Journal of Biotechnology, 14 (1990) 53-.

53

Elsevier BIOTEC 00482

Fluorometric determination of urea by flow injection analysis M.S. Abdel-Latif and G.G. Guilbault Department of Chemistry, University of New Orleans, New Orleans, Louisiana, U.S.A.

(Received 25 July 1989; accepted 1 November1989)

Summary Urea was determined using fluorometry with flow injection analysis. O-phthalaldehyde (OPA) reacts with enzymatically generated ammonia and sulfite in alkaline medium to give a highly fluorescent compound that has an excitation wavelength of 372 nm and an emission wavelength of about 430 nm. The method is more selective to ammonia than the one which uses mercaptoethanol in place of the sulfite. Urease was immobilized to a Pall Immunodyne membrane which is commercially available. The immobilization occurs through covalent bonding which results in a highly stable enzyme preparation. The enzymatic membrane was fitted in a 5 cm long, 0.125 inch o.d. Teflon tubing which served as the enzymatic reactor. The system is difficult to use for the analysis of urea in serum because some compounds normally present in serum fluoresce at the same wavelength. This results in higher values for urea. If the reaction system is to be used for the evaluation of urea in serum, a blank should be run so that urea concentration can be calculated by difference. Urea; Urease; Flow injection analysis; Immobilized enzyme; O-phthalaldehyde; Fluorescence

Introduction Urea, the end-product of nitrogen metabolism, has considerable significance in clinical chemistry, where blood urea nitrogen (BUN) analysis gives important indication of possible kidney disease. Increased levels of BUN occur in cases of

Correspondence to: G.G. Guilbault, Dept. of Chemistry, University of New Orleans, New Orleans, LA

70148, U.S.A. 0168-1656/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)

54

renal failure. The normal values of urea in serum range from 8 to 20 mg d1-1 of blood. The analysis of urea is also important in other fields like agricultural chemistry, where urea is used in fertilizer, for determination of water quality, and in sea water analysis. There are m a n y methods reported for urea analysis most of which involve the enzymatic hydrolysis of urea followed by the determination of H C O 3 or N H ~ / N H 3 liberated in the hydrolysis step (Eq. 1). O II

C H2N

+ 2H20 + H +

Urease

, H C O 3- + 2 N H ;

(1)

NH 2

Enzyme electrodes that measure the produced a m m o n i u m ions have been extensively described. The work of Guilbault and Montalvo (1970); Guilbault and H r a b a n k o v a (1970); Blaedel and Kissel (1975); and Papastathopoulos and Rechnitz (1975) should be consulted for details. Others which use the ammonia air-gap electrode have been reported by Ruzicka and Hansen (1974) and Guilbault and Trap (1974). This type of sensor overcomes the poblems caused by the interference of N a ÷ and K ÷ when the ammonium ion-selective electrode is used. Colorimetric and fluorometric methods have also been proposed and used in the urea analysis. Although not very specific, m a n y laboratories use a direct nonenzymatic spectrophotometric determination of urea. However, the use of enzymatic methods is of considerable importance, and both spectrophotometry and fluorometry methods depend on the detection of the ammonia generated in the hydrolysis step. The use of Nessler's reagent and the indophenol method are most common. In fluorometric methods, the most selective procedure involves a u r e a s e / g l u t a m a t e dehydrogenase bienzymatic reaction in which the urea is hydrolyzed using urease, with a m m o n i u m ions being produced. In the presence of reduced nicotinamideadenine dinucleotide (NADH), glutamate dehydrogenase converts a-ketoglutarate into glutamic acid when NH~- is present. The product of the reaction, nicotinamide-adenine dinucleotide (NAD) can be followed fluorometrically (RochRamel, 1967). The use of OPA reagent for the fluorometric enzymatic analysis of urea has been limited, mainly because the reaction system (which is OPAmercaptoethanol (ME) and ammonia at alkaline pH) is not selective for ammonia; m a n y amines and most amino acids interfere. Recently, Genfa and Dasgupta (1989) have described a system that substitutes sulfite for the ME. This system is much more selective for ammonia over amino acids. In this paper, urea is analyzed by coupling the urease reaction to the OPA-sulfite reaction at alkaline pH, using a flow injection system in which parameters were optimized to quantify urea in the physiological range. Materials and Methods

O-phthalaldehyde was obtained from Fluka Chemica-BioChemica. Solutions of OPA covering the concentration range from 1 x 10 -3 M to 5.8 x 10 -2 M were

55 prepared in 0.5 M N a O H directly before use, since the solution is not stable for more than a few hours. Sodium sulfite (Mallinckrodt, Inc.) solutions of 0.01 to 0.1 M were prepared in 0.5 M N a O H , containing an appropriate concentration of OPA. 0.05 M phosphate buffer was prepared from potassium dihydrogen phosphate, with p H adjusted to 7 using 0.2 M N a O H . Urea was obtained from J.T. Baker Chemical Co. and standard solutions were prepared in distilled water to cover the concentration range from 1 to 100 mM. A m m o n i u m chloride (EM Science) was used to prepare the a m m o n i u m standard solutions that were used in some optimization experiments. In all the work performed, doubly distilled deionized water was used. All chemicals were used as purchased without further purification.

