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A Combined Qualitative and Quantitative Procedure for the Chemical Analysis of Urinary Calculi

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A Combined Qualitative and Quantitative Procedure for the Chemical Analysis of Urinary Calculi J Clin Pathol: first published as 10.1136/jcp.24.2.147 on 1 March 1971. Downloaded from J. clin. Path., 1971, 24, 147-151 A combined qualitative and quantitative procedure for the chemical analysis of urinary calculi A. HODGKINSON From the MRC Mineral Metabolism Unit, The General Infirmary, Leeds SYNOPSIS A better understanding of the physico-chemical principles underlying the formation ofcal- culus has led to a need for more precise information on the chemical composition of stones. A com- bined qualitative and quantitative procedure for the 'chemical analysis of urinary calculi which is suitable for routine use is presented. The procedure involves five simple qualitative tests followed by the quantitative determination of calcium, magnesium, inorganic phosphate, and oxalate. These data are used to calculate the composition of the stone in terms of calcium oxalate, apatite, and magnesium ammonium phosphate. Analytical results and derived values for five representative types of calculi are presented. Many schemes have been described for the qualitative Brown (1939), Leonard and Butt (1955), Schneider chemical analysis of stones (McIntosh and Salter, (1968), Maurer (1969), and Thind and Nath (1969). 1942; Winer and Mattice, 1943; Holt and Tarnoky, However, some of the analytical procedures used by 1953; Kirby, Pelphrey, and Rainey, 1957; Henry, these authors are considered to be unreliable or too 1964; Varley, 1967). These methods have generally cumbersome for routine use. I have used the com- been adequate for clinical purposes but with a better bined qualitative and quantitative procedure de- understanding of the physico-chemical principles un- scribed in this communication for the analysis of derlying the formation of stone (Robertson, Peacock, over 400 urinary calculi and it has been found to be http://jcp.bmj.com/ and Nordin, 1968; Pak, 1969; Finlayson and Miller, reliable and suitable for routine use. Moreover it 1969) there is a need for more precise information, requires only apparatus which is currently available particularly with regard to the common calcium- in most modern hospital laboratories. Results containing stones. Qualitative chemical tests provide obtained from the first 200 analyses have already only a rough indication of therelative amounts of the been reported (Hodgkinson, Peacock, and Nichol- different constituents in a mixed stone and the results son, 1969). may be misleading (Leonard and Butt, 1955; on October 2, 2021 by guest. Protected copyright. Schneider, 1968). A number of physical methods Method have been used for the analysis of calculi including optical crystallography, x-ray diffraction, infrared PRELIMINARY EXAMINATION spectroscopy, x-ray spectroscopy, and thermo- Wash the stone with water to remove any blood or gravimetry, but these techniques require elaborate attached tissue and dry on a filter paper. Note the apparatus, are generally only semiquantitative and external appearance (single or multiple, rough, do not detect minor constituents of mixed calculi smooth, mulberry, horned, or waxy) and determine (Beeler, Veith, Morriss, and Biskind, 1964; Mathies the weight of the stone. Cut the stone into two equal and Lund, 1967; Pollack and Carlson, 1969). parts using a fine saw and examine for the presence Chemical analysis therefore remains the most of a nucleus. If the section shows a nucleus or other convenient procedure for routine use (Beeler et al, distinctive areas then analyse these separately. If the 1964). The method is relatively rapid, will detect stone has a homogeneous appearance then the minor components of mixed calculi, and can readily powder from the saw cut is often sufficient for the be made quantitative. subsequent analyses. Methods for the quantitative chemical analysis of stones have been described by Newcomb (1930), QUALITATIVE ANALYSIS Received for publication 30 July 1970. The scheme of analysis is summarized in Figure 1. 147 J Clin Pathol: first published as 10.1136/jcp.24.2.147 on 1 March 1971. Downloaded from 148 A. Hodgkinson POWDERED STONE 2N. HCI cover sslips QUALITATATIVE TESTS. QUANTITATIVE TESTS. 1. Heat on Pt. foil. 1. Calcium. 