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Cholesterol transport in thoracic duct lymph of the rabbit
Journal of A therosclerosis Research Elsevier P u b l i s h i n g C o m p a n y , A m s t e r d a m - P r i n t e d in T h e N e t h e r l a n d s
C H O L E S T E R O L T R A N S P O R T I N THORACIC DUCT L Y M P H OF T H E RABBIT D. B. Z I L V E R S M I T , F. C. C O U R T I C E AND R. F R A S E R
Department of Experimental Pathology, John Curtin School of Medical Research, Australian National University, Canberra (Australia) ( R e c e i v e d J u l y 10th, 1966)
SUMMARY
The method of transport of cholesterol in the thoracic duct lymph collected in unanaesthetized rabbits has been determined in three groups of animals fed a high cholesterol-low fat, high cholesterol-high fat or high fat diet. Three lipoprotein fractions in the lymph were separated b y the preparative ultracentrifuge, viz. (a) chylomicrons of Sf > 400, (b) very low-density lipoproteins (VLDL) of Sf 12-400 and d < 1.019, and (c) low and high density lipoproteins of d > 1.019. In those experiments in which the total protein as well as the lipoprotein fractions were determined in both lymph and plasma, the results suggest that in the thoracic duct l y m p h most of the cholesterol in the lipoprotein of d < 1.019 was derived b y absorption from the intestine. Most of the lymph cholesterol, 60-80 %, was carried in the chylomicron and V L D L fractions, i.e., d < 1.019. The distribution between these two fractions varied considerably, but compared with the rat and the dog, relatively more cholesterol was carried in the V L D L fraction. The cholesterol as a percentage of totat lipid in each of these two fractions was also greater in the rabbit than in the rat or dog on similar high cholesterol diets. A possible relationship between the tendency of the rabbit to develop hypercholesterolaemia and the form in which cholesterol is transported in thoracic duct lymph is suggested.
INTRODUCTION
Although much information is available on the composition and turnover of D. B. ZILVERSMIT: Career I n v e s t i g a t o r of t h e A m e r i c a n H e a r t Association. P e r m a n e n t a d d r e s s : G r a d u a t e School of N u t r i t i o n , CorneU U n i v e r s i t y , I t h a c a , N.Y. (U.S.A.).
J. Atheroscler. Res., 7 (1967) 3 1 9 - 3 2 9
320
D. B. ZILVERSMIT, F. C. COURTICE, R. FRASER
plasma lipids in the rabbit, practically nothing is known about the transport of lipids from the intestinal tract to the blood stream. Since rabbits are particularly prone to develop hypercholesterolaemia in response to cholesterol feeding, it seemed worthwhile to study the lymph lipids during cholesterol absorption to ascertain whether in this animal cholesterol is absorbed in a form which might account for the hypercholesterolaemia. In terminal experiments, PoPjAK 1 collected small samples of lymph from the cysterna chyli of three rabbits fed amorphous cholesterol for 1 month. The concentration of cholesterol in the lymph was less than one-third that of plasma. Although the animals did not receive appreciable amounts of fat in their diet, the neutral fat in the lymph was four times the concentration in the plasma. In another group of six rabbits which were fed a cholesterol-rich diet for 3 months and then a diet of only vegetable leaves for 7-10 days before l y m p h collection, MORRIS AND COORTICE2 found that on the average the concentration of total cholesterol in the lymph was also about one-third of the plasma level and the total f a t t y acids about one-half. In all of these experiments, the rabbits were under anaesthesia during lymph collection. One reason for the paucity of d a t a on the lipids in the thoracic duct l y m p h of rabbits is the difficulty involved in collecting lymph for several days after cannulation. YOUNG AND FREEMAN3 report the collection of thoracic duct lymph in rabbits for 48 h after recovery from anaesthesia, and the determination of its triglyceride content, but no details are given concerning the l y m p h flow or nutritional state of the animals. We have collected thoracic duct lymph from conscious rabbits for periods up to 7 days after cannulation. In the present paper we present data on the quantity and composition of lipoprotein fractions in thoracic duct lymph after feeding diets containing varying amounts of cholesterol and fat. METHODS
New Zealand white rabbits of either sex and 2.5-3.5 kg weight were maintained on ground rabbit chow containing 5 ~ triglyceride. For varying intervals before cannulation of the thoracic duct, as stated later, the experimental animals were kept on a diet of chow to which was added 0.8 % cholesterol* (high cholesterollow fat diet), 0.8 ~ cholesterol and 20 % fat* (high cholesterol-high fat diet) or20 ~ fat* (high fat diet). The thoracic duct was cannulated in the upper part of the mediastinum after anesthetizing the animal with N e m b u t a l | (about 30 mg/kg) injected into the marginal ear vein. In most cases an intravenous infusion of 0.9 % NaC1 (0.5-1 ml/min) was given during the course of the operation and for a varying time afterwards. A tracheal tube was inserted and connected with a Bird** respirator providing oxygen at inter* High cholesterol-low fat diet: powdered cholesterol was mixed with ground rabbit chow. High cholesterol-high fat diet: melted fat was mixed with above diet. T h e fat ( F r y m a s t a - V e g e t a b l e oils P t y Ltd. Australia) contained primarily p a l m i t a t e and oleate. ** Bird Corp., P a l m Springs, Calif. (U.S.A.). J. Atheroscler. Res., 7 (1967) 319-329
CHOLESTEROL TRANSPORT IN LYMPH
321
