Urinary Ionized Calcium and Renal Stone. An Interracial Study

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.J OURK~'l..L OF UROLOGY Copyright© 1967 by The Vt'illicnns & \Yilkins Co.

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l!RL'JARY IONIZED CALCH:1\I AND RENAL STONE. A~ INTERRACIAL STUDY M. MODLIN From lhc Endocrine Research Laboralory, Department of Jlleclicine, Jv[ecliml School, University of Cape Town, Cape Town, 8onlh Africa.

M o~t renal stones contain calcium. 1 - 2 The ionie calcium concentration in mine has been held to be rnspomible for the precipitation tendency of calcium ~alts which do not readily go into solution.'l Flocks investigatcxl the amount of ionized calcium 11rcscnt in urine and reported that 15 to 40 per cent of the calcium in normal urine was bound as a complexed ion carrying a charge. 4 Vermeulen and as~ociates concluded that calcium binding presumably plays a role in increa~ing the solubility of calcium salts in urine but found no difference in the proportion of ionized calciun1 ,Yhen normal urine ,Yas compared to urine from stone-forming patients. 5 Thomas and HmYard reported that ionized calcium constituted J. 0 to 22 per cent of the total minary calcium and that it was not raised in the urine of stone forn1ers. 6 Nordin concluded that calcium phosphate precipitation could probably not occur unles,; there was "free" calcium present but \Yas unable to reveal any cl iffcrence between "free" minary calciun1 in patients with renal 8tone and in those ,Yithout stone. 7 Raaflaub measured the concentration of Accepted for publication May 16, 1966. These investigations were supported by grants from the South African Council for Scientific and Industrial Research mid the Well come Trust Fund to the Endocrine Research Group and by a grant from the Staff Research Fuod of the University of Cape Town. 1 Prien, E. L.: Studies in urolithiasis: II. Relationships between pathogenesis, structure and composition of calculi. J. Urol., 61,821, 1949. 2 Lagergren, C.: Biophysical investigations of urinary calculi; crystallographic and microradiographic study. Radio!., supp., 133, 1956. '1 Ra.aflaub, J.: Kornplexchemische grundlagen der Harnsteingenese. Helv. Med. Acta., 30: 724, 1963. 4 Flocks, R. H.: St tidies on nature of urinary calciL1m: Its role in calcium urnlithiasis. J. Urol., 64: 633, 1950. 5 Yermeulen, C. W., ::\,filler, G. H. and Chapman, W. H.: Experimental urolithia.sis: X. On the state of the calcium in the urine. J. Urol., 75: 592, 1956. 6 Thomas, W. C., Jr. and Howard, J.E.: Studies on the mineralizing propensity of urine from patients with and without renal calculi. Trans. Ass. Amer. Phys., 72: 181, 1959. 7 Nordin, B. E.: The estimation of ''free" calcium in the urine and its relevance to calculus formation. Brit. J. Urol., 31: 404, 1959.

urinary ionized calcium a photometric metliod and demonstrated that it was smaller than that of total minary calcium." Nordin and Tribecli, using a similar technique, found that the proportion of ioniz('d calcium ranged from Oto 33 per ce11t of the total urinary calcium in normal and from O tP 100 per cent of the total urinary calcium i,, patients with renal stone. They concludl~cl IJrnL the proportion of minary ionized calcium was higher in patients with renal stone than in nonnu.l subjects. 9 Reports on nonnal values for ionrned calcinm are Moreover, in tions as to the importance of calciun1 in the genesis or renal stone, investigations been few and such data as have been are conflicting. This is possibly in part, due to the problems associated with the measurement of urinary ionized calcium. Therefore, the present study was done 1) to adapt an for the measurement of ionized calcium in biological fluids for use in the accurate mcasurnment of urinary ionized calcium, 2) to establisb normal values for ionized calcium in urine and 3) to investigate the significance of levels of ionized calcium in the genesis of renal stone.

