Increased Intestinal Permeability in Malnourished Patients with Liver Cirrhosis

ORIGINAL ARTICLE Increased intestinal permeability in malnourished patients with liver cirrhosis

K Norman1, M Pirlich1,2, J-D Schulzke1, C Smoliner1, H Lochs1, L Valentini1 and S Bu¨ hner1,3

BACKGROUND/OBJECTIVE: Malnutrition is a prominent feature in liver cirrhosis, with deleterious impact on clinical outcome. The objective of this study is to investigate whether malnutrition is associated with increased gastrointestinal permeability in liver cirrhosis reflected by altered urinary excretion of non-metabolizable sugar probes. SUBJECTS/METHODS: Patients with advanced liver cirrhosis (Child Pugh Score B or C) were recruited. Nutritional status was determined according to the Subjective Global Assessment. Intestinal permeability was assessed by measuring the urinary excretion of orally administered, non-metabolized sugar probe molecules. The lactulose/mannitol ratio served as marker for intestinal permeability and reflects non-carrier-mediated transcellular and paracellular transport of the small intestine during the first 5 h. Sucrose recovery in urine within the first 5 h reflects gastroduodenal permeability; sucralose recovery in urine 5–26 h after consumption reflects colonic permeability. RESULTS: Sixty-four patients (56.7±10.8 years; 33% female) were included in the study. Twenty-one patients were considered well nourished according to the Subjective Global Assessment, 23 moderately nourished and 20 patients severely malnourished; 74% had alcoholic liver disease and 67% had cirrhosis stage Child C. Gastroduodenal and colonic permeability was significantly increased in patients with liver cirrhosis compared with 63 healthy controls (0.23±0.22 and 1.37±1.42% vs 0.14±0.10 and 0.41±0.72% in controls), but not different between well and malnourished subjects. Small intestinal permeability (lactulose/ mannitol ratio) was increased in all patients (0.069±0.055%) and further increased in malnourished patients (0.048±0.031% vs 0.084±0.061%, P ¼ 0.004) due to decreased mannitol recovery only. CONCLUSIONS: Gastric, small intestinal and even colonic permeability was altogether increased in liver cirrhosis, and malnutrition was associated with further increased small intestinal permeability indicative of villous atrophy.

European Journal of Clinical Nutrition (2012) 66, 1116–1119; doi:10.1038/ejcn.2012.104; published online 8 August 2012 Keywords: liver cirrhosis; malnutrition; intestinal permeability; lactulose/mannitol ratio; sugar probe test

INTRODUCTION The lactulose/mannitol ratio (permeability index) is a marker for Malnutrition is a prominent feature in liver cirrhosis,1,2 with intestinal permeability corrected for differences in variable gastric deleterious impact on morbidity and mortality as the degree of emptying, intestinal transit time during the first 5 h and renal 14,15 nutritional impairment correlates inversely with survival.3,4 clearance. Sucralose reflects colonic permeability 5–26 h after Increased intestinal permeability occurs frequently in patients consumption. with liver cirrhosis, particularly in patients with ascites,5 and is Our aim was to investigate whether malnutrition is associated related to the progression of liver disease.6 It has been hypothe- with increased permeability as reflected by altered urinary sized to be not only a triggering factor in the pathogenesis excretion of non-metabolizable sugar probes in patients with of chronic liver disease6,7 but also in the development of liver cirrhosis. cachexia.8 Altered intestinal permeability and subsequent endotoxemia9 have been implicated in increased cytokine production and inflammatory processes observed in liver cirrhosis, PATIENTS AND METHODS which might subsequently trigger onset and progression of Patients cachexia.10 Antibodies to food proteins have been shown Patients admitted at the Department of Gastroenterology between 2003 in malnourished patients, which implies compromised gut and 2006 were considered for inclusion. They were recruited if they were barrier function.11 older than 18 years and had advanced liver cirrhosis, Child B or C, confirmed by ultrasound and/or biopsy. Main exclusion criteria were Measurement of urinary excretion of orally administered, non- 12 malignant comorbidity, renal insufficiency, coeliac disease or chronic metabolizable sugar probe molecules is one established method inflammatory bowel disease. All study participants gave written informed to test gastrointestinal permeability. Sucrose is considered a consent, and the Ethics Committee of our university clinic approved marker for the gastroduodenal permeability during the first 5 h.13 the study.

