AND PLASMA IN THE RAT. By D. S. ROBINSON and

THE ROLE OF ALBUMIN IN THE INTERACTION OF CHYLE AND PLASMA IN THE RAT. By D. S. ROBINSON and J. E. FRENCH. From the Sir William Dunn School of Pathology, Oxford. (Received for publication 27th July 1953.) WHEN rat chyle is added in vitro to the plasma of rats which have received an intravenous injection of heparin, there is clearing of the added particulate fat as shown by a progressive fall in the turbidity of the plasma [French, Robinson and Florey, 1953]. The rate at which this change occurs is determined by the concentration in the plasma of a factor formed following the injection of heparin. The extent of clearing, however, is independent of this heparin factor, and is determined by a second component present in normal as well as in heparinised plasma. This paper deals with investigations by plasma fractionation of this. second component of the clearing system. METHODS. Male Wistar strain albino rats, weighing from 240 g. to 300 g. each, were used. The techniques of chyle collection, of obtaining heparinised and normal plasma, and of turbidity measurement, and a definition of the terms clearing reaction, heparinised plasma, heparin factor and normal plasma factor, have already been given [French et al., 1953]. The dose of heparin used throughout was 200 units (Pularin-Evans) per kg. of body weight. The clearing reaction has been carried out at room temperature (16 C.). Fractions of rat plasma were prepared by the addition of solid ammonium sulphate at ph 7 to plasma diluted to six times its normal volume with 0-85 per cent sodium chloride solution. The fraction precipitated at 50 per cent saturation is referred to as the globulin fraction, and at 50 to 70 per cent saturation as the albumin fraction. Each fraction as recovered was dialysed at 40 C. against two changes of 0-3 per cent sodium citrate solution at ph 7, and sodium chloride then added to a concentration of 0-85 per cent. Before analysis each fraction was centrifuged and any precipitate discarded. In experiments to confirm the nature of the plasma factor, morehighly purified samples of plasma albumin were used. We are indebted 233

234 Robinson and French to Dr. R. A. Kekwick of the Lister Institute for one of these, containing by electrophoretic analysis 94 per cent albumin and 6 per cent fi globulin, and to Dr. J. T. Edsall for two others, one prepared by the ethanol fractionation procedure and the other a sample of the mercury dimer of mercaptalbumin [Hughes, 1947]. Protein nitrogen was estimated by the micro-kjeldahl method [Campbell and Hanna, 1937]. Analyses of total fatty acids were based on the titration method of Stoddard and Drury [1929], as modified by Man and Gildea [1932-33]. Free fatty acid was estimated by carrying out the Stoddard and Drury procedure but with the omission of any saponification step. Control analyses with stearic acid, glyceryl tristearin and mixtures of the two showed that, under these experimental conditions, there was less than two per cent hydrolysis of the glyceryl tristearin and complete recovery of added fatty acid: nor was a sample of lecithin hydrolysed under these conditions. The analyses for free fatty acid and for total fatty acid are considered accurate to + 5 per cent. RESULTS. The Nature of the Plasma Factor in the Clearing Reaction. Fig. 1 shows the increase in clearing capacity, as measured by change in turbidity, which follows the addition of equivalent amounts of different plasma fractions to heparinised plasma. There is increased clearing of added chyle in the tubes which contain either the additional normal plasma, whether dialysed or not, or the additional albumin fraction of normal plasma. The increase has occurred to an equal degree in each case. The globulin fraction, on the other hand, has caused no increase in the clearing capacity. Fig. 2 shows the results of a similar experiment in which the more highly purified plasma albumin fractions were added to the clearing system. Each albumin fraction has enhanced clearing in proportion to the amount added. This would indicate that the factor in normal plasma which controls the extent of clearing in the in vitro system is the plasma albumin, and not any additional component of the cruder fractions. The Distribution of Lipid in Protein Fractions following Clearing in vitro. Several in vitro systems were studied. In the first, 0 5 ml. chyle (17.5 mg. fatty acid per ml.) was added to 32 ml. heparinised rat plasma. This was less than the maximal amount of chvle which could be cleared by the system, so that when the reaction was complete, the original clarity of the plasma had been restored. Normal rat plasma, 30 ml., was used as a control. The globulin and albumin fractions obtained

Role of Albumin in Interaction of Chyle and Plasma in Rat 235 FIG. 1.-The increase in clearing capacity following the addition of plasma fractions in equivalent amounts to heparinised plasma. To 02 ml. of heparinised plasma and 0-2 ml. of 0-167 M phosphate buffer solution, ph 7 0, was added 05 ml. 0-85 per cent sodium chloride solution (A), 0-5 ml. plasma globulin fraction (B), 0 5 ml. normal plasma (C), 0.5 ml. dialysed normal plasma (D), or 0-5 ml. plasma albumin fraction (E). Excess chyle was added to each sample. FIG. 2.-The increase in clearing capacity following the addition of equivalent quantities of plasma albumin preparations to heparinised plasma. To 0*3 ml. of heparinised plasma and 0-2 ml. of 0-167 M phosphate buffer solution, ph 7i0, were added 0-5 ml. of a 0-85 per cent sodium chloride solution (A), 0-5 ml. of a 5 per cent solution of human plasma albumin (Kekwick) (B), 0*5 ml. of a 5 per cent solution of bovine plasma albumin (Edsall) (C), 0-5 ml. of a 5 per cent solution of the mercury dimer of human mercaptalbumin (D). Each of the albumin preparations was made up in 0-3 per cent sodium citrate solution and excess chyle added to each sample.