Apparatus A Rainin-HP p u m p and a Varian 500 Liquid Chromatograph were used to deliver the carrier solution and the OPA-sulfite solutions, respectively. A Rheodyne type manual injector, equipped with a 20 btl injection valve, was used. A Perkin-Elmer 650-10S fluorescence spectrophotometer with a 150 Xenon power supply and a digital multimeter readout were used. The slit widths were fixed at 10 nm each. The excitation and emission wavelengths were adjusted to 372 and 430 nm, respectively. An OmniScribe chart recorder was used to record the output signal.

Preparation of the enzyme reactor Two different solid supports were tried. Amberlite IRC-50, which is a weakly acidic type cationic exchange resin of the form R C O O - H ÷, was dried overnight at 70 ° C and then desiccated. A solution of urease (145 U mg -1, Sigma) was prepared by dissolving 3 mg of the enzyme in 3 ml of chloroform. Then, 0.7 g of the resin was added and the mixture was allowed to stay at room temperature for 10 min, after which it was kept at 4 ° C ovemight. The resulting resin was washed with 1 M NaC1 and a portion of it was packed in a Teflon column (0.125 inch o.d.). The other method used involved a commercially available preactivated I m m u n o d y n e membrane (Pall BioSupport Corporation, NY). These membranes are excellent supports to bind an enzyme chemically. They also have good mechanical strength, high loading capacity, as well as easy handling properties. A 6 cm long strip (3 m m wide) of the membrane was cut and placed in an enzymatic urease solution prepared by dissolving 2 mg of the enzyme in 1 ml of 0.05 M phosphate buffer, p H 6.8. The enzyme was allowed to react with the activated surface (most likely aldehyde functional groups) for 10 min. The enzymatic membrane was removed and washed in 1 M NaC1 for 30 min. Then the membrane was fitted inside a 5 cm Teflon tubing, 0.125 inch o.d. Both types of enzyme reactors were stored in 0.05 M phosphate buffer, p H 6.8.

Results and Discussion The OPA reaction has been used for the determination of ammonia (Genfa and Dasgupta, 1989), amino acids (Roth, 1971), and thiols (Nakamura and Tamura,

56

1981). The latter authors have also shown that sulfite reacts in a similar way as thiols. Simmons and Johnson (1976) reported that the reaction product is an isoindole derivative. Thus, the reaction of OPA with ammonia in the presence of sulfite can be represented by Eq. (2). O

H

+ NH3 + SO2-

' ~

N

H

(2)

O The reaction product is highly fluorescent and concentration quenching can easily be observed as the reaction proceeds toward completion. Stable preparations of OPA solutions are difficult to obtain. In our experience, OPA is almost indefinitely stable in pure acetone; however, problems of air bubbles due to the mixing of acetone dissolved OPA with buffer result in an inconvenient flow injection system. In addition, acetone was found to diminish the fluorescence signal. Buffered OPA solutions were also found to be unstable for more than a one-week period. However, dissolution of OPA in 0.1 M N a O H , followed by the addition of potassium dihydrogen phosphate to a final concentration of 0.1 M phosphate buffer, p H 7, gave the best stability. This preparation eliminated the need for an organic solvent. In our experiments, OPA and sulfite were dissolved in 0.5 M N a O H and were used in the same day. Although this preparation is not stable for more than a few hours, this method was chosen because of instrumental restrictions. Two pumps were used, one p u m p to deliver the carrier buffer solution while the other was used to deliver the alkaline OPA-sulfite solution. The flow rate of both p u m p s was adjusted to 1 ml min -~. The amount of enzyme loading, flow rate and the length of the tubings were chosen to fit a dynamic urea concentration range of 1 m M to 60 m M (the whole physiological range). Fig. 1 shows a schematic diagram of the flow injection system.

i

_

t

W

1 ml min -1 Fig. 1. S c h e m a t i c d i a g r a m of the F I A system; a = carrier solution; b = m a n u a l injector; c = O P A - s u l f i t e solution; d = detector.

