[cf. Pappenheimer, 1953] explain the extent of protein and lipoprotein

Transcription

1 THE ORIGIN OF CHYLOMICRONS IN THE CERVICAL AND HEPATIC LYMPH.' By BEDE MORRIS2 and F. C. COURTICE. From the Kanematsu Memorial Institute of Pathology, Sydney Hospital, Sydney. (Received for publication 14th December 1955) IT is known that the lipoproteins of the plasma can escape from the circulating plasma intact and enter the lymph in at least several regions of the body. Lipids in this form, therefore, undergo a tissue spacelymphatic circulation similar to that occurring with the plasma proteins in general. It has been shown [Courtice and Morris, 1955] that lymph collected from such areas as the head and neck and hind limbs, though macroscopically clear, contains fat particles which are similar in size and behaviour to the chylomicrons found in the intestinal lymph during fat absorption. The current concepts of capillary permeability [cf. Pappenheimer, 1953] explain the extent of protein and lipoprotein leakage through pores in the blood capillaries, but the chylomicrons are on an average many times larger than these pores. The permeability of different capillary beds is thought to vary, and the composition of liver lymph [Morris, 1956 a] suggests that the endothelial lining of the hepatic sinusoids is very permeable to both plasma proteins and lipoproteins. In view of the structure of the hepatic sinusoidal wall and the intimate association of the liver with fat storage and intermediary lipid metabolism, it was considered likely that much of the chylomicron fat would leave the circulation and enter the hepatic cells. This is in fact known to occur in rats following the intravenous injection of artificial fat emulsions [Waddell, Geyer, Clarke and Stare, 1953, 1954]. Our earlier report of the presence of chylomicrons in lymph from the cervical and leg ducts has been extended in an attempt to count these fat particles in the cervical and hepatic lymph and to ascertain whether they originate from the plasma as constituents of the capillary filtrate. METHODS Acute experiments were carried out on cats anaesthetized with intravenous nembutal (Abbott). The methods of lipid and protein analysis and zone electrophoresis have been described previously [Morris and Courtice, 1955]. Chylomicron counts were made using a standard 1 This work was carried out with the aid of a grant from the National Health and Medical Research Council, Canberra. 2 Present address: Sir William Dunn School of Pathology, Oxford. 341

2 342 Morris and Courtice dark field condenser, microscope and filtered light source. Postabsorptive plasma and lymph samples were counted without dilution, while hepatic lymph and plasma in absorptive samples were diluted 1 in 200 with 40 per cent urea solution before counting [Tidwell, 1950]. All counts were carried out immediately after collecting the sample. An ocular net was inserted into an eyepiece and sixteen randomly selected squares counted. The mean count of the sixteen squares in five different fields was recorded. In very fatty plasma and lymph samples, where chylomicron counting was impossible, opacities of samples were measured on a Beckman Quartz model D.U. spectrophotometer at a wavelength of 650 m,. Samples were diluted 1 in 26 with normal saline before reading. Fatty chyle for intravenous injection was obtained from the thoracic duct of the experimental animal itself, or from donor cats previously given a fatty meal. This lymph was always collected and transfused on the same day to avoid toxic reactions and chylomicron clumping. It had been found that stored fatty chyle produced hsemolysis when given intravenously in large volumes in cats, and this has previously been reported by Freeman and Johnson [1940]. In our experiments this did not occur with freshly collected fatty lymph. The intrahepatic sinusoidal pressure was raised by partial occlusion of the inferior vena cava above the liver [Morris, 1956 b]. Venous pressure measurements were made through an indwelling polythene catheter inserted through a splenic vein radicle into the portal system, using the method of Burch and Winsor [1943]. RESULTS The Chylomicron Content of Plasma and of Hepatic and Cervical Lymph Chylomicrons were found in the plasma, and in the hepatic and cervical lymph of all 10 cats examined (fig. la and b). The liver lymph contained relatively large numbers of these particles, and in postabsorptive cats the number present was of the same order as in the plasma. The cervical lymph by comparison contained many fewer such particles. Table I gives the number of chylomicrons counted in samples of plasma, hepatic and cervical lymph from a series of absorptive and post-absorptive cats. In animals absorbing fat the plasma was never very opalescent, and the counts could be made on 1 in 200 dilutions. The liver lymph was always clear, but the number of chylomicrons present was much greater than in post-absorptive liver lymph and it was necessary to dilute it before counting. In the cervical lymph, on the other hand, whilst there were more fat particles than in similar samples from post-absorptive cats, counts could be made without dilution.

