injection. triglyceride of the chylomicra is hydrolyzed to free fatty acid with the

Transcription

1 FURTHER STUDIES ON THE LIPOLYTIC SYSTEM INDUCED IN PLASMA BY HEPARIN INJECTION. By D. S. ROBINSON.' From the Sir William Dunn School of Pathology, Oxford. (Received for publication 26th December 1955) WHEN rat chylomicra are added in vitro to the plasma of rats which have received an intravenous injection of heparin a progressive clearing of the added chylomicra occurs [French, Robinson and Florey, 1953]. It has been shown that this clearing is a lipolytic process in which the triglyceride of the chylomicra is hydrolyzed to free fatty acid with the formation of a soluble fatty acid-albumin complex in the plasma [Robinson and French, 1953]. The addition of heparin to plasma in vitro does not produce a lipolytic system. Thus it appears that the lipolytic system induced by heparin injection must involve the participation of some component derived from the tissues. Korn [1955] has shown that extracts of heart tissue possess lipolytic activity similar to that induced by heparin injection. However, the function of heparin in the lipolytic process remains obscure. It was hoped that heparin could be removed from lipolytic plasma obtained after heparin injection by the use of anion exchange resins carrying strong basic groups. A study of the clearing activity of such plasma after treatment with the resin might then be made in the absence of heparin. The experiments to be reported, in which chyle was used as substrate for the clearing reaction, were carried out on such plasma after it had been passed down a column of a strong anion exchange resin. The results suggest that heparin may be responsible for maintaining the stability of the lipolytic system induced by its injection. METHODS Wistar strain albino rats, fed a laboratory stock diet containing 2 per cent fat, were used. All animals were starved for 16 hours before heparin was injected. In all the experiments reported, animals were bled 10 minutes after the intravenous injection of 40 International units (i.u.) of heparin (Pularin-Evans) per kg. of body weight. The plasma obtained in this way will be referred to as heparinized plasma. Heparin solutions used throughout this study were prepared by the dilution of a stock solution (1000 i.u. per ml.) with 0-9 per cent sodium chloride solution. Alan Johnston, Lawrence and Moseley Research Fellow of the Royal Society. 195

2 196 Robinson The techniques used for collecting chyle and for obtaining the plasma were described previously [French, Robinson and Florey, 1953]. The activity of a clearing system was determined by measuring the decrease in optical density which occurred when an active system was incubated at 370 C. in the presence of added chyle [Robinson and French, 1953]. A quantity of chyle in excess of that which could be cleared by the system was added in all experiments and the maximum initial rates of clearing were measured. Free fatty acids were determined as previously described [Robinson, Jeifries and French, 1954]. Use of Ion-exchange Resin Columns.-The experiments were carried out using a column of the resin "De-Acidite FF" (Permutit Co. Ltd.). Columns of ground resin ( mesh) were set up and washed successively with 10 to 20 bed volumes of 3N sodium chloride solution, 10 to 20 bed volumes of N sodium hydroxide solution, approximately 50 bed volumes of distilled water and, finally, with 01M sodium phosphate buffer solution of ph 7-2 to 7*4, until the effluent from the column had a ph of 7*2 to 7-4. Samples of the prepared resin were set up in a room at 40 C. in glass tubes of internal diameter 1 0 cm. The length of the resin column used varied between 7 and 12 cm. The resin was retained in the tube with a small cotton wool plug. Heparinized plasma, to be applied to the column, was dialysed for 4 hours at 4 C. against a large volume of a solution containing 0 3 per cent trisodium citrate, 0 9 per cent sodium chloride and 01M sodium phosphate buffer of ph 7-2 to 7T4. There was no appreciable loss in the clearing activity of this plasma during dialysis. In general about 20 ml. of the dialysed plasma were added to the resin column in stages so that a head of about 8 cm. of plasma was maintained on the column. Effluent samples were not collected until protein appeared, as indicated by the formation of a precipitate with 10 per cent trichloroacetic acid solution. A flow rate of 2 to 4 drops of effluent per minute was maintained. The ph of the effluent was the same as that of the plasma put on to the column. The effluent was stored at 00 C. until required. RESULTS Evidence that Heparin was removed from Heparinized Plasma by a Column of the Resin "De-Acidite FF" An experiment was carried out to determine whether heparin was retained by the resin column during the passage of heparinized plasma through it. Heparinized plasma (25 ml.) was passed through a column (11 x 1 cm.)