567

MET.HODS

A 24-hour collection of urine was obtained l'rorn 64 normal white male subjects, 66 normal Banta male subjects, and 31 paticntb (male and fornalc, white and colored) who had a proven calcium containing renal stone. All were on a self-selected diet. Specimens of urine were collected mlu YVinchester bottles containing toluene 8, preservative and stored at 4C. The initial and citric acid content of the urine were found to be satisfactorily preserved in this manner. This 8 Raaflaub, J.: -Uber die Beziehuugen dem totalgehalt von calcium und der ionenkonzentra.tion im Harn. Helv. Physiol Pharmacol. Acta, 18: C87, 1960. 9 Nordin, B. E. and Tribedi, K : Ionized calcium in normal and stone-forming nrine. Lancet, l: 409, 1962.

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FIG. 1. Buffer used in estimation of urinary ionized calcium. Imidazole (pH 6.5) used if sample pH is 6.5. or less; tris buffer used at specific pH of sample if more than 6.5. (Values for Kn after Walser. 11) insured that the physiologi.cal content of urinary ionized calcium would be measured. The 24-hour collections of urine were analyzed for total and ionized calcium. Total calcium estimations were performed by flame spectrophotometry according to the method of Jackson and Irwin.10 Recoveries of calcium added to urine gave recovery rates ranging from 93.6 to 105.6 per cent, with a mean recovery rate of 98.7 per cent. The standard error of the difference between duplicate estimations was ±0.09 meq. per liter. The estimations of ionized calcium were performed by the method of Walser,11 with certain modifications. On samples of urine of pH 6.5 and less, estimations were done at pH 6.5, using imidazole as a buffer to bring all solutions to a pH of 6.5. On samples of urine of pH more than 6.5, estimations were done at the specific pH of the sample, tris buffer being used to bring all solutions to the particular pH (fig. 1.) Measurements of Kn for the calcium murexide complex were carried out at different ionic strengths and the variations were observed (fig. 2). All estimations of ionized calcium were accordingly carried out at the specific ionic strength of each specimen of urine. The standard solutions and buffer were adjusted accordingly. The ionic strength of each sample of urine was estimated by the method of Isaacson.1 2 The 10 Jackson, W. P. U. and Irwin, L.: The estimation of calcium in urine by flame photometry with a note on the estimation of sodium and potassium. J. Clin. Path., 10: 383, 1957. 11 Walser, M.: Determination of free magnesium ions in body fluids. Improved methods for free calcium ions, total calcium, and total magnesium. Analyt. Chem., 32: 711, 1960. 12 Isaacson, L. C.: Ionic processes in urine. Ph.D. Thesis, University of Cape Town, 1964.

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FIG. 2. Variation of Kn with ionic strength for calcium-murexide complex (pH 7.5). method is based on the identically high correlations that he observed between urinary ionic strength and osmolality (r = 0.92), and urinary ionic strength and specific conductivity (r = 0.92), as seen in normal urines on which determinations of ionic strength were arrived at by calculation. The equation used was: I.S. = 0.000255 Osm. + 0.135 sp.K. + 0.0017 (where: I.S. = ionic strength; Osm. osmolality mOsm/kgH20; spK = specific conductivity = mHhos/cm. at room temperature). Urine diluted X25. Osmolality was determined on a Fiske osmometer. Specific conductivity was determined by standard methods. 13 The relationships in the equation were embodied in a nomogram for routine use. The standard error of the difference between duplicate estimations of ionized calcium was ±0.05 mg. per 100 ml. RESULTS

The results obtained are presented m the table. URINARY EXCRETION OF IONIZED CALCIUM

The urinary excretion of ionized calcium by 64 normal white male subjects ranged from 2.4 to 80.5 mg. with a mean value of 26.1 mg. and a standard deviation of 15.7 mg. The urinary excretion of ionized calcium by 66 normal Bantu male subjects ranged from 8.5 to 150 mg. with a mean value of 35.0 mg. and a standard deviation of 27 .8 mg. The urinary excretion of ionized calcium by 31 patients with stones ranged from 13 Findlay, A.: Practical Physical Chemistry. 8th ed. Revised and edited by J. A. Kitchener. New York: Longmans, Green & Co., 1954.

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URINARY IONIZED CALCIUM AND RENAL STONE

Mean values of total and ionized calcium Group

White subjects Bantu subjects Stone cases

No.

64 66 31

Significance of differences:

Ionized Calcium ·~·------(mg./100 ml.)

Total Calcium (mg./100 ml.)

(mg./24 hrs.)