2. Carbonate and 2. Magnesium. I_ A 3. ion. 77 oxalate ions. Phosphate - - - ion. 4. Oxalate ion. i i =WIF 3. Ammonium I microscope 4. Uric acid. powJered ash slide 5. Cystine. stone Fig. 1. Scheme for the combined qualitative and Fig. 2. Technique for the detection ofsmall quantities quantitative analysis ofcalculi. of CO2. Heat on a platinum foil with two glass microscope coverslips and place a Heat a small quantity of the powder to a dull red drop of 2N HCl at the edge of each coverslip with heat on a platinum foil. There is usually a little a Pasteur pipette (Fig. 2). Allow the acid to infiltrate charring since most calculi contain some organic under the coverslip. Any CO2 which is evolved is matter. If a deposit remains after heating, this trapped under the coverslip and is readily observed indicates the presence of inorganic salts. Analyse with the naked eye or with a hand lens. This useful these quantitatively as described below. technique was first described by Newcomb (1930) The absence of a deposit after heating indicates and subsequently by Brown (1939) and McIntosh the presence of uric acid, cystine, or, more rarely, and Salter (1942) but it is not mentioned in most of xanthine, fibrin, sulphonamide, or fats (urostealith). the modern laboratory manuals. Cystine burns with a pale blue flame having a sharp smell and fibrin with a yellow flame having a smell Ammonium ion of burnt feathers. Uric acid, ammonium urate, and Heat a little of the powdered stone with 10% (w/v) xanthine burn without producing a flame. potassium hydroxide solution. Evolution of am- monia shows the presence of magnesium ammonium http://jcp.bmj.com/ Carbonate and oxalate ions phosphate (triple phosphate) or, more rarely, Add a few drops of 2N HCI to a small portion of the ammonium urate. Ammonia may be detected by its powdered stone. An effervescence shows the presence smell or, better, by holding a piece of damp red of carbonate and this, in turn, suggests the presence litmus paper at the mouth of the test tube. of apatite since carbonate is invariably present as a carbonate-apatite in urinary calculi (Freeman and Uric acid (murexide test) Beeler, 1969). Add two or three drops of concentrated nitric acid on October 2, 2021 by guest. Protected copyright. If any ash remains after heating on a platinum to a small amount of the powdered stone in a foil then add a few drops of 2N HCl to the residue. porcelain evaporating dish and evaporate to dryness Any effervescence at this point when there was none by heating on a water bath or very carefully over a beforeheating showsthe presence of oxalate.This test small flame. The test is positive if a red or yellow depends upon the conversion of calcium oxalate to residue remains which changes to purplish red on calcium carbonate. Prolonged heating of the ash cooling and adding a drop of 2N ammonium should therefore be avoided since this results in the hydroxide solution. conversion of calcium carbonate to calcium oxide Xanthine does not give a murexide test. It and the latter does not effervesce with acid. dissolves in nitric acid leaving a yellow residue which The effervescence produced with HCI is frequently changes to orange on the addition of alkali and to difficult to detect with the naked eye and a greater red on warming. sensitivity can be achieved by trapping the gas bubbles under glass as follows. Cystine Place a small quantity of the powdered stone at Dissolve a small portion of the powdered stone in one end of a microscope slide. At the other end of the 1 ml of 2N HCl. Add 1 ml of 2N NaOH to neutralize, slide place a small quantity of the ash remaining followed by 1 ml of 5 % (w/v) sodium cyanide after heating on a platinum foil. Cover the samples solution. Allow to stand for a few minutes, then add J Clin Pathol: first published as 10.1136/jcp.24.2.147 on 1 March 1971. Downloaded from A combined qualitative and quantitative procedure for the chemical analysis of urinary calculi 149 a few drops of 5 % (w/v) sodium nitroprusside 2 ml of 2N H2SO4. Add one drop of an aqueous solution. A strong magenta colour indicates the solution of 5% (w/v) MnSO4, heat to between 70 presence of cystine. and 80°C, and titrate rapidly with 0-01 N KMnO4. A Rosenthal and Yaseen (1969) recommended 'control' sample of powdered calcium oxalate sodium borohydride in place of cyanide, as a (20 mg) when treated in the same manner should reducing agent for cystine, because of its lower give a recovery of 99 to 100% after allowing foi toxicity. However, the use of this more time- titration blanks (I ml of O-O1N KMnO4 is equiv- consuming procedure does not appear to be justified, alent to 0 45 mg of oxalic acid). providing that appropriate precautions are taken in The mechanism of the reaction between oxalate handling the cyanide reagent. This can be achieved and permanganate is complex (Kolthoff and by using one of the many automatic dispensing Belcher, 1957). The first few drops of permanganate devices that are now available commercially. react slowly with oxalic acid but after a small amount of manganous salt has been formed the QUANTITATIVE ANALYSIS reaction occurs almost instantaneously in hot The main elements and radicles of interest are solution. The manganous ions appear to catalyse the calcium, magnesium, inorganic phosphate, and reaction between permanganate and oxalic acid and oxalate. From a knowledge of these constituents it is the addition of a drop of MnSO4 solution therefore possible to calculate the composition of a stone in ensures a sharp endpoint from the start of the terms of calcium oxalate, calcium phosphate, and titration.
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