mittent pressures of 10-20 cm of water to prevent pneumothorax during the operative procedure. The thoracic duct was exposed b y partially severing the left sternocleidomastoid and pectoralis major muscles near their origin on the sternum, and then separating trachea and oesophagus from the left carotid sheath. The trachea, oesophagus and right common carotid sheath were displaced to the right and the left carotid sheath, the left external jugular vein and the left sternomastoid and sternohyoid muscles to the left. The tip of the sternum was then lifted with a retractor exposing the upper mediastinum on the left side. The thoracic duct was located as it passes dorsal and in close proximity to the aortic arch and was cannulated in this position with polyethylene or polyvinyl tubing of 0.5 m m internal and 0.8 m m external diameter. The free end of the tubing was then passed under the skin to a suitable exit along the back or side of the animal. Shortly after the completion of surgery, the rabbit was transferred to a restraining cage designed after the pattern of BOLLMAN et al. 4 for rats (Fig. 1). The cage allowed some freedom of movement
Fig. 1. Restraining cage for rabbits. back and forth and freedom to eat and drink. In prolonged experiments the animal was removed from the restraining cage periodically during which time the lymph was not collected. In some animals, cholesterol or corn oil was administered orally or b y duodenal tube, as stated later in the text. All lymph was obtained from conscious animals, the flow rate varying between 4 and 25 ml/h depending primarily on the degree of hydration of the animal. The samples were collected in chilled containers, allowed to clot and defibrinated b y repeated stirring and filtering through gauze. Blood samples were taken at intervals for the determination of plasma proteins or for the separation and analysis of the plasma lipoproteins. In the lymph samples a chylomicron fraction was isolated b y initial centriJ. Atheroscler. Res., 7 (1967) 319-329
322
D . B . ZILVERSMIT, F. C. COURTICE, R. FRASER
fugation in a Spinco SW 25.1 swinging bucket head for 1 h at 7-10~ and an average centrifugal force of 64,000 • g. The chylomicron fraction was centrifuged once at the same temperature and speed through a 5-cm layer of saline (d = 1.006) carefully pipetted on top of the chylomicrons resuspended in a salt solution of density approximately equal to 1.2. The second centrifugation for 2 h followed by removal of the top layer b y tube slicer yielded a fraction of S I > 400 (ref.5), henceforth called chylomicrons. The chylomicron-free sample was adjusted to density 1.019, centrifuged in a Spinco 40.3 or 50 rotor for 16 h at 100,000 • g or more and then separated into two fractions by slicing. The upper fraction containing lipoproteins of S s 12-400 (ref. 6) will be referred to as very low density lipoprotein (VLDL). The lower fraction of d > 1.019 contains low and high density lipoproteins. In some experiments plasma samples were fractionated at a density of 1.019 without prior separation of chylomicrons. All lipoprotein fractions were extracted with a minimum of 20 vol. chloroformmethanol (2 : 1), and washed b y equilibration with an excess of water 7. Total cholesterol was determined after saponification and extraction8 colorimetrically 9, and lipid phosphorus b y the method of BARTLETT10. A portion of the sample was chromatographed with a column of silicic acid supercel and triglycerides eluted with chloroform 11. Triglycerides were then determined b y the method of VAN HANDEL AND ZILVERSMIT12. Protein nitrogen in lymph and blood plasma samples was measured after micro-Kjeldahl digestion and subsequent titration 13 or nesslerization 14. RESULTS
The data in Tables I and I I summarize the findings on ten rabbits receiving various diets for differing periods of time. They are arranged in three groups depending on whether the animals were ingesting cholesterol in a low fat diet, cholesterol in a high fat diet, or a high fat diet without added cholesterol. Table I gives the time of collection of the lymph samples, the rate of flow and the concentrations of total protein and total lipid in the lymph. In the first group R4 and R5 received the diet of 0.8 % cholesterotin rabbit chow for 10 days, R9 for 20 days and R11 for only 2 days before cannulation. The duration of this diet was varied in an a t t e m p t to obtain animals with a relatively high level of cholesterol in the lymph at the lowest possible plasma concentration, thus avoiding too great a contribution to lymph of plasma cholesterol filtered through capillary beds drained b y the thoracic duct2,15,16. This same consideration also led to a somewhat different approach with rabbit 12. This animal had been kept on 20 ~o fat intake prior to cannulation so that the l y m p h collected during the first few days after cannulation was rather turbid (optical density at 650 m # about 2). 24 h after cannulation a 1 % cholesterol suspension was administered b y gastrostomy tube. From 42 to 66 h 4 g cholesterol on lettuce leaves was fed. At 66 h 2 g and at 73 h a further 2 g cholesterol was fed on lettuce leaves. L y m p h samples L1 and L2 from this animal were collected from 66 to 73 and 73 to 89 h respectively. They showed optical densities at 650 m/z of 0.8 and j . Atheroscler. Res., 7 (1967) 319-329
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0.2 and were considered to represent absorbed cholesterol without fat in the diet. The second group of animals received the high cholesterol-high fat diet. R8 was fed a diet containing 20 ~o fat for 12 days to which 0.8 ~ cholesterol was added for 3 days before the thoracic duct was cannulated. R12 had been used, as already stated, in the first group. On the fifth day (at 97 h) after cannulation the animal received 2 g cholesterol on lettuce b y mouth and 1 ml corn oil b y gastrostomy tube. When the lymph became quite milky L3 was collected for 8 h (O.D. at 650 m # = 3.0). At 113 h 1 g cholesterol dissolved in 5 ml corn oil was administered b y duodenal tube. A final lymph sample, L4, was collected from 116 to 125 h. Samples L3 and L4 from this animal thus represent lymph collected during absorption of cholesterol in the presence of corn oil. In the third group 20 % fat was added to the chow for several days before cannulation. R3 also received corn oil by mouth at intervals from 18 h after cannulation. The corn oil was placed on saline which this animal drank readily. R15 was given 6 ml corn oil by mouth in capsules before cannulation of the duct. The very high flow rate with relatively low protein concentration in the lymph of some animals, especially during the