CONCLUSIONS We have described a semi-automatic apparatus for the routine fabrication of microbulb electrodes with sensing tips in the 100- to 500-gm range. Use of the fabrication apparatus permits the routine and reproducible preparation of such electrodes with a high rate of success. The resulting electrodes generally have all of the desirable characteristics of conventional macroelectrodes and should find application for many analytical and biomedical purposes. Use of the inexpensive preamplifier described allows microelectrode measurements to be made in conjunction with any pH meter or pH recorder.

Works, for kindly supplying samples of the various glasses used in this investigation.

LITERATURE CITED (1) R. N. Khuri. in "Ion Selective Electrodes", R. A. Durst, Ed.. Nat. Bur. Stand. Spec. Pub/., 314, 1969, Chap. 8. (2) "Glass Microelectrodes", M. Lavallee, 0. Schanne, and N. Hebert, Ed., John Wiley and Sons, New York, 1969. (3) J. L. Walker, Jr., Anal. Chem., 43 (3). 89A (1971). (4) R. M. Fuller and J. B. Das, in "Ion Selective Microelectrodes", H. D. Berman and N. C. Hebert, Ed., Plenum Press, New York, 1974, Chap. 13. (5) Unpublished work in conjunction with Department of Biochemistry. (6) J. D. Czaban and G. A. Rechnitz, Anal. Chem., 45, 471 (1973).

RECEIVEDfor review February 24, 1975. Accepted June 4,

ACKNOWLEDGMENT Special thanks are due to Huvin Thompson, Electronic Instruments Limited, and LeRoy Morse, Corning Glass

1975. We gratefully acknowledge support of a grant from the National Institutes of Health, Institute of General Medical Sciences.

Enzymatic Cholesterol Determination Using ion-Selective Membrane Electrodes D.S.

Papastathopoulos and G. A. Rechnitz

Department of Chemistry, State University of New York, Buffalo, N. Y. 142 14

A potentiometric analysis method for cholesterol using a double enzyme procedure in an automated analysis system is described. The method is evaluated with synthetic standards, standard reference serum samples, and actual patient serum samples correlated with independent analyses. The clinical range, precision, and accuracy of the proposed method are attractive for routine determinations of total cholesterol in clinical serum samples. Operating variables are critically examined to define conditions for optimum linearity and sensitivity.

The determination of total cholesterol in blood serum plays an important role in the clinical diagnosis of disease states. Several authors (1-4) have reviewed the numerous classical methods of cholesterol analysis; these methods generally involve color-forming reactions of cholesterol with Lewis acids and suffer from significant drawbacks owing to the use of highly corrosive reagents as well as from chemical and optical interferences. Recently, enzymatic cholesterol analysis methods have been reported (5-9). These methods generally employ three enzymes to convert cholesterol into products which can be finally measured in a color reaction. Although superior to the non-enzymatic techniques, these methods still suffer from interferences by bilirubin and hemoglobin (7, 10) and from problems associated with the use of unstable and toxic reagents ( 9 , l l ) . The use of ion-selective membrane electrodes as sensors in enzymatic clinical analysis methods has been shown (12-15) to offer advantages of low cost, indifference to optical problems, and ready adaptability in automated systems. We now describe an ion electrode based enzymatic cholesterol analysis method utilizing the following reaction sequence C h o l e s t e r o l esters

+ H,O

cholescerol ester hydrolase

Free c h o l e s t e r o l 1792

c

+ Fatty

acids

(1)

Free c h o l e s t e r o l

+

O?

cholesterol oxidase

Cholest-4-en-3-one

+

H,O, (2) (3)

The enzymatic reactions 1 and 2 are carried out in an automated analysis system ( 1 6 ) under controlled conditions for a fixed time interval and a specially constructed flowthrough membrane electrode (17) is used to monitor the change in iodide concentration produced by the molybdenum(V1)-catalyzed indicator reaction. It will be seen that the proposed method yields excellent analytical results for the determination of total serum cholesterol over the clinically significant concentration range while eliminating most of the problems associated with the earlier enzymatic methods. Correlation studies, carried out using actual hospital samples, show good agreement for results obtained with this method in comparison to conventional optical methods.

