An AutomatedSystemfor KineticMultiple ... - CiteSeerX

An AutomatedSystemfor KineticMultiple-PointDeterminations Exemplifiedby Serum LacticDehydrogenaseDetermination Charles F. Fasce, Jr. and Robert Rej

A fully automated procedure is presented for kinetic determinations and for use of the AutoAnalyzer in their study; serum lactic dehydrogenase has been used to illustrate such an application. The LDH-catalyzed oxidation of L-lactate by NAD in separate, unequal timedelay coils is determined by measuring the natural fluorescence of reduced coenzyme. The normal range for LDH activity by this method was 63-169 U/liter at 37#{176}C. Additional

Keyphrases

other methods

D

.

optimization

AutoAnalyzer #{149} fluorometry normal range

OF human serum lactic dehydrogenase (LDH; L-lactate : NAD oxidoreductase, EC 1.1.1.27) activity has proved to be important as an indicator of myocardial infarction (1, 2) and as an aid in diagnosis of other conditions (3-5). The increased clinical demand for LDH determinations enhances the need for an accurate, reproducible automated method. LDH reversibly catalyzes the conversion of lactate to pyruvate in the presence of nicotinamide adenine dinucleotide (NAD) (6): ETERMINATION

L-lactate

+ NAD

+

LDH

pyruvate

+ NADH + H

+

Any reactant at optimum concentration is converted at a rate proportional to the LDH activity of the serum. The “forward” reaction, lactate to pyruvate, was chosen because of its merits as described by Thiers and Vallee (7) and others (8, 9), and its adaptability to an automated procedure. Several published AutoAnalyzer methods (1015) use either the lactate-to-pyruvate or pyruvateto-lactate reactions, most in conjunction with a colorimetric indicating reaction. Nearly all authors use the customary AutoAnalyzer technique for measuring extinction or fluorescence at a fixed time after reagent mixing. In such systems there is no guarantee that the point selected is on the linear plot of activity (16). Furthermore, serum blanks should be run to eliminate effects of From the Division of Laboratories and Research, State Department of Health, Albany, N.Y. 12201. Received April 11, 1970; accepted Sept. 14, 1970.

New York

972 CLINICAL CHEMISTRY, Vol. 16, No. 12, 1970

#{149}

comparison

with

substances in the serum that either absorb or fluoresce. To measure LDH activity more accurately, we believe that reaction rate must be determined. Henry et al. (17) described a serniautomated kinetic method in which a multiple-analysis system was used, and Pollard described an interrupted-flow system on the AutoAnalyzer (Pollard, A., “Studies on the Automation of Enzyme Reaction Rate Determinations,” presented at the 1964 Technicon International Symposium, London). The former method needs specialized equipment not available in most laboratories. Although Pollard’s method is an automated kinetic procedure, there are inherent procedural difficulties with this system-primarily the need for special timers and relays to interrupt the flow section of the apparatus. In addition, the mixture must he incubated in the cuvet, which leaves a relatively short

time

for reaction.

This study reports the development and evaluation of a rate-measuring assay to automatically determine serum LDH activity and the factors influencing such a procedure.

Materials and Methods Apparatus The instrumental system consists of a modified AutoAnalyzer (Technicon Corp., Tarrytown, N.Y.), comprising sampler, two proportioning pumps, and a dialyzer unit used as a constanttemperature water bath. Measurements were made

with

a Model 111 fluoroineter (G. K. Turner Associates, Palo Alto, Calif.), adapted for use in a flow system. In preliminary experiments a 10-in. linear recorder with scale expander (both from Beckman Instruments, Inc., Fullerton, Calif.) was used. The described method utilizes the standard AutoAnalyzer

recorder.

time-delay coils of differing nience, the incubation coils

Reagents Saline solution. Dissolve 9 g of NaCl in 1 liter of distilled water, and add 1 ml of Brij-35, a wetting agent

(described below) to allow simultaneous Sampling of serum and coenzyme. Serum samples are diluted with isotonic saline, segmented with air, and mixed into the buffered substrate to which the NAD solution has been added. The flow stream is then debubbled and split into three equal fractions, air is reintroduced, and these are pumped through

(Technicon).

