Korner, Morris and Courtice, 1954; Morris, 1954; Simmonds, 1954,

THE HEPATIC AND INTESTINAL CONTRIBUTIONS TO THE THORACIC DUCT LYMPH.1 By BEDE MORRIS.2 From the Kanematsu Memorial Institute of Pathology, Sydney Hospital, Sydney. (Received for publication 14th December 1955) THE lymph carried by the thoracic duct is derived largely from the capillary filtrate of the trunk, hind limbs and abdominal viscera. The volu'me of lymph contributed by the extensive muscular and subcutaneous capillary beds is small, especially in the resting or anaesthetized animal [Drinker and Yoffey, 1941]. Thus for practical purposes the thoracic duct lymph may be assumed to come from the abdominal viscera. The composition of this lymph may be modified by absorption of materials from the intestinal lumen or by substances formed in tissue cells, especially those of the intestinal mucosa and liver, and passed out into the tissue fluid [Bollman, Flock, Cain and Grindlay, 1950; Bollman and Flock, 1951; Borgstr6m, 1952; Borgstr6m and Laurell, 1953; Korner, Morris and Courtice, 1954; Morris, 1954; Simmonds, 1954, 1955]. Most studies on the exchange of protein and lipids between plasma and lymph have been concerned with the collection and analysis of thoracic duct lymph. In view of the anatomical and functional differences of the tissues from which this lymph originates, it seemed desirable to attempt to define the characteristics of lymph derived separately from the intestines and the liver. To this end, lymph has been collected from the hepatic, intestinal and thoracic lymph ducts and analysed for its protein and lipid constituents. METHODS All experiments were carried out on cats aneesthetized with intravenous nembutal (Abbott). The hepatic lymph duct was catheterized below the main hepatic lymph node, using a "Transflex" plastic tube (14 mm. external diameter). The duct here runs for a distance of 0 5 to 1 cm. before passing dorsally to join with the main intestinal lymphatic and empty into the cisterna chyli. The two ducts are readily distinguished in an animal absorbing food, as the hepatic lymph appears quite clear while that from the intestine is milky. Any accessory lymph ducts running across the mesentery were ligated and the catheter retained in place with stay sutures. The plastic tube was led out 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. 318

Liver and Intestinal Lymph through a stab wound in the lateral abdominal wall to deliver lymph by spontaneous flow into a graduated collecting vessel. The main intestinal lymphatic was similarly catheterized with plastic tubing after ligation of any accessory lymphatics. The thoracic duct was cannulated at the base of the neck using a glass lymphatic cannula. Except where specified, the samples of thoracic duct lymph collected represented the lymph carried by this duct with the hepatic and intestinal lymphatics intact. Blood samples were taken through an indwelling femoral polyethylene catheter, and supplementary doses of anaesthetic were delivered through a similar catheter inserted into the femoral vein. The anticoagulant used for blood and lymph collection was powdered heparin, although a sodium citrate-oxalate mixture was used to prevent clotting in some samples of fatty lymph. Chemical Analyses.-Protein and non-protein nitrogen estimations were carried out on plasma and lymph by micro-kjeldahl digestion and direct nesslerization. Albumin was determined after globulin precipitation with 21 per cent sodium sulphite according to the technique of Campbell and Hanna [1937]. Total esterified fatty acid was estimated by the method of Sterne and Shapiro [1953], phospholipid phosphorus by a micro modification of King's method [1932], and total cholesterol by the technique of Kingsley and Schaffert [1949]. Zone electrophoresis was carried out on samples of plasma and lymph in barbiturate buffer of ph 8-6. Protein patterns were stained with bromophenol blue and lipid patterns with Sudan black, as described by Swahn [1953]. Diagrams were constructed from optical density readings after eluting the dye from 0 5 cm. segments of the patterns, measurements being made on a Beckman spectrophotometer [Morris and Courtice, 1955]. RESULTS Hepatic, Intestinal and Thoracic Duct Lymph Flow and Protein Composition The lymph flow and protein concentration of the total thoracic duct, liver and intestinal lymph of cats is given in Table I. The flow of lymph obtained from the thoracic duct after cannulation of the intestinal TABLE I.-THE RATE OF FLOW AND PROTEIN CONCENTRATION OF THE TOTAL THORACIC DUCT, LIVER AND INTESTINAL LYMPH OF THE CAT Mean results are given together with their standard errors 319 Lymph Number of Flow, Protein concentration, animals ml./kg./hr. g. per cent Total thoracic duct. 66 2-42±0-12 4-53±0-1 Liver.... 38 073 4 006 6-06 ± 005 Intestinal... 30 1-54±t01 4-19±*01

