1907
Anal. Chem. 1982, 54. 1987-1990
for ascorbate. Using the standard spectrophotometric assay (6), we obtained an absorbance change of (2.3 f 0.3) X s-l averaged over seven measurements. This corresponds to an enzyme activity of ( 2 4 f 0.3) X lo5 units (mg of enzyme)-l, as specified earlier. It also agrees with the activity specified by the manufacturer (of 2.4 X lo5 units (mg of enzyme)-'. For comparison of tlhe spectrophotometric assay with the electrochemical assay, the former must also be expressed in terms of moles of substrate consumed per second. This conversion is simply accomplished by multiplying the enzyme activity by the extinction coefficient which we determined from a nine-point plot, of absorbance against ascorbic acid concentration in the ara~aymatrix. The value obtained was (1.92 k 0.02) X lo3 m2 mol-' with a correlation coefficient of 0.9994. Combining this figure with the measured rate of change of absorbance, we obtain for the spectrophotometric rate a value of (6.3 f 0.9) X mol s-l (g of enzyme)-'. It can be seen that the two techniques agree within the limits of experimental error but that the precision of the electrochemical method (5%)is substantially better than the spectrophotometric method (15%). We believe that this is due to the rapid mixing inherent in the design of the electrochemical cell. A maljor advantage of the electrochemical method is that it is independent of the optical properties of the solution and therefore may be used to assay turbid or opaque solutions which could not be assayed spectrophotometrically. The close agreement between the electrochemical assay for tyrosinase using 1,4-dihydroxybenzene as reductant and the standard spectrophotoinietric method using ascorbate as reductant are evidence that tyrosinase per se does not attack 1,4-dihydroxybenzene to any significant extent during the assay. When assaying any given plant or tissue extract for tyrosinase, the possibility that the extract might contain some species which reacts with 1,4-dihydroxybenzene must first be checked by a suitable bilank. The presence in the extract of electroactive species which can be oxidized or reduced at -0.15 V on the carbon electrode is not likely to be a problem since these species will only cause a current jump on injection of the extract and will not contribute to the rate of change of current on which the assay is based.
Comparison with Oxygen Uptake Method. Tyrosinase may also be assayed by measuring the rate of oxygen uptake. This may be most conveniently achieved by means of an oxygen electrode. However, the oxygen electrode is likely to be less satisfactory than the rotating disk electrode in several respects. The response of the oxygen electrode, which derives its selectivity from a membrane, is intrinsically slower than the rotating disk because of the membrane. Protein fouling of the membrane is also a known problem in enzyme assays using the oxygen electrode. Finally, it is difficult to carry out enzyme assays a t constant oxygen concentration using the oxygen electrode method. If it is also desired to keep the catechol substrate concentration constant, then a reductant such as 1,6dihydroxybenzene must be added. In a previous comparative study using oxygen uptake to follow the kinetics (7),it was found that the apparent rate with 1,Cdihydroxybenzene as reductant was substantially higher (40%) than the corresponding quantity using ascorbate as reductant. The authors of this paper suggest that there should be no intrinsic difference in the enzymic rate and ascribed the measured discrepancy to a difference in the stoichiometry of oxygen absorption. The present results confirm this point and illustrate the desirability of measuring enzyme kinetics by several different methods. ACKNOWLEDGMENT G.P.P. wishes to thank the University of Western Australia for a General Development Grant Fellowship. LITERATURE CITED (1) Ingraham, L. Anal. Chem. 1956, 28, 1177-1179. (2) Kamin, R. A.: Wilson, G. S . Anal. Chem. 1980, 52, 1196-1205. (3) Yuan, C.-L.; Kuan, S, S.; Guilbault, G. G. Anal. Chem. 1981, 53, 190- 193. (4) Bohinski, R. C. "Modern Concepts in Biochemistry"; Allyn and Bacon: Boston, MA, 1979. (5) . . Vielkind, U.: Schiaae, W.; Anders. F. A . Krebsforsch. Klin. Onkol. 1917, 90, 285. (6) "Catechol Oxidase (E.C. 1.14.18.1)"; Sigma Chemical Company Informatlon Bulletin: April 1979. (7) Kertesz, D.; Zlto, R. Blochlm. Blophys. Acta 1962, 64, 153.
