- Email: [email protected]
Interference and blood sample preparation for a pyruvate enzymatic assay
Clinical Biochemistry 39 (2006) 74 – 77
Interference and blood sample preparation for a pyruvate enzymatic assay Chih-Kuang Chuang a,d,1 , Tuen-Jen Wang b,1 , Chun-Yan Yeung c,e , Wen-Shyang Hsieh b , Dar-Shong Lin c,e , Shinn-Chamg Ho b , Shuan-Pei Lin a,c,e,⁎ a
Division of Genetics and Metabolism, Department of Medical Research, Taipei 10449, Taiwan b Department of Laboratory Medicine, Mackay Memorial Hospital, Taipei, Taiwan c Department of Pediatrics, Mackay Memorial Hospital, Taipei, Taiwan d College of Medicine, Fu-Jen Catholic University, Taipei, Taiwan e Mackay Medicine, Nursing and Management College, Taipei, Taiwan Received 17 June 2005; received in revised form 10 August 2005; accepted 14 October 2005 Available online 23 November 2005
Abstract Background: To assess the severity of circulatory failure, a pyruvate enzymatic assay was performed on whole blood using lactate dehydrogenase to catalyze the conversion of pyruvate to lactate. We investigated factors related to blood sample collection and preparation that might influence the results, including the timing of blood deproteinization, temperature of sample storage, and hemolysis. Method: A total of 25 whole blood specimens were collected for this study. Each sample was divided into 2 parts: one stored at room temperature (RT) and another kept on ice. The samples were deproteinizied by using 8% perchloric acid (PCA) at varying times after collection; the first deproteinization was immediately after the blood was drawn (0 h), then at 1 h intervals for 6 h and also in samples kept overnight. The supernatant samples were analyzed soon after deproteinization using a COBAS Centrifugal Analyzer. In another set of samples, the blood was immediately deproteinized, and the supernatants were stored at RT and 4°C and assayed for pyruvate at varying times, as above. Finally, the effect of hemolysis on the blood pyruvate enzymatic assay was also evaluated. Results: When samples were stored at RT, pyruvate levels remained constant until the third h after deproteinization, when there was an approximately 13.3% increase in pyruvate concentration. When whole blood samples were kept at 4°C before deproteinization, pyruvate levels were significantly reduced over time, ranging from 37.8% to 62.2% (paired t test showed a significant mean difference, P b 0.001). No significant differences in pyruvate concentration were observed in supernatant stored at either RT or 4°C. Hemolysis caused a 33.7% increase in the pyruvate concentration, equivalent to 0.18 mg pyruvate per gram per deciliter of hemoglobin. Conclusions: For a pyruvate enzymatic assay, keeping a whole blood sample at RT will not cause a significant difference in the pyruvate level as long as the sample is immediately deproteinized. Whole blood samples should not be stored in an ice bath for transport, nor should hemolyzed samples be used for a blood pyruvate enzymatic assay. © 2005 The Canadian Society of Clinical Chemists. All rights reserved. Keywords: Pyruvate; Deproteinization; Hemolysis; Centrifugal analyzer; Specimen stability
Introduction Pyruvate is the end product of glycolysis in cells with mitochondria in the presence of an adequate supply of oxygen. Pyruvate and lactate levels are used as biochemical indices of the Abbreviations: LDH, lactate dehydrogenase; RT, room temperature; PCA, perchloric acid. ⁎ Corresponding author. Division of Genetics and Metabolism, Department of Medical Research, MacKay Memorial Hospital, No. 92, Sec. 2, Chung-San N. RD., Taipei 10449, Taiwan. Fax: +886 2 253433642. E-mail address: [email protected] (S.-P. Lin). 1 The first two authors contributed equally to this work.
