MATERIAL AND METHODS Rabbit serum proteins were labelled with 131I (IBS3, The Radiochemical Centre,

Transcription

1 Quart. J. Exper. Physiol. (1966) 51, PLASMA AND BLOOD VOLUMES ESTIMATED BY THE SERUM 131I-PROTEINS METHOD IN NORMAL RABBITS OF VARYING BODY WEIGHT. By V. Bocci and A. VITI. From the Istituto di Fisiologia Generale, Universita di Siena, Italy. (Received for publication 26th November 1964) Plasma and blood volumes, haematocrit, the specific gravities of plasma and blood, and the protein content of plasma have been determined in thirty-five rabbits weighing from 585 to 364 g. As body weight increased over this range, the plasma volume (in g.) per 1 g. body weight decreased progressively from 4 94 to 3-38, and the blood volume decreased from 7-95 to 6-6 g. per 1 g. body weight. The haematocrit rose from 37-7 per cent to 41-3 per cent, plasma specific gravity from 1V37 to 1-43 g.fml., blood specific gravity from 1'46 to 1-72 g./ml., and plasma protein content from 55 to 72 mg. per ml. of plasma. Equations are calculated describing each quantity as a function of body weight within the range of body weights examined. As a preliminary to investigation of 131I-globulin metabolism in young and adult rabbits, plasma volume and protein oontent were required as a function of body weight over a wider range of body weight than has previously been reported. MATERIAL AND METHODS Rabbit serum proteins were labelled with 131I (IBS3, The Radiochemical Centre, Amersham) by the chloramine-t radioiodination procedure [Bocci, 1964 a and b]. Labelling efficiency was 83 per cent. Tungstophosphoric acid (TPA) soluble radioactivity was estimated by carrying out precipitation of the proteins with 1-7 per cent TPA in -68 N HC1 at the final concentrations, in the presence of carrier sodium iodide and bovine serum proteins. The value found was 2-1 per cent. The proteins (about 1-5 mc.) were concentrated by dialysis overnight to a volume of 5 ml. This volume was injected intravenously into a 642 g. rabbit. 4 min. were then allowed to elapse for the removal ofresidual unbound 1311 and of damaged proteins [McFarlane, 1956; Gordon, 1957; Dewey and Hunter, 196]. Thereafter, this rabbit was exsanguinated via the abdominal aorta; its serum was stored at C. and used for all experiments. The percentage of the original radioactivity recovered in its serum 1311 proteins was 26 per cent. TPA soluble radioactivity was down to 1 per cent and the specific activity was 3-1 uzc. per mg. protein. About 76 per cent of the total radioactivity was localized in the albumin region (starch gel electrophoresis as described by Smithies, 1959). Thirty-five male unansesthetized rabbits weighing between 585 g. and 364 g. were used. They were starved for 2-24 hr. before experiment and given water ad lib. A dose of serum proteins prepared as above was injected into one ear vein. A dose contained 35-4,zc. of total activity, and correction was made for loss as washings from syringe, needle and beaker. After appropriate warming of the opposite ear, three blood samples of about 1-5 ml. each were withdrawn from its vein over the period 5-16 min. following injection, and collected in tubes containing dry heparin. Five min. is considered adequate for intravascular mixing; degradation of the protein, and its transfer to an extravascular 27

