25(OH) vitamin D level and severity of chronic liver disease: A controlled study from Eastern India

 

Jyoti Paul¹, Gora Chand Bhattacharya^2*, Ila Bhattacharjee³

 

¹Consultant, ²Professor, Department of Biochemistry, Saroj Gupta Cancer Research Center, Thakurpukur, Kolkata-700041, West Bengal, INDIA

³Professor and HOD, Department of Biochemistry, MGM Medical College and LSK Hospital, Kishanganj-855 107, Bihar, INDIA.

Email: dr.jyotipaul@gmail.com, drgbhattacharya@gmail.com, prabir0209@gmail.com

 

Abstract               Background: Chronic Liver Disease (CLD) is defined as long term process of progressive degeneration and regeneration of liver cells and with advancement, leads to hepatic fibrosis, scarring, and cirrhosis. 25(OH) Vitamin D undergoes post synthesis processing in liver and in CLD, Vitamin D homeostasis is impaired. Active metabolite of Vitamin D 1,25 (OH)D3 is involved in calcium and phosphate metabolism. CLD is associated with cholestatic liver disease like primary biliary cirrhosis and many patients develop osteoporosis due to altered calcium and Vitamin D metabolism. Aims and Objectives: The aim of our study was to measure serum 25(OH) Vitamin-D in patients with chronic liver disease with an object to correlate the results with conventional hepatic markers for assessment of severity of the disease. Methods: In this study, we measured serum 25(OH) Vitamin D (Total) along with routine liver function test in 50 patients of CLD of various types, 50 healthy individuals were selected conveniently as controls. Results: We observed a deficient Vitamin D level in all the patients of CLD. Serum Vitamin D level was found to be inverse lyproportional with severity of liver damage and the result is statistically significant. Conclusion: Our study indicates that severity of liver disease is associated with hypovitaminosis D. We observed inverse relationship between hepatic enzyme levels and serum vitamin D concentrations in patients with severe type of CLD. We propose that estimation of vitamin D as a routine mighthelpin diagnostic and prognostic aspect of severe type of chronic liver disease.

Key Words: Chronic liver disease (CLD), 25(OH) vitamin D, Cirrhosis of liver, Hepatitis B, Hepatitis C, Hepatic

encephalopathy.

 

INTRODUCTION

Chronic liver disease (CLD) is defined as long term process of progressive degeneration and regeneration of liver cells and with advancement, ultimately leads tohepatic fibrosis, scarring and cirrhosis¹ and hepatonecrotic inflammation for more than 6 months is detectable by biochemical and histological findings.² Hepatocytes perform various functions that include the synthesis of serum proteins like albumin, coagulation factors, many hormonal and growth factors, production of bile, regulation of nutrients, metabolism and conjugation of bilirubin and excretion of drugs through bile.² Major site of Vitamin D synthesis is epidermal cells, where the UV radiation from sun exposure transforms 7dehydrocholesterol into pre-vitaminD₃andis finally transformed into the biological active form cholecalciferol (Vit-D3). Dietary source of Vitamin D in the form of vit-D₂ (ergocalciferol) and D₃ and are absorbed in intestine.^3,4. Then vitamin D from skin and dietary source are stored in adipocytes or undergo hepatic 25 hydroxylation and produces 25 (OH) D or calcidiol. Both Vitamin D₂and vitamin D₃ is hydroxylated in liver by 25 hydroxylase which is a cytochrome P450 enzyme family II. In circulation, 25(OH) D is mostly bound to DBP (vitamin D binding protein), which is simultaneously synthesized in liver.⁵ In the kidney,1,25-dihydroxyvitamin-D₃ is formed by the enzyme 1 α hydroxylase.⁶ 1,25 dihydroxy D₃ regulates approximately 2000 genes.⁷ Thisactive metabolite of Vitamin D is responsible for calcium and phosphate metabolism and exerts varieties of biological effects.⁸ Calcium metabolism is regulated with increase in intestinal calcium absorption and mobilizing calcium from the bones.⁹ Vitamin D regulates parathyroid hormone secretion, adaptive immunity, cell proliferation and promotes insulin secretion, and cellular differentiation.¹⁰ 25(OH) vitamin D is the inactive form and having a half-life of 2-3 weeks, and so, this is considered as an important parameter for estimation of vitamin D. This reflects total amount of vitamin D which is from dietary source, exposure to Sunlight and conversion from fatty deposit in liver, and thus plasmacon centration of 25 (OH) vitamin D is the most reliable indicator of vitamin D status of an individual.¹¹ 25 (OH) Vitamin D is synthesized in liver, and in CLD Vitamin D homeostasis is altered. Reports from researchers indicate altered level of 25 (OH) vitamin D in patients of CLD which extends infibrotic changes and hepatic cirrhosis and subsequently results in impaired hepatic function in the affected subjects.¹² About 43.6% of vitamin D deficient subjects develop CLD and neurological disturbances.¹³ Deficiency of vitamin D has been reported to be associated with cholestatic liver disease like primary biliary cirrhosis and some patients also develop osteoporosis due to altere dmetabolism of calcium and Vitamin D.¹⁴ This may be due to defective synthesis of Vitamin D in skin, lack of dietary intake, improper activation of the vitamin or resistance to biological effect of 1,25(OH) vitamin D. Vitamin D plays an important role in reducing the risk of chronic diseases including type 2 diabetes mellitus, several types of cancers, cardiovascular, autoimmune and infectious diseases. These functions occur mostly due to its autocrine and paracrine action in cellular proliferation and differentiation, apoptosis, insulin and renin secretion, synthesis of interleukins (IL) and bactericidal proteins.^{3,13,14,15,16} These effects might be relevant in pathogenesis of chronic liver disease.

