|Year : 2022 | Volume
| Issue : 2 | Page : 47-50
Regional differences in bile acid composition in gallbladder bile
J Ramana Ramya1, Mayank Jain2, M Mary Sheeba3, K Thanigaiarul1, R Karvembu3, Vijaya Srinivasan2, V Vaithiswaran2, S Narayana Kalkura1, Jayanthi Venkataraman2
1 Crystal Growth Centre, Anna University, Chennai, India
2 Department of Gastroenterology, Gleneagles Hospital, Chennai, India
3 Department of Chemistry, National Institute of Technology, Tiruchirappalli, Tamil Nadu, India
|Date of Submission||05-Jan-2022|
|Date of Decision||02-Feb-2022|
|Date of Acceptance||08-Feb-2022|
|Date of Web Publication||23-Mar-2022|
Crystal Growth Centre, Anna University, Chennai, Tamil Nadu
Source of Support: None, Conflict of Interest: None
Background: Chemical and structural analyses of gallstones (GS) from the Indian subcontinent has shown that the formation of GS type is dependent on regional and dietary factors. Aim of the Study: The aim is to determine the proportion of primary and secondary bile acids in gallbladder (GB) bile in patients with GS from South and North India using high-performance liquid chromatography (HPLC). Materials and Methods: Standards for primary and secondary bile acids were prepared and concentrations were determined by reversed-phase C18 HPLC column. Thirty-three GB bile samples from southern India and 28 samples from northern states of India were analyzed for differences in the proportion of primary and secondary bile acids. Ethics Committee of Gleneagles Global Health City, Chennai, approved the study. Statistical Analysis: concentration of bile acids (in mmol/L) were expressed as median and range. Chi-square test and Mann–Whitney U-test were applied. A P < 0.05 was considered as significant. Results: The median concentrations of cholic acid (CA) (P = 0.005) and its derivative deoxycholic acid (DCA) (P < 0.006) were significantly high in GB bile samples from South India with no differences in the concentration of chenodeoxycholic acid between the two samples. Furthermore, samples from North India had a significantly higher proportion of lithocholic acid (LCA) and low DCA compared to samples from South India. Conclusion: Primary bile acid CA and its derivative is high in GB bile from South; the proportion of hepatotoxic LCA is significantly high with low concentrations of DCA in bile samples from North India.
Keywords: Bile, bile acids, gall stones, gallbladder
|How to cite this article:|
Ramya J R, Jain M, Sheeba M M, Thanigaiarul K, Karvembu R, Srinivasan V, Vaithiswaran V, Kalkura S N, Venkataraman J. Regional differences in bile acid composition in gallbladder bile. Gastroenterol Hepatol Endosc Pract 2022;2:47-50
|How to cite this URL:|
Ramya J R, Jain M, Sheeba M M, Thanigaiarul K, Karvembu R, Srinivasan V, Vaithiswaran V, Kalkura S N, Venkataraman J. Regional differences in bile acid composition in gallbladder bile. Gastroenterol Hepatol Endosc Pract [serial online] 2022 [cited 2022 Aug 18];2:47-50. Available from: http://www.ghepjournal.com/text.asp?2022/2/2/47/340386
| Introduction|| |
Gallbladder (GB) bile consists of cholesterol, phospholipids, bilirubin, bile acid, and salts with inorganic solutes such as sodium, potassium, and calcium. The regional difference exists in the biochemical composition of gallstones (GS),,,,,, and GB bile in the Indian subcontinent. GS are predominantly pigment in south, whereas those from north are rich in cholesterol, the latter in a background of lithogenic bile. The cause for pigment GS in southern states of the Indian subcontinent is not known. Diet and genetic factors are considered as possible risk factors. Bacterial infection, an established risk factor for pigment GS is not a major risk factor for pigment GS from South India.
In lieu of our above observations, we undertook the present study to determine if there were regional differences in the proportion of primary and secondary bile acids in GB bile of patients with GS undergoing cholecystectomy.
