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Effects of glutamic acid and taurine on total parenteral nutrition
Journal of Pediatric Surgery (2006) 41, 1566 – 1572
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Effects of glutamic acid and taurine on total parenteral nutrition Takashi Tsuchioka*, Toshio Fujiwara, Masakatu Sunagawa First Department of Surgery, Dokkyo University School of Medicine, Mibu, Tochigi 321-0293, Japan Index words: Total parenteral nutrition (TPN); Glutamic acid; Taurine; Citrulline
Abstract Background: The objective of the present study was to ascertain whether simultaneous administration of glutamic acid (Glu) and taurine (Tau) to patients on total parenteral nutrition (TPN) could improve intestinal mucosal atrophy and suppress bacterial translocation. Methods: A 5-day TPN study was conducted in 5-week-old Sprague-Dawley rats. Commercially available Glu was used for TPN in group G and was enhanced with Tau (500 mg kg 1 day 1) in group GT. Oral nutrition was provided in group C controls. At 5 days, amino acid and cytokine levels in plasma and endotoxin levels in portal blood were measured. The histology of the small intestine, liver, and lung were analyzed. Results: Mucosal thickness and villus height in the small intestine were lower for group G than for groups C and GT. Taurine level in group GT was higher than in group G. Arginine and citrulline levels in groups G and GT were lower than in group C. Taurine level in the small intestine was greater in group GT than in group G. Citrulline concentration was lower in group G than in groups GT and C. Endotoxin level in portal blood and cytokine (tumor necrosis factor a, interleukin-1b, and interleukin-6) levels in blood tended to be lower for group GT than for group G, but no significant differences were noted. Immunostaining showed strong positive reactions to vascular cell adhesion molecule-1 in the liver and lung for group G, and milder reactions for group GT. Conclusions: Simultaneous administration of Glu and Tau improved small intestinal mucosal thickness and villus height during TPN. Levels of Tau in the small intestine and plasma increased, and the level of citrulline in the small intestine improved. Decreased expression of adhesion molecules in the liver and lung and improved microcirculation in the liver were also confirmed. D 2006 Elsevier Inc. All rights reserved.
The etiology of the hepatopathy caused by total parenteral nutrition (TPN) is considered multifactorial, involving factors such as the composition of carbohydrates and amino acids and complications by peritonitis, intestinal paralysis, or short gut syndrome [1 - 7]. In recent years,
* Corresponding author. Tel.: +81 282 86 1111; fax: +81 282 86 6213. E-mail address: [email protected] (T. Tsuchioka). 0022-3468/$ – see front matter D 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.jpedsurg.2006.05.021
bacterial translocation (BT) caused by intestinal mucosal atrophy during TPN has been closely examined [8 - 10]. Several compounds have been examined recently from the perspective of immunomodulation [11 - 13]. Some studies have reported that administration of glutamine (Gln), a substrate of intestinal mucosal metabolism, is effective in preventing BT [14 - 16]. However, because Gln tends to be unstable in solution, administration in clinical settings is difficult. The present study was therefore
Effects of glutamic acid and taurine on total parenteral nutrition Table 1
Small intestinal mucosal thickness and villous height
Mucosal thickness (Am) Villous height (Am)
Group C (n = 7)
Group G (n = 7)
Group GT (n = 7)
552 F 61*
421 F 56*
524 F 31*
334 F 34*
249 F 28*
315 F 23*
* P b .05 (C vs G, G vs GT).
conducted to ascertain whether simultaneous administration of glutamic acid (Glu) and taurine (Tau) could improve intestinal mucosal atrophy and suppress BT.
1. Subjects and methods In accordance with the guidelines for the care and use of laboratory animals, Dokkyo University School of Medicine, the present study was approved by the Animal Care and Use Committee at the university. In the present study, 5-week-old male Sprague-Dawley rats were used in a 5-day TPN study. Commercially available solution containing Glu was used in group G (n = 7), whereas the same solution enhanced with Tau (500 mg kg 1 day 1) was used in group GT (n = 7). For comparison, oral nutrition was provided for the same period in group C (n = 7). Experimental procedures for the 3 groups can be summarized as follows: group C, oral nutrition; group G, TPN including Glu (Glu + TPN) (Alimehl 400 ml + Moripron F 200 ml [Glu, 6.3%]); and group GT, TPN including Glu and Tau (Tau + Glu + TPN) (Alimehl 400 ml + Moripron F 200 ml [Glu, 6.3%)] + Tau [1%, 500 mg kg 1 day 1]).
1567
Next, 3 h after completion of the 5-day nutrition, blood samples were collected from the aorta to measure levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT; biochemical analysis), amino acids, and cytokines (tumor necrosis factor a [TNF-a], interleukin (IL)-1b, and IL-6). At the same time, a portal blood sample was collected to measure levels of endotoxin (Et) by using the endospecy method [17]. Furthermore, the small intestine, liver, and lungs were excised for histopathologic examination. Moreover, mucosal thickness and villus height of the small intestine were measured, and levels of amino acids were determined. Levels of AST and ALT were measured by the UV method using a 7150 Automatic Analyzer (Hitachi, Tokyo, Japan). For measurement of amino acid levels, plasma samples were deproteinized by using trichloroacetic acid and analyzed by the lithium method using an L-8500 High-Performance Amino Acid Analyzer (Hitachi). Levels of TNF-a, IL-1b, and IL-6 were measured by enzyme-linked immunosorbent assay (ELISA). In histopathologic analysis, samples were 0embedded in paraffin, stained using hematoxylin and eosin, and subjected to TdT-mediated dUTP-biotin nick-end labeling (TUNEL) staining using a Mebstain apoptosis detection kit (Medical & Biological Laboratories Co., Nagoya, Japan). Furthermore, samples were subjected to immunohistochemical staining for vascular cell adhesion molecule 1 (VCAM-1) and thrombomodulin (TM). VCAM-1 and TM staining was performed by labeled streptavidin–biotin method (LSAB) using 40 VCAM-1 (H-276) rabbit polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA) and 50 TM (H-300) rabbit polyclonal antibody (Santa Cruz Biotechnology). Numerical results were expressed as mean F standard deviation and statistically analyzed using either a Kruskal-
Fig. 1 Small intestinal mucosal thickness and villous height (original magnification 40). Mucosal thickness and villous height for group G was significantly lower than those for groups C and GT ( P b .05).