Buffered substrate: 0.14 inol of kictate and 0.1 mol of pyrophosphate per liter, pH 8.6. To 500 ml of warm distilled water add 12.0 ml of a solution of DL-lactic acid, 85 g/liter (Sigma Chemical Co., St. Louis, Mo.) and 45.4 g of Na4P2O7.10H20. With NaOH, electrometrically adjust the pH to 8.6 at 25#{176}C; dilute to 1 liter. This solution is stable for longer than six months when refrigerated. NAD, 41.0 mmol per liter. Weigh out 0.270 g of nicotinamide adenine dinucleotide (Grade III, Sigma), and place it in a large screw-capped tube. Add 10.0 ml of distilled water to which a drop of dilute acetic acid has been added. This quantity is sufficient for over 40 determinations. Stability when frozen is about two months; stored at 4#{176}C, it can he used for five days. Procedure A flow diagram for the kinetic deternunation of serum LDH is shown in Figure 1. The sampling rate is set at 10 specimens per hour with a specially constructed cam. The sampler is modified further

lengths. For conveare placed in a 37#{176}C

dialyzer unit with dialysis apparatus removed. The three flow lines then combine and are diluted with de-ionized water. The incubation system is phased so that the entrance times of each aliquot into the final stream differ by about 100 s, to ensure against intermixing of the three fractions. The stream is then debubbled and pumped through the flow cell of the fluorometer. The primary fluorometer filter is a Corning 7-60 (peak 360 nm); a Wratten 2A (passes light with wavelength greater than 415 nm) and a Wratten 47B (peak, 436 nm) serve

as secondary

filters.

Various

aperture

slits

and neutral-density filters can be chosen to vary the sensitivity. When the Beckman recorder is used, full-scale deflection is set at 10 mV with a 2X scale expansion. Procedural modifications. SAMPLER II. So that sufficient time is allowed to achieve desired sensitivity, sampling is restricted to 10 per hour with a duration of 40 s. A cam providing this ratio can be constructed from an existing cam or from thin sheet metal to the specifications of Figure 2. Since the wash time is relatively long it is uneconomical to aspirate NAD solution continuously. We constructed a dual-probe system that simultaneously samples serum and coenzyme by removing the usual probe holder and replacing it

Fig. 1. Flow diagram for automated kinetic determina. tion of lactic dehydrogenase

.STAP4OARDIZED TUSINO (a)PULIC SUPPACSSON 0. 005 I. 0. (b)PUL,c SU.P5,SIOR 0.01510.

o0 CLINICAL CHEMISTRY, Vol. 16, No. 12, 1970 973

Fig. 2. Cam for Sampler II module to provide 10-per-hour sampling

sprocket attached to the gear head of the motor and the drive chain are removed. These are replaced by a gear of larger diameter, with the same gear-tooth cut of the original and a longer drive chain. For the system described, a gear with a radius of 1.0 in. (which about doubles pumping rate) is sufficient. To allow for clearance of the larger gear, a small opening is made in the pump housing near the exhaust grating. TUBING STANDARDIZATION. After reagents and sample are mixed, the stream is debubbled and separated into three equal aliquots by a set of three 0.080-in. i.d. pump tubes. However, volumes pumped by tubing listed as being of the same size may vary considerably. Since the aliquots should be of equal volume, three tubes with comparable pumping rates must be found: one places several tubes on a manifold, each pumping water into volumetric flasks of the same size, and measures the individual filling times. A package of 12 pump tubes contains on the average two sets of three tubes having a variation of ±0.5% in volume delivered.