MATERIALS AND METHODS

A hospital based observational case control study was conducted jointly by the Department of Biochemistry and department of Medicine in M.G.M. Medical College and L.S.K. hospitals, Kishanganj, Bihar, betweenJuly2014 to June 2015.Patients with chronic liver disease attending the department of Medicine were selected as cases. Total 50 subjects suffering from chronic liver disease were selected as test group and 50 healthy individuals without any history of CLD or other diseases were selected conveniently as control group. Routine liver function test parameters were estimated and 25(OH) vitamin D level was measured in both test and control groups.

Inclusion Criteria: Patients suffering fromCirrhosis liver, Hepatitis B and Hepatitis C positive subjects, patients with Hepatic encephalopathy and Alcoholic Liver Disease were included as subjects of the study.

Exclusion Criteria: Patients suffering from tuberculosis, HIV infection, drug induced jaundice, diabetes mellitus were excluded from the study.

Study procedure: Collection of blood: Blood samples were collected in plain vials from participants. Two separate vials were used. For estimation of vitamin D, vials were covered with black paper to avoid exposure of light. The uncovered vials were used for liver function test. 5 ml blood was drawn from ante cubital vein and 2ml blood was taken into the vial covered with black paper and 3ml blood was taken into the uncovered vial. The vials were kept for serum separation. Serum total bilirubin, conjugated bilirubin, GPT, GOT, ALP, Total Protein, Albumin, and 25(OH) vitamin D were estimated.

All the parameters of LFT were estimated in full automated analyser (Selectra Junior ProS ELItech) clinical system: All analytical parameters were measured by standardised photometric methods. 25 OH Vitamin D (Total) estimation was performed by ELISA:

Immunoenzymetric-assay for the in vitro quantitative measurement of 25

hydroxyvitamin D2 and D3 (25 OH- D2 and 25OH –D3) in serum. (Merck

MIOS Mini)

Principle of the method: 25(OH) Vitamin D ELISA is a solid phase Enzyme Linked Immunosorbent Assay performed on microtiterplates. During first 2 hours of incubation step at room temperature, total 25(OH) Vitamin D (D₂ –D₃) present in calibrators, controls and samples are dissociated from binding serum proteins to fix on binding sites of a specific monoclonal antibody. After first washing step, a fixed amount of 25OH vitamin Dlabelled with biotin in presence of horseradish peroxidase (HRP), competes with unlabelled 25(OH) vitamin D₂ and D₃ present on the binding sites of the specific monoclonal antibody, then after 30 minutes incubation at room temperature, the microtiter plate is washed to stop the competitive reaction. The Chromogenic solution (TMB) is added and incubated for 15 minutes. The reaction is stopped with the addition of stop solution and the microtiterplate is then read at the appropriate wavelength. The amount of substrate turnover is determined by measuring the absorbance, and that is inversely proportional to the total 25(OH) vitamin D (D₂ and D₃) concentrations in samples, and are determined by interpolation from the calibration curve. The results were compared with control group for statistical analysis. Data was analysed in SPSS (20) software. Value of individual parameter was expressed as Mean ± Standard deviation. Significance of differences of the Means within groups was tested by Student ‘t’ test and p value of less than 0.05 was considered to be statistically significant.

 

RESULT

Table 1:

 

Table 2:

 

Table 3: Distributionof Serum Bilirubin among case and control

 

Table 4: Distribution of SGPT among case and control

Table 5: Distribution of SGOT among case and control

 

Table 6: Distribution of ALP among case and control

 

Table 7: Distribution of T. Protein among case and control

Table 8: Distribution of Albumin among case and control

 