RECEIVED for review August 21, 1981. Resubmitted June 7, 1982. Accepted June 14, 1982.
Amperometric Enzymic Determination of Triglycerides in Serum Handanl Wlnartasaputra, Shla S. Kuan,' and George G. Gullbault" Department of Chemistry, llniverslty of New Orleans, New Orleans, Louisiana 70 148
An amperatnetrlc three-electrode system has been developed for the assay of trlglycerldes. Two enzymes used in analysis, namely, glycerol dehydrogenase and diaphorase, are immoblllred on a collagen membrane. Serum samples are Incubated wlth a mlcroblal lllpase In phosphate buffer containing 2,6dlchlorophenollndophenol(DCPIP). The resulting glycerol Is oxldlred by NAD+ In the presence of glycerol dehydrogenase; the NADH produced is then oxldlred by DCPIP. The anodlc current generated by oxldatlon of the reduced form of the DCPIP at the surface of the worklng electrode at a constant of 0.300 V Is measured. Serum trlglycerides ranglng from 10 to 500 nng/dL can be easlly assayed In less than 15 mln by uslng only 25 pL of sample.
A fully enzymatic determination of triglyceride in biological samples has been reported mostly by spectroscopic methods (1-5). Electrochemical methods for measurement of enzymatic reactions have likewise been applied successfully (6-8). The fully enzymatic biamperometric determination of glycerol and triglycerides with open tubular carbon electrodes has been reported (9). This particular method employed soluble enzymes. This paper proposed the use of a three-electrode amperometric method and dual-immobilized enzymes on a collagen membrane method for determination of triglycerides in serum samples. The use of soluble enzymes in the reaction is also studied. The reactions of the proposed method are
'Food a n d Drug Admin.iutration, 4298 E l y s i a n Fields Orleans, LA 70148.
triglycerides
Av.,
New
lipase
glycerol
0003-2700/82/0354-1987$01.25/00 1982 American Chemical Society
+ free fatty acids
(1)
1988
ANALYTICAL CHEMISTRY, VOL. 54, NO. 12, OCTOBER 1982
glycerol
glycerol
+ NAD+, dehydrogenase ' dihydroxyacetone + E.C.1.1.1.6
NADH
+ H+ (2)
NAD+ (3) COlVNECTOR
The triglycerides were first converted to glycerol and fatty acids. This was accomplished by Rhizopus arrhizus lipase which hydrolyzed the triglycerides during the incubation period (10 min at room temperature). The resulting glycerol was oxidized by NAD+ in the presence of glycerol dehydrogenase. The NADH produced was in turn oxidized by 2,6-dichlorophenolindophenol(DCPIP) using diaphorase as the catalyst. As soon as the reduced form of DCPIP was formed, it was reoxidized on the surface of a rotating platinum electrode (RPE). The rate of anodic current generated at a constant potential of 0.300 V was measured.
EXPERIMENTAL SECTION Instrumentation. A PAR-174 A polarographic analyzer (Princeton Applied Research Co., Princeton, NJ) was used to apply a constant potential at 0.300 V and to measure the current. The current was recorded with a strip chart recorder, Model Eu-200-01 (Heath Co., Benton Harbor, MI). A rotating platinum electrode (RPE) was constructed from a platinum electrode, type 39273, obtained from Beckman Instrument, Inc. (Fullerton, CA) which is a flat platinum disk with a diameter of 0.5 cm housed in a glass body. The electrode was connected to the shaft of a 12-V motor by a connector (made from polyethylene) so a commutator (a brush spring) can be used as the electrical connector for the working electrode. A saturated calomel electrode (SCE) without ita filter was connected to a salt bridge with a resistance of about 2 kQ at 1000 Hz. A platinum electrode with an area of 1cm2was used as the auxiliary electrode. An H-cell with a fine fritted disk having a resistance about 400 0 at 1000 Hz was constructed. The frequency of the rotating electrode was controlled by a Heathkit power supply, Model 1P-2B. A sensor chip, having the ability to sense a magnetic field, was used to detect the frequency of rotation of the RPE. A small magnetic bar attached to the motor shaft served as the magnetic field source. The sensor chip was connected to a Heathkit frequency counter, Model 18-101. The complete arrangement of the instrumentation is shown in Figure 1. Reagents and Control Sera. Glycerol dehydrogenase (2.68 units/mg), NAD+, and 2,6-dichlorophenolindophenolwere obtained from Sigma Chemical Co., St. Louis, MO. Lipase from Rhizopus arrhizus (5300 units/mg) was a product of Fermco Biochemicals, Inc., Elk Grove Village, IL. Diaphorase (52 unita/mg) was from Worthington Biochemical .