39

Clinica Chimica Acta, @ Elsevier/North-Holland

CCA

98 (1979) 39-46 Biomedical Press

1128

INFLUENCE OF URINE ON “IN VITRO” CALCIUM OXALATE: DETERMINATION A [ 14C]OXALATE TECHNIQUE

A. LIGABUE

a**, M. FIN1

b and

W.G.

ROBERTSON

CRYSTALLIZATION RATE OF OF INHIBITORY ACTIVITY BY

’

a Department of Pathology, Ospedale M. Maipighi, 40100 b Department of Urology, Ospedale M. Malpighi, Bologna c M.R.C. Mineral Metabolism Research Unit, The General

(Received

March

Bologna (Italy) (Italy) and Infirmary, Leeds

(U.K.)

2, 1979)

Summary A simple radiochemical method is proposed for the in vitro assay of the inhibitory activity of urine with respect to calcium oxalate crystal growth using [ 14C] oxalate as a tracer. The method shows an improved sensitivity over existing methods and indicates that citrate, pyrophosphate and chondroitin sulphate are active inhibitors of calcium oxalate crystal growth down to concentrations of lo-“, lo-’ and lo-” mol/l respectively. The inhibitory activity in the urines of 12 recurrent calcium stone-formers was significantly lower than in the urines of matched control subjects (P < O.Ol), confirming the clinical usefulness of the test.

Introduction Calcium oxalate is the most common constituent of urinary calculi [ 1,2] and relatively large crystals of this salt are frequently found in freshly voided urine from patients with recurrent calcium-containing stones [3,4]. It seems likely, therefore, that the crystallisation of calcium oxalate is important in the genesis of the majority of stones. The rate of crystal growth and aggregation of this salt will determine whether or not a particle large enough to be trapped at some narrow point in the urinary tract can be formed within the transit time of urine through the urinary system. The chance of a particle being trapped depends partly on the size of the particle. The rate of crystal growth will also determine to a large extent the subsequent rate of growth of this nidus into a stone. The first factor controlling the rate of crystal growth of calcium oxalate in urine is its level of supersaturation [5--71. Most authors have shown that urine * To

whom

correspondence

should

be addressed.

is generally supersaturated with calcium oxalate [5,7,8]. Stone-former-s have higher levels of supersaturation than normals and this partly explains the larger crystals which they produce [9]. The second factor which determines the size to which the primary particles grow is the level of inhibitory activity in urine against the crystallisation of calcium oxalate crystals. It is well-known that several urinary constituents (e.g. pyrophosphate, glycosaminoglycans, citrate) can inhibit the rate of growth and aggregation of seed crystals of calcium oxalate in vitro [ 10.---151. Several techniques have been published for measuring the inhibitory activity of urine using metastable solutions of calcium oxalate seeded with crystals of the salt. The rate of growth of the added crystals may be followed by measuring the increase in crystal size ]ll], or the change in the concentration 01 stable [16] or radioactive [15] calcium in the supernatant. However there arc various disadvantages to these techniques such as the cost of equipment [ 111 and the relative insensitivity of measuring changes in calcium rather than oxalate concentration [ 15,161. Since the molar calcium/oxalate ratio in urinc~ is between 5 : 1 and 20 : 1, the rate of crystal growth may be determined much more sensitively by measuring the change in oxalate concentration. It is t,he object of this paper to describe a simple assay of crystal growth and inhibition based on the uptake of i4C-lahelled oxalate from solution. Materials

and methods

Patients. 24-h urine samples were collected with 5 ml hibitane as preservative from 12 recurrent calcium stone-formers (RSF) and 12 matched controls. The RSF consisted of patients with 2 or more stone episodes per year. The controls were drawn from members of staff. -411 subjects studied were on a free diet at home. Chemicals. Calcium oxalate monohydrate crystals (BDH 27609) were used for seeding. Analytical grade chemicals were used for preparing the stock solutions; CaCl, . 2 H,O (Merck 2382), Na,&O, (C. Erba 482065), sodium cacodylatr (Merck 820670), NaCl (C. Erba 479687). Anhydrous citric acid (Baker 0090), NaJPzO., . 10 HZ0 (Merck 6591) and the sodium salt of chondroitin sulphate (Sigma C-4134) were employed for the inhibition studies. Hibitane gluconate (20%) (I.C.I.) was used to inhibit bacterial growth in urine. “C-labelled oxalic acid (CFA.84) was purchased from the Radiochemical Centre, Amersham, U.K. For scintillation counting, Soluene (0.5 mol/l quaternary ammonium hydroxide in toluene) and Unisolve I (Koch Light 9972) were used. Solutions. The standard metastable calcium oxalate solution for the growth studies was prepared according to Robertson et al. [ 111. Essentially it consisted of a solution of CaCl, (1 mmol/l) and Na,C,O, (0.2 mmol/l) in NaCl (0.15 mol/l) and sodium cacodylate (5 mmol/l) at pH 6.0. The stock suspension of seed crystals consisted of 1 g/l of calcium oxalate crystals in 0.15 mol/l NaCl incubated with magnetic stirring for 48 h at 37°C before using. Solutions of citrate, pyrophosphate and chondroitin sulphate in 0.15 mol/l NaCl at pH 6.0 were used for the inhibition studies.

