Enzymatic photometric determination of lactate by ...

Fresenius J Anal Chem (1990) 338:t68 --171

Fresenius' Journal of

Only for private use

@ Springer-Verlag 1990

Enzymatic photometric determination of lactate by reversible reduction of methylene blue* Anja Rohen, J6rg Krause, and Karl Cammann Lehrstuhl ffir Analytische Chemie, Westf/ilische Wilhelms-Universit/it, Wilhelm-Klemm-Strasse 8, D-4400 Mtinster, Federal Republic of Germany

Summary. A method for lactate determination is described which is based upon oxidation to pyruvate catalyzed by L-lactic dehydrogenase (LDH) from baker's yeast. Methylene blue acts as an electron acceptor and is reduced to its colourless leuco form. The leuco dye is oxidized by oxygen present in the solution producing a reversible system. Reproducible signals are obtained if the solution is saturated with oxygen. The measuring range depends on the concentration of methylene blue and has to be adapted to the measuring problem. Lactate determinations in real samples by the new method agreed with determinations made by the classical N A D H method.

Introduction Lactate analysis is of increasing interest in many clinical, physiological, bio- and foodstuff technological fields. The lactate blood level is, for example, an important parameter in the diagnosis of shock and lactic acidosis. In sports medicine lactate is determined to check a sportman's condition. Lactic acid generation by micro-organisms in foodstuff technology during e.g. the production of dairy and meat products is an important quality factor. Most lactate determinations are carried out by enzymatic photometric tests based upon the following reaction: LDH lactate + N A D + > pyruvate + N A D H + H +

(I)

The amount of reduced coenzyme is equivalent to the lactate concentration, and the N A D H absorption is measured at 340 rim. A second reaction (2) is necessary to capture the pyruvate by reaction with glutamate catalyzed by glutamicpyruvic transaminase (GPT) to shift reaction (1) to the right side [1].

Some authors have described a lactate determination by photometric measurement of ferricyanide according to the reaction lactate + 2[Fe(CN)6] 3-

LDH ~ pyruvate

(3)

+ 2[Fe(CN)6] 4 + 2 H +

using yeast lactic dehydrogenase instead of L D H [2]. Recently some electrodes and optodes based upon the reactions mentioned above have been developed [ 3 - 6], but most of them still are in the experimental state and have not found widespread application yet. In this paper a photometric method for lactate determination is presented in which the decolourization of methylene blue resulting from oxidation of lactate by L D H is measured.

lactate + methylene blue + leuco methylene blue.

LDH ....~ pyruvate (4)

In a second step the leuco dye is reoxidized by oxygen present in the solution. The above reaction has already been used for determination of lactate. In the so-called Thunberg technique, oxygen is excluded from the reaction, the time until decolourization has occurred is measured and is used to quantify the lactate concentrations or to determine the activity of dehydrogenases [7]. Working under aerobic conditions leads to a more simple experimental set-up and to a reversible system in which several determinations of lactate are possible with one cuvette test solution.

Experimental Instrumentation

pyruvate + glutamate GPT> alanine + e-ketoglutarate. (2)

* Dedicated to Prof. Dr. G. Schulze on the occasion of his 60th birthday Offprint requests to: K. Cammann

Absorbance measurements were performed using a Zeiss PM 4 spectrophotometer and 1.0 cm quartz cuvettes. Signals were recorded by a chart recorder (BBC Goerz servogor 210) as well as by an integrator device (HP 3390 A). Oxygen measurements were carried out with the "Schott Oz-Meter CG 867".

169 0,6

absorbance

0,5 0,4

9 [ oxygen

~

concentration

[mg/l]

# lactate

7,5t

0,3

t

0,2

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L 1

~ 2

t [rninl

[ 7L--

3

~--

I

I

I

0,5

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1,5

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t Imin]

2,5

Fig. 1. Signal shape of the lactate determination by the methylene blue method (lactate addition at t = 0)

Fig. 2. Change in oxygen concentration during the enzymatic lactate oxidation (lactate addition at t = 0)