first few hours after recovery from anaesthesia, was due in part to the intravenous saline drip which was given during the operative procedure and for varying times postoperatively to all animals except R12. The concentration of lipid in the lymph would also be affected b y the extent of the infusion. In addition it would depend on the actual amount of lipid absorbed which would be related to the previous diet, the degree of emptying of the stomach and the amount of fat or cholesterol given b y mouth or b y duodenal tube. In these experiments the concentration of " t o t a l " lipid in the thoracic duct lymph was taken as the sum of total cholesterol (TC), triglyceride (TG) and phospholipid (PL) (calculated as lipid P • 25). This is not precisely total lipid since the f a t t y acid esterified to cholesterol is not included. Nevertheless, the values which were usually less than 1 % are well below those of similar preparations in the rat and dog. Table I I gives the total amount of lipid present in each of the three lipoprotein fractions expressed as mg/ml lymph, and the composition of each fraction expressed as a percentage of total lipid. The smallest lipoprotein fraction was that of d > 1.019. In the lipoproteins of d < 1.019 no consistent division of lipid between chylomicron and V L D L was evident which m a y reflect the rather arbitrary boundary between these two lipid classes. The composition of chylomicrons and very low-density lipoproteins appears to depend on the type of lipid in the diet. In the chylomicron fraction, the mean cholesterol concentration expressed as a percentage of total lipid was 14 % in the animals fed high cholesterol-low fat diet, 8 ~ in those fed high cholesterol-high fat diet and 3 ~ in those fed a high fat diet. Phospholipid, on the other hand, appeared to constitute a rather constant proportion of the chylomicron lipid, with a mean for all groups of 8 %. In the V L D L fractions the cholesterol and phospholipid concentrations were higher than in the chylomicrons and again the highest cholesterol concentration was present in the fraction from animals on a high cholesterol-low fat diet. In Fig. 2 the distribution of cholesterol within the three lipoprotein fractions J. Atheroscler. Res., 7 (1967) 319-329
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Fig. 2. The distribution of the total cholesterol content between the three lipopr~tein fractions, chylomicrons, VLDL and d > 1.019 of thoracic duct lymph in rabbits fed a high cholesterollow fat, high cholesterol-high fat and high fat diets. In the lower graphs cholesterol is expressed as mg/100 ml lymph and in the upper graphs as a percentage of the cholesterol ir~ whole lymph. in the lymph is depicted diagrammatically for the various groups of experiments. The lower graphs represent the actual amounts of cholesterol in each lipoprotein fraction expressed as mg/100 ml lymph. The levels for the group on a high fat diet without cholesterol are on the average about 20 mg/100 ml while in the two groups on diets to which cholesterol was added the levels were much higher, rising to 120 mg/ 100 ml, except in RSL1 in which case the lymph was apparently diluted by excessive filtration as shown by the high flow rate (Table I). The difference between the groups is probably due mainly to the degree of absorption of cholesterol from the intestinal lumen. The upper graphs in Fig. 2 show the amount of cholesterol in each of the three lipoprotein classes expressed as a percentage of the total cholesterol in each lymph sample. In most animals, irrespective of the levels of cholesterol or of triglyceride in the lymph, between 60 and 80 ~o of the cholesterol is present in the lipoprotein fraction of d < 1.019, i.e. the chylomicrons and VLDL. The question might be raised whether within the d < 1.019 lipoprotein class one can assign a predominant role as cholesterol carriers to chylomicrons or to the very low-density lipoprotein. Of the 8 lymph samples from animals on high cholesterollow fat diet, 2 had less than 10 % of the total lymph cholesterol in chylomicrons, 3 had j . Atheroscler. Res., 7 (1967) 319-329
CHOLESTEROLTRANSPORTIN LYMPH
327
TABLE III TOTAL PROTEIN
IN LYMPH AND PLASMA AND TOTAL CHOLESTEROL
LYMPH AND PLASMA IN RABBITS DURING
CHOLESTEROL
IN THE d >
1.019
FRACTIONS OF
ABSORPTION
Rabbit n u m b e r
Total protein Total cholesterol
d < 1,019 d > 1,019
lymph (g/100 ml) plasma (g/lO0 ml) 1/p ratio • 100 lymph (mg/lO0 ml) plasma (mg/lO0 ml) t/p ratio • 100 lymph (mg/lO0 ml) plasma (mg/100 ml) l/p ratio x 100
R5
R9
Rll
R8
1.5 5.0 30 0.44 0.62 71 0.08 0.46 17
2.1 4.9 48 0.53 1.20 44 0.35 3.40 10
1.7 4.6 37 0.30 0.72 42 0.27 2.10 13
3.0 6.7 45 0.48 1.20 40 0.29 1.80 16
between 10 and 20 %, 1 had nearly 30 % and 2 samples from rabbit R4 had 46 and 57 %. The last 2 samples had correspondingly less cholesterol in the very low-density fraction. The chylomicrons in lymph samples from animals on the other diets also showed a rather wide range in cholesterol carrying capacity when compared to the other lipoprotein fractions. While it is not possible in these experiments to divide the thoracic duct lymph lipid into two portions, that derived from the intestinal lumen and that derived from the plasma filtrate, a comparison of the concentrations in both plasma and lymph of total protein and of cholesterol in the lipoprotein fractions of d < 1.019 and d > 1.019, presented in Table III, gives an indication of this division. In four experiments the mean l y m p h : p l a s m a ratios, expressed as a percentage, were 40 for total protein, 49 for cholesterol in lipoprotein of d < 1.019 and 14 for cholesterol in lipoprotein of d > 1.019. DISCUSSION Most lipid absorbed from the intestinal lumen enters the lacteals and ultimately the thoracic duct. It is, however, also well-known that an appreciable but varying amount of the lipoprotein in the plasma is filtered from the circulation each day to be returned to the blood stream by the lymphatics 17. This applies particularly to the smaller lipoproteins of density > 1.019 and varies in extent in different tissues, being much greater in the liver than in a limb is-20. The thoracic duct collects lymph from most tissues of the body, but in our experiments, with the animal at rest, the intestines and liver would be the main sources. In the rabbit the lymph flow from the liver is about 1 ml/h (ref. 21) and thus represents a relatively small fraction of the total thoracic duct flow, although the leakage of the lipoproteins through the liver sinusoids is greater than in most other tissuesl9, 20. Taking these facts into consideration the results in Table I I I suggest that the cholesterol in lipoprotein of d > 1.019 could all or nearly all be derived from the j . Atheroscler. Res., 7 (1967) 319-329
