Administration of Chlorella sp. microalgae reduces endotoxemia, intestinal oxidative stress and bacterial translocation in experimental biliary obstruction

Clinical Nutrition 28 (2009) 674–678

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Original Article

Administration of Chlorella sp. microalgae reduces endotoxemia, intestinal oxidative stress and bacterial translocation in experimental biliary obstructionq Abdulkadir Bedirli a, *, Mustafa Kerem a, Ebru Ofluoglu b, Bulent Salman a, Hikmet Katircioglu c, Nurdan Bedirli d, Demet Yılmazer e, Murat Alper e, Hatice Pasaoglu b a

General Surgery, Gazi University, Turkey Biochemistry, Gazi University, Turkey Biology, Gazi University, Turkey d Anesthesiology, Diskapi Education and Research Hospital, Ankara, Turkey e Patholgy, Diskapi Education and Research Hospital, Ankara, Turkey b c

a r t i c l e i n f o

s u m m a r y

Article history: Received 15 April 2009 Accepted 1 June 2009

Rationale: Endotoxemia has long been documented in obstructive jaundice, and altered intestinal barrier function is considered to be one of the important mechanisms for this phenomenon. The aim of this study was to investigate the role of different microalgae (Chlorella sp. and Spirulina sp.) extracts in intestinal barrier function and oxidative stress in experimentally jaundiced rats.

Keywords: Microalgae Chlorella Obstructive jaundice Endotoxemia

Methods: A total of 60 male wistar rats were randomly divided into four groups of 15 each: I, sham operated; II, bile duct ligation (BDL); III, BDL þ Chlorella sp.; IV, BDL þ Spirulina sp. Rats were fed rat chow or microalgae extracts supplemented enteral diet ten days after sham operation or BDL. Main outcome measures were endotoxin concentrations in plasma, evidence of bacterial translocation (BT) in mesenteric lymph nodes (MLNs) and liver, oxidative stress, and histology. Results: Compared to the group I, a significant increase in contamined MLNs, liver, and spleen samples and increased endotoxemia were noted in group II (P < 0.01) but were significant reduced in group III (P < 0.05). There was no significant difference in BT rate between the group II and group IV (P > 0.05). Moreover, Chlorella sp. administration protected in jaundiced rats against oxidative stress, as demonstrated by reduction of intestinal lipid peroxidation, increase of the antioxidant reduced glutathione (GSH), and decrease of the oxidized glutathione (GSSG). The intestinal mucosa in control rats was atrophic with significantly decreased villous density and total mucosal thickness. Chlorella sp. caused a significant reduction in villous atrophy compared with controls. Conclusions: Chlorella sp. microalgae supplemented enteral diet has significant protective effects on intestinal mucosa barrier in obstructive jaundice, and reduces intestinal translocation of bacteria and endotoxin. Ó 2009 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.

1. Introduction Intestinal mucosa, apart from its function of nutritional absorption, displays complex defense mechanisms preventing potentially harmful intraluminal elements from migrating to extraintestinal tissues and entering the systemic circulation.1 Bacterial translocation (BT) has been defined as the passage of viable bacteria from the gut lumen to extraintestinal organs.2 The

q Presented at the 29th ESPEN Congress, September 8–11, 2007, Prague, Czech Repuplic. * Corresponding author at: A. Taner Kislali Mah. Siyasal Villari, No:44, 06810, Cayyolu, Ankara, Turkey. Tel.: þ90 312 2025724; fax: þ90 312 2150494. E-mail address: [email protected] (A. Bedirli).

pathogenesis of BT is unknown but major conditions contributing to bacterial translocation are a breakdown of the intestinal barrier, an impairment of host immune defense and a loss of the colonization resistance with bacterial overgrowth in the intestinal tract.3–5 BT and absorption of endotoxins, the lipopolysaccharides associated with cell membranes of Gram-negative bacteria, may result in endotoxemia and have severe systemic effects. Several reports describe spontaneous bacterial infections and sepsis in patients with obstructive jaundice.6–8 Surgery in patients with obstructive jaundice is associated with increased morbidity and mortality. Previous experimental studies have shown that obstructive jaundice promotes morphologic alterations in the intestinal mucosa indicative of atrophy, increases crypt epithelial apoptosis, and induces intestinal oxidative stress.4,5,9 The regulation of

0261-5614/$ – see front matter Ó 2009 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved. doi:10.1016/j.clnu.2009.06.001