3 Chylomicrons in Lymph The changes in the plasma and hepatic and cervical lymph chylomicron counts were followed in cats absorbing fat in which the thoracic duct had been cannulated. Diversion of thoracic duct chyle was followed by a fall in the number of chylomicrons in the plasma, and subsequently by a fall in the hepatic and cervical lymph chylomicron counts (Table II). Four hours after diversion of the chyle the counts in the hepatic and cervical lymph had fallen by half. This finding suggested a relationship between lymph and plasma chylomicrons. TABLE I.-CHYLOMICRON COUNTS IN PLASMA AND IN HEPATIC AND CERVICAL LYMPH OF CATS Mean figures are given for counts on 5 fields Cat No. Plasma Liver lymph Cervical lymph Post-absorptive-undiluted samples Absorptive-plasma and liver lymph diluted 1/200, cervical lymph undiluted TABLE II.-CHYLOMICRON COUNTS IN THE PLASMA AND HEPATIC AND CERVICAL, DUCT LYMPH OF A CAT PREVIOUSLY FED A FAT MEAL. AFTER INITIAL OBSERVATIONS OVER A PERIOD OF 3 HOURS, THE THORACIc DUCT WAS CANNULATED AND THE FATTY CHYLE DIVERTED FROM THE BLOOD STREAM Mean figures are given for counts on 5 fields Plasma 1/200 dilution Hepatic lymph 1/200 dilution Cervical lymph undiluted Before cannulating thoracic duct After cannulating duct: thoracic I hour hours ,, ,, The Effect of Fatty Chyle Transfusions on the Lymph Chylomicron Counts After cannulating the thoracic duct, fatty chyle was collected for some hours and then reinjected intravenously to create an acute lipaemic state. This procedure was carried out in three individual experiments VOL. XLI, NO

4 344 Morris and Courtice and the results were similar in each. A typical experiment is represented in Table III. Following the fatty chyle injection, the plasma TABLE III.-THE EFFECT OF THE INTRAVENOUS INJECTION OF 60 ML. FATTY CHYLE CONTAINING MEQ./L. TOTAL FATTY ACID INTO A FASTING CAT 2-6 KG. BODY WEIGHT. Before injection Immediately after 1st hour 2nd hour 3rd hour Plasma Total fatty acid (meq./l.) Chylomicron count (undiluted) 96.. Hepatic lymph Flow (ml./hr.) * Total fatty acid (meq./l.) Chylomicron count (undiluted) Cervical lymph Flow (ml./hr.) * * * *2 Chylomicron count (undiluted) became very milky, but the removal of the injected chylomicron fat proceeded at such a rapid rate that within 2 hours nearly all the injected lipid had left the circulating plasma. In this experiment, 60 ml. of chyle containing meq./l. of total fatty acids (1835 mg. of fat in all, expressed as tripalmitin) was injected, and the plasma total fatty acid concentration rose from 4-3 to 29-0 meq./l., but fell to 7*5 meq./l. within 1 hour. The number of chylomicrons in the plasma immediately following the injection was enormous and could not be counted. The chylomicron count in the hepatic lymph increased to about twice the pre-infusion level, and in the pooled sample of lymph collected for 3 hours after the infusion, the total fatty acid concentration (which represents the fatty acids in the lipoproteins as well as in the chylomicrons) rose from 3-9 to 5-8 meq./l. The total amount of lipid in the hepatic lymph, produced during this 3-hour period, however, was only 1 per cent of the amount injected, and the actual increase in the lymph lipids over the pre-infusion level was only one-third of this. The chylomicron count in the cervical lymph also rose, but in terms of total lipid this rise represented only a very small fraction of the fat injected, since the lymph flow from this duct was only 0*2 ml./hr. In this particular experiment, therefore, approximately 1835 mg. of fat, injected into the blood stream mainly in chylomicron form, passed from the circulation within 1 to 2 hours and only 0-4 per cent of it returned in the lymph. Over 99 per cent of this fat, then, on leaving the circulation was deposited in the tissues. The total amount of protein leaving the circulation per hour was 77 mg. in the liver, 142 mg. in the intestines and 11 mg. in the tissues drained by the cervical duct, that is, only 230 mg./hr. Since the amount

