LJM 1993-2820 1819-6357 Libyan Journal of Medicine Vol. 5, January 2010, pp. 1–6 sici xxx PUBMED Abbreviation AbdallaM.Jaraari PeelaJagannadharao

*Peela Jagannadharao, Department of Biochemistry, Faculty of Medicine, Al-Arab Medical UniversityBenghaziLibya

TrushakantN.Patil AbdulHai HayamA.Awamy SaeidO.El Saeity EzedinB.Abdel Kafi MaisoonN.El-Hemri MahmoodF.Tayesh Department of Biochemistry, Faculty of Medicine Al-Arab Medical University

BenghaziLibya

Department of Surgery, Faculty of Medicine Al-Arab Medical University

BenghaziLibya

Libyan Journal of Medicine ORIGINAL ARTICLE Quantitative analysis of gallstones Gallstone disease is one of the major surgical problems in the Libyan population; it is probably related to diet, especially excessive consumption of meat. The study was conducted to determine the composition of gallstones and their possible etiology in a Libyan population. The chemical composition of gallstones from 41 patients (six males and 35 females) was analyzed. The stones were classified into cholesterol, pigment, and mixed stones (MS). Cholesterol stones (CS) showed a significantly higher cholesterol content than pigment stones (PS) (p=0.0085) though not significantly higher than MS. Their phospholipid content and inorganic phosphates were higher than in the other types of stones and oxalate content was significantly elevated in comparison with MS (p=0.0471). In MS, the cholesterol, bile acids, and bilirubin were intermediate between cholesterol and PS, whereas triglycerides were significantly more than PS (p=0.0004). Bilirubin (0.0001) and bile acids (p=0.0009) were significantly higher than CS (p=0.0001). However, they contained the lowest amounts of sodium, potassium, magnesium, and oxalate. In PS, bilirubin (p=0.0001) was significantly higher than both groups. Bile acid content was significantly higher than CS (p=0.0001) but not significantly more than MS. They showed the highest values of calcium, sodium, potassium, magnesium, and chlorides compared to the other types of stones. High levels of cholesterol in stones and dyslipidemia associated with mixed as well as cholesterol gallstones suggest an etiological association and efforts to reduce dietary fat among the Libyan population may lead to decreased cholesterol and mixed gallstones. gallstones chemical composition Libya cholesterol Cholelithiasis or gallbladder stones are one of the major surgical problems in the Libyan population and account for many hospital admissions and surgical interventions. Most patients with gallstones present with severe abdominal colic requiring investigations and treatment. Many of them need surgical intervention by the time they are symptomatic. This problem is probably related to obesity, cardiovascular disorders (CVD), metabolic syndrome, and dietary habits, especially excessive consumption of meat, which is known to contain large amounts of cholesterol. Obese individuals with a BMI > 30 kg/m² have 95% cholesterol-dominant gallstones and are at a high-risk for cholesterol stones (CS) 1. The mechanism of stone formation has been the subject of extensive research for several years. However, there are no reports on the chemical composition of gallstones in Libya though a sizeable population suffers from gallstones. Earlier studies reported only on prevalence and case reports on gallstones in Libya 234. Studies on gallstone composition carried out in different parts of the world indicate a close link with dietary habits and ethnicity 56789101112131415. Studies have also shown that dietary intake of total calories in the form of carbohydrates and fats were associated with high triglyceride levels in gallstone patients 16. Gallstone formation is relatively increased with consumption of dietary fats rich in saturated fatty acids. Consequently, substitution of 18:1 with unsaturated fatty acids present in low-fat diets would reduce gallstone formation without affecting the lithogenic index 17. This study describes an extensive quantitative analysis of gallstones in Libyan patients, including cholesterol, triglycerides, phospholipids, bilirubin, bile acids, calcium, phosphorus, sodium, potassium, magnesium, oxalates, and chlorides. 