3 Lipolytic System Induced in Plasma by Heparin Injection 197 and the effluent from the column collected and retained. Sodium phosphate buffer solution (O1M) at ph 7*4 was then added to the column until the solution flowing from it no longer contained protein. This fraction was discarded. A solution of 3M sodium chloride was next passed through the resin and two successive 20 ml. fractions were collected. These fractions were dialysed for 5 hours against 5 litres of distilled water, reduced in volume by pervaporation, and then dialysed for a further period against 0*9 per cent sodium chloride solution. The heparin content of these two fractions was assessed by the injection of suitable volumes of each into starved rats. The rats were bled after 14 minutes and the clearing activities of their plasmas determined in the usual way. From these activities an approximate estimate of the amount of heparin injected could be made from a knowledge of the clearing activity to be expected after the injection of known amounts of heparin [Robinson, Jeffries and Poole, 1955]. The equivalent of about 16 units of heparin was found to be present in the first 20 ml. fraction and about 4 units in the second. This quantity of heparin (20 units) is very approximately what might be expected to be present in the 25 ml. of heparinized plasma added to the column originally. This plasma had been obtained from 4 rats weighing 200 g. each and injected with 40 units of heparin per kg. of body weight. The Effect of Heparin on the Clearing Activity of the Effluent from a Column of the Resin "De-Acidite FF" to which Heparinized Plasma had been added The results of the previous experiment suggest that heparin was removed from heparinized plasma by the resin. The clearing activity of the effluent from a resin column to which heparinized plasma had been added was determined, therefore, in the presence and absence of added heparin. The results of the experiment are shown in fig. 1. The activity of the effluent in the absence of heparin is high, but it is raised further by adding very small quantities of heparin. Maximal increase in activity is achieved with as little as 0-25 to 0 5 jig. of heparin per ml. in the test system used. This maximal activity is very similar to that of the dialysed heparinized plasma before it was added to the column. In the experiment quoted, the activity of the heparinized plasma, measured under comparable conditions, was only 10 per cent higher than the maximal activity of the effluent with added heparin. Adding heparin to the dialysed heparinized plasma does not affect its activity. The activating effect of heparin on the clearing activity of the effluent was also observed when chyle and effluent were mixed at 00 C. before incubation at 370 C.

4 198 Robinson /ho FINAL HEPARIN CONCENTRA TION(tVg/.ml) FIG. 1.-The effect of heparin on the clearing activity of the effluent. Each test sample consisted of 0 5 ml. of a 10 per cent solution of albumin in 0O1M sodium phosphate buffer, ph 7 4, 0.05 ml. of water or of heparin, 20,l. of chyle and 0*5 ml. of the effluent. The effluent was added last to the test mixture. Clearing was followed for 20 minutes at 370 C. and is expressed as the change in optical density during that time. The Effect of Incubation of the Effluent in the Presence and Absence of added Heparin Experiments were carried out to determine whether incubation of the effluent at 370 C., in the presence and absence of added heparin, produced any change in the clearing activity when this was subsequently tested in the presence of added chyle. The results of one experiment are shown in fig. 2. It is clear that in the absence of heparin all the activity of the effluent disappears rapidly on incubation at 370 C. Adding heparin to the effluent after incubation does not restore the clearing activity. In the presence of heparin, however, the decline in activity, on incubation, is much less marked. The experiment in fig. 3 shows that this slow rate of decline in activity of the effluent in the presence of added heparin is very similar to the rate of decline in activity of untreated heparinized plasma on incubation at 370 C. Fig. 4 shows the protective action of various concentrations of heparin. A concentration of heparin as low as 0 05,ug. per ml. affords some protection when the mixture is incubated at 370 C. for 30 minutes. Between 0.5 and 1*25,ug. of heparin per ml. affords maximum protection. With shorter periods of incubation, for example 5 minutes, whereas all the activity was lost in the absence of added heparin, some protection was afforded by as little as 0005,ug. of heparin per ml.

