/n European Review for Medical and Pharmacological Sciences Effect of Vitamin D supplementation in patients with liver cirrhosis having spontaneous bacterial peritonitis: a randomized controlled study A.A. MOHAMED¹, A.A. AL-KARMALAWY², A.A. EL-KHOLY³, D.A. EL-DAMAS+, H.M. ABOSTATE¹, S.M. MOSTAFA5, M. HAMADA, M.A. KHALIK ELKADY', Y. HASSAN, E. AL-HUSSAIN³, M.G. KHALIL', I. BADAWY¹0, D. ELEBEEDY¹⁰, B.A. ALSFOUK¹¹, M.M. SHAHEENL² 'Biochemistry, Molecular Biology and Biochemistry Department, National Hepatology and Tropical Medicine Research Institute, Egypt ²Department of Pharmaceutical Medicinal Chemistry, Faculty of Pharmacy, Horus University-Egypt, New Damietta, Egypt ³Clinical Pharmacy Department, Faculty of Pharmacy, Ain Shams University, Cairo, Egypt 4Microbiology, Faculty of Pharmacy, Egyptian Russian University, Cairo, Egypt 5Department of Hepatology, National Hepatology and Tropical Medicine Research Institute (NHTMRI), Cairo, Egypt 'Internal Medicine Department, Faculty of Medicine, Benha University, Cairo, Egypt 2021; 25: 6908-6919 7Gastroenterology and Hepatology Department, Theodor Bilharz Research Institute, Egypt Clinical Pathology Department, Faculty of Medicine, Faculty of Medicine, Cairo University, Cairo, Egypt 'Toxicology Department, Faculty of Pharmacy, Modern University for Technology and Information (MTI), Cairo, Egypt ¹ºPharmaceutical Biotechnology Department, Faculty of Biotechnology, Misr University for Sciences and Technology, Cairo, Egypt ¹¹Department of Pharmaceutical Sciences, College of Pharmacy, Princess Nourah bint Abdulrahman University, Riyadh, Saudi Arabia ¹²Internal Medicine and Nephrology Department, Cairo University, Cairo, Egypt Amal Ahmed Mohamed and Ahmed A. Al-Karmalawy contributed equally to this work Abstract. OBJECTIVE: Spontaneous Bac- terial Peritonitis (SBP) is one of the most seri- ous liver cirrhosis with ascites complications. Vitamin D (Vit D) deficiency has been associat- ed with a high risk of infection and mortality in cirrhotic patients. Herein, the assessment of Vit D level as a prognostic marker in SBP patients and the impact of Vit D supplementation on their treatment plan was studied as well. PATIENTS AND METHODS: Ascetic patients with SBP and Vit D deficiency were divided ran- domly into treatment and control groups. The control group received standard treatment with- out Vit D and the treatment group received stan- dard treatment plus Vit D. Clinical monitoring of Vit D was done over 6 months. RESULTS: At baseline, all patients in both groups revealed an elevated serum and ascetic TLC, AST, ALT, total and direct bilirubin, in addi- tion to elevation in INR and procalcitonin (PCT) level. Univariate regression analysis confirmed that deficiency of Vit D was an independent pre- 6908 dictor of infection and mortality (p < 0.01; Crude Hazard Ratio: 0.951). Over 6 months, the study revealed significant improvement in serum Vit D level in the treatment group (34.6 ± 9.2 and 18.3 ± 10.0 ng/mL; p < 0.001). Moreover, a statistical- ly significant increase in survival rate (64% vs. 42%; p<0.05) and duration (199.5 days vs. 185.5 days; p<0.05) were recorded as well. Univariate and multivariate regression analysis confirmed that Vit D supplementation was positively cor- related to survival over 6 months (p < 0.001; Ad- justed Hazard Ratio: 0.895). CONCLUSIONS: Vit D deficiency is prevalent in SBP cirrhotic patients and is used as an inde- pendent predictor of infection and death. There- fore, Vit D supplementation revealed improve- ment in their response to treatment. Key Words: Ascites, Spontaneous bacterial peritonitis, Vitamin D deficiency, Survival. Corresponding Author: Ahmed A. Al-Karmalawy, MD; e-mail: akarmalawy@horus.edu.eg Vit D supplementation in patients with liver cirrhosis and SBP Introduction Patients with liver cirrhosis had spontaneous bacterial peritonitis (SBP) infection that showed poor prognosis¹-³. Cirrhosis is the terminal stage of any chronic liver injury which accounts for the fourth major cause of mortality in central Eu- rope¹-6. Egypt has the