328
D . B . ZILVERSMIT, F. C. COURTICE, R. FRASER
plasma filtrate since the L : P ratio for this fraction, 14 %, is only about one-third that for total protein, 40 %. If we assume that all of the d > 1.019 lipoprotein cholesterol in the lymph is filtered from the plasma, the L : P ratio for the filtered portion of the lipoprotein cholesterol of d < 1.019 would be very much less than 14 %, since the size of these lipoproteins is greater. Since the actual L : P ratio for the d < 1.019 lipoprotein cholesterol is 49 %, it would seem t h a t practically all has been derived b y absorption from the intestinal lumen. Recent studies by ROHEIM et al. ~2 also show t h a t the dog in the postabsorptive state derives part of its d < 1.019 plasma lipoprotein from the intestine. The present investigation shows that in rabbits on the high cholesterol-low fat diet, less than 30 % of the lymph cholesterol was present in the chylomicrons in four out of five animals, while 30-70 % was present in the V L D L fraction. On the high cholesterol-high fat diet the animal receiving shortening carried 27 % of its lymph cholesterol in the chylomicrons and 35 % in the VLDL, whereas the animal receiving corn oil and cholesterol had 5 1 % of the lymph cholesterol in the chylomicrons and 33 % in the VLDL. These findings are rather different from those observed in the rat and dog. In rats fed 100 mg cholesterol and 3 ml olive oil, BYERS AND FRIEDMAN23 observed that 96 % of the lymph cholesterol was in the chylomicron fraction. Similarly HILLYARD et al. 24 observed in the dog fed 30 ml corn oil and 0.5 g cholesterol t h a t the thoracic duct lymph chylomicron fraction (Sf 103-105) had 10 times as much cholesterol as the V L D L (Sf 20-1000) fraction. Although our separation procedure of rabbit lymph did not exactly parallel that of HILLYARD et al., our chylomicrons include, if anything, a wider range of lipoprotein (Sf > 400) and the V L D L possibly somewhat less (Sr 12-400) and yet we find relatively more cholesterol in the V L D L and less in the chylomicrons than did HILLYARD et al. This same type of difference is also reflected in the composition of lymph lipoproteins of different cholesterol-fed animal species. BRAGDON25 reported t h a t in rats receiving olive oil and cholesterol the chylomicrons contained 1.9 % cholesterol. In the experiments of BYERS AND FRIEDMAN the ratio of cholesterol to oil which was fed to rats was very similar to ours whereas HILLYARD fed relatively somewhat less cholesterol. R a t chylomicrons were reported to contain 2.8 % cholesterol, dog chylomicrons about 3.5 % but the chylomicrons of rabbits in our study contained an average of 8 % cholesterol on the high cholesterol-high fat diet and 14 % on the high cholesterollow fat regimen. A similar difference is apparent in the V L D L fractions. HILLYARD reports 4 % cholesterol for the dog, while we find 15 % in the rabbit on high cholesterol-high fat intake and 23 % on the low fat regimen. I t seems that the lipoproteins in the thoracic duct lymph of cholesterol-fed rabbits are excessively loaded with cholesterol as compared to other species. One might speculate t h a t the tendency in the rabbit to develop hypercholesterolaemia and atherosclerosis is related to the form in which cholesterol is transported from the intestine to the bloodstream via the thoracic duct.
j . Atheroscler. Res., 7 (1967) 319-329
CHOLESTEROL TRANSPORT IN LYMPH
329
ACKNOWLEDGEMENTS The authors gratefully acknowledge the technical assistance of Miss Jacquetine Dempsey
and Miss Carolyn Podger.
REFERENCES 1 POPJ~.K, G., Biochem. J., 1946, 40: 608. 2 MORRIS, B. AND F. C. COURTICE, Quart. J. Exptl. Physiol., 1955, 40: 149. 3 YOUNG, N. AND N. K. FREEMAN, Proc. Soc. Exptl. Biol. Med., 1955, 90: 463. 4 BOLLMAN, J. L., J. c. CAIN AND J. H. GRINDLAY, J . Lab. Clin. Med., 1948, 33: 1349. 5 DOLE, V. P. AND J. T. HAMLIN III, Physiol. Rev., 1962, 42: 674. 8 HAVEL, R. J., H. A. EDER, AND J. H. BRAGDON, J. Clin. Invest., 1955, 34: 1345.
7 FOLCH, J., I. AscoLi, M. LEES, J. A. HEATH AND F. N. LEBARON, J. Biol. Chem., 1951, 191: 833. 8 ABELL, L. L., B. 13. LEVY, B. B. BRODIE AND F. E. KENDALL, J. Biol. Chem., 1952, 195: 3S7. 9 ZAK, ]~., N. Moss, A. J. BOYLE AND A. ZLATKIS, Analyt. Chem., 1954, 26: 776. 10 BARTLETT, G. R., J. Biol. Chem., 1959, 234: 466. 11 MINARI, O. AND D. B. ZILVERSMIT, J. Lipid Res., 1963, 4: 424. 12 VAN HANDEL, E., AND D. B. ZILVERSMIT J. Lab. Clin. Med., 1957, 50: 152. 18 CON~,VAY, E. J. AND E. O']V[ALLEY, Biochem. J., 1942, 35: 655. 14 MINARI, O. AND D. B. ZILVERSMIT, Analyt. Biochem., 1963, 6: 320. 15 PAGE, I. H., L. A. LEWIS AND G. PLAHL, Circulation Res., 1953, 1: 87. 16 COURTICE, F. C. AND B. MORRIS, Quart. J. Exptl. Physiol., 1955, 40: 138. a7 YOFEEY, J. M. AND F. C. COURTICE, Lymphatics, Lymph and Lymphoid Tissue, Arnold, London, and H a r v a r d University Press, Cambridge, Mass., 1956. 18 COURTICE, F. C. AND D. G. GARLICK, Quart. J. Exptl. Physiol., 1962, 47: 221. 19 COURTICE, F. C., G. WOOLLEY AND D. G. GARLICK, Australian f . Exptl. Biol. Med. Sci., 1962, 40: 111. 20 COURTICE, F. C., in: H. S. MAYERSON (Ed.), Symposium on "Lymph and Lymphatics", 1966. In press. 21 COURTICE, F. C., Australian J. Exptl. Biol. Med. Sci., 1960, 38: 403. 22 ROHE1M, P. S., L. I. GIDEZ AND H. A. EDER, J. Clin. Invest., 1966, 45: 297. 23 BYERS, S. O. AND M. FRIEDMAN, Am. J. Physiol., 1954, 179: 79. 24 HILLYARD, L. A., I. L. CHAIKOFF, C. ENTENMAN AND W. O. REINHARDT, J. Biol. Chem., 1958, 233 : 838. 25 BRAGDON, J., J. Lab. Clin. Med., 1958, 52: 564.
