The present observational study was conducted at Department of Biochemistry, North Eastern Indira Gandhi Regional Institute of Health and Medical Sciences (NEIGRIHMS), a tertiary care centre in North East India, from December 2009 to January 2014. The study was approved by the Institute Ethic Committee (IEC) of NEIGRIHMS. The study population included patients diagnosed with renal stones and who were operated in the Department of Urology in NEIGRIHMS. One hundred forty stones from a total of 110 individual (due to multiple stones in same individual or due to fragmentation of stones during the procedure) were studied over a period of four years irrespective of age, sex, and race. Questionnaires were completed covering the information pertaining to age, sex, dietary habits and health status. Renal stones were collected by surgical intervention and analysed chemically by protocols described in Varley’s Practical Clinical Biochemistry [12]. The cases were divided according to age and sex during the study. The study group composed of <18 years, 18-40 years, >40 years separately for male and female.

The specimen were first swabbed for microbial growth, washed carefully with deionized water and dried. The morphological features such as colour, shape and other characteristics were noted. The stones were subjected to X-ray exposure to determine the opacity. Using a scalpel the core and surface part of the kidney stone were cut. Then it was grounded with pestle and mortar to produce a fine homogeneous powder for qualitative estimation of different constituents.

The bacteriological study of the stones was conducted in the Department of Microbiology, NEIGRIHMS along with culture and sensitivity reaction. The swabs were incubated at 37°C in Brain Heart Infusion (BHI) broth overnight for 18-24 hours. If turbidity develops in the broth the growth were sub-cultured in MacConkey and blood agar. The isolated growth were then identified as per the standard protocol.

The data were analysed using the software Statistical Package for the Social Sciences (SPSS), version 16.0 (SPSS Inc., Chicago, USA). Data were expressed as count (with percentage). Bar diagram was used to express the frequency.

During the study period a total of 140 stones from 110 patients were analysed. It was found that 92 (65.71%) stones were from age group of 18-40 years, followed by 39 (27.85%) stones from the age group of greater than 40 years and 9 (6.42%) stones from age group less than 18 years. The incidence of renal stone was slightly more common in male with male to female ratio of 1.02:1. Chemical analysis of 140 stones showed that ammonia was the most frequent salt found in all age groups for both sexes (100% for males and 97.83% for females in 18-40 years age group). This was followed by magnesium (97.83%), oxalate for males (97.83%) and calcium for female (97.83%) [Table/Fig-1]. Multiple stones were found in 21 cases of which 17 cases were positive for inorganic, carbonate, oxalate, phosphate, ammonia, calcium and magnesium.

Microbiological analysis was carried out in all possible cases. The most common organism isolated in stone culture was Proteus spp., followed by Enterobacter species [Table/Fig-2]. On sub-analysis, it was found that renal stones are more common in non-vegetarian group [Table/Fig-3].

[Table/Fig-3]:

Dietary habit of patients with renal stones.

Calcium salts, uric acid, cysteine and struvite (MgNH₄PO₄) are the basic constituents of most kidney stones. Our study showed a highest occurrence of ammonia (100% for male and 97.83% for female in the age group of 18-40 years) in all age groups. This is followed by magnesium and relatively high amount of phosphate indicating the presence of struvite stones. The frequency of struvite stones was followed by stones containing calcium and oxalate which were the predominant renal stones after struvite stones. This is in contrast to the other observation where calcium oxalate stones are predominant [13,14].

Ansari MS et al., in their 1050 renal stone analysis from North Delhi showed calcium oxalate in 93.04% patients, uric acid stones in 0.95% and mixed pattern in 2.76% stones [15]. Similarly, Rao MVR et al., from Delhi has reported 96% of calcium oxalate stone in an analysis of 51 stones [16]. Urinary stone composition also reported predominance of calcium oxalate by Ahlawat R et al., and Sharma RN et al., as 97% and 86.1% respectively [17,18]. Kumari A et al., in her studies has shown higher incidence of uric acid pure stones (18%) and mixed stones containing uric acid (52.3%) [3].

The contrasting finding of the present study may be due to bacterial UTI which is usually caused by Proteous spp. Possessing the enzyme urease. This enzyme degrades urea to NH₃ and CO₂. The NH₃ is protonated to NH₄⁺ and raises the pH of urine to 8 or 9. The NH₄⁺ precipitates PO₄^3- and Mg⁺ to form MgNH₄PO₄(struvite). Chronic Proteus spp. infection can occur as a result of impaired urinary drainage. This finding is supported by the microbiological data which shows increased Proteus spp. infection especially in the older age group i.e., >40 years (35% for males and 36.8% for females) [Table/Fig-2]. In the age group of 18-40 years both sexes had relative increased frequency of Enterobacter species growth in comparison to other organism with a frequency of 8.7% for males and 10.9% for females.

In our study, the highest occurrence of renal stone in the age group of 18-40 may be because of relatively high incidence of calcium oxalate and calcium phosphate stones in this group of patients [Table/Fig-1]. The high intake of non-vegetarian food accompanied by lower intake of water contributes to stone formation. Non-vegetarian food promotes calcium oxalate stone formation by epitaxial growth [19]. This finding is supported by the fact that most of the patients (87%) in the study group were non-vegetarian in dietary habit [Table/Fig-3]. Robertson WG et al., have shown that prevalence of urinary stones was 40-60% amongst vegetarians, when compared to age/sex/social class matched general population [20].

One interesting observation noted in our study was the occurrence of multiple stones having the same chemical composition. The chemical composition of these stones revealed that carbonate was invariably present in all the stones although there was a difference in composition in individual stones in three cases. The high occurrence of carbonate stones may be due to urease- producing Proteus spp. which decomposes urea{(NH₂)₂CO} to Ammonia (NH₃) and carbon dioxide (CO₂). The ammonia increases the pH of the urine and CO₂ becomes hydrated to carbonic acid and then dissociates to carbonate ion that precipitates with calcium as calcium carbonate. As a result carbonate stones are usually admixed with struvite stones [21].

Small sample size; the method of determining the component of the renal stone in the present study was by chemical means which is inferior to standard methods like infrared spectroscopy, X-ray diffraction crystallography and scanning electron microscopy.

The study showed that MgNH₄PO₄ (struvite) stones were the most common variety of renal calculi encountered in patients, indicating Proteusspp. infection which can occur due to impaired urinary drainage resulting in chronic UTI. This factor, along with high intake of non-vegetarian diet may have contributed to the high frequency of struvite stones in our study population. However, this may be confirmed in future by a well-designed cohort study.