5 Chylomicrons in Lymph 345 of plasma protein leaving the circulation in regions of the body other than those drained by the hepatic and intestinal ducts is small, it is clear that, meight for weight, fat in chylomicron form disappears from the blood stream more rapidly than plasma protein. The Effect of increased Hep)atic Sinusoidal Pressure on Chylomiicron Leak'age in the Liver The endotheliumn of the liver sinusoids is so closely associated with the hepatic cells that any fat leaving the circulation imight be taken up by the parenchymal cells before reaching the lymph. It was thought, however, that by raising the pressure within the sinusoids and increasing !'~~~~~~~~ 7/AVE 1bPJ FiG. 2. The effect of increasing the intrasinusoidal pressure on the appearance of ehylomnicrons in the liver lymlph of the cat, followving the intravenous infusion of fatty ehyle. *: plasma. 0: liver lymlph. the filtration rate alnd liver lymlph flow, the Diss6 spaces wrould be dlistended and mnore chyloma:icron fat, having pasbsed through the wtalls of the sinusoids, wouldl enter the lvmphatics. By partial occlusionl of the imiferior vena cava above the liver, the portal venous pressure wals rajised byt Cmn. of w-ater. J{omologous fatty chyle wras injected intravrenously into 3 calts, anld plalsmla and hepatic lymph samlples collected throughout the ensuing 2 hours. The results of all three experimenlts wrere simlilar, and one experimlent is shown in fig. 2. On1 raising the intrahepatic venous pressure, the liver lymuph

6 346 Morris and Courtice flow rapidly increased [Morris, 1956 b]. Within 10 minutes of the chyle injection the hepatic lymph became grossly cloudy and after 20 minutes appeared milky. The number of chylomicrons in the lymph increased enormously, and could not be counted in a 1 in 200 dilution (fig. 3). After about half an hour the chylomicron content of the plasma and hepatic lymph, assessed by opacity measurements, came into equilibrium. The changes in the chylomicron content of the plasma and lymph were followed closely by changes in the plasma and liver lymph total fatty acid content. In no instance did any appreciable number of red cells appear in the liver lymph while the portal pressure was raised. These experiments suggest that when the filtration rate is raised by increasing the sinusoidal pressure, chylomicrons readily pass intact through the endothelial lining and those that are not taken up by the liver cells pass into the lymph. In actual amount, 16, 14 and 15 per cent of the injected fat was recovered in the hepatic lymph of 3 cats in 2 hours, lymph collection, compared with the normal animal, in which about 0 4 per cent was recovered. Plasma and Liver Lymph Electrophoretic Patterns Following Intravenous Injections of Fatty Chyle In the samples of fatty thoracic duct lymph collected for infusion, the electrophoretic patterns showed the presence of a pre-albumin component containing lipid. This fraction has been previously described [Morris and Courtice, 1955; Morris, 1956 a], and it is passed on to the plasma by the thoracic duct lymph, where it can sometimes be identified. This pre-albumin component is not found normally in post-absorptive cat plasma or hepatic lymph, but following the chyle transfusions, it appeared in the plasma electrophoretic patterns and later in samples of hepatic lymph. It was present in plasma and liver lymph up to 2 hours after the intravenous injection of fatty chyle. In those cats with raised intrahepatic sinusoidal pressures, it appeared in the liver lymph in the first 15 minutes following injection, and its concentration increased during the first 2 to 1 hour. DIsCuSSION Chylomicrons in Lymph The results of these experiments support the earlier findings of Courtice and Morris [1955] that lymph from regions remote from the alimentary tract contains some chylomicrons, and that the number of these particles in the lymph is a reflection of the number in the plasma. This suggests that some chylomicrons escape through the capillary membrane intact, and that when their numbers are increased in the plasma more appear in the lymph. This finding, however, does not