EXPERIMENTAL Reagents. All solutions were prepared using distilled-deionized water. Unless otherwise specified, all materials used were of reagent grade. Cholesterol ester hydrolase was obtained from Miles Laboratories, Inc. as freeze-dried powder with activities varying from 0.17 t o 0.23 units per milligram for different batches. Cholesterol oxidase was obtained from Beckman Microbics, Carlsbad, Calif., in powder form with activity of 3.75 units per milligram and from Boehringer Mannheim Corporation, New York, N.Y., as a buffered solution with an activity of 20 units per milliliter. Working solutions for the enzymatic analyses were prepared in two steps. First, a stock solution 5 X 10-*M in NaZHP04, 5 X 10-*M in NaH2P04, 6 X 10-3M in sodium cholate, 4 X 10-3M in sodium azide, and containing 5 ml/L of Triton X-100 surfactant was prepared. This stock solution, which has a p H of 6.8, is stable for a t least one week under refrigeration. Just before starting the analysis, cholesterol ester hydrolase and cholesterol oxidase were added to the stock solution to yield enzyme concentrations of 100 and 120 units per liter, respectively. This procedure minimizes loss of enzyme activity in storage.

ANALYTICAL CHEMISTRY, VOL. 4 7 , NO. 11, SEPTEMBER 1975

S a m p l e r I1

I_

- -1

Proportioning Pump Recorder

Meter

Figure 1. Schematic of automated analysis system H3, D1:tinings; SMC: mixing coil; G3: debubbler; SN: stainless steel ground contact; C: recorder ground input; FT: sensing electrode; R: reference electrode; VS: voltage suppressor 4 2 0 mg/d I d

353

l

'

!

I

30

/ k

1

M

b

I

1

I

1

L

-2Omin-I

Figure 2. Typical recording of peaks for total serum cholesterol 99-420 mg/di total cholesterol (Serachol-Versatol reference calibration) M:

Metrix serum control; a-l: unknown human serum samples-see other conditions

text for

For the indicator reaction, solutions 5 X lo-* in KI containing 1 g/l. of the (NH&Mo702~4H20catalyst and 1.6M HClOI were employed. Serum calibration curves for cholesterol were constructed using both Serachol and Versatol-A Alternate reference solutions obtained from General Diagnostics, Morris Plains, N.J. Actual patient serum samples for the correlation studies were kindly supplied by Buffalo General Hospital and St. Mary's Hospital, Rochester, N.Y. These samples were stored under refrigeration and used without pretreatment. Apparatus. Figure 1 shows a schematic diagram of the cholesterol analysis system employed in this study. The sampling and pumping components were of the AutoAnalyzer I1 type (Technicon Instruments, Tarrytown, N.Y.) while the flow-through memANALYTICAL CHEMISTRY, VOL. 47, NO. l l , SEPTEMBER 1975

*

1793

60

c

I 01

H202

ob

200

100

300

1

1

12

0

4

I

mmolll

400

cho erferal,mgidl

Figure 4. Calibration curve for free cholesterol using synthetic standards in isopropanol solutions

Figure 6. Comparison of indicator reaction in aaueous solutions (curve B)and biological samples (curve A )

60

I -

% 40

-

Lu

Q z

-

D

1

920-

-

0

01 50

,

200

100 Total

300

400

C h o l e s t e r o l , mg/dl

Figure 5. Effect of incubation period on total cholesterol determination

tween the reagent base line and the peak maxima and the known total cholesterol concentration (expressed as milligrams per deciliter) of the calibrating solutions, an excellent calibration curve can be constructed (Figure 3). Similarly, an excellent calibration curve (Figure 4) is obtained for synthetic free cholesterol standards in isopropanol. I t should be noted (Figure 2) that the actual analyses carried out for 220 mg/dl calibrating solutions a t the 20 samples per hour rate agree quite well (97%) with the steady state response obtained under continuous sample flow conditions; this indicates that electrode response times are not response-limiting.

RESULTS AND DISCUSSION The results of the calibration experiments using commercial reference standards and synthetic cholesterol solution standards (Figures 2-4) indicate the effectiveness and utility of the double enzyme method. Since actual patient samples are likely to show a wide range of variation in concentration levels (140-340 mg/dl) and composition, it is desirable to give careful attention to the optimalization of critical solution parameters. For example, it was found that a sodium cholate concentration of 6 X 10-3M is optimal because precipitation occurs by slow reaction with the molybdenum catalyst a t higher cholate concentration levels while lower cholate concentrations result in less efficient enzymatic hydrolysis with an attendant loss in analytical sensitivity. Sodium cholate acts as an emulsifier (18, 19) while the presence of the Triton X-100 nonionic surfactant serves 1794

*

ANALYTICAL CHEMISTRY, VOL. 47,

NO.

