normal and cholesterol-fed rabbits have been reported in a previous paper [Courtice and Munoz-Marcus, 1964]. To elucidate further the mechanisms

Transcription

1 THE PERMEABILITY OF THE BLOOD CAPILLARIES OF THE LEG TO THE LIPOPROTEINS IN VARIOUS HYPERLIPAEMIC STATES IN THE RABBIT. By F. C. COURTICE, M. MUNOZ-MARCUS* and D. G. GARLICK.t From The Department of Experimental Pathology, John Curtin School of Medical Research, Australian National University, Canberra, A.C.T. (Received for publication 17th March 1964) The transference of lipoproteins across the walls of blood capillaries was determined by measuring the concentration of their components in plasma and in lymph collected from the leg, both before and after injuring the paw by heat. These measurements were made in groups of normal rabbits in which the lipid levels were increased by Triton WR-1339, cortisone, alloxan or haemorrhage, and in cholesterol-fed animals given either Triton or cortisone. The transference of the lipoproteins in all these different hyperlipaemic conditions was considerably increased by injury, and, both before and after injury, varied inverselv with the size of the macromolecules concerned. The results showed that one way in which Triton, cortisone and alloxan might inhibit atherosclerosis in the cholesterol-fed rabbit was by changing the lipoprotein pattern, decreasing the amount at the lower end and greatly increasing the amount at the upper end of the spectrum in relation to size and Sf values, thereby decreasing the amount of lipoprotein filtered through the vascular endothelium. The composition and stability of lipid in the lymph and, therefore, in the tissue fluid, also varied considerably in the different conditions studied and these factors were also probably important in determining whether or not lipid was deposited in an atherosclerotic lesion. The experiments support the view that small, cholesterol-rich lipoproteins are the most likely to be deposited in the intima because they are more readily transferred across the endothelium, are rich in cholesterol, which cannot be broken down, and are probably less stable than the lipoproteins in conditions of hypertriglyceridw,mia. THE effects of the administration of cortisone, alloxan or Triton WR-1339 on the distribution pattern and composition of the plasma lipoproteins in normal and cholesterol-fed rabbits have been reported in a previous paper [Courtice and Munoz-Marcus, 1964]. To elucidate further the mechanisms concerned in the deposition of lipids in atherosclerotic lesions, experiments were made to determine the transference of the lipoproteins across the walls of the blood capillaries of the paw in rabbits with these different types of lipaemia. This investigation was carried out by measuring the lipid concentrations in plasma and in lymph from this region, as has been previously reported in cholesterol-fed animals [Courtice, 1961; Courtice and Garlick, 1962]. * Permanent address: Catedra de Fisiologia, Escuela de Quimica y Farmacia, Universidad de Chile, Santiago, Chile. t Permanent address: Department of Pathology, University of New South Wales, Sydney. 441

