Indi an Journal or Pure & Applied Physics Vol. 41 , March 2003, pp. 183- 192

Influence of inhibition of citric acid and lemon juice to the growth of calcium hydrogen phosphate dihydrate urinary crystals

Virna! S Joshi * & Mihir 1 Joshi

Solld State Phys ics and Materials Science Laboratory, Department or Physics, Saurashtra University, Rajkot 360 005

[E-mail: mshilp24@hotmail.com]

Received 2 Jul y 2002; revised 23 October 2002; accepted 23 January 2003

Calcium hydrogen phosphate dihydrate (CHPD) crystal is a well-known urinary crystal. Calcium phosphate is rresent in urinary ca lcul i (stones) as ei ther apatite or brushite. Crystal s or calcium hydrogen phosphate dihydrate were grown hy single diffusion gel growth technique in silica g~ l s. The supernatant solution containing combinations o r constituents, like onl y calcium chl oride, calcium chl oride + ci tric acid; calcium chloride + lemon juice; calcium chl ori de + lemon jui ce + artiticial refe rence urine (ARU ) and calcium ch loride + lemon juice + natural urine, were used to study the growt h and inhibition or CHPD crystals. The growth veloci ty measurements indicated maximum inh ibition in case or calcium chl oride + lemon jui ce + natural urine containing solution . SEM (scanning electron microscope) and FrlR (Fourier transform infrared ) spectroscopy were used to study the crystals. The results are discussed in terms or citrate inhi bition and inhibition due to natural urine.

1 Introduction

A uro li th is a po lyc rystalline concretion found in urinary tract, contammg primarily inorganic crysta ll oid and a sma ll amount of organic matrix . Uroliths have an organized regular pattern of crystal deposition on the organic matrix . Uroliths are a lso ca lled calcu li or stones. Urinary calcul i are among the o ldest affli ctions of men l.2 as well as anima l s ' -~.

As per one es timate, around 2 mi lli on people are treated world-wide with Extra Corporal Shock Wave Lithotripsy (ES WL)'. Around 12% of population in the European Uni on suffer at least one process re lated to uro lithiasis6

. About I adult in 1000 is hospitalized annually in the USA because of urinary calculi 7

• Whatever be the technique of extract ion , 70% of the cases generate new calcu li in 5 years. T hi s is regarded as due to the fact that, urinary alternations responsib le for the stone formation sti ll remain as it is. However, these alternations may be treated by means of dietary changes, medication, or both; minimizing the intensity of disease and reducing the number of re lapsesx.

Generally , calci um-contai ning stones occur as calcium phosphate or calcium oxalate or as a

Present Address: Physics Department , H. N.S .B. Science College, Hi mat nagar 383 0 II

mixture of the two. A lso, calcium phosphate is present in urinary calculi as ei ther apatite [Ca 111

(PO~),, (OH)"] or brushite [CaHP0~ .2H"O]. Many chemicals are found to have inhibitive acti on on the growth of urinary stones as we ll as crystals . Natarajan et aU have studi ed various inhibitors and promoters of urinary crystal s. Recentl y, Joshi 111 has tested various inhibitors such as, certain fruit juices, homogenate soluti ons of ayurvedic medicinal plants and different pu lses on the growth of calcium hydrogen phosphate and calcium oxalate crystals . Citrate is the most important complexor of calcium in urine and reduces ionic calcium concentrati on 11 ·n .

It inhibits both spontaneous and heterogeneous nucleation 11

•13

·14

•

The growth of crystal s by gel technique is we ll known 15

-17

, which can be treated as a simple model to grow the urinary type crystal 1u 1 and study the effect of various inhibitors in vitro~ · 1 11 211 · 2 1 • In the present investigation, the effect of c itric ac id , lemon juice and lemon j uice a long with natural and art ific ia l urine on the growth of calcium hydrogen phosphate (CHPD) crystals are di scussed and the ir inhibitive behaviour are studied . Thi s may be useful in the prevention of occurrence of urinary calcul i.

