Study
of gall s tone composition in North Karnataka
Kalghatgi S1, K Aravind2,
Mukund Kulkarni3, Vipin T4, Sanganal B5
1Kalghatgi S, Assistant Professor, Department of Surgical
Gastroenterology, Karnataka Institute of Medical Sciences, Hubli,
Karnataka, 2K Aravind, Associate Professor, 3Mukund Kulkarni, 4Vipin T, 5Sanganal B, All authors are affiliated with Department of Surgical
Gastroenterology, Karnataka Institute of Medical Sciences, Hubli,
Karnataka, India
Address for
correspondence: K Aravind, Email: dr.suhasrk@gmail.com
Abstract
Introduction:
Gall stone disease is common problem in Indian subcontinent. While
majority of gallstone disease are cholesterol in north India (similar
to western world) they are predominantly pigment or mixed type in south
India. There are recent reports of changing trends in composition of
gallstones with a shift towards cholesterol gall stones especially in
south Asian countries, and this has been attributed to changes in life
style and dietary factors. Material
and Methods: Descriptive analytic study were conducted
on39 gallstones from KIMS, HUBLI hospital were analysed by
semiquantitative titrimetric and colourimetric methods over a period of
18 months from dec 2014 to 2016.The proportion of different types of
gallstones was described using 95% confidence interval based on exact
method. Results:
The biliary calculi collected from 39 gallstone patients were divided
into 3 groups based on their colour: cholesterol calculi, mixed calculi
and pigment calculi. Out of the 39 stones collected , 15 were mixed
calculi, 14 were pigment calculi and 10 were cholesterol calculi
indicating the incidence of gallstones in the studied population from
KIMS, Hubli as follows : Mixed calculi (38%) > pigment calculi (
36%)> cholesterol calculi (26%). The incidence of gallstone was
higher in age group 41-50 yrs followed by 51- 60 yrs and 31-40 yrs in
which females were higher than males. Conclusion: A
quantitative chemical analysis of total cholesterol, total bilirubin,
fatty acids, triglycerides, phospholipids, bile acids, soluble
proteins, sodium potassium, magnesium, copper, oxalate and chlorides of
biliary calculi (10 cholesterol, 15 mixed and 14 pigment) retrieved
from surgical operation of 39 patients from KIMS, Hubli hospital was
carried out. Although total cholesterol was a major component of
cholesterol, mixed and pigment gall stone in KIMS, Hubli, the content
of most of the other lipids, cations and anions was different in
different gall stones indicating their different mechanism of formation.
Keywords:
Gall stone,Biliary calculi, Cholesterol, Calcium carbonate, Chloride
Manuscript Received:
4th October 2017, Reviewed:
14th October 2017
Author Corrected: 20th
October 2017, Accepted
for Publication: 25th October 2017
Introduction
Gallstone is one of the diseases prevalent in developed nations, but it
is less prevalent in the developing populations that still consume
traditional diets [1]. Gallstone chemical analysis gives important
evidences for the origin, aetiology, and the metabolic basis of its
formation, and helps in the identification of risk factors that
predispose certain individuals to the calculi formation.
Many studies to identify risk factors for biliary lithiasis in the West
have focused on hypersaturation of cholesterol in bile in the
nucleation process, a critical step in the genesis of bile stones
[2,3]. The high concentration of cholesterol in gallstones has been the
basis for the widespread use of bile acids, a nonsurgical treatment for
the dissolution of gall bladder stones. These stones account for as
much as 80% of Western stones[3]. Unfortunately, gall bladder stone
composition is heterogeneous, and differs within and without
populations around the world [4-9].
Gallstone disease is increasing in India. In the west, the majority of
stones can be dissolved with bile salts, since the major component is
cholesterol. This medical therapy is expensive and not readily
accessible to poor populations. It was therefore necessary to analyze
the chemical composition of biliary stones in a group of patients, so
as to make the case for introducing bile salt therapy.
Material
and Material
Patients admittedwith a diagnosis of gall stones in the department of
general surgeryKarnataka Institute of Medical Sciences, Hubli over a
period of 18 months from December 2014 to June 2016 will includes in
study.
Study Design- Descriptive
analytic design study was conducted using questionnaire to collect
required data.
Inclusion criteria-
patients with definitive diagnosis of gallstone based on ultra sonogram
of abdomen
Exclusion criteria-
Patients less than 15 yearsold, overt hemolysis, co morbidities like
pancreatitis and those who are not willing for participation in the
study.
