Modified Enzymatic Methods for the Determination of L-(+)-Lactic and Pyruvic Acids in Blood

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1 Modified Enzymatic Methods for the Determination of L-(+)-Lactic and Pyruvic Acids in Blood J. F. NEVILLE, JR., M.D., AND R. L. GELDER, CH(ASCP) Department of Surgery, Cardiovascular Laboratory, Upstate Medical Center, State University of New York, Syracuse, New York ABSTRACT Neville, J. F., Jr., and Gelder, R. L.: Modified enzymatic methods for the determination of L-(+)-lactic and pyruvic acids in blood. Amer. J. Clin. Path. 55: , Modifications of the enzymatic methods of analyses for L-(+)- lactic and pyruvic acids in whole blood result in average recoveries of 99% for lactate and 100% for pyruvate. The modified lactate procedure facilitates the handling of a large number of analyses. The pyruvate blood recovery studies demonstrated that proper centrifugation following deproteinization is a prime determinant for achieving 100% recovery from whole blood. The importance of technic in performing recovery experiments is emphasized. ENZYMATIC METHODS for analyses of L-(+)- relationship: lactic 3 > * a ' 8 ' 13 " 15 and pyruvic *> 2 < 5 " 7 ' 12 >"- 1B LDH acids in biologic materials have been described Lactate + NAD+ * NADH + H+ + pyruteva by various authors. In our investiga tions of anaerobic metabolism in dogs, we encountered the following problems in analyzing whole blood for these metabolites: nonlinear results were obtained with pyruvate standards; the pyruvate analyses failed to yield quantitative recoveries; the analytical range for lactate was inadequate for the levels to be measured; when large numbers of lactate analyses were to be performed, the time required for multiple pipettings and spectrophotometric readings became prohibitive. In the lactate analysis, conducted at an alkaline ph, the original pyruvate present is removed as the hydrazone or semicarbazide. Subsequent addition of LDH drives the reaction to the right, and continued removal of the pyruvate results in all of the lactate's being consumed, with an equimolar generation of NADH from NAD\ In the pyruvate analysis, by using a neutral ph, the addition of LDH converts all of the pyruvate to lactate with an equimolar loss of NADH. By measuring the change in NADH concentration We have modified the basic technics to spectrophotometrically at 340 obviate these problems. nm., the corresponding lactate and pyruvate concentrations can be calculated. Materials and Methods Preliminary Remarks Principle Enzymatic methods for lactate:pyruvate determinations depend upon the following If blood is allowed to stand for a short time, changes in pyruvate and lactate will occur due to glycolytic activity of the erythrocytes. 10 Therefore, free flow of blood into Received January 14, 1970; accepted for publication May 5, chilled perchloric acid is the preferred Supported by USPHS Grant HE method of sampling. 152

2 February 1971 DETERMINATION OF LACTIC AND PYRUVIC ACIDS 153 Our results are expressed as millimoles per liter of blood water, 10 which is calculated from the change in weight of a sample of blood following heating at 105 C. overnight. To compensate for changes in absorbance due to factors other than the enzyme-induced changes in NADH, a reagent blank of water and perchloric acid is utilized in both analyses. Centrifugation was carried out in an International Model 2K centrifuge with an I.E. 811 head at 4,200 X g. Spectrophotometry readings were taken with a Beckman DU monochromator, equipped with a Gilford Instrument Co. Model 220 Optical Density Converter (wavelength, 340 nm.; light path, 1 cm.). Reagents for Lactate Analysis Perchloric acid: (Allied Chemicals, 70%) ca. 6%. Dilute 50 ml. of 70% HC10 4 to 1 liter. Glycine buffer: (Fisher Scientific, mol. wt. = 75.07). 