2 442 4Courtice, Munoz-Marcus and Garlick METHODS Hutch-bred rabbits of various strains and of both sexes were used. Under nembutal anaesthesia lymph was collected from the main lymphatic trunks just below the popliteal fossa [Courtice, 1959]. These trunks drain the hind paw and lower part of the hind leg. After a sufficient volume of lymph was collected, usually over a period of 1-2 hr., the animal was temporarily very deeply anaesthetized with ether and the paw was injured by immersion in water at 7 C. for 1 min. This injury led to the development of considerable local cedema over the subsequent 2 hr. [Courtice, 1946]. Lymph was again collected for varying periods. Lymph propulsion was aided by passive flexion and extension of the hind legs, and uniform conditions were maintained by keeping the paw immersed in water at 43-45' C. throughout. Blood samples were taken from a catheter in a carotid artery and coagulation of both lymph and blood samples was prevented by powdered heparin. The methods of inducing hyperlipawmia and the analytical methods were those described by Courtice and Munoz-Marcus [1964]. In experiments in which the size of the lipoproteins was determined by electron microscopy, the method used was that described by Courtice and Garlick [1962]. In experiments in which Lipomul was infused intravenously, the preparation used was Upjohn's Lipomul I.V. (15 per cent cotton-seed oil stabilized with purified soyabean phosphatide and a synthetic surface-active agent, Pluronic F68) and the infusion was given with a Palmer's slow infusion apparatus through a catheter in an external jugular vein. RESULTS Triton Hyperlipaemia.-The concentrations of total esterified fatty acid (TEFA), phospholipid (PL), total cholesterol (TC) and total protein (PR), were determined in the plasma and in the lymph in each of a group of ten Triton hyperlipeemic rabbits. The effect of injury of the paw on these values in the lymph are shown in a typical experiment in fig. 1. With the type of injury used in these experiments, the concentrations of lipids as well as of total protein in the lymph rapidly reached an increased level which was maintained for the next 2 or 3 hr., the time of the experiment, during which gross cedema of the paw developed with a great increase in lymph formation. The plasma in these animals was quite milky in appearance: the lymph before injury was usually only very slightly opalescent, but it rapidly became milky in appearance after injury. During this post-injury period, the composition of the lymph and tissue fluid would resemble that of the capillary filtrate, whereas before injury the protein and lipoprotein concentrations of lymph and tissue fluid would probably be greater than that of the capillary filtrate. This fact together with the amount of tissue fluid and lymph formed under these conditions must be remembered when the transference of these lipoproteins from plasma to lymph is being considered. Fig. 2 shows the mean levels of TC, PL and TEFA in the lymph before and 1-1 hr. after injury plotted against the values in the plasma of two normal animals and of ten with varying degrees of lipoemia. It is evident that while the lipid levels in the lymph before injury were always higher in

3 Capillary Permeability to Lipoproteins 443 TRITON LIPAEMIC RABBIT PLASMA 6X 7 PLASMA 6 N, 2- u E LYMPH 2 C -~ VE OOQ PLASMAMAioo PA PLASMA 1 S 75 o 5 5 x 5 LYMPH LYMPH 25 5 O Iu INJURY HOURS INJURY HOURS 1FIG. 1.-The effect of thermal injury of the paw on the levels of TEFA, PL, TC and PR in the lymph from that region in a Triton lipaemic rabbit. 2 TOTAL CHOLESTEROL t -, x-x - - -, -' x FIG The levels of TEFA, PL and TC in the lymph from the paw before and after thermal E injury in relation to the levels in the plasma in I 4 PHOSPHOLIPIDtwo normal (furthest on left) and ten Triton x lipamic rabbits. 3 x *.* normal animals. z 2 x --.- x after injury to paw. ~. -.* -. z Ux u -X 15 - T.E.F.A. 1-5 'A C CONCENTRATION IN PLASMA MG/1ML

4 444 Courtice, Munoz-Marcus and Garlick the lipoemic than in the normal animals, there was only a very gradual increase in these values with increasing intensity of the lipaemia. In the lymph after injury, however, the rise in lipid levels with increasing levels in the plasma was more marked. It seems, therefore, that the lipoproteins in Triton hyperlipaemia are transferred to a small extent through the normal capillary wall, as are the lipoproteins of the plasma of the cholesterol-fed animal [Courtice, 1961; Courtice and Garlick, 1962], and that the changes in the ultrastructure of the endothelium produced by injury [cf. Majno and Palade, 1961] greatly accentuate this transfer. The results plotted in fig. 2 indicate that the lymph: plasma ratios of the various lipids decrease with increasing levels of lipid in the plasma. TABLE I.-THE LYMPH: PLASMA RATIOS OF THE LIPOPROTEINS IN TRITON HYPER- LIPAEMIA COMPARED WITH THOSE IN HYPERCHOLESTEROL2EMIC RABBITS, EXPRESSED AS PERCENTAGE. LYMPH WAS COLLECTED FROM A VESSEL IN THE LEG BEFORE AND AFTER THERMAL INJURY. Lipoprotein Triton Cholesterol-fed fraction Before injury After injury Before injury After injury d < ±4 3- ± ± ±2- d ± ± ± d ± ±4-1 Results are the means and S.E. of means in six Triton-administered and seven cholesterol-fed animals. This would be in keeping with the conclusions reached by Courtice and Munoz-Marcus [1964] that with rising levels of lipid there are increasing proportions of larger complexes. To elucidate further whether the transference of these lipids is related to the size of the lipoproteins, the lymph: plasma ratios of the d < 1.19 and d classes were determined in a group of six animals by measuring the levels of each of these fractions in samples of plasma and lymph. Because only relatively small volumes of lymph were available, 2 ml. samples were used for this purpose. The level of PL was measured in each case and the lymph: plasma ratio of PL was taken as a measure of the ratio of lipoprotein. Since the d class is so little in Triton hyperlipeemia, it was not possible to measure accurately the lymph: plasma ratio of this class in such small samples. The results of these experiments and, for comparison, the results previously obtained for similar experiments with cholesterol-fed rabbits [Courtice and Garlick, 1962] are given in Table I. These experiments indicate that the lymph: plasma ratios vary inversely with the size of the lipoproteins both before and after injury. To determine whether the same range of size of lipoproteins was present in the lymph as in the plasma, the diameters of the lipoproteins of the d < 1.19 class were determined after fixation in osmium tetroxide buffer by electron microscopy. The results of two typical experiments, one in an animal with Triton hyperlipaemia and one in a cholesterol-fed animal, are