1568 Table 2
T. Tsuchioka et al. Biochemical analysis (AST and ALT)
AST (IU/L) ALT (IU/L)
Group C (n = 7)
Group G (n = 7)
Group GT (n = 7)
83.5 F 21.7 30.6 F 5.8*
94.6 F 26.5 6.7 F 2.0*
81.6 F 23.8 6.9 F 2.9*
* P b .05 (C vs G, C vs GT).
Wallis test or 1-way analysis of variance first, and then a multiple comparison test according to the Bonferroni and Scheffe methods. Values of P b .05 were considered statistically significant.
2. Results 2.1. Small intestinal mucosal thickness and villus height Small intestinal mucosal thickness and villus height for group C were 552.1 F 61.0 and 333.4 F 33.6 Am, respectively, whereas those for group G were reduced at 420.6 F 56.4 and 248.9 F 27.6 Am, respectively ( P b .05 each). However, values for group GT were improved compared with group G, at 524.0 F 31.3 and 315.1 F 23.2 Am, respectively ( P b .05) (Table 1; Fig. 1).
2.2. Biochemical analysis (AST and ALT) No significant differences in AST levels were observed among the 3 groups. ALT levels were significantly lower for groups G and GT than for group C, but no significant difference was noted between groups G and GT (Table 2).
2.3. Plasma amino acids Plasma amino acid analysis showed that Tau levels for group GT were 394.7 F 85.1 nmol/mL, higher than levels for group G at 156.7 F 34.1 nmol/mL ( P b .05). No significant differences in level of Glu were observed among the 3 groups. Levels of arginine and citrulline for group G
Table 4 Amino acids in the small intestine (nanomoles per gram)
Tau Glu Gln Cit Cys Met Arg
Group C (n = 7)
Group G (n = 7)
Group GT (n = 7)
11768 F 1174 14344 F 1522 6092 F 849 228 F 32* 355 F 156 2781 F 434 6428 F 990
10266 F 2453* 14399 F 1154 6530 F 1030 135 F 38* 206 F 115 2965 F 571 7255 F 1647
13797 F 907* 15870 F 1859 7967 F 1513 232 F 114* 214 F 123 4179 F 1058 9819 F 2250
* P b .05 (Tau: G vs GT. Cit: C vs G, G vs GT).
were 138.1 F 2.7 and 59.9 F 15.6 nmol/mL, respectively, compared with 142.0 F 27.0 and 54.3 F 7.5 nmol/mL for group GT, respectively. These values were lower than those for group C ( P b .05) (Table 3).
2.4. Amino acids in the small intestine Level of Tau in the small intestine was 13,797 F 907 nmol/g for group GT, which was greater than that for group G at 10,266 F 2453 nmol/g ( P b .05). Levels of Gln, methionine, and arginine also tended to be higher for group GT, but no significant differences were identified. Although levels of citrulline for group G were low at 135 F 38 nmol/g, those for groups GT and C were higher at 232 F 114 and 228 F 32 nmol/g, respectively ( P b .05) (Table 4).
2.5. Endotoxin in portal blood Levels of Et in portal blood tended to be lower for group GT than for group G, but no significant difference was found (Table 5).
2.6. Blood cytokines Levels of cytokines TNF-a, IL-1b, and IL-6 in blood tended to be lower for group GT than for group G, but no significant differences between the 2 groups were noted (Table 6).
2.7. Histopathologic analysis Table 3 Plasma amino acid concentration (nanomoles per milliliter)
Tau Glu Gln Cit Cys Met Arg
Group C (n = 7)
Group G (n = 7)
Group GT (n = 7)
204.4 F 62.0* 141.3 F 31.6 664.6 F 68.2 101.9 F 15.7* 10.5 F 3.4 48.1 F 6.9 182.1 F 15.4*
156.7 F 34.1* 147.2 F 11.6 689.4 F 55.9 59.9 F 15.6* 20.5 F 8.7 48.6 F 6.0 138.1 F 22.7*
394.7 F 85.1* 129.6 F 26.3 682.7 F 112.4 54.3 F 7.5* 23.6 F 9.2 49.0 F 8.5 142.0 F 27.0*
* P b .05 (Tau: C vs GT, G vs GT. Cit: C vs G, C vs GT. Arg: C vs G, C vs GT).
In the liver, TUNEL staining showed no sign of apoptosis in any of the groups. VCAM-1 staining yielded positive reactions in the liver only for group G. Whereas strong reactions were seen in the
Table 5 Endotoxin level in portal blood (picograms per milliliter) Group C (n = 7)
Group G (n = 7)
Group GT (n = 7)
5F0
71.0 F 12.3
39.3 F 32.6
Effects of glutamic acid and taurine on total parenteral nutrition
1569
Table 6 Blood cytokines TNF-a, IL-1b, and IL-6 (picograms per milliliter)
TNF-a IL-1b IL-6
Group C (n = 7)
Group G (n = 7)
Group GT (n = 7)
4.5 F 3.8 62.5 F 0 62.5 F 0
44.0 F 49.0 75.2 F 23.3 195.9 F 276.1
24.3 F 17.6 62.5 F 0 87.0 F 53.4
bronchial epithelia for group G, only mild reactions were observed for group GT (Figs. 2 and 3). In TM staining, positive reactions were evenly observed throughout the liver for group C, whereas mild reactions
Fig. 3 VCAM-1 staining in the lung (original magnification 200). VCAM-1 staining strong reactions was seen in the bronchial epithelia for group G, only mild reactions were observed for group GT.
were seen around the central vein with stronger reactions around the hepatic vein for group G. In group GT, these changes were milder (Fig. 4).
3. Discussion
Fig. 2 VCAM-1 staining in the liver (original magnification 200). VCAM-1 staining yielded positive reactions in the liver only for group G.