0.5 M glycine, ca. 0.4 M hydrazine (as the hydrate, Arthur H. Thomas, mol. wt. = ), and M EDTA (as the disodium salt, dihydrate; Sigma, 99.5%, mol. wt. = ), final ph 9.5. Dissolve Gm. of glycine and 2.0 Gm. EDTA in ca. 500 ml. water. Add 20 ml. of hydrazine hydrate and mix; add 100 ml. of 2 N NaOH, mix, and dilute to 1 liter. The buffer is stable for 7 to 10 clays if kept in a refrigerator. LDH: (Sigma Chemical, Type III). Dilute the enzyme with 2.1 M (ca. 33%) ammonium sulfate to a final concentration of at least 3,000 units.t Nicotinamide adenine dinucleotide, beta form (Sigma Chemical, Grade III from yeast, mol. wt. = anhydrous). KOH: ca. 5 M. Dissolve Gm. of KOH and make up to 250 ml. Zinc lactate: (Mann Research Laboratories; L-(+)-lactic acid, mol. wt. = Grams o blood X % blood water per 100 = ml. of blood water. f International Units = mg. per ml. protein X activity (jumoles per min.). 11 without 12.97% water of hydration), 20.0 mm per 1. stock lactate solution. Dried zinc lactate, 887 Gm., is dissolved in, and diluted with, 6% HC10 4 to 250 ml. Standard solutions: 1.0 to 6.0 mm per 1. lactate. Make appropriate dilutions of the stock solution with 6% HC10 4. Reagents for Pyruvate Analysis Perchloric acid: same as described above. Triethanolamine (TEA) buffer: (Nutritional Biochemicals Corp., mol. wt. = , the hydrochloride); 0.4 M TEA, 0.004M EDTA; final ph 7.4. Dissolve 18.6 Gm. of TEA in ca. 200 ml. of water; 12.0 ml. of 2 N NaOH is added to this and mixed. Add 0.37 Gm. of EDTA-N 2 H 2-2 H 2 0, mix, and dilute to a final volume of 250 ml. The buffer is stable if kept in a refrigerator. NADH: (Sigma, disodium salt, Grade III, ca. 98%, mol. wt. = in anhydrous form). Dissolve either 1.4 mg. per ml. or 5.0 mg. per ml. in TEA buffer. Make fresh daily. LDH (Sigma Chemical, Type III): dilute the enzyme with 2.1 M ammonium sulfate to yield a minimum of ca. 2,500 units.t store in a refrigerator; during analysis, keep in an ice bath. KOH: same as described above. Sodium pyruvate: (Sigma, Type II, mol. wt. = 110.0); 2.0 mm per 1. stock pyruvate solution. Sodium pyruvate, 22.0 mg., is dissolved in, and diluted with, 6% HC10 4 to 100 ml. Standard solutions: 0.1 to 0.6 mm per 1. pyruvate. Make appropriate dilutions of the stock pyruvate solution with 6% HC10 4. All reagents used were of analytical grade; all solutions were made with "Water for Parenterals" (Abbott Laboratories) unless otherwise stated. Procedure A) Standard Preparation. Standard solutions of both lactate and pyruvate were diluted with water and 6% HC10 4 (1:1:3)

3 154 NEVILLE AND GELDER A.J.C.P. Vol. 55 to give a 1/5 dilution of standard to total volume. A reagent blank of water and 6% HC10 4 (1:4) was also prepared. The 5.0-ml. aliquots of standards and blank were subsequently chilled in an ice bath. B) Blood Deproteinization. Blood samples were obtained by allowing about 2 ml. of arterial blood to flow into preweighed centrifuge tubes containing 5.0 ml. of chilled 6% HCIO4, similar to the methods of Hohorst 8 and Bucher and associates. 2 After the denatured blood and perchlorate were equilibrated at room temperature, the tubes were reweighed and 6% HC10 4 was added X to give an approximate ratio of blood water to total volume of 1/5. The tubes were centrifuged at 4,200 x g. for 15 min., and the supernatants were decanted and recentrifuged for an additional 10 min. From these supernatants, 5.0 ml. aliquots were taken and chilled in an ice bath. C) Partial Neutralization. A predetermined amount of 5 M KOH was added to the chilled standard, blank, and the blood supernatants to yield ph values of 1.6 to 2.5. These were mixed on a vortex and centrifuged. While still chilled, the supernatants were decanted into tubes, with care so as not to resuspend and carry over the KCIO4 precipitate. The