Bile acids (BAs) are complex substances that are primarily synthesized from cholesterol within the hepatocytes. These are primary BAs, i.e., cholic acid (CA) and chenodeoxycholic acid (CDCA) and secondary BAs: deoxycholic acid (DCA) and lithocholic acid (LCA), the former, a derivative of CA and the latter of CDCA. Both primary and secondary BAs are conjugated to glycine and taurine in various proportions. The proportion of CA + CDCA: DCA: LCA in healthy bile is 75%: 20%: ~5%., Biliary BAs thus consist mostly (>90%) of the conjugates of CA and CDCA (primary BAs) and DCA (a secondary BA). Only trace amounts of the conjugates of LCA are present. Another BA, ursodeoxycholic acid (identical to ursodiol), is also present in trace amounts, and is identical in structure to CDCA, except that the hydroxyl group at C7 is in a β rather than an α configuration.
| Materials and Methods|| |
GB bile samples were collected from cholecystectomy specimens of GS patients and stored at −80°C. The samples were categorized as
- South India GB bile (Kerala, Tamil Nadu, Karnataka, Andhra Pradesh, and Telangana) (SI-GBB) and
- North India GB bile (NI-GBB).
Patients with diagnosis of GS and cholidocholithiasis, GB cancer, primary or secondary hepatolithiasis and empyema gall bladder and had cholecystectomy were excluded from the study.
The BA composition was assayed using high-performance liquid chromatography (Shimadzu SPD-M20A, 230V; Photodiode Array Detector) at the National Institute of Technology, Tiruchirappalli, Tamil Nadu, India [Figure 1]. The primers (Sigma, St. Louis MO, USA), i.e., sodium salts of CA, CDCA, DCA, LCA, were used as a reference for the study samples after appropriate preparation. Interpretation was done by a reversed-phase C18 column: PARTISIL 5 ODS-3, 5 μM, 250 mm × 4.6 mm (Whatman; [Singapore]). The final analysis was completed using Class VP software [Figure 1].
|Figure 1: Prototype chromatograms of primary and secondary bile acids in the study samples. Few bile samples showed taurine conjugates of LCA (higher concentration in samples from South India). DCA: Deoxycholic acid, TCA: Taurine conjugate of cholic acid, LCA: Lithocholic acid, TLCA: Taurine conjugate of lithocholic acid|
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The differences in primary and secondary BAs in southern GB bile and those from northern states were compared. Glycine and taurine conjugates of primary and secondary BAs although analyzed were not considered for analysis as the sample size was small.
This single-center study protocol conformed to the Declaration of Helsinki of 1975, as revised in 2008 and was approved by the Institutional Ethics Committee of Gleneagles Global Health City, Chennai. All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (institutional and national). Informed consent was obtained from all patients included in the study.
The concentration of BAs (in mmol/L) was expressed as median and range. Chi-square test and Mann–Whitney U-test were applied. A P < 0.05 was considered as significant.
| Results|| |
Thirty-three GB bile samples of patients who had undergone cholecystectomy for GS disease from SI-GBB (17 pigments, 14 mixed, and two cholesterol GS), and 28 samples from NI-GBB (20 cholesterol, seven mixed, and one pigment GS) were analyzed. The type of GS was classified based on our earlier validated publication. The median concentration of CA (P = 0.005) and DCA (P < 0.006) in the bile was significantly high in SI-GBB sample, with no difference in the concentration of CDCA between north and south [Table 1]. Among the secondary BAs, DCA was significantly high in SI-GBB compared to NI-GBB, whereas LCA showed a reverse trend with LCA being significantly high in North Indian bile samples (P = 0.02).
|Table 1: Composition of primary and secondary bile acids in North and Southern Indian states (concentrations in mmol/L)|
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| Discussion|| |
Our study for the first time has looked into the differences in the composition of primary and secondary BAs in GB bile in patients with GS from South and Northern states of India [Figure 2].
|Figure 2: Proportion of primary bile acids (CA + CDCA), DCA, LCA. In healthy, southern, and northern states in gallbladder bile sample of patients with GS disease. Numbers denote percentage; Healthy bile is a reference sample., GS: Gallstones, CDCA: Chenodeoxycholic acid, CA: Cholic acid, DCA: Deoxycholic acid, LCA: Lithocholic acid|
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Percentage concentration of LCA is significantly high (P = 0.002) in NI-GBB and low in DCA compared to South Indian GB bile sample. Whether these differences in BA concentration (high LCA) have a meaningful connotation to the high prevalence of cholesterol GS and GB malignancy in North India remains hypothetical and needs validation in a clinical setting. Experimental studies in animal models have demonstrated hepatotoxicity with LCA. Shukla et al. in their analysis of BAs in patients with GB cancer, GS disease, and healthy controls reported a mean ratio of primary to secondary BAs to be 3.5:1 that increased to 5.34:1 in those with GS disease (P < 0.001) and a significant increase in secondary BA in carcinoma GB (ratio 1:1; P < 0.001). Authors proposed a rise of biliary deoxycholate concentrations as a probable factor in pathogenesis of GB cancer. The study had not studied the lithocholate concentration in their sample.
