Acta Tropica 138 (2014) 28–37
Contents lists available at ScienceDirect
Acta Tropica journal homepage: www.elsevier.com/locate/actatropica
Clonorcis sinensis eggs are associated with calcium carbonate gallbladder stones Tie Qiao a,b,∗ , Rui-hong Ma c , Zhen-liang Luo c , Liu-qing Yang b,c , Xiao-bing Luo c , Pei-ming Zheng c a
The Second People’s Hospital of Panyu, Panyu, Guangzhou 511430, Guangdong Province, PR China Institute of Gallbladder Disease of Panyu, Guangzhou 511430, Guangdong Province, PR China c The Sixth People’s Hospital of Nansha, Nansha, Guangzhou 511470, Guangdong Province, PR China b
a r t i c l e
i n f o
Article history: Received 9 January 2014 Received in revised form 3 June 2014 Accepted 9 June 2014 Available online 16 June 2014 Keywords: Calcium carbonate stone Clonorchis sinensis Calcite type calcium carbonate Aragonite type calcium carbonate Scanning electron microscopy
a b s t r a c t Calcium carbonate gallbladder stones were easily neglected because they were previously reported as a rare stone type in adults. The aim of this study was to investigate the relationship between calcium carbonate stones and Clonorchis sinensis infection. A total of 598 gallbladder stones were studied. The stone types were identified by FTIR spectroscopy. The C. sinensis eggs and DNA were detected by microscopic examination and real-time fluorescent PCR respectively. And then, some egg-positive stones were randomly selected for further SEM examination. Corresponding clinical characteristics of patients with different types of stones were also statistically analyzed. The detection rate of C. sinensis eggs in calcium carbonate stone, pigment stone, mixed stone and cholesterol stone types, as well as other stone types was 60%, 44%, 36%, 6% and 30%, respectively, which was highest in calcium carbonate stone yet lowest in cholesterol stone. A total of 182 stones were egg-positive, 67 (37%) of which were calcium carbonate stones. The C. sinensis eggs were found adherent to calcium carbonate crystals by both light microscopy and scanning electron microscopy. Patients with calcium carbonate stones were mainly male between the ages of 30 and 60, the CO2 combining power of patients with calcium carbonate stones were higher than those with cholesterol stones. Calcium carbonate gallbladder stones are not rare, the formation of which may be associated with C. sinensis infection. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Cholecystolithiasis is a worldwide common disease with a mean prevalence rate of 10–20% in western countries (Kratzer et al., 1999). Most of patients (60–80%) with gallbladder stones do not have symptoms (Gibney, 1990). A small part of patients (20–40%) have symptoms may due to cholecystitis or migration of small gallstone from gallbladder to cystic duct or common bile duct, which may lead to major complications such as acute pancreatitis, even gallbladder cancer (Cho et al., 2010; Venneman et al., 2005a,b; Jain et al., 2013). For over 100 years, cholecystectomy has been the main treatment for cholecystolithiasis, although it may lead to complications such as bile duct injury, biliary leakage, injury of the gastrointestinal tract (Flum et al., 2003; Singh et al., 2010; Bano and
∗ Corresponding author at: The Second People’s Hospital of Panyu, No. 7 Gangdong Road, Dashi Town, Panyu, Guangzhou 511470, PR China. Tel.: +86 20 34999950; fax: +86 20 34999950. E-mail address: [email protected] (T. Qiao). http://dx.doi.org/10.1016/j.actatropica.2014.06.004 0001-706X/© 2014 Elsevier B.V. All rights reserved.
Kapoor, 2010), dyspepsia, and decreased appetite as well as other metabolic consequences (Girometti et al., 2010; Luman et al., 1996; Nervi and Arrese, 2013). Gallbladder-preserving cholelithotomy, a surgical treatment for gallbladder stones, has been advocated by Chinese doctors in recent years (Qiao et al., 2012a). The aim of this operation is to remove stones and retain the functional gallbladder. It is a safe, minimally invasive therapy, with good effect, quick recovery and no damage to organs as well as low recurrence rate (Qiao et al., 2012a; Zha et al., 2013). In addition, it provides important data for the research on cholecystolithiasis. As cholecystolithiasis not only causes suffering in patients, but also places a heavy economic burden on countries and societies (Shaffer, 2006), understanding the pathogenesis and reducing the incidence of gallbladder stones has become one of the main goals of modern medical science. Extensive work has been done to investigate the formation of gallbladder stones, and a number of studies have shown that different types of gallbladder stones have different formation mechanisms (Stolk et al., 2001; Matyja et al., 2013; Stender et al., 2013; Blázovics, 2007; Chuang et al., 2013; Wang et al., 2009; Abeysuriya et al., 2010; Nakeeb et al., 2002; Hattori et al., 2000).