57

Effect of the immobilization method Urease immobilized on cation exchange resin showed good stability and loading. However, the peaks which were recovered for urea injections were very broad with a long retention time in the enzymatic reactor. This behavior is most likely to be caused by the cation exchange process of the hydrogen ion with the ammonium generated as a result of the enzyme reaction with urea. The elution of NH~- ions with 0.05 M phosphate buffer seemed to be very inefficient. Although a good immobilized urease reactor had been achieved, the system proved to be inadequate to operate in the flow injection mode. The other enzymatic reactor, which utilizes the Pall Immunodyne membrane, did not have this problem. It showed considerable stability and activity for periods exceeding 2 months. The use of 0.125 inch o.d. enzyme reactor with the membrane fitted inside the tubing would certainly add to the band broadening due to diffusion, but it was used because efforts to mount the enzymatic membrane inside a 1 / 1 6 inch o.d. tubing were not successful. Although other immobilization methods are available in the literature, we preferred to use the above mentioned procedure because it is simple, fast and occurs through covalent bonding. Also, enzymes immobilized on such membranes were reported to be of very high stability (AbdelLatif and Guilbault, 1988). For these reasons, the Immunodyne based reactor was used throughout this study.

120 .._..---~

100

80

i

60

40

20

0 0.00

I

0.01

0.02

0.03

0.04

0.05

0.06

OPA (M) Fig. 2. Effect of O P A c o n c e n t r a t i o n o n the fluorescence intensity. 0.085 M sulfite s o l u t i o n was used. O t h e r c o n d i t i o n s are m e n t i o n e d in the text.

58

Effect of OPA concentration Since the reaction is expected to be highly dependent on the O P A concentration, solutions of a wide range of concentrations were prepared in 0.5 M N a O H containing 0.085 M sodium sulfite. A 1 x 10 -4 M a m m o n i a solution was used for the optimization experiments where standard reaction conditions were used (Fig. 2). As the concentration of O P A was increased, a large increase in the signal was observed at concentrations lower than 0.02 M. At concentrations of O P A above 0.02 M this increase becomes less significant, and the curve starts to plateau at 0.065 M OPA. This concentration was used in the remaining studies. It should be mentioned that it was not possible to readily dissolve a higher concentration of O P A in 0.5 M NaOH.

Effect of sulfite concentration It was observed that sulfite concentration has a great effect on the fluorescence signal (Fig. 3). As the concentration of sulfite was increased, the signal continued to increase until a m a x i m u m was obtained at about 0.085 M sodium sulfite. At higher sulfite concentration, the signal started to decline. Mixing the sulfite solution with the O P A solution did not produce difficulties such as baseline drift. A stable baseline was always obtained, contrary to the observations of others (Genfa and Dasgupta, 1989). A sodium sulfite concentration of 0.085 M was then mixed with

120

I O0

e-

80

t.-

o nr

60

M.

40

2O 0.00

I

I

I

I

I

0.02

0.04

0.06

0.08

0.10

0.12

Sodlum Sulflte (M)

Fig. 3. Effect of sulfite concentration on the fluorescence intensity. 0.065 M OPA solution was used.

59 0.065 M OPA in 0.5 M NaOH. These concentrations were routinely used through the rest of this study. Calibration curve f o r urea

Standard urea solutions covering the concentration range of 1 mM to 100 mM were prepared in distilled water. Routine reaction conditions were used to construct the calibration curve with the flow rate adjusted to 1 ml min -t each. This configuration resulted in a linear calibration curve that corresponds to urea concentrations from 1 mM to 60 mM (2% relative standard deviation for 10 replicates). The experimental conditions were carefully chosen to achieve this clinically useful physiological range. Fig. 4 shows that at high urea concentrations, the calibration curve deviates from linearity, but is still analytically useful because it does not flattering out. The system was found to be sensitive for some amino acids especially histidine. Glycine, if present in the mM range will also cause considerable interference. However, the system can tolerate many other amino acids (Genfa and Dasgupta, 1989). Proline, hydroxy-L-proline, and Tris buffer do not interfere. The above results imply that the system is only applicable for analysis of pure urea solutions. A multiplex system with a urease reactor on one channel and no reactor on the other channel, can be used to calculate the urea concentration of any sample by the difference between the two signals. The system is quite useful for the

80

60

=,, t,.-

t,.0

40

¢1 ii

20

I

I

I

I

I

20

40

60

80

100

Urea (mM) Fig. 4. Calibration curve for urea. Routine conditions were used.