To clarify the cause of hepatopathy associated with TPN, the composition and dosage of carbohydrates and amino acids were investigated, and some favorable findings were obtained [1 -7]. However, the precise cause has yet to be elucidated. In recent years, the involvement of BT in
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Fig. 4 TM staining (200). In TM staining, positive reactions were evenly observed throughout the liver for group C, whereas mild reactions were seen around the central vein with stronger reactions around the hepatic vein for group G. In group GT, these changes were milder.
intestinal mucosal atrophy associated with TPN has been closely examined [8- 10,18]. Furthermore, invasive stress during TPN has been shown to damage important organs, occasionally leading to multiple-organ injury [19]. We have previously reported that when peritonitis is induced during TPN, histologically severe damage can be seen in important organs despite low levels of Et and cytokines in blood [20]. This is because BT activates hepatic Kupffer cells and pulmonary macrophages during TPN, and additional stressors or secondary attack can lead to multiple-organ injury. Furthermore, we investigated some intracellular markers for
T. Tsuchioka et al. peripheral lymphocytes during TPN and documented that the defense mechanisms of the body could be depressed. In recent years, several compounds have been examined from the perspective of immunomodulation [11 -13]. Immunomodulation involves nutritional therapy that improves the condition of the patient by enhancing the defense mechanisms of the body, and compounds such as Gln, arginine, branched-chain amino acids, N3 fatty acids, nucleic acid, dietary fibers, and Tau have been used. In particular, several studies have reported that administration of Gln, which is a substrate of intestinal mucosal metabolism, is effective in preventing BT [15,16,21]. However, because Gln tends to be unstable in solution, administration in a clinical setting is difficult. Addition of Tau to amino acid solutions containing Glu is known to improve plasma Gln levels. Tau is a sulfur-containing amino acid that is known to protect hepatocytes and facilitate bile excretion. Furthermore, TPN causes Tau deficiency, and exogenous Tau administration is reportedly effective [22 -24]. Moreover, Tau has been studied as an immunomodulator [25]. In the present study, Tau and Glu were administered simultaneously to ascertain effects on intestinal mucosal atrophy, BT, immunocompetency, and damage to important organs. The control solution was TPN containing no Tau but with a composition that was otherwise identical to the experimental solution. The same amount of Glu was added to both solutions. Whereas small intestinal mucosal thickness and villus height improved with Tau, no significant differences in levels of plasma Gln were observed among groups C, G, and GT. However, levels of Gln in the small intestine for group GT were high. Also, levels of citrulline, which is synthesized in the small intestine, were low for group G, and levels of citrulline for group GT were comparable to that for group C. Citrulline is synthesized from Glu in the small intestine [26,27]. In short gut syndrome or when the small intestine mucosa is damaged by bolus chemotherapy or whole-body radiotherapy, level of citrulline synthase is reduced, thus lowering plasma levels of citrulline [28]. Simultaneous administration of Glu and Tau allowed intestinal functions to be conserved, thus maintaining citrulline synthesis. Tau administration appeared to conserve intestinal functions to maintain small intestinal mucosal thickness and villus height. However, the underlying mechanisms are unclear, and further investigation is necessary. By simultaneously administering Glu and Tau, small intestine function was maintained, and small intestinal mucosal thickness and villus height for group GT were comparable to those for group C. Although the 5-day time point seems rather short for a TPN study, we believe that this was sufficient, as the small intestine mucosa had atrophied by this time. Longer timescales would be ideal. Unfortunately, levels of Et in portal blood and levels of blood cytokines for group GT tended to be lower than those for group G, although no significant differences were apparent. We thought about
Effects of glutamic acid and taurine on total parenteral nutrition using levels of Et in the portal blood as an indicator for BT, but no significant differences in outcome were seen in the present study. This suggests that BT cannot be completely suppressed even when small intestinal mucosal thickness and villus height are maintained. Histopathologic analysis revealed no sign of apoptosis in the liver on TUNEL staining in any of the 3 groups, thus failing to clearly confirm DNA fragmentation. The present study used immunostaining. VCAM-1, which represents one of the adhesion molecules, is a glycoprotein with a molecular weight of about 110 kDa and belongs to the immunoglobulin superfamily [29 -31]. Expression of VCAM-1 is induced by inflammatory cytokines such as IL-1, TNF-a, and interferon c, and expression is enhanced when endothelial cells are damaged [32,33]. VCAM-1 is believed to be involved with cell adhesion in events such as lymphocyte invasion to inflamed areas. We have previously reported that TPN enhances expression of intercellular adhesion molecule 1 in hepatic sinusoidal endothelial cells [34]. In the present study, VCAM-1 staining showed positive reactions in the liver for group G. In addition, strong positive reactions were seen in the lung for group G. The degree of reaction in the lung was lower for group GT than for group G. TM, a glycoprotein found on the surface of vascular endothelial cells, binds to thrombin to inactivate thrombin and suppress platelet aggregation. Furthermore, the thrombin-TM complex activates protein C and inactivates factors Va and VIIIa to delay coagulation [35 -37]. TM also exists in hepatic sinusoidal endothelial cells and is involved in hepatic microcirculation. When hepatic sinusoidal endothelial cells are damaged and epithelial TM enters the circulation, intravascular coagulation is disrupted. The presence of TM in hepatic sinusoidal endothelial cells indicates maintenance of sinusoidal functions. The present study shows that in group C, TM was distributed evenly in the liver, and in group G, staining around the central vein was weak, but strong around the hepatic vein. In group GT, such changes were milder. In other words, TPN damaged the hepatic microcirculation and damage was more severe around the central vein. VCAM-1 and TM staining identify endothelial cell damage and represent microcirculation impairment in the liver and lung. Damage to hepatic sinusoidal endothelial cells caused by TPN was alleviated by administration of Tau. Tau is believed to have hepatoprotective and cell membrane– stabilizing effects. We believe that damage to sinusoid endothelial cells was alleviated by the addition of Tau. Also, as the level of citrulline in the small intestine improved, atrophy of the small intestine mucosa was prevented, and we believe this reduced BT and alleviated damage to sinusoid endothelial cells. Further investigation is necessary to elucidate whether this was attributable to the ability of Tau to protect hepatocytes and facilitate bile excretion, or the ability to maintain small intestine villi, thus somewhat suppressing BT.
1571
Acknowledgment The present study was partially funded by grant-in-aid for scientific research (C)(2) 13671871 provided by the Japan Society for the Promotion of Science.
