Indian Journal of Chemical TechnologyVol. 11, November 2004, pp. '764-768

Determination of serum and urinary urate with reusable uricase strip

Sushma, Rekha, Vijay Kumar, Suman & C S Pundir*Biochemistry Research Laboratory, Department of Bio-Sciences, M.D.University, Rohtak 124001, India

Received 7 January 2004; revised received 19July 2004; accepted 5 August 2004

Commercial uricase has been immobilized covalently onto alkylamine glass beads affixed on a plastic strip with aconjugation yield of 87mg/g and 11.5% retention of initial activity of free enzyme. Maximum activity of immobilized

enzyme was attained at pH 8.8, when incubated at 40°C for 20 min. Km for uric acid and Vmax of immobilized enzyme were'0.15mM and 0.68~moles H202/min respectively. A method for uric acid determination in serum and urine was developedusing the strip bound enzyme. The method is based on the measurement of H202 generated from uric acid by using a colourreaction consisting of 4-aminophenazone, p-hydroxybenzoic acid and horseradish peroxidase as chromogenic system. Theminimum detection limit of the method was 1.68mg/dl and recovery of added uric acid in urine was 90% .The coefficient ofvariation (CV) were <5 and <2% for within batch and between batch respectively. The immobilized enzyme lost 50% of its

initial activity after its regular uses for over 3 weeks, when stored in 0.05M glycine NaOH buffer, pH 8.8 at 4°C.

IPC Code: C03 C 17/28

Keywords: Urate, uricase strip, serum, urine, alkylamine glass beads

Determination of uric acid in serum and urine isrequired in the diagnosis and management of variousdiseases such as gout, leukemia, toxemia ofpregnancy, severe renal impairment and urinary stonedisease'. Among the various methods available foruric acid determination such as chemical'',colorimetric.', mass fragmantography", radiochemicalHPLC5

, enzyme electrode" and enzymic UVspectrophotometric", enzymic colorimetric methodemploying uricase is simple, sensitive, specific, rapidand does not require expensive apparatus hencesuitable for routine purposes. This method is sopopular that its commercial kit is available (MilesIndia Ltd, Baroda, Sigma Chemical Company, USA).However, enzymic colorimetric method becomesexpensive when used for a large number of samples.The high cost of uricase is a factor that limits thewidespread use of enzymic colorimetric method forroutine purposes. The immobilization of the enzymeonto insoluble support permits its reuse and hencereduces the cost of procedure. Among the variousinsoluble supports employed for immobilization ofuricase such as dextran and polyethylene glycol",nylon tubing!", polyamide tubing", elastin", cellulose

*For correspondence(E-mail: pundircs@rediffmail.com; Fax: (91)(1262) 74076)

acetate membrane", hornblende':' and inner wall ofglass beaker", alkylamine glass beads are generallyresistant to microbial attack, stable over a wide pHrange and in various solvents". The methods fordiscrete analysis of uric acid in serum and urineemploying free alkylamine glass beads bound uricaseand peroxidase have previously been reported'v".Although the methods were simple, sensitive, specificand economic, the handling of the glass beads wastedious, time consuming and always had a chance oftheir loss during transfer of reaction mixture fromreaction flask to the cuvette and their washing forreuse. This problem was overcome in the presentwork by affixing the alkylamine glass beads on aplastic strip prior to immobilization of enzyme.

Experimental Procedure

ChemicalZirconia coated alkylamine glass beads (pore

diameter 55nm) were received from Dr H.H WeetallEnvironment Protection Group, Las Vegas, NV, USA.Horse-radish peroxidase (RZ= 1.0), 4-aminophenaz-one (Sigma Chemicals Co., USA) and p-hydroxy benzoic acid (E. Merck, Germany) were used.The kit for enzymic colourimetric method for uricacid determination was obtained from Transasia Bio-

SUSHMA et al.: DETERMINATION OF SERUM AND URINARY URATE WITH REUSABLE URICASE STRIP 765

Medical Ltd., Daman, India (ERBA TEST). All otherchemicals used were of AR grade.Preparation of colour reagent

It was prepared as described earlier", and consistedof 61mg 4-amino phenazone, 1.035g p-hydroxyben-zoic acid and 300 unit of horse-radish peroxidase per100mL of 0.4 M sodium phosphate buffer (pH 7.0). Itwas stored in amber colored bottle at 4°C andprepared fresh every week.

