Effects of Laparoscopic Cholecystectomy on Apolipoproteins ( ApoA 1 , ApoB 100 , ApoE ) Status in Bangladeshi Patients with Cholelithiasis

Objectives: The present study was conducted on serum apolipoproteins (ApoA1, ApoB100, ApoE) status as scanty reports are available in Bangladeshi patients with cholelithiasis i.e. gallstone disease (GD). Patients and Methods: Fifty five adult patients (Pts) with cholelithiasis and 40 normal controls (NC) were included in the study carried out from October 2016 to March 2018. The blood samples were taken from fasting patients before cholecystectomy (Serum-I0), gall bladder biles during cholecystectomy (Bile-I0) and blood samples again after 2-3 months at follow-up (Serum-II0) and from fasting NC subjects. ApoA1, ApoB100 and ApoE levels were measured in serum and bile samples by enzyme immunoassay (EIA) method using commercially available research/diagnostic kits. The results were compared statistically by Student’s t-test and Chi-squared test using SPSS programme. Results: ApoA1 level (mg/dl) was reduced in the Pts which was raised significantly after 2-3 months of laparoscopic cholecystectomy, but not to the full extent similar to NCs (Mean ± SD → Serum I0: 50.89 ± 14.01, Serum II0: 60.75 ± 16.40) (p<0.001). ApoB100 level (mg/dl) was raised in Pts which was reduced significantly after cholecystectomy, but not to the full extent as NCs (Mean ± SD → Serum I0: 202.84 ± 53.42, Serum II0: 138.34 ± 30.63) (p<0.001). Similarly, ApoE level (mg/dl) was raised in Pts which was reduced significantly after laparoscopic cholecystectomy, but not to the full extent similar to NCs (Mean ± SD → Serum I0: 69.97 ± 13.50, Serum II0: 57.13 ± 17.38) (p< 0.001). Chi-squared (χ2) test revealed that large proportions of Pts had lower ApoA1, higher ApoB100 and higher ApoE levels compared to NCs and the proportions were changed towards improvement similar to NCs significantly, but not to the full extent after laparoscopic cholecystectomy) (p<0.001). Conclusions: Cholelithiasis i.e. GD had profound influence on ApoA1, ApoB100 and ApoE status suggesting their probable causative and protective roles in the aetiopathogenesis of the disease. Investigations related to leptin and apolipoproteins levels and their associations with metabolic syndrome in GD are needed in our future research endeavors to substantiate the findings. Short Title: Apolipoproteins (A1, B100, E) in Cholelithiasis.


Introduction
Lipoproteins are typically spherical particles with non polar neutral lipids, i.e. triglycerides (TG) and cholesterol ester (CE), in their core and more polar amphipathic lipids, i.e. phospholipids (PL) and free cholesterol (C), at their surfaces.Equally important is the fact that they also contain one or more specific proteins called apolipoproteins on their surfaces.Each class of lipoprotein, i.e.Chylomycron, VLDL, IDL, LDL, HDL & Lp(a), carries several apolipoproteins in different proportions such as ApoA1 in HDL and Chylomycron, http://dx.doi.org/10.16966/2578-9578.121involved in patients with myocardial infarction (MI) [3,4].Literature review indicated that some of the apolipoproteins such as ApoA1, ApoB100 and ApoE are involved in the process of Cholelithiasis i.e. gallstone disease (GD) as well [8][9][10].GD is a metabolic problem which correlates with lipid abnormalities, adiposity, diabetes mellitus, hypertension and metabolic syndrome.Some studies reported that ApoA1, ApoB100 and ApoE were striking higher in serum of patients with GD compared to individuals with no gallstones [11][12][13].Other factors such as cholesterol ester transfer protein (CETP) and mucin were also implicated in the aetiopathogenesis of GD.Suggestions were made that serum ApoA1, ApoB100 and ApoE levels would possibly be sensitive parameters as compared with serum lipids in distinguishing patients with gallstones from subjects without gallstones.The status of lipid profile, lipoprotein (a), mucin 1 and CETP in Bangladeshi patients with cholelithiasis i.e.GD had been published recently [13][14][15].However, literature survey indicated that various apolipoproteins status in Bangladeshi patients with GD is not known.In the present article, we have therefore reported effects of laparoscopic cholecystectomy on serum levels of ApoA1, ApoB100 and ApoE in Bangladeshi patients with cholelithiasis i.e.GD.