T. Qiao et al. / Acta Tropica 138 (2014) 28–37
Clonorchiasis is a common amphixenosis. Infection is caused by ingesting raw or undercooked freshwater fish or shrimp with living Clonorchis sinensis metacercaria (Kaewpitoon et al., 2008; Hong and Fang, 2012; Huang et al., 2012; Chen et al., 2010). The epidemiological data showed that more than 35 million people including 15 million Chinese are infected with C. sinensis in South-East Asia (Control of foodborne trematode infections, 1995). Meanwhile, annual incidence rate of clonorchiasis from 2006 to 2012 was 166.76, 191.55, 247.37, 213.82, 246.03, 274.71, and 239.63 (per 100 000), respectively in Guangzhou, China (Li et al., 2014). Previous studies have demonstrated that intrahepatic bile duct stones and gallbladder stones, especially pigment gallbladder stones, are associated with C. sinensis infection (Jang et al., 2007; Lim, 1991; Qiao et al., 2012b), but the relationship between C. sinensis infection and calcium carbonate stones has not been reported. It has been reported previously that the calcium carbonate stone is a rare type of stone, the formation of which is not clear (Goto et al., 1996; Beauregard and Ferguson, 1980; Wu et al., 2001; Phemister et al., 1931). The research data demonstrate that calcium carbonate stone is not a rare type and should never be neglected, and it is closely associated with C. sinensis infection. The aim of this study was to investigate the relationship between calcium carbonate gallbladder stone and C. sinensis eggs. 2. Materials and methods 2.1. Ethics statement A written informed consent was obtained from both adult subjects and parents/guardians on behalf of child subjects. This research was approved by the Medical Ethics Committee of The Second People’s Hospital of Panyu, Guangzhou. 2.2. Subjects and specimens Surgical indications for gallbladder-preserving cholelithotomies were as follows: (1) gallstones or gallbladder polyps with clinical symptoms, (2) gallbladder polyps with a diameter of 10 mm or more, and (3) gallbladder stones complicated by gallbladder polyps. All patients with gallbladder cancer or gallbladder atrophy and those who could not tolerate gallbladder surgery were excluded as demonstrated by Qiao et al. (2012a). Five hundred ninety-eight gallbladder stones from 598 cholecystolithiasis patients that had received gallbladder-preserving cholelithotomies in the Department of General Surgery of The Second People’s Hospital between May 2009 and October 2012 were obtained. All the recruited samples were gallbladder stones, most of them were isolated gallbladder stones and only about 15% combined with intrahepatic or common bile duct stones, but intrahepatic or common bile duct stones were not recruited in the present study. These patients consisted of 312 males and 286 females. The age range of male and female patients was 17–77 (mean = 44.90 ± 12.04) and 10–80 (mean = 46.04 ± 13.18) years old respectively. There was no significant difference when comparing the age of male vs. female groups by independent sample t-test. 2.3. Composition analysis of gallbladder stones by Fourier transform infrared (FTIR) spectroscopy The main components of the gallbladder stones were analyzed by FTIR spectroscopy and were used to identify the stone type. For the methods please refer to Qiao et al. (2012b). The main components were analyzed using a Bruker (TENSOR27, Germany) FTIR spectrometer. Gallstone composition was determined by comparison of gallbladder stones with standard control spectra (Sossé Djessou et al., 2010; Yoo et al., 2008). Control substances, which
29
were of high quality (99% pure), were purchased from Sigma Chemical Company (St. Louis, MO, USA), while the others were analytical reagents. 2.3.1. Identification of stone type Gallbladder stones were classified according to the appearance, profile structure, composition, microstructure, elemental composition and distribution. For the detailed methods please refer to Qiao et al. (2013). 2.4. Microscopic examination of ground gallbladder stone Gallbladder stones, obtained directly during the operation, were washed twice with distilled water, and then dried at 60 ◦ C for 12 h. For the method of microscopic examination of gallbladder stones please refer to Qiao et al. (2012b). 2.5. Detection of C. sinensis DNA in the gallbladder stones by real-time fluorescent PCR Gallbladder stones that were positive or negative for eggs (20 each) based on morphology were chosen randomly from the stone samples for DNA detection. For the method of extraction of DNA please refer to Qiao et al. (2012b). Fluorescent PCR: Through the sequence alignment in NCBI BLAST, the cytochrome C oxidase subunit 1 gene of C. sinensis (COX-1, GenBank: FJ965388.1), which is highly conserved and specific, was chosen as a target. The primers and Taqman probe were designed according to its sequence. These primers Cs-F (5 -GGTTTGGTATGATTAGTCACATTTG-3 ) and Cs-R (5 -ACCACCCTACCCAGACAAAC-3 ) amplify a 121-bp fragment of the COX-1 sequence. DNA detection of gallbladder stones was performed with an ABI 7300 fluorescence quantitative PCR instrument (Applied Biosystems, Foster City, CA, USA). The final concentration of the reaction solution included 1 l of both forward and reverse primers, 1 l of the TaqMan Probe, 25 l of Premix Ex TaqTM (Takara), 1 l ROX buffer, 2 l of DNA templates and was supplemented to 50 l with sterilized ultrapure water. The program for the amplifying reaction was as follows: pre-denaturation at 95 ◦ C for 30 s, denaturation at 95 ◦ C for 15 s, renaturation and elongation at 60 ◦ C for 31 s (40 cycles). 2.5.1. Observation of gallbladder stones with scanning electron microscopy (SEM) A total of 20 egg-positive stones, randomly selected as above, were observed using SEM. The stones were split and 1–2 pieces (about 3 mm in size) were sampled from each layer if the layered structures were distinct, otherwise 1–2 pieces, about 3 mm in size, were sampled directly from the amorphous stones. These stone samples were fixed on the sample table using an electro-conductive adhesive and were dried at 60 ◦ C overnight. Subsequently, the dried samples were sputter-coated with gold (ETD-2000, China) and observed using a ZEISS (EVO LS10, Germany) SEM and photographed. The EHT was 20 kV. 2.5.2. Analyses of blood biochemical indicator The corresponding blood from the above patients was obtained after 12 h fast: a total of 2 ml of blood was centrifuged at 1450 × g for 10 min. The blood plasma was transferred to another clean tube for biochemical indicator analysis. All samples were immediately analyzed for total cholesterol, CO2 combining power, Ca, total bile acid using automatic biochemical analyzer (TBA-120FR, Japan).
