Glycosylation Gap in Gallstone Patients

Background: Gall stones are one of the commonest biliary diseases. Interest in the formation and clinical management of the disease dates back to ancient times. Gall stones continue to be one of the major health problems in the world today, although the exact number of patients is unknown, because there are no signs and symptoms related to the disease when there are no complications. The majority of the patients are diagnosed incidentally. However, some patients come with acute complication of gall stones. Gall stones are classified into; cholesterol gall stones, pigment gall stones, and mixed gall stones. However, all stones even pure cholesterol gall stones usually contain bilirubin (conjugated and unconjugated bilirubin) and cholesterol. Aim: To determine the changes in glycosylation gap and glycation profile and the relation of this change to the changes in age and BMI on the measured parameters. Patients and methods: Serum samples were collected from 40 individuals included in this work. They are divided into 2 groups. The first group included 20 apparently healthy individuals (10 males and 10 females). The second group included 20 individuals (5 males and 15 females) with Gall stone diagnosed in out-patient Clinic at Al-Jamhoory General Hospital and Neinawa private Hospital. Gall stone patients with metabolic diseases, infections and other inflammatory diseases were excluded. Results: The study demonstrated a significant elevation in serum glucose, mean blood glucose, serum fructosamin, measured and predicated HbAc1 in GS patients in comparison with controls. Regarding the relation between age and measured parameters, the study showed a non-significant correlation between age and measured parameters in the control group, while in GS group there were a significant correlation (r = 0.05) between ages and predicted HbAc1 and BMI (p ≤ 0.03). Regarding the relation between BMI and measured parameters, the study showed a significant correlation between measured parameters and BMI in GS individuals, while in control group only serum glucose and MBG in control group (r = 0.01) (p ≤ 0.01) with MBI. Conclusion: The study shows that there is a significant change in glycosylation gap and glycation profile in gall stone patients.

The prevalence of gall stones continues to rise with age, and it is higher in women than men. This may be due to the increase of cholesterol content in the bile by the effect of estrogen 2 . Many hypotheses explain the reasons that can lead to gall stones, most of them may have a relation to lipid metabolism 3,4 .
Glycosylation is a non-enzymatic process in which proteins react with reducing sugar molecules causing impair of their function and change of their characteristics 5 . Glycosylation usually occurs in diabetes, aging and many other diseases where accumulation of glycosylation end products involve in pathogenesis of these diseases and their complications 6 . Glycated HbAc1 result from non-enzymatic concentration depen-dent covalent bonding of glucose to Hb within the erythrocytes, thereby HbAc1 is a good indicator for glycemic control over long period 2-3 month and also its good indicator for intracellular glycation process [7][8][9][10] .
The extracellular glycation process can also be evaluated using serum fructosamin, a glycated end product of serum protein 7 . Fructosamin is a measure of average glycemic control over a shorter period 2-3 week and its plasma concentration is much more stable than that of glucose itself and much more easier to measure than true mean blood glucose (MBG),which requires contagious blood glucose monitoring [8][9][10][11] . Glycosylation gap (GG) defined as the value that results from subtraction of direct measured HbAc1 value minus the predicted HbAc1 value obtained from equation using another indicator of glycemic control, fructosamin (FA) 7 . GG is use as clinical research tool for evaluating physiological sources of variation that affects glycemic control and it can also be used as a tool to define the direction of glycosylation process [6][7][8][9][10][11][12][13] . The aim of this study is to determine the changes in glycosylation gap and glycation profile and the relation of this change to the changes in age and BMI on the measured parameters.

PATIENTS AND METHODS
This study was conducted during the period from October 2011 to January 2013 in the department of clinical pharmacy -college of pharmacy in cooperation with the department of surgerycollege of medicine; under approval of scientific and ethics committee in Neinawa health office. Serum samples were collected from 40 individuals whose included in this work they are divided into 2 groups.
The first group included 20 apparently healthy individuals (10 males and 10 females). The second group included 20 individuals (5 males and 15 females) with Gall stone whose diagnosed in outpatient Clinic at Al-Jamhoory General Hospital and Neinawa private Hospital. Gall stone patients with metabolic diseases, infections and other inflammatory diseases were excluded.
Serum fasting glucose was assayed by glucose oxidase/peroxides colorimetric method 14 , FA by NBT-spectrophotometric method 15 and HbAc1% measured by Chromatographicspectrophotometric method 16 , While mean blood glucose (MBG) predicted HbAc1and Glycosylation gap were calculated using equations 13,17 . MBG =1.76×(HbAc1) -3.67mmol/L P-HbAc1 = 0.017×FA + 1.61 GG = M-HbAc1 -P-HbAc1 Data are presented as mean ±SD, 2-sample ttest was used to compare between measured parameters in test group and control group. The relationship between age or BMI and the measured parameters were determined by Pearson correlation.

RESULTS
The study demonstrated a significant elevation in serum glucose, mean blood glucose, serum fructosamin, measured and predicated HbAc1 in GS patients in comparison with controls Table 1.
The glycosylation gap shows a significant increase in GS patients when compared to controls.  Regarding the relation between age and measured parameters, the study showed nonsignificant correlation between age and measured parameters in the control group, while in GS group there were a significant correlation (r = 0.05) between ages and predicted HbAc1 and BMI ( p ≤ 0.03).
Regarding the relation between BMI and measured parameters, the study showed a significant correlation between measured parameters and BMI in GS individuals, while in control group only serum glucose and MBG in control group (r = 0.01) (p ≤ 0.01) with MBI.

DISCUSSION
Glycosylation gap is defined as the difference between the measured HbAc1 and HbAc1 predicted from glucose serum protein based on the HbAc1 from regression equation GG= HbAc1 measured-HbAc1 predicted. Glycation has both physiological and pathophysiological significance 18 .
Gallstone disease is one of the most prevalent gastrointestinal Diseases with a substantial burden to health. Etiology and pathogenesis of cholesterol gallstones are still not well defined, and strategies for prevention and efficient non-surgical therapies are missing 19 .
Based on the obtained results, it can be clearly stated that the glycosylation gap has elevated level in GS patients when compared to control group; furthermore, it demonstrated a significant correlation (r=0.05) between age, predicted HbAc1 and BMI (p≤0.03) among GS group 20 .
Thus we can hypothesize that significance correlation between predicted HbAc1 and BMI in GS may contribute to the increase in GG .This is in accordance with AL-kataan who described a significance correlation between MBG, serum fructoseamine, and GG with BMI in obese individuals 1 . as well as it may be associated with relation to an increase in insulin resistance that in turn associated with intravascular hyperglycemia and hypertriglyceridemia that associated with obesity and agrees with the results described by Ruderman et al.,Resnick et al and Boden et al. [21][22][23] The study showed a significant difference in mean blood glucose between GG patients and control group which is in accordance with AL-Kataan et al who demonstrated that there is a significance increase in fasting glucose (P< 0.01) in GS patient prior and posterior of Cholecystectomy 24 .
The study also revealed that the relation between age, BMI and measured parameters shows non-significance correlation between age and measured parameter in control group.

CONCLUSION
The study shows that there is a significant change in glycosylation gap and glycation profile in gall stone patients.