2 28 Bocci and Viti pool or to the lymphatic system, are unlikely to be significant till after 16 min. [Courtice, 1943; Wasserman and Mayerson, 1951; Huggins et at., 1963]. The hsematocrit ratios were determined on heparinized blood in a Wintrobe haematocrit tube which was centrifuged in a MSE 'Magnum' centrifuge for 3 min. at a radius of 2 cm. (2 g.). A trapped plasma correction factor of 4 per cent was applied [Gregersen, 1951]. No correction factor was employed to convert venous, E E ea 'U L --e x I T i me (min) FIG. 1. Typical extrapolation curves and plasma volumes at 'zero' time. Numbers refer to rabbit body weight (g.). haematocrit to whole body haematocrit [Veall and Vetter, 1958; Dewey, 196; Berman et al., 1964]; the large range of body weight covered in the present experiments made the use of any such factor precarious. Plasma was obtained by centrifugation at 2 g. for 3 min. Plasma and blood specific gravities were determined by weighing a known volume on an analytical balance. Proteins were estimated according to the biuret method of Gornall et al. TABLE I. REPRODUCIBILITY OF PLASMA VOLUME DETERMINATIONS AS ESTIMATED BY THE SERUM 131I-PROTEINS ME:THOD. PLASMA VOLUME WAS MEASURED WITH AN INTERVAL OF 7-8 DAYS. Difference (percentage) between two determinations 3.5 Experiment Body No. weight (g.) Plasma volume (ml.) Plasma volume (g.) as percentage body weight [1949]. A crystallized sample of bovine serum albumin (Sigma Co.) was used as a standard. Known volumes of plasma were diluted with saline up to 2 ml. and radioactivity was measured in a well-type scintillation counter. Plasma volume was calculated by extrapolation to zero time from the values obtained during the period 5-16 min. after injections (fig. 1); over this period plasma radioactivity decreased more slowly in the larger rabbits, suggesting lower transcapillary loss or depressed catabolism. Reproducibility was assessed in six rabbits by repeating the measurement of plasma volume 7-8 days later, with due allowance for radioactivity (about 7 per cent) persisting from the first measurement (Table I)

3 Rabbit Plasma and Blood Volumes 29 The mean percentage difference between the two measurements was 4-3 (range 1.5 to 7.6). Total blood volume was calculated from the measured values of plasma volume and haematocrit. Data were analyzed by standard statistical procedures [Barbensi, 1962]. The standard deviation of all determinations did not exceed ±1 per cent. RESULTS Forty-one plasma volume determinations are shown in fig. 2. The volume (g.) per 1 g. body weight falls from 4.94 in a 585 g. rabbit to 3.38 in a 5.85 ~ ~ E 4.95 ' E ' , A A 41 A AA A A A A8 A * A A A A A E 35., 32-4, ; , 8.3* E 755 t >'--, >68 &5 mm Body Weight (9) FIG. 2. Correlation between rabbit body weight and plasma volume, haematocrit and blood volume. Plasma and blood volumes are expressed as g./1 g. body weight. 364 g. rabbit, and the regression line y = x was calculated and is plotted together with lines corresponding to 2 S.D. Thirty-eight blood volume determinations are shown in fig. 2. The volume (g.) per 1 g. body weight falls from 7.95 in a 585 g. rabbit to 6.6 in a 364 g. rabbit. The regression line y = x was calculated AA

4 3 Bocci and Viti and is plotted together with confidence limits as before. Thirty-eight estimations only are given, since three blood samples were mislaid; they related to rabbits weighing 121, 2735 and 335 g., and their loss may explain in part the difference between the plasma and blood volume slopes, since heematocrit and specific gravities are slowly increasing with body 1D6 *> A; a = A A.- 1 3[ !5 L E ~~ O c..; , -.,,., -,, -, - - * Body weight (g) FIG. 3. Correlation between rabbit body weight and plasma specific gravity and protein concentration. weight. In fact when the three correspondent plasma volume results were temporarily excluded the regression line was y x. Similarly determinations of hawmatoerit are shown in fig. 2, and of specific gravities and plasma proteins in fig. 3. Regression lines are: Haematocrit: y = x Plasma specific gravity: y = x Blood specific gravity: y= x Plasma proteins: y= x DIscUSSION In this investigation, plasma volume was measured by the use of screened radiodinated homologous serum proteins, the screening (see Methods) serving

