Lymph and co1 protein concentration in initial ecting lymphatics of the rat D. C. ZAWIEJA AND B. J. BARBER Department of Physiology, The Medical College of Wisconsin, Milwaukee, Wisconsin 53226 ZAWIEJA, D. C., AND B. J. BARBER. Lymph protein concen- tration in initial and collecting lymphatics of the rat. Am. J. Physiol. 252 (Gastrointest. Liver Physiol. 15): G602-G606, 1987.- Lymph samples were obtained from villus initial and mesenteric prenodal collecting lymphatics of the anesthetized rat using standard micropuncture techniques. The protein con- centrations of the lymph samples were determined using a fluorometric microassay. These procedures were performed on preparations either superfused with a modified Krebs solution or covered with paraffin oil. The protein-concentrating ability of the intestinal lymphatics was evaluated to test the osmotic theory of lymph formation. The mean initial lymph protein concentration in superfused preparations was 2.08 g/d1 (SE = 0.20). The lymph protein concentration in collecting vessels in superfused preparations was 2.20 g/d1 (SE = 0.19). The protein concentration from initial lymphatics in oil-covered prepara- tions was 2.48 g/d1 (SE = 0.17). The lymph protein concentra- tion in collecting vessels in oil-covered preparations was 2.32 g/d1 (SE = 0.15). The difference between initial and collecting lymph protein concentration found was an order of magnitude lower than that predicted by the osmotic theory. These results cast serious doubts on the effectiveness of the osmotic mecha- nism of lymph formation as it is presently defined. lymph formation; fluid exchange; microcirculation; microvas- cular exchange THE DEBATE over the mechanism of lymph formation has continued for more than a century. Two basic theo- ries that are currently used to describe lymph formation are the hydraulic and the osmotic mechanisms. The osmotic theory of lymph formation (5-9) postulates a protein-concentrating mechanism within the initial lym- phatic. According to this hypothesis, only 10-40s of the fluid present in the initial lymphatic at the beginning of a lymphatic compression is expressed centrally, whereas 50-70s is filtered out of the vessel as protein-free fluid. This causes a protein concentration gradient between the initial lymphatic fluid and the surrounding tissue fluid. Thus an osmotic pressure gradient across the lym- phatic wall is formed. This osmotic pressure gradient in turn pulls interstitial fluid into the initial lymphatic during the relaxation or decompression phase. This hy- pothesis requires the mean lymph protein concentration within the initial lymphatic to be about two or three times the protein concentration in the surrounding in- terstitial fluid. This corresponds to an initial lymph protein concentration that is -1.0-1.5 times the plasma protein concentration (8). However, most reported meas- urements of lymph protein concentration give values less than the normal plasma protein concentrations. To ac- count for this discrepancy, Casley-Smith includes a cor- ollary in his hypothesis of osmotic lymph formation (5- 9, 12). This corollary states that the concentrated fluid from the initial lymphatics is rapidly rediluted as the lymph flows along what Casley-Smith terms the remote collecting vessels. The cycle of compression and relaxa- tion that caused the concentration gradient in the initial lymphatics must be absent in the remote lymphatics. Experimental evidence exists to support this theory of lymph formation (3, 6-9, 15, 24, 27). The second theory asserts that lymph formation is due to the balance of hydraulic forces across the initial lym- phatic. This theory relies on the functioning of one-way valves in the initial lymphatic wall and between lym- phangion segments for lymph formation and flow. Be- cause lymph formation is dependent on the hydraulic forces across the lymphatic, a lymphatic protein-concen- trating mechanism is not needed. Experimental evidence also exists to support the hydraulic theory of lymph formation (1, 2, 4, 10, 17, 19, 21-23, 25, 26, 28). Thus there are two clearly conflicting hypotheses for the lymph formation mechanism with abundant experi- mental evidence to support each concept. At present the existing data is insufficient to reject either hypothesis. What is lacking is a definitive measurement of lymph protein concentration from initial and collecting lym- phatics using direct micropuncture methods. This ab- sence of data can be partially attributed to the lack of a sensitive, reliable protein assay for nanoliter- and sub- nanoliter-size lymph samples. These measurements can now be accomplished using a protein fluorometric mi- croassay that we have developed to routinely measure proteins in samples of this size (29). The purpose of the present study is to experimentally test the osmotic hypothesis of lymph formation. This was done by measuring the protein concentration of lymph, obtained by standard micropuncture techniques from villus initial lymphatics and from various points along the subsequent prenodal collecting and transport lymphatics in the rat. Mesenteric collecting vessel lymph is a mixture of fluid derived from all portions of the lymphatic drainage proximal to the collection site. This includes any initial lymphatics in the mucosa, submu- cosa, muscularis, and serosa of the intestine as well as initial lymphatics in the mesentery itself. There is little evidence for the existence of many initial lymphatics or G602 0193-1857/87 $1.50 Copyright 0 1987 the American Physiological Society