12.0 ± 5.2 4.5 ± 2.7 13.6 ± 6.6

26.1 ± 15.7 35.0 ± 27.8 26.4 ± 21,0

2.4 ± 1.3 1.8 ± 1.3 1.6 ± L2

21.l ± lOJi 44.9 ± 23 12.6 ± 7 5

1:2 P < 0.001 1:3 P < 0.2 2:3 P < 0.001

1:2 P < 0.05 1:3 P < 0.2 2:3P
1:2 P < 0.01 1:3 P < 0.01 2:3 P < 0.4

:2 P < O. O(Jl 1:3 P < 0.001 2:3 P < 0.001

0 to 75 mg. with a mean value of 26.4 mg. and a standard deviation of 21.0 mg. The average 24-hour urinary ionized calcium in the Bantu subjects was significantly higher than in the white subjects (P < 0.05). The average 24-hour urinary ionized calcium in patients with stones did not differ significantly from that in the white subjects or in the Bantu subjects (fig. 3).

White vs. Bantu White VS. Stones Bantu vs. Stones

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40 The urinary concentration of ionized calcium in 64 normal white male subjects ranged from 0.4 to 7 .2 mg. per 100 ml. with a mean value of 2.4 mg. and a standard deviation of 1.3 mg. The urinary concentration of ionized calcium in 66 normal Bantu male ,mbjects ranged from 0.4 to 6.8 mg. per 100 ml. with a mean value of 1.8 mg. and a standard deviation of 1.3 mg. The urina.ry concentration of ionized calcium in 31 patients with stones ranged from O to 4.3 mg. per 100 ml. with a mean value of 1.6 mg. and a standard deviation of 1.2 mg. The average urinary ionized calcium concentration vrns significantly higher in the white subjects than in the Bantu ~ubjects (P < 0.01). The average urinary ionized calciw11 concentration was also signifieantly higher in the white subjects than in patients with stones (P < 0.01). The average urinary ionized calcium concentration in patients 1Yith stones did not differ significantly fron1 that in the Bantu. The average total urine calcium concentration in the white subjects did not differ significantly from that in patients with stones. The average total urine calcium concentration was significantly higher in the white subjects than in the Bantu subjects (P < and also significantly higher in patients with stones than in the Bantu subjects (P < 0.001) (fig. 4).

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URINARY CONCENTRATION OF IONIZED CALCIUM

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The proportion of ionized calcium in the urine of 64 white male subjects ranged [rorn 3.G to 52.2 per cent with a mean value of 21.l per cen and a standard deviation of 10.9 per cent. The proportion of ionized calcium in the urine of 66 Bantu male subjects ranged from 13.8 to 100 per cent with a mean value of 44.9 per cent and a standard deviation of 23.2 per cent. The proportion of ionized calcium in the urine of 31 with stones ranged from O to 33.5 per cent with a mean value of 12.6 per cent and a standard deviation of 7.5 per cent. The average proportion of ionized calcium in the urine of the Bantu subjects was

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Fm. 6. Mean value of percentage of urinary ionized calcium in stone cases, white subjects and Bantu subjects (S.D. = standard deviation.) higher than in the white subjects (P < 0.001). The average proportion of ionized calcium in the urine of patients with stones was significantly lower than in the white subjects (P < 0.001) (figs. 5 and 6) . DISCUSSION

In the present study a photometric method based on the change of color of a metal indicator murexide (acid ammonium purpurate) has been used for the estimation of urinary ionized calcium. The principles and the theory of the method were originally outlined by Schwarzenbach and Gysling,14 and Raaflaub. 16 Rose16 and Fanconi and Rose17 used a similar method to 14 Schwarzenbach, G. and Gysling, H.: Murexid als Indikator auf calcium und andere metallionen. Komplex-bildung und Lichtabsorption. Helv . Chim. Acta, 32: 1314, 1949. 16 Raaflaub, J.: Uber ein photometrisches verfahren zur Bestimmung des ionisierten calciums. Hoppe-Seyler Z. Physiol. Chem., 288: 228, 1951. 16 Rose, G. A.: Determination of the ionized and ultra-filterable calcium of normal human plasma. Clin. Chim. Acta, 2: 227, 1957. 17 Fanconi, A. and Rose, G. A.: The ionized, complexed and protein-bound fractions of calcium in plasma. An investigation of patients with

