Oxalate Determination in Urine Using an Immobilized Enzyme on Sorghum vulgare Seeds in a Flow Injection Conductimetric System

Desenvolveu-se um método por injeção em fluxo para determinação de oxalato em urina, baseado na utilização da oxalato oxidase (E.C. 1.2.3.4) imobilizada em sementes trituradas de Sor ghum vulgare, variedade BR-303. Amostras de 200 μL contendo oxalato são introduzidas num fluxo de água deionizada que passa por um reator, em forma de coluna, preenchido com o ma te rial enzimático ativado. O dióxido de carbono produzido pela reação enzimática é conduzido, pelo fluxo, até uma cela de permeação, contendo uma membrana de PTFE, onde permeia para um outro fluxo de água deionizada. Este fluxo passa por uma cela de condutividade. A presença de dióxido de carbono provoca uma diferença na condutividade, proporcional à concentração de oxalato originalmente presente na amostra. Os resultados obtidos mostram uma faixa de linearidade en tre 0,05 e 0,50 mmol dm. O método proposto, quando comparado com o procedimento enzimático da Sigma, mostra uma boa correlação (Y = 0,006( ±0,016) + 0,98( ±0,019) X, r = 0,9995, Y = condutividade em μS e X = concentração em mmol dm), seletividade e sensibilidade. O novo procedimento de imobilização promove grande aumento de estabilidade da enzima permitindo a determinação de oxalato por cerca de seis meses. Cerca de 13 determinações podem ser realizadas por hora. A precisão do método proposto é bastante satisfatória (d.p.r. = ± 3,2 %).


In tro duc tion
In re cent years a large num ber of meth ods [1][2][3][4][5][6][7] have been de vel oped to im mo bi lize en zymes on solid ma tri ces.The most pre dom i nant method is that of car rier bind ing, and many com mer cially avail able im mo bi lized en zymes are found with syn thetic ma tri ces.Crude ma te ri als like Sorghum vulgare may con tain many or ganic com pounds, mainly poly sac cha rides, pro teins, and lipids.In this work we have used nat u ral seeds ac ti vated with glutaraldehyde.The ionic in ter ac tion, hydroxyl groups from the poly saccha rides, and pro teins may be at tracted by hy dro philic regions of the en zyme mol e cule, while lipid groups may sup ply ad di tional at trac tion to the hy dro pho bic re gions of the en zyme.In ad di tion, the amino groups of the pro teins may link with glutaraldehyde mol e cules and yield a cross link ing in ter ac tion with the en zyme.With this kind of immo bi li za tion, the frag ile en zyme mol e cules may be protected by an ap pro pri ate mi cro en vi ron ment, dif fer ent from those ob served when ar ti fi cial sup ports are em ployed.Nature was the first to re al ize this, since liv ing or gan isms have many en zymes in the im mo bi lized form.
The de ter mi na tion of ox a late in urine has been very impor tant for the clin i cal di ag no sis of var i ous forms of hyperoxaluria and uri nary tract stones 8,9 .Cur rent meth ods for ox a late de ter mi na tions in clude sol vent ex trac tion and pre cip i ta tion 9 , as well as colorimetric 10 , fluorimetric 11 , and chro mato graphic meth ods [12][13][14] .All of these meth ods require la bo ri ous sam ple pre treat ment due to prob lems of inter fer ence.In ad di tion, the ma jor ity of chro mato graphic meth ods in volve HPLC, which pres ents high costs.On the other hand, an im mo bi lized en zyme pro ce dure has many ad van tages, such as se lec tiv ity and sen si tiv ity, low cost and speed 15 .Gen erally, the pu ri fied en zymes are im mo bi lized on in or ganic ma tri ces or poly sac cha rides.In all of them, ma trix ac ti va tion, sam ple prep a ra tion, and FI man i fold demand com pli cated steps, in clud ing analyte sep a ration 1,2,[16][17][18][19][20][21] .For ox a late anal y sis, the most used en zyme has been the ox a late oxidase (E.C. 1.2.3.4) from dif fer ent veg eta bles sources such as bar ley 3 , ba nanas 4 , amarantus 5 , sorghum 6 , beets 7 , etc.
As far as we know, there are no meth ods that use the bio log i cal sup ports for en zyme im mo bi li za tion that are or not em ployed in flow in jec tion anal y sis de scribed in the lit er ature.As Sor ghum vulgare seeds pres ent nat u rally im mo bilized ox a late oxidase 6 , the pure en zyme can be eas ily im mo bi lized onto its sur face, in creas ing the en zyme ac tivity, al low ing the use of this ma te rial in the pro posed FI method for ox a late de ter mi na tion.The use of such ma te rial pres ents ad van tages such as high sta bil ity and low cost.
This work de scribes a flow in jec tion (FI) sys tem for the de ter mi na tion of ox a late in urine, us ing a new sup port for en zyme im mo bi li za tion and a flow conductimetric de tector.The use of this nat u ral sup port in creases the sta bil ity and activity of the enzyme reactor.The carbon di ox ide pro duced was de tected us ing a conductimetric meth od ol ogy.

