Chemical analysis of human urinary calculi. Mehdi S. Hamed, Dept. of Biochemistry, College of Dentistry- Tikrit University

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1 Chemical analysis of human urinary calculi Mehdi S. Hamed, Dept. of Biochemistry, College of Dentistry- Tikrit University Abstract This study included (25) patients in Al_dhuloiya in salahaldeen from April 2007 to June 2007 and (20) looked healthy individual as control group. The study was case series study male to female ratio was almost 2:1. chemical analysis of urinary stone revealed that mixed stone from about (84%) of the urinary stone and the percentage of ions in the urinary stones was highest for Phosphate (88%) and lowest for Carbonate (12%). The serum values of Ca +2 and uric acid were significantly higher than that of control group while serum Mg +2 was significantly lower than that of control group Introduction Urinary stones in humans are known since the earliest records of civilization. Advances in the surgical treatment of urinary stones have outpaced our understanding of their etiology (1). Most urinary calculi generally are composed primarily of a poorly soluble salt with a small amount of protein, containing calcium (Ca +2 ) as a main constituent (2). The direct cause of calculi is unknown and likely to be multifactor, but urinary physiological abnormalities can be identified in more than 60%of patients (3). Hypercalciuria is the most common of these abnormalities, increases the risk of stone formation by raising saturation of stone forming salt and reducing the endogenous stone inhibitors (4). The prevalence of urolithiasis is approximately 2-3% of general population (5). Once kidney stone forms the probability that a second stone will form within 5 7 years is approximately 50% (6). Stone disease is 2-3 times more common in men than in women (7). A family history of kidney stones insulin resistant state, history of hypertension, chronic metabolic acidosis and surgical menopause are all risk factors (8). In postmenopausal women the occurance of stone is associated with history of hypertension and low dietary intake of Mg +2 and Ca +2 (9). Incidence of stone is higher in patients with anatomical abnormality of urinary tract and neurological disease that may result in urinary stasis (1).The are many varieties of urinary stones which are either calcium calculi or non-calcium calculi (1).The interplay between Mg +2 and Ca +2 is complex and crucially influences Ca +2 Homeostasis. Hypomagnesemia is relatively common and often overlooked cause of ionic disturbances such as hypocalcemia and hypokalemia. Although its causes are diverse, if chronic it can induce plastic changes in the parathyroid hormone (PTH)-Ca +2 regulatory axis (10). Management of a kidney stone depends on its size, location, and composition and the presence of anatomical malformation and complications. The goals of treatment are to control symptoms, render the patient stone free, and prevent recurrence (11).This study aimed to investigate the chemical composition of urinary stone and serum values of Ca +2, Mg +2 and uric acid in both patients and control groups. Materials and Methods The study was conducted in the urology department wards and outpatient clinics in Al-dhilouiya in Salah Aldeen governerate, from April to June A total of 25 patients with urolithiasis were submitted to hospital, 17 of them were males and 8 females were examined. 25 urinary stones were collected and submitted to chemical analysis. Blood samples were collected from those patients who underwent lithotomy or passed their urinary stones spontaneously and also from the 20 control subjects to evaluate their serum Ca +2, Mg +2 and uric acid (UA). 5 ml of blood were 114

