496

Journal of Crystal Growth 89 (1988) 496-500 North-Holland, Amsterdam

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POLYHYDROXYCARBOXYLIC ACIDS AS INIllBITORS OF CALCIUM OXALATE CRYSTAL GROWTH; RELATION BETWEEN INIllBITORY CAPACITY AND CHEMICAL STRUCTURE F. GRASES, A. MILLAN Departament

01 Chemistry,

and A. GARCIA-RASO

University 01 Balearic Islands, 07071 Palma de Mallorca, Spain

Received 5 February 1988

The kinetics of crystal growth of calcium oxalate monohydrate seed crystals were investigated potentiometrical!y in the presence of several polyhydroxycarboxylic acids: etylenediaminetetraacetic acid, citric acid, isocitric acid, malic acid and succinic acid, and it was found that they inhibited crystal growth, except in the case of isocitric acid that manifested no-effects. An apparent rate order of 2 in the presence of al! the inhibitors, suggested a spiral growth mechanism. Application to a kinetic Langmuir-type model suggested that adsorption of the polyhydroxycarboxylic acids, at the active growth sites, is the cause of the reduction in the crystal growth rates. The inhibitory action of the different substances assayed was comparatively'evaluated. Relations between chemical structure and inhibitory capacity were established.

1. Introduction Cítrate, as well as other polyhydroxycarboxilic acids, have been cited as effective inhibitors of calcium oxalate crystallization [1-3]. In spite of this, several autors postulate that the effects of such substances in the prevention of urolithiasis is doubtful [4-6]. However, in view of their potential application in the urolithiasis therapy, the study of these processes can be of great importance. In the present paper, a potentiometric method (using a selective calcium electrode) has been used to investigate the crystallization of calcium oxalate in the presence of several polyhydroxycarboxylic acids with the aim to go deeply into such processes and to be able to establish relations between the chernical structure of this substances and its inhibitory capacity on the calcium oxalate crystallization.

pared using Reagent Grade chernicals (PANREAC) with triply distilled deionized water. Calcium oxalate monohydrate seed crystals were obtained from Panreac. 2.1. Crystal growth measurements Growth experiments were made in a Pyrex glass vessel of approximately 250 mI capacity. The solutions were maintained at an appropriate temperature by circulating thermostatted water through the corresponding containers (0.10 C). Working solutions were always magnetically stirred. The precipitation reaction was initiated by the introduction of a weighted amount (200 mg) of calcium oxalate monohydrate seed crystals in the supersaturated solution. Crystal growth experiments were followed through potentiometric measurements. The potentiometric measurements were performed by means of a Crison 501 potentiometer using a calcium specific ion electrode (Ingold) coupled with a silver/ silver-chloride electrode, which was separated from the cell solution by an intermediate junction containing potassium nitrate. Rates of crystallization were calculated

2. Experimental Solutions of calcium chloride, sodium oxalate, ammonium chloride, malic acid, citric acid, isocitric acid, tartaric acid, succinic acid and etylenediarninetetraacetic acid (EDT A) were pre0022-0248/88/$03.50 © EIsevier Science Publishers (North-Holland Physics Publishing Division)

BY

F. Grases et al. / Polyhydroxycarboxylic

from the experimental curves (potential (m V) versus time) by the use of the following equation: d[Ca] dt

R=

=

exp[(mV-b)¡a] a

d(mV) dt

'

(1)

where a and b are constant parameters deduced from the calibration graph (potential versus ln[Ca2+]). Thus eq. (1) was deduced assuming that in the experimental work range the [Ca2+] and m V are related by the expression: mV= a ln[Ca2+]

+ b,

(2)

and by simple derivation and operation on eq. (2), eq. (1) is obtained. The pH of each experiment was previously adjusted to the adequate value by addition of ammonium hydroxide or hydrochloric acid. 2.2.

t Potential

measurements

The measurements of the t potential were performed by use of a Micromeritics Zeta potential analyzer, model 1202. A suspension of calcium oxalate monohydrate dry powder of 20%, in synthetic urine, was used to carry out the measurements. 3. Results and discussion

K¡

H+ + C20¡-

'=7

(3)

HC20i , K2

C20¡-(aq)

+ Ca2+(aq)

'=7

CaC204(aq),

(4)

K

CaC204(aq)

+ C20¡-(aq)

~ Ca(C204);-(aq), (5)

K,p

Ca2+(aq)

+ C20¡-(aq)

'=7

CaC204•

Ca2+(aq) + R(OH)n-(COOH)m

H20(s),

(6)

(aq)