A. Bedirli et al. / Clinical Nutrition 28 (2009) 674–678

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gastrointestinal mucosal response to injury is thus have clinical importance as well as biological relevance. Many methods have been used to reduce complications resulting from biliary obstruction, but no treatment produced clinically effective results. Chlorella is a green single-celled microalgae that contains the highest concentrations of chlorophyll. The nutrient profile is subject to much variation between habitats and harvest procedures which influences the content of vitamins and antioxidants delivered in the final product.10 Certain features are common in all green algae, including a high content of bioavailable amino acids and minerals, including zinc, selenium, and magnesium.11 Among green algae, many species have been documented biomodulatory effects. In the present study, we investigated the effect of different microalgae (Chlorella sp. and Spirulina sp.) extracts on small intestinal histopathology, the degree of BT in a rat model of obstructive jaundice. In addition, we examined the potential effect of these algae on biochemical parameters of oxidant intestinal injury by determining lipid peroxidation and thiol redox state.

centrifugation and pellets were dried at 60  C for 24 h. After separating the extraction phase, all of the extracts were preserved at 4  C. The dose of algae extract used was 50 g/kg body weight/day.12,13 The suspension was given via oral gavage three times a day in equal doses (each dose was less than 5 ml).

2. Materials and methods

2.5. Determination of bacterial translocation

2.1. Animals Male Wistar rats weighing 230–280 g were housed individually in cages under constant temperature (22  2  C) and humidity. The animals were made to fast overnight before the experiments but were given free access to water. The experimental protocols were conducted with the approval of the Animal Research Committee at Gazi University, Ankara. All animals were maintained in accordance with the recommendations of the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals.

Mesenteric lymph nodes (MLNs), portions of liver and spleen were harvested for culture. Each of the first set of organ samples was weighed and suspended in 3.0 ml of Ringer’s solution. The second set of samples was homogenized in 4 ml of sterile Ringer’s solution and 200 ml of each homogenate was cultured on McConkey’s agar plates. Anaerobic cultures were performed by using an anaerobic blood agar in Gas-Pak Jars at 37  C for 24 h. The culture was considered positive if any bacterial growth was found in either the aerobic or the anaerobic culture, and the incidence of BT was calculated by determining the number of rats with positive bacterial culture divided by the total number of rats studied.

2.2. Experimental design

2.6. Intestinal malondialdehyde activity assay

A total of 60 male wistar rats were randomly allocated to four groups. Group I (n ¼ 15) underwent sham operation with standard rat chow. Group II (n ¼ 15) underwent common bile duct ligation and division with standard rat chow. Group III (n ¼ 15) underwent common bile duct ligation and division with Chlorella sp. via oral gavage in addition to the standard rat chow. Group IV (n ¼ 15) underwent common bile duct ligation and division with Spirulina sp. via oral gavage in addition to the standard rat chow. Rats were anesthetized by ketamine 100 mg/kg and xylazine 20 mg/kg body weight, intraperitoneally. They breathed spontaneously throughout the procedures. The abdominal skin was disinfected with 70% alcohol. All procedures were performed under sterile conditions. A upper midline incision was made approximately 2 cm long, sufficient to expose the gastroduodenal ligament. The gastroduodenal ligament was isolated and the common bile duct was identified. In groups II, III, and IV, the common bile duct was further double-ligated with 4-0 silk, and divided between the two ligatures. Sham-operated animals had the common bile duct freed from the surrounding soft tissue without ligation and transection. The abdominal incision was closed in two layers with chromic 4-0 cat gut and 4-0 silk. On the 10th day, all animals were reoperated. Samples were obtained according to the experimental protocol, after which the rats were sacrificed by exsanguinations. Main outcome measures were bilirubin and endotoxin concentrations in plasma, determination of BT, tissue MDA activity, oxidative stress, and histology.

For determination of malondialdehyde (MDA) contents ileal mucosa was scrubbed of and weighed. All specimens were immediately frozen in liquid nitrogen until assay. MDA was determined as an index of ileal mucosal lipid peroxidation according to the method of Ohkawa et al.15 Briefly, 0.1 ml of homogenate was mixed with 0.1 ml of 8.1% sodium dodecyl sulfate, 0.75% 0.8% thiobarbituric acid and 0.3 ml of distilled water and kept in a boiling water bath for 60 min. After cooling, 0.5 ml of distilled water and 2.5 ml 15/1 (v/v) n-butanol/pyridine were added. After centrifugation at 4000 rpm, the absorbance of the supernatant at 532 nm was measured with spectrophotometry (Shimadzu Corp., Tokyo, Japan).

2.3. Preparation and administration of algae extract

For histological analysis of the mucosal damage, a section of terminal ileum, was immediately fixed in 10% buffered formalin subsequently embedded in paraffin, cut into slices of 5–7 mm thickness and stained with hematoxylene and eosin (H&E).