References [1] Peden VH, Witzleben CL, Skelton MA. Total parental nutrition (letters to the editor). J Pediatr 1971;78:180 - 1. [2] Touloukian RJ, Downing SE. Cholestasis associated with long term parenteral hyperalimentation. Arch Surg 1973;106:58 - 62. [3] Preisig R, Rennert O. Biliary transport and cholestatic effects of amino acids. Gastroenterology 1977;73:1240. [4] Beale EF, Nelson RM, Bucciarelli RL, et al. Intrahepatic cholestasis associated with parenteral nutrition in premature infants. Pediatrics 1979;64:342 - 7. [5] Bell RL, Ferry GD, Smith EO, et al. Total parenteral nutrition-related cholestasis in infants. JPEN J Parenter Enteral Nutr 1986;10:356 - 9. [6] Drongowski RA, Coran AG. An analysis of factors contributing to the development of total parenteral nutrition induced cholestasis. JPEN J Parenter Enteral Nutr 1989;13:586 - 9. [7] Quigley EH, Marsh MN, Shaffer JH, et al. Hepatobiliary complication of total parenteral nutrition. Gastroenterology 1993;104:286 - 301. [8] Alverdy JC, Aoys E, Moss GS. Total parenteral nutrition promotes bacterial translocation from the gut. Surgery 1988;104:185 - 90. [9] Deitsh EA, Winterton J, Li M, et al. The gut as portal of entry for bacteria. Ann Surg 1987;205:681 - 92. [10] Illing KA, Ryan CK, Handy DJ, et al. Total parenteral nutritioninduced changes in gut mucosal function; atrophy alone is not the issue. Surgery 1992;112:631 - 7. [11] Beale RJ, Bryg DJ, Bihari DJ. Immunonutrition in the critically ill; a systematic review of clinical outcome. Crit Care Med 1999;27: 2799 - 805. [12] Grimble RF. Nutritional modulation of immune function. Proc Nutr Soc 2001;60:389 - 97. [13] Koretz RL. Immunonutrition; fact, fantasy, and future. Curr Gastroenterol Rep 2002;4:332 - 7. [14] Gianotti L, Alexander JW, Gennari R, et al. Oral glutamine decreases bacterial translocation and improves survival in experimental gutorigin sepsis. JPEN J Parenter Enteral Nutr 1995;19:69 - 74. [15] Li J, Kudsk KA, Janu P, et al. Effect of glutamine-enriched total parenteral nutrition on small intestinal gut-associated lymphoid tissue and upper respiratory tract immunity. Surgery 1997;121:542 - 9. [16] Saito H, Furukawa S, Matsuda T. Glutamine as an immunoenhancing nutrient. JPEN J Parenter Enteral Nutr 1999;23:59 - 61. [17] Obayashi T, Tamura H, Tanaka S, et al. A new chromogenic endotoxin-specific assay using recombined limulus coagulation enzymes and its clinical application. Clin Chim Acta 1985;149: 55 - 65. [18] MacFie J. Enteral versus parenteral nutrition; The significance of bacterial translocation and gut-barrier function. Nutrition 2000;16: 606 - 11. [19] Fukushima R, Alexander JW, Gianotti L, et al. Bacterial translocationrelated mortality may be associated with neutrophil-mediated organ damage. Shock 1995;3:323 - 8. [20] Tsuchioka T, Urao M, Fujiwara T, et al. The effect of total parenteral nutrition and enteral nutrition on LPS-induced sepsis in the rat. J Jpn Soc Pediatr Surg 2000;36:1034 - 41. [21] Souba WW, Smith RJ, Wilmore DW. Glutamine metabolism by the intestinal tract. JPEN J Parenter Enteral Nutr 1985;9:608 - 17. [22] Cook RJ, Whittington PF, Kelts D. Effect of taurine supplementation on hepatic function during short-term parenteral nutrition in the premature infant. J Pediatr Gastroenterol Nutr 1984;3:234 - 8.
1572 [23] Giggle HS, Aments ME, Heckenlively JR, et al. Nutritional requirement for taurine in patients receiving long-term parenteral nutrition. N Engl J Med 1985;312:142 - 6. [24] Cooper A, Betts JM, Pereira GR, et al. Taurine deficiency in severe hepatic dysfunction complication total parenteral nutrition. J Pediatr Surg 1984;19:462 - 6. [25] Chesney RW, Helms RA, Christensen M, et al. An update view of the value of taurine in infant nutrition. Adv Pediatr 1998;45:179 - 200. [26] Windmueller HG, Spaeth AE. Source and fate of circulating citrulline. Am J Physiol 1981;241:E473 - 80. [27] Morris SM. Regulation of enzymes of urea and arginine synthesis. Annu Rev Nutr 1992;12:81 - 101. [28] Wakabayashi Y, Yamada E, Yoshida T, et al. Arginine becomes an essential amino acid after massive resection of rat small intestine. J Boil Chem 1994;269:32667 - 71. [29] Hynes RO. Integrins; a family of cell surface receptors. Cell 1987;48: 549 - 54. [30] Hynes RO. Integrins; versatility, modulation, and signaling in cell adhesion. Cell 1992;69:11 - 25. [31] Osborn L, Hession C, Tizard R, et al. Direct expression cloning of vascular cell adhesion molecule 1, a cytokine-induced endothelial protein that binds to lymphocytes. Cell 1989;59:1203 - 11.
T. Tsuchioka et al. [32] Walsh GH, Moermod JJ, Hartnell A, et al. Human eosinophil, but not neutrophil, adherence to IL-1–stimulated human umbilical vascular endothelial cells is a4b1 (very late antigen-4) dependent. J Immunol 1991;146:3419 - 23. [33] Wellicome SM, Thornhill MH, Pitalis C, et al. A monoclonal antibody that detects a novel antigen on endothelial cells that is induced by tumor necrosis factor, IL-1, or lipopolysaccharide. J Immunol 1990; 144:2558 - 65. [34] Tsuchioka T, Fujiwara T, Sunagawa M. Immunohistochemical study of adhesion molecule in liver receiving total parenteral nutrition. Dokkyo J Med Sci 1997;24:11 - 9. [35] Esmon CT. The regulation of natural anticoagulation pathways. Science 1987;235:1348 - 52. [36] Suzuki K, Kusumoto H, Deyashiki Y, et al. Structure and expression of human thrombornodulin, a thrombin receptor on endothelium acting as a cofactor for protein C activation. EMBO J 1987;6: 1891 - 7. [37] Maruyama I, Bell CE, Majerus PW. Thrombomodulin is found on endothelium of arteries, veins, capillaries and lymphatics, and on syncytiotrophoblast of human placenta. J Cell Biol 1985;101: 363 - 71.