K4 '=7

The equilibria (3) and (5) can be neglected when using the experimental conditions of this paper. This assumption was justified from the pH and Ca2+/C20¡ratios used. The values of the thermodynamic solubility product (Ksp), association constant of calcium and oxalate ion s (K2), and formation constants of metal complexes between calcium ion s and the corresponding polyhydroxycarboxylic acids were obtained from refs. [7-9]. The results of the crystal growth experiments are surnmarized in table 1. Typical crystallyzation rate curves are shown in fig. 1. As can be seen, the precipitation rate decreases by the presence of several polyhydroxycarboxylic acids, in the following order: ethylenediaminetetraacetic acid (more effective inhibition was caused), citric acid, malic acid, tartaric acid, and succinic acid (less effective inhibition was observed). Isocitric acid did not cause any inhibitory effect under the conditions studied. The kinetic data were adjusted to the rate equation:

R= -d[CaL/dt = k {([ Ca 2+] t [ C2O¡ - ] t ) 1/2 - ([ Ca 2+] eq [ C2042-]

The crystallization of calcium oxalate was studied in the presence of several polyhydroxycarboxylic acids, with the aim to be able to establish relations between the inhibitory capacity on crystal growth and its chemical structure. The following equilibria must be considered in the presence of polyhydroxycarboxylic acids:

Ca-R(OH)n-(COOH)m(aq).

(7)

497

acids as inhibitors 01 calcium oxalate growth

eq

f/2r

.

(8)

where n is the apparent arder of the crystal growth reaction. Logarithmic plots of the rate, R, in eq. (8) yielded straight lines that permit the evaluation of the apparent order n. Fig. 2 shows several logarithmic plots corresponding to kinetics of crystal growth of calcium oxalate monohydrate seed crystals, obtained in the absence and presence of the polyhydroxycarboxylic acids assayed. As can be seen, an apparent order of n = 2 was assessed in all cases. These data confirm that the surface-controlled mechanism - where a spiral step is centred at the screw dislocation - for calcium oxalate previously found also holds in the presence of the polyhydroxycarboxylic acids studied. The application of a simple kinetic-Langmuir model yielded the straight lines shown in fig. 3 and suggested that adsorption of the polyhydroxycarboxylic acids at the active growth sites in the cause of the reduction in the crystal growth rates.

498

F. Grases el al.

I Polyhydroxycarbo;q/ic

acids as inhibilors 01 calcium oxalate growth

Table 1 Crystallization of calcium oxalate fram supersaturated acids (T= 28°C, 1= 0.15 mol/I and pH = 5.8) Tea (0 C) Experiment

[Ca2+ ]Xl04 (mol/I)

solutions

in the absence and in the presence

[C20¡-]

Toxalate (0 C)

(mol/I)

X

104

of diverse polyhydroxycarboxylic

- LlG

R

(kJ/mol)

(mol/l·

X 106

s)

3.23 6.84 2.21 6.69 1.73 1.07 6.21 1.23 1.57 1.44 1.79 6.48 6.95 1.34 1.91 1.96 2.31 1.56 2.04 7.25 6.68 7.02 7.38 6.70 2.30 2.81 5.06 3.79 7.50 7.26 7.68 1.74 2.29 2.07 2.50 2.47 2.46 2.73 1.92 1.51 1.23 7.21 6.23 6.51 6.47 1.92 6.36 2.64 2.04 1.72 1.13 1.43 2.20 6.13 6.43 7.20 1.80 3.08 1.06 6.56 7.48 1.60 1.95 2.63 6.78 6.41 1.76 6.99 6.53 2.26 1.59 1: without any6.25 additive

2.48 7.29 2.50 2.68

= - ~RT In([CaH L [oxalateL/[CaH]eq [oxalate]eq)' where the braekets denote aetivities at time I and at equilibrium; = 0.511522[11/2/(1 + 11/2)_ 0.31], where /2 is the aetivity eoeffieient of a divalent ion, 1 is the iome strength and -log(f2) valenee of the ion.

LlG

mV

15

3 4 2

I

5 '----

>--

-;

3 mino

_ t

Fig. 1. Typical

ealeium

oxalate

crystallization

runs with

an

without addition of several earboxylie aeids. T = 28 ° C, pH = 5.8, [Caja = 2.5 X 10-4M, [oxalate]a = 7.5 X 10-4M. lome additives; (2) [sueeinie] = strength = 0.15. (1) Without 0.0862M; (3) [malie] = 0.0862M; (4) [tartarie] = 0.0862M.