Algal mass from an axenic exponential culture of the microalgae strains grown in BG11 were separated from the culture medium by

2.4. Bilirubin AST, ALT and endotoxin measurements For the determination of total bilirubin, aspartate transaminase (AST), and alanine transaminase (ALT), 0.5 ml of blood was collected from all animals, by transecting the tip of their tail. Then the laparotomy was performed and in all groups, the portal vein and abdominal aorta were punctured and samples were obtained for estimation of endotoxins and BT. Bilirubin, AST and ALT concentrations were assayed using a standard biochemical technique and expressed in mg dl1, IU/l, respectively. Endotoxin concentration was determined by the Limulus Amebocyte Lysate test and expressed in EU ml1.14

3. Quantification of GSH and GSSG Endothelial cell GSH and GSSG levels were determined by the method of Reed et al.16 Briefly, the assay is based on the initial formation of S-carboxymethyl derivatives of free thiols followed by the conversion of free amino groups to 2,4-dinitrophenyl derivatives. Separation of GSH and GSSG derivatives is achieved by reverse-phase ion-exchange HPLC. GSH and GSSG were expressed as nmol/mg protein. The GSH/GSSG ratio was calculated as an index of the glutathione pool redox state. 3.1. Histology

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Table 1 Description of histological grading scheme.a

4.4. Bacterial translocation

Grade Histologic changes

There was significantly more bacterial translocation detected in bile duct-ligated animals than in sham-operated animals (P ¼ 0.006) (Table 3). In group II, 30 of the 45 samples demonstrated aerobic bacterial growth. Eleven of these were in the MLN, ten in the spleen, and nine in the liver. Only one of 45 samples from the sham-operated group demonstrated live aerobic bacteria, in the liver. In group II, 28 of the 45 samples demonstrated anaerobic bacterial growth. In group III, the incidence of BT was reduced up to approximately 50% in the MLN, liver, and spleen, in both aerobic and anaerobic culture (P < 0.05). When compared with group II, the incidence of bacterial translocation in group IV was reduced, however, not significantly.

0 1

Normal mucosal villi Development of a subepithelial space, usually at the tip of the villus, with capillary congestion Extension of the subepithelial space with moderate lifting of the epithelial layer Massive epithelial lifting down the sides of villi Denuded villi with lamina propria and dilated capillaries exposed. Increased cellularity of the lamina propria Digestion and disintegration of the lamina propria, hemorrhage and ulceration

2 3 4 5 a

From Chiu et al.17

Histologic assessment was carried out in blinded fashion by one pathologist. The grading scheme has been adapted from Chiu et al.17 Thus, injury was classified using a semiquantitative grading system where a numerical score was assigned based on the type of mucosal and submucosal damage (Table 1). 3.2. Statistics Results are expressed as means  SD. Differences for histologic grading were evaluated by the Mann-Whitney U test. A one-way analysis of variance and the Fisher exact test protected the least significant difference. Post-hoc tests were used to determine the significance for the number of the bacteria. Statistical evaluation was carried out by using SPSS 10.0 software (SPSS, Chicago, IL, USA). Values of P < 0.05 were accepted as significant. 4. Results 4.1. Animal outcomes All animals survived and were in good health throughout the experiment. There were no significant differences in body weight between sham-operated and bile duct-ligated rats.

Ten days after the surgical procedure, the serum bilirubin, AST, ALT levels in groups II, III, and IV significantly increased (P < 0.01). However, there were no significant difference between groups II, III, and IV (Table 2). 4.3. Plasma endotoxin Ten days after the surgical procedure, there was highly significant change in endotoxin levels between group I and group II (P < 0.01). Similar results were observed in the Spirulina-treated group. In contrast, Chlorella sp. algae extract achieved a significant reduction of plasma endotoxin concentration when compared with groups II and IV (Fig. 1).

Table 2 Bilirubin and liver enzyme activities determined in serum. Group

Total bilirubin (mg/dl)

AST (IU/l)

ALT (IU/l)

I II III IV

0.5 9.3 8.7 11.1

27 181 197 205

35 162 145 160

Values are means (SD). *P < 0.01 vs group I (Sham).