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Effects of glutamic acid and taurine on total parenteral nutrition Takashi Tsuchioka*, Toshio Fujiwara, Masakatu Sunagawa First Department of Surgery, Dokkyo University School of Medicine, Mibu, Tochigi 321-0293, Japan Index words: Total parenteral nutrition (TPN); Glutamic acid; Taurine; Citrulline
Abstract Background: The objective of the present study was to ascertain whether simultaneous administration of glutamic acid (Glu) and taurine (Tau) to patients on total parenteral nutrition (TPN) could improve intestinal mucosal atrophy and suppress bacterial translocation. Methods: A 5-day TPN study was conducted in 5-week-old Sprague-Dawley rats. Commercially available Glu was used for TPN in group G and was enhanced with Tau (500 mg kg 1 day 1) in group GT. Oral nutrition was provided in group C controls. At 5 days, amino acid and cytokine levels in plasma and endotoxin levels in portal blood were measured. The histology of the small intestine, liver, and lung were analyzed. Results: Mucosal thickness and villus height in the small intestine were lower for group G than for groups C and GT. Taurine level in group GT was higher than in group G. Arginine and citrulline levels in groups G and GT were lower than in group C. Taurine level in the small intestine was greater in group GT than in group G. Citrulline concentration was lower in group G than in groups GT and C. Endotoxin level in portal blood and cytokine (tumor necrosis factor a, interleukin-1b, and interleukin-6) levels in blood tended to be lower for group GT than for group G, but no significant differences were noted. Immunostaining showed strong positive reactions to vascular cell adhesion molecule-1 in the liver and lung for group G, and milder reactions for group GT. Conclusions: Simultaneous administration of Glu and Tau improved small intestinal mucosal thickness and villus height during TPN. Levels of Tau in the small intestine and plasma increased, and the level of citrulline in the small intestine improved. Decreased expression of adhesion molecules in the liver and lung and improved microcirculation in the liver were also confirmed. D 2006 Elsevier Inc. All rights reserved.
The etiology of the hepatopathy caused by total parenteral nutrition (TPN) is considered multifactorial, involving factors such as the composition of carbohydrates and amino acids and complications by peritonitis, intestinal paralysis, or short gut syndrome [1 - 7]. In recent years,
* Corresponding author. Tel.: +81 282 86 1111; fax: +81 282 86 6213. E-mail address: [email protected] (T. Tsuchioka). 0022-3468/$ – see front matter D 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.jpedsurg.2006.05.021
bacterial translocation (BT) caused by intestinal mucosal atrophy during TPN has been closely examined [8 - 10]. Several compounds have been examined recently from the perspective of immunomodulation [11 - 13]. Some studies have reported that administration of glutamine (Gln), a substrate of intestinal mucosal metabolism, is effective in preventing BT [14 - 16]. However, because Gln tends to be unstable in solution, administration in clinical settings is difficult. The present study was therefore
Effects of glutamic acid and taurine on total parenteral nutrition Table 1
Small intestinal mucosal thickness and villous height
Mucosal thickness (Am) Villous height (Am)
Group C (n = 7)
Group G (n = 7)
Group GT (n = 7)
552 F 61*
421 F 56*
524 F 31*
334 F 34*
249 F 28*
315 F 23*
* P b .05 (C vs G, G vs GT).
conducted to ascertain whether simultaneous administration of glutamic acid (Glu) and taurine (Tau) could improve intestinal mucosal atrophy and suppress BT.
1. Subjects and methods In accordance with the guidelines for the care and use of laboratory animals, Dokkyo University School of Medicine, the present study was approved by the Animal Care and Use Committee at the university. In the present study, 5-week-old male Sprague-Dawley rats were used in a 5-day TPN study. Commercially available solution containing Glu was used in group G (n = 7), whereas the same solution enhanced with Tau (500 mg kg 1 day 1) was used in group GT (n = 7). For comparison, oral nutrition was provided for the same period in group C (n = 7). Experimental procedures for the 3 groups can be summarized as follows: group C, oral nutrition; group G, TPN including Glu (Glu + TPN) (Alimehl 400 ml + Moripron F 200 ml [Glu, 6.3%]); and group GT, TPN including Glu and Tau (Tau + Glu + TPN) (Alimehl 400 ml + Moripron F 200 ml [Glu, 6.3%)] + Tau [1%, 500 mg kg 1 day 1]).
1567
Next, 3 h after completion of the 5-day nutrition, blood samples were collected from the aorta to measure levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT; biochemical analysis), amino acids, and cytokines (tumor necrosis factor a [TNF-a], interleukin (IL)-1b, and IL-6). At the same time, a portal blood sample was collected to measure levels of endotoxin (Et) by using the endospecy method [17]. Furthermore, the small intestine, liver, and lungs were excised for histopathologic examination. Moreover, mucosal thickness and villus height of the small intestine were measured, and levels of amino acids were determined. Levels of AST and ALT were measured by the UV method using a 7150 Automatic Analyzer (Hitachi, Tokyo, Japan). For measurement of amino acid levels, plasma samples were deproteinized by using trichloroacetic acid and analyzed by the lithium method using an L-8500 High-Performance Amino Acid Analyzer (Hitachi). Levels of TNF-a, IL-1b, and IL-6 were measured by enzyme-linked immunosorbent assay (ELISA). In histopathologic analysis, samples were 0embedded in paraffin, stained using hematoxylin and eosin, and subjected to TdT-mediated dUTP-biotin nick-end labeling (TUNEL) staining using a Mebstain apoptosis detection kit (Medical & Biological Laboratories Co., Nagoya, Japan). Furthermore, samples were subjected to immunohistochemical staining for vascular cell adhesion molecule 1 (VCAM-1) and thrombomodulin (TM). VCAM-1 and TM staining was performed by labeled streptavidin–biotin method (LSAB) using 40 VCAM-1 (H-276) rabbit polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA) and 50 TM (H-300) rabbit polyclonal antibody (Santa Cruz Biotechnology). Numerical results were expressed as mean F standard deviation and statistically analyzed using either a Kruskal-
Fig. 1 Small intestinal mucosal thickness and villous height (original magnification 40). Mucosal thickness and villous height for group G was significantly lower than those for groups C and GT ( P b .05).
