Rapid Kinetic Measurement of Lactate in Plasma ... - Clinical Chemistry

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to lactate concentra- tion. The assay is complete in 4 mm and absorbance is linearly related to concentration from 0.625 to 15 mmol/ liter. Analytical recoveries of ...

CLIN. CHEM. 21/13, 1932-1934 (1975)

Rapid Kinetic Measurement of Lactate in Plasma with a Centrifugal Analyzer Michael A. Pesce, Selma H. Bodourian, and John F. Nicholson

In this method, blood is collected in ammonium heparinized microhematocrit mined in the plasma,

tubes and lactate separated within

15 mm from the erythrocytes. Lactate is assayed by mixing 10 zl of sample with NAD and lactate dehydrogenase in tris(hydroxymethyl)aminomethane hydrazine buffer. The rate

of increase in absorbance of the NADH formed, measured at 340 nm, is proportional to lactate concentrais complete in 4 mm and absorbance is linearly related to concentration from 0.625 to 15 mmol/ liter. Analytical recoveries of lactate added to plasma averaged 104% (range, 91-116%). Results compared tion. The assay

well for plasma

samples

analyzed

by this method

with

the CentrifiChem and the Du Pont aca. Addftlonal

Keyphrases:

glycogen storage disease ugal analyzer #{149} stability

metabolic

of lactate

#{149} diabetes

acidosis

#{149}hypoxemic

shock

#{149}

#{149}centrif-

in plasma

Clinically, lactate in whole blood or plasma is determined in cases of metabolic acidosis of unknown etiology, in certain carbohydrate disorders such as type I glycogen storage disease, during phenformin therapy of diabetes mellitus, and in cases of hypoxemic shock (1). Most enzymatic methods for determining lactate require immediate deproteinization of whole blood and are end-point analyses (2-7). These methods require unusually careful handling of the blood by the laboratory technologist and are timeconsuming. Recently, an end-point assay was developed for directly measuring lactate in plasma (8). We considered that lactate could be measured if the absorbances could be precisely recorded at short inter-. vals. In this paper, we present a fixed-time rate analysis for measuring lactate in plasma with a centrifugal analyzer. The method is based on the oxidation of lactate to pyruvate by lactate dehydrogenase (LD; Llactate:NAD oxidoreductase, EC 1.1.1.27) in the presence of NAD+. The reaction scheme is: Division of Clinical Chemistry, Babies Hospital, The Childrens Medical and Surgical Center of New York at Columbia Presbyterian Medical Center; and The Department of Pediatrics, College of Physicians and Surgeons of Columbia University, 3959 Broadway, New York, N.Y. 10032. Presented at the 9th International Congress on Clinical Chemistry, Toronto, Canada, July 13-18, 1975. Received Aug. 6, 1975; accepted Sept. 25, 1975.

1932

CLINICAL CHEMISTRY. Vol. 21, No. 13, 1975

+ NAD

L-(+)-Lactate

is directly deter-

LD pH9.

pyruvate

+ NADH

+ W

The equilibrium favors reduction of pyruvate to lactate. If hydrazine is added, it removes the pyruvate as it is formed. An alkaline pH shifts the equilibrium in the direction of pyruvate. If the rate of increase in absorbance of NADH that is being formed is measured at 340 nm, it is directly and linearly proportional to the lactate concentration up to 15 mmol/ liter.

Materials We (Union

and Methods

used Carbide

a CentrifiChem Corp.,

centrifugal

analyzer

Rye, N.Y. 10580).

Reagents Buffer: Tris(hydroxymethyl)aminomethane buffer (0.5 mol/liter, pH 9.8) with added disodium ethylenediaminetetraacetate (10 mmol/liter) and hydrazine hydrate (0.2 mol/liter). Dissolve 60.5 g of tris(hydroxymethyl)aminomethane (Fisher Scientific Co., Pittsburgh, Pa. 15219), 4.0 g of disodium ethylenediaminetetracetate (Sigma Chemical Co., St. Louis, Mo. 63178), and 11.0 ml of hydrazine hydrate (Matheson, Coleman and Bell, Norwood, Ohio 45212) in 800 ml of distilled water. Adjust the pH to 9.8 with potassium hydroxide and dilute to 1 liter with distilled water. NAD, 27 mmol/liter. Dissolve 19.8 mg of NAD (grade III, Sigma) in 1 ml of distilled water. Lactate dehydrogenase, from rabbit muscle, 2750 U/ml (Boehringer Mannheim Corp., New York, N. Y.