Results Factors Influencing

Fig. 3. Modified Sampler II module with a metal rod, 40-cm in length and of similar diameter. Two probes were soldered at positions along the rod, enabling the serum probe to sample from a cup on the sampler tray and the other probe to sample NAD solution from a vessel in front of the sampler (Figure 3). Serum and coenzyme transmission-tube lengths must be adjusted to ensure that the two streams arrive at the juncture concurrently. The NAD solution was placed in a small test tube fastened inside a beaker that was filled with distilled water as a probe rinse. This was then placed in an open container packed with ice to keep the heat-labile NAD and surrounding water cool. PROPORTIONING PUMP NO. 2. The conventional AutoAnalyzer proportioning pump was unsuitable for forcing a sufficient volume of reaction mixture into the time-delay coils within optimum time of the reaction. Since reproducibility is sacrificed in using tubes with a large inside diameter, pump speed was instead increased by using a larger gear head. This also has the advantages of decreasing the time the reaction mixture is nonisothermal and unsegmented by air, thus giving increased precision and decreased sample duff usion. To do this, the base plate and top-chain housing are removed from the pump module. Then the

974 CLINICAL CHEMISTRY, Vol. 16, No. 12, 1970

Procedure

Fluorometry. Applying fluorometric technique to a serum assay introduces problems of light scattering, inner filter effects, and quenching by protein and serum macromolecules (18). The automated technique of Passen and Gennaro (19) eliminates these effects by use of dialysis. When dialysis was used with our system, we encountered phasing difficulties and a marked decrease in sensitivity. However, if small quantities of serum in relation to the total volume are used, the amount of interfering materials would be negligible (20). To determine the proper volumes, we used a wide range of serum dilutions and recorded the change in fluorescence due to LDH activity. Activities were derived kinetically on an earlier manifold, after multiple dilutions were made of lyophilized and patient sera of known LDH activities. Each dilution was then run as a regular specimen, corrected for dilution, and a factor derived to convert the effective fluorescence change to the previously determined activity. To ensure against nonfluorescent artifacts-e.g., substrate or coenzyme depletion because of greater LDH activities in undiluted specimens-spectrophotometric determinations were performed manually with use of the reagent concentrations of the fluorometric assay. None of the effects shown in the fluorometric measurement (Figure 4) could be observed. In Figure 4, the variation in the factor derived to convert fluorescence change (SF) per unit time to enzyme activity is shown as a function of dilution. At low serum dilutions, fluorescence quench and

3O

I2O9O DILUT ION

60’

Fig. 4. Effects of serum quenching on 37#{176}C other

factors

The dilution provides

can result factor

N.UMI

interference

180’

fluorescence,

in errors greater

in the present

negligible

50-I

than 40%.

manifold and

(1: 150) excellent

sensitivity. Optimization

Studies

Optimum concentrations of coenzyme and substrate were determined by the method of continuous variation as described by Ryland (1964 Technicon International Symposium, New York, N.Y.), which enables a continuous graph of fluorescence as a function of reagent concentration in the AutoAnalyzer system For one study, the activities of patient sera were measured while NAD concentration was increased. A broad concentration range was found in which sensitivity would be maximum. Concentration of reagent NAD was 41.0 mmol/liter. A variation up to ± 10% in this concentration resulted in only a slight change in measured LDH activity. Substrate concentration is least critical, showing a relatively broad plateau, once minimum concentration is reached. The less expensive DL-lactic acid was used rather than the L-isomer, with no significant inhibition observed due to a possible competitive effect of the nonreactive D-lactiC acid. A reagent concentration of 0.14 mol of DL-lactate per liter is sufficient substrate even for sera with greatly supranormal activities. Some authors (21, 22) suggest that small amounts of hydroxylamine or semicarbazide be used to trap the generated pyriivate, which inhibits the NAD NADH reaction (23. 24). When we used these, results and reproducibility were unaffected, although they reportedly increase reagent stability (10). Pronounced variations in pH optima and serum LDH activity have been shown when different buffer systems are compared (23, 25), especially when glycine buffer was compared to tris(hydroxymethyl)aminomethane or pyrophosphate. In the glycine buffer system a pH optimum at 20#{176}C of greater than 9.5 was observed in the activities of