severity of circulatory failure [1,2]. Increased blood pyruvate levels are reported in a number of disorders including shock, liver disease, congestive heart failure, diabetes mellitus, muscular dystrophy, thiamine deficiency, and neoplastic disorders. An enzymatic method to assay pyruvate uses lactate dehydrogenase (LDH) with NADH as a cofactor to convert pyruvate to lactate, with the formation of NAD+ being proportional to the concentration of pyruvate [3–5]. This method is assayed either by using a manual procedure or an automatic COBAS-MIRA Centrifugal Analyzer (Roche Diagnostic, Montclair, NJ, USA) [6,7]. Whole blood is considered the best specimen for assessing physiological levels of pyruvate. Nevertheless, many factors
0009-9120/$ - see front matter © 2005 The Canadian Society of Clinical Chemists. All rights reserved. doi:10.1016/j.clinbiochem.2005.10.007
C.-K. Chuang et al. / Clinical Biochemistry 39 (2006) 74–77
affect the rate of pyruvate formation. There have been few reports of analytical variables that may influence the results from this assay [7–9]. We designed this study to examine the effect of three such factors on the results of the assay, including the timing of deproteinization vis-à-vis sample analysis, sample storage temperature, and hemolysis. Materials and methods Specimens Blood samples (4.5 mL) were collected in EDTA from 25 healthy adults (14 males and 11 females; 22 to 46 years of age) in the resting state after a minimum 12 h fast. Samples that were hemolyzed when they arrived in the laboratory were excluded. All subjects gave signed informed consent. Each sample was divided into two aliquots: one stored on ice and one kept at room temperature (RT). Sample deproteinization The samples were deproteinized by a method described previously [10] by mixing blood and 8% perchloric acid (PCA 69% to 72%; J.T. Baker, Phillipsburg, NJ, USA) in a 2:1 ratio with a vortex mixer for about 30 s. The mixture was kept cold for an additional 5 min to ensure complete protein precipitation. This procedure was performed on samples stored both at RT and 4°C immediately after the blood was drawn (0 h), at 1 h intervals after drawing for 6 h, and finally on samples stored overnight. In each case after immediate or delayed deproteinization, samples were centrifuged for 10 min at approximately 1500 × g, and the supernatant was collected for analysis. In addition to the above series of samples treated with delayed deproteinization, another series was immediately deproteinized and the supernatants were then stored at either RT or 4°C for varying intervals, as described above for the whole blood samples, after which the pyruvate assay was performed.
75
Roche Diagnostic (Montclair, NJ, USA). The parameters of COBAS-MIRA settings for the pyruvate assay in whole blood filtrates were programmed according to the instrument settings offered by Instruchemie. The Start Reagent 1 (R1) and Reagent 2 (R2) were β-NADH and LDH, respectively. The principle of pyruvate enzymatic assay is an endpoint method. In the presence of excess NADH, substantially all pyruvate is converted to lactate. The reduction of absorbance at 340 nm due to oxidation of NADH to NAD becomes a measure of the amount of pyruvate originally present. Results The within-run and between-run precisions (CV%) of the pyruvate enzymatic assay were investigated by using the pyruvate/lactate control purchased from Instruchemie (Delfzijl, The Netherlands). The pyruvate mean value equals 1.69 mg/dL (0.19 mmol/L) and the range is from 1.24 to 2.13 mg/dL (0.14 to 0.24 mmol/L). Ten control samples were sequentially assayed. The within-run and between-run precisions were 3.7% and 4.3%, respectively. Analytical recoveries of pyruvate added (0.089–0.89 mg/dL) to blood samples (n = 20) ranged from 95.9% to 99.1% (average, 97.3%). The sensitivity of the pyruvate enzymatic method is as low as 0.1 mg/dL. In whole blood samples kept at RT, the pyruvate levels were slightly decreased (by about 6.7%) during the first 2 h of delayed deproteinization, followed by a slight increase in the 3rd through 6th hours and overnight. The magnitude of the increases ranged from 13.3% to 4.4%, with the highest at the 3rd hour. However, the pyruvate levels at each hour of delayed deproteinization were not statistically significantly different compared to the result after immediate deproteinization. In samples kept at 4°C before deproteinization, pyruvate levels were markedly reduced, ranging from 37.8% to 62.2% lower than those in samples maintained at RT, and a paired t test showed significant differences between the results obtained from samples stored at RT and those stored at 4°C (P b 0.001) (Fig. 1).