10017). Working reagent. Mix bO ml of the buffer, 1 ml of the NAD reagent, and 4 1 of the enzyme as supplied. Stock lactate standard, 20 mmol/liter. Dissolve 192 mg of lithium L-(+)-lactate (Sigma) in 100 ml of distilled water. Working standards, with distilled water, dilute stock standard to give concentrations of 0.625, 1.25, 2.5, 5, 10, and 15 mmol/liter.

CURVE

C

CURVE

Table 1. Stability

8

of Lactate in Heparinized

Plasma

Blood in Plasma separated from blood without

Blood immediately

A

deproteinized

ice-bath for 15 mm before plasma

delay

separated

Lactic acid, mmol/liter

LACTIC

Fig. 1. Linearity tate in plasma

ACID STANDARD NMOL/LITrR

of the kinetic assay for measurement

0.86 9.3 1.02

0.77 9.3 1.00

0.86 9.1 0.96

1.31 1.59

1.30 1.60

1.41

2.56 1.56

3.22

of lac-

1.56 2.33 3.22 4.67

2.11 3.22

Procedure

4.22

With a CentrifiChem diluter, pipet 10 l of plasma or standard and 50 l of water into the sample cavities and 350 l of working reagent into the reagent cavities of the transfer disc. In the reference position place working reagent, because this mixture of NAD+ and hydrazine forms a complex with an absorbance of 0.380 at 340 nm, which increases slowly with time. At each time interval, the absorbance reading for the working reagent is subtracted from the absorbance reading for the other cuvettes. In this way, the absorbance reading of the working reagent is blanked out. Use a temperature of 30 #{176}C and a wavelength of 340 nm. An initial absorbance reading is stored at 3.0 s after starting the rotor. The change in absorbances between 3.0 s and 1, 2.5, and 4 mm are used to calculate the lactate concentration by comparison with data for simultaneously determined standards.

Results and Discussion Figure 1 shows that concentration and absorbance are linearly related from 0.625 to 15 mmol of lactate per liter. In this assay, linearity is observed at different intervals of time. During the first minute, linearity is achieved for the standards in the range of 5 to 15 mmol of lactate per liter (curve A). At this time sensitivity for samples containing less than 5 mmol/liter of lactate is poor. As the reaction proceeds, the linear relationship for standards containing 8 to 15 mmol/ liter of lactate is lost. At the same time, the relation for the 1.25 to 5 mmol/liter standards, gradually becomes more nearly linear so that by 2.5mm it is linear with these standards (curve B). To increase sensitivity for samples containing less than 1.25 mmol of lactate per liter, absorbance values are measured at 4 mm, at which time a linear relationship is observed for samples containing 0.625 to 1.25 mmol/liter of lactate (curve C). The initial rate of reaction is rapid at.pH’s between 8.5 and 9.0, so that samples with lactate concentrations greater than 9 mmol/liter cannot be measured at this pH range. At pH’s between 9.5 and 10.0, the rate is slow enough to allow measurement of lactate in concentrations of 15 mmol/liter, but also fast

1.60 3.00

1.07

1.01

1.09 1.26 1.36 1.46

1.15

1.32 1.52

1.53 1.50 2.07

1.53

2.21

Table 2. Recovery of Lactate Added to Plasma Lactate in sample

Added

Found

Expected

Recovery, %

mmol/liter 4.0

1.25

5.27

5.25

102

4.0

2.5

6.39

6.50

96

4.55 4.55 4.55

1.25 5.0 10.55

5.87 10.0 14.11

5.80 9.55 15.10

106

7.22 7.22

2.5 5.0

10.12 12.67

9.72 12.22

116 109

109

91

enough to allow quantitation of lactate in concentrations as low as 625 smo1/liter. At pH’s greater than 10.0, the reaction is very slow. Therefore, in our working reagent, we use a pH of 9.8. Under our conditions, hydrazine concentration has little effect on the rate of reaction. We use a 0.2 mol/ liter concentration of hydrazine in the buffer. If the hydrazine concentration is doubled the reaction rate is the same but the absorbance of the reagent is increased; if it is halved there is a slight decrease in reaction rate and reagent absorbance. The reaction rate is not affected by NAD concentrations between 2.7 and 5.4 mmol/liter in the working reagent; we use a NAD concentration of 2.7 mmol/liter in the working reagent. The LD activity is critical in obtaining rate measurements and must be carefully controlled so that samples containing 0.625 to 15 mmol/liter of lactate CLINICAL CHEMISTRY, Vol. 21, No. 13, 1975