236 Robinson and French from each were analysed for total fatty acids. in Table I. The results are shown TABLE I.-DISTRIBUTION OF LIPIDS IN PLASMA FOLLOWING CLEARING in vitro. (a) Using a quantity of chyle which could be completely cleared. Mg. of fatty acid per ml. of original plasma. Group., ^ Globulin. Albumin. Test... 034 057 Control.. 030 031 (b) Using excess chyle. Test... 040 073 Control.. 0-39 0*33 The test plasma, when compared with the control, contains an excess of lipid in the albumin fraction only. The lipid which has become soluble during the clearing reaction is therefore associated entirely with this fraction. Based on protein nitrogen analysis of the albumin fraction, an excess of 3x1 molecules of fatty acid was associated with each albumin molecule, assuming a molecular weight of 70,000 for albumin and 277 for the fatty acid. There was good agreement between the quantity of chyle added (8.75 mg.) and that recovered in association with the albumin (8-32 mg.). Thus all the lipid cleared remained with albumin throughout the fractionation procedure. In the second system, 1-48 ml. chyle was added to 46 ml. heparinised rat plasma and the clearing reaction followed turbidimetrically until it had slowed to a very low rate. Since the quantity of chyle added was in excess of that which could be cleared by this volume of heparinised plasma, the plasma had a residual turbidity. This residual turbidity was balanced in 42 ml. of a control normal plasma by the addition of chyle. Both control and test plasmas were spun at 23,000 g for two hours at 00 C. to separate the residual particulate lipid as a cream layer. The infranatants were then recovered as free as possible from this lipid layer and globulin and albumin fractions prepared from each. These were analysed for total fatty acids. The results are shown in Table I. The heparinised plasma again contained an excess of lipid which was confined to the albumin fraction. In this case 4-8 molecules of fatty acid were associated with each albumin molecule. In other similar experiments, where clearing was allowed to proceed to its maximum extent, an excess of 5-7 to 6-2 molecules of fatty acid was found in association with each molecule of albumin. The lipid contents of the globulin fractions of both test and control are somewhat higher than those quoted for the previous experiment.

Role of Albumin in Interaction of Chyle and Plasma in Rat 237 This is because it was not possible to remove the residual turbidity of the test plasma completely by the method of centrifugation used, and the particulate lipid which remained in the infranatant was recovered with the globulin fraction. It is for this reason that the residual turbidity in the test had to be balanced in the control by addition of chyle before carrying out the centrifugation and fractionation. The Nature of the Lipid associated with Plasma Albumin following Clearing in vitro. It has been shown that in the in vitro clearing systems described, the final acceptor of the lipid was plasma albumin. An experiment was therefore carried out to determine the form in which this lipid was combined. The accepting ability of the system was enhanced by carrying out the reaction in the presence of an excess of the albumin provided by Dr. Kekwick. Chyle, 1-5 ml., was added to a system containing 5 0 ml. heparinised rat plasma, 4 0 ml. phosphate buffer, 0-166 M, ph 7 0, and 16 ml. 5 per cent plasma albumin in 0-85 per cent sodium chloride solution. The clearing reaction was followed until near completion as shown by a marked fall in the rate. The residual turbidity was then balanced in a control sample of the same composition by the addition of chyle. Both control and test plasmas were spun at 23,000 g for two hours at 00 C. to separate the residual particulate lipid as a cream layer. The infranatants were recovered as free as possible from this cream layer and separated into globulin and albumin fractions. Aliquots of each were analysed both for total fatty acids and for free fatty acids. The results are shown in Table II. TABLE II.-DISTRIBUTION OF LIPIDS IN PLASMA FOLLOWING CLEARING in vitro. The reaction has been carried out with albumin in excess. Mg. of fatty acid per ml. of original system. Group. Test Total. Free. Globulin.. 010 005 Albumin.. 0-92 094 Control Globulin.. 004 004 Albumin.. 015 008 The test system contained a large excess of lipid associated with the albumin fraction. On the basis of protein nitrogen analysis of the albumin fraction, 5-0 molecules of fatty acid were associated with each molecule of plasma albumin. Moreover, all of this albumin-associated VOL. XXXVIII, NO. 4.-1953. 17