30
T. Qiao et al. / Acta Tropica 138 (2014) 28–37
2.6. Statistical analysis The experimental data analysis was executed by SPSS v.16.0 software. The detection rate was analyzed using a chi-square test. P < 0.05 was regarded as statistically significant. A contingency table chi-square test was used for multiple comparisons, and the reinspection standard was ˛| = ˛/N (N = n(n − 1)/2 + 1), and n was the number of groups participating in test. An unpaired Student’s t-test was used for comparisons of mean total cholesterol, CO2 combining power, Ca, total bile acid among different gallstone groups. 3. Results 3.1. The infrared spectrum features of calcium carbonate stones According to the analytic results of FTIR spectroscopy, all of the gallbladder stone samples were classified into five groups, including cholesterol stones, pigment stones, calcium carbonate stones, mixed stones and other-kinds of stones groups. The other-kind of stones group consisted of phosphate, calcium stearate, protein and cystine stone types and were very few in number. The numbers and proportions of cholesterol, pigment, calcium carbonate, mixed and other-kind of stone types were 234 (39.13%), 133 (22.24%), 112 (18.73%), 86 (14.38%) and 33 (5.52%), respectively. The analytic results of FTIR spectroscopy showed that there were 3 types of calcium carbonate stones, including calcite, aragonite and calcite–aragonite mixed, the number and proportion of which were 49 (43.75%), 55 (49.11%), and 8 (7.14%) respectively. The characteristic infrared absorption peaks were different among different types of calcium carbonate stones. In the region of 1419–1497 cm−1 , both calcite type and aragonite type calcium carbonate had a very strong and broad absorption peak. Calcite type had a strong absorption peak at 875 cm−1 , and a moderate one at 712 cm−1 , while aragonite type had a strong absorption peak at 855, as well as a moderate one at 712 and a weak one at 699 constituting double peaks. The characteristic absorption peaks of calcite type and aragonite type were both strong in the calcite–aragonite mixed type stones. The infrared spectrogram of the calcium carbonate standard control and calcium carbonate gallbladder stones are shown in Fig. 1. 3.2. External appearance of calcium carbonate stones Calcium carbonate stones were mainly black, followed by green, brown and white (Fig. 2). The external appearance of black calcium carbonate stones was cinder-like, hedgehog-like, coralliform, piniform or mulberry-like, while the green and brown stones were mud-like and white ones were clay-like (Fig. 2). Calcium carbonate stones with hedgehog-like, coralliform and piniform appearances had a hard texture, while those with mud-like, clay-like, cinderlike and mulberry-like appearances were hard or brittle. Calcite type calcium carbonate stones were mostly hard (71.43%, 35/49), while aragonite type and mixed type stones were approximately half hard and half brittle (50.91%, 28/55; 50.00%, 4/8 respectively). Calcite type calcium carbonate stones were significantly harder than aragonite type ones (P < 0.05). 3.3. The detection rate of C. sinensis eggs was highest in calcium carbonate stones Light microscopy of split gallbladder stones revealed that the detection rate of eggs was highest in calcium carbonate stones, reaching 60% (Table 1). Real-time fluorescent PCR was used to detect some of the egg-positive stones, the results showed a positive amplification curve emerging in the DNA of the positive control and egg-positive gallbladder stones and no positive amplification curve (a straight line) in the negative control and DNA
of egg-negative gallbladder stones, confirming that the eggs in these stones were actually C. sinensis eggs (Fig. 3). C. sinensis eggs were seen adhered to different forms of calcium carbonate crystals, mucoid matter and bilirubinate granules as well (Fig. 4). 3.4. C. sinensis eggs adhered to different forms of calcium carbonate crystals under SEM SEM revealed that the microstructure of calcium carbonate stones took many forms, the majority of which was bulbiform, followed by fusiform, dumbbell-shaped, chrysanthemum-like, button-like, cuboid, rod-like, acicular, broken firewood-like, and lamellar. C. sinensis eggs were also adhered to bulbiform, hawthornlike, dumbbell-shaped and fusiform calcium carbonate crystals, as well as mucoid matter and bilirubinate granules (Fig. 5). 3.5. The clinical and blood biochemical indicator characteristics of calcium carbonate stones Patients with calcium carbonate and pigment stones were mainly male between the age of 30 and 60, while patients with cholesterol stones were mainly female between the age of 30 and 50. Most of the patients have symptoms and signs, with no difference among gallstone groups. The proportion of overweight patients in cholesterol stones was higher than that in pigment stones (P < 0.05). The blood total cholesterol, Ca and total bile acid had no significant difference among different gallstone groups, while the CO2 combining power of patients with calcium carbonate and mixed stones were higher than those with cholesterol stones, as shown in Table 2. 3.6. The clinical and blood biochemical indicator characteristics of patients with or without C. sinensis infection As shown in Table 3, patients with C. sinensis infection were mainly male between the age of 30 and 60, while patients without C. sinensis infection were mainly female between the age of 30 and 60. The proportion of overweight patients had no significant difference between the groups with and without C. sinensis infection (P > 0.05). The HCO3 − and pH value of egg-positive bile is remarkably higher than that of egg-negative bile. 4. Discussion In the present study, FTIR spectrometry was used to identify stone types, accordingly, 598 gallbladder stones were classified into five groups, including cholesterol stones, pigment stones, calcium carbonate stones, mixed stones and other-kinds of stones groups. Many researches were focused on common stone types such as cholesterol, pigment and mixed stones, while calcium carbonate stones, which were considered ‘rare’, were less studied. Calcium carbonate stone is a stone type with calcium carbonate as its main component. It has previously been reported that this type of stone is rare (Goto et al., 1996; Phemister et al., 1931; Yang et al., 1987), and has a relationship with milk of calcium bile and limewater bile (Beauregard and Ferguson, 1980; Wu et al., 2001). It has been found to account for a low proportion in gallstones of adults, but a higher proportion in those of kids (Stringer et al., 2007). In the present study, the results indicated that calcium carbonate stones were not rare and accounted for a high proportion of gallbladder stones in adults, especially egg-positive stones. One possible reason that calcium carbonate stones were considered rare may be that calcium carbonate stones presented more than ten kinds of morphologies and were always adhered to or wrapped by bilirubinate granules. As a result, according to the classification methods based on stone appearance and microscopic morphology
T. Qiao et al. / Acta Tropica 138 (2014) 28–37
31
Fig. 1. The spectrogram of calcium carbonate standard control and calcium carbonate stone type. (A) Calcium carbonate standard control; (B) calcite type calcium carbonate stone; (C) aragonite type calcium carbonate stone; (D) calcite–aragonite mixed type calcium carbonate stone.