5 Capillary Permeability to Lipoproteins 445 shown in fig. 3 a and b. It is evident that the complexes in the lymph both before and after injury cover approximately the same range as in the plasma in both cases. TRITON-LIPAEMIA FRACTION D(1.19 HYPERCHOLESTEROLAEIA FRACTION D<1.19 z co 4 LYMPH BEFORE INJURY z 3 go.- 43 z U 3 - PLASMA (2 z C. 2 r 1 v PLASM,k IX DIAMETER OF LIPOPROTEINS A (a) DIAMETER OF LIPOPROTEINS A (b) FIG. 3.-The range of diameters of the low-density lipoproteins in the plasma and in the lymph before and after injury. (a) Triton lipaemic rabbit. (b) Cholesterol-fed rabbit. In studying the deposition of lipid in the thickened intima of an injured portion of the carotid artery, Courtice and Schmidt-Diedrichs [1962 and 1963] suggested that the finding that lipid was not deposited in Triton hyperlipaemic TABLE II.-THE MEAN LEVELS OF TOTAL CHOLESTEROL IN THE PLASMA AND IN THE LYMPH FROM THE LEG BEFORE AND AFTER INJURY IN A GROUP OF TEN TRITON HYPERLIPAEMIC RABBITS AND A GROUP OF NINE CHOLESTEROL-FED RABBITS. Lymph Plasma, (mg./1 ml.) Before injury After injury (mg./1 ml.) (mg./1 ml.) Triton group Cholesterol-fed group 597 ± ±6 76 ±8 148 ± ±29 animals might be due to the composition of the lipoproteins rather than to their permeability through the endothelium. If the actual level of cholesterol in the tissue fluid is important, Table II shows that this is much less in the present experiments than in similar experiments in cholesterol-fed rabbits VOL. XLIX, NO

6 446 Courtice, Munoz-Marcus and Garlick [from Courtice, 1961]. In each group the mean levels of TC in the plasma were approximately the same, but the levels in the lymph both before and after injury in the Triton group were approximately only half the level in the cholesterol-fed group. Cortisone, Alloxan and Haemorrhagic Lipcemia.-The lipid and protein levels in the plasma and in the lymph from the leg before and after injury in typical experiments in cortisone and alloxan lipaemia are shown in fig ( -1)( )( V 12( * CORTISONE * ALLOXAN PLASMA 7 -PLASMA 6 LYMPH 5 >J 4 s 1( I.- LYMPH 3Z ui oc X o 8( E. 4C ) 4 s 3PLASMA PLASMA 3 E ) -+ 2= I 2C O LYMPH LYMPH 1 U I INJURY HOURS INJURY HOURS FIG. 4.-The effect of thermal injury of the paw on the levels of TEFA, PL, TC and PR in the lymph from that region in hyperlipaemia following the administration of cortisone and of alloxan. As with the Triton experiments there was an increase in the level of all the lipids in the lymph after injury, showing the increase in the permeability to the lipoprotein complexes. For comparison with Triton lipeemia it is necessary to use experiments in which the level of hyperlipaemia is about the same in each group. For this purpose the group of eight cortisone, three alloxan and two haemorrhagic lipaemias have been placed in one group and six animals with Triton lipaemia in another. The mean levels of TEFA in the plasma were 225 mg./1 ml. in the Triton group and 22 mg./1 ml. in the second group. The results are shown in Table III. These figures show that the lymph: plasma ratios of TEFA, PL and TC before injury were about 1 per cent in each group, but after injury the ratios were about 3 per cent in the Triton group and 2 per cent in the other group. When two different groups are being compared in this way,