~ Co., Fieehold, NJ. Control sera, Lipid-Trol and Monitrol I and 11,were obtained from Dade Division, American Hospital Supply Co., Miami, FL. Preparation of Reagents. Hydrolysis reagent: 21 200 units of liDase/l.O mL of uhomhate buffer (DH 8.0, 50 mmol/L). - Giycerol dehydrogenase (soluble): - 10.7 units/l.O mL of phosphate buffer (pH 7.5, 50 mmol/L). NAD+was dissolved in doubly distilled water to give 10 mg/mL solution. Diaphorase (soluble): 156 units/ 1.0 mL of phosphate buffer (pH 8.0, 50 mmol/L). 2,6-Dichlorophenolindophenol: 4 mg of DCPIP was dissolved in 1L of phosphate buffer (pH 8.0,0.05 M) containing 0.1 M KC1 as the supporting electrolyte. Standard Triglyceride Solutions: 62, 124, 248, and 498/100 mL working standard solutions were prepared by appropriately diluting the control sera with doubly distilled water. Immobilization of Glycerol Dehydrogenase and Diaphorase on the Collagen Membrane. Coulet et al. (IO)reported
--
U
O-RING I COLLAGEN 4EMBRAIIE
\
\ FRITTED DISK
Figure 1. Instrumental setup.
the use of a highly polymerized collagen, prepared under industrial conditions, as the binding site of various immobilized enzymes, and discussed the advantages using the collagen membrane as matrix. We followed the same technique of immobilization with minor modification. Collagen membranes (a gift from Centre Technique du Cuir, Lyon, France), with diameter of 2.5 cm and 0.1 mm thick in the dry state and 0.3 to 0.5 mm thick when swollen, were immersed in 60 mL of methanol containing 0.2 N HC1 for 3 days at room temperature. Then the membranes were rinsed with doubly distilled water thoroughly. The membranes were transferred into 100 mL of 1% hydrazine at room temperature and immersed for 12 h. After another thorough washing in distilled water at 0 OC, the membranes were immersed into a mixture of 0.5 M KNOz and 0.3 N HCl for 15 rnin at 0 OC. The membranes in turn were washed with buffer solution, 50 mmol/L, and pH 7.5 phosphate buffer at 0 OC. Finally, five activated membranes were dipped into a solution containing 10.7 units of glycerol dehydrogenase and 104 units of diaphorase in 2 mL of phosphate buffer (50 mmol/L, pH 7.5) and stored overnight in a refrigerator (4 "C). The membranes were washed with phosphate buffer and stored in the same buffer until used. Procedure. (A) Soluble Enzyme System. Into each side of the H-cell3 mL of DCPIP solution was pipetted. Then 25 p L of serum sample and 25 pL of lipase solution were added into one side of the H cell into which the rotating platinum electrode and SCE were introduced. The auxiliary electrode was placed into the other side of the H-cell. The solution was incubated for 10 min at room temperature to accomplish the hydrolysis of triglycerides. During this time a constant potential of 0.300 V was applied to the rotating electrode. After 10 min, 25 p L of glycerol dehydrogenase and 25 pL of glycerol and 25 pL of diaphorase were added. At last 25 pL of NAD+ was added to trigger the reactions. The rate of current generated at the RPE was measured for about 1 or 2 min. (B) The Immobilized Enzyme System. The collagen membrane (now retaining the immobilized enzymes) and an O-ring on top of it were placed into the H-cell. Three milliliters of DCPIP solution was pipetted into each side of the H-cell. The hydrolysis procedure described above was repeated on the side of the H-cell containing the collagen membrane. Then 25 p L of NAD' solution was injected. The rate of current generated in the reaction was monitored for 1 or 2 min. To assay for the free glycerol that exists prior to the hydrolysis step, we omitted the addition of lipase and repeated the remaining procedure. After each analysis the collagen membrane was washed several times with phosphate buffer.