human and bovine LDH. Work by Mezey et al. (26) on the action of LDH on endogenous lactate (“Nothing Dehydrogenase”) confirms this pH optimum. We are now studying this highly basic pH optimum, which may result from a specific-ion effect or an increase in the rate at which product is removed from the enzyme complex. Hankiewicz (27) has reported the dependence of NADH absorbance upon pH; however, we were unable to show any significant dependence of NADH fluorescence on pH. We reconfirmed the optimum pH as reported by Gay et at. (28) and recommend the use of 0.1 mol/liter pyrophosphate buffer for the reagent substrate, pH 8.6 at 25#{176}C. Because of its availability, we used the bath for the AutoAnalyzer dialyzer to carry out the reaction at 37#{176}C. At this temperature the activity is greater, hence, sensitivity is greater than that at lower temperatures. It has also been reported that the optima of the respective LDH isoenzymes are less dissimilar near physiological temperatures (24, 29-32). Denaturation of serum LDH at 37#{176}C is reported by some authors (33); however, we saw no substantial denaturation even when incubations were several times longer than those described above. Calibration The standard unit for reporting LDH activity is the International Unit (U); for this expression, one must measure the change in concentration of substrate in Mmol/min for a given volume of serum. Since the change in NADH concentration is equivalent to the change in lactate concentration, LDH activity for this system could be defined as equal to (NADH) (in mol/min) for a volume of serum at 37#{176}C. The correlation, however, between the change in fluorescence and LDH activity must be experimentally determined because of variations in AutoAnalyzer tube sizes and in fluorometer sensitivity, which varies greatly from system to system. A convenient method exists for measuring this conversion factor. About 5 ml of a NADH solution is prepared (about 1 g of fl-dihydronicotinamide adenine dinucleotide per liter, Grade III, Sigma) having a a pH of 8.6 at 25 #{176}C. NADH purity should he checked by the method of Klingenberg (34). The absorbancies at 340 nm of 25- and 50-fold dilutions of this solution are then recorded [a Model DU-2 or DB-G ultraviolet spectrophotometer (Beckman Instruments Corp., Fullerton, Calif.) was satisfactory for these measurements]. Photometric accuracy was checked with solutions of acid dichromate and acid cobalt ammonium sulfate (“Ultrex special product,” J. T. Baker Co., Phillipsburg, N.J.); wavelength was calibrated at the 334.15- and 365.01-nm lines of a mercury dis-

CLINICAL CHEMISTRY, Vol. 16, No. 12, 1970 975

*ith1T1

if

T1

t

1k$tl1T

‘:::

if

JjB’fIJi;.

.

H

.:,

t

1HT.Tl tlii 1 J1r.j.rfl

.

..

-

Iii

TH

.

i:::

-

It[!1..

. -

I

I. Ifflh11]1IT’’#{149} Ittt±ii#{149}

I.,

. -

r

III

-

.

-

-t

-

#{149}11-

-

-

-.

ttt-

-

. +-

P1dIJT

ii.itLr’r’

1ll1i1L

T

-

#{231}

U1j

TifTH:

if

#{149}1 . If.

.

Fig. 5. Recorder tracings for five replicate analyses charge lamp (35). The original NADH solution is then placed in a sample cup and run as a specimen in the automated procedure. A water blank should also be run to ensure against fluorescence originating from possible disintegration or contamination of the NAD reagent. From the accepted molar absorptivity of NADH at 340 nm, 6.2 X 10 cm2/mole (36), one can calculate the concentration of NADH from A, the absorbance measured 0.161 A

=

(NADH),

in jimol/ml

Dilutions of NADH solution and serum sample are equal and a conversion factor, C’, can then be calculated. -

A X 0.161 %F’

x

d

where d is the dilution factor for extinction measurements, and %F’ is the fluorescence (peak heights) of the NADH solution recorded. Since the standard AutoAnalyzer recorder chart drives at 18 in./h and LDH activity is reported in U/liter of serum, the above factor can be multiplied by 300 ml in./liter mm to derive the more useful factor of C in U/liter per %F/ in. So for any specimen the LDH activity in U/ liter of specimen can be stated as U/liter

=

C(%F,/in.)

where %F,/in. is the change in percentage fluorescence per inch of chart paper. Although this value will vary among systems, it remains constant for any one manifold over a long time [we measured it on 15 successive days and found a value of 13.06 ± 0.28 (SD)]. While the authors recommend recalibration at the start of each set of determinations, the average value of C determined previously should be used if the recalibrated value is within 2 SD of the mean.