184 INDIAN J PURE & APPL PHYS , VOL 41 , MARCH 2003

2 Experimental Technique

The CHPD crysta ls were grown in sili ca gel medium by incorporat ing the single diffu sion react ion tec hnique. The gel was prepared by mixing aqueous so luti on of sodium meras ilicate of 1.03 specifi c grav ity with di luted ortho-phosphoric ac id , so that the pH of 6.0 cou ld be set for thi s mi xture. This mixture was transferred into g lass test tubes of 150 mm length and 25 mm diameter. After ge lation took place, an aqueous so lution of ca lcium chloride was poured . After a few days, c rys tals were fou nd to be grow ing and growth was completed with in a month. A total of 18 Li esegang rings were formed. A reg ion of 5 mm thi ck wh ite prec ipitate occurred just be low the ge l li quid inte rface. Ca lc ium hydrogen phosphate dihydrate crysta ls were found to be growin g within the Li esegang rings. A total of 20 crys ta ls were grown ha ving e longated needles, p la tes and star-shaped morpho logies. The largest needl e was found to have a length of 18 mm. The type of c rys ta l growth is shown in F ig. I . Henisch 15

&,cusses the forma ti on of Liesegang rings in detail.

The e ffect of c itric ac id , le mon juice, artifi c ial urine and natural urine on the growth of crysta ls was studied by adding them in specific amoun ts a long with the calci um chloride sol uti ons. The apparen t lengths of growing crystals were measured by trave lling microscope, having the least capacity of measurement of 0.00 I em, at different time inte rva ls.

The FTIR spectra of powdered sampl es in KBr medium were recorded in the range 400-4000 cnr 1

,

by using a NICOLET MAGNA - TR550 SERIES II FTIR spec trometer.

Scanning e lectron micrographs were recorded on LEfCA S 400 OXFORD model 7060 m1croscope.

3 Results

3.1 Effect of citric acid concentration on growth of CHPD crystals

In the present sec tion, the effect of concentration of c itric acid on the growth of CHPD crystal is reported by choosing citric acid molarities from 0 . 1 to 0.9 mol ar. The growth observations are as follows:

When supernatant solution, containing 0.1 molar c itric acid and I molar calc ium chloride solution in

equa l amounts ( 12.5 ml each) was poured on the set gel , two broad Liesegang rings and two parti a ll y formed discs were observed. Initi a ll y, the di scs were formed ; thereafter, they convertvd into the Liesegang rings . Near the li qu id-ge l in terface, co loured precipitates were found , which may be due to direct reaction. Snowflake shaped cry~ta l s having a maximum length of 1.5 mm and need le-shaped crysta ls having a maximum length 4 mm were grown in the ge l. The crystals were found to remain stab le in the ge l. F ig. 2 is a photograp h depi ct ing this type of crys tal growth.

Fi g. I- Photograph showing the growth ol C HPD crystal s in pure CaCI2 conlaining supernatant solut ion

Supernatant so lution containi ng 0 .2 molar c itric acid and I molar calcium chl oride each in equal amounts (12.5 ml each), initially , produced two Liesegang rings and two partly fo rmed di scs . After some time, these two rings formed a broad band of finely di spersed precipitates . In aiL seven crystals were grown, hav ing sta r and needle shaped morphologies, with a maximum length of 3.5 mm. The crysta ls remained stable for a long time . F ig . 3 shows this type of growth.