RESULTS AND DISCUSSIONS The use of 2,6-dichlorophenolindophenolas redox mediator and electroactive species has been reported by Smith and Olson (8). In this study a constant potential of 0.300 V was chosen for analysis. At this potential the current observed
ANALYTICAL CHEMISTRY, VOL. 54, NO. 12, OCTOBER 1982
25
1989
I/ 16
L L - -
150
300
600
450
STIRRING SPEED,
I
I
I
I
200
400
600
aoo
UNITIAssaj
rpm
LIPASE
Flgure 2. Effect of stirring speed on reaction rate.
loo 75
t
Flgure 4. Effect of lipase concentration on the rate of hydrolysis of serum triglycerides.
P
I
8.0
7.5
8.5 I
PH
I .@
Flgure 3. Effect of pH Ion reaction rate.
I
2.0
I
I
3.0
4.0
I
5.0
U N I T / As say
Table I. Reagents Used for the Assay of Serum Triglyceride vol and name of substance/assay 25 p L of glycerol 25 M Lof glycerol dehydrogenase 2 5 pL of diaphorase 25 MLNAD+ 3 mL of DCPIP solution containing 0 . 1 M KCl
Diaphorase
concn 20 mg/100 mL 13.5 units/mL 234 units/mL 8 mg/mL 8 mg/mL
is the anodic current of the reduced form of the DCPIP. This potential was chosen from the study of a single-sweep voltamogram toward cathodlic and anodic potentials, respectively ( 5 mV/SCE scan speed and 450 rpm of RPE). The change in reaction rate increased with the change of the stirring speed of the R P E linearly up to about 350 rpm and then it levels off (Figure 2). For maximum reproducibility a stirring rate at 450 rpm was used in all assays. Reagents used for this study are shown in Table I which gave fast response and good measurable signal. Figure 3 indicates tlhe dependence of the reaction rate on the pH value, the reaction rate increases with the increase in pH. A p H 8.0, 50 mmol/L phosphate buffer solution containing 0.1 M KCl was chosen for analysis. The units of lipase needed in analyzers was also studied by using the standard serum as reference containing 496 mg/100 mL triglyceride. Without the presence of lipase, a small amount of free glycerol presently in the serum before hy-
Flgure 5. Optimization of diaphorase. drolysis gives a relatively weak signal (Figure 4). The relative rate of reaction increases with increase of lipase concentration, leveling off at about 500 units per assay. Hence, we used approximately 550 units per assay which was sufficient for complete hydrolysis of the triglycerides in 10 min a t room temperature. Grossman et al. ( 4 ) used the same lipase for hydrolysis of triglycerides and found that 400 units of lipase were enough to hydrolyze completely as much as 500 mg/100 mL triglycerides in 5 pL of serum sample. The condition of reaction for our study is also listed in Table I except that the glycerol was formed from hydrolysis in this case. The optimum concentrations of soluble glycerol dehydrogenase and diaphorase were studied. The rate of reaction increased linearly with the increase in glycerol dehydrogenase concentration. A 0.25 unit amount of glycerol dehydrogenase per assay was found to be sufficient (Figure 5). The presence of 4 units of diaphorase per assay was chosen as the optimum. These studies were carried out by using the condition listed in Table I. The standard triglyceride serum (248 mg/100 mL) was hydrolyzed by 550 units of lipase. These results were also used for estimation of the concentration of immobilized glycerol dehydrogenase and diaphorase on the collagen membrane. Since it is difficult to measure the absolute activities of each enzyme actually retained on the membrane, therefore, by assuming that the ratio of immo-
1990
ANALYTICAL CHEMISTRY, VOL. 54, NO. 12, OCTOBER 1982
Table 11. Analytical Recovery for the Amperometric Method (Both Systems)
100
triglycerides, mg/dL added found
75
recovery, %
Immobilized c Y LL
50
3. M
c
0 62
101
124 248
221
98
339
97
165
10 1
_I
c
Nonimmobilized 25
I
I
I
I
I
0.05
0.10
0.15
0.20
0.25
mol elilssay, D C P I P
Flgure 6. Effect of DCPIP on reaction rate.