2 aspirated from upper limb veins after disinfected the skin over the vein with 70% ethanol for each candidate of patients and control group the blood was collected in disposable plane tubes and transported to the laboratory within 30 minutes. Urinary stones specimens were collected in disposable screw-capped containers. Calculi analysis tests were done according to Gowenlock et al 1988 (12) The stone was washed and dried then crushed in a mortar to fine powder. This powder was analyzed for determination of Ammonia, Ca +2, Mg +2,carbonate, cystine, oxalate, phosphate, uric acid and ammonium urate (12). To determine serum Ca +2, Mg +2 and UA. for the patients who underwent lithotomy or passed their stones spontaneously and for control subjects to compare between them, 5 ml of blood was aspirated from the cubital fossa veins under aseptic technique and separation of serum was done by centrifugation of clotted blood and the serum was deeply freezed in deep freezer under 20ºC for less than a week (13) and then serum analysis was done to determine the serum Ca +2 by spectrophotometric determination using Moorhead and Briggs derived CPC method with absorbance measured at 570 nm. was measured. Serum Mg +2 was measured by spectrophotometric determination using Mg +2 -Xylidyl blue complex method with absorbance measured at 520 nm, and serum UA was measured by spectrophotometric determination using uricase method with absorbance measured at 520 nm.statistical analysis of the results in this study were done by using SPSS version 7.5 computer software (Statistical Package for Social Sciences (14, 15). Results In this study, 25 patients were included, all of them were diagnosed to have urinary stones by means of Clinical, radiological, ultrasound and laboratory investigations, it was shown that (17) of them were males and (8). 25 stones obtained from the patients of the study were assessed for qualitative and quantitative chemical analysis. The cations found in these stones were calcium, ammonium and magnesium and their percentages were 84 %, 20% and 20% respectively. While the anions types were phosphate, oxalate, urate and carbonate and their percentages were 88%, 68%, 44% and 12% respectively. Table (1) shows the chemical composition of the 25 urinary stones. It was found that the stones that had mixed chemical composition had the highest percentage (84%). The majority among those mixed stones were composed of a mixture of calcium oxalate and calcium phosphate (24%). Other stones were as a mixture of calcium oxalate and calcium phosphate and uric acid. which came in the second rank (20%) but the other stones types were rare with variable rates of frequency. Calcium oxalate and /or phosphate stones account for most of the urinary stones obtained in this study. Statistical analysis showed no significant difference in stone composition distribution between males and females (P >0.05) using Chi-square test. In this present study a comparison of some serum values like S. Ca+2, S. U A and S. Mg+2 between the patients and the controls groups was shown in table (2). These serum values were distributed among patients who passed stones or underwent removal of their stones and according to the chemical composition of the stones. The serum values abnormalities included an increase in means of S. UA., S. Ca+2 and their values were 0.366mmol/l and mmol/l. and decrease in S. Mg mmol/l. Table 2 shows a comparison of serum values of Ca+2, U A and Mg+2 between patients and control groups. Statistically there was significant difference in the distribution of S.UA, S. Ca+2and S. Mg+2 between the patients and control groups, was significant p = 0.05 using T test. Discussion In this study urolithiasis composed mainly of the cations, Ca+2 and ammonium and Mg+2 and their percentages were 84%, 20% and 20% respectively, while the anions were phosphate, oxalate, urate and carbonate and their percentages were 88%, 68%, 44%, and 12% respectively. These results were similar to those of, Fahad 2001(16),Qaader 2002(17) and Al-Jebouri (2007) (18). 115

3 This study also showd in table 1, that the majority of them were composed of calcium oxalate and calcium phosphate and uric acid, and this indicates the multiplicity of environmental factors. This was followed by pure calcium oxalate, calcium phosphate and uric acid stones and their percentages were 8%, 8% and 4% respectively. Statistically, there was no significant difference of urinary stones distribution according to their chemical composition between males and females. These results were almost agreed with those of Qaader 2002(17) and Fahad 2001(16).Calcium oxalate and/or phosphate available in most of urinary stones observed in this study. The prevalence of this type of stones varies depends on environmental factors, especially dietary intake and life style(19). Calcium oxalate urolithiasis occurred more frequent in males than in females. This observation suggests that sex hormones play a role in the pathogenesis of urinary stones, estrogen, progesterone and testosterone modulate the synthesis of 1,25- dihydroxyvitamin D3 and the intestinal absorption of calcium by stimulating 1 α- hydroxylase in the kidney(20). In the present study, three serum components were measured and compared them between patients and control groups. These serum components were S. U A, S. Ca+2 and S. Mg+2 as seen in table 2. The comparison between the means values of these serum components of patients and control groups there was significant difference in the means between both groups at level of significant α =0.05 using T test. There were high S. UA and S. Ca+2 and low S. Mg+2 means in patients group if they compared with control group. Mean of serum uric acid (S. UA) in patients group was mmol /l and in control group was 0.32 mmol /l and this result was agreed with those of Al-Jebouri et al 2007(28).The mean of S.Ca+2 in patients group was mmol/l and in control group was mmol /l and this result was agreed with those of Al-Naas et al 2001(31). while the mean of S. Mg+2 in patients group was 0.73 mmol /l and in control group was mmol /l. This result was supported by Barakat et al 2004(20). From these results one can conclude that the increment in S. Ca+2 might lead to stone formation due to increase urinary Ca+2 excretion and the urine become super saturated with Ca+2, and Ca+2 salts rendered less soluble in urine(32). While increment in S. UA might lead to stone formation via increasing uric acid excretion in urine and also it decrease the solubility of calcium oxalate salts in urine(15).. On the other hand, the decrease in serum Mg+2 might lead to stone formation by stimulation of parathyroid hormone secretion due to inhibition of Ca+2 / Mg+2 sensing receptors by longstanding hypomagnesemia (20). Chemical analysis of urinary stones revealed that urinary stones of mixed chemical composition were the most common type and the most common elements available in urinary stones were calcium 84% from the cations, and phosphate, oxalate and urate from the anions and the percentage of availability were 88%, 68% and 44% respectively. References 1. Marshall L, Stoller M D. Urinary stone disease. In: Emil A Tanagho, Jack W McAninch, editors. Smith s general urology. 16th ed. New york: Lange medical books/ McGraw- Hill medical publishing division; Mandel N S, Mandel G S. Urinary tract stone disease in the U S. veteran population II. Geographical variation in composition. J Urol. 1989; 142: Levy F L, Adams-Huet B and Pak C Y C. Ambulatory evaluation of nephrolithiasis: an update of a 1980 protocol. Am. J. Med. 1995; 98: Pak C Y C and Holt K. Nucleation and growth of brushite and CaOx in urine of stone formers. Metabolism 1976; 25: Menon M, Parul kav B C and Darch W. Urinary lithiasis: etiology, diagnosis and medical management. In: Walsh, Retik, Vaughan and Wein, editors. Campbell s Urology. 7th ed. Vol. 2. Philadelphia: Saunders;