(Ren, 1986), the black calcium carbonate stones may have been mistaken for black pigment stones and the brown calcium carbonate stones may have been mistaken for brown pigment stones and other colors of calcium carbonate stones may have been mistaken for other stone types. It was descriptively as black stone were hard and brown stones were soft (Cahalane et al., 1988), while the present data indicated that some black stones were hard but some were brittle, and brown stones were brittle. A second reason may have been that the gallstones were classified into three types by traditional classification methods (Ravnborg et al., 1990), according to
cholesterol content, but calcium carbonate stones were excluded. A third reason could be that there were few calcium carbonate stones in the gallstone samples selected (without infection factors like C. sinensis). Three types of calcium carbonate stones, including calcite, aragonite and vaterite-types, have heretofore been identified in gallstones in human body (Been et al., 1979; Bogren and Larsson, 1963), and the vaterite-type calcium carbonate is rare in nature (Rege and Moore, 1986). It has been reported that calcium carbonate in human gallstones usually presents as a structure of
Table 1 The detection of eggs in different types of gallbladder stones. Stone types
Pos. no.
Neg. no.
Rate
Total
P value
Calcium carbonate stone Pigment stone Total
67 59 126
45 74 119
60% 44% 51%
112 133 245
0.016
Calcium carbonate stone Cholesterol stone Total
67 15 82
45 219 264
60% 6% 24%
112 234 346
0.000
Calcium carbonate stone Mixed stone Total
67 31 98
45 55 100
60% 36% 49%
112 86 198
0.001
Calcium carbonate stone Other stone Total
67 10 77
45 23 68
60% 30% 53%
112 33 145
0.003
Cholesterol stone Pigment stone Total
15 59 74
219 74 293
6% 44% 20%
234 133 367
0.000
Cholesterol stone Mixed stone Total
15 31 46
219 55 274
6% 36% 14%
234 86 320
0.000
Cholesterol stone Other stone Total
15 10 25
219 23 242
6% 30% 9%
234 33 267
0.000
Note: The divided inspection standard was ˛ = 0.05/(5(5 − 1)/2 + 1) = 0.0045; P < 0.0045 was regarded as statistically significant.
32
T. Qiao et al. / Acta Tropica 138 (2014) 28–37
Fig. 2. Different kinds of external appearances of calcium carbonate stones. (A) Coralliform; (B) hedgehog-like; (C) piniform; (D and E) mulberry-like; (F) brown mud-like; (G) white clay-like; (H) green mud-like. (The ruler used in these photos is a centimeter ruler, the minimum grid represents 1 mm.)
calcite (Zeng et al., 2001), yet the findings showed that the main crystal form of calcium carbonate gallbladder stones was both calcite-type and aragonite-type, and the physical and chemical significance of different calcium carbonate types in human gallstones remains unknown. In the current study we found that the detection rate of C. sinensis eggs was highest in calcium carbonate stones. Calcium carbonate stones accounted for 37% (67/182) of egg-positive stones, remarkably higher than that found for egg-negative stones.
The association between pigment stones and C. sinensis infection had been confirmed previously (Jang et al., 2007; Lim, 1991; Qiao et al., 2012b; Guo et al., 1990), and calcium carbonate stones are also closely related to C. sinensis infection. Patients with calcium carbonate and pigment stones were mainly male between the age of 30 and 60 in this study, so were those with C. sinensis infection, which may be due to that males are infected much more severely in most endemic areas (Yu et al., 2003).