320.Morris and hepatic lymph ducts and ligation of any accessory mesenteric lymphatics was usually less than 0 1 ml./kg./hr. This showed that when the liver lymph was collected, the thoracic duct carried lymph almost entirely from the intestines. The liver lymph contributed about 30 per cent of the total volume of thoracic duct lymph and had a protein content of approximately 90 per cent of the plasma levels. The plasma and hepatic lymph protein concentrations were significantly correlated (fig. 1). The PL coj MPROTEN' GZ FIG. 1.-The relationship between the protein concentration (g. per cent) in the plasma, liver and intestinal lymph of the cat. 0: liver lymph. 0: intestinal lymph. The calculated regression equations are: Liver lymph protein = 6-06 + 0-66 (plasma protein-6 82), P= <0-001. Intestinal lymph protein= 4-19 + 0-29 (plasma protein- 6-71), P =0-01. FIG. 2.-The relationship between the protein output (mg./kg./hr.) of the total thoracic duct and liver lymph of cats and lymph flow (ml./kg./hr.). *: liver lymph. 0: thoracic duct lymph. The calculated regression equations are: Liver lymph protein output = 430+595 (lymph flow-0-73), P=< 0-001. Thoracic duct protein output= 109 +41-1 (lymph flow-2.42), P=< 0-001. protein concentration of the intestinal lymph was more variable, and was between 50 and 60 per cent of the plasma levels. This variability was probably due to absorption of fluid from the intestinal lumen [Simmonds, 1954]. The relationship between the plasma and intestinal lymph protein concentration is shown in fig. 1. The mean protein output in the liver lymph was 43-0 mg./kg./hr. or about 40 per cent of the total thoracic duct output, and was significantly related to the lymph flow. The same estimate on a series of total thoracic duct lymph results showed a mean protein output of 109 mg./kg./hr. Fig. 2 shows the relationship between protein output and lymph flow in the liver and thoracic duct lymph. It is seen that

Liver and Intestinal Lymph 321 the protein output increases linearly with flow in the hepatic lymph and all results fall closely along the calculated regression line. The scatter with the thoracic duct lymph results was much wider, particularly at the higher rates of lymph flow found in cats absorbing food. PLA5A CtlQLE5TRL GZ PiASA PHtOSPHOJIPID P MGZ FIG. 3.-The relationship between the cholesterol and phospholipid P concentrations (mg. per cent) in the plasma and liver lymph of cats. The calculated regression equations are: Liver lymph cholesterol = 100 + 0-74 (plasma cholesterol- 118), P = < 0.001. Liver lymph phospholipid P = 6*92 + 0*86 (plasma phospholipidp-8.00), P = < 0.001. Hepatic and Intestinal Lymph Lipids The total cholesterol and phospholipid content of the liver lymph was estimated in 18 cats. The liver lymph cholesterol was about 85 per cent of the plasma concentration, whilst the phospholipids were 87 per cent of the plasma levels. The concentration of these lipids in the lymph was significantly related to their plasma concentrations (fig. 3). The liver lymph total fatty acid concentrations were more variable than the cholesterol and phospholipid estimates, and in several samples the level of fatty acids in this lymph was slightly higher than in the plasma. The mean liver lymph total fatty acids were about 92 per cent of the plasma concentration. In a series of estimates on the intestinal lymph of post-absorptive cats, the concentration of cholesterol and phospholipid was also found to be related to the levels of these substances in the plasma. In a group of 10 cats, the mean plasma cholesterol was 122 4S.E.M. 6-9 per cent and the phospholipid phosphorus 8-34 ±S.E.M. 0-43 mg. per cent. The corresponding intestinal lymph estimates were 62 ±S.E.M. 4*6 and 4@62 ±S.E.M. 0-30 mg. per cent. The mean plasma concentration of