7 Capillary Permeability to Lipoproteins the ratios after injury are probably the ratios of the levels of capillary filtrate through fairly uniformly damaged capillaries. If this can be assumed, the findings would be in conformity with the view that on the average the complexes in the cortisone group were slightly larger than in the Triton group. The fact that the lymph: plasma ratio before injury was the same as in the Triton group could be due to a possible lower capillary pressure in the cortisone group with a lower filtration rate and more reabsorption of tissue fluid with a low lymph flow. Although the actual lymph production could not be accurately determined, it was noted that the cortisone animals were often what have been termed "dry" animals in which only a small amount of lymph could be collected. TABLE III-.---THE MEAN LEVELS OF TEFA, PL AND TC IN THE PLASMA AND LYMPH IN Two GROUPS OF ANIMALS EACH WITH APPROXIMATELY THE SAME TOTAL PLASMA LIPID CONTENT (i) TRITON (6) AND (ii) CORTISONE (8), ALLOXAN (3) HACMORRHAGE (2). Triton 447 Cortisone, alloxan, hiemorrhage Lymph Lympli Plasma r Plasma -'-~ Before After Before After injury injury injury injury TEFA mg./1 m ± ±3 595 ±78 22 ± ± ±4 PL mg./1 ml. 783 ±16 79 ± ±41 42 ±74 48 ±4 14 ±7 TC mg./1 ml. 498 ±76 48 ± ±31 18 ±1 3 ±4 To see if there were any gross changes in permeability in the various groups, the protein concentrations of the lymph before and after injury were plotted against the level in the plasma in a group of eight normal, twenty-four cholesterol-fed, seven Triton, eight cortisone and three alloxanhyperlipanmic animals (fig. 5). The mean levels of protein for the entire group were 6.81 ±.8 g./1 ml. in the plasma, 2.65 ±.11 g./1 ml. in the lymph before injury and.5.21 ±.8 g./1 ml. in the lymph after injury. The scatter in the results with lymph before injury was probably due to the fact that in so-called "dry" animals the formation of lymph was very small with resultant increase in the protein concentration, whereas in some animals with a very rapid filtration, the protein concentration was low. However, on the whole, there was no gross difference between the different groups, especially after injury. Before injury, most of the cortisone animals fell above the general regression line. Table IV shows that while the protein level in the cortisone group was higher than normal, the lymph: plasma ratio was also higher than in normal animals and in those in the other groups before injury but not after injury. While the lymph: plasma ratios of different macromolecules give an indication of their relative permeability in the same animal, it is difficult to compare degrees of permeability in different animals without a measure of the volume of lymph formation. What can be measured accurately, however, are the concentrations of the various lipids in the lymph, and therefore in the tissue fluid, in the different conditions. Fig. 6 is a composite diagram