5 Rabbit Plasma and Blood Volumes to prevent overestimates of plasma volume due to early capture [Gordon, 1957] of denatured labelled protein by the liver. The values for blood volume here reported may be slightly too high, since no correction has been applied for the difference between venous and whole body hawmatocrit (see Methods). However, certain previous workers [Went and Drinker, 1929; Kunde et al., 1932] reported considerably higher values for rabbit blood volume than those given here. They used Vital Red, and the resultant errors are discussed by Ancill [1956]. Armin et al. [1952] found an average blood volume of ml./kg. body weight in brown rabbits of a mean body weight of 2.77 kg.; this figure can be converted (by using our determinations of the specific gravity of blood) to 6.89 g./1 g. body weight, which is close to 6.65, our mean value (for rabbits of mixed breed). Courtice [1943] found blood volume proportional to body weight, but used only one sample, drawn 6 min. after injection, and examined a weight range more limited than ours. The present results show a decline in blood and plasma volumes per 1 g. body weight as body weight increases. Similar declines were shown by Kunde et al. [1932] in the rabbit from age 3 weeks to age between 5 and 28 weeks; by Constable [1963] in guinea pigs, and by Ormond and Rivera-Velez [1965] in the male rat. ACKNOWLEDGMENTS This work was supported in part by a grant from the Consiglio Nazionale delle Ricerche. The helpful technical assistance of Mr. M. Fedeli and Mr. A. Vanni is gratefully acknowledged. 31 REFERENCES ANcILL, R. J. (1956). 'The blood volume of the normal guinea-pig', J. Physiol. 132, ARMIN, J., GRANT, R. T., PELS, H. and REEVE, E. B. (1952). 'The plasma, cell and blood volumes of albino rabbits as estimated by the dye (T. 1824) and 32P marked cell methods', J. Physiol. 116, BARBENsI, G. (1962). 'Metodologia statistica applicata alle scienze biologiche', Valsalva Editrice, Firenze. BERMAN, I., CARR, R. and MALONE, E. (1964). 'Determination of total blood volume from measurements of total red blood cell mass and plasma volume, using simultaneously injected isotopes', Nature, 22, Bocci, V. (1964 a). 'Marcatura di sieroproteine con 1131 mediante cloramina T', Boll. Soc. it. Biol. Sper. 4, Bocci, V. (1964 b). 'Efficient labelling of serum proteins with iodine-131 using chloramine T', Int. J. appl. Rad. Isotopes, 15, CONSTABLE, B. J. (1963). 'Changes in blood volume and blood picture during the life of the rat and guinea-pig from birth to maturity', J. Physiol. 167, COURTICE, F. C. (1943). 'The blood volume of normal animals', J. Physiol. 12, DEWEY, W. C. (196). 'Distribution and hematocrit ratios of blood in the rat', Am. J. Physiol. 198,

6 32 Bocci and Viti DEWEY, W. C. and HUNTER, J. D. (196). 'Inhomogeneity of iodinated human serum albumin as detected with rat liver', J. Appl. Physiol. 15, GORDON, A. H. (1957). 'The use of the isolated perfused liver to detect alterations to plasma proteins', Biochem. J. 66, GORNALL, A. G., BARDAWILL, C. J. and DAVID, M. M. (1949). 'Determination of serum proteins by means of the biuret reaction', J. Biol. Chem. 177, GREGERSEN, M. I. (1951). 'Blood volume', Ann. Rev. Physiol. 13, HuGGINS, R. A., SMITH, E. L. and DEAVERS, S. (1963). 'Volume distribution of Evans blue dye and iodinated albumin in the dog', Am. J. Physiol. 25, KUNDE, M. M., GREEN, M. F., CHANGNON, E. and CLARK, E. (1932). 'Variations in the blood of rabbits from birth to maturity', Am. J. Physiol. 99, McFARLANE, A. S. (1956). 'Labelling of plasma proteins with radioactive iodine', Biochem. J. 62, ORMOND, A. P., Jr. and RIVERA-VELEZ, J. M. (1965). 'Blood volume in relation to body weight of the male rat using radio-iodinated serum albumin', Proc. Soc. Exp. Biol. and Med. 118, SMITHIES,. (1959). 'An improved procedure for starch-gel electrophoresis: further variations in the serum proteins of normal individuals', Biochem. J. 71, VEALL, N. and VETTER, H. (1958). 'Radioisotope techniques in clinical research and diagnosis', 231. London: Butterworth & Co. (Publishers) Ltd. WASSERMAN, K. and MAYERSON, H. S. (1951). 'Exchange of albumin between plasma and lymph', Am. J. Physiol. 165, WENT, S. and DRINKER, C. K. (1929). 'A micromethod for determination of the absolute blood volume, with data upon the blood volume of the guinea pig, white rat, rabbit and cat', Am. J. Physiol. 88,