bilized glycerol dehydrogenase and diaphorase was 1:1,the rate of reaction obtained by the immobilized enzyme system indicates that approximately 0.2 unit of glycerol dehydrogenase and 3 units of diaphorase were immobilized on each membrane. The rate of reaction also increased with an increase in the NAD+ concentration; 0.35 pmol of NAD+per assay was found to be sufficient. Finally, the optimum concentration of DCPIP was also investigated; 0.1 pmol of DCPIP per assay was chosen (Figure 6). The optimization of NAD+ and DCPIP was carried out using optimal concentrations described above for glycerol dehydrogenase and diaphorase. Under the optimized assay conditions, the reaction rate was linear for serum triglycerides in the range of 10-500 mg/dL and interference due to the presence of serum protein during assay was not noted. Analytical Variables. Reproducibility and Precision. A standard solution of serum containing 248 mg/dL of triglycerides was assayed 10 times each using soluble and immobilized enzyme systems. Coefficients of variation of 2.8 and 4.9%, respectively, were obtained. Recovery Study. On addition of control sera to serum sample that had been previously assayed, both procedures, immobilized and soluble, gave analytical recoveries ranging from 97 to 101% to 98%, respectively. The results are tabulated in Table 11. Interference Study. To control serum (triglyceride concentration of 101 mg/dL) several common interfering substances were added (25 pL). An interference was found only by addition of a high concentration of ascorbic acid. This was expected because this substance is a good reducing agent. The concentrations of substances that were added to the sample and yet gave interference of less than 5% are 20 mg/dL uric acid, 1000 mg/dL glucose, and 80 mg/dL ethanol.
0
101
62 124 248
160 221
98 98
238
96
The stability of the immobilized enzymes was also studied. The immobilized glycerol dehydrogenase and diaphorase membrane still retained about 82% of its original activities after 27 days and 105 analyses. Thereafter, with storage in buffer solution at 4 "C for about 2 months, without use, the membrane still retained 70% of its original activities. Thus, the projected number of assays could reach several hundred. Comparison Study. Twenty-four fresh serum samples with known values obtained from a local hospital (Ochsner Clinic, New Orleans, LA) were analyzed by using the immobilized enzyme system. The results obtained showed a good correlation between the two methods. The linear regression analysis of the triglycerides value gave a correlation coefficient of 0.995. The equation for the comparison was Y(amperometric) = 0.976 X 7.52 mg/dL. We conclude from these results that the application of immobilized enzymes, glycerol dehydrogenase and diaphorase,on a collagen membrane provides a good electroanalytical procedure for the determination of triglycerides in serum samples.
LITERATURE CITED (1) Winartasaputra, H.; Mallet, V. N.; Kuan, S. S.; Gullbault, G. G. Clin. Chem. (Winston-Salem, N . C ) 1980, 2 6 , 613. (2) Hinsch, W.; Ebersbach, D.; Sundaram, P. V. Clh. Chim. Acta 1980, 104, 95. ( 3 ) Hercules, D. M.; Sheehan, T. L. Anal. Chem. 1978, 5 0 , 22. (4) Grossrnan, S. H.; Mollo, E.; Ertlnghausen, G. Clin. Chem. (WinstonSalem, N. C . ) 1976, 22, 1310. (5) Bowie, L.; Cochrnan, N. Clln. Chem. (Winston-Salem, N.C.)1973, 19, 656. (6) Blaedel, W. J.; Wang, J. Anal. Chem. 1980, 5 2 , 1426. (7) Chen, F. S.;Christian, G. D. Clin. Chem. (Winston-Salem, N.C.) 1978, 2 4 , 621. (8) Smith, M. D.; Olson, C. L. Anal. Chem. 1975, 4 7 , 1974. (9) Attiyat, A. S.; Christian, G. D. Anal. Chlm. Acta 1979, 106, 225. (IO) Coulet, P. R.; Julllard, J. H.; Gautheron, D. C. Biotechnol. Bioeng., 1974, 16, 1055.
RECEIVED for review December 7, 1981. Accepted June 21, 1982.