The crystalline [ “Cloxalic acid was dissolved in 0.15 mol/l NaCl to produce f &i/l00 pl activity. All solutions were filtered through a 0.45 i;lm pore size Millipore filter before using. Standard technique. Flasks were set up containing 100 ~1 (1 FCi) of [ ‘“C]oxalie acid and 30 ml of metastable solution. To the reference flask was added 1 ml 0.15 mol/l NaCl and to the test flask 0.6 ml of millipore-filtered urine diluted to 1 ml with 0.15 mol/l NaCl. An aliquot of 1.2 ml of the caIcium oxalate crystal suspension was added to’each flask at 37°C and the flasks incubated with stirring for 210 min. At selected times, samples of the suspension were removed, centrifuged quickly at 1000 X g and ZOO+1 aliquots of the supernatant mixed with 1 ml Soluene and 10 ml Unisolve. The samples were counted for 2 min in a Isocap 300 (Searle Nuclear Chicago) fi counter. Calculation of the inhibition index (I.I.) The inhibitory activity was expressed as the inhibition

at 210 min.

(a) Standard procedure (151 Under the conditions employed the growth curve followed there was no necessity to correct for isotopic exchange [ 151. -dm/dt

the eqn. 1 and

= K(m -- m+-J2

(I)

where m is the total concentration of oxalate concentration, and K is a growth constant. We define 1.1. =

at time t, m, is the eqLIilibrium

(2)

I -K/K,

where Ki and K, are the growth constants in the solution containing inhibitor and in the saline control solution respectively. Assuming that the concentration of stable oxalate is proportional to the residual radioactivity (R) in the solution then I-1. = 1 - (R, - Ri)(R,

- Rc,)/(Ro

- R,)(Ri

- RiM)

(3)

where Ri is the radioactivity in the inhibited system, R, that in the saline control, R, the initial radioactivity, Ri and R, the radioactivity in the inl~ibited and control systems after 210 min and R, that at equilibrium. For simplicity R, is taken to be the radioactivity after 24 h incubation. (b) Simplified procedure An approximate measure

of 1.1. is given by:

1.1. = 1 - (R, - Ri)/(RO --R,)

(4)

where RO is the initial radioactivity and Ri and R, are the radioactivities min for the inhibited and control systems respectively.

at 210

Results growth curves Fig. 1 shows typical

Crystal

examples

of calcium oxalate

crystal growth expressed

as

150

90

30

270

210

Fig. 1. The % residual radioactivity of [ 14~ loxalate in the supernatant in relation to tune. mrtastablr solution +l ml 0.15 mol/l N&l: ‘.------ --- ( metastabk solution +2% RSF urme: metastable urine +‘P; normal urine. hlran S.E.M. of within-dav range for 8 measurements.

•m-~ 0. ~~~~~ ~~~

the fractional radioactivity remaining in solution up to 300 min. The reference curve represents the maximal uptake of [ “C]oxalate in the absence of inhibitors. The other curves show that urine contains some inhibitor(s) of the crystal growth of calcium oxalate, which are active in low concentration (i.e. 2% v/v). Fig. 1 also shows that under the conditions of the study the fractional radioactivity curves are virtually at equilibrium after 210 min incubation. We have adopted this time for the standard 1.1. calculation. Four flasks with saline and 10 containing 2% (v/v) of the same urine incubated simultaneously showed the within-run coefficient of variation to be 4%. Fig. 2 demonstrates the dependence of the growt,h (eqn. 5) rate on the integrated form of a second order kinetic law, both in the absence and presence of

I

.

.

.

.

.

.

.

30

60

90

120

150

180

210

Fig. 2. Rate of disappearance *resence of 2% urine (A----activity at time t and R, that

..r

240

from of [ ‘4C]oxalatr A) and in the presence at 24 h).

270

300

Minutes

the metastable solution of 1 ml 0.15 molil NaCl

(--l/(fi (a--

-K,)) in thr 0). (R = radio-

43 100

i

80

8

T

60.