JOSHI & JOSHI: URINARY CRYSTALS 185

Fig. :2- Photograph of crystal growth in CaCI2 and 0.1 M citric acid containing supernatant soluti on

ln the case of supernatant so lu tion contain ing 0.3 molar c itric ac id and I molar calcium ch loride in equal amounts ( 12.5 ml each) , three bands of di spersed precipitates were found. A total of s ix crysta ls hav ing spikey, spherulitic and needle shaped morphologies were grown with a maximum length of 3 mm. A few crystals were observed to be di sso lved after some time and , at the same time, a few crystals were found to be grown at some other sites towards the bottom of the test tube. The CHPD crystal s dissolve in c itric ac id solution , because, the diffusion of high concentration of citric acid into the gel further d isso lved the grown crystals . But, at some other places, for exampl e, at the bottom of a test tube, where the concentrati on of citric acid was not enough to dissolve the grown crystals, nucleation was poss ible, which grew into CHPD crystal s. Initially, when c itric ac id did not diffuse sufficiently, the growth of CHPD crystal s took place. After passage of some time, the amount of diffused citric acid increased, which di ssolved the grown crystals.

Supernatant so lutions compnsmg 0.4 molar citric acid and I molar calcium chloride solution in

equal amounts ( 12 .5 ml each) yie lded one lightl y di spersed ring and two partia lly formed discs in the gel. Four crystals were grown whic h di ssolved after three weeks. Maximum length of crystal obtained was I .0 mm.

Table I - Assignments of ab~orption s bands of FTIR spectra for CHPD crystals grown in cases of pure calcium chl oride as well as calci um chloride+ citr ic acid containin g supernatant solut ion s

Assignments of absorption peak

Inter-mo lecu lar and weakly H bonded 0- H due to water of crystalli zat ion

HP04.2 ions

H -0 - H bend ing vibrations

P = 0 associated stretching vibrati ons

P = 0 stretching vibrati ons

P-0-P asymmetric stretching vibrations

( H -0-) P=O

Acid phosphates

Peak position (in cm.1) for

C HPD crystal

Pure calcium chloride so luti on

3545 3490 3284 3168

2375 1722

1651

1217 11 37

1061

987 874 794

665

577 525

Calcium chl oride +Citri c acid solu ti o n

3546 3489 3283 3170

2372

1651

1211 11 36

1063

987 872 794

661

577 525

When 0.5 molar citric acid was added to one molar calcium chloride solution in equal amoun ts ( 12.5 ml each), initially, two Liesegang rings were found, which diffused later to form one band of precipitates. Four crystal s were grown having a max imum length of I mm, which dissolved after 3 weeks.

I X6 INDIAN J PURE & APPL PHYS, VOL 4 1, MARCH 2003

The supernatant solut ion compn s tng o f 0 .6 molar c itri c acid and I molar calcium chloride gave two li ght ly dispersed Li esegang rings, which su bsequent ly di ffused into one band afte r some time . Four c rysta ls were grown, which di ssolved after two weeks; subsequentl y a new crysta l of 0 .2 mm max imum length appeared, whi ch a lso d isso lved after three weeks.

Fig. :l- Photograph of crysta l growth in CaC1 2 and 0.2 M citric acid comaining supernatant solution

In the case of supernatant soluti on hav ing 0 .7 mo lar c itri c ac id and I molar calcium chloride soluti on in equa l amounts (12.5 ml each), one Liesegang ring was observed . O ne spheruliti c crys ta l o f 0.5 mm dimension was observed. The crystal was di sso lved afte r th ree weeks. Peri odi ca ll y dispe rsed prec ip itates were observed in the ge l, its dens ity was fo und to be decreas ing on movmg towards the bottom of the test tu be.

T he supernatant solu tion containing 0 .8 molar c itri c ac id and I molar ca lc ium chl oride so luti on in equa l amounts ( 12.5 ml each) gave one lightl y d ispersed Li esegang ring. One crysta l of very sma ll di mension grew and di sso lved within a short time.

From the liquid-ge l inte rface up to 42 mm width , peri od ically dispersed prec ipitates were observed .

