Zinc deficiency correlates to spontaneous bacterial peritonitis in patients with cirrhotic ascites

Background Zinc deficiency in patients with liver cirrhosis has been frequently studied. Although spontaneous bacterial peritonitis (SBP) is one of the most serious complications of liver cirrhosis, few studies addressed the relationship between zinc deficiency and SBP. Aim This study aims to detect the effect of zinc deficiency in the development of SBP in patients with liver cirrhosis. Patients and methods The current study included 306 patients with liver cirrhosis and ascites who underwent diagnostic paracentesis for exclusion of SBP during admission to the Gastroenterology and Hepatology Unit, Ain Shams University Hospital, Egypt. Of these, 79 patients diagnosed with SBP by means of ascitic fluid polymorph nuclear leukocytic count more than or equal to 250 cells/mm3 were assigned to group 1. The remaining 227 patients with cirrhotic ascites were assigned to group 2. Routine laboratory tests, in addition to, serum zinc concentrations were assessed in all patients. Results Patients with SBP showed lower serum zinc in comparison to patients with cirrhotic ascites (49.11±11.84 vs. 79.27±9.58 μg/dl) with a statistically significant difference between the two study groups (P<0.001). Conclusion Zinc deficiency might be related to the development of SBP in patients with cirrhotic ascites.


Introduction
A variety of mechanisms result in necroinflammation and fibrogenesis of the liver tissue ending in cirrhosis. Cirrhosis is defined as diffuse nodular regeneration of hepatocytes with dense fibrous tissue septa, leading to hepatic parenchymal extinction and architectural collapse [1].
Regardless of the etiology of cirrhosis, individuals with chronic liver disease and cirrhosis are more prone to complications such as ascites, hepatic encephalopathy, variceal hemorrhage, and hepatocellular carcinoma [2].
Ascites is a hallmark of liver cirrhosis. It can be attributed to a series of pathophysiological events including portal hypertension and progressive vascular dysfunction. The presence of ascites is associated with a high 3-year mortality rate. Following the development of ascites, there is an increased risk of certain complications including hyponatremia, spontaneous bacterial peritonitis (SBP), and progressive renal impairment [3].
SBP is a frequent complication of ascites in patients with liver cirrhosis. It is defined as an infection of ascitic fluid that cannot be attributed to any abdominal inflammation or surgically correctable condition where the polymorph nuclear leukocytic count in ascetic fluid is more than or equal to 250 cells/mm 3 [4]. SBP needs prompt recognition and treatment. SBP has an estimated prevalence of 1.5-3.5% in outpatients and 10-30% in hospitalized cirrhotic patients [5].
Typically, Gram-negative bacteria are the main causative organisms of SBP in patients with cirrhotic ascites; however, there is a constant rising number of SBP cases due to Gram-positive bacteria [6].
Zinc as an essential micronutrient is needed for the development and integrity of monocytes and macrophages as well as regulation of their functions such as phagocytosis and proinflammatory cytokine production. Zinc deficiency was reported in many disorders like chronic liver disease, chronic alcoholism, malabsorption syndrome, chronic renal disease, and other chronic diseases including malignancy [7].
Zinc homeostasis is mandatory for many catalytic, regulatory and defensive mechanisms. It was noticed to be disturbed in patients with chronic liver disease [8].
Zinc plays a vital role in the early response and recovery after acute illnesses or major surgical procedures. During acute stress, zinc is transferred from the circulation to specific tissues such as the liver and the spleen with increased turnover in those organs, and increased disposal in the urine and injured tissues. Severe zinc deficiency is manifested by delayed recovery and increased mortality following major surgery, peritonitis, and sepsis [9].
SBP is a known predictor of mortality in patients with liver cirrhosis and ascites. Zinc deficiency has also been linked, in few studies, to an increased risk of developing SBP [10].

Patients and methods
The current study included 306 patients with liver cirrhosis and ascites who underwent ascitic fluid sampling for the exclusion of SBP during admission to the Gastroenterology and Hepatology Unit, Ain Shams University Hospital, Egypt, from March 2017 to March 2018. Of these, 79 patients were diagnosed with SBP by means of ascitic fluid polymorph nuclear leukocytic count more than or equal to 250 cells/mm 3 and were assigned to group 1. The remaining 227 patients with cirrhotic ascites were assigned to group 2.
The inclusion criteria were liver cirrhosis and age over 18 years. Patients with less than 18 years, secondary bacterial peritonitis, sepsis, hepatocellular carcinoma, as well as all patients with local causes of ascites such as tuberculosis or malignancy were excluded from the start.
The following information was gathered from the patients' medical records on the date of the diagnosis: age, sex, alcohol intake, history of diabetes, hypertension, cardiovascular disorders, viral hepatitis, cirrhosis, hepatic encephalopathy, hematemesis, SBP, chronic kidney disease, nonalcoholic steatohepatitis, primary biliary cirrhosis, hepatic focal lesions, as well as a full review of all past medical records including previous admissions and diagnoses and previous laboratory and imaging studies.
Laboratory investigations included liver function tests (serum aspartate aminotransferase, alanine aminotransferase, total and direct bilirubin, total protein, albumin, alkaline phosphatase, and alphafetoprotein), renal function tests (serum sodium, potassium, creatinine, and blood urea nitrogen), coagulation profile (prothrombin time, partial thromboplastin time, and international normalized ratio), complete blood count, and inflammatory markers (C-reactive protein) by standard laboratory tests. Serum zinc concentration was measured using the colorimetric end point method using zinc assay kit.
Diagnostic paracentesis was performed to obtain 15 ml of ascitic fluid under complete aseptic conditions; 10 ml were immediately inoculated in blood culture bottles. The rest was sent for chemical and cytological examination in tubes containing EDTA and was analyzed within 3 h of aspiration. Ascitic fluid protein was measured by spectrophotometry. Ascitic neutrophil count was detected by microscopic examination after centrifugation.
All patients signed a written informed consent prior to inclusion into this study. The study protocol conforms to the ethical guidelines of the 1975 Declaration of Helsinki (6th revision, 2008) as reflected in a prior approval by the institution's ethics committee.