322 3Morris CAT 25 TIME HOURS IMTESTIUJAL LYMPH FIG. 4. The changes in the intestinal and FIG. 5). The electrophoretic pattern hepatic lymph lipids of the absorption. cat, duiring fat, of a sample of fat,ty cat's intestinal lyinph stained for protein and lipid. 0: liver lymph. protein. 0: intestinal lymph lipid. total fatty acids was 78 ±S.E.MI. 0 3 mieq./l., whilst in the lymph it was 6*9 +S.E.MI. 0-3 meq./l. Some intestinal lymph samples had higher levels of fatty acid than the correspondin, plasma samples even though these animals had been without food for 36 hours. The protein and lipid content of lyim1ph collected from various regions of the body are given in Table II, expressed as percentages of the concentrations of these substances in the plasma. TABLE II. THE CONCENTRATIONS OF PROTEIN, PHOSPHOLIP ID AND CHOLESTERZOL IN THE LYMIPH OF CATS EXPRESSED AS PERCENTAGCES OF THE PLAS-MA LEVELS Plasma Hepatic Intestinal Total p)roteil 100 89 62 Albutimin. 100 92 73 (lobulin 100 86 52 Cholesterol. 100 84 51 Phospholipid 10() 88 53 Lymph Lipids duri?iy Fat Absorption.-Following a fat meal, the intestinal lymph lipids rose to very high levels and all three lipid fractions were involved. There w-as no significaint change in the lipids of the hepatic lymph, however, durinog fat absorption. This is shown in fig. 4.

Liver and Intestinal Lymph The electrophoretic patterns of samples of liver lymph examined during this absorptive phase revealed no alteration in the protein or lipid pattern. Fatty samples of intestinal lymph, however, showed characteristic changes. The post-absorptive lipoprotein pattern was masked by the presence of large amounts of chylomicron fat. A large proportion of this lipid was localized at the point of application, but there was a trail of fat-staining material extending some distance along the pattern. Characteristic of all these fatty samples was the presence of a "lipoprotein" fraction with a high proportion of lipid to protein migrating in advance of the albumin (pre-albumin component). This component was found in fatty samples of lymph collected in citrate or heparin and in lymph that had been allowed to clot (fig. 5). DISCUSSION 323 These results indicate that assessments of changes in thoracic duct lymph volume and composition must take into account the principal origins of this lymph. The large volume derived from the liver, with its high concentrations of proteins and lipids, provides a significant proportion of the total thoracic duct lymph content. Again, during fat absorption, little change takes place in the liver lymph, but the intestinal lymph shows large increases in its lipid components. Changes in circulatory function, whether localized or systemic, may well produce very different responses in these two regions, where the anatomical arrangement and architecture of the blood and lymph capillaries show striking differences. Morris [1954] has shown that in the post-absorptive animal, protein and lipids of thoracic duct lymph originated largely in the capillary filtrate. The results presented here indicate a similar origin for these components in the liver and intestinal lymph of post-absorptive cats. The lymph from the liver has long been known to contain a high protein content [Starling, 1894]. The results reported here show mean concentrations of 90 per cent of the plasma levels, in agreement with McCarrell, Thayer and Drinker [1941] working with cats, and Nix, Mann, Bollman, Grindlay and Flock [1951] working with dogs. The filtration pressure responsible for the formation of liver lymph is low, and the high protein content and large lymph flow point to a high degree of permeability of the endothelium of the liver sinusoids. About 40 per cent of the total intravascular protein, cholesterol and phospholipid is transported per day in the liver lymph of the postabsorptive cat. This amount of cholesterol and phospholipid probably represents lipoprotein and, in the cat. the principal lipoprotein is one associated with the faster migrating alpha globulins [Morris and Courtice, 1955]. Estimates of lymph flow and protein transport from the liver, expressed in terms of organ weight, give an indication of the large amounts