120

60 d e t e r m i n a t i o n of a q u e o u s urea in a m a t r i x where glycine a n d histidine are absent, or at a m u c h lower c o n c e n t r a t i o n t h a n urea. T h e sensitivity of the m e t h o d can be increased, if necessary, b y s i m p l y r e d u c i n g the flow rate, increasing the e n z y m e l o a d i n g in the e n z y m a t i c reactor, a n d insertion of a d e l a y coil t h e r m o s t a t e d at a b o u t 8 5 ° C . S u r f a c t a n t s d i d n o t result in a n y a t t r a c t i v e advantages, a n d n o n e was used.

Conclusion A simple m e t h o d for the d e t e r m i n a t i o n of urea using flow injection analysis is possible. T h e e n z y m a t i c r e a c t o r p r o v e d to be very stable a n d easy to handle. T h e m e t h o d is fast, where 40 s a m p l e s h -1 can be a n a l y z e d with a relative s t a n d a r d d e v i a t i o n of 2% for 10 replicates. D e p e n d i n g on the c o n c e n t r a t i o n range of urea, the flow rate can be easily a d j u s t e d to achieve the r e q u i r e d p e r f o r m a n c e . However, it s h o u l d be r e m e m b e r e d that very low flow rates m a y result in severe b a n d b r o a d ening due to diffusion, especially if solutions of low viscosity were used. It is n o t e w o r t h y to m e n t i o n that d e p e n d i n g on the c o n d i t i o n s utilized, a wide variety of c o m p o u n d s with different p r o p e r t i e s can b e o b t a i n e d . F o r example, at high p H values a n d p r o l o n g e d times of reactions ( s o m e t i m e s less t h a n 1 min) an intense green r e a c t i o n p r o d u c t is formed. Also, d e p e n d i n g on the thiol used, if an acid is a d d e d to the r e a c t i o n m i x t u r e to b r i n g the p H to < 2, c o m p o u n d s of different colors can be o b t a i n e d . Some of these reactions m a y be useful for selective d e t e r m i n a t i o n of e n z y m a t i c a l l y g e n e r a t e d a m m o n i a . P r e l i m i n a r y results show that 2-thiazoline-2-thiol, when used as the thiol at n e u t r a l p H , can result in a very selective system for a m m o n i a over a m i n o acids at a final p H < 2.

Acknowledgement T h e financial assistance f r o m the L o u i s i a n a E d u c a t i o n Q u a l i t y S u p p o r t F u n d ( L E Q S F - R D - B - 1 7 ) is gratefully a c k n o w l e d g e d .

References Abdel-Latif, M.S. and Guilbault, G.G. (1988) Fiber optic sensor for the determination of glucose using micellar enhanced chemiluminescence of the peroxyoxalate reaction. Anal. Chem. 60, 2671-2674. Blaedel, W.J. and Kissel, T.R. (1975) Reactor-separator membrane combining immobilized urease and an anion exchange membrane. Anal. Chem. 47, 1602-1605. Genfa, Z. and Dasgupta, P.K. (1989) Fluorometric measurement of aqueous ammonium ion in a flow injection system. Anal. Chem. 61,408-412. Guilbault, G.G. and Hrabankova, E. (1970) Determination of urea in blood and urine with a urea sensitive electrode. Anal. Chim. Acta 52, 287-294. Guilbault, G.G. and Montalvo, J.G. (1970) An enzyme electrode for the substrate urea. J. Am. Chem. Soc. 92, 2533-2538.

61 Guilbault, G.G. and Trap, M. (1974) A specific enzyme electrode for urea. Anal. Chim. Acta 73, 355-365. Nakamura, H. and Tamura, Z. (1981) Fluorometric determination of thiols by hquid chromatography with postcolumn derivatization. Anal. Chem. 53, 2190-2193. Papastathopoulos, D.S. and Rechnitz, G.A. (1975) A urea-sensing membrane electrode for whole blood measurements. Anal. Claim. Acta 79, 17-26. Roch-Ramel, F. (1967) An enzymic and fluorophotometric method for estimating urea concentrations in nanoliter specimens. Anal. Biochem, 21, 372-381. Roth, M. (1971) Fluorescence reaction for amino acids. Anal. Chem. 43, 880-882. Ruzicka, J. and Hansen, E.H. (1974) A new potentiometric gas sensor-the-air-gap electrode. Anal. Chim. Acta 79, 17-26. Simmons, S.S. and Johnson, D.F. (1976) The structure of the fluorescent adduct formed in the reaction of o-phthalaldehyde and thiols with amines. J. Am. Chem. Soc. 98, 7098-7099.

Fluorometric determination of urea by flow injection analysis.

Urea was determined using fluorometry with flow injection analysis. O-phthalaldehyde (OPA) reacts with enzymatically generated ammonia and sulfite in ...
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