-0

.E E

.P 2

40,

A 201

. 1

*/ 100

10

Fig. 3. The relationship urines (overall r = 0.75;

1000 Urine volume

between the inhibition P < 0.001).

index

and the concentration

of urine added

in 7 random

urine : = K,(R

--dR/dt

-R,)’

(5)

where R is the residual radioactivity and K, is the rate constant. Effect

of concentration

at time t, R,

that after 24 h incubation

of urine

Several 24-h urine samples were assayed at various concentrations for inhibitory activity using the above technique and 1.1. calculated. Fig. 3 shows the relationships between 1.1. and log (volume of urine added). The overall correlation coefficient was 0.75 (P < 0.001). When the concentration of urine fell below 0.03 to 0.1% (v/v), there was no detectable inhibitory activity. The proposed 600~~1 sample of urine (2% v/v), meets two conditions; it means that

10

‘

210

’

5.10 ’

IO’

2.10

5.10

1

2 mmol/l Citrate

Fig. 4. The relationship between the inhibition index and the final concentration range” of citrate concentration corresponds to that in 2% urine.)

of citrate.

(The “normal

11

20 nclrmal "rlne t-c

oi IO‘

Fig.

10'

5. The

““or”Ial

IO'

relationship

range”

betxvcrn

of pvrophosphat(,

IO

the

10

inhibition

10

index

concentration

and

mmol/l

the

corresponds

final to

concrntration

that

in

of pvrr~phosphdt~~.

(7‘11~.

Zp; urine.)

the calcium and oxalate concentrations of metastable solution are not significantly altered by the calcium and oxalate in the urine itself, and secondly it allows a reliable measure of 1.1. in urines with low inhibitory activity.

Inhibitory

activity

of some urinary constituents

Figs. 4-6 show the dose-response curves of inhibitory activity against concentration of citrate, pyrophosphate and chondroitin sulphate respectively. Also shown are the normal ranges of the concentrations of these inhibitors in urine [12]. From the mean urinary concentrations of these ions, the relative magnitude of 1.1. is in the order chondroitin sulphate (52%) > pyrophosphate (36%) > citrate (34%).

!irinary inhibitory The

inhibitory

uctivity activity

of RSF and controls of twelve 24-h urine

samples

from

matched

pairs

1001

-cE_-o--o-x -

60

i

L 1.10R

1.w

IW”

1.w

l.lo-”

I.&

mmol/l

Chondroitin sulfate Fig. (The

6.

The

“normal

relationship range”

between of chondroitin

the

inhibition sulphatc

index

and

concentration

the

final

concentration

corresponds

to

that

of chondroitin in

2% urine.)

sulphate.

of

8 . .

0

Fig. 7. The inhibition controls (I’ < 0.01).

index

in the

24-h urines

of matched

pairs of recurrent

stone-farmers

and normal

controls and RSF are shown in Fig. 7. Two samples from the RSF were lost during the study. Statistical analysis shows a mean (+S.D.) 1.1. of 65% ? 17 for controls and 43% + 23 for RSF. The mean values are significantly different (P< 0.01). Discussion This paper describes a simple, reliable method for determining the inhibitory activity of urine with respect to the growth of calcium oxalate crystals in vitro. The method is shorter and more sensitive than existing methods for the measurement of inhibitory activity [ll-161. The improvement in sensitivity can be attributed to measuring changes in oxalate concentration, rather than calcium concentration, in the system. This increased sensitivity allows lower concentrations of urinary inhibitors to be measured than by previously published methods. With the present method, it has been possible to confirm the difference in urinary inhibitory activity between stone-formers and normals previously observed by some workers [ 17,181 but not by others [ 191. References 1 2 3 4 5 6

Prien, E.L. and Frond& C. (1947) Studies in urolithiasis. I. The composition of urinary calculi. .I. Ural. 57, 949 Hodgkinson. A.. Peacock. M. and Nicholson, M. (1969) Q uantitative analysis of calcium-containing urinary calculi. Invest. Ural. 6. 549 Sengbusch, R. and Timmermann, A. (1957) Das kristalline Calciumoxalat in menschlichen Ham und seine Beziehung zur Oxalatstein-Bildung. Urol. Int. 4, 76 Robertson, W.G.. Peacock, M. and Nordin, B.E.C. (1969) Calcium crvstalluria in recurrent renal stoncfarmers, Lancet ii, 2.1 Robertson, W.G.. Peacock, M. and Nordin, B.E.C. (1968) Activity products in stone-forming and non-stone-forming urine. Clin. Sci. 34, 579 Elliot, J.S. and Ribeiro. M. (1967) Calcium oxalate solubility in urine: the state of relative saturation. Invest. Urol. 5, 239

46

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C.Y.C.,

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and

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clertrolrtes

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Robertson,

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and

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Smith,

L.H.

and

L.H.

(1975)

Tht J.

6.

stale

of

Clin.

saturation

Med.

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of

891

428

oxaldt