11, SEPTEMBER 1975

I

1

100

200

200

400

Table 111. Analysis of Patient Serum Samples

Table I. Total Serum Cholesterol Determinations Using Electrode MethodQ

T o t a l cholesterol, m o / d l

T o t a l cholesterol ( m g l d l ) Taken

1:ow.d

(a\- of 4 detn)

99 102 i 2 118 117 i 4 168 166 8 221 226 i 9 2 54 251 5 8 3 09 312 5 3 3 54 364 i 10 420 415 5 8 0 Analyses carried out a t 20 samples/hr using ratio with Serachol-Versatol mixtures.

*

+3 .O -0.9 -1.2 +2.3 -1.2 +1.0 -2.3 -1.2 1:2 sample-to-wash

Table 11. Reproducibility of Total Serum Cholesterol DeterminationsQ Cholesterol, rng dl L o . ot amples

105 6 210 6 3 09 6 420 6 a Conditions as in Table I

\lean of psak

heights,

* *

Sample no,

S.MA 12/60 method

Electrode method

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26

206 108 153 253 14 5 187 274 209 224 155 155 221 265 140 206 121 253 280 163 193 248 24 1 176 134 214 294

211 90 150 260 158 195 280 170 221 151 163 225 277 132 201 115 241 270 158 186 257 237 181 121 217 277

Iteiative error

TI\

5.2 0.13 19.6 0.42 30.5 i 1.10 43.7 j; 1.0

5td dL\,

2.4 2.1 3.6 2.3

what higher catalyst levels than those employed in our previous study (13). The indicator reaction requires an acid medium which was provided by the perchloric acid (0.36M final concentration) used to terminate the enzymatic reactions. Serum proteins are precipitated at such low pH values but are held in solution as a colloidal suspension. We found it desirable to wash out the system with a 1M NaOH wash solution after 40-50 serum samples to minimize any build up of precipitates in the flow tubing or a t the electrode. Since the reaction sequence also consumes oxygen, we investigated the behavior of both pure oxygen and air as segmenting gases in the sample stream. As seen in Figure 7 , the use of oxygen improves the linearity of the response, especially a t the higher cholesterol concentrations where the reaction sequence might be rate-limited by the availability of oxygen. Using the optimized conditions just discussed, we tested the analytical utility of the proposed method on commercial reference samples and actual hospital samples. In addition to the calibration curve (Figure 3) which is linear over the total cholesterol concentration range of 80-420 mg/dl, with a correlation coefficient of 0.9996, the reference samples were taken as unknowns in recovery studies with the excellent results shown in Table I. The reproducibility of the method, a t four different concentration levels prepared from reference samples, is illustrated in Table 11. While evaluation of the proposed method with reference standards is important, the real test of the method must be carried out with actual patient samples. For this purpose, we were fortunate to obtain 26 patient samples from the Buffalo General Hospital and 27 further samples from St. Mary’s Hospital, Rochester, N.Y. The Buffalo General Hospital samples had been freshly analyzed using the standard SMA 12/60 method (20-22) while the samples from St. Mary’s Hospital were analyzed by the enzymatic method (9-11) using optical readout. Thus, our measurements of these samples using the electrode method can be checked against two independent techniques. A comparison of the analytical results obtained is given in Tables I11 and IV for the SMA 12/60 and enzymatic

Table IV. Analysis of Patient Serum Samples T o t a l cholesterol, rn!g,‘dl

Sample no.

C olorim e p i c e n q m e method

3 524 3529 3531 3554 3559 3560 3562 3566 3577 3569 3583 3585 3 584 3586 3591 3601 3613 3614 362 5 3624 3645 3656 3557 3660 3662 3675 3677

348 150 318 214 155 229 175 81 128 253 216 309 232 203 197 153 187 2 04 175 167 153 230 165 175 178 236 224

Electrode method

336 153 310 221 143 207 167 82 120 2 54 217 318 220 200 195 160 179 192 170

177 150 221 163 171 200 249 223

measurements, respectively. The agreement between the electrode method and the two independent methods is highly encouraging. The results are also plotted as correla-