976 CLINICAL CHEMISTRY, Vol. 16, No. 12, 1970

Recordings

and Reproducibility

Results consist of a set of three peaks for each specimen (Figures 5 and 6). The best straight line is constructed through the three maxima, and the slope of this line, in %F/in., is recorded. This value times the constant determined above is the measured activity of the serum. Figure 5 shows the results of five consecutive determinations on the same sample. Figure 6 shows the results of runs on serum diluted with isotonic saline. Slopes are in a ratio of 0.41:0.59:1.00 for serum diluted in the ratio 0.40:0.60:1.00, respectively. This high degree of precision is characteristic of the system. Table 1 shows the LDH activity of a lyophilized serum as measured on 13 of 18 successive days; such data for 30 days had a mean and standard deviation of 188 ± 6.0 U/liter,

U

z

U U, lJ

Ui

0 -J U.

.U;LffiB Fig. 6. Recorder tracings for serum dilutions

Table 1. LDH Activity of a Lyophilized Serum Reconstituted Daily, as Determined by the Automated Kinetic Technique Plate Day

1 2 3 7 8 9 10 11 14 15 16 17

18 Mean±1SD

position 20

10

185 185 195 177 185 198 188 185 185 186 200 188 185 188±6.3

189 182 188 177 192 199 188 ...

193 184 193 186 187 188±5.8

30

186 180 192 172 191 201 187 190 191 188 ...

185 188 188±7.0

40 U)

z

LU

30

0 LU

a.

U)

20

tj

0 Lii I0 #{163}0

z

0 100

140

U /L

Fig. 7. Serum

LDR

value distribution

Broken line: manually determined activities. tivities by the automated kinetic technique

Solid line:

ac-

:

MO.77

U, Ui

2io

180

at 37#{176}C.

SC

-j

indicating this procedure to have a coefficient of variation (cv) of 3.2%, which was also observed on a sample having a mean value of 256 U/liter at 37#{176}C. This is significantly lower than the cv published for the manual methods by Weinberg (37) although greater than the 1.3% reported by Amador et al. (8). After extended use, accuracy decreases because of variations in tube wear. Worn tubes should be replaced, and the conversion constant must then be redetermined.

Comparison of Methods Results of automated tests were compared with those obtained by the method of Amador and Wacker (38) with a Model 2000 (Gilford Instrumentation Labs, Inc., Oberlin, Ohio). A plot of patient specimens vs. LDH activity in Wacker units at 30#{176}C for both methods is shown in Figure 7. Tests run on two days using 142 patient sera produced results showing that our automated method was more precise. Figure 8 shows the correlation between the described method and values reported by the Technicon SMA 12/60 [NAD-diaphorase procedure (39), in U/liter at 37#{176}C]. The correlation measured is described by the equation V = 0.77 X, where Y = SMA 12/60-determined values, and X = values reported by the present method. This difference in reported units led us to examine LDH activity as measured by the SMA 12/60 vs. the manual spectrophotometric method. Five lyophilized sera prepared by our laboratories were used and data were compiled from three different laboratories that used the SMA 12/60 and manual method determinations performed by us. These data (Table 2) agree with the derived relationship, and the values fall close to the curve in Figure 8.

>

U 120

::

z

‘;.

80

Uat37C

.5

z

.5

0

40 .5

Cl

0

.

40

82

20

60

S MA)2/60

200

_______

240

282

VALUES

Fig. 8. Correlation between automated kinetic and those from the Technicon SMA 12/60

results

Table 2. LDH Activity of Five Lyophilized Sera as Determined by Two Different Methods in Three Laboratories LDH activity SMA 12/60

106± 7 237 ± 8 280 ± 12 196±10 164 ± 8

In U at 37#{176}C1 mean ± 1 SD Manual lactate-topyruvate method (8)

78± 8 181 ± 14 211 ± 21 146±16 121 ± 12

RatIo

0.736 0.764 0.754 0.745 0.738

We also compared the manual method of Amador and Wacker (38) and the automated kinetic system (Figure 9). There is poor agreement among published accounts of normal ranges for LDH activity by the lactate-to-pyruvate reaction (1, 8, 40, 41). With the temperature conversion factors of Amador et at. (8) and the range published by Bell (1), the normal range by our method would be 63-169 U/liter at 37#{176}C. Accepting this normal range, we compared data from the SMA 12/60 [normal range, 90 to 200 U/liter (Technicon AutoAnalyzer LDH Meth-