Statistical analysis
Statistical analysis of the present study was conducted using statistical package for the social sciences, version 23 (IBM SPSS Statistics for Windows, Version 23.0, Armonk, NY: IBM Corp, USA). The normality of the distribution was checked by Shapiro-Wilk test. Numerical variables were expressed as mean, SD, median, and interquartile range. For comparison between two groups, t test was used for parametric data, while Mann-Whitney test was used for nonparametric data. The cutoff point with the highest sensitivity and specificity rates was chosen using the receiver operating characteristic (ROC) curve. A P value less than 0.05 was considered statistically significant.

Results
This cross-sectional study was conducted between March 2017 and March 2018 in the Gastroenterology and Hepatology Unit, Internal Medicine Department, Ain Shams University, Egypt. The study included 306 patients with liver cirrhosis who were selected from the Ain Shams University Hospital and were assigned to two groups. Seventy-nine patients, who were diagnosed with SBP, were assigned to group 1. The remaining 227 patients, who were diagnosed with cirrhotic ascites, were assigned to group 2.
Group 1 included 52 (65.8%) male patients and 27 (34.2%) female patients, while group 2 included 149 (65.6%) male patients and 78 (34.4%) female patients. There was no statistically significant sex difference between the two groups (P=0.546). The mean age for group 1 patients was 55.89±6.7, while the mean age for group 2 patients was 55.5±7.5 with no statistically significant age difference between the two groups (P=0.174). Table 1 shows univariate analysis of data from patients with SBP (group 1) and patients with cirrhotic ascites (group 2), as well as a comparison between laboratory parameters and ascitic fluid parameters between the the two groups.
In group 1, cultures of ascetic fluid samples were positive for Escherichia coli in nine patients (11.4%), Staphylococcus spp. in nine (11.4%) patients, and Klebsiella spp. in six (7.6%) patients. The rest of group 1 patients were culture negative (69.6%).
Patients with SBP showed insignificantly lower zinc/ serum ascites albumin gradient ratio than patients with cirrhotic ascites (36.8±30.9 vs. 38.9±8 with P=0.55). Eta-squared value was calculated using serum zinc as an independent variable for comparing patients with cirrhotic ascites to those with SBP aiming to estimate the effect size of serum zinc concentration on the development of SBP. The Eta-squared value was 0.80 which indicates that serum zinc concentration strongly correlates to SBP. Serum zinc concentration accounts for 80% of variance between patients with cirrhotic ascites and patients with SBP.
The ROC curve was obtained to determine the sensitivity and specificity of serum zinc concentration in differentiating SBP patients from those with cirrhotic ascites, as well as in determining the best cutoff value at which serum zinc levels could be used for such a purpose. Serum zinc concentration below 70.5 μg/dl predicted SBP in patients with cirrhotic ascites with a sensitivity of 94.9% and specificity of 84.1% (Fig. 1).

Discussion
Zinc plays an important role in the immune system serving as an enzyme cofactor in a number of cellular and metabolic processes. This micronutrient has a role in oxidative stress and in anti-inflammatory effects [11]. SBP prophylaxis has become an important issue in the era of the increasing resistance to antibiotics. If zinc deficiency proves to be an important predictor of SBP, zinc supplementation in patients with cirrhotic ascites might alter the risk of infection and decrease the need for unnecessary antibiotic administration. This might, then, significantly reduce morbidity and mortality in such a risky population.
The hallmark of the current study is that low serum zinc concentration is linked to the development of SBP in patients with cirrhotic ascites. The prevalence of SBP in the current study was 25.82%. According to the  Sengupta and colleagues concluded that serum zinc concentrations were inversely correlated with ascites and SBP. Low serum zinc leads to poorer clinical outcomes and shorter transplant-free survival. Zinc was considered to be an independent predictor of SBP in patients with cirrhotic ascites. They also stated that zinc might serve as a useful diagnostic and prognostic marker in such patients [11].Zinc deficiency in patients with liver cirrhosis could be attributed to various mechanisms. Ascites causes mechanical compression on the stomach resulting in early satiety and malnutrition. In SBP, this process is enhanced by infection that leads to anorexia and decreased appetite. In addition, liver cirrhosis is associated with limited intestinal absorption and limited portal venous extraction of zinc [12]. Finally, increased portosystemic shunting is associated with higher zinc excretion and thus increased urinary loss [13]. Previous studies addressed the possible effect of zinc on immunodeficiency states such as T-cell defects. Zinc appears to influence both lymphocyte and neutrophil function. Thus, zinc deficiency remains an important suspect is cases of SBP [14].

Conclusion
Zinc deficiency might be strongly correlated to the development of SBP in patients with cirrhotic ascites. Zinc supplementation might play an important role in prophylaxis against SBP.

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Conflicts of interest
There are no conflicts of interest.