Hemolyzed sample preparation A total of 10 blood samples (about 5 mL for each sample) were used in this part of the study. Hemolyzed samples were prepared by using the cell lysis method reported by Yulcel and Dalva [11]. Blood samples (2 mL) were hemolyzed by stirring with a metal bar for 1 or 2 min. The degree of hemolysis was determined by automated plasma hemoglobin measurement (Gen. S System, Beckman-Coulter, Fullerton, CA, USA). The remaining 3 mL from each blood sample without hemolysis was used for control. The samples were then deproteinizied as described above. Assay procedure A commercial pyruvate kit was purchased from Instruchemie (Delfzijl, The Netherlands) that contained pyruvate standard, control, Tris-buffer reagent (stabilizers), β-NADH, and LDH reagent. The COBAS-MIRA centrifugal analyzer was from
Fig. 1. Comparisons of pyruvate levels in whole blood stored at RT or in an ice bath, and supernatants stored at RT or at 4°C immediately after the blood was drawn (0 h), at 1 h intervals after drawing for 6 h, and finally on samples stored overnight (paired t test showed a significant mean difference between whole blood stored at RT or in an ice bath (P b 0.001), as well as between whole blood and supernatant stored at RT (P b 0.005); no significant mean difference was found between supernatants stored at RT or at 4°C).
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C.-K. Chuang et al. / Clinical Biochemistry 39 (2006) 74–77
In the supernatant stored at RT and 4°C for varying intervals after immediate deproteinization, the pyruvate was slightly reduced at 1 h and then remained constant from the 2nd hour to the end. The supernatant stored at RT was reduced by 15.6% and that at 4°C by 11.1%, with no statistically significant difference between them (Fig. 1). There was a significant difference by a paired t test between the results for whole blood and for supernatant stored at RT (P b 0.005). For overall comparison of the results after delayed deproteinization with those after immediate deproteinization, there was a significant correlation only in the supernatants stored in RT (r = 0.753 to 0.871), and the whole blood sample stored at RT at 1 h after delayed deproteinization (r = 0.697). The averaged pyruvate levels on non-hemolytic and hemolytic samples were 0.424 (±0.088) and 0.567 (±0.127) mg/dL, respectively. The mean degree of hemolysis was 0.9 g Hb per deciliter (0.3–1.4 g/dL). Hemolysis resulted in a mean increase in the pyruvate level of 29.7% (±15.8). The magnitude of increase was equivalent to 0.1782 mg pyruvate per gram per deciliter of hemoglobin. In an overall comparison with the original data, the correlation was significant (P = 0.011; r = 0.728). There was a significant difference in a paired t test between the hemolyzed and non-hemolyzed samples (P b 0.001). Discussion Lactate and pyruvate are physiological blood components, the levels of which reflect the equilibrium between their cytoplasmic production from glycolysis and their consumption by various tissues. That is, the blood lactate and pyruvate concentrations are affected by the rate of production as well as the rate of metabolism. These substances indicate the severity of circulatory failure and the state of tissue oxidation. Pyruvate in the blood is extremely unstable, so careful attention must be paid to blood sample preparation and storage to avoid errors in the results. According to the literature, if blood samples are not treated immediately, lactate will increase rapidly as a result of glycolysis, whereas the blood pyruvate will be significantly reduced [12,13]. This explains why we wanted to investigate the influence of delayed deproteinization on the results of a blood pyruvate enzymatic assay. Delays in processing are frequent because of problems in transporting the samples to the laboratory or from hemolysis resulting from improper venipuncture technique or handling, particularly for pediatric patients. It may be that correction factors can be derived to help reduce the magnitude of laboratory error. In our study, immediate deproteinization did not appear to be necessary, as delay resulted in only slight decreases in the measured pyruvate level. Maintaining a blood sample at RT also resulted in only insignificant differences. It appears that whole blood samples should not be iced before deproteinization. If the sample is immediately deproteinized, however, the supernatant should be kept at 4°C rather than at RT. According to another report, pyruvate in metaphosphoric acid filtrates of blood remains stable for 6 days at RT and for 8 days at 4°C [14]. Hemolysis influences most biochemical test values, either because it dilutes the plasma or releases intracellular constitu-