T. Qiao et al. / Acta Tropica 138 (2014) 28–37
33
Fig. 3. Detection of C. sinensis DNA in gallbladder stones by real-time PCR. (A) DNA of C. sinensis adults; (B) DNA of egg-positive gallbladder stones; (C) DNA of egg-negative gallbladder stones; (D) sterilized water
The formation mechanism of calcium carbonate stones may be as follows: The C. sinensis infection of biliary tree associates with inflammation around intrahepatic duct and poor gallbladder contraction and the changes of bile composition. Poor contraction of gallbladder and the abnormal metabolism of bile raises CO2 in common bile duct, and accordingly changes the HCO3 − and pH value (Sutor and Wilke, 1978). The HCO3 − and pH value of egg-positive
bile is remarkably higher than that of egg-negative bile. Cholecystitis caused by C. sinensis infection or other factors induces pathological changes in gallbladder mucosa, thereby reducing the absorption of calcium ions, causing calcium retention in the bile since calcium in the gallbladder is passively absorbed by the electrochemical gradient of the membrane at the junction of cells (Shen and Xiao, 2000). With the increase in pH, the alkaline condition makes ionization
Table 2 The clinical and blood biochemical indicator characteristics of calcium carbonate stones. Stone type Age (years) and gender Male
Contents lists available at ScienceDirect
Acta Tropica journal homepage: www.elsevier.com/locate/actatropica
Clonorcis sinensis eggs are associated with calcium carbonate gallbladder stones Tie Qiao a,b,∗ , Rui-hong Ma c , Zhen-liang Luo c , Liu-qing Yang b,c , Xiao-bing Luo c , Pei-ming Zheng c a
The Second People’s Hospital of Panyu, Panyu, Guangzhou 511430, Guangdong Province, PR China Institute of Gallbladder Disease of Panyu, Guangzhou 511430, Guangdong Province, PR China c The Sixth People’s Hospital of Nansha, Nansha, Guangzhou 511470, Guangdong Province, PR China b
a r t i c l e
i n f o
Article history: Received 9 January 2014 Received in revised form 3 June 2014 Accepted 9 June 2014 Available online 16 June 2014 Keywords: Calcium carbonate stone Clonorchis sinensis Calcite type calcium carbonate Aragonite type calcium carbonate Scanning electron microscopy
a b s t r a c t Calcium carbonate gallbladder stones were easily neglected because they were previously reported as a rare stone type in adults. The aim of this study was to investigate the relationship between calcium carbonate stones and Clonorchis sinensis infection. A total of 598 gallbladder stones were studied. The stone types were identified by FTIR spectroscopy. The C. sinensis eggs and DNA were detected by microscopic examination and real-time fluorescent PCR respectively. And then, some egg-positive stones were randomly selected for further SEM examination. Corresponding clinical characteristics of patients with different types of stones were also statistically analyzed. The detection rate of C. sinensis eggs in calcium carbonate stone, pigment stone, mixed stone and cholesterol stone types, as well as other stone types was 60%, 44%, 36%, 6% and 30%, respectively, which was highest in calcium carbonate stone yet lowest in cholesterol stone. A total of 182 stones were egg-positive, 67 (37%) of which were calcium carbonate stones. The C. sinensis eggs were found adherent to calcium carbonate crystals by both light microscopy and scanning electron microscopy. Patients with calcium carbonate stones were mainly male between the ages of 30 and 60, the CO2 combining power of patients with calcium carbonate stones were higher than those with cholesterol stones. Calcium carbonate gallbladder stones are not rare, the formation of which may be associated with C. sinensis infection. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Cholecystolithiasis is a worldwide common disease with a mean prevalence rate of 10–20% in western countries (Kratzer et al., 1999). Most of patients (60–80%) with gallbladder stones do not have symptoms (Gibney, 1990). A small part of patients (20–40%) have symptoms may due to cholecystitis or migration of small gallstone from gallbladder to cystic duct or common bile duct, which may lead to major complications such as acute pancreatitis, even gallbladder cancer (Cho et al., 2010; Venneman et al., 2005a,b; Jain et al., 2013). For over 100 years, cholecystectomy has been the main treatment for cholecystolithiasis, although it may lead to complications such as bile duct injury, biliary leakage, injury of the gastrointestinal tract (Flum et al., 2003; Singh et al., 2010; Bano and
∗ Corresponding author at: The Second People’s Hospital of Panyu, No. 7 Gangdong Road, Dashi Town, Panyu, Guangzhou 511470, PR China. Tel.: +86 20 34999950; fax: +86 20 34999950. E-mail address: [email protected] (T. Qiao). http://dx.doi.org/10.1016/j.actatropica.2014.06.004 0001-706X/© 2014 Elsevier B.V. All rights reserved.
Kapoor, 2010), dyspepsia, and decreased appetite as well as other metabolic consequences (Girometti et al., 2010; Luman et al., 1996; Nervi and Arrese, 2013). Gallbladder-preserving cholelithotomy, a surgical treatment for gallbladder stones, has been advocated by Chinese doctors in recent years (Qiao et al., 2012a). The aim of this operation is to remove stones and retain the functional gallbladder. It is a safe, minimally invasive therapy, with good effect, quick recovery and no damage to organs as well as low recurrence rate (Qiao et al., 2012a; Zha et al., 2013). In addition, it provides important data for the research on cholecystolithiasis. As cholecystolithiasis not only causes suffering in patients, but also places a heavy economic burden on countries and societies (Shaffer, 2006), understanding the pathogenesis and reducing the incidence of gallbladder stones has become one of the main goals of modern medical science. Extensive work has been done to investigate the formation of gallbladder stones, and a number of studies have shown that different types of gallbladder stones have different formation mechanisms (Stolk et al., 2001; Matyja et al., 2013; Stender et al., 2013; Blázovics, 2007; Chuang et al., 2013; Wang et al., 2009; Abeysuriya et al., 2010; Nakeeb et al., 2002; Hattori et al., 2000).