Patients and Methods
Fifty five adult patients (Gender: 10 males, 45 females; Age range: 25-65 years, Mean age ± SD: 45.5 ± 12.2 years) with cholelithiasis i.e.GD and 40 healthy adult normal controls (Gender: 16 males, 24 females; Age range: 28-60 years; Mean age ± SD: 42.5+10.5years) were included in this case-control prospective interventional study.The patients with GD were diagnosed as having cholelithiasis according to standard clinical and laboratory criteria as practiced in hospital and patients not fulfilling the criteria for our study i.e.Patients with diabetic mellitus, renal disease, thyroid disease, liver disease, history of taking anti-hypertensive or anti-hyperlipidemic drugs and smoking habits were excluded from the study [14][15][16].After obtaining consent, patient's demographic details and clinical findings such as pain (severity, duration, location), merphy's sign, ultrasonogram (USG), etc were recorded as per 'PROFORMA' at diagnosis.The fasting blood samples were taken at diagnosis before cholecystectomy, and conducted routine laboratory tests.The serum separated was aliquoted and stored frozen at -80°C as first degree serum sample (I 0 ).At the time of cholecystectomy, gall bladder bile was also collected from the same patient, centrifuged, aliquoted and stored frozen at -80°C as first degree bile sample (I 0 ).After Cholecystectomy, treatments/ medications were given as required for the patients.After 2-3 months at follow-up, fasting blood samples were taken again from the same patient, serum separated, aliquoted and stored frozen at -80°C as second degree serum samples (II 0 ) until analyzed for the lipid profile (i.e.TG, TC, HDL-C, LDL-C), Lp(a) and apolipoproteins (ApoA1, ApoB100, ApoE) related to our research interest.All quantitative estimations in serum and bile were made by standard medical laboratory methods using research/diagnostic kits from internationally reputed companies and LDL-C calculated by Friedwald formula [14,16].ApoA1, ApoB100 and ApoE levels in serum were determined by enzyme-linked immunosorbent assay (ELISA) research/diagnostic kits based on biotin double antibody sandwich technique taking optical density readings at 450 nm in a Microplate EIA Reader (Model: RE-4.0 Microplate Reader, IDCS Ltd Briloner Landstrasse 4-6, 34497 KORBACH-Germany).The apolipoprotein kits were obtained from Shanghai Yehua Biological Technology Co Ltd, Shanghai, China (Ref: ApoA1, Cat No.YHB3610Hu; ApoB100, Cat No.YHB0379Hu; ApoE, Cat No.YHB0371Hu).The results of laboratory analyses in biological specimens of patients (I 0 ,II 0 ) and controls (NCs) were compared statistically by Student's t-test and Chi-squared (χ 2 ) test using SPSS programme in computer [17].The study was carried out at Medical Research Unit (MRU), The Medical and Health Welfare Trust (MHWT), Uttara, Dhaka-1230, Bangladesh in collaboration with Department of Surgery, Medical College for Women and Hospital (MCW and H), Uttara, Dhaka-1230, Bangladesh from October 2016 to March 2018.

Results
The results of ApoA1, ApoB100 and ApoE status in our study subjects and their statistical analyses are stated in (Tables 1-4).ApoA1 level was significantly reduced in our Pts which was raised significantly after 2-3 months of laparoscopic cholecystectomy, but not to the full extent similar to NCs and bile (Pts) contained lower level of ApoA1 (p<0.001) (Table 1).ApoB100 level, on the other hand, was significantly raised in Pts which was reduced significantly after cholecystectomy, but not to the full extent as NCs and bile (Pts) contained lower levels of ApoB100 (p 0.001) (Table 2).Similarly, ApoE was significantly raised in Pts which was reduced significantly after 2-3 months of laparoscopic cholecystectomy, but not to the full extent similar to NCs and bile (Pts) contained lower level of ApoE (p<0.001) (Table 3).The analysis by Chi-squared (χ 2 ) test revealed that large proportion of Pts had lower ApoA1, higher ApoB100 and higher ApoE levels compared to NCs and the proportions were changed towards improvement significantly similar to NCs, but not to the full extent in 2-3 months after laparoscopic cholecystectomy (p<0.001)(Table 4).

