtained. At distances of 5, 10, and 15 cm these plateaus were 8, 10, and 12 cm in breadth, respectively. The outer opening of the other insert was 85 mm from the surface of the crystal, and had a diameter of 94 mm. The plateaus of the scan-curves were 13, 17, and 21 cm in breadth at distances of 5, 10, and 15 cm, respectively.

    Apparatus for radioactivity measurements of samples

    Blood samples in amounts of 5 ml were measured in the low-background well- scintillation detector described in Chap- ter 111.

    Urine samples were measured for radio- activity with a Tracerlab P-20 BQG scintillation detector with a 1x 1 solid crystal, described in Chapter 111. Urine, or dilutions thereof, and standards in amounts of 200 ml were kept in identical glass vessels, and placed 147 mm from the crystal in the symmetry line .during the measurements.

    7-2 0 1 7 6 I 9 7


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