ents into plasma from erythrocytes. The errors can be slight or severe depending on the relative concentrations of erythrocyte constituents [11,15]. The measuring error can be avoided if the effect of hemolysis on a particular test is known. Red blood cells use anaerobic glycolysis for energy delivery and contain large amounts of LDH [11,16]. Hemolysis causes a false increase in the pyruvate concentration due to LDH release, thus accelerating the reaction between pyruvate and lactate. The optimum concentrations of LDH and its effects on blood lactate and pyruvate enzymatic measurement have been investigated by Marbach and Weil [3]. The magnitude of the change in pyruvate is not directly proportional to the degree of hemolysis. The reference interval for whole blood pyruvate concentration ranges from 0.3 to 0.7 mg/dL [7,14]. Given this relatively small amount in the blood, it seems likely that hemolysis could introduce a substantial error in measurement. The cell lysis method we used was intended to mimic the mechanical disruption of erythrocytes which typically happens when venipuncture is difficult or the sample is improperly handled. Improper handling or storage of blood samples and sample hemolysis will affect the results of a blood pyruvate enzymatic assay. In conclusion, whole blood samples kept at room temperature are acceptable, as long as the sample is immediately deproteinized; whole blood sample should not be stored in an ice bath for transport prior to deproteinization; and hemolyzed samples are not acceptable for a blood pyruvate enzymatic assay.
Acknowledgments We are grateful to the Mackay Memorial Hospital (MMH # 9330) and the National Science Council, Taiwan (NSC-922314-B-195-032) for supporting research grants and necessary instrumentations for this study. In addition, we would like to express our sincere thanks to Dr. Mary Jeanne Buttrey for her revision of this article. References [1] Huckabee WE. Relationships of pyruvate and lactate during anaerobic metabolism: I. Effects of infusion of pyruvate or glucose and of hyperventilation. J Clin Invest 1958;37(2):244–54. [2] Huckabee WE. Relationships of pyruvate and lactate during anaerobic metabolism: II. Exercise and formation of O-debt. J Clin Invest 1958;37 (2):255–63. [3] Marbach EP, Weil MH. Rapid enzymatic measurement of blood lactate and pyruvate. Use and significance of metaphosphoric acid as a common precipitant. Clin Chem 1967;13(4):314–25. [4] Neville Jr JF, Gelder RL. Modified enzymatic methods for the determination of L-(+)-lactic and pyruvic acids in blood. Am J Clin Pathol 1971;55(2):152–8. [5] Artuch R, Vilaseca MA, Farre C, Ramon F. Determination of lactate, pyruvate, beta-hydroxybutyrate and acetoacetate with a centrifugal analyser. Eur J Clin Chem Clin Biochem 1995;33(8):529–33. [6] Bonora R, Pagani F, Panteghini M. Pyruvate measured in whole blood with the Cobas Bio analyzer. Clin Chem 1989;35(2):325. [7] Hansen JL, Freier EF. Direct assays of lactate, pyruvate, beta-hydroxybutyrate, and acetoacetate with a centrifugal analyzer. Clin Chem 1978; 24(3):475–9.
C.-K. Chuang et al. / Clinical Biochemistry 39 (2006) 74–77 [8] Panteghini M, Pagani F. Biological variation of lactate and pyruvate in blood. Clin Chem 1993;39(5):908. [9] Chariot P, Ratiney R, Ammi-Said M, Herigault R, Adnot S, Gherardi R. Optimal handling of blood samples for routine measurement of lactate and pyruvate. Arch Pathol Lab Med 1994;118(7):695–7. [10] Segal S, Blair AE, Wyngaarden JB. An enzymatic spectrophotometric method for the determination of pyruvic acid in blood. J Lab Clin Med 1956;48(1):137–43. [11] Yucel D, Dalva K. Effect of in vitro hemolysis on 25 common biochemical tests. Clin Chem 1992;38(4):575–7. [12] Alderman JA, Cross RE. Adaptation to the centrifugal analyzer of an
[13]
[14] [15] [16]
77
enzymatic method for the measurement of lactate in plasma and cerebrospinal fluid. Clin Chem 1977;23(10):1917–20. Chariot P, Ratiney R, Ammi-Said M, Herigault R, Adnot S, Gherardi R. Optimal handling of blood samples for routine measurement of lactate and pyruvate. Arch Pathol Lab Med 1994;118(7):695–7. Caraway WT, Watts NB. Carbohydrates. In: Tietz NW, editor. Textbook of Clinical Chemistry. Philadelphia, PA: W.B. Saunders; 1986. pp. 814–8. Brydon WG, Roberts LB. The effect of haemolysis on the determination of plasma constituents. Clin Chim Acta 1972;41:435–8. Caraway WT. Chemical and diagnostic specificity of laboratory tests. Am J Clin Pathol 1961;37:445–64.
