A comparative study of gallstones from children and adults using FTIR spectroscopy and fluorescence microscopy

Expert surgeons from the department of Pediatric surgery extracted the gallstones during surgery. Initially, the gallbladder from patients was surgically removed in the department of Pediatric surgery using a Laparoscopic Cholecystectomy procedure. The gallstones were extracted from the gallbladder and preserved under sterile conditions. The patients consisted of both children and adults. Gallstones samples were collected from 67 adult and 21 child patients for this study. GSD (Gall Stone Disease) is relatively less common among children in comparison to adult population and hence the number of child patients taken for this study was limited. Helsinki agreement was obtained to carry out this project at SUMC (Soroka University Medical Center) and consent was obtained from all patients to use the isolated stones for research purpose. All stone samples were stored in sterile dried condition and later used for FTIR and Fluorescence microscopic analysis.

The FTIR measurements were performed using JASCO615 FTIR spectrometer in the frequency range 400–4000 cm^-1 at 4 cm^-1 resolution. To obtain a high signal/noise ratio 128 scans were accumulated for each sample. Initially, the spectra were fitted to parabolic function. Then the RMS (Root Mean Square) was calculated between the original spectrum and the one, fitted by the function. The area under the fitted spectrum was divided by the noise RMS, which is reported as Signal/ Noise Ratio (SNR). The Evaluate menu provided in the OPUS software performed the entire process automatically. As prescribed by Bruker, SNR was calculated in the range 1900–2100 cm^-1

The stone samples were visualized on an Olympus SZX 12 fluorescent research stereomicroscope adapted with two standard filter sets. One allowed blue excitation for observation in the green (filter1) (GFP, YFP etc. excitation filter BP460–490 nm, absorption filter BA510IF, dichroic mirror DM505) and the other allowed green excitation for observation in the red (filter2) (TRITC dying etc., excitation filter 460–560 nm, absorption filter O-590, dichroic mirror DM580). The total magnification range was between 11 × – 144×. A 100 W mercury lamp made illumination of samples. Photographs were taken with an Olympus Camedia C-3030 Zoom digital camera adapted to the microscope. The image size of each photo was 2048 × 1536 pixels.

Average FTIR spectra of black (12 stones) and brown (10 stones) stones from adults are shown in Figure 1 (1a,1b &1c). Even though FTIR data was collected on 88 patients (67 adults and 21 children), only representative spectra from 35 patients are presented in this study. Our results showed that there were two families of black stones (labeled a & b) observed by the FTIR method. Both types of black stones showed characteristic bands in the region between 1500–1700 cm^-1 due to the stretching vibrations of C=C, CO and C-N at 1570, 1626, 1661 and 1700 arising from bilirubinate salts [7, 16]. To make the identification of IR bands simpler, the reference spectra of cholesterol, bilirubin and calcium carbonate are presented in Figure 2 (2a,2b &2c). Also, the triplet between 1500–1700 cm^-1 is an indication of bilirubinate in the metal complexed form whereas a doublet characterizes the unconjugated bilirubin. The type of stone, the principal chemical components of each type and its corresponding IR bands from our study along with literature values are given in Table 1. The strong bands around 1050 cm^-1 from one of the brown stones (Figure 1c) as well as the black stone (type II) shown in the figure (Figure 1b) confirm the existence of cholesterol. It can be a mixed stone by chemical composition having cholesterol along with pigment components Cholesterol in the brown and black stones was characterized by the bands between 2800–3000 cm^-1 due to asymmetric and symmetric stretching vibrations of CH₂ and CH₃ groups [17]. The other black stone (type II)) contained less cholesterol as the absorbance of the CH₂ and CH₃ vibrations was relatively much lower compared to the other two shown in Figure 1.

