462
A Comparative Study of Blood Lactate Analytic Methods F A. Rodriguez" 2, M. Banquells V Pons, F Drobnic, R A. Galilea1 Centre d'Alt Rendiment (CAR), Departament de Fisiologia i Valoració Funcional, Sant Cugat del Vallés, Barcelona Nacional d'Educació Fisica de Catalunya (INEFC), Departament de Ciències Mediques, Barcelona, Spain
2 Institut
F A. RodrIguez, M Ban quells, V Pons, F Drobnic and P A. Galilea, A Comparative Study of Blood Lactate Analytic Methods. Tnt J Sports Med, Vol 13, No 6, pp 462—466, 1992.
Accepted after revision: April 21, 1992
Three different blood lactate analytic methods were tested for precision, accuracy, linearity, and intermethod comparison: a photoenzymatic assay (PHE), and three electroenzymatic (EE) semiautomatic assays (EE 1,
EE2, EE3). Reference standards and duplicate capillary blood samples from the earlobe were used. Precision and accuracy of the three techniques, when measuring L-lactate standards, were good in the whole range of measurement
(mean variation coefficient, VC = 1.78 — 3.38 %; mean difference = 1.81 —3.38 %). Correlation between the three methods was high (r = 0.9 13 — 0.946), but all three electroenzymatic techniques systematically measured lower values as compared to the PHE tests. The differences ranged from 0.1—1.2 (Smmoll-' PHE level), to 3.4—5.7 (20mmol 1 I PHE level). These differences were drastically reduced when a hemolyser and a glycolitic inhibitor were added to the sample prior to the assay. The measurements obtained in capillary blood by the three techniques are not equivalent.
The differences are partially attributed to the fact that the PHE technique measures total blood lactate, while the EE methods only measure plasmatic-extraerythrocytic lactate. Some regression equations are presented that may be used to convert values measured by the PHE technique, to EE values and vice versa.
Key words _______________________________________ L-lactate, blood lactate, enzymatic analysis, exercise, physiological testing, lactate threshold
of the anaerobic lactic energy sources to intense exercise (14, 21) have increased the demand for these techniques. The use of micro-analytic techniques proved to be an important qualitative step, as they allow the assays with vety small volumes of arterialized capillary blood, obtained by puncture of the earlobe or fingertip (4,9), instead of venous or arterial blood, thus minimizing the trauma to the subject. At the present time, the most widely used micro-techniques for the determination of L-lactate in whole blood are:
(i) Photoenzymatic methods (PHE), based on the photometric determination of NADH increase in the enzymatic reaction lactate-pyruvate. The amount of NADH formed is proportional to the concentration of L-lactate (18,24). (ii) Electroenzymatic — enzymopolarographic
—
methods (EE),
based on the linear proportion between the lactate concentration in a sample and the production of hydrogen peroxide (H202), yielding a current which is registered by a specific electrode (3,4). (iii)Flow injection analysis (FIA) is based on the same principle as the PHE method, that is the reaction between lactate and NAD with the formation of pyruvate and NADH. The trans-
port system of small sample aliquots is based on the controlled dispersion of an injected sample into a continuously moving, nonsegmental carrier or reagent stream (12,23). The increase in NADH is measured fluorometrically. The method has been validated by comparison to colorimetric and manual enzymatic fluorometric methods using microsamples — 25 j.tl — in an exercise laboratory (12). The purpose of this study was to verif' precision, accuracy and linearity of three different blood lactate analytical micromethods very commonly used in exercise laboratories, and to compare their measurements, using both L-lactate
standards obtained by dilution, and blood capillary samples obtained during and after exercise.
Material and Methods Introduction
One PHE and two EE analytical methods — plus a third EE modified method — have been tested and com-
The determination of blood lactate concentration is nowadays a routine test in most exercise laboratories. Far from being of recent interest (7), the consolidation of the con-
cepts relative to the anaerobic threshold (8,25), lactate threshold (5,6,9), and OBLA (10,11), as well as to the contribution Int. J. Sports Med. 13 (1992) 462—466 Georg Thieme Verlag Stuttgart- New York
pared. The test principle and the basic characteristics of each method will be briefly described. The photoenzymatic method (PHE) is based on the photometric determination of NADH increase — absorbance — in the enzymatic reaction lactate-pyruvate. In the presence of
L-lactate dehydrogenase (LDH), nicotinamide-adenine dinucleotide (NAD) oxidizes L-lactic acid (L-lactate) to pyruvate.