highest age-standardized mortality rate of 72.7 deaths for every 100,000 patients. Approximately one-fifth (18.1%) of most deaths in males of age ranged from 45 to 54 years were caused by liver cirrhosis7-10. Cirrhosis manifests with the presence of vascular fibrosis and distortion resulting in increased hepatic re- sistance, which may cause portal hypertension if there was splanchnic hyperperfusion¹¹-13. Com- plications like variceal bleeding and renal dys- function may be due to portal hypertension¹4-16 Renal dysfunction and splanchnic vasodilation contribute to the development of ascites¹7-19. As- cites are frequently complicated by SBP20,2¹, most commonly due to intestinal bacterial transloca- tion, which can trigger renal failure and is associ- ated with increased mortality. New inflammation biomarkers are needed in clinical practice for the early diagnosis of SBP, such as procalcitonin (PCT)22,23. Meanwhile, the pathophysiology of SBP is not understood, since translocation of bac- teria from the gastrointestinal tract to the ascetic fluid (AF) is thought to be a crucial mechanism behind the development of SBP. Also, SBP is encouraged by impaired defensive mechanisms in cirrhotic patients. It is diagnosed by counting polymorphonuclear (PMN) cell numbers in AF of more than or equal to 250 cells/mm³, where the infectious organism is isolated in about 70% of cases24,25 Procalcitonin (PCT) is a polypeptide precursor for calcitonin synthesized in the thyroid C cells. Microbial toxins and pro-inflammatory media- tors, such as IL-1B, IL-6, and TNF-a, triggered PCT release26-28. In decompensated cirrhosis, se- rum and ascetic fluid PCT were examined and we found they were not effective as a marker in the detection of acute bacterial infection (such as SBP) compared to patients without hepatic impairment ²9-32. PCT, however, appears to be a strong marker for bacterial translocation, a well-established mechanism for decompensat- ed cirrhosis infection that can lead to bacterial infection 33,34 Combining procalcitonin (PCT), the difference in hemoglobin concentration be- tween newly formed and mature red blood cells (dCHC) and mean fluorescence intensity of ma- ture neutrophils (sNFI) in patients with cirrhosis is a valuable predictive composite marker for early detection of ascites infection ³5. The acute stage reactant proteins, such as C-reactive pro- tein (CRP) and procalcitonin (PCT), have been assessed in many clinical situations as indicators for early diagnosis of SBP. CRP and PCT are well known to rise rapidly in response to bacterial infection 36,37. So, they are used as sensitive pre- dictive and monitoring parameters for bacterial infections and controlling the use of antibiotics. In addition, cirrhotic patients usually have Vit D deficiency 38,39 The liver is a crucial organ for Vit D bio- transformation, where it is the only organ that generates the (25-OH) Vit-D3 from Vit D40,41 Low hepatocytes, reduced exposure to sunlight, low adipose tissue, malabsorption of Vit D, and altered hydroxylation of Vit D in the liver are the normal reasons for the decreased levels of Vit D in patients with cirrhosis 42. It was found that low Vit D in the ascetic fluid is related to the higher risk of AF bacterial infection in cirrhotic patients with ascites 43,44 There are few studies available concerning the impact of Vit D supplementation on people with Vit D deficiencies, particularly those with decom- pensated cirrhosis 45,46. The effect of Vit D sup- plementation on mortality rate in those patients is still unclear. The main purpose of the present work is to assess Vit D and procalcitonin levels as prognostic markers in SBP patients, besides, evaluating the effect of Vit D administration on the response of SBP patients for treatment from liver cirrhosis. Patients and Methods Study Design and Setting This study was a prospective randomized con- trolled study that included 328 ascetic patients with spontaneous bacterial peritonitis (SBP) who were admitted to the National Hepatology and Tropical Medicine Research Institute