Reagents

8l

The following enzymes and chemicals were used: L-lactic dehydrogenase (LDH EC 1.1.2.3) from baker's yeast in a 6.7 ml suspension containing 100 units. L-lactic dehydrogenase (LDH EC 1.1.1.27) from porcine muscle, 10,000 units in 1.1 ml. Glutamic pyruvic transaminase (GPT EC 2.6.1.2) from porcine heart, 1,000 units in 1.1 ml. Phosphate buffer solution: 0.05 tool/1Na~P207 • 10 H 2 0 solution was adjusted to pH 7.4 by addition of HC1 and a 0.1 tool/1 solution of ethylenediaminetetraacetate was added up to a concentration of 10 -4 tool/1. Dye solution: 10 ml of a 10-3 tool/1 solution of methylene blue was made up with water to 100 ml. Glycylglycinebuffer-NAD solution: 6 ml solution contained 60 mg glycylglycine, 70 mg glutamic acid and 40 mg nicotinamideadeninedinucleotide (NAD). The solution was adjusted to pH 10 by adding a 5 tool/1 K O H solution dropwise. The buffer was prepared fresh daily. Lactate standard solution: 190rag Li-lactate were dissolved in 100 ml water and diluted daily to 3.3 • 10 -3 tool/1. Only twice distilled water was used. All chemicals were purchased from Sigma Chemical Co.

Procedure For comparison two different absorption photometric methods were carried out. In one case tile absorption of methylene blue at 669 nm was measured, in the other case the absorption of N A D H at 340 rim. For absorbance measurements of methylene blue, 2 ml of phosphate buffer, 200 ~1 methylene blue solution and 200 gl of L D H suspension (EC 1.1.2.3) were pipetted into the 1 era-cell and mixed. An aliquot of lactate solution was added and the change in absorbance was registered. The next addition of lactate followed when the original absorbance was re-adjusted. Between measurements the cuvette had to be shaken gently to make sure that sufficient oxygen was always present. Lactate determination by measurement of N A D H absorption was carried out according to a modified procedure based upon the UV-test described by Boehringer [1]. 1,2ml glycylglycine-buffer-NAD solution, 0.9 ml of bi-

signal height

[cm]

6 -t .....

1

2

3

4

5

6

7

Fig. 3. Increase of signal height caused by lack of oxygen (addition of 30 pl lactate standard solution every 5 rain)

distilled water, 20 gl GPT suspension and lactate solution were mixed. A second cuvette was prepared containing no lactate. Absorbances were measured after 5 min (At). Then the reaction was started by addition of 20 pl L D H (EC 1.1.1.27) suspension. The reaction was complete after 20 min and the absorbances were determined again (A2). Absorbance differences AA -- A2 - A j were calculated. The reference measurement was subtracted from the AA of the lactate containing cell and lactate concentrations were calculated. All measurements were carried out at room-temperature.

Results and discussion

Influence of Oxygen The two competing reactions: reduction of methylene blue and reoxidation of leuco methylene blue result in a peakshaped signal (Fig. 1). At the beginning of the reaction when lactate has just been added and is oxidized to pyruvate, methylene blue is reduced to the colourless leuco dye resulting in a decrease of absorbance. At the same time the oxidation of leuco methylene blue by oxygen present in solution starts to fall

170 2,5

1 0 -

s i g n a l h e i g h t [cm]

2

peak height [om]

...............

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peak area

25 '20

,

Ni

1,5

.......... ~\\\~....

...........

15

peak height ~Z~

1

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.................................

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added volume

60

80

100

[p[

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1 2 3 4 6 7 5 g to tt 12 13 14 Fig. 4. Course of peak height with increasing number of measurements in an oxygen saturated solution (addition of 30 rtl lactate standard solution every 5 rain)

,o [0.k heightomJ

.e.k re 125

0

20

40

120

Fig. 6. Calibration curve for the methylene blue method; plot of peak area (arbitrary units) and peak height versus added volume of lactate standard solution

14

peak height [cm]

10 6[~-~ helg~-~t peak

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140

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Fig. 5. Calibration curve for the methylene blue method; plot of peak area (arbitrary units) and peak height versus lactate concentration in the cuvette

Fig. 7. Calibration curve for the lactate determination in blood

behind the enzymatic reaction. When lactate has been nearly completely oxidized, reoxidation of leuco methylene blue becomes the decisive reaction leading to an increase in absorbance ending up with the starting value. Considering this complex reaction scheme it is obvious that the concentration of oxygen in the solution is an important factor in determining the shape of the resulting signals. The course of oxygen concentration during the reaction was observed by a "Schott Oz-Meter". Figure 2 shows the decrease of 02 content when the reaction proceeds. If the next addition of lactate occurs when the original absorbance value is reached, the subsequent change in absorbance will be higher because the reoxidation of leuco methylene blue will be slow and decolourization by the enzymatic reaction will predominate for a longer period of time (Fig. 3). For this reason it is necessary to shake the cuvette gently between measurements to supply sufficient oxygen to obtain reproducible results.