Downloaded by: East Carolina University. Copyrighted material.
Abstract _________________________________________
mt. J Sports Med. 13 (1992) 463
A Comparative Study of Blood Lactate Analytic Methods
pyruvate + NADH + H
The equilibrium of this reaction is almost completely on the side of lactate. However, by trapping the pyruvate in a subsequent reaction catalyzed by the enzyme glutamate-pyruvate transaminase (GPT) in the presence of L-glutamate, the equilibrium can be displaced in favour of pyruvate and NADH. (eq. 2)
GB) instrument was used. The reagents used were contained in the standard Lactate solution (Analox Instruments Ltd., London, GB, ref. GMRD 090/091/092), a cocktail containing buffer and the lactate-oxidase enzyme (1). Capillary tubes containing heparin, sodium fluoride, and sodium nitrite were used in
obtaining the blood samples. The EE2 and EE3 assays were performed with an YSI 23L electroenzymatic lactate analyzer (Yellow Springs Instruments Co., Ohio, USA). The buffer and the lactate-oxidase enzyme are also contained in the standard
pyruvate + L-glutamate _____ L-alanine + a-oxoglu-
reagent solution (Yellow Springs Instruments Co., Ohio, USA) (26). The blood samples were collected by capillary tubes with
rate
heparin. In the EE3 assay, a solution of octyl-phenoxi-polyethoxiethanol as a hemolysing agent (Triton X- 100, Sigma
The amount of NADH formed in reaction (1) is stoichiometric with the concentration of L-lactic acid (18,24). The electroenzymatic — enzymopolarographic
—
(EE) are based on the linear proportion between the lactate concentration in a sample and the production of hydromethods
gen peroxide (H202) (2,3). When a sample is injected, L-lactate diffuses through a membrane and the following reaction occurs:
(eq. 3) L-lactate + 02
Chemical Co. T-6878) and sodium fluoride (NaF) as a glycolitic
inhibitor was used; 501.11 of this solution and 25 fl blood samples were assayed. The solution was prepared as follows: 25 ml buffer, SOLd Triton X-100, and 0.5mg NaF. For the assay,
blood samples were directly injected into the analyzer: 7 Ill whole blood (EEl), 25 Id whole blood (EE2), 25 Il hemolyzed blood solution (EE3). Readings were direct in all cases, except in EE3, in which case direct reading was multiplied by factor 3 because of the preliminary dilution.
For the comparative study capillary blood
H202 + pyruvate
samples (20 s.d) were obtained by simultaneous and bilateral The oxygen required for the enzymatic oxidation is supplied via an air-permeable membrane used for stirring. The hydrogen peroxide produced diffuses through a second membrane and comes into contact with a polarographic enzyme electrode — platinum anode — yielding the reaction: (eq. 4) H202 ————---* 2H +02+ 2e
This reaction yields a current which is linearly proportional to
the concentration of lactate in the sample. The circuit is completed by a reference cathode. The validity of this method has been tested in comparison with the PHE method used as a
puncture of the hyperhaemic earlobe from eight subjects, before, during, and after exercise. The subjects were athletes from different sports who had been tested for maximal aerobic power and endurance and who had given their informed consent to perform a regular incremental treadmill test in the laboratory
in which blood samples were taken. The sampling technique was as follows: the earlobe was frictioned, disinfected with alcohol, cleaned with distillated water, and wiped with a dry tissue prior to puncturing with a microlancet; the first drop of blood was wiped and then 25 j.il of blood were collected in the capillary tubes.
Quality Control, Precision, Accuracy, and Linearity
reference (2).