2 Gallstones from 41 patients with cholelithiasis were collected after cholecystectomy at the Department of Surgery, Seventh October Hospital, Benghazi, Libya, between March and September 2008. This study was approved by the ethics committee of the Al-Arab Medical University, Benghazi, Libya. The stones were classified into three types depending on their color and degree of hardness. Yellow and whitish stones were identified as CS, black and dark brown as pigment stones (PS), and brownish yellow or green as mixed stones (MS). Demographic data on age and sex was also collected. The stones were powdered using a mortar and dissolved in different solvents depending on the type of chemical constituent to be analyzed. To determine total cholesterol and total bilirubin, 30 mg stone powder was dissolved in 3 ml chloroform in a test tube. The tube was kept in boiling water bath for 2 min. Aliquots from these samples were used for determination of total cholesterol and total bilirubin. To determine calcium, oxalate, inorganic phosphate, magnesium, chloride, triglycerides, sodium, and potassium, 30 mg of the powdered stone was dissolved in 3 ml of HCl in a graduated 10-ml tube and the volume was made up to 10 ml with distilled water. The tubes were kept in a boiling water bath for one hour. To analyze phospholipids, 20 mg of powdered stone was dissolved in 15 ml of a 2:1 mixture of chloroform and methanol containing 1 N HCl. To measure bile acids, the stones were dissolved in chloroform–methanol (2:1) mixture. The solutions were preserved at 4°C until they were used.Total cholesterol was estimated by a colorimetric enzymatic method (Biocon Diagnostics, Germany) 18, total bilirubin by Accurex Biomedicals 19, triglycerides by an enzymatic colorimetric method of Biocon Diagnostics 20, oxalate by the method described by Satyapal and Pundir based on colorimetric enzymatic method 21, calcium by an o-cresolphthalein complexone (OCPC) kit (Biocon Diagnostics) 22. Phospholipid and inorganic phosphate were determined according to Fiske and Subba Rao 23, magnesium by xylidyl blue (Biocon Diagnostics) 24, chloride by the method of Schoenfeld 25, sodium and potassium by flame photometry (Clinical flame photometer, Evano Electro Selenium Halsted, Essex, UK), and bile acids by the colorimetric method of Carey 26. For colorimetric procedures, we used reagents from Biocon Diagnostics, Germany, and a spectrophotometer from Labomed Inc., USA (UV-Vis-1179, RS Spectrophotometer). Statistical analyses were performed with GraphPad software (GraphPad Software Inc., USA). The unpaired t-test was used for comparison of group means. A p-value of <0.05 was considered significant. 3 All 41 patients (six males and 35 females) had multiple gallstones. CS were bigger than MS and PS. Fourteen of the patients (34%) had MS, 16 (39%) had PS, and 11 (27%) had CS (Table 1). The incidence of gallstones was highest in age group of 41–50 years (Table 2). Cholesterol stones Mixed stones Pigment stones Sex Females 11 12 12 Males 0 2 4 Shape Round 3 Irregular 8 Round 4 Irregular 10 Round 6 Irregular 10 Color Yellow 9, White 2 Green 12, Greenish brown 2 10 brown, 6 black Surface Smooth 6 Rough 5 Smooth 5 Rough 5 Smooth 12 Rough 4 Weight (g, mean) 5.40 1.78 3.00 Character Soft 9, hard 2 Soft 11, hard 3 Soft 10, hard 6 Size (cm, mean) 0.8×0.9 0.6×0.7 0.9×0.9 Age group (years) CS MS PS Total Note: CS, cholesterol stones; MS, mixed stones; PS, pigment stones. ≤20 0 0 1 1 21–30 2 2 5 9 31–40 5 5 2 11 41–50 2 4 6 12 51–60 1 3 1 5 ≥61 1 0 1 2 11 14 16 41 CS had the highest composition of cholesterol, while MS had a high content of triglycerides and PS were comprised mostly of bilirubin (Tables 3 and 4). Stone type Cholesterol (mg/gm) Triglycerides (mg/gm) Bilirubin (mg/gm) Bile acids (mg/gm) PL (mg/gm) Note: CS, cholesterol stones; MS, mixed stones; PS, pigment stones; PL, phospholipids. Values are the mean and standard deviation. CS 608±173 51±14 0.5±0.30 16.5±2.60 8.0±2.70 MS 518±125 56±10 2.0±0.30 20.0±2.00 5.7±0.90 PS 466±87 40.5±11 4.0±0.90 22.0±3.60 6.0±1.40 Groups Cholesterol Triglycerides Bilirubin Bile acids Phospholipids Note: CS, cholesterol stones; MS, mixed stones; PS, pigment stones. CS vs. MS 0.1472 0.3186 0.0001 0.0009 0.0080 CS vs. PS 0.0085 0.0263 0.0001 0.0001 0.0170 MS vs. PS 0.1983 0.0004 0.0001 0.8521 0.5101 The concentrations of different ions also varied with the type of stone. PS had the highest concentrations of most of the ions, including calcium, sodium, potassium, magnesium, and chloride, whereas CS contained higher