CLINICAL CHEMISTRY, Vol. 16, No. 12, 1970 977

M

C’,

Ui -j .5 >

.00

60

U IUi z

S

‘2

Iii N -j .5

Sc

z

U ot37C

.5

0 C8

4

0

0

40

80

‘20

MANUAL

60

KINETIC

200

VALUES

_________ 240

280

Fig. 9. Correlation between automated kinetic method and those from the spectrophotometric method of Amador and Wacker (38)

odology File N-60 I-IT, Technicon Corp.)] and our method and found 129 of 131 sera that were classified as “normal” or “abnormal” (Figure 8) by both methods.

Discussion In our opinion, the preferred method for measurement of enzyme activity is a kinetic assay of at least three points (16, 42). Since automated analyses are generally more precise than manual techniques, our automated kinetic assay was developed. The data presented indicate that optimum lactate concentration for this method (during incubation) is the same as used by Babson and Phillips (43) and recommended by Gay et al. (28). The concentration of NAD in our method (2.1 mmol/liter) is in the same range as used by Capps et al. (44) and Babson and Phillips (43), but significantly lower than in classical spectrophotometric methods (28, 38). NAD concentration was studied further in a semiautomated spectrophotometric system (“Kintrac VII,” Beckman) with the conditions of the automated method. With the above NAD concentrations, the mean activity of 17 freshly drawn human sera was 97% as great as that measured with use of the greater NAD concentrations of the manual method (38). Since this reagent is expensive and the reaction was found to be linear for the time required for the automated procedure, a reagent concentration of 41.0 mmol/liter was used. The p11 optimum determined for LDH in the described system is near that recommended by Gay et al. (28) and is the pH used by Amador and Wacker (38). This optimum is shifted to a more basic pH if a glycine buffer system is used (45) and is consistent with Schwert’s data on initial velocities for beef heart LDH (25). Brooks and Olken’s findings (10) on activity-pH dependence could not

978 CLINICAL CHEMISTRY, Vol. 16, No. 12, 1970

be confirmed in any of the buffer systems investigated. Our results show excellent correlation with data obtained by the manual procedure (38). Both manual and automated methods showed a consistent, although not 1: 1, relationship to activities determined by the Technicon SMA 12/60. The normal range for the described procedure was 63169 U/liter at 37#{176}C. The system can assay 10 samples per hour, and with minor modifications-e.g., shortening the differences in incubation coil lengths-can perform 20 determinations per hour, but with some loss in reproducibility. In such modifications one becomes limited by recorder drive time although precision can be improved with a different gear assembly to increase chart speed. We also used an ultraviolet spectrophotometer (Beckman Model DB-G, equipped with flow cell) successfully. The advantages of spectrophotometry in this system can be readily appreciated in the direct conversion of absorbance change to increase in NADH concentration. However, this approach was discontinued because its sensitivity was lower in comparison with fluorometry and its precision was decreased for sera with lower activities. Fluorometry is expected to become more useful in applying this system to other enzyme determinations. We are now studying a related technique for a kinetic assay of serum GOT. We think this system of differential incubation time on the AutoAnalyzer is valuable not only for enzyme determinations, but also for the thorough study of any kinetic reaction requiring a large number of analyses that are too cumbersome to perform manually. We thank the staff of the Clinical Chemistry Laboratory of the Albany Medical Center, Albany, N.Y., for providing the vast number of specimens and data from the SMA 12/60. We also thank Barbara Graffunder and Barbara Robertson, who assisted with the determinations by the manual method.

References 1. Bell, R. L., Separation of serum lactic dehydrogenase ing in myocardial and hepat.ic ti*ue, by means of heat ation. Amer. J. Clin. Pat ho!. 40, 216 (1963).

originatfraction-

2. Wroblewski, F., The mechanisms of alteration in lactic dehydrogena.se activity of body fluids. Ann. N.Y. A cad. Sci. 75, 322 (1958). 3. Glick, J. H., Jr., Serum lactate dehydrogena.se isoenzyme and total lactate dehydrogenase values in health and disea.se, and clinical evaluation of these tests by means of discriminant analysis. Amer. J. Clin. Pathol. 52, 320 (1969). 4. Erickson, dehydrogenase.