Instruments and Reagents
Precinorm S (Boehringer Mannheim, FRG, ref. 125130) and L-lactate standards from 2.39 to 24.40 mmol I-i, prepared by dilution of 1.0 mol 1 standard L-lactate with 0.33 mol . - 1 perchloric acid, were used for quality control of the PHE method. L-lactate 8.0, SOy 15.0 mmol .1-1 standards were used for calibration of EE instruments. All instruments were properly calibrated before and during the measurements accord-
For the PHE assays, a Photometer 4020 (Hitachi/Boehringer Mannheim, Tokyo, Japan) with a wavelength 340 nm filter and a 1 cm light path microcuvette was used. The additional laboratory instruments were semiautomatic fixed volume pipettes (Eppendorf, FRG), disposable capillary tubes (20, 25 y 50 j.tl), and regular material for obtaining capillary ing to the manufacturer's instructions. All assays were perblood samples. The reagent solution — NAD, GPT, LDH and a formed in the same laboratory under similar temperature conbuffer (eq. 1,2) — was the Test Combination Lactate assay for sports medicine (Boehnnger Mannheim, FRG, ref. 1178750). ditions (23±2'C). The blood samples were deproteinized immediately before the For the linearity test, eleven reference standard
assay using perchioric acid 0.33 mol l (Boehringer Mann-
heim, FRG, ref. 125369), and then well mixed and centrifuged for 4 mm at 12,000 rpm. The separated supernatant was immediately assayed or stored up to 72 hours at 4 'C. For the assay,
500 l reagent solution were added to 25 j.tl perchloric acid (sample blank), or 25 jd supematant (sample). Incubation time was 30 mm at room temperature (21—25 'C). One sample blank was used for each assay series.
The EE methods were performed using two different instruments and reagents. For the first technique (EEl), a Micro Stat PLM4 (Analox Instruments Ltd., London,
samples were assayed: 2.39, 4.97, 7.93, 9.90, 11.85, 14.78, 16.71, 17.68, 19.61, 22.48, 24.40mmol1'. All measurements were duplicated, and the mean value was used for the statistical calculations, including the linear regression equation.
Precision and accuracy were tested using L-
lactate reference standard samples — lactate concentrations = 2.39,
4.97, 14.78, and 19.61 nmol11 —,each of them being
assayed 20 times (n = 20). Precision is expressed as the value in percentage of the variation coefficient (VC, %), and accuracy is
expressed as the differences between the mean value for the series and the standard sample's value (duff., %).
Downloaded by: East Carolina University. Copyrighted material.
(eq. 1) L-lactate + NAD
F A. Rodriguez, M. Ban quells, V Pons, F Drobnic, P A. Galilea
464 mt. .1 Sports Med. 13 (1992) Table 1 Precision and accuracy of three blood lactate analytic methods, measured with four L-lactate standards. lAll differences between the mean values are significant (p
A Comparative Study of Blood Lactate Analytic Methods F A. Rodriguez" 2, M. Banquells V Pons, F Drobnic, R A. Galilea1 Centre d'Alt Rendiment (CAR), Departament de Fisiologia i Valoració Funcional, Sant Cugat del Vallés, Barcelona Nacional d'Educació Fisica de Catalunya (INEFC), Departament de Ciències Mediques, Barcelona, Spain
2 Institut
F A. RodrIguez, M Ban quells, V Pons, F Drobnic and P A. Galilea, A Comparative Study of Blood Lactate Analytic Methods. Tnt J Sports Med, Vol 13, No 6, pp 462—466, 1992.
Accepted after revision: April 21, 1992
Three different blood lactate analytic methods were tested for precision, accuracy, linearity, and intermethod comparison: a photoenzymatic assay (PHE), and three electroenzymatic (EE) semiautomatic assays (EE 1,
EE2, EE3). Reference standards and duplicate capillary blood samples from the earlobe were used. Precision and accuracy of the three techniques, when measuring L-lactate standards, were good in the whole range of measurement
(mean variation coefficient, VC = 1.78 — 3.38 %; mean difference = 1.81 —3.38 %). Correlation between the three methods was high (r = 0.9 13 — 0.946), but all three electroenzymatic techniques systematically measured lower values as compared to the PHE tests. The differences ranged from 0.1—1.2 (Smmoll-' PHE level), to 3.4—5.7 (20mmol 1 I PHE level). These differences were drastically reduced when a hemolyser and a glycolitic inhibitor were added to the sample prior to the assay. The measurements obtained in capillary blood by the three techniques are not equivalent.
The differences are partially attributed to the fact that the PHE technique measures total blood lactate, while the EE methods only measure plasmatic-extraerythrocytic lactate. Some regression equations are presented that may be used to convert values measured by the PHE technique, to EE values and vice versa.