j . Atheroscler. Res., 7 (1967) 319-329
C H O L E S T E R O L T R A N S P O R T I N THORACIC DUCT L Y M P H OF T H E RABBIT D. B. Z I L V E R S M I T , F. C. C O U R T I C E AND R. F R A S E R
Department of Experimental Pathology, John Curtin School of Medical Research, Australian National University, Canberra (Australia) ( R e c e i v e d J u l y 10th, 1966)
SUMMARY
The method of transport of cholesterol in the thoracic duct lymph collected in unanaesthetized rabbits has been determined in three groups of animals fed a high cholesterol-low fat, high cholesterol-high fat or high fat diet. Three lipoprotein fractions in the lymph were separated b y the preparative ultracentrifuge, viz. (a) chylomicrons of Sf > 400, (b) very low-density lipoproteins (VLDL) of Sf 12-400 and d < 1.019, and (c) low and high density lipoproteins of d > 1.019. In those experiments in which the total protein as well as the lipoprotein fractions were determined in both lymph and plasma, the results suggest that in the thoracic duct l y m p h most of the cholesterol in the lipoprotein of d < 1.019 was derived b y absorption from the intestine. Most of the lymph cholesterol, 60-80 %, was carried in the chylomicron and V L D L fractions, i.e., d < 1.019. The distribution between these two fractions varied considerably, but compared with the rat and the dog, relatively more cholesterol was carried in the V L D L fraction. The cholesterol as a percentage of totat lipid in each of these two fractions was also greater in the rabbit than in the rat or dog on similar high cholesterol diets. A possible relationship between the tendency of the rabbit to develop hypercholesterolaemia and the form in which cholesterol is transported in thoracic duct lymph is suggested.
INTRODUCTION
Although much information is available on the composition and turnover of D. B. ZILVERSMIT: Career I n v e s t i g a t o r of t h e A m e r i c a n H e a r t Association. P e r m a n e n t a d d r e s s : G r a d u a t e School of N u t r i t i o n , CorneU U n i v e r s i t y , I t h a c a , N.Y. (U.S.A.).
J. Atheroscler. Res., 7 (1967) 3 1 9 - 3 2 9
320
D. B. ZILVERSMIT, F. C. COURTICE, R. FRASER
plasma lipids in the rabbit, practically nothing is known about the transport of lipids from the intestinal tract to the blood stream. Since rabbits are particularly prone to develop hypercholesterolaemia in response to cholesterol feeding, it seemed worthwhile to study the lymph lipids during cholesterol absorption to ascertain whether in this animal cholesterol is absorbed in a form which might account for the hypercholesterolaemia. In terminal experiments, PoPjAK 1 collected small samples of lymph from the cysterna chyli of three rabbits fed amorphous cholesterol for 1 month. The concentration of cholesterol in the lymph was less than one-third that of plasma. Although the animals did not receive appreciable amounts of fat in their diet, the neutral fat in the lymph was four times the concentration in the plasma. In another group of six rabbits which were fed a cholesterol-rich diet for 3 months and then a diet of only vegetable leaves for 7-10 days before l y m p h collection, MORRIS AND COORTICE2 found that on the average the concentration of total cholesterol in the lymph was also about one-third of the plasma level and the total f a t t y acids about one-half. In all of these experiments, the rabbits were under anaesthesia during lymph collection. One reason for the paucity of d a t a on the lipids in the thoracic duct l y m p h of rabbits is the difficulty involved in collecting lymph for several days after cannulation. YOUNG AND FREEMAN3 report the collection of thoracic duct lymph in rabbits for 48 h after recovery from anaesthesia, and the determination of its triglyceride content, but no details are given concerning the l y m p h flow or nutritional state of the animals. We have collected thoracic duct lymph from conscious rabbits for periods up to 7 days after cannulation. In the present paper we present data on the quantity and composition of lipoprotein fractions in thoracic duct lymph after feeding diets containing varying amounts of cholesterol and fat. METHODS
New Zealand white rabbits of either sex and 2.5-3.5 kg weight were maintained on ground rabbit chow containing 5 ~ triglyceride. For varying intervals before cannulation of the thoracic duct, as stated later, the experimental animals were kept on a diet of chow to which was added 0.8 % cholesterol* (high cholesterollow fat diet), 0.8 ~ cholesterol and 20 % fat* (high cholesterol-high fat diet) or20 ~ fat* (high fat diet). The thoracic duct was cannulated in the upper part of the mediastinum after anesthetizing the animal with N e m b u t a l | (about 30 mg/kg) injected into the marginal ear vein. In most cases an intravenous infusion of 0.9 % NaC1 (0.5-1 ml/min) was given during the course of the operation and for a varying time afterwards. A tracheal tube was inserted and connected with a Bird** respirator providing oxygen at inter* High cholesterol-low fat diet: powdered cholesterol was mixed with ground rabbit chow. High cholesterol-high fat diet: melted fat was mixed with above diet. T h e fat ( F r y m a s t a - V e g e t a b l e oils P t y Ltd. Australia) contained primarily p a l m i t a t e and oleate. ** Bird Corp., P a l m Springs, Calif. (U.S.A.). J. Atheroscler. Res., 7 (1967) 319-329
CHOLESTEROL TRANSPORT IN LYMPH
321
mittent pressures of 10-20 cm of water to prevent pneumothorax during the operative procedure. The thoracic duct was exposed b y partially severing the left sternocleidomastoid and pectoralis major muscles near their origin on the sternum, and then separating trachea and oesophagus from the left carotid sheath. The trachea, oesophagus and right common carotid sheath were displaced to the right and the left carotid sheath, the left external jugular vein and the left sternomastoid and sternohyoid muscles to the left. The tip of the sternum was then lifted with a retractor exposing the upper mediastinum on the left side. The thoracic duct was located as it passes dorsal and in close proximity to the aortic arch and was cannulated in this position with polyethylene or polyvinyl tubing of 0.5 m m internal and 0.8 m m external diameter. The free end of the tubing was then passed under the skin to a suitable exit along the back or side of the animal. Shortly after the completion of surgery, the rabbit was transferred to a restraining cage designed after the pattern of BOLLMAN et al. 4 for rats (Fig. 1). The cage allowed some freedom of movement