from June 2019 to December 2019. Subject Selection The inclusion criteria were: patients with liv- er cirrhosis, ascites with SBP, and aged over 18 years. The excluded patients from the study were: those with non-peritoneal infections (septicemia, skin infection, chest infection, biliary tract in- fection, urinary tract infection, gastroenteritis, 6909 A.A. Mohamed, A.A. Al-Karmalawy, A.A. El-Kholy, D.A. El-Damas, et al. dental infection, meningitis), administration of non-absorbable antibiotics in the prior 6 weeks, infection with HIV, hepatoma or other forms of malignancy, hepatorenal syndrome on admis- sion, significant cardiac and respiratory disease, pregnancy, patients prepared for transplantation, and signs of gastrointestinal bleeding or bacterial infection in the prior 6 weeks. Clinical Evaluation Detailed patient's clinical history and physi- cal examination were performed. Patients were screened for complete blood count (using Phoe- nix NCC-2310 Hematology Analyzer Device), random blood sugar, liver profile [aspartate ami- notransferase (AST), alanine aminotransferase (ALT), direct bilirubin, total serum bilirubin (TSB), alkaline phosphatase (ALP), serum al- bumin by OLYMPUS biochemistry autoanalyz- er (USA), coagulation profile (prothrombin time (PT), and international normalized ratio (INR)] by kcl delta, renal function tests (urea and serum creatinine), viral markers (HBsAg; hepatitis C vi- rus antibodies (HCV Abs) by ELISA, alpha-feto- protein (AFP) by Eliza according to manufactur- ing instructions. Values were considered normal if > 10 ng/ml. In addition, abdominal ultrasonog- raphy was done for grading of ascites. All stud- ied patients underwent diagnostic paracentesis to obtain AF samples for total leucocyte count (TLC) (polymorph nuclear leucocytes equal or more than 250 cells/mm³ considered as sponta- neous bacterial peritonitis), malignant cells, total protein, albumin, and glucose. Serum Procalcitonin and Vit D Measurements Serum procalcitonin (PCT) was measured us- ing the PCT enzyme-linked immunosorbent as- say kit for the in vitro assessment of serum PCT (Chongqing Biospes Co. Ltd, Chongqing City, China; biopsies). The Vit D level was also mea- sured by ELISA (by direct competitive chemi- luminescence immunoassay using commercially available kit Unicel® DxI 800 immunoassay sys- tems (Beckman Coulter, Inc., CA, USA) within 24 hours of admission. Patient with serum Vit D concentration < 25 ng/mL was considered as Vit D deficient or insufficient. Medication Exposure The total number of patients who participated in this study with Vit D deficiency was 328 pa- tients, randomly divided into control and treat- 6910 ment groups. The control group included patients who received standard treatment without Vit D (n=168). Whereas the treatment group included patients who received standard treatment plus Vit D (n=160). Patients in the treatment group received a dose of 300000 IU of cholecalciferol as a loading dose by intramuscular injection, fol- lowed by a maintenance dose of 800 IU/d orally with oral calcium supplementation in a dose of 1000 mg. Monitoring of Vit D, serum calcium, and phosphorus was measured every month for 6 months or until death, whichever happened first. Administration of Vit D was discontinued if signs of hypercalcemia, renal stone, or Vit D level above 80 nmol/L (> 32 ng/mL) were detected. Ethical Considerations Informed consent was received from all partic- ipants included in the study. The study protocol complied with the Ethical Guidelines set out in the Declaration of Helsinki (sixth revision, 2008) and Good Clinical Practice guidelines. The National Hepatology and Tropical Medicine Research Institute Committee approved the study (Serial: 5-2020). Statistical Analysis The data were analyzed using a statistical package for the social sciences, version 23 (IBM SPSS, Statistics for Windows, Version 23.0, IBM Corp, Armonk, NY, USA). Numerical variables were expressed as mean±SD (standard devia- tion) and median (range). Comparison between treatment and control groups was conducted