Interference and blood sample preparation for a pyruvate enzymatic assay Chih-Kuang Chuang a,d,1 , Tuen-Jen Wang b,1 , Chun-Yan Yeung c,e , Wen-Shyang Hsieh b , Dar-Shong Lin c,e , Shinn-Chamg Ho b , Shuan-Pei Lin a,c,e,⁎ a
Division of Genetics and Metabolism, Department of Medical Research, Taipei 10449, Taiwan b Department of Laboratory Medicine, Mackay Memorial Hospital, Taipei, Taiwan c Department of Pediatrics, Mackay Memorial Hospital, Taipei, Taiwan d College of Medicine, Fu-Jen Catholic University, Taipei, Taiwan e Mackay Medicine, Nursing and Management College, Taipei, Taiwan Received 17 June 2005; received in revised form 10 August 2005; accepted 14 October 2005 Available online 23 November 2005
Abstract Background: To assess the severity of circulatory failure, a pyruvate enzymatic assay was performed on whole blood using lactate dehydrogenase to catalyze the conversion of pyruvate to lactate. We investigated factors related to blood sample collection and preparation that might influence the results, including the timing of blood deproteinization, temperature of sample storage, and hemolysis. Method: A total of 25 whole blood specimens were collected for this study. Each sample was divided into 2 parts: one stored at room temperature (RT) and another kept on ice. The samples were deproteinizied by using 8% perchloric acid (PCA) at varying times after collection; the first deproteinization was immediately after the blood was drawn (0 h), then at 1 h intervals for 6 h and also in samples kept overnight. The supernatant samples were analyzed soon after deproteinization using a COBAS Centrifugal Analyzer. In another set of samples, the blood was immediately deproteinized, and the supernatants were stored at RT and 4°C and assayed for pyruvate at varying times, as above. Finally, the effect of hemolysis on the blood pyruvate enzymatic assay was also evaluated. Results: When samples were stored at RT, pyruvate levels remained constant until the third h after deproteinization, when there was an approximately 13.3% increase in pyruvate concentration. When whole blood samples were kept at 4°C before deproteinization, pyruvate levels were significantly reduced over time, ranging from 37.8% to 62.2% (paired t test showed a significant mean difference, P b 0.001). No significant differences in pyruvate concentration were observed in supernatant stored at either RT or 4°C. Hemolysis caused a 33.7% increase in the pyruvate concentration, equivalent to 0.18 mg pyruvate per gram per deciliter of hemoglobin. Conclusions: For a pyruvate enzymatic assay, keeping a whole blood sample at RT will not cause a significant difference in the pyruvate level as long as the sample is immediately deproteinized. Whole blood samples should not be stored in an ice bath for transport, nor should hemolyzed samples be used for a blood pyruvate enzymatic assay. © 2005 The Canadian Society of Clinical Chemists. All rights reserved. Keywords: Pyruvate; Deproteinization; Hemolysis; Centrifugal analyzer; Specimen stability
Introduction Pyruvate is the end product of glycolysis in cells with mitochondria in the presence of an adequate supply of oxygen. Pyruvate and lactate levels are used as biochemical indices of the Abbreviations: LDH, lactate dehydrogenase; RT, room temperature; PCA, perchloric acid. ⁎ Corresponding author. Division of Genetics and Metabolism, Department of Medical Research, MacKay Memorial Hospital, No. 92, Sec. 2, Chung-San N. RD., Taipei 10449, Taiwan. Fax: +886 2 253433642. E-mail address: [email protected] (S.-P. Lin). 1 The first two authors contributed equally to this work.