T. Qiao et al. / Acta Tropica 138 (2014) 28–37
Clonorchiasis is a common amphixenosis. Infection is caused by ingesting raw or undercooked freshwater fish or shrimp with living Clonorchis sinensis metacercaria (Kaewpitoon et al., 2008; Hong and Fang, 2012; Huang et al., 2012; Chen et al., 2010). The epidemiological data showed that more than 35 million people including 15 million Chinese are infected with C. sinensis in South-East Asia (Control of foodborne trematode infections, 1995). Meanwhile, annual incidence rate of clonorchiasis from 2006 to 2012 was 166.76, 191.55, 247.37, 213.82, 246.03, 274.71, and 239.63 (per 100 000), respectively in Guangzhou, China (Li et al., 2014). Previous studies have demonstrated that intrahepatic bile duct stones and gallbladder stones, especially pigment gallbladder stones, are associated with C. sinensis infection (Jang et al., 2007; Lim, 1991; Qiao et al., 2012b), but the relationship between C. sinensis infection and calcium carbonate stones has not been reported. It has been reported previously that the calcium carbonate stone is a rare type of stone, the formation of which is not clear (Goto et al., 1996; Beauregard and Ferguson, 1980; Wu et al., 2001; Phemister et al., 1931). The research data demonstrate that calcium carbonate stone is not a rare type and should never be neglected, and it is closely associated with C. sinensis infection. The aim of this study was to investigate the relationship between calcium carbonate gallbladder stone and C. sinensis eggs. 2. Materials and methods 2.1. Ethics statement A written informed consent was obtained from both adult subjects and parents/guardians on behalf of child subjects. This research was approved by the Medical Ethics Committee of The Second People’s Hospital of Panyu, Guangzhou. 2.2. Subjects and specimens Surgical indications for gallbladder-preserving cholelithotomies were as follows: (1) gallstones or gallbladder polyps with clinical symptoms, (2) gallbladder polyps with a diameter of 10 mm or more, and (3) gallbladder stones complicated by gallbladder polyps. All patients with gallbladder cancer or gallbladder atrophy and those who could not tolerate gallbladder surgery were excluded as demonstrated by Qiao et al. (2012a). Five hundred ninety-eight gallbladder stones from 598 cholecystolithiasis patients that had received gallbladder-preserving cholelithotomies in the Department of General Surgery of The Second People’s Hospital between May 2009 and October 2012 were obtained. All the recruited samples were gallbladder stones, most of them were isolated gallbladder stones and only about 15% combined with intrahepatic or common bile duct stones, but intrahepatic or common bile duct stones were not recruited in the present study. These patients consisted of 312 males and 286 females. The age range of male and female patients was 17–77 (mean = 44.90 ± 12.04) and 10–80 (mean = 46.04 ± 13.18) years old respectively. There was no significant difference when comparing the age of male vs. female groups by independent sample t-test. 2.3. Composition analysis of gallbladder stones by Fourier transform infrared (FTIR) spectroscopy The main components of the gallbladder stones were analyzed by FTIR spectroscopy and were used to identify the stone type. For the methods please refer to Qiao et al. (2012b). The main components were analyzed using a Bruker (TENSOR27, Germany) FTIR spectrometer. Gallstone composition was determined by comparison of gallbladder stones with standard control spectra (Sossé Djessou et al., 2010; Yoo et al., 2008). Control substances, which
29
were of high quality (99% pure), were purchased from Sigma Chemical Company (St. Louis, MO, USA), while the others were analytical reagents. 2.3.1. Identification of stone type Gallbladder stones were classified according to the appearance, profile structure, composition, microstructure, elemental composition and distribution. For the detailed methods please refer to Qiao et al. (2013). 2.4. Microscopic examination of ground gallbladder stone Gallbladder stones, obtained directly during the operation, were washed twice with distilled water, and then dried at 60 ◦ C for 12 h. For the method of microscopic examination of gallbladder stones please refer to Qiao et al. (2012b). 2.5. Detection of C. sinensis DNA in the gallbladder stones by real-time fluorescent PCR Gallbladder stones that were positive or negative for eggs (20 each) based on morphology were chosen randomly from the stone samples for DNA detection. For the method of extraction of DNA please refer to Qiao et al. (2012b). Fluorescent PCR: Through the sequence alignment in NCBI BLAST, the cytochrome C oxidase subunit 1 gene of C. sinensis (COX-1, GenBank: FJ965388.1), which is highly conserved and specific, was chosen as a target. The primers and Taqman probe were designed according to its sequence. These primers Cs-F (5 -GGTTTGGTATGATTAGTCACATTTG-3 ) and Cs-R (5 -ACCACCCTACCCAGACAAAC-3 ) amplify a 121-bp fragment of the COX-1 sequence. DNA detection of gallbladder stones was performed with an ABI 7300 fluorescence quantitative PCR instrument (Applied Biosystems, Foster City, CA, USA). The final concentration of the reaction solution included 1 l of both forward and reverse primers, 1 l of the TaqMan Probe, 25 l of Premix Ex TaqTM (Takara), 1 l ROX buffer, 2 l of DNA templates and was supplemented to 50 l with sterilized ultrapure water. The program for the amplifying reaction was as follows: pre-denaturation at 95 ◦ C for 30 s, denaturation at 95 ◦ C for 15 s, renaturation and elongation at 60 ◦ C for 31 s (40 cycles). 2.5.1. Observation of gallbladder stones with scanning electron microscopy (SEM) A total of 20 egg-positive stones, randomly selected as above, were observed using SEM. The stones were split and 1–2 pieces (about 3 mm in size) were sampled from each layer if the layered structures were distinct, otherwise 1–2 pieces, about 3 mm in size, were sampled directly from the amorphous stones. These stone samples were fixed on the sample table using an electro-conductive adhesive and were dried at 60 ◦ C overnight. Subsequently, the dried samples were sputter-coated with gold (ETD-2000, China) and observed using a ZEISS (EVO LS10, Germany) SEM and photographed. The EHT was 20 kV. 2.5.2. Analyses of blood biochemical indicator The corresponding blood from the above patients was obtained after 12 h fast: a total of 2 ml of blood was centrifuged at 1450 × g for 10 min. The blood plasma was transferred to another clean tube for biochemical indicator analysis. All samples were immediately analyzed for total cholesterol, CO2 combining power, Ca, total bile acid using automatic biochemical analyzer (TBA-120FR, Japan).