Key words _______________________________________ L-lactate, blood lactate, enzymatic analysis, exercise, physiological testing, lactate threshold
of the anaerobic lactic energy sources to intense exercise (14, 21) have increased the demand for these techniques. The use of micro-analytic techniques proved to be an important qualitative step, as they allow the assays with vety small volumes of arterialized capillary blood, obtained by puncture of the earlobe or fingertip (4,9), instead of venous or arterial blood, thus minimizing the trauma to the subject. At the present time, the most widely used micro-techniques for the determination of L-lactate in whole blood are:
(i) Photoenzymatic methods (PHE), based on the photometric determination of NADH increase in the enzymatic reaction lactate-pyruvate. The amount of NADH formed is proportional to the concentration of L-lactate (18,24). (ii) Electroenzymatic — enzymopolarographic
—
methods (EE),
based on the linear proportion between the lactate concentration in a sample and the production of hydrogen peroxide (H202), yielding a current which is registered by a specific electrode (3,4). (iii)Flow injection analysis (FIA) is based on the same principle as the PHE method, that is the reaction between lactate and NAD with the formation of pyruvate and NADH. The trans-
port system of small sample aliquots is based on the controlled dispersion of an injected sample into a continuously moving, nonsegmental carrier or reagent stream (12,23). The increase in NADH is measured fluorometrically. The method has been validated by comparison to colorimetric and manual enzymatic fluorometric methods using microsamples — 25 j.tl — in an exercise laboratory (12). The purpose of this study was to verif' precision, accuracy and linearity of three different blood lactate analytical micromethods very commonly used in exercise laboratories, and to compare their measurements, using both L-lactate
standards obtained by dilution, and blood capillary samples obtained during and after exercise.
Material and Methods Introduction
One PHE and two EE analytical methods — plus a third EE modified method — have been tested and com-
The determination of blood lactate concentration is nowadays a routine test in most exercise laboratories. Far from being of recent interest (7), the consolidation of the con-
cepts relative to the anaerobic threshold (8,25), lactate threshold (5,6,9), and OBLA (10,11), as well as to the contribution Int. J. Sports Med. 13 (1992) 462—466 Georg Thieme Verlag Stuttgart- New York
pared. The test principle and the basic characteristics of each method will be briefly described. The photoenzymatic method (PHE) is based on the photometric determination of NADH increase — absorbance — in the enzymatic reaction lactate-pyruvate. In the presence of
L-lactate dehydrogenase (LDH), nicotinamide-adenine dinucleotide (NAD) oxidizes L-lactic acid (L-lactate) to pyruvate.
Downloaded by: East Carolina University. Copyrighted material.
Abstract _________________________________________
mt. J Sports Med. 13 (1992) 463
A Comparative Study of Blood Lactate Analytic Methods
pyruvate + NADH + H
The equilibrium of this reaction is almost completely on the side of lactate. However, by trapping the pyruvate in a subsequent reaction catalyzed by the enzyme glutamate-pyruvate transaminase (GPT) in the presence of L-glutamate, the equilibrium can be displaced in favour of pyruvate and NADH. (eq. 2)
GB) instrument was used. The reagents used were contained in the standard Lactate solution (Analox Instruments Ltd., London, GB, ref. GMRD 090/091/092), a cocktail containing buffer and the lactate-oxidase enzyme (1). Capillary tubes containing heparin, sodium fluoride, and sodium nitrite were used in
obtaining the blood samples. The EE2 and EE3 assays were performed with an YSI 23L electroenzymatic lactate analyzer (Yellow Springs Instruments Co., Ohio, USA). The buffer and the lactate-oxidase enzyme are also contained in the standard
pyruvate + L-glutamate _____ L-alanine + a-oxoglu-
reagent solution (Yellow Springs Instruments Co., Ohio, USA) (26). The blood samples were collected by capillary tubes with
rate
heparin. In the EE3 assay, a solution of octyl-phenoxi-polyethoxiethanol as a hemolysing agent (Triton X- 100, Sigma
The amount of NADH formed in reaction (1) is stoichiometric with the concentration of L-lactic acid (18,24). The electroenzymatic — enzymopolarographic
—
(EE) are based on the linear proportion between the lactate concentration in a sample and the production of hydromethods
gen peroxide (H202) (2,3). When a sample is injected, L-lactate diffuses through a membrane and the following reaction occurs:
(eq. 3) L-lactate + 02
Chemical Co. T-6878) and sodium fluoride (NaF) as a glycolitic
inhibitor was used; 501.11 of this solution and 25 fl blood samples were assayed. The solution was prepared as follows: 25 ml buffer, SOLd Triton X-100, and 0.5mg NaF. For the assay,
blood samples were directly injected into the analyzer: 7 Ill whole blood (EEl), 25 Id whole blood (EE2), 25 Il hemolyzed blood solution (EE3). Readings were direct in all cases, except in EE3, in which case direct reading was multiplied by factor 3 because of the preliminary dilution.