supernatants were allowed to warm to room temperature. D) Reaction Mixture for Lactate Analysis. Depending upon the expected number of analyses, a reaction mixture was prepared just prior to use, as follows: 3.0 ml. of glycine buffer X number of analyses 4.0 mg. of NAD+ X number of analyses 10.0 units of LDH X number of analyses ^Assuming 80% blood water, to achieve a 1:5 dilution of blood water:perchlorate, multiply 0.8 X 4 x weight o blood. This is the total amount of 6% HCIO, needed. The total ml. of perchlorate less original 5.0 ml. = ml. of perchlorate to be added. The exact amounts necessary in the above have to be determined for each batch of 6% HClOj and KOH. This is usually about 0.56 ml. for standards and blank, and about 0.46 ml. for blood supernatants. E) Lactate Analysis. 0.3-ml. amounts of the partially neutralized supernatants (step C) were pipetted into test tubes. To each was added 3.0 ml. of the freshly prepared reaction mixture. The solutions were mixed and incubated at 37 C. for 1 hr. After remixing, the contents of each unknown and standard were transferred to a cuvette and the absorbance (E 2 ) measured. The absorbance of the blank (E,) was remeasured with each set of unknowns. All analyses, including the blank, were done in duplicate. F) Pyruvate Analysis. The following were pipetted H into 10 by 40 mm. cuvettes in the given order: 2.0 ml. of supernatant (step C) 1.0 ml. of TEA buffer 0.1 ml. of NADH solution The contents of the cuvettes were mixed with a stirring rod I and the Ej^ absorbance read after about 2 min. The readings were repeated until a stable value was obtained. The reaction was started by the addition and mixing of 0.01 ml. of LDH in each cuvette. Starting about 3 to 4 min. after the addition of LDH, the E 2 absorbance was read until a stable value was achieved. The reagent blank was analyzed in order to determine the absorbance change due to the dilution by LDH of the Et value. This is subtracted from the AE of the unknowns and standards to eliminate a systematic error in calculations. G) Calculations. mm lactate: pyruvate per liter = AE X K where: lactates Ei = absorbance of reagent blank Ej = absorbance of the unknown AE = E2 Ei _, dilution factor K O^d pyruvates Ei = absorbance before addition of LDH E2 = absorbance after addition of LDH AE = (Ei E2)-correction of blank fl The volumetric additions of NADH and LDH were carried out using Hamilton Co. Microliter syringes with Chaney adaptors. Polypropylene cell stirring rod with flared end, Kopp Scientific, Inc.

4 February 1971 DETERMINATION OF LACTIC AND PYRUVIC ACIDS 155 and: e = extinction coefficient of NADH at 340 nm. (6.22 sq. cm. per /am). 9 d = light path of the cuvette, 1 cm. dilution factor = reciprocal of all dilutions. X* 9.0 mm/1.standard Example of the Pyruvate Calculations 1) Original volume of HClOi 2) Weight of blood added 3) Total HC10 4 = 0.8 X 4 X ) Total volume = (0.8 X 3.000) 5) Blood water dilution = 2.4/12.0 6) Neutralization dilution 7) Analysis dilution = ( ) 8) K = 5/1 X 5.46/5 X 3.11/2 X 1/6.22 = 5.0 ml. = Cm. = 9.6 ml. = /5 = 5.0/5.46 (blood) = 5.0/5.56 (standard) = 2.0/3.11 = 5.45:4 = for a blood factor. 9) Upon analysis: Ei = = absorbance units Ej = = absorbance units Z m O 'SI (0 < C.400 AE = = absorbance units and correction of blank = absorbance units corrected AE = absorbance units 10) Corrected AE X factor = mm pyruvate per 1. blood water X = mm pyruvate per 1. blood water. Quality Control: The percentage recovery of the standards, which were analyzed in conjunction with the blood unknowns, was the quality control of the procedures. m mm of lactate/pyruvate obtained... % recovery = %r,, '-^- ; X 100 mm of lactate/pyruvate expected Conversion of mm to mg. per 100 ml. mg. per 100 ml. pyruvate X mm per 1. pyruvate. mg. per 100 