Fig. 1. Restraining cage for rabbits. back and forth and freedom to eat and drink. In prolonged experiments the animal was removed from the restraining cage periodically during which time the lymph was not collected. In some animals, cholesterol or corn oil was administered orally or b y duodenal tube, as stated later in the text. All lymph was obtained from conscious animals, the flow rate varying between 4 and 25 ml/h depending primarily on the degree of hydration of the animal. The samples were collected in chilled containers, allowed to clot and defibrinated b y repeated stirring and filtering through gauze. Blood samples were taken at intervals for the determination of plasma proteins or for the separation and analysis of the plasma lipoproteins. In the lymph samples a chylomicron fraction was isolated b y initial centriJ. Atheroscler. Res., 7 (1967) 319-329
322
D . B . ZILVERSMIT, F. C. COURTICE, R. FRASER
fugation in a Spinco SW 25.1 swinging bucket head for 1 h at 7-10~ and an average centrifugal force of 64,000 • g. The chylomicron fraction was centrifuged once at the same temperature and speed through a 5-cm layer of saline (d = 1.006) carefully pipetted on top of the chylomicrons resuspended in a salt solution of density approximately equal to 1.2. The second centrifugation for 2 h followed by removal of the top layer b y tube slicer yielded a fraction of S I > 400 (ref.5), henceforth called chylomicrons. The chylomicron-free sample was adjusted to density 1.019, centrifuged in a Spinco 40.3 or 50 rotor for 16 h at 100,000 • g or more and then separated into two fractions by slicing. The upper fraction containing lipoproteins of S s 12-400 (ref. 6) will be referred to as very low density lipoprotein (VLDL). The lower fraction of d > 1.019 contains low and high density lipoproteins. In some experiments plasma samples were fractionated at a density of 1.019 without prior separation of chylomicrons. All lipoprotein fractions were extracted with a minimum of 20 vol. chloroformmethanol (2 : 1), and washed b y equilibration with an excess of water 7. Total cholesterol was determined after saponification and extraction8 colorimetrically 9, and lipid phosphorus b y the method of BARTLETT10. A portion of the sample was chromatographed with a column of silicic acid supercel and triglycerides eluted with chloroform 11. Triglycerides were then determined b y the method of VAN HANDEL AND ZILVERSMIT12. Protein nitrogen in lymph and blood plasma samples was measured after micro-Kjeldahl digestion and subsequent titration 13 or nesslerization 14. RESULTS
The data in Tables I and I I summarize the findings on ten rabbits receiving various diets for differing periods of time. They are arranged in three groups depending on whether the animals were ingesting cholesterol in a low fat diet, cholesterol in a high fat diet, or a high fat diet without added cholesterol. Table I gives the time of collection of the lymph samples, the rate of flow and the concentrations of total protein and total lipid in the lymph. In the first group R4 and R5 received the diet of 0.8 % cholesterotin rabbit chow for 10 days, R9 for 20 days and R11 for only 2 days before cannulation. The duration of this diet was varied in an a t t e m p t to obtain animals with a relatively high level of cholesterol in the lymph at the lowest possible plasma concentration, thus avoiding too great a contribution to lymph of plasma cholesterol filtered through capillary beds drained b y the thoracic duct2,15,16. This same consideration also led to a somewhat different approach with rabbit 12. This animal had been kept on 20 ~o fat intake prior to cannulation so that the l y m p h collected during the first few days after cannulation was rather turbid (optical density at 650 m # about 2). 24 h after cannulation a 1 % cholesterol suspension was administered b y gastrostomy tube. From 42 to 66 h 4 g cholesterol on lettuce leaves was fed. At 66 h 2 g and at 73 h a further 2 g cholesterol was fed on lettuce leaves. L y m p h samples L1 and L2 from this animal were collected from 66 to 73 and 73 to 89 h respectively. They showed optical densities at 650 m/z of 0.8 and j . Atheroscler. Res., 7 (1967) 319-329
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0.2 and were considered to represent absorbed cholesterol without fat in the diet. The second group of animals received the high cholesterol-high fat diet. R8 was fed a diet containing 20 ~o fat for 12 days to which 0.8 ~ cholesterol was added for 3 days before the thoracic duct was cannulated. R12 had been used, as already stated, in the first group. On the fifth day (at 97 h) after cannulation the animal received 2 g cholesterol on lettuce b y mouth and 1 ml corn oil b y gastrostomy tube. When the lymph became quite milky L3 was collected for 8 h (O.D. at 650 m # = 3.0). At 113 h 1 g cholesterol dissolved in 5 ml corn oil was administered b y duodenal tube. A final lymph sample, L4, was collected from 116 to 125 h. Samples L3 and L4 from this animal thus represent lymph collected during absorption of cholesterol in the presence of corn oil. In the third group 20 % fat was added to the chow for several days before cannulation. R3 also received corn oil by mouth at intervals from 18 h after cannulation. The corn oil was placed on saline which this animal drank readily. R15 was given 6 ml corn oil by mouth in capsules before cannulation of the duct. The very high flow rate with relatively low protein concentration in the lymph of some animals, especially during the first few hours after recovery from anaesthesia, was due in part to the intravenous saline drip which was given during the operative