8 448 Courtice, Munoz-Marcus and Garlick showing the mean lipid levels in the leg lymph before and after injury of three groups of hyperlipasmic rabbits-cholesterol-fed, Triton and cortisone, x NORMAL - CHOLESTEROL-FED * TRITON + CORTISONE + ALLOXAN a Xc I,'C * -o., I O. * 5.OF 4A. z z Lu 4 De 5K 11 f I - + I + t AFTER INJURY -o x ' o kx 2.1 n - -, + X 1 X / *X,'O BEFORE INJURY 1.5L - X PROTEIN IN PLASMA G/1ML FIG. 5.-The protein content of the lymph from the paw, before and after thermal injury, in normal and cholesterol-fed rabbits and in animals made lipwmic by the administration of Triton, cortisone or alloxan. alloxan, haemorrhage-in which the mean level of the total lipids (TG, FC+EC, PL) was about the same. In these experiments in the last two groups measurements of TEFA, TC and PL were made in plasma and lymph and triglyceride calculated on the assumption that 25 per cent of the +

9 Capillary Permeability to Lipoproteins 449 cholesterol was esterified. In the cholesterol-fed group the levels of TC and of PL were determined in plasma and lymph. The level of total lipid in the plasma, however, was extrapolated from another group of seventeen TABLE IV.-THE MEAN PROTEIN LEVELS IN PLASMA AND LEG LYMPH BEFORE A] ATE:R INURY TO THE LEG IN GROUPS OF NORMATL, CHOLESTEROL-FED, TRrroN, COIRTISONE AND ALLOXAN HYPERLIPpEMIC RABBITS. Leg lymph Plasma (g./1 ml.) Before Bfr After fe (g/ I) injury inljury (g./1 ml.) (g./1 ml.) Normal (8). 6-7 ± ± Cholesterol-fed (24) ± ± ±-13 Triton (7) 6-45 ± ± ±-1 Cortisone (8) ± ± ±-1 Alloxan (3) experiments in which all the lipids (TG, FC, CE and PL) in plasma but not in lymph were measured and in which the mean cholesterol and phospholipid values were 1195 mg./1 ml. and 485 mg./1 ml. respectively, i.e. nearly the same as in the group depicted in fig. 6. PLASMA TOTAL LIPID 251 LEVELS MG/1ML TC PL TOTAL LIPID 279 TOTAL LIPID 238 TC 498 TC 175 PL 783 PL 42 FIG. 6.-The mean concentrations of the various lipids in the lymph, before and after thermal injury of the paw, in three groups of animals, each with approximately the same mean total lipid level in the plasma. Before injury of the paw. * After injury of the paw. The lipids-tc, PL and TG-in the lymph in the experiments with Triton and with cortisone, alloxan and hasmorrhage would represent all the lipid except a small amount of fatty acid as cholesterol ester and as albuminfatty acid complex. In the cholesterol-fed group, however, about 73 per cent

10 45 Courtice, Munoz-Marcus and Garlick of the cholesterol would be esterified so that to the TC and PL that were actually measured and plotted, fatty acid attached to 73 per cent of the cholesterol, and a very small amount of triglyceride as well as the free fatty acid-albumin complex should be added. TABLE V.-THE TOTAL LIPID IN THE PLASMA AND LYMPH OF THREE GROUPS OF RABBITS IN WHICH THE LEVELS IN THE PLASMA WERE APPROXIMATELY THE SAME IN EACH GROUP. TOTAL LIPID INCLUJDES TC INCLUDING ESTER, PL AND TG. Plasma mg./1 ml. Lymph before injury{ mg./1 ml. L/P ratio percentage Lymph after f mg./1 ml. pae injury il/p ratio Cholesterol-fed Triton Cortisone, alloxan (24) (6) haemorrhage (13) These figures have been derived on certain assumptions explained in the text. If these calculations are made the total lipid in plasma and in lymph and the mean lymph: plasma ratios of total lipid would be as shown in Table V. These figures show that while the values in the lymph for the Triton and cortisone groups are less than for the cholesterol-fed group, the most striking difference is in the cholesterol levels as shown in fig. 6. TABLE VI.-THE LEVELS OF TEFA, PL AND TC, EXPRESSED AS mg./1 ml., IN THE PLASMA AND LYMPH OF FOUR RABBITS CHOLESTEROL-FED FOR SEVERAL WEEKS, Two OF WHICH, C AND G, HAD BEEN GIVEN CORTISONE DAILY FOR 7 DAYS AND Two, D AND E, TRITON 3 DAYS BEFORE THE EXPERIMENT. Lymph Lymph Lymph Lymph Plasma before after Plasma before after injury injury injury injury A. N Cortisone + Cholesterol-fe( C G r A - I _As TEFA PL TC Triton + Cholesterol-fed TEFA PL TC D E The composition of the plasma lipoproteins of these animals were given in Courtice and Munoz-Marcus [1964] (fig. 4). The Effect of Triton or Cortisone Administration to Cholesterol-fed Rabbits.- It has been shown [Courtice and Munoz-Marcus, 1964] that the increase in TG in the plasma following the administration of Triton or of cortisone to cholesterol-fed rabbits decreased the amount of the smaller lipoproteins. The composition of the lymph from the leg in those experiments quoted in this paper are given in Table VI.