5 Lipolytic System Induced in Plasma by Heparin Injection O.5 0-0O.3 O 25 5 /O /NCUBATION PERIOD rn). FIa. 2.-The clearing activity of the effluent after incubation for varying periods in the presence and absence of heparin. Each test sample con. sisted of 0 5 ml. of the effluent incubated at 370 C. in the absence (U) or presence (E) of 0*05 ml. of heparin (10 units per ml.). After incubation, 0-5 ml. of a 10 per cent solution of albumin in 01M sodium phosphate buffer, ph 7*4, and 20,ll. of chyle were added to each test mixture and clearing followed for 30 minutes. Activity is expressed as the change in optical density occurring in that time. The possible influence of phosphate ions, which are present in the effluent, on this protective function of heparin was investigated by studying the activity of the effluent after its dialysis for 4 hours at 0 C. against a 0 9 per cent sodium chloride solution. Such a dialysed effluent showed the same rapid decline in activity on incubation at 370 C. in the absence of heparin as did the undialysed effluent and, in the presence of heparin, this rapid decline in activity was prevented. Other Properties of the Effluent Experiments were carried out to determine whether clearing of chyle by the effluent was associated with the production of free fatty acid. Table I shows analyses of the free fatty acid content of four different test systems. When the effluent was tested immediately, slightly more

6 200 Robinson 0o Robinson6 QC) ~O2 0 7 / INCUBATI/ON PERIOD (mm). FIG. 3.-The clearing activity of: A, the effluent after incubation at 370 C. for varying periods in the presence of heparin (O), and B, heparinized plasma after incubation at 370 C. for varying periods (in). Test samples A contained 0-25 ml. of the effluent, 0-25 ml. of 01M sodium phosphate buffer solution, ph 7 4, and 01 ml. of heparin (20 units per ml.). Test samples B contained 0-15 ml. of heparinized plasma, 0 35 ml. of 01M sodium phosphate buffer solution, ph 7-4, and 01 ml. of water. After incubation, 0-5 ml. of a 10 per cent solution of albumin in 01M sodium phosphate buffer, ph 7-4, and 20 /A. of chyle were added to each test mixture and clearing followed for 15 minutes. Activity is expressed as the change in optical density occurring in that time. fatty acid was produced in the presence of added heparin than in its absence. After the effluent had been incubated at 37 C. for 10 minutes in the absence of heparin, very little fatty acid was released when the activity of the system was subsequently tested with added chyle. When the effluent was incubated for the same period in the presence of heparin, however, a good deal of free fatty acid was subsequently released from added chyle. These analyses show that the clearing of chyle by the effluent is accompanied by the release of fatty acid. Protamine and bile salts were found to inhibit completely the clearing of chyle by the effluent. This occurred at concentrations of protamine and bile salts very similar to those required to inhibit completely heparinized plasma of comparable activity. The dependence of the activity of the effluent on ph was tested, and the relationship between the activity and the ph was found to be similar to that of heparinized plasma. This relationship was not altered by the addition of heparin. Maximum rates of clearing occurred between ph 7-35 and 7-5 [cf. Robinson, Jeffries and French, 1954].

7 Lipolytic System Induced in Plasma by Heparin Injection (1) 144J q Q~ /k I.= a I I-a. a */ FINAL HEPARIN CONCENTRATION (,ijg/rnl) FIa. 4.-The clearing activity of the effluent after its incubation at 370 C. in the presence of varying concentrations of heparin. Effluent samples (0.2 ml.) were incubated in the presence of 0 05 ml. of various concentrations of heparin for 30 minutes at 370 C. To each sample 0-8 ml. of a 10 per cent solution of albumin in 0O1M sodium phosphate buffer, ph 7.4, and 20,il. of chyle were then added and clearing followed for 15 minutes. Activity is expressed as the change in optical density occurring in that time. TABLE I.-THE RELEASE OF FATTY ACID DURING THE CLEARING OF CHYLE BY FOUR TEST SYSTEMS System Effluent (Ml.) 1-8 1* Water Heparin Albumin Chyle Incubation (ml.) solution (ml.) (ml.) period (M l)(l.) (hrs.) * * * Free fatty acid released during incubation (mg.) *60 System 3 differs from system 1, and system 4 differs from system 2, only in their being incubated for 10 minutes at 370 C. before adding albumin and chyle. Albumin was added as a 10 per cent solution in 0O IM sodium phosphate buffer at ph 7.4. The heparin solution added contained 10 units of heparin per ml. Each system was analysed after 2 hours' incubation at 370 C. for its free fatty acid content [Robinson, Jeffries and French, 1954]. Evidence that the Resin Column was saturated with Heparin It is to be expected that, under given experimental conditions, a resin column of a given volume will have a limited capacity for retaining heparin. When this is exceeded, then heparin will presumably escape