20

.. v 16 c 0 --E IJI c 0 .... I-

D

' D

- 0·4 ';;';;---:-:::::----::~::------1--~~--.U 4000 3000 2000 1000

Wave numbers km1)

Fig. 4- FTIR spectrum of CHPD crys tals for pure CaCI2

containing supernatant solution

~ 0

(\) u c:: 0 ... ... E Ill c:: 0 !... 1-

24 t. 0':-:0 0~3::-::0'::':0 0::--2::-::0700:----::15::'-:-0-:-0 ----,,0..._00--5~00

Fi g. 5-- FTIR spectrum of CHPD crystals containi ng CaC12

and citric ac id in supernatant so lution

F inally, a supernatant so lution co mpri sing 0.9 molar c itric ac id and I molar calc ium chl oride solution, in equal amounts (12.5 ml each) gave one lightly di spersed Liesegang ring . Interes tingly, no crys ta ls were grown in the gel because of higher concentration o f c itric ac id . F rom liquid-ge l inte rface up to 50 mm depth in the ge l, periodically di spersed precipitates were observed .

JOSHI & JOSHI : URINARY CRYSTALS 187

Fig. 6- Photograph depicting the growth of CHPD crystals for CaC I2 and lemon juice contain ing supernatant soluti ons

It can be noticed from the growth rate observations that, the size of grown CHPD crystals decreases as the concentration of citric acid in the supernatant soluti on increases. The maximum lengths are far less than 5 mm, which can easily pass through the urinary tract. Also, the increased amount of citric acid disso lves the grown crystal and for 0.9 molar concentration of citric acid, the crystal is not allowed to grow.

The FfiR spectra of powdered samples were recorded in KBr medium for crystal growth in pure calcium chloride containing supernatant solution and calcium chloride + citric acid containing supernatant solution, which are presented in Figs 4 and 5, respectively. Comparing these two spectra and assigning the absorption bands li sted in Table I, one can say that , both the spectra are identical and indicate the same compound, that is, CHPD crystals. Thus, the crystals grown in the presence of citric acid were also CHPD crystals.

3.2 Effect of lemon juice on the growth of CHPD crystals

Lemon juice was extracted by squeezing lemons in the conventional way and filtered by Whatman filter papers. After gelation took place, the supernatant solution containing I molar calcium

chloride and lemon juice in equal amounts ( 12.5 ml each) was added . Three broad Li esegang rings were obtained. A total of 28 small c rystals of the snowflake type , were grown . The maximum length of the crystal was up to 6 mm. The crystal growth is depicted in the photograph of Fig. 6. The pH of the supernatant solution was found to be 2.88 . The 6 mm length calculi can st ill pass through the urinary tract and the maximum length of the crystal is within this limit.

0

' .!.. (>I

u c 0 .. .. 'E Ill c 0 ..

1-

40

10

4000 3000 2000 1500 -1

Wove numbers !em )

noo 500

Fig. 7- FfiR spectrum of CHPD crystals for pure CaC I2 and lemon juice containing supernatan t solution

Fig. 7 is the FfiR spectrum of the crystals grown using thi s supernatant solution. Comparing this spectrum with the spectrum of Fig. 4 for pure calcium chloride containing supernatant solution, it can be noticed that , both spectra are s imilar and indicate the same crystal.

The crystals grown in the case of pure calcium chloride were needle shaped and platelet shaped . However, in the presence of lemon juice, their morphology changed into snow-flaked shape and lengths were also reduced. Fig. 8 is the scanning electron micrograph of snowflake type crystal, which was recorded at 594 x magnification, indicating need les emanating from a single point. The micrograph of Fig. 9 is recorded at 759x magnification of the same region .

The apparent lengths of the crystals were measured at different time intervals during the growth. From the plots of apparent length versus

188 INDIAN J PURE & APPL PHYS , VOL 4 1, MARCH 2003

...

)

Fig. 8- Electron micrograph o r snowfl ake type CHPD crystals grown in the supern atant sol uti on containing CaC12 and lemon juice at a magnificati on 594 x

, ..

·"'· . .

', . . , ~ ' ~ t;.

;, .. , \ ~ .....

. · .. , · ... l; .... ...

