Disparity between phenol red and diodrast clearances in the dog.

The clearance of radioiodinated Diodrast and that of phenol red were compared in anesthetized dogs in an attempt to determine the reason why the former is roughly twice the latter, even though the two substances are secreted by the same mechanism. The infusion of sodium acetate increases both clearances but does not alter the ratio of the two. Hence availability of substrate is not likely to be a significant factor in restricting the clearance of phenol red relative to Diodrast. Replacement of plasma proteins, which bind phenol red, with dextran, which does not, increases the clearance of phenol red relative to Diodrast. This increase in clearance ratio is a result of an increase both in the quantity of dye filtered and in the quantity secreted. These experiments show that either rate of diffusion of free phenol red from peritubular capillaries into tubular cells or rate of dissociation of phenol red bound by plasma proteins is a limiting factor in secretory transport. However, the Diodrast clearance exceeds the phenol red clearance in isolated kidneys perfused with dextran solutions in which all dye is free. Thus, factors other than protein binding play some role in causing disparity of clearances. TIS GENERALLYBELIEVED thatphenolred, I We have investigated both hypotheses in Diodrast and para-aminohippurate are anesthetized dogs. In order to avoid any secreted by a single mechanism in the possibility of competitive inhibition of secrerenal tubular cells. However, even at low tory transport, the clearance of radioiodinated plasma levels, the clearance of phenol red is Diodrast has been used as a standard of considerably lower than that of the other two reference. Utilizing a well-type scintillation substances (1-3). Shideman, Rathbun and counter, activity was such that accurate Stoneman (4) observed an increase in phenol clearances could be measured at plasma conred clearance after the intravenous infusion of sodium acetate and maintained that availability of acetate might well be the rate limiting factor in the secretory transport of phenol red. Smith (s), on the other hand, suggested that centrations of IO-~ to IO-~ mg % Diodrast. The results of our investigation show that the availability of acetate is not a significant factor in determining the relatively low phenol red/Diodrast clearance ratio. In contrast, this difference might be due to the extensive high protein binding of phenol red is a factor of binding of phenol red by the plasma proteins, significance and contributes to the low ratios retarding the diffusion of the dye from the observed normally. However, other still unperitubular capillaries and reducing its extraction relative to that of a less bound substance such as Diodrast or para-aminohippurate. know plete n factors are involved for even in the comabsence of plasma proteins, i.e., i n experiments on isolated dextran perfused kidneys, the Dhenol red clearance is only about threeReceived for publication January 3, 1956. 1 Aided by research grants from the Life Insurance quarters that of Diodrast. d Medical Research Fund and the National Heart Institute, National Institutes of Health. METHODS 2 Rockefeller Foundation Fellow (19$+-1955) from Dog Experiments. Clearance observations, prior the Dept. of Physiology, University of G-ijttingen, to and following plasmapheresis, were made on monGermany. Present address: Physiologische Abteilung, grel female dogs, anesthetized with sodium pentoMedizinische Forschungsanstalt, Max-Planck-Gesellbarbital. Plasma levels of creatinine, phenol red and schaft, Giittingen, Germany. 318 radioiodinated Diodrast were stabilized by constant by 10.0.33.4 on July 0, 2017 http://ajple.physiology.org/ D ow nladed fom PHENOL RED AND DIODRAST CLEARANCES 39 intravenous infusion. Blood samples were obtained at the mid-point of each period from an indwelling needle in the femoral artery and urines were collected by catheter and bladder washout. The following solution was used for plasmapheresis: 5% dextran,3 0.637~ NaCl, 0.0377~ KC], 0.25% NaHC03, 0.1% glucose, plus phenol red, creatinine and radioiodinated Diodrast in appropriate amounts. Heparin was used as an anticoagulant both in an intravenous dose prior to the start of plasmapheresis and added to each sample of blood as drawn. After the removal