8 202 Robinson from the column into the effluent. In consequence, from the experimental evidence already presented, the effluent will not lose all its clearing activity on incubation at 370 C. Table II shows that this situation actually occurred when large amounts of heparinized plasma were added to the resin column. In this case progressively greater amounts of clearing activity remained in successive effluent samples when they were incubated in the absence of any added heparin. TABLE II.-THE CLEARING ACTIVITY OF SUCCESSIVE EFFLUENT FRACTIONS BEFORE AND AFTER THEIR INCUBATION AT 370 C. FOR 15 MINUTES Length of column = 8 cm. Volume of heparinized plasma added to column =25 ml. Rate of flow =4 drops per minute. Clearing activity (change in optical Effluent Effluent density occurring in 15 minutes) number volume Before incubation After incubation 1 10 ml ,, *15 3 2,, ,, *27 5 2,, *30 Before incubation. To 0 5 ml. of a 10 per cent solution of albumin in 01M sodium phosphate buffer, ph 7*4, and 20,ul. of chyle was added 05 ml. of the effluent sample. Clearing was followed at 370 C. for 15 minutes. After incubation. Effluent samples (0.5 ml.) were incubated for 15 minutes at 370 C. Then 0a5 ml. of a 10 per cent solution of albumin in O1M sodium phosphate buffer, ph 7-4, and 20,l. of chyle were added to each sample and clearing followed at 370 C. for 15 minutes. The Stability of the Activity of the Effluent in the Presence of Chyle and the Association of the Lipolytic Activity of the Effluent with Chyle It has been shown that the activity of the effluent declines very rapidly on incubation at 370 C. in the absence of heparin. However, when its activity is measured immediately with added chyle, clearing continues for a long period. It thus appears that during clearing the enzyme system is protected from destruction by the presence of its substrate, the chylomicron. Previous studies showed that when plasma obtained from animals injected with heparin was stored at 00 C. with chyle and then centrifuged at a high speed so that a clear infranatant solution free from chylomicra was obtained, then this infranatant solution did not clear chyle at 370 C. at an appreciable rate in the presence of albumin. The lipid layer, recovered by the centrifugation procedure, was, on the other hand, cleared by albumin solutions. These results were interpreted as showing an adsorption of the lipolytic system of the heparinized plasma on to its substrate, the chylomicron [Robinson, Jeffries and Poole, 1955].