Based on our experimental study, we hypothesize that the rarity of cholesterol GS and protection against GB malignancy in South India may be related to the BA composition that is similar to the healthy BA composition quoted in world literature., Is DCA protective for GB cancer? Probably, yes. The high incidence of both cholesterol GS and GB malignancy in North India is likely to be due to high concentrations of hepatotoxic LCA. This hypothesis needs confirmation in larger sample size.
In future, for a better understanding of the influence of BAs in health and disease such as GS and GB malignancy, it is important that multicenter studies are undertaken across the Indian subcontinent to determine the BA profile in different regions of the country. Further, the wide dietary and ethnic variations among different states in India, it is highly likely that there may be other risk factors that further enhance the formation of GS and also heighten the risk of GB cancer.
Limitations of the study
The limitation of this study is the small sample size.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Jayanthi V. Pattern of gall stone disease in Madras city, south India – A hospital based survey. J Assoc Physicians India 1996;44:461-4.
Ashok M, Nageshwar Reddy D, Jayanthi V, Kalkura SN, Vijayan V, Gokulakrishnan S, et al.
Regional differences in constituents of gall stones. Trop Gastroenterol 2005;26:73-5.
Ashok M, Krishnan A, Choudhury G, NarayanaKalkura S, Jayanthi V. Regional differences in composition of cholesterol gallstones in India. J Med Sci Res 2012;3:3-5.
Tandon R. Pigment gallstones. Indian J Gastroenterol 1988;7:5-6.
Tandon RK, Thakur US, Basaki AK, Lal K, Jayanthi V, Nijahawan S. Pigment gallstone predominant in South India. Indian J Gastroenterol 1994;12:A18.
Amin AM, Ananthakrishnan N, Nambinarayanan TK. Composition of gallstone and sequential event in biliary lithognensis – Is it different in South India compared to north? J Assoc Physicians India 2000;48:885-90.
Selvaraju R, Ganapathi Raman R, Thiruppathi G, Valliapan R. Epidemiological study of gallstone in Cuddalore district. Int J Pharm Tech Res 2010;2:1061-7.
Jayanthi V, Sarika S, Varghese J, Vaithiswaran V, Sharma M, Reddy MS, et al.
Composition of gallbladder bile in healthy individuals and patients with gallstone disease from north and South India. Indian J Gastroenterol 2016;35:347-53.
Jayanthi V, Anand L, Ashok L, Srinivasan V. Dietary factors in pathogenesis of gallstone disease in southern India – A hospital-based case-control study. Indian J Gastroenterol 2005;24:97-9.
Ravikanth VV, Rao GV, Govardhan B, Sasikala M, Subramanyam C, Vivekananda Murthy HV, et al.
Polymorphisms in UG1A1 gene predispose south Indians to pigment gallstones. J Clin Exp Hepatol 2016;6:216-23.
Pavithra S, Rao U, Mohan P, Venkataraman J. Bactibilia in pigment gallstone disease: A report from the Indian subcontinent. Dig Liver Dis 2010;42:231-2.
Hofmann AF. The continuing importance of bile acids in liver and intestinal disease. Arch Intern Med 1999;159:2647-58.
Hofmann AF. Bile acids: The good, the bad, and the ugly. News Physiol Sci 1999;14:24-9.
Gokulakrishnan S, Murugesan R, Mathew S, Prasanthi R, Ashok AC, Ramesh H, et al.
Predicting the composition of gallstones by infrared spectroscopy. Trop Gastroenterol 2001;22:87-9.
Hofmann AF. Detoxification of lithocholic acid, a toxic bile acid: Relevance to drug hepatotoxicity. Drug Metab Rev 2004;36:703-22.
Shukla VK, Tiwari SC, Roy SK. Biliary bile acids in cholelithiasis and carcinoma of the gall bladder. Eur J Cancer Prev 1993;2:155-60.
[Figure 1], [Figure 2]