1Medizinische Klinik fu¨r Gastroenterologie, Infektiologie und Rheumatologie (einschl. Arbeitsbereich Erna¨hrungsmedizin), Charite´ Universita¨tsmedizin Berlin, Campus Charite´ Mitte, Berlin, Germany; 2Abteilung fu¨r Innere Medizin, Evangelische Elisabeth Klinik, Berlin, Germany and 3Lehrstuhl fu¨r Humanbiologie, Technische Universita¨tMu¨nchen, Mu¨nchen, Germany. Correspondence: Dr K Norman, Klinik fu¨r Gastroenterologie, Infektiologie und Rheumatologie, Sektion Erna¨hrungsmedizin, Charite´ – Universita¨tsmedizin Berlin, 10117 Berlin, Germany. E-mail: [email protected] Contributors: KN and SB conceived the study and performed the analysis; KN, MP and CS recruited the patients; KN, MP and SB wrote the manuscript; JS, LV and HL contributed significant advice. Received 9 March 2012; revised 10 July 2012; accepted 11 July 2012; published online 8 August 2012 Increased intestinal permeability in malnutrition K Norman et al 1117 Data from 63 healthy controls (mean age 42±14.8 years; 68% female) Laboratory parameters recruited for internal reference values were used for comparison. Controls All laboratory parameters were determined with standard measurements. had to be free of gastrointestinal or hepatobiliary disease, severe neurological, endocrine, cardiovascular, pulmonary, or renal disease, gastrectomy, colectomy, or extensive resection of the small bowel, cancer, Statistics rheumatoid-based diseases, acute infections, acute urticaria, or pregnancy. Statistical analysis was carried out using the software package PASW Drug intake, including glucocorticosteroids, antimicrobial agents, immu- (version 18 SPSS Inc., Chicago, IL, USA). All data are presented as mean and nosuppressives and regular consumption of non-steroidal anti-inflamma- s.d. if not indicated otherwise. Multiple comparisons between more than tory drugs (for example, for arthropathies) were not allowed. Substances two groups were conducted using one-way between-groups analysis of influencing gut functions, including laxatives and anti-diarrhoeal agents, variance. Student’s t-test was then used for comparison between patients were not allowed for 24 h before the test. Smoking was strictly forbidden in and controls, malnourished and well nourished patients, as well as the morning before the test and during the test. Controls were also between Child Pugh Classes B and C. One sided t-tests were used to test asked to record alcohol and cigarette consumption and medicine intake sugar recovery values against published cut-off values. Spearman’s for 1 week before the test. correlation was calculated to assess the relationship between variables; w2-tests were used to analyse percentages. An acceptable level of statistical significance was established a priori Nutritional status at Po 0.05. The Subjective Global Assessment was carried out using the protocol developed by Detsky et al.16 to asses non-oedematous weight loss and nutritional status. On the basis of nutritional and weight history, and RESULTS on physical examination, patients were classified as well nourished (A), We recruited 64 patients (mean age 56.7±10.8 years; 33% female) moderately malnourished (B) or severely malnourished (C). admitted to the Department of Gastroenterology at the Charite´ University Hospital (Table 1). The main aetiology of cirrhosis was alcohol abuse (74%). Twenty-one patients were considered well Intestinal permeability nourished according to the Subjective Global Assessment, 23 Intestinal permeability was assessed using a sugar-drink test as previously moderately nourished and 20 patients severely malnourished. 14 described in detail. After an overnight fast, each subject drank a solution There were no differences between well-nourished and malnour- containing 10 g lactulose, 5 g mannitol and 20 g sucrose dissolved in ished patients regarding liver enzymes or prothrombin time. 100 ml of water, and received six pills containing 5 g sucralose. Subjects Albumin and cholinesterase were, however, significantly reduced were in a fasting state during the first 5 h and were encouraged to drink water 2 h after test start. Three urine samples were taken: a pre-test and C-reactive protein was significantly increased in malnourished sample, one collection sample during the first 5 h after test start and one patients (Table 1). collection sample of the following period until 26 h after consumption. Alcohol or non-steroidal anti-inflammatory drugs intake was not allowed within 24 h before the test. Intestinal permeability Sodium acid was used as a preservative of urine. Total urine volume was Sucrose, indicating gastric as well as duodenal permeability, and recorded on completion of the test and a 10-ml aliquot of each urine sucralose, reflecting colonic permeability were significantly higher sample was stored at 20 1C. For urine analysis, protein content was in patients than healthy controls, but were not significantly removed using sulphosalicylic acid. Urine was then desalted with different between well- and malnourished patients (Table 1). amberlite-mixed bed-3 resin. Sugars were separated using meso-erythritol A correlation between C-reactive protein and sucralose excretion and turanose as internal standards, analysed and quantified by was observed (r ¼ 0.418, P ¼ 0.013). When comparing sucralose high-performance liquid chromatography with pulsed electrochemical 17 detection (chromatography module: 250, Dionex, Idstein, Germany); excretion to suggested reference values, mean excretion was 40 mm Carbopac PA-1 column (Dionex; eluent 150 mmol NaOH) at a also higher than the mean excretion in healthy controls, but lower flow of 1 ml/min. Results were expressed as percentage recovery of the than the proposed cut-off value of 2.11%, which represents the ingested dose. 90th percentile.