Dissolution of commercial uricaseThe powdered reagent 1 (5 mg) of commercially

available enzo kit for uric acid determinationcontaining uricase, 3,5-dichloro-2-hydroxy benzenesulphonate (DHBS), peroxidase and potassiumferricyanide was dissolved in 3.0 mL aqua (distilledwater) supplied with the kit. It was stored at 4°C untilused.

Assay of free uricaseThe assay of uricase was carried out as described

earlier", The enzyme assay was carried out in a 15mL test tube wrapped with black paper. Assaymixture contained 1.8 mL of O. 05 M glycine NaOHbuffer (pH 8.8), 0.1 mL of uric acid (1 roM) and 0.1mL of uricase solution (0.2 unit/mL). The control tubecontained 1.9 mL of glycine NaOH buffer (pH 8.8)and 0.1 mL of uric acid (1 mM). After incubation at37°C for 5 min in a water bath, 1 mL of colourreagent was added to reaction mixture and kept atroom temperature for 15 min in dark to develop thecolour. A520 was read in Spectronic-20 and the contentof H202 generated in the reaction was determinedfrom the standard curve of H20z prepared in 0.4 Msodium phosphate buffer of pH 7.0. One unit ofenzyme is defined as the amount of enzyme requiredto produce one nmole of H202 per min under thestandard assay conditions. The soluble protein contentin uricase solution was estimated".

Preparation of reusable enzyme strip

Affixation of alkylamine glass beads on a plastic stripA plastic strip of 15x1 em was cut from a plastic

sheet and its one end was made round. A layer of. 1mm thickness of a non reactive fixative i.e. Lakmenail paint (no.754, dew drops) was applied uniformlyonto both sides of the round end of the strip up to theheight of 2cm. Alkylamine glass beads (50mg) weresprinkled over the fixative layer on the stripuniformly. The strip was kept in a 15mL test tube at

room temperature undisturbed for overnight fordrying.Immobilization of uricase onto affixed alkylamine glass beads

This was carried out as described elsewhere" withsome modifications. In a 15 mL test tube, 0.3 mL ofglutaraldehyde (25%) was mixed in 2.7 mL of 0.1 Msodium phosphate buffer (pH 7.0) and the stripcontaining affixed glass beads was dipped in thissolution and allowed to stand for 2 h at roomtemperature with occasional shaking. The strip wastaken out of glutaraldehyde solution and dippedrepeatedly into distilled water until the pH of thewashing discard was 7.0 to ensure the completeremoval of glutaraldehyde. The beads were washedfinally in 0.05 M sodium phosphate buffer (pH 7.0).The end of plastic strip containing glutaraldehydeactivated glass beads was dipped in uricase (2 ml.)solution in a 15 mL test tube and allowed to stand at4°C for 48 h with occasional stirring. Afterimmobilization, the strip was taken out and theremaining enzyme solution was tested for activity andprotein. The strip was dipped in distilled water 5-6times until no activity was detected in the washingdiscard.

Assay of immobilized uricaseThe assay of uricase immobilized onto alkylamine

glass beads affixed on one end of the plastic striptermed as Enzyme strip, was carried out in a 15 mLtest tube wrapped with black paper. The reactionmixture consisting of 1.9 mL of 0.05 M glycineNaOH buffer (pH 8.8) and enzyme strip waspreincubated at 37°C for 5 min. The reaction wasstarted by adding 0.1 mL uric acid solution to thereaction mixture and the mouth of test tubes wascovered with aluminium foil. After incubation at 37°Cfor 15 min in a temperature controlled water bathunder constant stirring, the enzyme strip was takenout from the test tube and 1.0 mL colour reagent wasadded to it and kept at room tempetrature for 15 minto develop the colour. A520 was read against the blankand content of H202 generated in the reaction wasextrapolated from standard curve of H202. The blankwas prepared in the same manner as described forassay except that it lacked enzyme strip and forcontrol, the enzyme strip was replaced by a plasticstrip containing the same quantity of glutaraldehydeactivated affixed glass beads termed as Control strip'(Fig. la & l b), Both enzyme strip and control stripwere stored separately at 4°C in reaction buffer, when