2 the

It is very interesting to compare and discuss the different inhibitory capacities of the organic acids studied. Thus, tetracarboxylic acids c1early manifested a greater inhibitary capacity than tricarboxylic acids, the latter being superior to those exhibited for the dicarboxylic acids. This can be explained as a consequence of EDT A causing a more effective adsorption on the calcium oxalate crystals, and its active sites being more effectively blocked, resulting in the weakest adsorption that corresponded to the dicarboxylic acids. The most active adsorption could be due to the greatest negative charge of the respective organic acids (see the pK values of these substances in table 2). Nevertheless, when the inhibitory capacities of the different dicarboxylic acids assayed was compared, the majar inhibitory capacity (major ad-

Citrie aeid Tartarie aeid eeid aeid

F. Grases et a/. / Polyhydroxycarboxylic

3

Table 2 pK values (25 ° C) of the polyhydroxyearboxylie aeids studied (after ref. [9])

-Ln R

3

16

/ //

14

6.384 4.761 4.68 6.16 2.67 4.366 5.635 pK3 pK2 tetraaeetie aeid

3.128 3.26 3.036 1.99 4.207

499

acids as inhibitors 01 calcium oxalate growth

Ethylenediamine-

pK¡

10.26

9.2

9.6

10

-Ln e

Fig. 2. Aparent order for erystal growth kineties. T = 28 ° C, ionie strength = 0.15, pH = 5.8, [Ca]o = 2.5 X 10-4M, [oxalate]o = 7.5 X 10-4M. (1) Without additives; (2) [malie] = 3.73 X 1O-4M; (3) [eitrie] = 1.7 X 1O-4M; (4) [EDTA] = 3.36 X 10-5M.

sorption) was found that it did not correspond to the substances with the greatest pK values (table 2). Thus, when comparing the inhibitory capacity of malic acid, tartaric acid and succinic acid, the first exhibited the most effective inhibitory action and the last the weakest. This demonstrates that structural factors can also play an important role in the adsorption mechanism. In this way, in the absence of OH groups (succinic acid), the molecular rigidity was minimum and in the presence of two OH groups (tartaric acid), it was maximum. Precisely in the presence of only one OH group 3 4

(intermediate rigidity), the inhibitory capacity was maximum, this indicating that a certain molecular rigidity can favour the adsorption process. The absence of inhibitory capacity of the isocitric acid can be attributed to its easily attainable lactonization process in aqueous media, in which a carboxylic group is lost and a ring of five members is generated (the rigidity is notably increased). Finally, it must be pointed out that the study of the ~ potential of a ca1cium oxalate monohydrate-synthetic urine slurry was practically not affected by the presence of the polyhydroxycarboxylic acids, at concentrations similar to those found in real urine. These findings demonstrate that, in spite of the potential importance of some polyhydroxycarboxylic acids in the inhibition of crystal growth in urine, they scarcely prevent the crystal agglomeration. Consequently, if it is considered that the two processes of growth and agglomeration are both crystal-surface-related processes, they may react in opposite directions, as in the case of glycosaminoglycans [10,11], aggregation phenomena would probably occur as a consequence of the presence of promoters rather than to the absence of inhibitors.

3 2

Acknowledgement

2

10

20

30

Financial support by the Dirección General de Investigación Cientifica y Técnica, Spain (Grant No. 86-0002) is gratefully acknowledged.

40 [inhib

J

x 104

Fig. 3. Langmuir adsorption kinetie model applied to ea1cium oxalate growth. T=28°C, pH=5.8, ionie strength = 0.15, [oxalate]o = 7.5 X 10-4M. (1) In the [Ca]o = 2.5 X 10-4M, presenee of malie aeid; (2) in the presenee of eitrie aeid; (3) in the presenee of EDTA.

References [1] P.A. Curreri, G. Onoda and B. Finlayson, J. Crystal Growth 53 (1981) 209.

500

F. Grases el al.

I

Polyhydroxycarboxylic

[2] G.A. Rose and P.e. Hallson, Fortschr. Urol. Nephrol. 22 (1984) 484. [3] S. Bisac, R. Felix, W.F. Newman and H. Fleisch, Mineral. Electrolyte Metab. 1 (1978) 74. [4] D.H. Hosking, J.W.L. Wilson, R.R. Liedtke, L.H. Smith and D. Wilson, J. Lab. Clin. Med. 106(6) (1985) 682. [5] W.G. Robertson, M. Peacock, P.J. Heyburn, D.H. Marshall and P.B. Clark, Brit. J. Urol. 50 (1978) 449. [6] R.W. Marshall and H. Barry, Urine Saturation and Formation of Calcium Containing Calculi: The Effects of Various Forms of Therapy (Karger, Basel, 1973).

acids as inhibilors o[ calcium oxalate growth

[7] B. Tomazic and G.H. Nancollas, J. Crystal Growth 355 (1979) 46. [8] e.W. Davies, Ion Association (Butterworths, London, 1962). [9] L. Meites, Handbook of Analytical Chemistry (McGrawHill, New York, 1963). [10] D.J. Kok, L.J. Blomen, P. Westbroek and O.L.M. Bijvoet, European J. Biochem. 158 (1986) 167. [11] F. Grases, J.J. Gil and A. Con te, in preparation.

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