ml. lactate X mm per 1. lactate. Results Lactates Zinc lactate standards (0.5 to 6.0 mm per 1. lactate before dilution) in 6% HC10 4 yielded an average recovery of 100.1%, (N = 93, SD = 3.1%). In blood recovery studies, blood was added to perchlorate containing known amounts of lactate; an average recovery of 99.0% (N = 24, SD = 2.6%) was obtained. i_l TIME 1.0 mm/1.standard FIG. 1. Rate of absorbance change in the reagent blank and lactate standards (9.0 mm and 1.0 mm) vs. time. Partially neutralized supernatant (0.3 ml.; ph 1.6 to 2.5 with 5 M KOH) plus 3.0 ml. of reaction mixture. When lactate is the only metabolite to be analyzed, the deproteinization ratio of blood water to the total volume may be adjusted to a smaller ratio than used here, so long as the amount of KOH added is adjusted to maintain the ph of the supernatant in the range of 1.6 to 2.5 prior to addition of the reaction mixture. We have used 0.1 to 0.5-ml. aliquots of supernatant in the method described, as well as de-

5 156 NEVILLE AND GELDER A.J.C.P. Vol. 55 Table 1. Percentage Recoveries of Pyruvate Standards in 6% HC10 4 * mm per 1. 5 M K 2 C0 3 5 M K 3 PO, 5 M KOH * Standard supernatants partially neutralized to ph values of 1.6 to 2.5 with various 5 M K + reagents. proteinization ratios of both 1/5 and 1/4, without bias in recovery or loss of linearity. We found from time studies that by using sufficient excess LDH (> 15 units per reaction mixture tube), the analysis could be completed within an incubation period of an hour for lactate concentrations as high as 9.0 mm per 1. (Fig. 1). Using hydrazine sulfate instead of the hydrate or decreasing the molarity of the hydrazine by half did not appreciably decrease the absorbance of the reagent blank. However, when the NAD + concentration was halved, the recovery of the higher concentrations of lactate was suppressed. Pyruvates Utilizing the method described, sodium pyruvate standards (0.1 to 0.6 mm pyruvate before dilution) in 6% HCIO4 yielded an average recovery of 100.2% (N = 123, SD = 1.4%). In blood recovery studies, blood was added to perchlorate containing known amounts of pyruvate; the average recovery was 100.1% (N = 26, SD = 2.6%). Standards. When either 5 M K 3 P0 4 or 5 M K 2 CO s was used for neutralization, the percentage recoveries of the standards decreased as the concentration of the pyruvate increased (Table 1). When we used 0.04 M EDTA in the 0.4 M TEA buffer, 2 the percentage recoveries of the pyruvate standards decreased as the concentrations of standards increased (Table 2). Using 0.4 M TEA without EDTA, 0.4 M TEA with M EDTA, and 0.4 M Tris" linear and quantitative recoveries of standard were obtained, provided that the ph of the supernatant after addition of KOH was in the range of 1.6 to 2.5. B'.ood Recoveries. When recovery experiments used pyruvate added to plasma or to deproteinized supernatants, 100% recovery was achieved routinely. Addition of the pyruvate to the HCIO4 used for deproteinization resulted in only 80 to 90% recovery, which could not be improved by different methods of deproteinization It was noted that the radial centrifugal force of our centrifugation was lower than recommended. 2 Subsequent centrifugation of the blood perchlorate mixture at 4,200 X g. or more corrected this problem. Re-examination of K 3 P0 4 vs. KOH for use in the partial neutralization step of analyzing blood supernatants was undertaken. Recovery of pyruvate utilizing K 3 P0 4 was in the 80 to 90% range, whereas " Tris = 2-amino-2-(hydroxymethyl)-l,3-propancdiol, Eastman Organic. Table 2. Percentage Recoveries of Pyruvate Standards in 6% HC10V mm per M TEA 0.04 M EDTA 0.4 M TEA M EDTA 0.4 M TEA 0.4 M Tris * Standard supernatants partially neutralized with 5 M KOH to ph values of 1.6 to 2.5 and analyzed utilizing various buffers.