11 Capillary Permeability to Lipoproteins 451 Rabbits C and G received cortisone and in rabbit G the level of TG was much higher than in rabbit C with a decrease in the amount of,b-lipoprotein. The results show that the lipids in the lymph both before and after injury were considerably lower in G and in C. Rabbits D and E received Triton and the level of triglyceride was greater in E than in D suggesting a greater proportion of larger complexes. The lipids in the lymph both before and after injury were somewhat lower in E than in D indicating that this is so. NORMAL RABBIT 14- PLASMA PLASMA E LYMPH M4 <,6i 3 u., 2- P LASMWA O O 1 1 L 8 LYMPH PLASMA, IPOMULLYMPH J INJURY HOURS INJURY HOURS FIG. 7.-The effect of thermal injury of the paw followed by an intravenous infusion of Lipomul I.V., 1 ml./kg. body weight, on the levels of TEFA, PL, TC and PR in the lymph of a normal rabbit. The Effect of Infusion of Lipomul on the Transference of Lipoproteins from Plasma to Lymph.-When an emulsion of Lipomul I.V., with particles about the size of chylomicrons, was infused intravenously into a normal rabbit, there was no increase in lipid in the lymph from the leg, even after injury. Fig. 7 shows a typical experiment in which lymph was collected from the paw of a normal rabbit, the leg was injured and lymph was further collected. Immediately after injury there was a rise in the lipids in the lymph as well as in the protein, showing that the normally circulating lipoproteins as well as the uncomplexed protein were more readily transferred across the capillary wall. Lipomul I.V., 1 ml./kg., was then slowly infused over a period of 1 hr. This increased the amount of TG and PL in the plasma (the chief constituents of Lipomul), yet the lipid levels in the lymph fell and the lymph remained clear. A similar phenomenon was found after the infusion of Lipomul into cholesterol-fed rabbits [Courtice and Garlick, 1962]. In further experiments Lipomul was infused into Triton lipaemic rabbits. Figs. 8 and 9 show that 5 per cent dextrose had no significant effect on the level of lipids in the

12 452 Courtice, Munoz-Marcus and Garlick lymph after injury whereas Lipomul infusion caused a decrease in the levels in the lymph in spite of a considerable increase in the TEFA level of the plasma. One explanation for this phenomenon could be that the large Lipomul particles might block the gaps in the injured capillary endothelium through which the smaller lipoproteins are passing. In all these experiments, INJURY HOURS INJURY HOURS FIG. 8.-The effect of thermal injury of the paw followed by an intravenous infusion of 5 per cent dextrose, 1 ml./kg. body weighb, on the levels of TEFA, PL, TC and PR in the lymph of a Triton lipwmic rabbit. however, the total protein in the plasma and lymph was also determined. The results of typical experiments in which Lipomul or the same quantity of isotonic dextrose or sodium chloride was infused are given in fig. 1. The concentrations in the lymph have been expressed as a percentage of the corresponding plasma level. The results show no significant difference in the transference of total protein between the Lipomul-infused or control animals. It seems unlikely, therefore, that this phenomenon can be explained by blocking the gaps in the endothelium by the Lipomul particles. Courtice and Garlick [1962] suggested that some of the smaller lipoproteins were adsorbed onto the larger Lipomul particles thus decreasing their filtration. To test this hypothesis experiments were made in vitro. Plasma was collected from a cholesterol-fed rabbit. To one portion of this, Lipomul

