Sun XQ, Fu XB, Rong-Zhang, Lü Y, Deng Q, Jiang XG, Sheng ZY. Relationship between plasma D(-)-lactate and intestinal damage after severe injuries in rats. World J Gastroenterol 2001; 7(4): 555-558
Corresponding Author of This Article
Dr. Xiao-Qing Sun, Trauma Center, 304th Hospital, Beijing 100037, China. email@example.com
Article-Type of This Article
Open-Access Policy of This Article
This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/
Xiao-Qing Sun, Xiao-Bing Fu, Rong-Zhang, Yi Lü, Xiao-Guo Jiang, Zhi-Yong Sheng, Burn Institute, 304th Hospital, Beijing 100037, China
Qun Deng, Department of General Surgery, Chinese PLA 304 Hospital, Beijing 100037, China
ORCID number: $[AuthorORCIDs]
Author contributions: All authors contributed equally to the work.
Supported by the Fund for National Outstanding Young Researchers of China, No. 39525024
Correspondence to: Dr. Xiao-Qing Sun, Trauma Center, 304th Hospital, Beijing 100037, China. firstname.lastname@example.org
Telephone: 0086-10-66867391 Fax: 0086-10-68429998
Received: April 6, 2001 Revised: May 5, 2001 Accepted: May 12, 2001 Published online: August 15, 2001
AIM: To explore the kinetic changes in plasma D(-)-lactate and lipopolysaccharide (LPS) levels, and investigate whether D(-)-lactate could be used as a marker of intestinal injury in rats following gut ischemia/reperfusion, burn, and acute necrotizing pancreatitis (ANP).
METHODS: Three models were developed in rats: ① gut ischemia/reperfusion obtained by one hour of superior mesenteric artery occlusion followed by reperfusion; ② severe burn injury created by 30% of total body surface area (TBSA) full-thickness scald burn; and ③ ANP induced by continuous inverse infusion of sodium taurocholate and trypsin into main pancreatic duct. Plasma levels of D(-)-lactate in systemic circulation and LPS in portal circulation were measured by enzymatic-spectrophotometric method and limulus amebocyte lysate (LAL) test kit, respectively. Tissue samples of intestine were taken for histological analysis.
RESULTS: One hour gut ischemia followed by reperfusion injuries resulted in a significant elevation in plasma D(-)-lactate and LPS levels, and there was a significant correlation between the plasma D(-)-lactate and LPS (r = 0.719, P < 0.05). The plasma concentrations of D(-)-lactate and LPS increased significantly at 6 h postburn, and there was also a remarkable correlation between them (r = 0.877, P < 0.01). D(-)-lactate and LPS levels elevated significantly at 2 h after ANP, with a similar significant correlation between the two levels (r = 0.798, P < 0.01). The desquamation of intestine villi and infiltration of inflammatory cells in the lamina propria were observed in all groups.
CONCLUSION: The changes of plasma D(-)-lactate levels in systemic blood paralleled with LPS levels in the portal vein blood. The measurement of plasma D(-)-lactate level may be a useful marker to assess the intestinal injury and to monitor an increase of intestinal permeability and endotoxemia following severe injuries in early stage.
Citation: Sun XQ, Fu XB, Rong-Zhang, Lü Y, Deng Q, Jiang XG, Sheng ZY. Relationship between plasma D(-)-lactate and intestinal damage after severe injuries in rats. World J Gastroenterol 2001; 7(4): 555-558
Apart from their major functions of digestion and absorption of nutrients, the intestines also act as a barrier to prevent micro-organisms and toxins contained within the lumen from spreading to distant tissues and organs. Failure of intestinal barrier function often occurs in many clinical conditions, including hemorrhage shock, severe burn injury, and the surgically critical illness, resulting in the increased intestinal permeability and subsequent translocation of bacteria or/and endotoxin from gut. It is clear that increased gut permeability and bacteria with or without endotoxin translocation play a key role in the development of severe complications such as systemic inflammatory response syndrome (SIRS), sepsis, multiple organ dysfunction syndrome (MODS) and multiple organ failure (MOF)[13-20]. Therefore, it is important to know the intestinal injuries following a variety of insults (shock, burn injury, sepsis, and some critically surgical illness)[21,22]. D(-)-lactate is a product of bacterial fermentation. It is produced by many of the bacteria found in the human gastrointestinal tract. Tissues in mammalian do not produce it and its metabolism is very slow. In this study, we investigated the changes of plasma D(-)-lactate and lipopolysaccharide (LPS) levels and their correlation in gut ischemia/reperfusion, burn injury and acute necrotizing pancreatitis (ANP); and explored whether the changes of D(-)-lactate levels could be used as a predictor of increased intestinal permeability and endotoxemia following severe injuries.