Table 9: Distribution of 25(OH) Vitamin D among case and control

DISCUSSION

In this study 50 CLD patients were selected, we observed 44% of Alcoholic Liver Disease (ALD) patients were between the age group of 15-30 years and45-60 years. ALD occupies first position among the study population. In the second position, hepatitis B affected patients claim for 20% of the study group. Cirrhosis of liver stands third in rank and constituting 16% of the study subjects. A significant increase in serum bilirubin levels and the hepatic enzymes levels confirms the diagnosis of liver diseases for all the test cases. All the patients with CLD had significantly higher SGOT, SGPT, ALP levels. Our findings are similar to the observations of other researchers.^17,18. Serum total protein and albumin concentration were significantly decreased in the test group. Serum 25(OH) vitamin D concentration was in deficient range in all the patients of CLD. The deficiency of vitamin D is more in subjects of ALD. Patients with positive HBsAg also had a lower concentration of 25(OH) D. Reports regarding the deficiency of 25(OH) vitamin D and severe histological changes in Non Alcoholic Fatty Liver disease have been observed by Researchers who found the link between deficient level of Vitamin D and severe fibrosis in patients with genotype I Hepatitis C¹⁹. Study from Israel reveals that Vitamin D supplementation causes improvement of sustained virologic response in chronic hepatitis ‘C’ genotype 2, 3 patients.²⁰ In non-cirrhotic liver disease Vitamin-D deficiency is more as reported by Fisher L, Fisher A,¹². A decreased level of 25(OH) D and serum albumin have been observed in patients with severe liver disease. The reason is thought to be due to impaired hydroxylation reaction and impaired synthesis of albumin and D binding protein. In non-cirrhotic liver disease Vitamin-D deficiency is more as reported by Fisher L, Fisher A,¹².

 

CONCLUSION

We carried out the study with an objective to assess vitamin D status in subjects of CLD in eastern part of India, which is also adjacent to North-Eastern states. All patients with CLD in our study, had a deficient level of 25(OH) Vitamin D in their blood and finally we conclude that this study reveals a deficient level of Vitamin D in all 50 patients of chronic liver disease and estimation of Vitamin D concentration in serum might assist in detection of severity of CLD which would provide a better management to the patients.

 

REFERENCES

1. Hernandez-Gea V, Friedman SL. Pathogenesis of liver fibrosis. Annu Rev Pathol 2011; 6: 425–56.
2. Harrison’s Principles of Internal Medicine, 16^th edition; vol.2: 1844.
3. Holick MF. Vitamin D deficiency. N Engl J Med. 2007; 357:266-8.
4. Holick MF. Vitamin D: evolutionary, physiological and health perspectives. Curr Drug Targets.2011;12:4–18
5. Ho A-S, Cheng C-C, Lee S-C, et al. Novel biomarkers predictliver fibrosis in hepatitis C patients: alpha 2 macroglobulin,vitamin D binding protein and apolipoprotein AI. J Biomed Sci 2010; 17: 58.
6. Lin R, White JH. The pleiotropic actions of vitamin D. BioEssays 2004; 26: 21-8.
7. Bikle D. Nonclassic actions of vitamin D. J ClinEndocrinolMetab 2009; 94: 26–34.
8. Plum LA, Deluca HF. Vitamin D, disease and therapeutic opportunities. Nat Rev Drug Discov 2010; 9: 941–55.
9. Nagpal S, Na S, Rathnachalam R. Noncalcemic actions of vitamin D receptor ligands. Endocr Rev.2005; 26:662–687. 
10. Heaney RP. The Vitamin D requirement in health and disease. J Steroid BiochemMol Biol. 2005; 97:13–19. 
11. Arteh J, Narra S, Nair S. Prevalence of vitamin D deficiency in chronic liver disease. Dig Dis Sci 2010; 55: 2624–8.
12. Fisher L, Fisher A (2007) Vitamin D and parathyroid hormone in outpatients with noncholestatic chronic liver disease. ClinGastroenterolHepatol 5: 513–520.
13. Messa P, Alfieri C, Rastaldi MP. Recent insights into vitamin D and its receptor. J Nephrol. 2011; 24 Suppl 18:S30–S37. 
14. Ramagopalan SV, Heger A, Berlanga AJMaugeri NJ, Lincoln MR, Burrell A, Handunnetthi L, Handel AE, Disanto G, Orton SM, et al. A ChIP-seq defined genome-wide map of vitamin D receptor binding: associations with disease and evolution. Genome Res. 2010;20,:1352–1360.
15. Van Belle TL, Gysemans C, Mathieu C. Vitamin D in autoimmune, infectious and allergic diseases: a vital player? Best Pract Res ClinEndocrinolMetab. 2011; 25:617–632. 
16. Mathieu C, Badenhoop K. Vitamin D and type 1 diabetes mellitus: state of the art. Trends EndocrinolMetab. 2005; 16:261–266. 
17. Johnston DE (1999). “Special consideration in interpreting liver function test.” Am Fam Physician 59 (8):2223-30.
18. McClatchy, Kenneth D (2002) Clinical laboratory medicine. Lippincott Williams andWilkins.pp.288.
19. Satheesh Nair, “Vitamin D deficiency and liver disease”, Gastroenterology and Hepatology; vol-6, Issue 8, Aug 2010:491-492.
20. Kitson MT, Dore GJ, George J, Button P, McCaughan GW, Crawford DH, Sievert W, Weltman MD, Cheng AS, Roberts SK, Vitamin D status does not predict sustained virologic response or fibrosis stage in chronic hepatiis C genotype 1 infection. J Hepatol 2013; 58:467-472.