Collection of data- Controls
are age and sex matched asymptomatic subjects with a normal gall
bladder from same population coming to KIMS Hubli for regular health
check up or admitted in other department, taking ultra sonogram for
purposes not related with gall stone. Cases (n = 39) from all age
groups and both sexes with sonographically proven gallstones were
recruited over a duration of 18 months from the surgical wards of a
KIMS, HUBLI Hospital from December 2014 to 2016.
The stones were divided
into 3 groups depending upon their colour: pale yellow and
whitish stones as cholesterol calculi, black and blackish brown as
pigment calculi and brownish yellow or greenish with laminated features
as mixed calculi. The other relevant information about the patients
such as age, sex and number of calculi were obtained from hospital
records. The various physical parameters of stones such as number,
shape, size, texture and cross-section were noted. The stones were
powdered in a pestle and mortar and dissolved in different solvents
depending upon the type of chemical constituent to be analyzed. To
determine total cholesterol and total bilirubin, 30mg stone powder was
dissolved in 3 ml chloroform in a test tube. The tube was kept in
boiling water bath for 2 min. The stone solution thus obtained was used
for determination of total cholesterol and total bilirubin. To
determine calcium, oxalate, inorganic phosphate, magnesium, chloride,
soluble protein, triglycerides, iron, copper, sodium and potassium, 30
mg stone powder was dissolved in 3 ml IN HCl in graduated 10 ml tube
and its final volume was made up to 10 ml with distilled water. The
tube was kept in boiling water bath for 1 hr. To analyze phospholipids,
stone powder (20 mg) was dissolved in 15 ml CHCl3+CH3OH in 2:1 ratio,
containing 1N HCl. To measure bile acids and fatty acids, the stones
were dissolved in chloroform-methanol (2:1) mixture and ethyl
alcohol-solvent ether in (3:1 mixture) respectively. Total cholesterol
by enzymic colorimetric method of Bayer Diagnostic India Ltd [10],
total bilirubin by colorimetric method method of Accurex Biomedical
Pvt. Ltd [11], triglycerides by enzymatic colorimetric method of Bayer
Diagnostics India Ltd [11], soluble protein by colorimetric method of
Lowry et al [12].
Statistical analysis-
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.Descriptive analysis was done using, mean and standard
deviation for quantitative variables, frequency and percentages for
categorical variables. The comparison between explanatory and outcome
variables was assessed by comparing the means for quantitative
variables and proportions for categorical variables. Independent sample
t-test and chi square test were used appropriately to assess the
statistical significance. Appropriate graphs like, bar diagram and pie
diagram were used to visually represent the data. IBM SPSS version 21
was used for statistical analysis
Results
The biliary calculi collected from 39 gallstone patients were divided
into 3 groups based on their colour: cholesterol calculi, mixed calculi
and pigment calculi. Out of the 39 stones collected , 15 were mixed
calculi, 14 were pigment calculi and 10 were cholesterol calculi
indicating the incidence of gallstones in the studied population from
KIMS, Hubli as follows : Mixed calculi (38%) > pigment calculi (
36%)> cholesterol calculi (26%).
Table No.-1:Incidence of
different types of gallstones in relation to age
|
Age group(yrs)
|
Cholesterol
|
Mixed
|
Pigment
|
Total gallstones
|
|
1-10
|
-
|
-
|
-
|
-
|
|
11-20
|
1
|
-
|
1
|
2
|
|
21-30
|
1
|
3
|
1
|
5
|
|
31-40
|
2
|
2
|
4
|
8
|
|
41-50
|
4
|
4
|
3
|
11
|
|
51-60
|
2
|
2
|
4
|
8
|
|
>60
|
1
|
1
|
2
|
4
|
|
Total
|
10
|
15
|
14
|
39
|
Out of the total number of stones collected, the incidence of gallstone
was higher in age group 41-50 yrs followed by 51- 60 yrs and 31-40 yrs.
Table-2: Descriptive
analysis of cases & controls in study group (N=79)
|
Cases & Controls
|
Frequency
|
Percent
|
|
Cases
|
39
|
49.4
|
|
Controls
|
40
|
50.6
|
Out of the 79 subjects, 39 were cases and remaining 40 were controls.
Table-3: Comparison of
Age and gender parameters across the two study groups (N=79)
|
Parameter
|
Cases(N=39)
|
Controls(N=40)
|
P-value
|
|
Age
|
45.56±14.65
|
40.20±15.79
|
0.12
|
|
Female: Male
|
1:0.25
|
1:0.29
|
0.83
|
Above table shows mean age was 45.56 ±14.65 for gall stones
and females were higher than males.