Table 1. Clinical characteristics and intestinal permeability in the study population

Parameter Healthy All liver P-value Wellnourished Malnourished P-value Controls cirrhosis (controls vs (SGA A) (SGA B þ C) (SGA A vs patients patients) SGA B þ C)

Number (m/f) 63 (20/43) 64 (43/21) o0.0001a 21 (13/8) 43 (30/13) n.s. Age (years) 42±14.8 56.7±10.8 o0.0001 53.1±12.6 58.2±9.9 n.s. Alcoholic liver disease (%) 74% 57% 81% n.s. Child Score C (%) 76% 47% 76% n.s. Presence of ascites (%) 78% 53% 88% 0.03 Albumin (mg/dl) 3.0±0.6 3.3±0.7 2.8±0.5 0.015 C-reactive protein (mg/dl) 2.72±2.17 1.55±1.50 3.20±2.25 0.007 Cholinesterase (kU/l) 2.5±1.34 3.30±1.91 2.23±1.00 0.023 Small intestinal permeability 0.018±0.007 0.069±0.055 o0.0001 0.048±0.031 0.084±0.061 0.004 (lactulose/mannitol ratio) Lactulose 0.29±0.15 0.54±0.52 o0.0001 0.53±0.45 0.58±0.57 n.s. (% urine recovery) Mannitol (% urine recovery) 15.71±6.00 8.91±5.2 o0.0001 11.69±7.19 7.72±4.53 0.044 Gastroduodenal permeability 0.14±0.10 0.23±0.22 0.002 0.22±0.16 0.25±0.26 n.s. (% urine recovery of sucrose) Colonic permeability 0.41±0.72 1.37±1.42 o0.0001 0.96±0.77 1.56±1.61 n.s. (% urine recovery of sucralose) Abbreviations: SGA, Subjective Global Assessment; n.s., not signiﬁcant. aw2-test.