13 1 8k ::2 6k 4 F TRITON LIPAEMIC RABBIT PLASMA - I / r LYMPH X 3-z 2-2 LYMPH - C 1,,- PLASMA PLASMA E 8 6 f 4 ' I 2 o F LYMPH Io- LYMPH IN. IURY HOURS INJURY HOURS lfia. 9.-The effect of thermal injury of the paw followed by an intravenous infusion of Lipomul I.V., 1 ml./kg. body weight, on the levels of TEFA, PL, TC and PR in the lymph of a Triton lipaemic rabbit. X 1 (.1n z W 8 :~ i 6 1/) 4.) z m 2 - II I- -< Itl%JIURY I I- I- I- I^ * In,,\ I O NORMAL LIPOMUL A NORMAL 5% DEXTROSE * HYPERCHOLESTEROLAEMIA LIPOMUL + HYPERCHOLESTEROLAEMIA.9% NaCI x TRITON HYPERLIPAEMIA LIPOMUL + TRITON HYPERLIPAEMIA 5% DEXTROSE HOURS FIG. 1.-The relation between the concentrations of proteins in the lymph and in the plasma following thermal injury of the paw and an) intravenous infusion of either Lipomul I.V. or an isotonic solution of dextrose or NaCl, 1 ml./kg. body weight, in normal, cholesterol-fed and Triton hyperlipaemic rabbits. Duration of infusion. 4 e) 2 U

14 4,54 Courtice, Munoz-Marcus and Garlick was added in the same proportion as that infused in vivo, assuming a plasma volume of 4 ml./kg. To the other portion 5 per cent dextrose was added in the same proportion. These mixtures were then mixed continuously in a vessel at 37 C. for 1 hr. The same volume of each mixture was then spun in Lusteroid tubes in a Spinco Model L preparative ultracentrifuge at 2, r.p.m. for 3 min. and the supernatant and infranatant separated by a Beckman tube slicer. The amount of cholesterol in the supernatant and in the infranatant in each case was then measured. The results of three experiments are shown in Table VII. These results show that an appreciable TABLE VII. THE CHOLESTEROL IN THE SUPERNATANT, EXPRESSED AS PERCENTAGE OF TOTAL, WHEN PLASMA FROM THREE CHOLESTEROL-FED RABBITS WAS SHAKEN in vitro WITH EITHER LIPOMUL OR AN EQUIVALENT AmOUNT OF 5 PER CENT DEXTROSE AND SPUN AT 2, r.p.m. FOR 3 min. Rabbit TC in plasma Lipomul +plasma 5 per cent dextrose ± plasma No. (mg./1 ml.) amount of the cholesterol of the smaller lipoproteins floated with the Lipomul suggesting some sort of adsorption. These experiments with Lipomul would, therefore, support the view that the transference of plasma lipoproteins from plasma to lymph in the paw of the rabbit is related to the size of these macromolecules. DIscussIo:Nr The object of these experiments was to clarify the mechanisms whereby such agents as Triton, alloxan and cortisone, while accentuating the degree of lipaemia in cholesterol-fed rabbits, inhibit the development of atherosclerosis. Since these agents do not reduce an existing atherosclerosis [Duff and McMillan, 1949], the inhibition of the development of experimental atherosclerosis must depend on interference with the process of deposition of lipids. If we subscribe to the theory of filtration and deposition, it would seem that the administration of alloxan, cortisone or Triton to a cholesterolfed rabbit must either decrease filtration across the endothelium or decrease deposition once the lipoproteins have traversed the endothelium. The permeability of the vascular endothelium to these macromolecules has been shown in the present and in previous experiments [Courtice and Garlick, 1962] to depend on its structure and on the size of the lipoproteins. The transference of proteins across the capillary wall indicates that there is no gross difference in the structure of the endothelial barrier in the various lipaemic states investigated, either before or after injury. The transference of lipoproteins across the capillary wall would, therefore, depend mainly on their size and concentration and on the filtration pressure. The evidence shows when there is a considerable increase in triglyceride in the plasma, the lipids are transported as larger, less dense macromolecules and that the smaller