Ma te rials
All re ac tants used were of an a lyt i cal grade.Ox a late urine con trol, N, and E, ox a late oxidase (E.C.1.2.3.4), were ob tained from Sigma (St.Louis).Stan dard ox alic acid solu tions, with con cen tra tions from 0.05 to 1.0 mmol dm -3 , were pre pared in a 0.05 mol dm -3 so dium dihydrogenophosphate/phos pho ric acid so lu tion (pH = 3.0).Cal i bra tion curves were made daily with these so lutions.A 10 mmol dm -3 ox alic acid so lu tion was pre pared, and suit able vol umes were added to pre-treated urine spec imens by the stan dard ad di tion method.All mea sure ments were per formed us ing deionized wa ter ob tained from a NANOPURE ® deionizer (0.056 µS).All data were expressed in millimol (mmol) of ox alic acid in 24 h urine spec i mens.
Con duc tiv ity me ter -Micronal model B-331 con nected to a chart re corder, Cole-Palmer 9375 se ries Spectrophotometer -Sin gle beam Micronal model B-382.
Conductimetric flow cell -as pre vi ously de scribed 22 .
CO2 Per me ation cell -al ready pub lished 23 .

Sam ple prep a ra tion
Sev eral 24 h urine spec i mens were col lected, and then ap pro pri ate di lu tions were made in 0.05 mol dm -3 so dium dihydrogenophosphate pH 3.0 so lu tion ad justed with phospho ric acid.Urine spec i mens were frozen when not in use.
The re sults were com pared with those ob tained by the en zy matic spec tro pho to met ric method (Sigma Cat a log no.591C/94).

En zyme im mo bi li za tion
Sor ghum vulgare seeds (BR-303) from EMBRAPA/BRAZIL were ground with a mor tar and pestle and passed through a sieve to get ho mo ge neous par ti cles of about 1mm in size.A 5 g amount was im mersed in aqueous 25% (w/v) glutaraldehyde so lu tion over night at 5 °C.The supernatant was re jected and the pieces were washed ten times with deionized wa ter and four times with 0.10 mol dm -3 glycine so lu tion.Fur ther wash ing with deionized wa ter was per formed to elim i nate the ex cess glycine.Two batches of 1.2 g of ac ti vated Sor ghum vulgare seeds were sub mit ted to im mo bi lize the ox a late oxidase (Sigma O-4127); one with 0.42 µkatal and the other with 0.17 µkatal of the en zyme, and then 2 mL of a 0.05 mol dm -3 succinate buffer (pH 3.8) were added.Then, the mix tures were stored for a week in a re frig er a tor at 5 °C.Finally these ma te ri als, af ter be ing washed with a 0.05 mol dm -3 succinate buffer so lu tion (pH 3.8), were used for the construc tion of the en zyme re ac tors.

Method
The en zyme re ac tor (a 100 mm length/2.5mm i.d.polyeth yl ene tube) filled with the nat u ral ma te rial, onto which ox a late oxidase was im mo bi lized, was used in a typ i cal FI set-up (Fig. 1).A conductimetric flow cell, with an in ter nal vol ume of 25 µL and a con stant of 0.186 S, was used in all mea sure ments.
When in jected (200 µL sam ple) into the car rier stream which passes through the en zyme re ac tor, the ox alic acid re acts with the en zyme to pro duce car bon di ox ide.This CO 2 per me ates through the stretched polytetrafluoroethylene (PTFE) mem brane into the sec ond wa ter stream chang ing the con duc tiv ity (CO2 + H2O = HCO3 -+ H + ).In or der to ob tain a damp ing sys tem for the flow, the waste tips were im mersed at the same depth in a beaker com pletely filled with wa ter.For com par i son, a non-enzymatic re ac tor was em ployed, us ing a tube with the same di men sions, but hav ing pieces of Sty ro foam® as an in ert filler ma te rial with a size sim i lar to that of the Sorghum vulgare.