Table 1. Results of the lactate determination (average of 5 repeated measurements); values of the photometric NADH- and of the methylene blue depending method are compared

Stability of lactic dehydrogenase (EC 1.1.2.3) To test the stability of the yeast lactic dehydrogenase the reproducibility of the signals was measured by adding, repeatedly, a constant volume of lactate standard solution.

Sample

NADH method lactate concentration (mmol/1)

Methyleneblue method lactate concentration (mmol/l)

Beer Blood Sauerkraut-juice Yoghurt

1.24 + 0.04 2.1 +0.2 53 -+3 58 +4

1.0 +_0.07 2.0-+ 0.2 56 +3 59 _+4

Figure 4 shows changes in peak-height with increasing number of measurements in an oxygen saturated solution. Peak height increases for the first two measurements, remains roughly constant for the next 10 and then decreases. The stability of the enzyme is not only dependent on the number of measurements but also on time and on the amount of added lactate. The enzyme is sufficiently active for 90 to 120 rain. Addition of higher concentrations leads to a lower number of possible reproducible measurements.

171 When adding an amount of 30 ~tl lactate standard solution, up to ten determinations with a standard deviation of 8% are possible, a usual value for enzymatic reactions.

Calibration curve Because of the complexity of the signal producing reactions it is not possible to obtain lactate concentrations simply from the absorbance differences. Therefore a calibration curve has to be set up. Three values can be determined from the peaks and related to the added amount of lactate. These are: the initial slope, the area and the height of the peak. The evaluation of the initial slope requires a computer because the reaction starts rapidly. However, such an experimental set-up is in contrast to the demand for a simple and cheap method for lactate determination so this was not persued. Evaluation of the peak area determined by an integrator gives a calibration plot consisting of two linear ranges with different slopes (Fig. 5); the first part covering 1.5' 10 -5 tool/1 to 8" 10 -5 tool/1 lactate, the second extending up to 12.10 -5 mol/1. A plot of the signal height versus the concentration of lactate in the cell results in a linear curve from 2 to 10- 10- 5 tool/1 (Fig; 5). This range is limited by the concentration of methylene blue in the cuvette and may be varied according to concentration of analyte solution. The calculation of the added amount of lactate becomes easier if the added volume of lactate solution is plotted rather than the concentration (Fig. 6). This is especially important when measurements are not carried out with constantly increasing or decreasing amounts of lactate but with randomly varying amounts, as it is the case when analysing real samples. When plotting the added volume of lactate solution a proper control of the actual total volume in the cuvette is not necessary.

Determination of lactate concentration in real samples To compare the new method for lactate determination with the standard N A D H method, lactate contents of beer, sauerkraut-juice, blood and yoghurt were determined. Sample preparation was as follows: beer and sauerkrautjuice were filtered, and the sauerkraut-juice was diluted

1:100; blood was deproteinized by addition of perchloric acid and centrifuged. The clear solution was neutralized with K O H solution and filtered [1]. 1 ml yoghurt was mixed with 49 ml of water and filtered. Measurements were taken using the procedures described. In case of the methylene blue dependent reaction, different volumes of lactate standard solution and of sample solution were added to one cuvette to obtain the calibration curve. Figure 7 shows as an example the calibration curve for the lactate determination in blood. The evaluated lactate concentrations of the different samples are listed in Table 1. The results obtained with the different methods were not significantly different except for the values for lactate in beer. This difference may be due to some matrix effects.

Conclusions

The feasibility of a new method for lactate determination by means of yeast L D H and methylene blue has been demonstrated. Results obtained with this new method and the N A D H method show an acceptable agreement. An advantage of the methylene blue method is the possibility of measuring in the visible range instead of the ultraviolet region. Furthermore, the new method is economically more efficient. One determination can be done in 5 min whereas the N A D H method takes about 20 min to obtain a single value. Costs are much lower when using the methylene blue method instead of the familiar N A D H dependent process due to the reversibility of the methylene blue reaction which allows the enzyme to be used for several determinations.

References

t. Boehringer Mannheim (1989) Methoden der biochem. Analytik und Lebensmittelanalytik, p 104 2. Durliat H, Comtat M, Baudras A (1976) Clin Chem 22:1802 3. Wangsa J, Arnold MA (t988) Anal Chem 60:1080 4. Schelp C, Scheper T, Biickmann AF (1989) GBF Monogr 13 : 263 5. Trettnak W, Wolfbeis OS (1989) Fresenius Z Anal Chem 334:427 6. Blaedel WJ, Jenkins RA (1976) Anal Chem 48:1240 7. Franke W (1955) In: Hoppe-Seyler/Thierfelder, Handbuch der physiol.- und pathol, chem. Analyse, 10. Aufl., Bd 311 Received January 25, 1990