concentrations of phosphates and oxalates (Tables 5 and 6). Type Ca^2 + PO₄⁻ Na⁺ K⁺ Mg^2 + Cl⁻ Oxalate Note: CS, cholesterol stones; MS, mixed stones; PS, pigment stones. Values are the mean and standard deviation. CS 11.4±2.40 14.0±1.60 1.4±0.50 0.5±0.17 8.5±1.80 19.5±1.50 7.0±1.18 MS 18.0±4.70 10.5±1.90 1.15±0.18 0.32±0.06 7.75±0.90 21.5±1.90 6.0±1.20 PS 21.75±3.60 9.0±1.50 3.5±0.73 0.65±0.17 10.8±1.25 33.5±3.60 6.5±0.90 Comparison Ca^2 + PO₄⁻ Na⁺ K⁺ Mg^2 + Cl⁻ Oxalate Note: CS, cholesterol stones; MS, mixed stones; PS, pigment stones. CS vs. MS 0.0002 0.0001 0.1044 0.0016 0.1952 0.0080 0.0471 CS vs. PS 0.0001 0.0001 0.0001 0.0291 0.0005 0.0001 0.2140 MS vs. PS 0.0218 0.0248 0.0001 0.0001 0.0001 0.0001 0.2105 4 4.1 Cholesterol content was found to be highest in CS. This is because the cholesterol saturation index is more than 1 between cholesterol and bile salts 27. The finding that the highest cholesterol content was in CS reiterates that these type of stones are formed primarily because of supersaturation of cholesterol in the bile, which precipitates as a stone. The pathogenesis of cholesterol gallstones is rooted in altered lipid metabolism, e.g. hyperlipidemia type IIa and type IV give rise to a greater proportion of cholesterol relative to other bile lipids secreted from the liver into bile 2829. The co-existence of nucleating factors, gallbladder hypomotility 28, and mucus hyper secretion also contribute to cholesterol precipitation leading to the development of gallstones 3031. 4.2 Bile salts were significantly lower in CS than in MS and PS, while bile acids were significantly higher in PS. Supersaturation of bile with calcium bilirubinate is inhibited by bile salts, which bind calcium, reducing the activity of free calcium ions. When supersaturation occurs, usually due to increased concentrations of bilirubinate anion, nucleation may be initiated by binding of calcium bilirubinate to mucin glycoproteins in bile 3132. Similarly, the phospholipid content was marginally higher in cholesterol than PS and MS. Cholesterol has to be in relative proportion with the bile salts and phosphatidyl choline to remain soluble in the bile and thereby avoid stone formation 3334. About 5% of cholesterol is barely soluble in 20% phosphatidyl choline and this should require approximately 60% of bile salts. Any concentration above this level would cause precipitation of cholesterol, as shown by the presence of the highest amount of cholesterol in CS is an indicator of this fact 35. PS contained less cholesterol than the other two types of stone. Cholesterol in PS is mostly because of co-precipitation with bilirubin and other compounds but not due to its supersaturation in the bile 36. In contrast to this, MS have less cholesterol than CS but more than PS. This might be due to the presence of both cholesterol and bile pigment in these stones. 4.3 Triglyceride content was higher in MS than in the other two types of stones, but the difference was significant only compared to PS (p=0.0004). Triglycerides accumulate along with cholesterol salts to form gallstones. The higher content of triglycerides in MS or CS compared to PS might be due to a higher deposition of calcium salts of cholesterol and esters of fatty acids in MS and CS when compared to PS in which calcium bilirubinate is the major salt 37. 4.4 Phospholipid content was highest in CS and lowest in MS. There were significantly more phospholipids in CS than in MS (p=0.0080) and PS (p=0.0170).This might be due to accumulation of phospholipids along with cholesterol during CS formation. 4.5 As expected, the bilirubin content was highest in PS. This has been demonstrated in many earlier studies 32. The bilirubin in these stones is mostly unconjugated because the conjugated form is water soluble and excreted through bile 38. Decreased secretion of biliary acids, increased secretion of unconjugated bilirubin into the bile, and infection of the biliary tract are the most important causative factors 3940. 4.6 Calcium content was highest in PS, while phosphorus was lowest. It is known that bilirubin combines with calcium to form a precipitate of calcium bilirubinate 41. Since PS have excess bilirubin, calcium forms calcium bilirubinate 42. 