15 Capillary Permeability to Lipoproteins 4,55 lipoproteins are decreased in amount. This change in distribution in the broad spectrum of plasma lipoproteins also occurs when a hypertriglyceridaemia is imposed on an existing hypercholesterolhemia. It would seem, therefore, that alloxan, cortisone and Triton inhibit atherosclerosis in a cholesterol-fed rabbit mainly by decreasing the amount of lipoprotein in the lower end of the lipoprotein spectrum and increasing the amount in the upper end in relation to size or Sr values. While it is clear that this is one factor involved, the experiments in the present paper do show that an appreciable amount of lipoprotein may be transferred across the vascular endothelium in the various lipaemic states studied. The composition of the lipids in the tissue fluid and lymph, however, varies greatly as has been strikingly shown in fig. 6. It would seem, therefore, that in these cases another important factor in determining whether or not lipid is deposited in an area of thickened intima of an artery is the composition of the lipids concerned. Courtice and Schmidt--Diedrichs [1962 and 1963] showed that lipid was deposited in a thickened area of intima resulting from injury to an artery, in cholesterol-fed animals but not in Triton-induced lipaemia. One reason for this could be that the high cholesterol content of the lipoproteins in the cholesterol-fed animals led to deposition because the cholesterol, once ingested by macrophages, could not be broken down and the macrophages seem unable to remove the cholesterol from such an area very quickly. Another factor, however, may be the stability of the lipoproteins. Duff and Payne [195] in studying the mechanism of the inhibition of experimental atherosclerosis by alloxan concluded that the instability of the lipoproteins rather than the hypercholesterolhemia per se was important in the deposition of lipid in the development of atherosclerosis. The present paper shows that, in the paw, the lipoproteins filter to some extent through the blood capillaries and are returned to the blood stream by the lymphatics. In the arterial wall, the lipoproteins would have to filter through the internal elastic lamina into the media before being taken up by lymphatics. If some lipoproteins were less stable because of a high cholesterol and a relatively low phospholipid content, they might be more readily precipitated in an area of thickened intima. The inhibition of atherosclerosis in these cholesterol-fed rabbits by Triton, cortisone and alloxan might, therefore, be due, in part, to an increased stability of the lipoproteins produced by these agents. The experiments presented in this paper support such a view by clarifying the importance of the relation of the size of the lipoproteins in transference across the capillary wall and in the composition of the lipids in the tissue fluid and lymph in the various lipeemic states. They support the view that relatively small, cholesterol-rich lipoproteins are the most likely to be deposited in an area of thickened intima, because they are more readily transferred across the endothelial barrier, are rich in cholesterol.and are probably less stable than the lipoproteins in conditions of hypertriglycerid&emia.

16 456 Courtice, Munoz-Marcus and Garlick REFERENCES COURTICE, F. C. (1946). J. Physiol. 14, 321. COURTICE, F. C. (1959). Au8t. J. exp. Biol. and Med. Sci. 37, 451. COURTICE, F. C. (1961). J. Phy8iol. 155, 456. COIJRTICE, F. C. and GARLICK, D. G. (1962). Quart. J. exp. Physiol. 47, 221. COuRTICE, F. C. and MUNOZ-MARCUS, M. (1964). Quart. J. exp. Physiol. 49, 432. COuRTICE, F. C. and SCHMIDT-DIEDRICHS, A. (1962). Quart. J. exp. Physiol. 47, 228. COURTICE, F. C. and SCHMIDT-DIEDRICHS, A. (1963). Brit. J. exp. Path. 44, 339. DUFF, G. L. and MCMILAN, G. C. (1949). J. exp. Med. 89, 611. DUIFF, G. L. and PAYNE, T. P. B. (195). J. exp. Med. 92, 299. MAJNO, G. and PALADE, G. E. (1961). J. biophys. biochem. Cytol. 11, 571.