30
T. Qiao et al. / Acta Tropica 138 (2014) 28–37
2.6. Statistical analysis The experimental data analysis was executed by SPSS v.16.0 software. The detection rate was analyzed using a chi-square test. P < 0.05 was regarded as statistically significant. A contingency table chi-square test was used for multiple comparisons, and the reinspection standard was ˛| = ˛/N (N = n(n − 1)/2 + 1), and n was the number of groups participating in test. An unpaired Student’s t-test was used for comparisons of mean total cholesterol, CO2 combining power, Ca, total bile acid among different gallstone groups. 3. Results 3.1. The infrared spectrum features of calcium carbonate stones According to the analytic results of FTIR spectroscopy, all of the gallbladder stone samples were classified into five groups, including cholesterol stones, pigment stones, calcium carbonate stones, mixed stones and other-kinds of stones groups. The other-kind of stones group consisted of phosphate, calcium stearate, protein and cystine stone types and were very few in number. The numbers and proportions of cholesterol, pigment, calcium carbonate, mixed and other-kind of stone types were 234 (39.13%), 133 (22.24%), 112 (18.73%), 86 (14.38%) and 33 (5.52%), respectively. The analytic results of FTIR spectroscopy showed that there were 3 types of calcium carbonate stones, including calcite, aragonite and calcite–aragonite mixed, the number and proportion of which were 49 (43.75%), 55 (49.11%), and 8 (7.14%) respectively. The characteristic infrared absorption peaks were different among different types of calcium carbonate stones. In the region of 1419–1497 cm−1 , both calcite type and aragonite type calcium carbonate had a very strong and broad absorption peak. Calcite type had a strong absorption peak at 875 cm−1 , and a moderate one at 712 cm−1 , while aragonite type had a strong absorption peak at 855, as well as a moderate one at 712 and a weak one at 699 constituting double peaks. The characteristic absorption peaks of calcite type and aragonite type were both strong in the calcite–aragonite mixed type stones. The infrared spectrogram of the calcium carbonate standard control and calcium carbonate gallbladder stones are shown in Fig. 1. 3.2. External appearance of calcium carbonate stones Calcium carbonate stones were mainly black, followed by green, brown and white (Fig. 2). The external appearance of black calcium carbonate stones was cinder-like, hedgehog-like, coralliform, piniform or mulberry-like, while the green and brown stones were mud-like and white ones were clay-like (Fig. 2). Calcium carbonate stones with hedgehog-like, coralliform and piniform appearances had a hard texture, while those with mud-like, clay-like, cinderlike and mulberry-like appearances were hard or brittle. Calcite type calcium carbonate stones were mostly hard (71.43%, 35/49), while aragonite type and mixed type stones were approximately half hard and half brittle (50.91%, 28/55; 50.00%, 4/8 respectively). Calcite type calcium carbonate stones were significantly harder than aragonite type ones (P < 0.05). 3.3. The detection rate of C. sinensis eggs was highest in calcium carbonate stones Light microscopy of split gallbladder stones revealed that the detection rate of eggs was highest in calcium carbonate stones, reaching 60% (Table 1). Real-time fluorescent PCR was used to detect some of the egg-positive stones, the results showed a positive amplification curve emerging in the DNA of the positive control and egg-positive gallbladder stones and no positive amplification curve (a straight line) in the negative control and DNA
of egg-negative gallbladder stones, confirming that the eggs in these stones were actually C. sinensis eggs (Fig. 3). C. sinensis eggs were seen adhered to different forms of calcium carbonate crystals, mucoid matter and bilirubinate granules as well (Fig. 4). 3.4. C. sinensis eggs adhered to different forms of calcium carbonate crystals under SEM SEM revealed that the microstructure of calcium carbonate stones took many forms, the majority of which was bulbiform, followed by fusiform, dumbbell-shaped, chrysanthemum-like, button-like, cuboid, rod-like, acicular, broken firewood-like, and lamellar. C. sinensis eggs were also adhered to bulbiform, hawthornlike, dumbbell-shaped and fusiform calcium carbonate crystals, as well as mucoid matter and bilirubinate granules (Fig. 5). 3.5. The clinical and blood biochemical indicator characteristics of calcium carbonate stones Patients with calcium carbonate and pigment stones were mainly male between the age of 30 and 60, while patients with cholesterol stones were mainly female between the age of 30 and 50. Most of the patients have symptoms and signs, with no difference among gallstone groups. The proportion of overweight patients in cholesterol stones was higher than that in pigment stones (P < 0.05). The blood total cholesterol, Ca and total bile acid had no significant difference among different gallstone groups, while the CO2 combining power of patients with calcium carbonate and mixed stones were higher than those with cholesterol stones, as shown in Table 2. 3.6. The clinical and blood biochemical indicator characteristics of patients with or without C. sinensis infection As shown in Table 3, patients with C. sinensis infection were mainly male between the age of 30 and 60, while patients without C. sinensis infection were mainly female between the age of 30 and 60. The proportion of overweight patients had no significant difference between the groups with and without C. sinensis infection (P > 0.05). The HCO3 − and pH value of egg-positive bile is remarkably higher than that of egg-negative bile. 4. Discussion In the present study, FTIR spectrometry was used to identify stone types, accordingly, 598 gallbladder stones were classified into five groups, including cholesterol stones, pigment stones, calcium carbonate stones, mixed stones and other-kinds of stones groups. Many researches were focused on common stone types such as cholesterol, pigment and mixed stones, while calcium carbonate stones, which were considered ‘rare’, were less studied. Calcium carbonate stone is a stone type with calcium carbonate as its main component. It has previously been reported that this type of stone is rare (Goto et al., 1996; Phemister et al., 1931; Yang et al., 1987), and has a relationship with milk of calcium bile and limewater bile (Beauregard and Ferguson, 1980; Wu et al., 2001). It has been found to account for a low proportion in gallstones of adults, but a higher proportion in those of kids (Stringer et al., 2007). In the present study, the results indicated that calcium carbonate stones were not rare and accounted for a high proportion of gallbladder stones in adults, especially egg-positive stones. One possible reason that calcium carbonate stones were considered rare may be that calcium carbonate stones presented more than ten kinds of morphologies and were always adhered to or wrapped by bilirubinate granules. As a result, according to the classification methods based on stone appearance and microscopic morphology
T. Qiao et al. / Acta Tropica 138 (2014) 28–37
31
Fig. 1. The spectrogram of calcium carbonate standard control and calcium carbonate stone type. (A) Calcium carbonate standard control; (B) calcite type calcium carbonate stone; (C) aragonite type calcium carbonate stone; (D) calcite–aragonite mixed type calcium carbonate stone.