766 INDIAN J CHEM. TECHNOL., NOVEMBER 2004

Fig. l-(a) Blank: strip containing alkylamine glass beads affixed with nail paint; Control: strip containing glutaraldehyde activatedaffixed alkylamine glass beads; Test: strip containing affixed alkylamine glass beads coupled to uricase. (b) Control: strip containingactivated alkylamine glass beads; Test: strip containing activated alkylamine glass bound uricase; reaction buffer, 1.9 mL; uric acidsolution, 1.0mL; and colour reagent, 1.0 mL

not in use. One unit of immobilized enzyme is definedas the amount of enzyme bound to glass beads, whichproduces lnmole of H202 from urate per min understandard assay condition'"

Kinetic properties of immobilized uricaseVarious kinetic properties of immobilized uricase

viz., pH optima, incubation temperature for maximumactivity, energy of activation (Ea), time of incubationand effect of substrate concentration. To determinethe pH optima, the pH of reaction buffer was variedfrom pH 8.6 to 10 using the glycine-NaOH buffereach at final concentration of 0.05 M. Energy ofactivation (Ea) was calculated from Arrhenius plotbetween reciprocal of temperature (T) in Kelvin andlog of enzyme activity (v). Km and Vmax values werecalculated from Lineweaver-Burk plot.

Preparation of standard curve for uric acidA standard curve between uric acid concentration

ranging from 0.05 to 4 mM and A520 was preparedunder optimal assay conditions as described for assayof immobilized uricase.

Determination of serum uric acid

Collection and pretreatment of serumBlood (2 mL) was withdrawn intravenously from

apparently healthy adults and gout patients with thehelp of a sterilized needle and syringe at local PGIMSHospital and transferred to a glass vial and kept atroom temperature for lh. After centrifugation at 5000rpm for 5 min, the supernatant (serum) was collected.Solid potassium ferrocyanide and sodium nitrite (35mg/dL) were added in the serum sample to avoidpossible interference by bilirubin and ascorbaterespectively.

SUSHMA et at. : DETERMINATION OF SERUM AND URINARY URATE WITH REUSABLE URICASE STRIP 767

Assay of serum uric acidThe uric acid in pretreated serum was determined

as described for standard curve of uric acid excepturic acid solution was replaced by 0.1 mL pretreatedserum. The concentration of uric acid in serum wasextrapolated from standard curve of urate preparedbetween uric acid concentration versus AS20.

Determination of urinary urate

Collection and pretreatment of urine samplesUrine samples (24h) were collected in plastic

bottles containing 15mL concentrated HCI, fromapparently healthy adults. To 1 mL acidified urinetaken in a 15 mL graduated centrifuge tube, wasadded 1 mL CaCh (5g/L) solution and final pH wasadjusted around 7.0 by adding NaOH. Absolute ethylalcohol (8.0 mL) was added immediately and tubeswere covered with aluminium foil and kept at roomtemperature for precipitation. After 24 h, the tubeswere centrifuged at 3000 rpm for 5 min and theprecipitate was dissolved in 1.0 mL O.IN HCl.

Assay of urinary uric acidUric acid in urine was determined as described for

standard curve of uric acid except that uric acidsolution was replaced by 0.1 mL of pretreated urine.The concentration of uric acid in urine wasextrapolated from standard curve for uric acid.

Storage stabilityThe enzyme strip was kept dipped in reaction

buffer (0.05 M glycine NaOH buffer, pH 8.8) at 4°Cwhen not in use.

Reuse of immobilized enzymeTo reuse the immobilized enzyme, the 'enzyme

strip' was dipped in the test tube containing 3.0 mL of

reaction buffer (0.05 M glycine NaOH buffer, pH 8.8)and shaken gently for 10-15 s. The strip was taken outand the same process was repeated 3-4 times prior toits use in the next assay.