4.7 Phosphorus content was low in PS. The reason for this is not clear but may be related to the availability of calcium, which is more in these stones in the form of calcium bilirubinate. Phosphorus content was significantly higher in CS and MS than PS. It is likely that phosphorus may play a more important role than calcium in CS formation by forming a salt with calcium, which might be responsible for the hardness of the CS 6. 4.8 Sodium content was higher in PS than in the other types. It was not surprising that bile acids were more in PS because an increase in sodium content facilitates excessive formation of bile salts. Potassium was also higher in PS. It is presumed that the sodium to potassium ratio will be maintained in the bile. Hence, higher sodium content is associated with higher potassium content, although the increase in the latter was not as much as that of the sodium content. 4.9 Chlorides were higher in PS than MS and CS. Chlorides are among the important constituents of bile, and they may form salts with sodium, thereby contributing to stone formation. 4.10 Magnesium was higher in PS and CS than MS. This is in conformity with observations made by Chandran et al. 13. 4.11 Since the higher oxalate content in CS is likely to be associated with higher magnesium content, the formation of magnesium oxalate may be responsible for the hardness of PS.The Libyan population seems to be more susceptible to cholelithiasis when compared with other countries according to earlier studies in Libya 23. The observation that CS are the most prevalent type of gallstones is in agreement with studies performed in northern India 6711, Japan 14, and Singapore 15, but differs from sub-Saharan countries and southern India, where PS are more prevalent, with a higher bilirubin and calcium content and lower cholesterol content when compared with stone composition in Libya 4344.The potential reasons for stone formation may be attributed to (a) dietary factors, (b) multiple pregnancies with less spacing between pregnancies, (c) metabolic syndrome, (d) familial, and (e) ethnic 454647. It is known that foods that are rich in lipids result in development of hyperlipidemias and subsequent increase in all lipid constituents. There is no corresponding increase in substances such as bile salts and phospholipids, which are responsible for solublization of cholesterol and other biliary constituents. Consequently, there is a precipitation of cholesterol and bilirubin leading to stone formation 40.Comparison of the chemical composition of gallstones in Libyan individuals with those in other countries is summarized in Table 7. Though cholesterol is a major component of gallstones, the composition of gallstones varies from country to country and region to region. One study in sub-Saharan Africa showed that majority of stones were of the pigment type, in which the contribution of cholesterol is less than in other stones 43. Similarly, a study in southern India reported predominance of pigment and mixed gallstones with reduced cholesterol and increased bilirubin and calcium concentrations in the stones 44. Country and reference No. of stones Cholesterol (%) Triglycerides (%) Bilirubin (%) Calcium (%) PO₄⁻_, Mg, Oxalates, Na⁺, K⁺, and Cl⁻ (%) Australia 49 5 93 3.5 3.5 England 51 11 66 17 17 Germany 1 1,025 93.3 5.5 4.8 India 13 200 56.6 5.7 0.3 2.2 35.2 Kuwait 51 10 60 37 3 South Africa 51 11 66 29 5 Sweden 51 27 94 4 2 USA 51 42 88 9 3 Bolivia 50 66 93.3 4.1 Libya (present study) 41 53 4.9 2.16 1.70 38.4 CS are more prevalent than PS in north India, Australia, Bolivia, Germany, England, Kuwait, USA, Sweden, and South Africa 48495051. Although all types of gallstones are prevalent among the Libyan population, it is interesting to note that cholesterol is the major component of all stones. 5 An interesting finding of our study is that although PS was the most common type of gallstones, cholesterol seemed to be the major component in all types of stones. High cholesterol content in CS especially suggests supersaturation of cholesterol in bile consequent to dyslipidemia (excessive cholesterol and altered lipid metabolism) is an etiological factor. Higher triglyceride content in MS also suggests that dyslipidemic changes contribute to etiology. Our findings suggest that dyslipidemia consequent to high intake of fats by Libyan population may be responsible for gallstones, and dietary modification might reduce the incidence of gallstones. 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