1568 Table 2
T. Tsuchioka et al. Biochemical analysis (AST and ALT)
AST (IU/L) ALT (IU/L)
Group C (n = 7)
Group G (n = 7)
Group GT (n = 7)
83.5 F 21.7 30.6 F 5.8*
94.6 F 26.5 6.7 F 2.0*
81.6 F 23.8 6.9 F 2.9*
* P b .05 (C vs G, C vs GT).
Wallis test or 1-way analysis of variance first, and then a multiple comparison test according to the Bonferroni and Scheffe methods. Values of P b .05 were considered statistically significant.
2. Results 2.1. Small intestinal mucosal thickness and villus height Small intestinal mucosal thickness and villus height for group C were 552.1 F 61.0 and 333.4 F 33.6 Am, respectively, whereas those for group G were reduced at 420.6 F 56.4 and 248.9 F 27.6 Am, respectively ( P b .05 each). However, values for group GT were improved compared with group G, at 524.0 F 31.3 and 315.1 F 23.2 Am, respectively ( P b .05) (Table 1; Fig. 1).
2.2. Biochemical analysis (AST and ALT) No significant differences in AST levels were observed among the 3 groups. ALT levels were significantly lower for groups G and GT than for group C, but no significant difference was noted between groups G and GT (Table 2).
2.3. Plasma amino acids Plasma amino acid analysis showed that Tau levels for group GT were 394.7 F 85.1 nmol/mL, higher than levels for group G at 156.7 F 34.1 nmol/mL ( P b .05). No significant differences in level of Glu were observed among the 3 groups. Levels of arginine and citrulline for group G
Table 4 Amino acids in the small intestine (nanomoles per gram)
Tau Glu Gln Cit Cys Met Arg
Group C (n = 7)
Group G (n = 7)
Group GT (n = 7)
11768 F 1174 14344 F 1522 6092 F 849 228 F 32* 355 F 156 2781 F 434 6428 F 990
10266 F 2453* 14399 F 1154 6530 F 1030 135 F 38* 206 F 115 2965 F 571 7255 F 1647
13797 F 907* 15870 F 1859 7967 F 1513 232 F 114* 214 F 123 4179 F 1058 9819 F 2250
* P b .05 (Tau: G vs GT. Cit: C vs G, G vs GT).
were 138.1 F 2.7 and 59.9 F 15.6 nmol/mL, respectively, compared with 142.0 F 27.0 and 54.3 F 7.5 nmol/mL for group GT, respectively. These values were lower than those for group C ( P b .05) (Table 3).
2.4. Amino acids in the small intestine Level of Tau in the small intestine was 13,797 F 907 nmol/g for group GT, which was greater than that for group G at 10,266 F 2453 nmol/g ( P b .05). Levels of Gln, methionine, and arginine also tended to be higher for group GT, but no significant differences were identified. Although levels of citrulline for group G were low at 135 F 38 nmol/g, those for groups GT and C were higher at 232 F 114 and 228 F 32 nmol/g, respectively ( P b .05) (Table 4).
2.5. Endotoxin in portal blood Levels of Et in portal blood tended to be lower for group GT than for group G, but no significant difference was found (Table 5).
2.6. Blood cytokines Levels of cytokines TNF-a, IL-1b, and IL-6 in blood tended to be lower for group GT than for group G, but no significant differences between the 2 groups were noted (Table 6).
2.7. Histopathologic analysis Table 3 Plasma amino acid concentration (nanomoles per milliliter)
Tau Glu Gln Cit Cys Met Arg
Group C (n = 7)
Group G (n = 7)
Group GT (n = 7)
204.4 F 62.0* 141.3 F 31.6 664.6 F 68.2 101.9 F 15.7* 10.5 F 3.4 48.1 F 6.9 182.1 F 15.4*
156.7 F 34.1* 147.2 F 11.6 689.4 F 55.9 59.9 F 15.6* 20.5 F 8.7 48.6 F 6.0 138.1 F 22.7*
394.7 F 85.1* 129.6 F 26.3 682.7 F 112.4 54.3 F 7.5* 23.6 F 9.2 49.0 F 8.5 142.0 F 27.0*
* P b .05 (Tau: C vs GT, G vs GT. Cit: C vs G, C vs GT. Arg: C vs G, C vs GT).
In the liver, TUNEL staining showed no sign of apoptosis in any of the groups. VCAM-1 staining yielded positive reactions in the liver only for group G. Whereas strong reactions were seen in the
Table 5 Endotoxin level in portal blood (picograms per milliliter) Group C (n = 7)
Group G (n = 7)
Group GT (n = 7)
5F0
71.0 F 12.3
39.3 F 32.6
Effects of glutamic acid and taurine on total parenteral nutrition
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Table 6 Blood cytokines TNF-a, IL-1b, and IL-6 (picograms per milliliter)
TNF-a IL-1b IL-6
Group C (n = 7)
Group G (n = 7)
Group GT (n = 7)
4.5 F 3.8 62.5 F 0 62.5 F 0
44.0 F 49.0 75.2 F 23.3 195.9 F 276.1
24.3 F 17.6 62.5 F 0 87.0 F 53.4
bronchial epithelia for group G, only mild reactions were observed for group GT (Figs. 2 and 3). In TM staining, positive reactions were evenly observed throughout the liver for group C, whereas mild reactions
Fig. 3 VCAM-1 staining in the lung (original magnification 200). VCAM-1 staining strong reactions was seen in the bronchial epithelia for group G, only mild reactions were observed for group GT.
were seen around the central vein with stronger reactions around the hepatic vein for group G. In group GT, these changes were milder (Fig. 4).
3. Discussion
Fig. 2 VCAM-1 staining in the liver (original magnification 200). VCAM-1 staining yielded positive reactions in the liver only for group G.