URINARY IONIZED CALCIUM AND RENAL STONE

determine ionized calcium in plasma ultrafiltrate. The accuracy and specificity of the method for the measurement of ionized calcium in biological fluids have been satisfactorily demonstrated. 11 • 15 As applied in the present study with the modifications described the method is valid for the estimation of urinary ionized calcium and yielded reproducible results. The range of normal urinary ionized calcium values observed in this study is greater than those quoted by Thomas and Howard, 6 and by Nordin and Tribedi. 9 However, the methods used by these investigators for the estimation of urinary ionized calcium are unsatisfactory and the conclusions reached are therefore unlikely to be valid. The ethnic difference in urinary ionized calcium in normal subjects has not been investigated previously and no published data are available for comparison with the present findings. The population of the Republic of South Africa consists of two main groups, the white in whom renal stones occur with no less frequency than in other Western communities and the Bantu in whom renal stones are extremely rare. It seems reasonable to conclude that the present comparative study of urinary ionized calcium in normal white and Bantu subjects and in patients with stones, provides suitable data for evaluating the significance of urinary ionized calcium in the genesis of renal stone. The average 24-hour urinary ionized calcium in patients with stones did not differ significantly from that in the white subjects. This observation suggests that the daily level of urinary ionized calcium is not an important factor in the genesis of renal stone. Moreover, the observation that the average daily urinary ionized calcium in the Bantu subject did not differ significantly from that in patients with stones provides further evidence in support of this conclusion. There was no significant difference between the average urinary ionized calcium concentration in patients with stones and in the Bantu subjects. It is concluded from this evidence that renal various diseases which affect calcium metabolism with an additional study of the role of calcium ions in the prevention of tetany. Quart. J. Med., 27: 463, 1958.

571

stone formation is not related to the concentration of urinary ionized calcium. The observation that there is a higher average urinary ionized calcium concentration in the normal white subjects than in the stone cases or Bantu subjects supports this conclusion. In the three groups of subjects studied, the average proportion of urinary ionized calcium was significantly higher in the white subjects than in the stone cases, and significantly higher in the Bantu than in the white subjects. Thus, the proportion of urinary ionized calcium was highest in a gToup in whom renal stone formation is extremely rare and lowest in the group of stone formers. It is concluded from this evidence that a high proportion of urinary ionized calcium does not appear to be associated with a tendency to renal stone formation. The finding of a significantly lower average proportion of urinary ionized calcium in patients with stones than in the normal subjects is at variance with that of Nordin and Tribedi, who reported that the proportion of urinary ionized calcium was higher in patients with renal stone than in normal persons. 9 However, in their study the estimations of urinary ionized calcium were all carried out at a pH of 7 .4, regardless of the pH of the sample being analyzed, and variations in the ionic strength of the urines were ignored. It is apparent from discussion earlier in this report that the values for urinary ionized calcium obtained under the conditions of their study are unlikely to represent the physiological amounts of ionized calcium present in the urines analyzed. It therefore follows that any conclusions based on these observations are invalid. The observations in the present study are also at variance with those of Vermeulen and associates 5 and Thomas and Howard. 6 The methods that they used for the estimation of urinary ionized calcium would appear to be unsatisfactory and the conclusions that they reached are consequently probably invalid. SUMMARY

An existing photometric method for the measurement of ionized calcium in biological fluids has been adapted for the measurement of ionized calcium in urine. The method and the modifications introduced in this study are discussed.

572

MODLIN

Urinary ionized calcium has been studied in 64 normal white subjects, in 66 normal Bantu subjects and in 31 patients with stones. Ethnic differences in urinary ionized calcium have been demonstrated. The average daily excretion of urinary ionized calcium and the average daily proportion of urinary ionized calcium were found to be greater in the I3antu than in the white subjects. The average urinary

ionized calcium concentration was found to be lower in the Bantu subjects. The implications of the findings in this study in regard to the genesis of renal stone are discussed. The evidence indicates that the daily level, or concentration, of urinary ionized calcium is not an important factor in renal stone formation, nor does a high proportion of urinary ionized calcium appear to be associated with a tendency to renal stone formation.