MATERIALS AND METHODS
Male Wistar rats were used in this serial studies. They were housed in individual cages. The room temperature was maintained at 22 °C-24 °C with a 12 h light-dark cycle, and free access to a commercial laboratory rodent chow and fresh water were allowed. Twelve hours prior to experiment, the rats were fasted, but allowed free access to water.
Rat models of gut ischemia/reperfusion
Rats weighing 190 g-250 g were divided into three groups. Gut ischemic group (n = 20): Animals were anesthetized with an intraperitoneal injection of 0.3 mL 30 g·L-1 pentobarbital sodium. Through a middle abdominal incision, intestinal ischemia was produced by occluding the superior mesenteric artery for 1 and 1.5 h with an automatic microvascular clamp. Animals were sacrificed at the end of gut is chemia. Gut ischemia/reperfusion group (n = 50): superior mesenteric artery was occluded for 1 h and then the vascular clamp was removed to produce gut reperfusion. Animals were sacrificed at 0.5, 1, 2, 6 and 24 h after gut reperfusion. Sham-operated control (n = 10): animals were treated identically omitting the superior mesenteric artery occlusion. Blood samples were collected aseptically from cervical artery and portal vein for D(-)-lactate and LPS assay before animals were killed at each time point.
Rat models of burn
Male Wistar rats weighing 190 g-250 g were used. Animals were divided into two groups. In thermal group, they were subjected to a 30% total body surface area (TBSA) full-thickness scald burn injury (n = 40). They were anesthetized with an intraperitoneal injection of 30 g·L-1 pentobarbital sodium (60 mg·kg-1) and then the dorsal hair was shaved. A 30% TBSA full-thickness burn was created on the back of the rats in boiling water at 98 °C-100 °C for 12 s. Rats were resuscitated immediately after thermal injury with 50 g·L-1 glucose saline solution (50 mL·kg-1) intraperitoneally. In the control group (n = 10), rats were exposed to the room-temperature water. Animals in thermal group were killed at 3, 6 12 and 24 h after burn. Blood samples were collected aseptically from cervical artery and portal vein before the rats were killed at each time point.
Rat models of acute necrotizing pancretitis (ANP)
Male Wistar rats weighing 270 g-330 g were randomly divided into two groups. In the ANP group (n = 27), animals were anesthetized with 30 g·L-1 pentobarbital sodium (60 mg·kg-1, ip). After medium laparotomy, the duodenum was mobilized and the pancreatic duct was identified at its duodenal junction. ANP was induced by a continuous inverse infusion of sodium taurocholate (50 g·L-1, 1 mL·kg-1) and trypsin (1.67 × 105 U·kg-1) into the main pancreatic duct. Animals were immediately given saline (50 mL·kg-1) subcutaneously after injury. In control group (n = 6), animals were treated identically with infusion saline. Blood samples were taken aseptically from cervical artery and portal vein at 2, 8, 24 and 48 h after injury.
The plasma from systemic blood samples was obtained and subjected to a deproteination and neutralization process by acid/base precipitation using perchloric acid and potassium hydroxide. The protein-free plasma was then assayed for D(-)-lactate concentration by enzymatic-spectrophotometric method with minor modification.
Lipopolysaccharide (LPS) determination
The plasma from portal vein blood was also obtained and subjected to a deproteination and neutralization process by acid/base precipitation using perchloric acid and sodium hydroxide. The LPS levels of portal vein blood were assayed by the chromogenic limulus amebocyte lysate (LAL) test with a kinetic modification according to the test kit procedure.
Tissue samples of intestines were taken for morphologic study. Biospies were fixed in 100 mL·L-1 neutral buffered formalin, embedded in paraffin, microtome sectioned at 4 μm-6 μm thickness, and stained with hematoxylin and eosin. Sections were examined under light microscope.
Data were expressed as means ± SD. The statistical significance of mean values between groups was evaluated by the Student’s t test. The relationship between circulating systemic D(-)-lactate and portal vein LPS concentrations was determined by the calculation of Pearson correlation coefficient. P < 0.05 was considered to be significant.