1933

Table 3. Lactate in 10 Plasma Samples as Measured by the Rate Method with the CentrifiChem and by Use of the Du Pont aca CentrifiChem

ace mmolfliter

9.0 4.2 2.8 4.0 3.7 3.3 4.2 13.3 4.3 2.9

8.7

4.3 3.1 4.0

3.4 2.8 4.0 12.9 3.8 3.0

can be analyzed. Our method specifies 1.0 U of LD per milliliter in the working reagent. When less LD activity is present, the reaction rate is slower, and linear rates can be obtained for samples with lactate concentrations of 10 to 20 mmol/liter for as long as 5 mm. However, sensitivity for samples containing 0.625 to 8 mmol/liter of lactate is poor. When LD activity in the working reagent is increased to 3.2 U/ml, the reaction rate is much greater but linearity is lost for samples containing more than 9 mmol/liter of lactate. In this case, sensitivity for the samples containing 0.625 to 5 mmol/liter of lactate is increased. The use of an LD activity of 1.0 U/mi in the working reagent allows lactate concentrations ranging from 0.625 to 15 mmol/liter of sample to be measured with reasonable sensitivity. Since LD loses stability on exposure to the atmosphere, the LD should be withdrawn from the vial with a syringe just before each analysis. In this way, the entire contents of a vial of LD can be used without significant loss of activity. The activity of each bottle of LD should be measured before use, because different bottles of the same lot of LD may contain different activities. The stability of lactate in blood was investigated. Blood was collected in ammonium heparinized microhematocrit tubes and treated in three ways. One set of samples were immediately deproteinized with sodium hydroxide zinc sulfate solutions and lactate was determined on the filtrates. The second set of samples were centrifuged within 1 mm after collection of blood and the plasma was separated from the erythrocytes. The third set of samples were left on ice for 15 mm, then centrifuged, and lactate determined on the separated plasma. As shown in Table 1, if

1934

CLINICAL CHEMISTRY, Vol. 21, No. 13, 1975

whole blood is kept on ice for 15 mm there is no significant change in lactate. After the plasma is separated from the erythrocytes, lactate is stable for at least 4 h at 4 #{176}C. When whole blood remains on ice for 1 h, a significant increase in lactate is observed. Table 2 shows the results obtained when lithium lactate was added to plasma. Recoveries ranged from 91 to 116%. Within-run precision was established by simultaneously performing 22 determinations on three plasma samples. Mean values were 1.64, 3.35, and 3.96 mmol/liter, with coefficients of variation of 5.24, 3.82, and 2.58%. Day-to-day precision for plasma could not be measured because of the instability of the lactate. Good comparisons were obtained when results by our kinetic method was compared to those by another discrete analyzer system, Du Pont’s aca (Table 3). Both methods are for lactate in plasma. For quality control, it is desirable that a control serum be analyzed with each lactate determination. When General Diagnostics Versatol acid-base control was used with our kinetic system, we obtained values lower than stated by the manufacturer. This may be due to an inhibitor in the control that decreases the rate during the initial phase of reaction. We have not tested this method with other controls. The advantages of this assay over the classical methods for lactate determination are that deproteinization of the blood is avoided and that the results are obtained quickly.

This Corp.

work

was supported

by a grant

from

the

Union

Carbide

References 1. Olvia,

P. B., Lactic

acidosis.

Am. J. Med. 48, 209 (1970).

2. Olson, G. F., Optimal conditions for the enzymatic tion of L-lactic acid. Clin. Chem. 8, 1 (1962).

determina-

3. Rosenberg, J. C., and Rush, B. F., An enzymatic-spectrophotometric determination of pyruvic and lactic acid in blood. Chem. 12,299 (1966).

Clin.

4. Marbach, E. P., and Weil, M. H., Rapid enzymatic measurement of blood lactate and pyruvate. Clin. Chem. 13, 314 (1967). 5. Hadjivassiliou, pyruvic and lactic 357 (1968).

A. G., and Rieder, S. V., The enzymatic acids. A definite procedure. Clin. Chim.

assay of Acta 19,

6. Tfelt-Hansen, P., and Siggaard-Andersen, 0., Lactate and pyruvate determination in 50 l of whole blood. Scand. J. Clin. Lab. Invest. 27, 15 (1971). 7. Neville, J. F., Jr., and Gelder, R. L., Modified enzymatic methods for the determination of L-(+)-lactic and pyruvic acids in blood. Am. J. Clin. Pathol. 55,152 (1971). 8. Westgard, J.0.,Lahmeyer, B. L., and Birnbaum, M. L., Use of the Du Pont “Automatic ClinicalAnalyzer” in direct determination of lactic acid in plasma stabilized with sodium fluoride. Clin. Chem. 18, 1334 (1972).

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