& 2012 Macmillan Publishers Limited European Journal of Clinical Nutrition (2012) 1116 – 1119 Increased intestinal permeability in malnutrition K Norman et al 1118 P<0.004 lactulose excretion therefore implies pathological paracellular gut 0.35 permeability. In our study population, lactulose excretion was P<0.0001 significantly increased in all patients and further increased in 0.30 advanced disease (Child C), but not significantly different between well-nourished and malnourished patients. 0.25 The permeating pathway of mannitol, a monosaccharide alcohol, is however not yet perfectly understood. In vivo recovery 0.20 of mannitol is about 50 times that of lactulose, which suggests transcellular permeation.12,18 Reduced mannitol excretion 0.15 indicates villous atrophy.19–23 In alcoholic liver disease, villous atrophy is frequently observed; reduction in villous height in 0.10 relation to crypt depth and lower mucosal surface have been detected by light microscopy in alcoholics, even in the absence of (lactulose/mannitol ratio) 24–26 Small intestinal permeability 0.05 cirrhosis. Following the introduction of capsule endoscopy, mucosal abnormalities of the small intestine in liver cirrhosis 0.00 patients with portal hypertension have been well documented Controls Patients with liver cirrhosis and include inflammatory-like abnormalities (oedema, erythema, SGA A SGA B & C granularity and friability) as well as vascular lesions (cherry red wellnourished malnourished spots, telangiectasias or angiodysplasia-like lesions and varices).27 Figure 1. Small intestinal permeability (lactulose/mannitol ratio) in The reduced recovery of mannitol in all our study patients would be consistent with these findings. patients with liver cirrhosis, stratified according to nutritional state 28 and compared with controls. Data presented as boxplots displaying Elia et al. observed decreased urinary excretion of orally minimum, maximum, and the 25th, 50th and 75th percentiles. administered mannitol in short-term starvation and concluded that it probably reflects changes in small intestinal mucosa during early starvation. Periods of starvation or reduced nutritional intake are common in liver cirrhosis,29,30 particularly in alcohol-related liver disease, but the aetiology is still not completely clear.8 These Small intestinal permeability, however, reflected by the periods have been related to anorexia,29 impaired gastric lactulose/mannitol ratio (permeability index) was significantly accommodation in alcoholic liver disease,31,32 reduced gastric elevated in all patients and further increased in malnourished relaxation33 in portal hypertension and disturbed relation patients (Table 1 and Figure 1). The elevated permeability index in between gastric accommodation and energy intake in cirrhotic malnourished patients was due to significantly reduced mannitol patients.34 Interestingly, chronic caloric restriction alone excretion, whereas lactulose excretion was not significantly altered has moderate effect on intestinal structure and permeability. in malnutrition. One study in anorexia nervosa reported decreased permeability, No differences regarding lactulose/mannitol ratio, sucralose or and similarly in animal models, hypocaloric feeding had little or no sucrose were found between Child B and C class (data not shown), effect on intestinal structure.35 These findings indicate adaptation only lactulose excretion was significantly increased in Child C to long-term caloric restriction. (0.31±0.12 vs 0.68±0.60, Po0.01). Mannitol excretion was When malnutrition occurs in disease, however, several factors however significantly lower in patients with ascites (8.1±4.5 vs may further interact to adversely affect the structure and function 11.2±7.2, P ¼ 0.042) and lactulose recovery was higher (0.6±0.6 of the gut mucosa. Welsh et al.36 likewise observed increased vs 0.3±1.2, P ¼ 0.043), although the difference regarding lactulose/mannitol ratio in malnourished cancer patients with the lactulose/mannitol ratio was not significant (0.05±0.07 vs normal intestinal morphology, but did not report the excretion 0.08±0.04). rates of lactulose and mannitol, which hampers comparison. We used the SGA for the classification of malnutrition in the study patients, as it is a method based not only on weight change DISCUSSION but on clinical examination as well, which focuses on muscle and We investigated intestinal permeability in patients with liver fat mass, ascites and hydration. Relying solely on body mass index cirrhosis and found an increased lactulose/mannitol ratio, sucrose for the classification of nutritional status in liver cirrhosis patients and sucralose permeability in all patients compared with healthy is not possible, as ascites increases weight and masks weight loss controls, indicating altered gastric and small intestinal perme- and body mass index values are frequently increased. ability. Colonic permeability was increased compared with healthy When analysing intestinal permeability according to the subjects used as controls, which has hitherto not been investi- presence of ascites, we found significantly decreased mannitol gated in liver cirrhosis, but was not elevated above suggested recovery and increased lactulose recovery in patients with pathological values.17 Moreover, we were able to demonstrate ascites. As malnourished patients exhibited ascites significantly that lactulose/mannitol ratio was even further increased in more often, this may partly explain the finding of altered sugar malnourished patients compared with well-nourished patients, discovery in liver cirrhosis-related malnutrition. However, lactu- which is indicative of altered small intestinal barrier function or lose/mannitol ratio was not significantly different between morphology. Although it is known that intestinal permeability patients with or without ascites, which indicates that the might be generally impaired in patients with nutritional deficits, significantly increased lactulose/mannitol ratio in malnourished in our study population, the altered ratio was principally due patients at least partly stem from another cause. to decreased mannitol excretion, whereas lactulose excretion was Taken together, we have found increased gastric, small not significantly different between malnourished and well- intestinal and even increased colonic permeability in patients nourished patients. This has not been reported before and with liver cirrhosis. Advanced disease stage and inflammation was merits attention, since depending on the recovered sugar probe associated with higher tight junction permeability (lactulose diverse pathogenesis mechanisms are implied. excretion), which might facilitate bacterial translocation. Malnutri- Lactulose, a synthetic disaccharide, is interpreted as a tight tion on the other hand was associated with increased small junction marker, as due to its size, it is believed to permeate intestinal permeability, exclusively due to decreased mannitol exclusively through tight junctions of epithelial crypts. Increased excretion, which implies altered gut morphology.

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& 2012 Macmillan Publishers Limited European Journal of Clinical Nutrition (2012) 1116 – 1119