R.

J.,

and

Morales,

D.

R.,

Clinical

use of lactic

New Engi. J. Med. 265, 478, 531 (1961).

5.

Cohen, L., Djordjevich, J., and Ormiste, V., Serum lactic dehydrogenase isoenzyme patterns in cardiovascular and other diseases, with particular reference to acute myocardial infarction. J. Lab. Clin. Med. 64, 355 (1964). 6. Wroblewski, F., and LaDue, activity in blood. Proc. Soc. Exp. 7. Thiers, R. E., and Vallee, serum enzyme determinations. (1958).

J. S., Lactic dehydrogenase Biot. Med. 90, 210 (1955).

B. L., Analytical considerations Ann. N.Y. Aced. Sd. 75,

in 214

8. Amador, E., Dorfinan, L. E., and Wacker, lactic dehydrogenase activity: An analytical current assays. CLIN. CHJ:M. 9, 391 (1963).

W. E. C., Serum assessment of

9. Snodgrass, P. J., Wacker, W. E. C., Eppinger, E. L., and Vallee, B. L., Metalloelizymes and myocardial infarction. III. Lactic dehydrogenase activity of serum-a determinate diagnostic measure. New Engi. J. Med. 261, 1259 (1959). 10. Brooks, L., and Olken, method for determination CLIN. CHEM. 11, 748 (1965).

H. (1., An automated of lactic dehydrogenase

fluorometric in serum.

11. Morgenstern, S., Floe, It., Kessler, G., and Klein, B., The automated determination of NAD coupled enzymes. I. l)etermination of lactic dehydrogenase. In Automation in Analytical C’hemistry; Skeggs, L. T., Jr., Ed. Mediad, Inc., New York, N.Y., 1965, p 545. 12. Schwartz, M. K., Kessler, U., and Bodansky, 0., Automated assay of activities of enzymes involving the diphosphopyridine nuc1eotide-reduced diphosphopyridine nucleotide reaction. J. Biol. Chem. 236, 1207 (1961). 13. Levy, A. L., Dalmasso, C., and l)aly, J., Automated colonmetric determination of lactic dehydrogenase. In Automation in Analytical Chemistry; Skeggs, L. T., Jr., Ed. Mediad, New York, N.Y., 1965, p 531. 14. Runstedler, B. F., Automated of lactate dehydrogenase activity 28, 217 (1966).

method in serum.

for the determination Can. J. Med. Technol.

25. Schwert, dehydrogenase.

The mode of action of heart muscle N.Y. Aced. Sci. 75, 311 (1958).

26. Mezey, E., Slater, nothing dehydrogenase Acta2S, 11(1969).

lactic

K. C., and Holt, P. R., The source activity in normal serum. Clin.

27. Hankiewicz, J., Wplyw niektorych oznaczania aktywnosci dehydrogenazy Chem. Anal. 14, 537 (1969).

of the Chim.

czynnikow na mleczanowej

wyniki (LDH).

28. Gay, R. J., McComb, H. B., and Bowers, G. N., Jr., Optimum reaction conditions for human lactate dehydrogenase isoenzymes as they affect total lactate dehydrogenase activity. CLIN. CHEM. 14, 740 (1968). 29. Krieg, A. F., Gorton, S., and Henry, J. B., Effect of temperature on activity and lactate optima of LDH isoenzymes. Clin. Chin,. Acta 17, 363 (1967). 30. Kaplan, N. 0., substrate inhibition 151, 400 (1968).

Everse, J., and Admiral, J., Significance of dehydrogenases. Ann. N.Y. Aced.

31. Stambaugh, R., and Post., D., Effects temperature on lactate dehydrogenase Biophys. Acta 122, 541 (1966).

of Sci.

of tissue extracts and isoenzymes. Biochim.

32. Vesell, E. S., Lactate debydnogenase isoenzyrnes: Substrate inhibition in various human tissues. Science 150, 1590 (1965). 33. Hess, Academic

15. Cloutier, L.-A., Determination fluorimetrique de la deshydrogenase de l’acide lactique totale (LDt) et stable a l’ur#{233}e (L.Di) par uii proc#{233}d#{233} automatise. Clin. Biochem. 2, 81(1968).