9 Lipolytic System Induced in Plasma by Heparin Injection 203 A similar association of enzyme system and substrate was shown to occur when the effluent from the resin column was stored with chyle at 00 C. In one experiment 6 ml. of the effluent were stored at 00 C. for 2 hours with 0-7 ml. of chyle and then spun at 40,000 g for 45 minutes at 00 C. The infranatant solution (0.5 ml.) to which albumin (0.5 ml.) was added, cleared added chyle at 370 C. at the rate of 0-08 optical density units per hour. A control system, consisting of 6 ml. of the effluent stored at 00 C. with chyle for 2 hours, but not centrifuged, cleared chyle, under comparable conditions in the presence of albumin, at the rate of 0-56 optical density units per hour. A sample (70 pkl.) of the lipid layer, recovered from the test effluent by the centrifugation process and suspended in 0 7 ml. of water, was cleared by albumin solutions at a rate of 0-8 optical density units per hour. These results suggest that a close association of the lipolytic system with chylomicra occurs with the effluent as it does with heparinized plasma. It is possible that the stability of the enzyme system in the effluent in the presence of chyle is due to this close association of enzyme and substrate. DIscusSION The results presented suggest that the stability of the lipolytic system in heparinized plasma in vitro is due to the presence of heparin. It has been shown that the removal of heparin from heparinized plasma by a suitable anion exchange resin yields a system which loses its activity extremely quickly on incubation at 370 C. Although there seems little doubt that the anion exchange resin column does remove heparin from heparinized plasma, there is no evidence that both free and combined heparin are removed. For this reason it cannot be claimed that the sole function of heparin in the clearing reaction in vitro lies in its ability to protect the enzyme system from destruction. It may be that only the free heparin in the plasma is retained by the resin column and that it is this free heparin which exerts a protective effect. Combined heparin may not be removed by the resin column and may still form part of the clearing system. The significance of the protective function of heparin in the production of clearing activity in vivo cannot be decided from these experiments. While it is possible that injected heparin protects a lipolytic enzyme in the plasma from destruction in vivo, the present experiments do not rule out the possibility that heparin has also a more direct role in the formation of the lipolytic system. A lipolytic system similar to that induced by injection of heparin has been demonstrated in rat plasma during fat absorption [Jeffries, 1954; Robinson, Jeffries and French, 1954; Robinson, Jeffries and Poole, 1955]. The ability of chyle to maintain the lipolytic activity of heparinized plasma, after heparin has been removed by a resin column, VOL. XLI, NO

10 204 Robinson suggests that the presence of lipid particles in plasma after a fat meal may result in a stabilization of the plasma lipolytic activity and so allow its demonstration in vitro. It may be that if the formation of an enzyme-substrate complex between lipase and lipid particle occurs [Robinson, Jeffries and Poole, 1955], such a complex is more stable than the enzyme alone. The quantities of heparin required for the protection of the lipolytic system of heparinized plasma at 370 C. after its passage down the resin column are extremely small. The absolute quantity required for full protection depends on the amount of enzyme activity present and upon the period for which the enzyme is incubated; but it is probable that an extremely sensitive method for the assay of free heparin could be devised on the basis of these experiments. The smallest amount of heparin detectable by conventional blood-clotting techniques is of the order of 10,ug. per ml., whereas 01,tg. of heparin per ml. could be easily detected in the present system. SUMMARY 1. When plasma, obtained from rats previously injected with heparin, is passed through a column of a strong anion exchange resin, heparin is retained by the column. 2. The clearing activity of the effluent from the column, as determined with added chyle, is slightly increased by the addition of heparin. 3. When the effluent is incubated for very short periods at 370 C. its clearing activity is destroyed. Incubation at 370 C. in the presence of very small quantities of heparin does not result in rapid destruction of the clearing activity. 4. During the clearing of added chyle by the effluent no marked destruction of clearing activity occurs. The author would like to thank Sir Howard Florey for his continued advice and encouragement, and Dr. J. E. French, Dr. G. G. F. Newton and Dr. J. C. F. Poole for many useful discussions. The provision of samples of rat chyle by Dr. J. E. French is gratefully acknowledged. The author is indebted to Miss P. M. Harris and Mr. H. W. Wheal for their technical assistance. A personal grant from the Albert and Mary Lasker Foundation Inc. to Miss P. M. Harris is gratefully acknowledged. REFERENCES FRENCE, J. E., ROBINSON, D. S. and FLOREY, H. W. (1953). Quart. J. exp. Phy8iol. 38, 101. JEFFRIES, G. H. (1954). Quart. J. exp. Physiol. 39, 77. KORN, E. D. (1955). J. biol. Chem. 215, 1. ROBINSON, D. S. and FRENCH, J. E. (1953). Quart. J. exp. Physiol. 38, 233. ROBINSON, D. S., JEFFRIES, G. H. and FRENCH, J. E. (1954). Quart. J. exp. Physiol. 39, 165. ROBINSON, D. S., JEFFRIES, G. H. and POOLE, J. C. F. (1955). Quart. J. exp. Physiol. 40, 297.