324 Morris of lymph and protein passing from this tissue. Forty-eight ml. of fully elaborated lymph are formed daily per 100 g. of liver tissue, and its protein content represents about 3 per cent of the liver weight. The results obtained confirm the findings of Bollman, Flock, Cain and Grindlay [1950], who showed in dogs increases in intestinal lymph fatty acid and phospholipid concentration during fat absorption, but no change in hepatic lymph lipid content. Studies on the thoracic duct lymph of cats before and during fat absorption [Morris, 1954] have revealed increases in all lipid fractions, and it is evident that the intestinal lymph is the source of these lipids. The changes in the thoracic duct lymph electrophoretic patterns during fat absorption have been described in rats [Borgstrom and Laurell, 1953] and in cats [Courtice and Morris, 1955]. The latter authors recorded the presence of a pre-albumin component in some samples of fatty cat and rat plasma and thoracic duct lymph. Here, in the intestinal lymph of the cat during fat absorption, a pre-albumin component was a characteristic feature. The amount of lipid in this fraction was small in relation to the very large amounts of particulate fat present in the lymph. The nature of this protein-lipid association has not been investigated further, though it may be related to a fatty acid albumin complex. It is unlikely that the pre-albumin lipid component is concerned to any extent with the transport of absorbed fat in the lymph. SUMMARY 1. In the anaesthetized cat, the liver lymph contributes about 30 per cent of the total thoracic duct lymph volume and approximately 40 per cent of the total thoracic duct protein. The remaining thoracic duct lymph is almost entirely derived from the intestines. 2. In the post-absorptive state, liver lymph contains about 90 per cent of the plasma concentrations of protein, phospholipid and cholesterol, whilst the intestinal lymph contains 50-60 per cent. There is a significant relationship between the concentrations of these substances in the plasma and lymph. 3. During fat absorption no significant change occurs in the liver lymph composition, whereas the intestinal lymph total fatty acid, phospholipid and cholesterol show large increases. During fat absorption an additional pre-albumin lipoprotein component appears in the intestinal lymph. ACKNOWLEDGMENTS I should like to acknowledge the assistance of Dr. F. C. Courtice in the preparation of the manuscript, and the technical assistance of Miss Marianne Kearns.

Liver and Intestinal Lymph 325 REFERENCES BOLLMAN, J. L. and FLOCK, E. V. (1951). Amer. J. Physiol. 164, 480. BOLLMAN, J. L., FLOCK, E. V., CAIN, J. C. and GRINDLAY, J. H. (1950). Amer. J. Physiol. 163, 41. BORGSTROM, B. (1952). Acta physiol. scand. 25, 315. BORGSTROM, B. and LAURELL, C. B. (1953). Acta physiol. scand. 25, 291. CAMPBELL, W. R. and HANNA, M. J. (1937). J. biol. Chem. 119, 15. COURTICE, F. C. and MORRIS, B. (1955). Quart. J. exp. Physiol. 40, 138. DRINKER, C. K. and YOFFEY, J. M. (1941). Lymphatics, Lymph and Lymphoid Tissue. Cambridge, Mass.: Harvard University Press. KING, E. J. (1932). Biochem. J. 26, 292. KINGSLEY, G. R. and SCHAFFERT, R. R. (1949). J. biol. Chem. 180, 315. KORNER, P. I., MORRIS, B. and COURTICE, F. C. (1954). Aust. J. exp. Biol. and med. Sci. 32, 301. MCCARRELL, J. D., THAYER, S. and DRINKER, C. K. (1941). Amer. J. Physiol. 133, 79. MORRIS, B. (1954). Aust. J. exp. Biol. and med. Sci. 32, 763. MORRIS, B. and COURTICE, F. C. (1955). Quart. J. exp. Physiol. 40, 127. NIX, J. T., MANN, F. C., BOLLMAN, J. L., GRINDLAY, J. H. and FLOCK, E. V. (1951). Amer. J. Physiol. 164, 119. SIMMONDS, W. J. (1954). Aust. J. exp. Biol. and med. Sci. 32, 285. SIMMONDS, W. J. (1955). Aust. J. exp. Biol. and med. Sci. 33, 305. STARLING, E. (1894). J. Physiol. 16, 224. STERNE, I. and SHAPIRO, B. (1953). J. clin. Path. 6, 158. SWAHN, B. (1953). Scand. J. clin. Lab. Invest. 5, Supplementum 9.