238 Robinson and French lipid was in the form of free fatty acid. The small quantity of excess lipid in the globulin test fraction was present almost entirely as neutral fat. This artificial system was designed to exaggerate the accepting role of the plasma albumin. However, in the previously quoted in vitro systems, small aliquots of each globulin and albumin fraction were analysed for free fatty acid as well as for total fatty acid, and the results showed that in these systems also the excess lipid was combined as free fatty acid. DIscussIoN. When chyle is added to heparinised plasma in vitro the amount of chyle which can be cleared is determined by the quantity of plasma albumin present. This suggests that the albumin may function as an acceptor in the clearing reaction, and such a role has been confirmed by the quantitative recovery of the cleared lipid from the albumin fraction of the plasma. The recovery of the added lipid as free fatty acid makes it necessary to suppose that hydrolysis of neutral fat is an essential part of the clearing reaction; and indeed Shore, Nichols and Freeman [1953] have recently obtained evidence that heparinised plasma has the ability to liberate fatty acid from egg lipoprotein preparations. It is now recognised that plasma albumin can combine with a variety of negatively charged molecules [Klotz, Walker and Pivan, 1946; Putnam, 1948; Goldstein, 1949; Karush, 1950; Teresi, 1950; Glassman, 1950-51; Lewin, 1951], and, in particular, association between fatty acids and albumin has been demonstrated [Boyer, Lum, Ballou, Luck and Rice, 1946; Luck, 1949; Westphal, Stets and Priest, 1953]. The determination of the extent of the clearing reaction by the quantity of plasma albumin present in the system is an expression of the ability of the albumin molecule to combine with a fixed number of fatty acid molecules. The value of 5-7 to 6-2 molecules fatty acid in excess per molecule albumin cannot be considered too critically as it relates solelv to the conditions of the present experiments in vitro. Recent isotope studies have suggested that phospholipid is not the major vehicle for fat transport in the plasma [Goldman, Chaikoff, Reinhardt, Entenman and Dauben, 1950; Harper, Neal and Hlavicek, 1953; Pihl and Bloch, 1950]. Bennhold [1932] considered that the function of plasma proteins as carriers of small molecules might be exceedingly important. The present results give some support to the possibility that plasma albumin may act as a carrier of lipid as fatty acid in the blood.

Role of Albumin in Interaction of Chyle and Plasma in Rat 239 SUMMARY. 1. When chyle is added to the plasma of rats which have received an intravenous injection of heparin, the extent of clearing is determined by the quantity of plasma albumin present. 2. After clearing, the lipid which has become soluble can be recovered quantitatively from the albumin fraction of the plasma proteins in the form of fatty acid. ACKNOWLEDGMENTS. The authors wish to thank Mr. H. W. Wheal and Mr. M. B. Oldfield for technical assistance. REFERENCES. BENNHOLD, H. (1932). Ergebn. inn. Med. Kinderheilk, 42, 273. BOYER, P. D., LUM, F. G., BALLOU, G. A., LUCK, J. M., and RICE, R. G. (1946). J. biol. Chem. 162, 181. CAMPBELL, W. R., and HANNA, M. I. (1937). J. biol. Chem. 119, 1. FRENCH, J. E., ROBINSON, D. S., and FLOREY, H. W. (1953). Quart. J. exp. Physiol. 38, 101. GLASSMAN, H. N. (1950-51). Ann. N.Y. Acad. Sci. 53, 91. GOLDMAN, D. S., CHAIKOFF, I. L., REINHARDT, W. O., ENTENMAN, C., and DAUBEN, W. G. (1950). J. biol. Chem. 184, 727. GOLDSTEIN, A. (1949). Pharmacol. Rev. 1, 102. HARPER, P. V., NEAL, W. B., and HLAVICEK, G. R. (1953). Metabolism, 2, 69. HUGHES, W. L. (1947). J. Amer. chem. Soc. 69, 1836. KARUSH, F. (1950). J. Amer. chem. Soc. 72, 2705. KLOTZ, I. M., WALKER, F. M., and PIVAN, R. B. (1946). J. Amer. chem. Soc. 68, 1486. LEWIN, J. (1951). J. Amer. chem. Soc. 73, 3906. LUCK, J. M. (1949). Disc. Faraday Soc. 6, 44. MAN, E. B., and GILDEA, E. F. (1932-33). J. biol. Chem. 99, 43. PIHL, A., and BLOCH, K. (1950). J. biol. Chem. 183, 431. PUTNAM, F. W. (1948). Advanc. Protein Chem. 4, 80. SHORE, B., NICHOLS, A. V., and FREEMAN, N. K. (1953). Proc. Soc. exp. Biol. N. Y. 83, 216. STODDARD, J. L., and DRURY, P. E. (1929). J. biol. Chem. 19, 840. TERESI, J. D. (1950). J. Amer. chem. Soc. 72, 3972. WESTPHAL, U., STETS, J. F., and PRIEST, S. G. (1953). Arch. Biochem. 43, 463.