To clarify the cause of hepatopathy associated with TPN, the composition and dosage of carbohydrates and amino acids were investigated, and some favorable findings were obtained [1 -7]. However, the precise cause has yet to be elucidated. In recent years, the involvement of BT in
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Fig. 4 TM staining (200). In TM staining, positive reactions were evenly observed throughout the liver for group C, whereas mild reactions were seen around the central vein with stronger reactions around the hepatic vein for group G. In group GT, these changes were milder.
intestinal mucosal atrophy associated with TPN has been closely examined [8- 10,18]. Furthermore, invasive stress during TPN has been shown to damage important organs, occasionally leading to multiple-organ injury [19]. We have previously reported that when peritonitis is induced during TPN, histologically severe damage can be seen in important organs despite low levels of Et and cytokines in blood [20]. This is because BT activates hepatic Kupffer cells and pulmonary macrophages during TPN, and additional stressors or secondary attack can lead to multiple-organ injury. Furthermore, we investigated some intracellular markers for
T. Tsuchioka et al. peripheral lymphocytes during TPN and documented that the defense mechanisms of the body could be depressed. In recent years, several compounds have been examined from the perspective of immunomodulation [11 -13]. Immunomodulation involves nutritional therapy that improves the condition of the patient by enhancing the defense mechanisms of the body, and compounds such as Gln, arginine, branched-chain amino acids, N3 fatty acids, nucleic acid, dietary fibers, and Tau have been used. In particular, several studies have reported that administration of Gln, which is a substrate of intestinal mucosal metabolism, is effective in preventing BT [15,16,21]. However, because Gln tends to be unstable in solution, administration in a clinical setting is difficult. Addition of Tau to amino acid solutions containing Glu is known to improve plasma Gln levels. Tau is a sulfur-containing amino acid that is known to protect hepatocytes and facilitate bile excretion. Furthermore, TPN causes Tau deficiency, and exogenous Tau administration is reportedly effective [22 -24]. Moreover, Tau has been studied as an immunomodulator [25]. In the present study, Tau and Glu were administered simultaneously to ascertain effects on intestinal mucosal atrophy, BT, immunocompetency, and damage to important organs. The control solution was TPN containing no Tau but with a composition that was otherwise identical to the experimental solution. The same amount of Glu was added to both solutions. Whereas small intestinal mucosal thickness and villus height improved with Tau, no significant differences in levels of plasma Gln were observed among groups C, G, and GT. However, levels of Gln in the small intestine for group GT were high. Also, levels of citrulline, which is synthesized in the small intestine, were low for group G, and levels of citrulline for group GT were comparable to that for group C. Citrulline is synthesized from Glu in the small intestine [26,27]. In short gut syndrome or when the small intestine mucosa is damaged by bolus chemotherapy or whole-body radiotherapy, level of citrulline synthase is reduced, thus lowering plasma levels of citrulline [28]. Simultaneous administration of Glu and Tau allowed intestinal functions to be conserved, thus maintaining citrulline synthesis. Tau administration appeared to conserve intestinal functions to maintain small intestinal mucosal thickness and villus height. However, the underlying mechanisms are unclear, and further investigation is necessary. By simultaneously administering Glu and Tau, small intestine function was maintained, and small intestinal mucosal thickness and villus height for group GT were comparable to those for group C. Although the 5-day time point seems rather short for a TPN study, we believe that this was sufficient, as the small intestine mucosa had atrophied by this time. Longer timescales would be ideal. Unfortunately, levels of Et in portal blood and levels of blood cytokines for group GT tended to be lower than those for group G, although no significant differences were apparent. We thought about
Effects of glutamic acid and taurine on total parenteral nutrition using levels of Et in the portal blood as an indicator for BT, but no significant differences in outcome were seen in the present study. This suggests that BT cannot be completely suppressed even when small intestinal mucosal thickness and villus height are maintained. Histopathologic analysis revealed no sign of apoptosis in the liver on TUNEL staining in any of the 3 groups, thus failing to clearly confirm DNA fragmentation. The present study used immunostaining. VCAM-1, which represents one of the adhesion molecules, is a glycoprotein with a molecular weight of about 110 kDa and belongs to the immunoglobulin superfamily [29 -31]. Expression of VCAM-1 is induced by inflammatory cytokines such as IL-1, TNF-a, and interferon c, and expression is enhanced when endothelial cells are damaged [32,33]. VCAM-1 is believed to be involved with cell adhesion in events such as lymphocyte invasion to inflamed areas. We have previously reported that TPN enhances expression of intercellular adhesion molecule 1 in hepatic sinusoidal endothelial cells [34]. In the present study, VCAM-1 staining showed positive reactions in the liver for group G. In addition, strong positive reactions were seen in the lung for group G. The degree of reaction in the lung was lower for group GT than for group G. TM, a glycoprotein found on the surface of vascular endothelial cells, binds to thrombin to inactivate thrombin and suppress platelet aggregation. Furthermore, the thrombin-TM complex activates protein C and inactivates factors Va and VIIIa to delay coagulation [35 -37]. TM also exists in hepatic sinusoidal endothelial cells and is involved in hepatic microcirculation. When hepatic sinusoidal endothelial cells are damaged and epithelial TM enters the circulation, intravascular coagulation is disrupted. The presence of TM in hepatic sinusoidal endothelial cells indicates maintenance of sinusoidal functions. The present study shows that in group C, TM was distributed evenly in the liver, and in group G, staining around the central vein was weak, but strong around the hepatic vein. In group GT, such changes were milder. In other words, TPN damaged the hepatic microcirculation and damage was more severe around the central vein. VCAM-1 and TM staining identify endothelial cell damage and represent microcirculation impairment in the liver and lung. Damage to hepatic sinusoidal endothelial cells caused by TPN was alleviated by administration of Tau. Tau is believed to have hepatoprotective and cell membrane– stabilizing effects. We believe that damage to sinusoid endothelial cells was alleviated by the addition of Tau. Also, as the level of citrulline in the small intestine improved, atrophy of the small intestine mucosa was prevented, and we believe this reduced BT and alleviated damage to sinusoid endothelial cells. Further investigation is necessary to elucidate whether this was attributable to the ability of Tau to protect hepatocytes and facilitate bile excretion, or the ability to maintain small intestine villi, thus somewhat suppressing BT.
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Acknowledgment The present study was partially funded by grant-in-aid for scientific research (C)(2) 13671871 provided by the Japan Society for the Promotion of Science.