Kinetics of D(-)-lactate and lipopolysaccharide concentrations in plasma after gut ischemia/reperfusion in rats
One hour of gut ischemia alone induced a slight increase in systemic blood D(-)-lactate and portal vein blood LPS concentrations (Table 1). Either D(-)-lactate or LPS concentrations had a further significant increase at 0.5 h-2 h after gut reperfusion (P < 0.05-0.01), and decreased to normal at 6 h. Meanwhile, correlation analysis revealed a significant correlation between systemic blood D(-)-lactate levels and portal vein blood LPS concentrations (r = 0.719, P < 0.05).
Table 1 The plasma contents of D(-)-lactate and lipopolysac charide in rats after gut ischemia/reperfusion insults (mean ± SD).
Compared with sham-operated control, respectively:
aP < 0.05;
bP < 0.01.
Alterations in plasma D(-)-lactate and LPS levels in thermal rats
Results presented in Table 2 indicated that there was a significant increase both in circulating blood D(-)-lactate and portal vein blood LPS concentrations at 6h after injury, and kept significantly increasing to the end of our observation period (72 h, P < 0.01). In addition, correlation analysis revealed that there was a strong positive correlation between plasma levels of D(-)-lactate and LPS after injury (r = 0.877, P < 0.01).
Table 2 Changes in systemic blood D(-)-lactate levels and portal blood LPS content in thermal rats (mean ± SD).
Changes in plasma D(-)-lactate and LPS levels in ANP rats
In rats subjected to ANP, the levels of D(-)-lactate in systemic blood and LPS in portal vein blood began to increase at 2 h after ANP (P < 0.01) (Table 3), and peaked at 24 h after injury. Furthermore, a marked correlation was noted between the changes in contents of plasma D(-)-lactate and LPS (r = 0.798, P < 0.01).
Table 3 Alterations in systemic blood D(-)-lactate levels and portal blood LPS content in ANP rats (mean ± SD).
Mucosal edema, necrosis, and the loss of the epithelium in mucosa, as well as vascular dilution, congestion, edema and inflammatory cell infiltration in the lamina propria were observed in small intestinal biopsies in three groups. The intestinal injury paralleled with the changes of plasma D(-)-lactate levels.
The present study showed that the intestinal damage caused by gut ischemia caused a slight increase in plasma concentrations of D(-)-lactate in systemic blood and LPS in portal vein blood. After gut ischemia followed by reperfusion, the plasma levels of D(-)-lactate and LPS significantly elevated, but declined to normal rapidly at 6 h after reperfusion[27-29]. The intestinal damage mediated by burn injury or ANP displayed a more severe damage than that in gut ischemia/reperfusion. A remarked increase of plasma D(-)-lactate and LPS concentrations occurred at 6 h, and 3 h after insult, respectively, and persisting to the end of our observation. Moreover, the elevation of plasma D(-)-lactate levels in systemic blood was associated with increased plasma LPS contents in portal vein blood, and histological examination also exhibited intestinal injury in those three rodent models.
D(-)-lactate is produced by some bacteria including Klebsilla, Escherichia coli, Lactobacillus species, and Bacteroides species. It is an indigenous products in gut. Normally, serum levels of D(-)-lactate in mammals are quite low. During the event that an ischemia/reperfusion insults, the mucosa is injured and intestinal permeability is increased, leading to an efflux of bacteria and the products of their metabolism[30-33], including D(-)-lactate into the circulation. Otherwise, the gut ischemic insult leads to a loss in normal host defenses against bacterial overgrowth, resulting in increased numbers of bacteria within the lumen of the infected intestine[34-36]. This bacterial proliferation would be expected to cause an increased bacterial metabolism with increased production of D(-)-lactate. Mammals do not possess the enzyme system to rapidly metabolize D(-)-lactate, thus, it passes through the liver with unchanged way and enters the peripheral blood early in the disease process. Thus, D(-)-lactate accumulation in the systemic circulation can generally be considered as a result of bacterial over growth and increase in gut permeability induced by some gastrointestinal disorders. Therefore, D(-)-lactate levels could be used as a predictor of intestinal injury. In fact, the elevation of plasma D(-)-lactate levels has been used as the predictor of bacterial infection in patients with short-bowel syndrome. In rat model of acute mesenteric ischemia, D(-)-lactate was significantly elevated after gut ischemia, and the histopathological evaluation scores of intestinal injury were remarkably correlated to the plasma D(-)-lactate levels[38,39]. Recently, in clinical study, it has also been demonstrated that patients with mesenteric ischemia at laparotomy had significantly elevated D(-)-lactate levels in systemic circulation as compared with patients operated on for an acute abdomen or normal abdomen.