G. W., Ann.

B., and Henley, Press, New York,

34. Bergmeyer, Academic Press 35.

K. S., Enzymes in Blood N.Y. 1963, p 140.

H.-U., Ed., Methods Inc., New York, N.Y.,

Rand, H. N., Practical 15, 839 (1969).

Plasma.

of Enzymatic 1965, p 531.

spectrophotometric

Analyses.

standards.

CLIN.

CHEM.

16. Henry, J. B., Fundamentals of clinical enzymology. In Workshop on Clinical Enzymology: Pre-workshop Manual 1. American Society of Clinical Pathologists, Chicago, 1964, p 6. 17. Henry, J. B., Cestaric, E. S., and Goodwin, A., automated system for clinical assays of enzymes. Amer. Pathol. 40, 252 (1963).

A semiJ. Clin.

18. Phillips, it. E., and Elevitch, F. R., Fluorometric technics clinical pathology and their interpretation. Prog. Clin. Pathol. 62 (1966). 19. Passen, S., and Gennaro, W., Au automated fluorometric determination of serum lactate Amer. J. Clin. Pat hot. 46, 69 (1966). 20. Udenfriend, cine. Academic 21. Loomis, determination (1961).

S., Fluorescence Press, New York,

Assay N.Y.,

in 1,

system for the dehydrogenase.

in Biology 1962, p 108.

and

M. E., Aui enzymatic fluorometric method of lactic acid in serum. J. Lab. Clin. Mcd.

Medifor the 57, 966

22. Hakala, M. T., Glaid, A. J., and Schwert, G. W., Lactic dehydrogena.se. II. Variation of kinetic and equilibrium constants with temperature. J. Biol. Chem. 221, 191 (1956). 23. Winer, A. D., and Schwert, G. W., Lactic dehydrogena.se. IV. The influence of pH on the kinetics of the reaction. J. Biol. Chein. 231, 1065 (1958). 24. Latner, A. L., Siddiqui, S. A., and Skillen, inhibition of lactate dehydrogenase activity extracts. Science 154, 527 (1966).

A. W., Pyruvate in human tissue

36. Horecker, of the reduced 385 (1948).

B. L., and Kornberg, A., The extinction band of pyridine nucleotides. J. Biol.

coefficients Chem. 175,

37. Weinberg, M. S., and Adler, D. H., Lactic dehydrogenase: comparison of two methods. CLIN. CHEM. 10, 749 (1964). 38. Amador, for diagnosis.

E., and Wacker, W. E. C., Enzymatic methods Methods Biochem. Anal. 13, 265 (1965).

39. Hochella, N. H., lactate dehydrogenase 40. Marymont, A clinical and genase methods.

and Weinhouse, in urine. Anal.

used

S., Automated assay Biochem. 13, 322 (1965).

of

J. H., Jr., Cawley, L. P., and Hoffmann, H. G., laboratory comparison of four lactate dehydroAmer. J. Clin. Pathol. 49, 431 (1968).

41. Wacker, W. E. C., Ulmer, D. D., and Vallee, enzymes and myocardial infarction. II. Malic hydrogenase activities and zinc concentrations Engl. J. Mcd. 255, 449 (1956). 42. Dixon, New York,

A

M., and Webb, N.Y., 1958, p 62.

E.

43. Babson, A. L., and Phillips, for serum lactic dehydrogenase.

C.,

Enzymes.

B. L., Metalloand lactic dein serum. New Academic

Press,

G. E., A rapid colorimetric assay Clin. Chins. Acta 12, 210 (1965).

44. Capps, R. D., II, Batsakis, J. U., Brieno, R. 0., and Calam, R. G., An automated colorimetric (tetrazolium salt) assay for serum lactate dehydrogenase. CLIN. CHEM. 12, 406 (1966). 45. King, J., Practical Clinical New York, N.Y., 1965, p 31.

Enzymology.

Van

Nostrand,

CLINICAL CHEMISTRY, Vol. 16, No. 12, 1970 979