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9 Chylomicrons in Lymph4 347 prove that all or even a large proportion of the chylomicrons entering the blood following a fat meal pass through the capillary membrane in this way. When fat emulsions or fatty chyle are injected intravenously into man or animals, the fat rapidly disappears from the blood stream [cf. Little, Harrison and Blalock, 1942; Meng and Freeman, 1948; Berry and Ivy, 1948; Lerner, Chaikoff and Entenman, 1949]. When artificially stabilized emulsions were injected, most of the fat was deposited in the liver cells or in the spleen [Waddell et al., 1953, 1954]. Attention was therefore focused on the liver. It was found that after the intravenous injection of fresh, homologous fatty chyle only about 04 per cent of the particulate fat disappearing from the blood stream appeared in the hepatic lymph, although histological examination showed the liver cells contained large amounts of fat (fig. 4). The behaviour of chylomicron fat was thus quite different from that of the lipoprotein fat, for, whereas the lipoproteins escaped from the blood stream and recirculated in the lymph, chylomicrons once having left the circulation were taken up by the tissue cells. When the pressure in the sinusoids was raised by partially occluding the inferior vena cava above the liver, the filtration rate through the sinusoidal walls and the perisinusoidal spaces increased and the lymph flow rose. Under these conditions the size of the interstitial pool in the liver increases [Morris, 1956 b], and it is reasonable to assume that there is a distension of the Disse spaces. This, together with the increased lymph flow, would decrease the likelihood of the chylomicrons being taken up by the liver cells, and in these animals as much as 15 per cent of the injected fat was collected in the hepatic lymph in 2 hours. This recovered fat was present as chylomicrons and the lymph became milky. Under these conditions of increased filtration pressure, there was no doubt that at least a considerable proportion of the injected fat left the circulation by passing through the sinusoids as chylomicrons. Whilst the increase in venous pressure may have been responsible for some stretching of the endothelial lining of the hepatic sinusoids, it does appear that these fat particles may escape in quite large numbers through anatomical or functional pores in the walls of the liver capillaries. In this regard, the hepatic sinusoids and possibly those of the spleen may possess a functional degree of permeability intermediate to that seen in lymphatic capillaries and blood capillaries found in other parts of the body. While lymph vessels are readily permeable to chylomicrons and red blood cells [Courtice and Simmonds, 1954; Courtice and Morris, 1955], the blood capillaries allow only an occasional red cell, and in most tissues probably only a few chylomicrons, to escape. The endothelium of a lymphatic capillary is continuous like that of a typical blood capillary, and the way in which such large particles pass through it so readily is not known.

10 348 Morris and Courtice SUMMARY 1. The chylomicron content of the plasma, hepatic and cervical lymph of cats has been examined. Clear lymph coming from the head and neck and the liver contains numerous chylomicrons, the number present in the hepatic lymph being much greater than in the cervical lymph. 2. The number of chylomicrons in the liver and cervical lymph varies with the number present in the plasma, and can be increased by the intravenous injection of fatty chyle, and decreased in absorptive cats by diverting the thoracic duct lymph from the plasma. Following the intravenous infusion of fatty chyle, about 04 per cent of the amount of lipid injected is returned by the lymph during the 2-hour period taken for the plasma lipids to return to normal. 3. Raising the intrahepatic sinusoidal pressure during the fatty chyle transfusions increases the rate of liver lymph flow and the liver lymph rapidly becomes milky. In these animals large amounts of lipid were carried in the liver lymph, and about 14 per cent of the total amount of fat injected was recovered in the lymph in the 2-hour post-injection period. It is Kearns. ACKNOWLEDGMENT a pleasure to acknowledge the technical assistance of Miss Marianne REFERENCES BERRY, I. M. and Ivy, A. C. (1948). Fed. Proc. 7, 7. BURCH, G. E. and WINSOR, T. (1943). J. Amer. med. Assoc. 123, 90. COURTICE, F. C. and MORRIS, B. (1955). Quart. J. exp. Physiol. 40, 138. COURTICE, F. C. and SIMMONDS, W. J. (1954). Phys. Revs. 34, 419. FREEMAN, L. W. and JOHNSON, V. (1940). Amer. J. Physiol. 130, 723. LERNER, S. R., CHAIKOFF, I. L. and ENTENMAN, C. (1949). Proc. Soc. exp. Bios. and Med. 70, 388. LITTLE, J. M., HARRISON, C. and BLALOCK, A. (1942). Surgery, 11, 392. MENG, H. C. and FREEMAN, S. (1948). J. Lab. and clin. Med. 33, 689. MORRIS, B. (1956 a). Quart. J. exp. Physiol. 41, 318. MORRIS, B. (1956 b). Quart. J. exp. Physiol. 41, 326. MORRIS, B. and COURTICE, F. C. (1955). Quart. J. exp. Physiol. 40, 127. PAPPENHEIMER, J. R. (1953). Physiol. Revs. 33, 387. TIDWELL, H. C. (1950). J. biol. Chem. 182, 405. WADDELL, W. R., GEYER, R. P., CLARKE, E. and STARE, F. J. (1953). Amer. J. Physiol. 175, 299. WADDELL, W. R., GEYER, R. P., CLARKE, E. and STARE, F. J. (1954). Amer. J. Physiol. 177, 90.