/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) in the treat-
ment group (Table I). In addition, blood test
parameters and ascetic fluid composition were
comparable in the control and treatment groups
(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
> 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 <0.001 for each group after 6 months,
*p<0.001 vs. the control).
Treatment group
(N = 160)
283.1 ± 89.1
0.95 ± 0.16
0.76 ± 0.19
0.15 +0.07
57.8 ± 22.4
66.3 ± 26.3
2.84 +0.36
0.54 ± 0.32
0.98 +0.07
6 (2.9-9)
35.5 (25-59)
7.5 (2.5-12.0)
124 (90-3400)
0.65± 0.57
34.6±9.2
64:36
199.5 (85-535)
p-value
< 0.001
< 0.001
< 0.001
< 0.001
<0.05
< 0.001
> 0.05
<0.05
<0.001
< 0.001
< 0.001
<0.001
< 0.001
< 0.001
< 0.001
Discussion
<0.05
<0.05
patients (p>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