Re sults and Dis cus sion
The im mo bi lized sor ghum seeds en zyme gave a re action that showed rea son able sta bil ity for six months, i.e. car ry ing out about 20 as says per day, it re mained at 65% rel a tive ac tiv ity.Ap par ently, the glutaraldehyde used in the im mo bi li za tion pro cess helps pre vent the de com po si tion of the bi o log i cal ma te rial by mi cro or gan isms.
The re ac tor with 0.42 µkatal of en zyme gave a good per for mance and was used in all ex per i ments.As says were car ried out us ing stan dard so lu tions to de ter mine the best FI con di tions.In Fig. 2, the in flu ence of pH on the peak height is shown.The best en zyme ac tiv ity oc curs at pH 3.0.Be low this value, the en zyme lost ac tiv ity, al though at higher hydrogenionic con cen tra tions the car bon di ox ide per meation through the PTFE mem brane is fa vored.At pH val ues above 3.0, the equi lib rium is dis placed to the right, de creasing the amount of CO 2 that per me ates through the PTFE mem brane.As a con se quence, a low er ing of the sig nal was ob served.
Ta ble 1 shows that the flow rate of 1.1 mL min -1 and 3.5 min for wash ing time were the best con di tions es tab lished ex per i men tally, when a poly eth yl ene loop with a vol ume of 200 µL, (0.7 mm i.d.) was used.De spite the fact that with a flow rate of 0.67 mL min -1 higher sig nals are ob tained than with 1.11 mL min -1 , the wash ing time is in creased 3.4 times, de creas ing the sam pling rate to a max i mum of 4 per hour.
Fig ure 1.The conductimetric FI man i fold : A and B, deionized wa ter car rier streams; P, peri stal tic pump; S, sam ple in let loop; ER, en zyme re ac tor; NR, non-enzymatic re ac tor; RI, ion ex change resin clean-up col umn; V, se lec tion valve; SV, sam pling valve sys tem; PM , per meation cell; FC, con duc tance flow cell; C, con duc tiv ity me ter; R, chart re corder; and W, waste.
Fig ure 2. The ef fect of pH on the en zyme re ac tor for 0.50 mmol dm -3 ox a late stan dard so lu tion in NaH 2PO 4/H 3PO 4 me dia.Tem per a ture con trolled at 25.0 ± 0.5 °C.
Ta ble 1.The re la tion ship be tween flow rate, peak height, and washing time for the FI method.A typ i cal conductimetric FI pro file and the re peat ability for 0.10 mmol dm -3 are shown in Fig. 3.The cal i bra tion curve for ox a late de ter mi na tions, ob tained un der the con ditions de scribed, pres ents a use ful range be tween 0.05 and 0.50 mmol dm -3 of ox alic acid.This range can be fit ted by a lin ear func tion [Y = 1.1 ( ± 0.77) + 229 ( ± 3) X, r = 0.9997 for n = 5], with a rel a tive stan dard de vi a tion (r.s.d.) of ± 3.2%.
Since urine ma tri ces usu ally change sig nif i cantly, the stan dard ad di tion method was used for all de ter mi na tions, mak ing di lu tions of the sam ples to the work ing range.The ox a late con cen tra tions pres ent in the sam ple can be obtained graph i cally by in ter po la tion.
Urine spec i mens from 3 adult male do nors, and E and N con trols from Sigma were an a lyzed, us ing the es tab lished Sigma spec tro pho to met ric method and the method proposed in this study.The re sults are shown in Ta ble 2. The sta tis ti cal t-test was used to com pare the re sults ob tained by the two meth ods.It can be ob served that there are no sig nifi cant dif fer ences be tween the re sults at a 95% con fi dence level.
The pro posed FI method is sim pler and less ex pen sive.In ad di tion, the sam ple pre treat ment is elim i nated, as is the in ter fer ence from ascor bic acid.These fea tures are not com monly found in other pro ce dures 2,[4][5][6][7] .The new im mobi li za tion ap proach ap pears to be very con ve nient since it pre serves the en zyme ac tiv ity at use ful lev els for a lon ger pe riod of time.
The de vel oped flow in jec tion sys tem for ox a late de termi na tion pre sented a use ful con cen tra tion range from 0.05 mmol dm -3 up to 0.50 mmol dm -3 .Al though the main spectro pho to met ric and amperometric meth ods pub lished show lower de tec tion lim its, they pres ent sev eral in ter ference prob lems for bi o log i cal sam ple anal y sis.The proposed method shows good per for mance for the de ter mi na tion of ox alic acid in urine with out in ter fer ence, pre sent ing good op er a tional ad van tages.Fig ure 3. FI cal i bra tion runs for the de ter mi na tion of ox alic acid.From left to right: trip li cate sig nals for ox alic acid stan dard so lu tions (0.05, 0.10, 0.25, 0.50, 0.75, 1.0 mmol dm -3 , and the in verse or der), with several ad di tional sig nals from the 0.10 mmol dm -3 so lu tion for a re peat abil ity Ta ble 2. A com par i son be tween the val ues ob tained by the Sigma spec tro pho to met ric and the pro posed FI meth ods for ox a late in urine spec i mens us ing the sta tis ti cal stu dent's t-test 24 .Values pre sented in mmol/24 h.* Lyophilized urines re con sti tuted in 25 and 20 mL for N and E con trols re spec tively.Daily fi nal vol ume of these sam ples is 1.48 dm -3 .* Tab u lated t value for the de gree of free dom (ν) 4 is 2.776 (α = 0.05); ν = n1 = n2 = 3 in this in stance.