In conclusion, our data in these rat models suggest that the changes in D(-)-lactate concentrations paralleled with LPS concentrations, and correlated similarly with the intestinal histopathological alterations as well. Therefore, plasma D(-)-lactate in systemic circulation measurement would be a useful marker to evaluate intestinal injury and endoxemia following severe injuries.
Edited by Ma JY
Liu CH, Liu C, Liu P, Xu LM. Seropharmalogical effects of Fuzheng Huayu decoction on rat Ito cell morphology and functions in culture.China Natl J New Gastroenterol. 1997;3:263-265.
Li ZL. Diagnosis and treatment of multiple organ disorder and failure induced by severe infections.Shijie Huaren Xiaohua Zazhi. 1999;7:1074-1076.
Li Y, Li ZL. Influence of gastric distension on jejunal fluid absorption and transmural potential difference in rats.Xin Xiaohuabingxue Zazhi. 1997;5:684-686.
Dong HL. Intestinal permeability test and its clinical significance.Shijie Huaren Xiaohua Zazhi. 2000;8:562-563.
Luo H, Wang LF, Imoto T, Hiji Y. Inhibitory effect and mechanism of acarbose combined with gymnemic acid on maltose absorption in rat intestine.World J Gastroenterol. 2001;7:9-15.
Ruan CP, Wang YH, Wang LG, Wang YX. Changes of neurotensin and endotoxin in rats with intestinal ischemia.China Natl J New Gastroenterol. 1996;2:200-202.
Qin RY, Zou SQ, Wu ZD, Qiu FZ. Influence of splanchnic vascular infusion on the content of endotoxins in plasma and the translocation of intestinal bacteria in rats with acute hemorrhage necrosis pancreatitis.World J Gastroenterol. 2000;6:577-580.
Swank GM, Deitch EA. Role of the gut in multiple organ failure: bacterial translocation and permeability changes.World J Surg. 1996;20:411-417.
Langkamp-Henken B, Donovan TB, Pate LM, Maull CD, Kudsk KA. Increased intestinal permeability following blunt and penetrating trauma.Crit Care Med. 1995;23:660-664.
Koike K, Moore FA, Moore EE, Poggetti RS, Tuder RM, Banerjee A. Endotoxin after gut ischemia/reperfusion causes irreversible lung injury.J Surg Res. 1992;52:656-662.
Wang XJ, Luo XD, Luo Q, Yang ZC. Effects of sera from burn patients on human hepatocytic viscoelasticity.World J Gastroenterol. 1998;4:60.
Ren JY, Ojeas H, Lightfoot SA, Harty RF. Effects of capsaicin on stress-induced duodenal injury.World J Gastroenterol. 1998;4:53.
Baue AE, Durham R, Faist E. Systemic inflammatory response syndrome (SIRS), multiple organ dysfunction syndrome (MODS), multiple organ failure (MOF): are we winning the battle?Shock. 1998;10:79-89.
Ammori BJ, Leeder PC, King RF, Barclay GR, Martin IG, Larvin M, McMahon MJ. Early increase in intestinal permeability in patients with severe acute pancreatitis: correlation with endotoxemia, organ failure, and mortality.J Gastrointest Surg. 1999;3:252-262.
Baker JW, Deitch EA, Li M, Berg RD, Specian RD. Hemorrhagic shock induces bacterial translocation from the gut.J Trauma. 1988;28:896-906.
Li JY, Sheng ZY, Lu Y, Yu Y, Hu S, Zhou BT. Severe trauma induced intestinal barrier function injury and protection.Shijie Huaren Xiaohua Zazhi. 2000;8:1093-1096.
Yue MX. Management of digestive diseases complicated with multi-organ dysfunctional failure.Huaren Xiaohua Zazhi. 1998;6:277-279.
Zhang P, Yang WM, Shui WX, Du YG, Jin GY. Effect of Chinese herb mixture, shock decoction on bacterial translocation from the gut.World J Gastroenterol. 2000;6:74.
Wu CT, Huang XC, Li ZL. Increased intestinal permeability and intestinal bacterial transposition.Shijie Huaren Xiaohua Zazhi. 1999;7:605-606.
Zhang QH, Ni QX, Cai D, Zhang YL, Jiang YF, Wu SQ, Xiang Y, Yin BB, Zhang N, Hou LD. Somatostatin and growth hormone protection on multiple organ injury in acute necrotizing pancreatitis.Huaren Xiaohua Zazhi. 1998;6:185-188.