ANALYTICAL CHEMISTRY, VOL. 47, NO. 11, SEPTEMBER 1975

1795

1 4001

300-

y

I /

= 2 434

rl"0

"-26

n

100

300

400

tion diagrams in Figures 8 and 9 with standard statistical parameters; the scatter and negative intercept may be due to optical interferences and lack of specificity in the SMA method. It should be noted that the 53 patient samples tested cover a wide clinical range of cholesterol concentration levels. On the basis of these comparison analyses, the excellent calibration experiments of Figures 3 and 4, and the peakbase-line characteristics shown in Figure 2 for various standards and patient samples, we conclude that the proposed method provides a satisfactory potentiometric alternative to conventional cholesterol analysis methods. The combination of enzymatic selectivity with the continuous automated analysis. capabilities of the flowthrough electrode would appear to offer competitive advantages over classical methods.

ACKNOWLEDGMENT We thank Buffalo General Hospital and St. Mary's Hospital, Rochester, for kindly providing the patient samples used.

(1) G. Vanzetti, Clin. Chim. Acta, 10, 389 (1964). (2) D. B. Tonks, Clin. Biochem., 1, 12 (1967). (3) S.S. Brown, Ann. Clin. Biochem., I O , 146 (1973).

1796

100

mpldl

Flgure 8. Comparison of patient sample data for electrode and SMA 12/60 methods (Data from Table Ill).

LITERATURE CITED

0 978x

:27

/ SMA 12/60 m e ) h o d l i , r b c r m . n - n u r r h a r d l .

+

987

1

200

B M C ~ E ~ ~ ~ ~ m. e t! r

300

CI

method,

400

mgldl

Figure 9. Comparison of patient sample data for electrode and colorimetric enzyme methods (Data from Table W) (4) "Clinical Chemistry. Principles and Techniques", 2nd ed., R. J. Henry, D. C. Cannon, and J. W. Winkelman, Ed., Harper and Row, Hagerstown, Md., 1974. (5) H. F. Flegg, Ann. Clin. Biochem., 10, 79 (1973). (6) W. Richmond, Clin. Chem., I O , 1350 (1973). (7) C. C. Allein, L. C. Poon, C. S.G. Chan, W. Richmond, and P. C. Fu. Clin. Chem., 20, 470 (1974). ( 8 ) P. N. Tarbunon and C. R. Gunther, Clin. Chem., 20, 724 (1974). (9) P. Roschiau, E. Bernt, and W. Gruber, 2.Klin. Biochem., 12, 403 (1974). (10) D. L. Wine, D. A. Barren, and D. A. Wycoff, Clin. Chem., 20, 1282 (1974). (11) "Cholesterol (Enzymatic Method) Procedure Manual", BoehringerMannheim Corp., New York, N.Y., 1974. (12) R. A. Llenado and G. A. Rechnitz, Anal. Chem., 45, 826 (1973). (13) R. A. Llenado and G. A. Rechnitz, Anal. Chem., 45, 2165 (1973). (14) R. A. Llenado and G. A. Rechnitz, Anal. Chem., 48, 1109 (1974). (15) H. Thompson and G. A. Rechnitz, Anal. Chem., 48, 246 (1974). (16) "Technicon Principles of Continuous Flow Analysis", Technicon instruments Co., Tarrytown, N.Y.. 1969. (17) H. Thompson and G. A. Rechnitz, Chem. Instrum., 4, 239 (1972). (18) H. H. Hernandez and I. L. Chaikoff, J. Biol. Chem., 228 447 (1957). (19) J. Hyun, H. Kothari, E. Herm, J. Mortenson, C. R. Treadwell, and G. V. Vahouny, J. Biol. Chem., 244, 1937 (1969). (20) T. C. Huang, C . P. Chen, V. Wefler, and A. Raftery, Anal. Chem., 33, 1405 (1961). (21) J. Levine, S. Morgenstern, and D. Vlastelice, "Technicon Symposium", Vol. 1, Technicon Instruments, Tarrytown, N.Y., 1967. (22) Technicon Methods File, Cholesterol N-77, Technicon instruments, Tarrytown, N.Y., 1970.

RECEIVEDfor review April 18, 1975. Accepted June 9, 1975. We gratefully acknowledge financial support of this work by a grant from the National Institutes of Health.

ANALYTICAL CHEMISTRY, VOL. 47, NO. 11, SEPTEMBER 1975