6 February 1971 DETERMINATION OF LACTIC AND PYRUVIC ACIDS 157 FIG. 2. Recovery of pyruvate from blood after partial neutralization of blood supernatants with either 5 M K 3 PO< or 5 M KOH. KOH neutralization yielded an average recovery of 100%; K 3 P0 4 neutralization yielded 80 to 90% recovery rail Pyruvate Expected 100% recovery was achieved utilizing KOH (Fig. 2). Discussion Lactates The reaction mixture technic for lactate analysis was developed primarily because of the large numbers of samples which we were analyzing and the extended range of analysis that we needed. Validity of the reaction mixture is predicated upon the assumption that the reagent blank (containing NAD-LDH-hydrazine, with an absorbance in itself) changes at the same rate as in the unknown sample, independent of the absorbance change due to the conversion of NAD + to NADH (Fig. 1). Lundholm and colleagues 13 observed that as the lactate concentration in the cuvette increased, the ph of the reaction solution fell, due to an increase of H + ions as the lactate was oxidized to pyruvate. This subsequently led to a difference between the absorption of the blank and that of the unknown, giving high recoveries. We have not observed this phenomenon, perhaps because we partially neutralize the supernatant prior to analysis and include EDTA in the buffer. For lactates, the enzyme and coenzyme are in excess. The procedure as described permits large A absorbance per mm of lactate (resulting from a small dilution factor), thereby minimizing analytical errors. We have only suggestive evidence that recovery of lactate from blood is dependent upon high centrifugation speeds. Hohorst 8 mentions a minimum of 3,000 X g. Pyruvate The original discrepancy between recoveries when pyruvate was in the perchlorate used to precipitate protein and when pyruvate was added to the protein-free supernatant emphasizes the need for proper controls in evaluating an analytical method. It

7 158 NEVILLE AND GELDER A.J.C.P. Vol. 55 is not valid to assume that the supernatant contains all of the original substrate just because substrate added to the supernatant can be recovered quantitatively. Closter and Harris, 5 and Landon and associates 12 have offered the view that low recoveries, or inability to recover pyruvate added to the perchloric acid prior to denaturization, were due to the inability to deproteinize the blood completely and thereby inactivate the LDH. We consistently had quantitative recoveries of pyruvate added to blood supernatants which would not have been possible with residual LDH present. Our studies indicate that the need for centrifugation at high radial centrifugal force cannot be overemphasized as part of the pyruvate analysis. This may be the reason that Gloster and Harris 5 failed to obtain accurate blood recoveries when perchloric acid was used as the deproteinizing agent. Using 5 mg. per ml. of NADH in the pyruvate analysis, the E x value lies between and absorbance units, permitting a wide range of pyruvate to be analyzed. The small dilution factors provide a large A absorbance per mm pyruvate and increase the accuracy of a given determination. For those spectrophotometers in which accuracy of reading above a absorbance is a factor, the concentrations of NADH for pyruvate analysis and NAD+ for lactate analysis must be decreased. The dilution of supernatants will have to be increased to encompass the same range of unknowns. Acknowledgment. Mr. Donell Bacon and Mrs. Anna Marie Fehling gave generous interest and technical assistance. References 1. Biicher Th. et al.: Unpublished investigations. In pamphlet "Enzymatic determination of the pyruvate concentration in blood. ETC-C973, 1.5. MGD 1. Biochemica Boehringer, C. F. Boehringer & Soehne, Mannheim, West Germany, Biicher T, Czok R, Lamprecht W, et al.: Pyruvate, Methods of Enzymatic Analysis. Edited by HU Bergmeyer. Academic Press, 1963, pp Friedland IM, Dietrich LS: A rapid enzymic determination of L-(+)-lactic acid. Anal Biochem 2: , Gercken G: Die quantitatin enzymatisiche Dehydrierung von L-(+)-Lactate fur die Mikroanalyse. Z Physiol Chem 320: , Gloster JA, Harris P: Observation on an enzymic method for the estimation of pyruvate in blood. Clin Chim Acta 7: , Hadjivassiliou AG, Rieder SV: The enzymatic assay of pyruvic and lactic acids. A definitive procedure. Clin Chim Acta 19: , Hess B: Bererkungen zur Bestimmung der Brenztraubensaure im menschlichen Serum. Klin Wschr 33: , Hohorst HJ: L-(+)-lactatc determination with lactic dehydrogenase and DPN, Methods of Enzymatic Analysis. Edited by HU Bergmeyer. Academic Press, 1963, pp Horecker BL, Romberg A: The extinction coefficients of the reduced band of pyridine nucleotides. J Biol Chem 175: , Huckabee WE: Control of concentration gradients of pyruvate and lactate across cell membranes in blood. J Appl Physiol 9: , King EJ, Campbell DM: International enzyme units an attempt at international agreement. Clin Chim Acta 6: , Landon J, Fawcett JK, Wynn V: Blood pyruvate concentration measured by a specific method in control subjects. J Clin Path 15: , Lundholm L, Mohmc-Lundholm E, Vamos N: Lactic acid assay with L-(+)-lactic acid dehydrogenase from rabbit muscle. Acta Physiol Scand 58: , Marbach EP, Weil MH: Rapid enzymatic measurement of blood lactate and pyruvate. Clin Chem 13: , Rosenberg JC, Rush, BF: An enzymatic-spectrophotometric determination of pyruvic and lactic acid in blood. Clin Chem 12: , 1966