Table-4: Comparison of
bilirubin parameters across the two study groups (N=79)
|
Parameter
|
Cases(N=39)
|
Controls(N=40)
|
P-value
|
|
Total protein
|
8.25±8.54
|
7.88±8.50
|
0.85
|
|
Albumin
|
3.75±0.64
|
3.48±0.733
|
0.08
|
|
Total Bilirubin
|
1.42±2.17
|
0.68±0.33
|
0.03
|
|
Direct Bilirubin
|
0.67±1.03
|
0.31±0.22
|
0.03
|
|
AST
|
45.40±36.73
|
34.57±17.51
|
0.09
|
|
ALT
|
40.23±49.34
|
24.63±17.69
|
0.06
|
|
ALP
|
131.20±120.07
|
77.37±33.65
|
0.008
|
The mean total protein at cases was 8.25±8.54 and
7.88±8.50 was at controls which was statistically not
significant. The mean albumin was 3.75±0.64 at cases and
3.48 at controls with a p value 0.08 which was not significant.
Table-5: Quantitative
analysis of metabolites in different types of biliary calculi expressed
as mg/gm dry stone powder.
|
Parameter
|
Mean±STD
|
Median
|
Max
|
Min
|
95%
C.I.for EXP(B)
|
|
Lower
|
Upper
|
|
Weight in Gms
|
5.58±13.82
|
2.00
|
59.0
|
1.0
|
-1.51
|
12.69
|
|
Cholesterol
|
13.45 ±30.45
|
1.46
|
110.00
|
0.12
|
-2.20
|
29.10
|
|
TG mg/dl
|
7.04±18.23
|
1.90
|
76.90
|
0.11
|
-2.33
|
16.41
|
|
Bilirubin
|
0.81 ± 0.75
|
1.00
|
2.10
|
0.01
|
0.42
|
1.20
|
|
Protein
|
2.01 ±1.40
|
2.10
|
4.8
|
0.2
|
1.29
|
2.74
|
|
Chloride
|
98.84 ±73.17
|
63.00
|
196.0
|
0.72
|
61.22
|
136.46
|
|
Magnesium
|
2.66 ± 1.84
|
2.15
|
6.08
|
0.82
|
1.72
|
3.61
|
|
Phosphorous mg/dl
|
31.93±61.56
|
2.90
|
200.18
|
0.00
|
0.28
|
63.58
|
|
Calcium
|
16.20 ±8.28
|
20.05
|
29.00
|
0.04
|
11.95
|
20.46
|
|
Copper
|
444.50±721.11
|
179.90
|
2535
|
14.40
|
73.73
|
815.26
|
|
Iron
|
530.83 ±414.57
|
732.50
|
998.6
|
6.4
|
317.67
|
743.99
|
The mean weight of the gall stone was 5.58 ±13.01 in study
population. The mean cholesterol was 13.45±30.45 and
7.04±18.23 was triglycerides in study population. The mean
bilirubin was 0.81±0.75 and the mean protein was
2.01± 1.40respectively. The mean Chloride, magnesium and
phosphorouswere 98.84 ± 73.17, 2.66±1.84 and
31.93±61.56 respectively in study population.
Discussion
The biliary calculi collected from 39 gallstone patients were divided
into 3 groups based on their colour: cholesterol calculi, mixed calculi
and pigment calculi. Out of the 39 stones collected, 15 were mixed
calculi, 14 were pigment calculi and 10 were cholesterol calculi
indicating the incidence of gallstones in the studied population from
KIMS, Hubli as follows : Mixed calculi (38%) > pigment calculi (
36%)> cholesterol calculi (26%), which is similar to study done
by chandran et al [13] where they have reported quantitative chemical
analysis of total cholesterol bilirubin, calcium, iron and inorganic
phosphate in 120 gallstones from haryana. To extend this chemical
analysis of gall stones by studying more cases and by analyzing more
chemical constituents. A quantitative chemical analysis of total
cholesterol, total bilirubin, fatty acids, triglycerides,
phospholipids, bile acids, soluble proteins, sodium potassium,
magnesium, copper, oxalate and chlorides of biliary calculi (52
cholesterol, 76 mixed and 72 pigment) retrieved from surgical operation
of 200 patients from Haryana state was carried out. Total cholesterol
as the major component and total bilirubin, phospholipids,
triglycerides, bile acids, fatty acids (esterified), soluble protein,
calcium, magnesium, iron, copper, sodium, potassium, inorganic
phosphate, oxalate and chloride as minor components were found in all
types of calculi.