(Ren, 1986), the black calcium carbonate stones may have been mistaken for black pigment stones and the brown calcium carbonate stones may have been mistaken for brown pigment stones and other colors of calcium carbonate stones may have been mistaken for other stone types. It was descriptively as black stone were hard and brown stones were soft (Cahalane et al., 1988), while the present data indicated that some black stones were hard but some were brittle, and brown stones were brittle. A second reason may have been that the gallstones were classified into three types by traditional classification methods (Ravnborg et al., 1990), according to
cholesterol content, but calcium carbonate stones were excluded. A third reason could be that there were few calcium carbonate stones in the gallstone samples selected (without infection factors like C. sinensis). Three types of calcium carbonate stones, including calcite, aragonite and vaterite-types, have heretofore been identified in gallstones in human body (Been et al., 1979; Bogren and Larsson, 1963), and the vaterite-type calcium carbonate is rare in nature (Rege and Moore, 1986). It has been reported that calcium carbonate in human gallstones usually presents as a structure of
Table 1 The detection of eggs in different types of gallbladder stones. Stone types
Pos. no.
Neg. no.
Rate
Total
P value
Calcium carbonate stone Pigment stone Total
67 59 126
45 74 119
60% 44% 51%
112 133 245
0.016
Calcium carbonate stone Cholesterol stone Total
67 15 82
45 219 264
60% 6% 24%
112 234 346
0.000
Calcium carbonate stone Mixed stone Total
67 31 98
45 55 100
60% 36% 49%
112 86 198
0.001
Calcium carbonate stone Other stone Total
67 10 77
45 23 68
60% 30% 53%
112 33 145
0.003
Cholesterol stone Pigment stone Total
15 59 74
219 74 293
6% 44% 20%
234 133 367
0.000
Cholesterol stone Mixed stone Total
15 31 46
219 55 274
6% 36% 14%
234 86 320
0.000
Cholesterol stone Other stone Total
15 10 25
219 23 242
6% 30% 9%
234 33 267
0.000
Note: The divided inspection standard was ˛ = 0.05/(5(5 − 1)/2 + 1) = 0.0045; P < 0.0045 was regarded as statistically significant.
32
T. Qiao et al. / Acta Tropica 138 (2014) 28–37
Fig. 2. Different kinds of external appearances of calcium carbonate stones. (A) Coralliform; (B) hedgehog-like; (C) piniform; (D and E) mulberry-like; (F) brown mud-like; (G) white clay-like; (H) green mud-like. (The ruler used in these photos is a centimeter ruler, the minimum grid represents 1 mm.)
calcite (Zeng et al., 2001), yet the findings showed that the main crystal form of calcium carbonate gallbladder stones was both calcite-type and aragonite-type, and the physical and chemical significance of different calcium carbonate types in human gallstones remains unknown. In the current study we found that the detection rate of C. sinensis eggs was highest in calcium carbonate stones. Calcium carbonate stones accounted for 37% (67/182) of egg-positive stones, remarkably higher than that found for egg-negative stones.
The association between pigment stones and C. sinensis infection had been confirmed previously (Jang et al., 2007; Lim, 1991; Qiao et al., 2012b; Guo et al., 1990), and calcium carbonate stones are also closely related to C. sinensis infection. Patients with calcium carbonate and pigment stones were mainly male between the age of 30 and 60 in this study, so were those with C. sinensis infection, which may be due to that males are infected much more severely in most endemic areas (Yu et al., 2003).
T. Qiao et al. / Acta Tropica 138 (2014) 28–37
33
Fig. 3. Detection of C. sinensis DNA in gallbladder stones by real-time PCR. (A) DNA of C. sinensis adults; (B) DNA of egg-positive gallbladder stones; (C) DNA of egg-negative gallbladder stones; (D) sterilized water
The formation mechanism of calcium carbonate stones may be as follows: The C. sinensis infection of biliary tree associates with inflammation around intrahepatic duct and poor gallbladder contraction and the changes of bile composition. Poor contraction of gallbladder and the abnormal metabolism of bile raises CO2 in common bile duct, and accordingly changes the HCO3 − and pH value (Sutor and Wilke, 1978). The HCO3 − and pH value of egg-positive
bile is remarkably higher than that of egg-negative bile. Cholecystitis caused by C. sinensis infection or other factors induces pathological changes in gallbladder mucosa, thereby reducing the absorption of calcium ions, causing calcium retention in the bile since calcium in the gallbladder is passively absorbed by the electrochemical gradient of the membrane at the junction of cells (Shen and Xiao, 2000). With the increase in pH, the alkaline condition makes ionization
Table 2 The clinical and blood biochemical indicator characteristics of calcium carbonate stones. Stone type Age (years) and gender Male