References [1] Peden VH, Witzleben CL, Skelton MA. Total parental nutrition (letters to the editor). J Pediatr 1971;78:180 - 1. [2] Touloukian RJ, Downing SE. Cholestasis associated with long term parenteral hyperalimentation. Arch Surg 1973;106:58 - 62. [3] Preisig R, Rennert O. Biliary transport and cholestatic effects of amino acids. Gastroenterology 1977;73:1240. [4] Beale EF, Nelson RM, Bucciarelli RL, et al. Intrahepatic cholestasis associated with parenteral nutrition in premature infants. Pediatrics 1979;64:342 - 7. [5] Bell RL, Ferry GD, Smith EO, et al. Total parenteral nutrition-related cholestasis in infants. JPEN J Parenter Enteral Nutr 1986;10:356 - 9. [6] Drongowski RA, Coran AG. An analysis of factors contributing to the development of total parenteral nutrition induced cholestasis. JPEN J Parenter Enteral Nutr 1989;13:586 - 9. [7] Quigley EH, Marsh MN, Shaffer JH, et al. Hepatobiliary complication of total parenteral nutrition. Gastroenterology 1993;104:286 - 301. [8] Alverdy JC, Aoys E, Moss GS. Total parenteral nutrition promotes bacterial translocation from the gut. Surgery 1988;104:185 - 90. [9] Deitsh EA, Winterton J, Li M, et al. The gut as portal of entry for bacteria. Ann Surg 1987;205:681 - 92. [10] Illing KA, Ryan CK, Handy DJ, et al. Total parenteral nutritioninduced changes in gut mucosal function; atrophy alone is not the issue. Surgery 1992;112:631 - 7. [11] Beale RJ, Bryg DJ, Bihari DJ. Immunonutrition in the critically ill; a systematic review of clinical outcome. Crit Care Med 1999;27: 2799 - 805. [12] Grimble RF. Nutritional modulation of immune function. Proc Nutr Soc 2001;60:389 - 97. [13] Koretz RL. Immunonutrition; fact, fantasy, and future. Curr Gastroenterol Rep 2002;4:332 - 7. [14] Gianotti L, Alexander JW, Gennari R, et al. Oral glutamine decreases bacterial translocation and improves survival in experimental gutorigin sepsis. JPEN J Parenter Enteral Nutr 1995;19:69 - 74. [15] Li J, Kudsk KA, Janu P, et al. Effect of glutamine-enriched total parenteral nutrition on small intestinal gut-associated lymphoid tissue and upper respiratory tract immunity. Surgery 1997;121:542 - 9. [16] Saito H, Furukawa S, Matsuda T. Glutamine as an immunoenhancing nutrient. JPEN J Parenter Enteral Nutr 1999;23:59 - 61. [17] Obayashi T, Tamura H, Tanaka S, et al. A new chromogenic endotoxin-specific assay using recombined limulus coagulation enzymes and its clinical application. Clin Chim Acta 1985;149: 55 - 65. [18] MacFie J. Enteral versus parenteral nutrition; The significance of bacterial translocation and gut-barrier function. Nutrition 2000;16: 606 - 11. [19] Fukushima R, Alexander JW, Gianotti L, et al. Bacterial translocationrelated mortality may be associated with neutrophil-mediated organ damage. Shock 1995;3:323 - 8. [20] Tsuchioka T, Urao M, Fujiwara T, et al. The effect of total parenteral nutrition and enteral nutrition on LPS-induced sepsis in the rat. J Jpn Soc Pediatr Surg 2000;36:1034 - 41. [21] Souba WW, Smith RJ, Wilmore DW. Glutamine metabolism by the intestinal tract. JPEN J Parenter Enteral Nutr 1985;9:608 - 17. [22] Cook RJ, Whittington PF, Kelts D. Effect of taurine supplementation on hepatic function during short-term parenteral nutrition in the premature infant. J Pediatr Gastroenterol Nutr 1984;3:234 - 8.
1572 [23] Giggle HS, Aments ME, Heckenlively JR, et al. Nutritional requirement for taurine in patients receiving long-term parenteral nutrition. N Engl J Med 1985;312:142 - 6. [24] Cooper A, Betts JM, Pereira GR, et al. Taurine deficiency in severe hepatic dysfunction complication total parenteral nutrition. J Pediatr Surg 1984;19:462 - 6. [25] Chesney RW, Helms RA, Christensen M, et al. An update view of the value of taurine in infant nutrition. Adv Pediatr 1998;45:179 - 200. [26] Windmueller HG, Spaeth AE. Source and fate of circulating citrulline. Am J Physiol 1981;241:E473 - 80. [27] Morris SM. Regulation of enzymes of urea and arginine synthesis. Annu Rev Nutr 1992;12:81 - 101. [28] Wakabayashi Y, Yamada E, Yoshida T, et al. Arginine becomes an essential amino acid after massive resection of rat small intestine. J Boil Chem 1994;269:32667 - 71. [29] Hynes RO. Integrins; a family of cell surface receptors. Cell 1987;48: 549 - 54. [30] Hynes RO. Integrins; versatility, modulation, and signaling in cell adhesion. Cell 1992;69:11 - 25. [31] Osborn L, Hession C, Tizard R, et al. Direct expression cloning of vascular cell adhesion molecule 1, a cytokine-induced endothelial protein that binds to lymphocytes. Cell 1989;59:1203 - 11.
T. Tsuchioka et al. [32] Walsh GH, Moermod JJ, Hartnell A, et al. Human eosinophil, but not neutrophil, adherence to IL-1–stimulated human umbilical vascular endothelial cells is a4b1 (very late antigen-4) dependent. J Immunol 1991;146:3419 - 23. [33] Wellicome SM, Thornhill MH, Pitalis C, et al. A monoclonal antibody that detects a novel antigen on endothelial cells that is induced by tumor necrosis factor, IL-1, or lipopolysaccharide. J Immunol 1990; 144:2558 - 65. [34] Tsuchioka T, Fujiwara T, Sunagawa M. Immunohistochemical study of adhesion molecule in liver receiving total parenteral nutrition. Dokkyo J Med Sci 1997;24:11 - 9. [35] Esmon CT. The regulation of natural anticoagulation pathways. Science 1987;235:1348 - 52. [36] Suzuki K, Kusumoto H, Deyashiki Y, et al. Structure and expression of human thrombornodulin, a thrombin receptor on endothelium acting as a cofactor for protein C activation. EMBO J 1987;6: 1891 - 7. [37] Maruyama I, Bell CE, Majerus PW. Thrombomodulin is found on endothelium of arteries, veins, capillaries and lymphatics, and on syncytiotrophoblast of human placenta. J Cell Biol 1985;101: 363 - 71.