The MDA levels of all groups subjected to bile duct ligation were significantly higher than the MDA levels of the sham group (P < 0.05) (Fig. 2). Administration of chlorella caused significant decreases in the ileal MDA levels in comparison with the control group (268  37 vs 146  17 nM/g protein) (P < 0.01). There were no significant differences in MDA levels between control and Spirulina-treated groups (P > 0.05). 4.6. Mucosal glutathione redox state GSH, the most important endogenous agent for cellular defense against oxidative stress, is vital for the integrity of the gut. The ratio of reduced-to-oxidized glutathione, GSH/GSSG, in the intestinal tissue was significantly reduced in bile duct-ligated animals (6.2  0.8 vs 3.6  0.7), which indicated accumulation of oxidative radicals (P < 0.01) (Fig. 3). Chlorella-treated rats resulted in GSH/GSSG ratios that were significantly higher in intestinal segments (P < 0.05). Thus, chlorella treatment resulted in a more reduced glutathione pool at both levels of refeeding and prevented the oxidation of the glutathione pool. 4.7. Histology

4.2. Serum levels of bilirubin, AST and ALT

(0.2) (2.1)* (1.6)* (1.8)*

4.5. Tissue MDA levels

(2) (13)* (29)* (32)*

(4) (22)* (16)* (17)*

The histopathological examination of the terminal ileum showed submocosal edema in groups II, III, and IV, whereas no pathological change was observed in the sham-operated animals. The villous height of the terminal ileum in groups II and IV was significantly lower than that in the sham-operated group, whereas the villous height in the group III was almost the same as that in the sham-operated group. The villous width in the groups II and IV was significantly wider than that in the sham-operated group. 5. Discussion Our study showed that Chlorella sp. microalgae enteral diet plays an important role as a protective factor of intestinal oxidative stress secondary to experimental bile duct ligation. Although obstructive jaundice is a systemic disease where several important organs can be damaged, we focused our study to evaluate the sepsis in the biliary obstruction. In bile duct-ligated animals, the incidence of positive bacterial cultures increased, and bacteria of enteric origin could be cultured in MLNs, liver, and spleen. High plasma concentrations of lipid peroxides in obstructive jaundice are believed to be an important mediator for intestinal bacterial colonization and neutrophil infiltration into the bowel.18 Chlorella treatment improved BT and intestinal MDA activities.

A. Bedirli et al. / Clinical Nutrition 28 (2009) 674–678

4

2,5 2 1,5 1 0,5

250 200

100 50 0

BDL + Spirulina

Sham

Fig. 1. Effects of Chlorella and Spirulina on plasma endotoxin concentration. *P < 0.05 vs sham group. yP < 0.05 vs BDL and BDL þ Spirulina groups.

Chlorella, a green single-celled microalgae, is able to enhance the nutritional content of conventional food preparations and hence, to positively affect the health of humans and animals. The high protein content of chlorella is one of the main reasons to consider them as an unconventional source of protein.11 Algal lipids are composed of glycerol, sugars or bases esterified to saturated or unsaturated fatty acids. Among all the fatty acids in microalgae, some fatty acids of the omega-3 and omega-6 families are of particular interest. Microalgae and fish oil have about the same amount of omega-3 PUFAs, 41% and 34%, respectively.19 Chlorella also represent a valuable source of nearly all essential vitamins. Thus, their composition gives microalgae interesting qualities, which can be applied in human and animal nutrition. In experimental studies, chlorella has been supplemented to enteral feeding in various models.12,13 In fact, the mucosa of the gastrointestinal tract provides an effective barrier to entry of intestinal bacteria and endotoxin in healthy individuals. Altered nutritional status could be due to poor appetite and decreased food intake associated with obstructive jaundice. Up to date, no studies have investigated the role of microalgae in intestinal barrier function and oxidative stress in obstructive jaundice. In obstructive jaundice, the absence of intestinal bile flow, impaired reticuloendothelial system function and immunity, intestinal bacterial overgrowth, and physical disruption of the gut mucosal barrier explain the high incidence of infectious complications.1,3,4 Endotoxaemia is implicated in the development of these and it is currently postulated that there are two major contributing factors responsible for the morbidity and mortality seen in jaundiced patients. The first is impairment of gut barrier function, allowing the passage of bacteria and their toxins into the portal circulation and the second is impaired mononuclear phagocytic function, allowing ‘spillover’ of

Table 3 The positive bacterial culture in cultured organs. Group

MLN (%)

Liver (%)

Spleen (%)

(0) (73) (40) (67)

1/15 9/15 4/15 8/15

(7) (60) (27) (53)

0/15 10/15 4/15 8/15

(0) (67) (27) (53)

Anaerobic bacteria (n ¼ 15) I 0/15 (0) II 11/15 (73) III 6/15 (40) IV 10/15 (67)

0/15 9/15 4/15 8/15

(0) (60) (27) (53)

0/15 8/15 4/15 7/15

(0) (53) (27) (47)