,·. .,

Fig. 9- Electron mi crograph of the same crys tal or Fig. 8 at a magnilica ti on of 1.02 kx

time, the growth rates were deduced from the tangents drawn at different time va lues. The plots of growth rate versus time are shown in Fig. I 0 for supernatan t solution with pure calcium chloride and calc ium chloride with lemon juice. It is noticed that , the growth of CHPD crystals started late in the case of le mon juice. Also, the maximum length of crystal s measured was 1.83 1 em for pure calcium chloride and 0 .232 em for lemon juice containing so luti on. As lemon juice contains c itric acid, this further supports the earlie r theories on c itrate

inhibition 1. 14 and indi cates that, consumpti on of lemon juice may he lp to inhibit the growth of CHPD containing urinary calculi in body.

3.3 Effect of artificial reference urine and natural urine with lemon juice on CHPD c1·ystals

Artificial reference urine (ARC) was prepared by mixing in equal amounts the so luti ons of 0.1055 M sodium chloride, 0.0323 M sodi um dihydrogen phosphate, 0.00321 M sod ium c itrate, 0.00385 M magnesium sulphate, 0.01695 M -odium sulphate

JOSHI & JOSHI: URINARY CRYSTALS

and 0.0637 M potass ium chl oride. The pH was adjusted to 6.5 by using ammonium hydroxide or hydrochloric ac id as per requirement21

.

Urine of healthy, adult person was collected and filtered twice before using, as an additive to the supernatant so lutions. Urine was tested in pathological laboratory and normal , natural urine was used for the study .

When the supernatant solution compris ing 7 ml calcium chloride so lution, 7 ml ARU and 7 ml lemon juice was used, it exhibited strong, inhibiti ve action due to lemon juice. Variation of growth velocity with time is presented in Fig. l I. Fig. 12 is a plot of growth velocity versus time for the supernatant so lution containing 7 ml each of calcium chloride, natural urine and lemon juice. Table 2 compares the growth velocity data at 120 h after pouring the supernatant solutions . Minimum growth velocity is observed in the case of supernatant solution containing lemon juice and natural urine.

Table 2- Growth velocity data of CHPD crystals at 120 h for different supernatant so lutions

Supernatant sol utions Growth velocity clx/d1

(in mm/ h x l0.3)

at 120 h

Calcium chloride 5. 1 1 Calcium ch loride+ lemon juice 2.08 Calcium ch loride+ lemon juice+ AR U 1.78 Calci um chl oride+ lemon juice+ NU 1.5 1

4 Discussions

An estimation of the state-of-saturation of urine with the stone-forming salts, is of prime importance in clinical practice. Usuall y, the state-of-saturati on indicates the free energy of a given solution, which by entropy tends to decrease, resulting into the precipitation of the super-saturated so lid phase2

' . In general, the satu rati on increases with the concentrations of ionic components, and espec iall y, it depends on the activities of the constituent (non­complexed) free ions . To calcul ate the state of saturat ion (~) , the free ion concentrati on must be es tabli shed from the difference between the measured total concentrati on and the sum of concentrations of all the relevant complexesD

7,--~------------------~

Vi ... ::;, 0

.s::.

' E E

7~ 4 ..-)( -"0

' ~ 3

Time (Hours)

Fig. I 0- Plots of growth velocity of CHPD versus time for pure CaCI 2 and lemon juice

The amount of soluble complexes depends on the ir stability constants. The free ions in so lution interact in such a way that , their e lectrochemica l act ivity decreases by a coefficient that is in verse ly rel ated to the ion ic strength . In other words, in concentrated urine, the activity coefficient tends to decrease, and for a given ion concentrati on the activities are lower at higher ioni c strength. The state of saturation ulti mate ly results from the activity product of constituent ions and ca n be expressed as relative super-saturati on, by ca lcu lating the ratio between the activity product and the solubility product of a given sa lt. This has been discussed by Marangell et aF1 in detail by exemplifying for calcium oxalate. A CD-ROM has been made on Turin and Mess ina model, which offers a computer programme named URSUS requiring input variab les of some cations (Na, K: Ca) and anions (oxa late, phosphate, citrate, etc.). The results obtained include, concentrati ons of main soluble complexes, state-of-saturation with ca lc ium

190 INDIAN J PURE & APPL PHYS, VOL 4 1, MARCH 2003

oxa late monohydrate, brushite, etc. in terms of indicati ons of saturation or super-saturati on.