procedure and for varying times postoperatively to all animals except R12. The concentration of lipid in the lymph would also be affected b y the extent of the infusion. In addition it would depend on the actual amount of lipid absorbed which would be related to the previous diet, the degree of emptying of the stomach and the amount of fat or cholesterol given b y mouth or b y duodenal tube. In these experiments the concentration of " t o t a l " lipid in the thoracic duct lymph was taken as the sum of total cholesterol (TC), triglyceride (TG) and phospholipid (PL) (calculated as lipid P • 25). This is not precisely total lipid since the f a t t y acid esterified to cholesterol is not included. Nevertheless, the values which were usually less than 1 % are well below those of similar preparations in the rat and dog. Table I I gives the total amount of lipid present in each of the three lipoprotein fractions expressed as mg/ml lymph, and the composition of each fraction expressed as a percentage of total lipid. The smallest lipoprotein fraction was that of d > 1.019. In the lipoproteins of d < 1.019 no consistent division of lipid between chylomicron and V L D L was evident which m a y reflect the rather arbitrary boundary between these two lipid classes. The composition of chylomicrons and very low-density lipoproteins appears to depend on the type of lipid in the diet. In the chylomicron fraction, the mean cholesterol concentration expressed as a percentage of total lipid was 14 % in the animals fed high cholesterol-low fat diet, 8 ~ in those fed high cholesterol-high fat diet and 3 ~ in those fed a high fat diet. Phospholipid, on the other hand, appeared to constitute a rather constant proportion of the chylomicron lipid, with a mean for all groups of 8 %. In the V L D L fractions the cholesterol and phospholipid concentrations were higher than in the chylomicrons and again the highest cholesterol concentration was present in the fraction from animals on a high cholesterol-low fat diet. In Fig. 2 the distribution of cholesterol within the three lipoprotein fractions J. Atheroscler. Res., 7 (1967) 319-329
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Fig. 2. The distribution of the total cholesterol content between the three lipopr~tein fractions, chylomicrons, VLDL and d > 1.019 of thoracic duct lymph in rabbits fed a high cholesterollow fat, high cholesterol-high fat and high fat diets. In the lower graphs cholesterol is expressed as mg/100 ml lymph and in the upper graphs as a percentage of the cholesterol ir~ whole lymph. in the lymph is depicted diagrammatically for the various groups of experiments. The lower graphs represent the actual amounts of cholesterol in each lipoprotein fraction expressed as mg/100 ml lymph. The levels for the group on a high fat diet without cholesterol are on the average about 20 mg/100 ml while in the two groups on diets to which cholesterol was added the levels were much higher, rising to 120 mg/ 100 ml, except in RSL1 in which case the lymph was apparently diluted by excessive filtration as shown by the high flow rate (Table I). The difference between the groups is probably due mainly to the degree of absorption of cholesterol from the intestinal lumen. The upper graphs in Fig. 2 show the amount of cholesterol in each of the three lipoprotein classes expressed as a percentage of the total cholesterol in each lymph sample. In most animals, irrespective of the levels of cholesterol or of triglyceride in the lymph, between 60 and 80 ~o of the cholesterol is present in the lipoprotein fraction of d < 1.019, i.e. the chylomicrons and VLDL. The question might be raised whether within the d < 1.019 lipoprotein class one can assign a predominant role as cholesterol carriers to chylomicrons or to the very low-density lipoprotein. Of the 8 lymph samples from animals on high cholesterollow fat diet, 2 had less than 10 % of the total lymph cholesterol in chylomicrons, 3 had j . Atheroscler. Res., 7 (1967) 319-329
CHOLESTEROLTRANSPORTIN LYMPH
327
TABLE III TOTAL PROTEIN
IN LYMPH AND PLASMA AND TOTAL CHOLESTEROL
LYMPH AND PLASMA IN RABBITS DURING
CHOLESTEROL
IN THE d >
1.019
FRACTIONS OF
ABSORPTION
Rabbit n u m b e r
Total protein Total cholesterol
d < 1,019 d > 1,019
lymph (g/100 ml) plasma (g/lO0 ml) 1/p ratio • 100 lymph (mg/lO0 ml) plasma (mg/lO0 ml) t/p ratio • 100 lymph (mg/lO0 ml) plasma (mg/100 ml) l/p ratio x 100
R5
R9
Rll
R8
1.5 5.0 30 0.44 0.62 71 0.08 0.46 17
2.1 4.9 48 0.53 1.20 44 0.35 3.40 10
1.7 4.6 37 0.30 0.72 42 0.27 2.10 13
3.0 6.7 45 0.48 1.20 40 0.29 1.80 16
between 10 and 20 %, 1 had nearly 30 % and 2 samples from rabbit R4 had 46 and 57 %. The last 2 samples had correspondingly less cholesterol in the very low-density fraction. The chylomicrons in lymph samples from animals on the other diets also showed a rather wide range in cholesterol carrying capacity when compared to the other lipoprotein fractions. While it is not possible in these experiments to divide the thoracic duct lymph lipid into two portions, that derived from the intestinal lumen and that derived from the plasma filtrate, a comparison of the concentrations in both plasma and lymph of total protein and of cholesterol in the lipoprotein fractions of d < 1.019 and d > 1.019, presented in Table III, gives an indication of this division. In four experiments the mean l y m p h : p l a s m a ratios, expressed as a percentage, were 40 for total protein, 49 for cholesterol in lipoprotein of d < 1.019 and 14 for cholesterol in lipoprotein of d > 1.019. DISCUSSION Most lipid absorbed from the intestinal lumen enters the lacteals and ultimately the thoracic duct. It is, however, also well-known that an appreciable but varying amount of the lipoprotein in the plasma is filtered from the circulation each day to be returned to the blood stream by the lymphatics 17. This applies particularly to the smaller lipoproteins of density > 1.019 and varies in extent in different tissues, being much greater in the liver than in a limb is-20. The thoracic duct collects lymph from most tissues of the body, but in our experiments, with the animal at rest, the intestines and liver would be the main sources. In the rabbit the lymph flow from the liver is about 1 ml/h (ref. 21) and thus represents a relatively small fraction of the total thoracic duct flow, although the leakage of the lipoproteins through the liver sinusoids is greater than in most other tissuesl9, 20. Taking these facts into consideration the results in Table I I I suggest that the cholesterol in lipoprotein of d > 1.019 could all or nearly all be derived from the j . Atheroscler. Res., 7 (1967) 319-329