For the comparative study capillary blood
H202 + pyruvate
samples (20 s.d) were obtained by simultaneous and bilateral The oxygen required for the enzymatic oxidation is supplied via an air-permeable membrane used for stirring. The hydrogen peroxide produced diffuses through a second membrane and comes into contact with a polarographic enzyme electrode — platinum anode — yielding the reaction: (eq. 4) H202 ————---* 2H +02+ 2e
This reaction yields a current which is linearly proportional to
the concentration of lactate in the sample. The circuit is completed by a reference cathode. The validity of this method has been tested in comparison with the PHE method used as a
puncture of the hyperhaemic earlobe from eight subjects, before, during, and after exercise. The subjects were athletes from different sports who had been tested for maximal aerobic power and endurance and who had given their informed consent to perform a regular incremental treadmill test in the laboratory
in which blood samples were taken. The sampling technique was as follows: the earlobe was frictioned, disinfected with alcohol, cleaned with distillated water, and wiped with a dry tissue prior to puncturing with a microlancet; the first drop of blood was wiped and then 25 j.il of blood were collected in the capillary tubes.
Quality Control, Precision, Accuracy, and Linearity
reference (2).
Instruments and Reagents
Precinorm S (Boehringer Mannheim, FRG, ref. 125130) and L-lactate standards from 2.39 to 24.40 mmol I-i, prepared by dilution of 1.0 mol 1 standard L-lactate with 0.33 mol . - 1 perchloric acid, were used for quality control of the PHE method. L-lactate 8.0, SOy 15.0 mmol .1-1 standards were used for calibration of EE instruments. All instruments were properly calibrated before and during the measurements accord-
For the PHE assays, a Photometer 4020 (Hitachi/Boehringer Mannheim, Tokyo, Japan) with a wavelength 340 nm filter and a 1 cm light path microcuvette was used. The additional laboratory instruments were semiautomatic fixed volume pipettes (Eppendorf, FRG), disposable capillary tubes (20, 25 y 50 j.tl), and regular material for obtaining capillary ing to the manufacturer's instructions. All assays were perblood samples. The reagent solution — NAD, GPT, LDH and a formed in the same laboratory under similar temperature conbuffer (eq. 1,2) — was the Test Combination Lactate assay for sports medicine (Boehnnger Mannheim, FRG, ref. 1178750). ditions (23±2'C). The blood samples were deproteinized immediately before the For the linearity test, eleven reference standard
assay using perchioric acid 0.33 mol l (Boehringer Mann-
heim, FRG, ref. 125369), and then well mixed and centrifuged for 4 mm at 12,000 rpm. The separated supernatant was immediately assayed or stored up to 72 hours at 4 'C. For the assay,
500 l reagent solution were added to 25 j.tl perchloric acid (sample blank), or 25 jd supematant (sample). Incubation time was 30 mm at room temperature (21—25 'C). One sample blank was used for each assay series.
The EE methods were performed using two different instruments and reagents. For the first technique (EEl), a Micro Stat PLM4 (Analox Instruments Ltd., London,
samples were assayed: 2.39, 4.97, 7.93, 9.90, 11.85, 14.78, 16.71, 17.68, 19.61, 22.48, 24.40mmol1'. All measurements were duplicated, and the mean value was used for the statistical calculations, including the linear regression equation.
Precision and accuracy were tested using L-
lactate reference standard samples — lactate concentrations = 2.39,
4.97, 14.78, and 19.61 nmol11 —,each of them being
assayed 20 times (n = 20). Precision is expressed as the value in percentage of the variation coefficient (VC, %), and accuracy is
expressed as the differences between the mean value for the series and the standard sample's value (duff., %).
Downloaded by: East Carolina University. Copyrighted material.
(eq. 1) L-lactate + NAD
F A. Rodriguez, M. Ban quells, V Pons, F Drobnic, P A. Galilea
464 mt. .1 Sports Med. 13 (1992) Table 1 Precision and accuracy of three blood lactate analytic methods, measured with four L-lactate standards. lAll differences between the mean values are significant (p