as follows: for parametric data, a two-sample t-test was used while for the nonparametric data, the Mann-Whitney test was used. For analysis of survival, the log-rank test was used for binary variables while continuous variables were ana- lyzed using the Cox-proportional hazard method. A p-value below 0.05 was considered statistically significant. Results Demographic Data Out of 328 patients, 160 patients received stan- dard SBP therapy plus Vit D (treatment group) and 168 patients received only SBP standard therapy (control group). The age was expressed as mean±SD in the treatment group (51.4± 12.1), and in the control group (55.0+ 8.5) with a ratio of male to female 85:75 and 81:86, respectively. Vit D supplementation in patients with liver cirrhosis and SBP Regarding the baseline characteristics, no sig- nificant difference (p>0.05) was observed be- tween treatment and control groups, except sig- nificantly higher smoking (p 0.05), except for HBsAg positive and AST levels, which were significantly higher in the con- trol group than in the treatment group (p< 0.05) (Table 1). Treatment and control groups showed an elevation in PCT level (1.78± 0.49), (1.84+ 0.78) and deficiency of Vit D (22.5± 11.5), (20.2+ 11.0), respectively. For both groups, there was no significant difference in those values. Effect of Vit D Supplementation After 6 months of treatment and follow-up, most of the blood test parameters and ascetic fluid composition were significantly improved in both groups. However, the level of improvement Parameters Table I. Baseline characteristics, blood tests, and ascetic fluid composition of control and treatment groups. Control group (N = 168) Age in years (Mean ± SD) Gender (Male: Female) BMI (Kg/m²) (Mean ± SD) Smoking [No. (%)] Bleeding [No. (%)] Ascites [No.(%)] Encephalopathy [No.(%)] Fever [No.(%)] Jaundice [No.(%)] HBsAg positive [No.(%)] HCV Ab positive [No.(%)] Hb (g/dL) (Mean ± SD) Serum glucose (mg/dL) (Mean ± SD) Ascetic glucose (mg/dL) (Mean ± SD) Platelet ( 109/L) (Mean ± SD) Serum Creatinine (mg/dL) (Mean ± SD) Total bilirubin (mg/dL) (Mean ± SD) Direct bilirubin (mg/dL) (Mean ± SD) ALT (IU/L) (Mean ± SD) AST(IU/L) (Mean ± SD) Serum albumin (g/dL) (Mean ± SD) Ascetic albumin (g/dL) (Mean ± SD) INR (Mean ± SD) was significantly higher in the treatment group compared to the control group (p< 0.001) (Table II). The PCT, which was investigated as an early diagnostic marker for ascites infection, showed a highly significant reduction in the treatment group than the control group (p< 0.001) (Figure 1). Furthermore, patients in the control group demonstrated an average 9.4% decrease in the mean serum Vit D level (20.2 ng/mL vs 18.3 ng/ mL) at the end of 6 months. On the other hand, patients who received Vit D showed a 53.8% in- crease in mean serum Vit D level (22.5 ng/mL vs. 34.6 ng/mL). Also, a highly significant elevation of Vit D in the treatment group in comparison with the control group (p< 0.001) was observed (Table III) (Figure 2). AFP (ng/mL) [Median (Range)] GGT (IU/L) [Median (Range)] Blood TLC ( 10¾μµL) [Median (Range)] Ascetic TLC (x 103/µL) [Median (Range)] Procalcitonin (ng/mL) (Mean ± SD) Sr.Vitamin D level (ng/mL) (Mean ± SD) Survival Rate Analysis The survival rate was significantly higher in the treatment group (64%) compared to the con- trol group (42%) (p< 0.05). The mean surviv- 55.0+ 8.5 81:86 26.02.2 62 (37.1%) 35 (21%) 167 (100%) 36 (21.6%) 43 (25.7%) 35 (21%) 24 (14.4%) 13 (7.8%) 11.8 ± 2.0 104.2 + 8.5 47.3 ± 9.2 135.5 + 27.02 1.61 ± 0.49 3.87 ± 0.81 0.93 ± 0.40 89.0 ± 23.1 149.0 + 48.3 1.86 ± 0.30 0.73 +0.23 1.32 ± 0.31 7.9 (3.1-9.2) 81.73 (12-187) 11.43 (3.71-23.37) 600 (140-7000) 1.84 ± 0.78 20.2 ± 11.0 Treatment group (N = 160) 51.4 12.1 85:75 25.9 ± 1.9 65 (40.6%) 32 (20%) 160 (100%) 31 (19.4%) 38 (23.8%) 31 (19.4%) 19 (11.9%) 10 (6.3%) 12.0+ 1.5 101.6 15.0 45.0 10.2 138.0 + 71.1 1.56 ± 0.42 3.18±0.73 0.80 ± 0.49 83.1 ± 22.2 138.0 + 46.9 1.90 ± 0.37 0.70 ±0.29 1.29 ± 0.27 8.0 (2.0-8.0) 75.4 (3.4-176.0) 12.10 (4.71-21.73) 587 (120-4300) 1.78 ± 0.49 22.5 11.5 p-value 0.05 > 0.05 0.05 > 0.05 > 0.05 > 0.05 > 0.05 0.05 > 0.05 > 0.05 > 0.05 > 0.05 > 0.05 > 0.05 0.05 > 0.05 > 0.05 > 0.05 > 0.05 > 0.05 > 0.05 > 0.05 > 0.05 > 0.05 > 0.05 AFP, alpha-fetoprotein; ALT, alanine transaminase; AST, aspartate transaminase; BMI, body mass index; GGT, gamma-glutamyl transferase; Hb, Hemoglobin; HBsAg, Hepatitis B surface antigen; HCV Ab, Hepatitis C virus antibodies; INR, international normalized ratio; SBP, spontaneous bacterial peritonitis; TLC, total leukocytic count. 