Tu WF, Li JS, Zhu WM, Li ZD, Liu FN, Chen YM, Xu JG, Shao HF, Xiao GX, Li A. Influence of Glutamine and caecostomy/ colonic irrigation on gut bacteria/endotoxin translocation in acute severe pancreatitis in pigs.Shijie Huaren Xiaohua Zazhi. 1999;7:135-138.
Ci XL, Wang BE, Zhang SW, Zhang NN. Alterations of gas-trointestinal motility and mucosal barrier in shock rat model induced by endotoxin plus TNF-α.Shijie Huaren Xiaohua Zazhi. 1999;7:510-512.
Yao YM, Yu Y, Wu Y, Lu LR, Sheng ZY. Plasma D(-)-lactate as a new marker for diagnosis of acute intestinal injury following ischemia-reperfusion.China Natl J New Gastroenterol. 1997;3:225-227.
Smith SM, Eng RH, Buccini F. Use of D-lactic acid measurements in the diagnosis of bacterial infections.J Infect Dis. 1986;154:658-664.
Brandt RB, Siegel SA, Waters MG, Bloch MH. Spectrophotometric assay for D-(-)-lactate in plasma.Anal Biochem. 1980;102:39-46.
Yao YM, Tian HM, Wang YP, Yu Y, Shi ZG. Microassay for quantification of endotoxin in blood with new PCA treatment using chromogenic limulus amebocyte lysate.Shanghai Fenxi Zazhi. 1993;8:31-33.
Zhu L, Yang ZC, Li A, Cheng DC. Protective effect of early enteral feeding on postburn impairment of liver function and its mechanism in rats.World J Gastroenterol. 2000;6:79-83.
Xu GH, Shi BJ, Liu HY. Analysis of prognostic factors in 178 patients with acute pancreatitis.Xin Xiaohuabingxue Zazhi. 1997;5:723-724.
Xie CG, Wang XP. Endotoxin and pancreatic damage.Shijie Huaren Xiaohua Zazhi. 2000;8:1039-1041.
Ruan CP, Wang YH, Wang LG. Bacterial translocation from the gastrointestinal tract in rats with intestinal ischemia.Xin Xiaohuabingxue Zazhi. 1996;4:304-305.
Yu Y, Tian HM, Shi ZG, Yao YM, Wang YP, Lu LR, Yu Y, Chang GY, Ma NS, Sheng ZY. Relationship between endotoxemia and dysfunction of intestinal immuno-barrier after scald in rats.Huaren Xiaohua Zazhi. 1998;6:703-704.
Zhang WZ, Han TQ, Tang YQ, Zhang SD. Rapid detection of sepsis complicating acute necrotizing pancreatitis using polymerase chain reaction.World J Gastroenterol. 2001;7:289-292.
Horton JW, Walker PB. Oxygen radicals, lipid peroxidation, and permeability changes after intestinal ischemia and reperfusion.J Appl Physiol (1985). 1993;74:1515-1520.
Fu XB, Yang YH, Sun TZ, Gu XM, Jiang LX, Sun XQ, Sheng ZY. Effect of intestinal ischemia-reperfusion on expressions of endogenous basic fibroblast growth factor and transforming growth factor betain lung and its relation with lung repair.World J Gastroenterol. 2000;6:353-355.
Fu XB. Growth factors in treatment of digestive system organ injuries.Xin Xiaohuabingxue Zazhi. 1997;5:663-664.
Yang YH, Fu XB, Sun TZ, Jiang LX, Gu XM. bFGF and TGFbeta expression in rat kidneys after ischemic/ reperfusional gut injury and its relationship with tissue repair.World J Gastroenterol. 2000;6:147-149.
Li YS, Li JS, Li N, Jiang ZW, Zhao YZ, Li NY, Liu FN. Evaluation of various solutions for small bowel graft preservation.World J Gastroenterol. 1998;4:140-143.
Murray MJ, Barbose JJ, Cobb CF. Serum D(-)-lactate levels as a predictor of acute intestinal ischemia in a rat model.J Surg Res. 1993;54:507-509.
Marcos MA, Vila J, Gratacos J, Brancos MA, Jimenez de Anta MT. Determination of D-lactate concentration for rapid diagnosis of bacterial infections of body fluids.Eur J Clin Microbiol Infect Dis. 1991;10:966-969.
Murray MJ, Gonze MD, Nowak LR, Cobb CF. Serum D(-)-lactate levels as an aid to diagnosing acute intestinal ischemia.Am J Surg. 1994;167:575-578.