In the literature, the reported colors of pigmented stones observed extensively in patients are black and brown. For the first time, our group has identified a green color gallstone from an adult patient. It was green in color all over the surface. The FTIR spectra of green and black (type II) gallstones obtained from adults are shown in Figure 3a, 3b &3c. The spectrum for the green stone in the region between 1400–1800 cm^-1 was entirely different from the black stone. This is due to the varying cholesterol content of these stones. Black stones contained high amounts of cholesterol and lower quantities of bilirubin. In contrast, the cholesterol was a major component of the green stone, with much less content of bilirubin. This is evident in the spectra in the region between 2800–3000 cm^-1 shown in Figure 3b &3c. Also, the FTIR spectra of the outer surface and the inner core of the green stone were measured. The results showed that the inner core had slightly more cholesterol content than the outside surface.

FTIR-Attenuated Total Reflection (FTIR-ATR) has become a powerful tool in the studies of the structure of biological materials and biomaterial surfaces [18, 19]. Figure 4a &4b shows the FTIR-ATR and regular FTIR spectra of the entire and part of the surface of a brown stone. There were significant differences in the spectra by these two different methods. The IR bands between 1300–1500 and 1000–1100 cm^-1 showed that the inner core as indicated by FTIR-ATR method was richer in carbonate and salts of the fatty acid contents than the outer surface of the stone. But, the cholesterol contents of the outer surface and the entire stone were equal as indicated by the characteristic cholesterol bands in the region between 2800–3000 cm^-1 by FTIR and FTIR-ATR respectively. Bilirubin content of the surface and the entire stone were similar as showed by both the FTIR-ATR and regular FTIR methods. It is important to note that the two different aforementioned measurement methods did not demonstrate any significant changes in the cholesterol contents.

Comparison of average FTIR spectra of black stones (12 stones) from children and adults (black Type I) is shown in Figures 5a &5b and 6a &6b. Our results showed that the spectra of black stones from children (Figure 5a : i) matched with type I obtained from adults (Figure 5a : ii). Type II black stones from adults were rich in cholesterol and less in bilirubin content. The main difference between the children and adults (type II) was the variation in the absorbance in the region between 1000–1100 cm^-1. Apart from this variation, the chemical composition of the black stones from children was similar to the adults. The cholesterol content in type I black stones from adults was similar to that of black stones from children (Figure 6a). But, type II stones from adults showed higher cholesterol content compared to the children as indicated in Figure 6b.

FTIR spectra of a mixed stone and the average of 12 black stones from children are shown in Figure 7a &7b. The spectrum of black stone showed characteristic IR bands for bilirubin in the region between 1500–1700 cm^-1 as reported in the earlier results. The higher wavenumber region presented in Figure 7b indicated that it had reduced cholesterol content. In contrast, the spectra of the mixed stone showed the presence of a large cholesterol content evident by the drastic reduction in the absorbance of bilirubin IR bands (1500–1700 cm^-1) and a higher absorbance of the IR bands pertaining to cholesterol in the region between 2800–3000 cm^-1.

Fluorescence microscopy (FM) has made essential contributions in modern cell biology and medicine [20, 21]. In this article, we present the use of this technique in the analysis of gallstones to measure its intrinsic fluorescence. The stones were cut under sterile environment into equal halves to investigate the inner core using fluorescence microscopy. Figure 8a,8b,8c,8d,8e shows the fluorescent micrographs of white and green stones using blue and green filters. The green and blue filters have the excitation range between 480–560 and 480–490 nm respectively. Figures 8a &8b show the FM picture of a white stone using green and blue filters. The white stone emitted red and green fluorescence using green and blue filters. Figure 8c shows a light micrograph of a green stone.

The inner core of the green stone was dark and the outer surface was relatively brighter. This indicated that the inner core may contain a pigment like bilirubin and the outer surface could be made up of cholesterol. The green stone emitted red fluorescence using both filters. But, the intensity of red fluorescence was higher using a green filter than with a blue filter. Interestingly, ring patterns were clearly observed in the green stone using both filters (Figure 8d &8e).