of the first 300-500 ml of blood, three-quarters of this volume of dextran solution was immediately reinfused. The removed blood was then centrifugated, the red cells suspended in an amount of dextran solution slightly less than that of the plasma and reinfused immediately after the next bleeding. A total of 1200-2000 ml blood, depending on the size of the animal, was removed and reinfused. Isolated Kidneys. Ten liters of a solution of 37” dextran in balanced salt solution (see above) containing phenol red, creatinine and radioiodinated Diodrast was equilibrated with 5% CO2 in oxygen at 37OC under a total pressure of 900 mm Hg. Thus, this solution contained about 2.5 vol, y0 02. Samples were analyzed for creatinine, phenol red and radioiodinated Diodrast. The kidneys were removed from the dog and perfused with a constant ‘arterial’ pressure of about IOO mm Hg. Total venous outflow and urine during each clearance period were collected, measured and aliquots were analyzed for creatinine, phenol red and radioiodinated Diodrast . Determination of the Free Phenol Red. For the determination of the percentage of free phenol red in relation to the plasma albumin concentration, fresh plasma (oxalated) from four dogs, undiluted and diluted with different amounts of 5% dextran in saline, was dialyzed against a bicarbonate-Ringer-solution at 37°C. During the dialysis, a hydrostatic pressure of 35 mm Hg was applied so that no appreciable shift of water into the plasma occurred. Equilibration with 5% CO2 stabilized the PH at 7.4. The equilibrium between free and bound dye can be described by the adsorption isotherm x/m = Kc1 in where x is the milligram of dye bound by m grams of albumin, c is the equilibrium concentration of free dye in milligrams per IOO ml of plasma water and K and r/n are constants (6). For dog plasma, the equilibrium constant in this equation has been determined by Shannon (I) as r/n = 0.83. Using this value, K was found to have an average value of 0.83 (range 0.73-1.03) in 27 samples. By inserting this value in the equation, the percentage of free dye (c/(c + x)) for the condition c = 1.0 mg 70 has been calculated for various values of m, thus yielding the smooth curve in figure I. The observed values for the samples of dextran-diluted plasma (albumin < 2.0 gm yO) agree sufficiently well with the calculated curve for K = 0.83 to permit the conclusion that K has the same value in the presence of dextran and that dextran does not bind phenol red. Analytical Methods. Creatinine was determined in urine and protein-free filtrates of plasma (ferric 3 We are indebted to Commercial Solvents, Inc. for the gift of a generous supply of clinical dextran powder which made these experiments possible. sulfate) by the method of Phillips (7). In experiments in which phenol red was given, 5 ml of 1% NaOH instead of 5 ml of alkaline picrate were added to a second set of the same urine dilutions and plasma filtrates. The color was read with the same filter (no. 520 in the Evelyn-calorimeter) employed in the creatinine determination and the value so obtained was subtracted from the total (creatinine + phenol red) in order to determine the true creatinine concentration. The error of the creatinine determination due to the presence of phenol red (plasma phenol red concentration < I mg yO) was less than 2ojo. Phenol red was determined by an acetone extraction method in which 3 ml of acetone were added to I ml of plasma or diluted urine. At phenol red concentrations below 0.4 mg yO, 5 ml of acetone were added to 2 ml of plasma. The mixture was shaken vigorously and centrifuged in stoppered tubes. Three drops of 10% NaOH were added and the solution recentrifuged in stoppered, matched tubes which could be inserted directly into the Coleman calorimeter. Readings were taken at awavelength of 555 mg. The plasma blank was negligible; furthermore hemolysis did not affect the determination. Total plasma proteins were determined with the microXjeldah1 method, the albumin concentration after precipitation with saturated NazSOA (0.5 ml plasma + 9.5 ml 24.5% NazS04 at 37°C). R adioiodinated Diodrast , obtained from the Abbott Laboratories, Oak Ridge, was determined in plasma and appropriately diluted urine with a well-type scintillation counter. The method of Smith et at. (8) was employed for the determination of para-aminohippurate.