Aerobic bacteria (n ¼ 15) I 0/15 II 11/15 III 6/15 IV 10/15

BDL

BDL + Chlorella

BDL + Spirulina

Fig. 2. Malondialdehyde (MDA) levels in intestinal tissue. Values are expressed as mean  SD. *P < 0.05 vs sham group. yP < 0.01 vs BDL and BDL þ Spirulina groups.

endotoxin into the systemic circulation.9 MDA is an end product of peroxidative decomposition of polyenic fatty acids in the lipid peroxidation process. MDA accumulation in tissues is an indicative of the extent of lipid peroxidation and oxidative stress.20 The reduced glutathione (GSH) is an important cellular antioxidant because of its elevated intracellular concentration and also for being the substratum of essential scavenger enzymes for the maintenance of oxidative balance.21 Excessive production and reduced elimination of active oxygen play an important role in the development of sepsis during biliary obstruction, mainly through direct destruction of intestinal epithelial cells and promotion of inflammatory reaction.22 Chlorella was effective in inhibiting intestinal mucosal lipid peroxidation in bile ductligated rats, which resulted in decreased MDA levels in the ileal mucosa. Subsequently, chlorella treatment resulted in a more reduced glutathione pool at both levels of refeeding and prevented the oxidation of the glutathione pool. Both decreased mucosal MDA and oxidized glutathione (GSSG) levels were associated with a significantly lower endotoxin levels. Primary site for de nova GSH synthesis is the liver, which supplies approximately 90% of the circulating plasma GSH.23 Oxidation of hepatic glutathione occurred during biliary obstruction since the concentration of GSSG increased significantly. In this work, chlorella was able to prevent lipid peroxidation induced by biliary obstruction. However, it failed to protect the liver from cholestatic injury as evaluated by conventional biochemical markers of liver damage. It is possible that a homeostatic response

7

†

6

The ratio of GSH/GSSG

BDL + Chlorella

*,†

150

0 BDL

*

* MDA (nM/g protein)

Endotoxin (EU/mL)

*,†

3

Sham

*

300

*

3,5

677

5

* 4 3 2 1 0 Sham

BDL

BDL + Chlorella BDL + Spirulina

Fig. 3. The ratio of reduced-to-oxidized glutathione, GSH/GSSG, in the intestinal tissue. Values are expressed as mean  SD. *P < 0.01 vs sham group. yP < 0.05 vs BDL group.

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of the liver cells, up regulates the synthesis of GSH, preventing further damage in bile duct-ligated rats. Histologic alterations in bile duct-ligated rats are believed to occur at the subcellular levels in the intestinal mucosa. Alterations of cell membranes and disruption of tight junctions may initiate an increased bacterial adherence to the mucosa cell, which is an important step in the translocation process, and promote the intraand intercellular escape of intestinal bacteria.24,25 Out of these considerations, in bile duct-ligated rats, intestinal mucosal lipid peroxidation might play an associated role required for BT. The mechanism leading to BT is probably a disruption of the intestinal barrier at subcellular levels, e.g. tight junctions, or at other levels of the intestine by lipid peroxidation or by toxic metabolites released from activated polymorphonuclear neutrophils. Our experimental results indicated that chlorella could significantly elevate the ratio of reduced-to-oxidized glutathione (GSH/GSSG) in bile duct-ligated rats, as well as decrease the levels of tissue MDA. Moreover, the results from pathological scoring also showed that pathological changes in rats in chlorella-treated group on 10 days were improved, suggesting that chlorella is capable of improving the body’s ability to remove oxygen free radicals, mitigate membrane lipid peroxidation, and thereby effectively protect intestinal epithelial cells. Considering this, bacterial overgrowth itself cannot be the principle mechanism responsible for BT in bile duct-ligated animals. In this study, significant BT, intestinal mucosal lipid peroxidation, and mucosal damage have been found in small intestine of bile duct-ligated rats. But administration of Chlorella, a green single-celled microalgae, significantly reduced BT, intestinal mucosal lipid peroxidation. In addition, the intestinal oxidative stress status observed in experimental obstructive jaundice was significantly ameliorated by chlorella administration. Conflict of interest All authors have made disclose any financial and personal relationships with other people or organisations that could inappropriately influence (bias) their work. Examples of potential conflicts of interest include employment, consultancies, stock ownership, honoraria, paid expert testimony, patent applications/ registrations, and grants or other funding. References 1. Deitch EA. The role of intestinal barrier failure and bacterial translocation in the development of systemic infection and multiple organ failure. Arch Surg 1990;125:403–4. 2. Berg RD, Garlington AW. Translocation of certain indigenous bacteria from the gastrointestinal tract to the mesenteric lymph nodes and other organs in a gnotobiotic mouse model. Infect Immun 1979;23:403–11.

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