-~ ...c:. ......... E E

("'l-

10 -X -"0 ......... )(

"0

3·0

216

Tirn~(Hours)

Fig. II -Plots of growth velocity of CHPD versus time fo r rurc CaCI 2, lemon juice and ARU .

Saturati on and so lubility product in water are simple to define, but urine is a more complex so lution. In urine, when the concent ra ti on of a substance reaches a point at which the saturati on woul d take place in water, crystalli zati on does not occur as ex pected . However, the urine has the ability to hold more so lute in the solution, than does pure water. Although, all the molecul es of vari ous substances in urine are present in water, the mtxture of many electrica ll y ac ti ve ions in urine causes interac tion that changes the solubility of their components. Moreover, many organic molecules such as urea, uric ac id, cit rate and complex mucoproteins of urine, naturall y, affect the so lu bi li ty of other substances, which has been discussed by Paru lka r er a/.1 in detai l. The inhi bit ive effect due to natural inhi bitors present in urine, to the o-rowth of CHPD crystals can be veri fied from

e . . . Table 2. The growth rate is fo und to be :mn mwm 111

the presence of lemon juice and natu ral un ne_ conta in ing supernatant so lu tion. The absence of natura l inh ibitors in arti fic ial reference urine does not provi de that much inh ibition as natura l urine.

Many chemi ca ls are found to have inhibiti ve ac tions on the growth of urinary calcu li (s tones) and crys tals; for examp le, tartrates have been found as good inhibi tors in natural urine and arti ficial urine medi a24 as well as in tamarind so lutions""- The influence of some amino acids on the spontaneous prec ipitation of calcium oxalate depends on the type and concentrati on of amino ac id used2r' .

1·5

-... .1:. ....... E E ,...,- 1·0 10

X ... "0 ....... X "0

0·5

120 216

Tim ll (Hour s)

Fig. 12- Plots of growt h velocity of C HPD versus time lo r rurc CaCJ2, lemon juice and natu ra l urine

Citrate inhibits nucleati on and growt h steps of ca lcium oxa late monohydrate crys ta lli za ti on27 The c itrate inhi bi ti on suggests that, sod ium citrate, citri c ac id and other cit rate compounds are ac ting as alka linizing agents indicated for systemic metabo lic ac idosis (renal tubul ar ac idos is), whil e urinary alka li zati on or hypoc itraturi a contai ns el i-sod ium citrate. Usuall y, it is admini stered orall y, and it is metaboli zed to bicarbonate by li ver. lt is also used to increase urinary pH and/or provide increased ci trate in urine in persons with hypocitritur ia. Both h:lYe tendency to increase so lu· ility of some mineral s. In hypoc itratu ria, the urina ry citrate is <350 mg/day . Along with other disorders, it promotes ca lculi , because citrate no rma ll y binds Ca as a so luble calc ium ci trate. Pak er of2x reported successful management of uric ac id nep hro li thiasis with potass ium citrate. Potass ium citrate red uces urinary saturation of ca lc ium salts by complex ing calcium and reducing ionic ca lciu m concentration. Apart from th is, due to its alka lini zing effect , it also

JOSHI & JOSHI: URINARY CRYSTALS 19 1

increases the di ssoc iation of uric ac id , lowers the amount of poorl y so luble undi ssociated uric acid, reduc ing its propensity to form uric ac id stones . Ettinger et a/2~ reported potass ium magnesium c itrate as an e ffecti ve prophylaxi s against recurrent ca lc ium oxalate nephrolithiasi s (stones in kidney).