4 6. Asplin J R, Favus M J and Coe F L. Nephrolithiasis. In: Brenner B M, editor. Brenner and Reclors The kidney. 7th ed. Vol. 2. Philadelphia: Saunders; Curhan G C, Willett W C, Rimm E B and Stampfer M J. A prospective study of dietary Ca+2 and other nutrients and the risk of symptomatic kidney stone. N. Engl. J. Med. 1993; 328: Curhan G C, Willett W C, Rimm E B and stampfer M J. Family history and risk kidney stones. J. Am. Soc. Nephrol. 1997; 8: Hall W D, Pettinger M, Oberman A, Watts N B, Johnson K C, Paskett E D, et al. Risk factors for kidney stones in older women in southern U S. Am. J. Med. Sci. 2001; 322: Barakat, Ashrafian, Jeannie F, et al. Renal Mg loss causing hypomagnesemia and autonomous hyperparathyroidism. Nephrol. Dial. Transplant. [serial on line] 2004; 19(4): Available from URL Malvinder S Parmar. Kidney stone disease. B M J. 2004; 328: Gowenlock A H, McMurray J R and McLauchan D M, editors. Practical clinical biochemistry. 6th ed. London: Heinemann Medical Books; J G Collee, R S Miles, B Watt. Test for identification of bacteria. In: J G Collee, Barrie P, Marmion, Andrew G Fraser and Anthony Simmons, editors. Makie and McCartney Practical Medical Microbiology. 14th ed. New York: Churchill- Livingstone; Beth Dawson and Robert G Trapp, editors. Basic and clinical biostatistics.4th ed. New York: Lange Medical Books/ McGraw-Hill Medical Publishing Division; , and Duncan, Knapp and Miller III. Introductory Biostatistics for health sciences. 2nd ed. New York: Wiley Medical Publication John Wiley and Sons; Fahad H G. A study on bacteria associated with kidney stones. [Ph.D. thesis]. Iraq, submitted to Baghdad University/ College of Medicine Qaader D. S. Nanobacteria, a novel bacterium isolated from urinary stones. [M Sc. thesis]. Iraq, submitted to the Saddam University/ College of Medicine Al-jobouriy Omar," The relationship between urinary calculi typs and urinary tract infection among patient in Tikrit district ".Iraq, submitted to Tikrit Univ. Coll. of Med Fellstorm B, Danielson B G, Britakarlstorm, Lithell H, Ijungall S and Vess B. Dietary habits in renal stone patients compared with healthy subjects. Br. J. Urol. 1989; 63: Robertson W G, Peacock M and Marshal D H. The prevalence of urinary stone disease in practicing vegetarian. Fortchr. Urol. Nephrol. 1981; 17: Al-Naas Thuraya, Al-Uqaily Salim and Othman Basheer. Urinary calculi: bacteriological and chemical association. 2001; 7 (4/5): Asplin J R, Parks J H, Nakagawa Y and Coe F L. Reduced crystallization inhibition by urine from women with nephrolithiasis. Kidney Int. 2002; 61:

5 Table (1) The assessment of chemical composition of the stones from urolithiasis patients NO Type of Stones Male Female Total NO. % NO. % NO. % 1 CaOx CaPO CaPO 4 Ox CaPO 4 Ox Uric acid CaPO 4 Uric acid Cystine Uric acid AMP CaPO 4 CO CaPO 4 CO 3 Ox Uric a CaPO 4 Ox Uric a.amp Total AMP: ammonium magnesium phosphate Table (2) Distribution of some serum components among urolithiasis patients and control group. NO. Variables Controls ± SD Patients ± SD Male Female Average Male Female Average Total S.Ca +2 ±0.04 ±0.07 ±0.54 ±0.14 ±0.19 ± S.Mg +2 ±0.09 ±0.11 ±0.095 ±0.13 ±0.09 ± S.UA ±0.05 ±0.05 ±0.05 ±0.05 ±0.05 ± Total Unit of concentration (mmole/l) 118