Jaraari AM et al [14] 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, which is similar to
our study.
In our study, Out of the total number of stones collected, the
incidence of gallstone was higher in age group 41-50 yrs followed by
51- 60 yrs and 31-40 yrs. mean age was 45.56 ±14.65 for gall
stones and females were higher than males. similarly Hussain SM et
al[15] found that the highest incidence of gallstones in the age group
40-49 was 13 cases followed by 11, 8 and 4 cases for age groups 30-39,
50-59, 20-29 and 60 and above, respectively. The chemical analysis
showed the majority of gallstones were mixed, 38 containing calcium
followed by 37 cases with uric acid, 28 with magnesium, and 25 and 22
stones with oxalate and phosphate, respectively.
Atamanalp SS et al [16] found that High serum cholesterol and LDL
levels were associated with high cholesterol stone rates (86.7% vs.
40.0%, P < 0.001; 75.0% vs. 48.9%, P < 0.05,
respectively). Similarly, high serum cholesterol and LDL levels were
correlated with high gallbladder stone cholesterol concentrations
(63.6% vs. 44.4%, P < 0.001; 62.3% vs. 46.0%, P < 0.001,
respectively). In contrast, low serum HDL levels do not seem to affect
the occurrence of gallbladder cholesterol stones (60.0% vs. 58.3%,
respectively, P > 0.05) or gallbladder stone cholesterol
concentrations (50.8% vs. 52.4%, respectively, P > 0.05).
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 [17]. 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 co-existence of nucleating factors, gallbladder hypomotility
[18], and mucus hyper secretion also contribute to cholesterol
precipitation leading to the development of gallstones [18].
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 [19]. The incidence of
gallstones increases with an increase in age, with females more likely
to form gallstones than males. Age 50 – 65 approximately 20%
of women and 5% of men have gallstones. Overall, 75% of gallstones are
composed of cholesterol, and the other 25% are pigmented. Despite the
composition of gallstones the clinical signs and symptoms are the
same[20].
Ramana Ramya J et al [21] foundRepresentative gallstones from north and
southern parts of India were analyzed by a combination of
physicochemical methods: X-ray diffraction (XRD), infrared spectroscopy
(IR), scanning electron microscopy (SEM), energy-dispersive X-ray
spectroscopy (EDX), CHNS analysis, thermal analysis and Nuclear
Magnetic Resonance (NMR) spectroscopy (1H and 13C). The stones from
north Indian were predominantly consisting of cholesterol monohydrate
and anhydrous cholesterol which was confirmed by XRD analysis. FTIR
spectroscopy confirmed the presence of cholesterol and calcium
bilirubinate in the south Indian gallstones.
Jayanthi V et al [22]Gallstones (GS) in south India (SI) are
predominantly pure pigment or mixed, while in North India (NI), these
are either pure cholesterol or mixed. While cholesterol rich
gallbladder (GB) bile predicts cholesterol GS, constituent of bile in
primary pigment GS is not known. Gallbladder bile in controls and
patients with GS from north India had significantly high cholesterol
concentration. In south India, patients with mixed GS had cholesterol
rich bile while pigment GS had higher concentrations of bile salts.
Conclusion
Total cholesterol as the major component and total bilirubin,
phospholipids, triglycerides, bile acids, fatty acids (esterified),
soluble protein, calcium, magnesium, iron, copper, sodium, potassium,
inorganic phosphate, oxalate and chloride as minor components were
found in all types of calculi. The cholesterol stones had higher
content of total cholesterol; phospholipids, fatty acids (esterified),
inorganic phosphate and copper compared to mixed and pigment stones.
Further, considering that cholesterol levels in the gallstones mirrors
the serum cholesterol levels, health issues associated with increased
cholesterol levels, such as cardiovascular diseases, might be
associated. However, larger randomized studies are required to study
this association and to confirm these observations.
Funding:
Nil, Conflict of
interest: None initiated.
Permission from IRB:
Yes
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How to cite this article?
Kalghatgi S, K. Aravind, Mukund Kulkarni, Vipin T, Sanganal B. Study of
gall stone composition inNorth Karnataka. Surgical Update: Int J surg
Orthopedics. 2017;3(4):93-98.doi:10.17511/ijoso.2017.i4.01.