6911 Table II. Univariate analysis comparing the control and treatment groups regarding blood tests and ascetic fluid composition after 6 months. Platelet ( 10%/L) (Mean ± SD) Serum Creatinine (mg/dL) (Mean ± SD) Total bilirubin (mg/dL) (Mean ± SD) Direct bilirubin (mg/dL) (Mean ± SD) ALT (IU/L) (Mean ± SD) AST(IU/L) (Mean ± SD) Serum albumin (g/dL) (Mean ± SD) Ascetic albumin (g/dL) (Mean ± SD) INR (Mean ± SD) AFP (ng/mL) [Median (Range)] GGT (IU/L) [Median (Range)] Blood TLC (x 10/µL) [Median (Range)] Ascetic TLC ( 10³/µL) [Median (Range)] Procalcitonin (ng/mL) (Mean ± SD) Sr.Vitamin D level (ng/mL) (Mean ± SD) Survival at 6 months: ● • Ratio (alive:dead) (%) Survival (days) [Median (Range)] A.A. Mohamed, A.A. Al-Karmalawy, A.A. El-Kholy, D.A. El-Damas, et al. SERUM PROCALCITONIN (NG/ML) 2.5 Parameters 2 1.5 0.5 al duration was (199.5 days) in the treatment group vs. (185.5 days) in the control group (p< 0.05) (Table II). For detection of the different factors that were associated with the 6 months survival of the patients, univariate and multivar- iate analyses were used. The univariate analysis confirmed that higher TLC, higher creatinine, higher bilirubin level, and higher AST level were nonsignificant predictors of death in ascetic SBP 0 6912 Control Treatment Control group (N = 168) AFP, alpha-fetoprotein; ALT, alanine transaminase; AST, aspartate transaminase; BMI, body mass index; GGT, gamma-glutamyl transferase; Hb, Hemoglobin; HBsAg, Hepatitis B surface antigen; HCV Ab, Hepatitis C virus antibodies; INR, international normalized ratio; SBP, spontaneous bacterial peritonitis; TLC, total leukocytic count. Baseline 141.0±66.8 1.30 ± 0.49 2.05 ± 0.83 0.62 ± 0.38 65.0+ 28.8 104.3 ± 53.4 After 6 months 2.76 ± 0.44 0.68 ± 0.32 1.25 +0.24 7.1 (2-32) 51 (2.9-189) 8.8 (4.5-17.0) 231 (189-291) 1.41 ± 0.86 18.3 ± 10.0 42:58 185.5 (85-535) Figure 1. Change in serum Procalcitonin level in both con- trol and treatment groups over 6 months. Results presented as the mean ± SD. (p 0.05 0.05). However, low Vit D level in those patients was the only significant predictive mortality marker (p< 0.01). On the other hand, for assessment of the effect of Vit D supplemen- tation on 6 months survival, both univariate and multivariate analysis revealed a highly significant association (p<0.001) (HR=0.895) (Table IV). Cirrhosis has a noticeable correlation with morbidity and mortality rate in developed coun- tries 47-49. Mortality is 4 times higher in a patient with cirrhosis and infection with a poor progno- sis; 30% of patients die within a month of infec- tion in addition to another 30% within a year 50,51 Spontaneous bacterial peritonitis (SBP) is one of the most frequently diagnosed infections. The ascetic fluid infection happens when there is no visceral perforation or inflammatory focus in the abdominal cavity. For SBP diagnosis, the poly- morphonuclear leukocytic count (PMNLs) from the ascetic fluid obtained through paracentesis must be more than 250 cells/mm52,54. In addition, procalcitonin (PCT) appears to be a sensitive diagnostic indicator that can be used to predict diagnosis, monitor bacterial infections, and guide the clinical use of antibiotics. Vit D may enhance

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/n European Review for Medical and Pharmacological Sciences Effect of Vitamin D supplementation in patients with liver cirrhosis having spontaneous bacterial peritonitis: a randomized controlled study A.A. MOHAMED¹, A.A. AL-KARMALAWY², A.A. EL-KHOLY³, D.A. EL-DAMAS+, H.M. ABOSTATE¹, S.M. MOSTAFA5, M. HAMADA, M.A. KHALI