Discussion
Apolipoproteins were reported to be associated with CHD showing that some of them are involved in MI in many studies [3][4][5][18][19][20].Multiple associations between gallstones and dislipidaemias, CHD and stroke have been reported [21][22][23][24].Our findings on serum levels of ApoA1 (Table 1) and ApoB100 (Table 2) in Bangladeshi patients with cholilithiasis were similar to those reported by Sarac, et al. [8].These results were consistent with those of other previous studies showing lower ApoA1 and higher ApoB100 serum levels in patients with cholilithiasis [25,26].These findings could be useful in understanding the pathophysiology of GD.Leptin, a hormone produced by adiposities, was shown to modulate the activity of enteric inhibitory and excitatory neurons in the proximal colon and it seems to have a regularly function in gastrointestinal motility [8,27].Sarac et al showed further that levels of ApoA1 were negatively correlated while ApoB100 levels were positively corrected with leptin [8].Our findings on ApoE levels in patients with GD had similar trends as ApoB100 i.e.ApoE was increased in serum I 0 , followed by decrease in Serum-II 0 , significantly, but not to the full extent as NCs (p<0.001) (Table 3).
ApoE is one of the key regulatory protein in cholesterol and lipoprotein metabolism [1,27].ApoE has three common isoforms, E2, E3, and E4, which are coded by the alleles e2, e3 and e4, respectively, at a single locus in chromosomal region 19q13.These alleles define six apo E phenotypes: E2/2, 2/3, 2/4, 3/3, 4/3, and 4/4.Apo E4 has been associated with several diseases, such as coronary heart disease and Alzheimer's disease [28][29][30].The E2 allele is associated with low and the E4 allele with high serum total and LDL cholesterol concentrations in various populations and with altered enterohepatic metabolism of cholesterol and bile acid.It was reported that the cholesterol content of gallstones tends to be low in subjects with the E2 allele and high in subjects with the E4 allele suggesting that ApoE2 is a genetic factor providing protection against GD, while ApoE4 isoform may be a risk factor for cholilithioasis [28]      ApoE level could mean either a protective or a susceptibility/risk factor role in GD.To resolve this vital issue, study on ApoE polymorphism is required to be done in patients with GD.Also in recent times, antioxidant defense system has been implicated with the prevalence of GD and thought to play a role in the pathophysiology of GD [31][32][33][34][35].Among them, higher circulating vitamin E and vitamin C levels have been suggested that might provided protection against GD, and area of research that needs to be further investigated in longitudinal studies [34,35].
Our findings were important and interesting in the sense that serum levels of significantly large proportion of patients were altered in GD which were reversed towards NCs after 2-3 months of laparoscopic cholecystectomy, but not to the full extent (p<0.001)(Table 4).The reasons for this situation need some careful, relevant and logical explanations.In recent times, GD is considered to be a metabolic disease as Mendez-Sanchez, et al. and others reported a strong relationship between GD and metabolic syndrome [8][9][10]23].They reported that leptin dysfunction and insulin resistance might In conclusion, cholelithiasis i.e.GD had profound influence on ApoA1, ApoB100 and ApoE status suggesting their cause and effect roles in the aetiopathogenesis of the disease.It is important to further investigate leptin and apolipoproteins levels and their associations and also to study metabolic syndrome in Bangladeshi patients with GD in our future research endeavors to substantiate the findings.

Citation:4
Jhuma KA, Giasuddin ASM, Choudhury MAM, Haq AMM (2018) Effects of Laparoscopic Cholecystectomy on Apolipoproteins (ApoA1, ApoB100, ApoE) Status in Bangladeshi Patients with Cholelithiasis.J Clin Lab Med 3(1): dx.doi.org/10.16966/2578-9578.121Journal of Clinical and Laboratory Medicine Open Access Journal be responsible for reduced gallbladder motility, which in turn may contribute to cholesterol gallstone formation.In the light of these reports, we are now in the process of evaluating our results for the association between Lipid profile and ApoA1, Lipid profile and ApoB100, Lipid profile and ApoE, Lp(a) and ApoA1, Lp(a) and ApoB100 and Lp(a) and ApoE.

Table 1 :
Apolipoprotein A1 levels in patients (Pts) and controls (NCs) and their statistical analyses by student's t-test.

Table 2 :
Apolipoprotein B100 levels in patients (Pts) and controls (NCs) and their statistical analyses by student's t-test.

Table 3 :
Apolipoprotein E levels in patients (Pts) and controls (NCs) and their statistical analyses by student's t-test.