Results and DiscussionUricase employed in reagent 1 of commercial enzo

kit for uric acid determination has been immobilizedcovalently onto alkylamine glass beads affixed on aplastic strip with a fixative. The conjugation yield ofplastic strip bound uricase was 87 mg/g, which ishigher than that on free alkylamine glass beads 4mg/gI5.16, alkylamine salinized glass wall 10 rng/g"and glass column 25.6 mg/g'". However, the stripbound enzyme retained only 11.5 % of the initialspecific activity of free enzyme, which is lower thanthat of enzyme bound to free alkyl amine glass beads(83.3 %)16, controlled pore glass (70 %io, elastin (50%) 14 and protamine (85 %)21. The low acti vity ofplastic strip bound enzyme could be due to eitherhydrophobic environment provided by the plastic striparound the enzyme or leakage of some metal ionsfrom the adhesive, which might interfere in theenzyme activity. A comparison of kinetic propertiesof free and enzyme bound to free/affixed alkylamineglass beads is given in Table I.

A simple, sensitive and specific method for discreteanalysis of serum and urinary urate was developedemploying uricase immobilized to alkylamine glassbeads affixed onto plastic strip termed as 'enzymestrip'. The method is based on quantification of H202generated from serum/urinary urate by plastic stripbound uricase with a color reaction consisting of4-aminophenazone, p-hydroxy benzoic acid andperoxidase as chromogenic system. The advantage ofthis method is that it not only provides very easy

Table 1- A comparison of kinetic properties of free uricase, uricase bound to free and affixed 2 lkylamine glass beads(pore diameter 55 nm) on a plastic strip

Uricase

Kinetic properties Free Bound to affixed alkyamine glass beadsBound to free alkyamine glassbeads

Optimum pHOptimum temp. (0C)

Time of incubation (min)Km(mM)

Vmax (11 molH202/minlmL)

Stability after storage at 4°C

for three weeks

*The initial activities of free and immobilized uricase were considered as 100%.

8.5

37

20

0.70

0.033

100*

8.8

40

250.13

0.029

8.8

40

20

0.15

0.068

50*85*

768 INDIAN J CHEM. TECHNOL., NOVEMBER 2004

reuse of alkylamine bound uricase but also avoids itsexposure to color reaction. The following analyticalparameters were studied to evaluate the method.

LinearityThe relationship between A520 and uric acid

concentrations ranging from 0-48/lg/2mL reactionmixture was linear up to an absorbance of 0.16, whichis comparable to that obtained by employing freealkylamine glass".

Detection limit

The lower detection limit was 11.0 /lg uric acid/O.lmL urine which is lower than those usingimmobilized uricase in closed loop system (19ug/sample) mass fragmentography method (126ug/sample) but higher than that using uricase coupledto protamine bound glass beads (2 ug /sample). Theminimum detection limit for serum uric acid was 25ug/ml., which is higher than that reported foremploying uricase and peroxidase immobilized ontofree alkyl amine glass beads".

Percent recoveryTo test the reliability of the method, solid uric acid

was added to urine (22 mgIL). The recovery of addeduric acid was 90 % which is comparable to thatreported for free alkylamine glass beads immobilizeduricase (91 %)15.

PrecisionThe within and between batch coefficient of

variation (CV) for uric acid determination in serum bythe present method were <5 and <2 % respectivelywhich is comparable to those obtained by employingfree alkyl amine glass beads immobilized uricase':',

AccuracyTo check the accuracy, the total uric acid value in

serum as determined by the present method (y) werecompared with those obtained by commerciallyavailable Enzo-kit method employing solubleenzymes (x). The values obtained by both themethods showed good correlation with r=:0.88.

Uric acidThe uric acid value in the serum of apparently

healthy persons (20) and gout patients (15) weredetermined by the present method and was found tobe in the range 25 to 50 ug/ml, with a mean of 37ug/ml, and 84 to 168 ug/ml, with a mean of 116ug/rnl, in healthy and gout patients respectively. Uric

acid values in the urine samples of apparently healthyadults as determined by present method were in therange 50 to 100 mg/l00mL with a mean of 72.8mg/lOOmL which were comparable to that reportedfor free alkylamine beads immobilized uricase (44 to92 mg/dl.)". The uric acid value in the urine of goutpatients was in the range 148 to 200 mg/dl, with amean of 164 mg/dL.

Reusability and storageThe enzyme strip lost 50% of their initial activity

after its use 50 times during the span of three weekswhen stored at 4°C in reaction buffer. The lowreusability of enzyme strip might be due to gradualdeposition of reaction product onto plastic strip withits reuses. Effects are being made in our laboratory toovercome this problem.

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