Moreover, c itrus juices like, orange and lemon are expected to prevent the stone formati on due to their high citrate content. One preliminary study fo und that, drinking 2 litres of lemonade per day improved the quality of urine in ways that are associated with stone prevention·10

. The effect of orange juice consumpti on on urinary stone ri sk facto r is a lso reported31

• The present in vestigation gives further proof of c itrate inhibition to the growth of calcium containing urinary crys ta ls, that is, CHPD crystals . Both c itric acid as well as lemon ju ice ex hibits inhibition of CHPD crys tal s . In artific ia l urine and natural urine media as well as without ARU and NU in ge ls, lemon juice gives inhibitions. The inhibition is found maximum and hence the growth ve loc ity of CHPD crystals is found to be minimum in case of lemon juice and natural urine, because natural urine also possesses the natural inhibitors and ex tra inhibition IS

produced .

Ge ls are ne ither liquid nor so lid. Ge ls consist of sheet-like stmctures of vary ing degrees of surface roughness and poros ity , forming inte r-connected cell s. They are not simpl e three-dimensional networks. The cell and pores are having different s izes. At so me stage in gel, as the concentration of diffusant increases, a few nuc le i begin to form . Through the diffusion process the nuclei are supplied by nutri ents and the nuc le i grow . Various authors have discussed this in deta il 15

.17

·32

. Joseph & Jos hi 11

, recentl y di scussed , e laborate ly, the formation of Liesegang rings in the gel growth o f C HPD crysta ls and the parameters affect ing the Li esegang rings . Therefore, de ta iled di scuss ions are avo ided here. There fore, ge l growth is found to be suitab le for growing urinary crysta ls and study of the inhibition behavi our of cit ri c acid and lemon juice under d ifferent medi a. T his can be he lpful to simulate the growth and inhibiti ons of urinary calcu li.

5 Conclusions

Add iti on of c itn c ac id to ca lc ium chl oride contai ning supernatant soluti on reduced the length

of grown CHPD c rystal s and modified their morphology . On increas ing the molarity of citric acid from 0 . 1 to 0 .3 molar, the c rysta l length s decreased appreciab ly. For 0.4-0.8 molar so luti on of citric acid, the grown crysta ls di sso lved afte r some days. Finally, for 0 .9 and 1.0 molar c itri c acid , no growth of CHPD crysta ls was observed , that is, complete inhibition .

Lemon juice contai ns citric ac id . When le mon juice was added to the calc ium chl oride contain ing supernatant solution, the morpho logy o f CHPD crysta ls was changed and the dimensions were reduced. Snowflake type crystals were grown , which were investigated by scanning e lectron microscopy and fo und to be having needles coming out from a si ngle point.

The FTIR spectra obtained for the crysta ls grown by applying c itric acid and lemon juice containing supernatant soluti ons exhibited same natu re as that of CHPD crystals, therefore, confirmed the CHPD crysta l growth.

The growth velocity measure ments for pure calcium chloride, calcium chl oride + le mon juice ; calcium chl oride + lemon juice + ARU; and finally calcium chloride + lemon juice + NU were made . The minimum growth ve loc ity and hence the max imum inhibition was observed in the case of lemon juice and natural urine containing so lution s. Natural urine contains certai n inhibiting substances and the inhibiti ve action of lemon juice added up to the maximum inhibition . Thi s furth er supports the citrate inhibition to CHPD crysta ls, wh ich can be extended to urinary calculi inhibitio n.

Acknowledgement

The authors are thankful to Prof B S Shah , Head , Phys ics Department , Dr Ashok B Vaidya, SPARC, Mumhai; and Dr Jayant Vyas, Director, Forensic Sc ience Laboratory, Ahmedabad , for fmitful discuss ions and the ir inte res t in the present work. They are thankful to the Direc tor, Regional Sophi sti cated In stmmentation Centre, Nagpur, for FTIR spec tra . One of the autho rs (VSJ) is th ankful to the Univers ity Grants Commiss ion, New De lhi , for fund ing minor research project.

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