328
D . B . ZILVERSMIT, F. C. COURTICE, R. FRASER
plasma filtrate since the L : P ratio for this fraction, 14 %, is only about one-third that for total protein, 40 %. If we assume that all of the d > 1.019 lipoprotein cholesterol in the lymph is filtered from the plasma, the L : P ratio for the filtered portion of the lipoprotein cholesterol of d < 1.019 would be very much less than 14 %, since the size of these lipoproteins is greater. Since the actual L : P ratio for the d < 1.019 lipoprotein cholesterol is 49 %, it would seem t h a t practically all has been derived b y absorption from the intestinal lumen. Recent studies by ROHEIM et al. ~2 also show t h a t the dog in the postabsorptive state derives part of its d < 1.019 plasma lipoprotein from the intestine. The present investigation shows that in rabbits on the high cholesterol-low fat diet, less than 30 % of the lymph cholesterol was present in the chylomicrons in four out of five animals, while 30-70 % was present in the V L D L fraction. On the high cholesterol-high fat diet the animal receiving shortening carried 27 % of its lymph cholesterol in the chylomicrons and 35 % in the VLDL, whereas the animal receiving corn oil and cholesterol had 5 1 % of the lymph cholesterol in the chylomicrons and 33 % in the VLDL. These findings are rather different from those observed in the rat and dog. In rats fed 100 mg cholesterol and 3 ml olive oil, BYERS AND FRIEDMAN23 observed that 96 % of the lymph cholesterol was in the chylomicron fraction. Similarly HILLYARD et al. 24 observed in the dog fed 30 ml corn oil and 0.5 g cholesterol t h a t the thoracic duct lymph chylomicron fraction (Sf 103-105) had 10 times as much cholesterol as the V L D L (Sf 20-1000) fraction. Although our separation procedure of rabbit lymph did not exactly parallel that of HILLYARD et al., our chylomicrons include, if anything, a wider range of lipoprotein (Sf > 400) and the V L D L possibly somewhat less (Sr 12-400) and yet we find relatively more cholesterol in the V L D L and less in the chylomicrons than did HILLYARD et al. This same type of difference is also reflected in the composition of lymph lipoproteins of different cholesterol-fed animal species. BRAGDON25 reported t h a t in rats receiving olive oil and cholesterol the chylomicrons contained 1.9 % cholesterol. In the experiments of BYERS AND FRIEDMAN the ratio of cholesterol to oil which was fed to rats was very similar to ours whereas HILLYARD fed relatively somewhat less cholesterol. R a t chylomicrons were reported to contain 2.8 % cholesterol, dog chylomicrons about 3.5 % but the chylomicrons of rabbits in our study contained an average of 8 % cholesterol on the high cholesterol-high fat diet and 14 % on the high cholesterollow fat regimen. A similar difference is apparent in the V L D L fractions. HILLYARD reports 4 % cholesterol for the dog, while we find 15 % in the rabbit on high cholesterol-high fat intake and 23 % on the low fat regimen. I t seems that the lipoproteins in the thoracic duct lymph of cholesterol-fed rabbits are excessively loaded with cholesterol as compared to other species. One might speculate t h a t the tendency in the rabbit to develop hypercholesterolaemia and atherosclerosis is related to the form in which cholesterol is transported from the intestine to the bloodstream via the thoracic duct.
j . Atheroscler. Res., 7 (1967) 319-329
CHOLESTEROL TRANSPORT IN LYMPH
329
ACKNOWLEDGEMENTS The authors gratefully acknowledge the technical assistance of Miss Jacquetine Dempsey
and Miss Carolyn Podger.
REFERENCES 1 POPJ~.K, G., Biochem. J., 1946, 40: 608. 2 MORRIS, B. AND F. C. COURTICE, Quart. J. Exptl. Physiol., 1955, 40: 149. 3 YOUNG, N. AND N. K. FREEMAN, Proc. Soc. Exptl. Biol. Med., 1955, 90: 463. 4 BOLLMAN, J. L., J. c. CAIN AND J. H. GRINDLAY, J . Lab. Clin. Med., 1948, 33: 1349. 5 DOLE, V. P. AND J. T. HAMLIN III, Physiol. Rev., 1962, 42: 674. 8 HAVEL, R. J., H. A. EDER, AND J. H. BRAGDON, J. Clin. Invest., 1955, 34: 1345.
7 FOLCH, J., I. AscoLi, M. LEES, J. A. HEATH AND F. N. LEBARON, J. Biol. Chem., 1951, 191: 833. 8 ABELL, L. L., B. 13. LEVY, B. B. BRODIE AND F. E. KENDALL, J. Biol. Chem., 1952, 195: 3S7. 9 ZAK, ]~., N. Moss, A. J. BOYLE AND A. ZLATKIS, Analyt. Chem., 1954, 26: 776. 10 BARTLETT, G. R., J. Biol. Chem., 1959, 234: 466. 11 MINARI, O. AND D. B. ZILVERSMIT, J. Lipid Res., 1963, 4: 424. 12 VAN HANDEL, E., AND D. B. ZILVERSMIT J. Lab. Clin. Med., 1957, 50: 152. 18 CON~,VAY, E. J. AND E. O']V[ALLEY, Biochem. J., 1942, 35: 655. 14 MINARI, O. AND D. B. ZILVERSMIT, Analyt. Biochem., 1963, 6: 320. 15 PAGE, I. H., L. A. LEWIS AND G. PLAHL, Circulation Res., 1953, 1: 87. 16 COURTICE, F. C. AND B. MORRIS, Quart. J. Exptl. Physiol., 1955, 40: 138. a7 YOFEEY, J. M. AND F. C. COURTICE, Lymphatics, Lymph and Lymphoid Tissue, Arnold, London, and H a r v a r d University Press, Cambridge, Mass., 1956. 18 COURTICE, F. C. AND D. G. GARLICK, Quart. J. Exptl. Physiol., 1962, 47: 221. 19 COURTICE, F. C., G. WOOLLEY AND D. G. GARLICK, Australian f . Exptl. Biol. Med. Sci., 1962, 40: 111. 20 COURTICE, F. C., in: H. S. MAYERSON (Ed.), Symposium on "Lymph and Lymphatics", 1966. In press. 21 COURTICE, F. C., Australian J. Exptl. Biol. Med. Sci., 1960, 38: 403. 22 ROHE1M, P. S., L. I. GIDEZ AND H. A. EDER, J. Clin. Invest., 1966, 45: 297. 23 BYERS, S. O. AND M. FRIEDMAN, Am. J. Physiol., 1954, 179: 79. 24 HILLYARD, L. A., I. L. CHAIKOFF, C. ENTENMAN AND W. O. REINHARDT, J. Biol. Chem., 1958, 233 : 838. 25 BRAGDON, J., J. Lab. Clin. Med., 1958, 52: 564.
j . Atheroscler. Res., 7 (1967) 319-329