severity of circulatory failure [1,2]. Increased blood pyruvate levels are reported in a number of disorders including shock, liver disease, congestive heart failure, diabetes mellitus, muscular dystrophy, thiamine deficiency, and neoplastic disorders. An enzymatic method to assay pyruvate uses lactate dehydrogenase (LDH) with NADH as a cofactor to convert pyruvate to lactate, with the formation of NAD+ being proportional to the concentration of pyruvate [3–5]. This method is assayed either by using a manual procedure or an automatic COBAS-MIRA Centrifugal Analyzer (Roche Diagnostic, Montclair, NJ, USA) [6,7]. Whole blood is considered the best specimen for assessing physiological levels of pyruvate. Nevertheless, many factors
0009-9120/$ - see front matter © 2005 The Canadian Society of Clinical Chemists. All rights reserved. doi:10.1016/j.clinbiochem.2005.10.007
C.-K. Chuang et al. / Clinical Biochemistry 39 (2006) 74–77
affect the rate of pyruvate formation. There have been few reports of analytical variables that may influence the results from this assay [7–9]. We designed this study to examine the effect of three such factors on the results of the assay, including the timing of deproteinization vis-à-vis sample analysis, sample storage temperature, and hemolysis. Materials and methods Specimens Blood samples (4.5 mL) were collected in EDTA from 25 healthy adults (14 males and 11 females; 22 to 46 years of age) in the resting state after a minimum 12 h fast. Samples that were hemolyzed when they arrived in the laboratory were excluded. All subjects gave signed informed consent. Each sample was divided into two aliquots: one stored on ice and one kept at room temperature (RT). Sample deproteinization The samples were deproteinized by a method described previously [10] by mixing blood and 8% perchloric acid (PCA 69% to 72%; J.T. Baker, Phillipsburg, NJ, USA) in a 2:1 ratio with a vortex mixer for about 30 s. The mixture was kept cold for an additional 5 min to ensure complete protein precipitation. This procedure was performed on samples stored both at RT and 4°C immediately after the blood was drawn (0 h), at 1 h intervals after drawing for 6 h, and finally on samples stored overnight. In each case after immediate or delayed deproteinization, samples were centrifuged for 10 min at approximately 1500 × g, and the supernatant was collected for analysis. In addition to the above series of samples treated with delayed deproteinization, another series was immediately deproteinized and the supernatants were then stored at either RT or 4°C for varying intervals, as described above for the whole blood samples, after which the pyruvate assay was performed.
75
Roche Diagnostic (Montclair, NJ, USA). The parameters of COBAS-MIRA settings for the pyruvate assay in whole blood filtrates were programmed according to the instrument settings offered by Instruchemie. The Start Reagent 1 (R1) and Reagent 2 (R2) were β-NADH and LDH, respectively. The principle of pyruvate enzymatic assay is an endpoint method. In the presence of excess NADH, substantially all pyruvate is converted to lactate. The reduction of absorbance at 340 nm due to oxidation of NADH to NAD becomes a measure of the amount of pyruvate originally present. Results The within-run and between-run precisions (CV%) of the pyruvate enzymatic assay were investigated by using the pyruvate/lactate control purchased from Instruchemie (Delfzijl, The Netherlands). The pyruvate mean value equals 1.69 mg/dL (0.19 mmol/L) and the range is from 1.24 to 2.13 mg/dL (0.14 to 0.24 mmol/L). Ten control samples were sequentially assayed. The within-run and between-run precisions were 3.7% and 4.3%, respectively. Analytical recoveries of pyruvate added (0.089–0.89 mg/dL) to blood samples (n = 20) ranged from 95.9% to 99.1% (average, 97.3%). The sensitivity of the pyruvate enzymatic method is as low as 0.1 mg/dL. In whole blood samples kept at RT, the pyruvate levels were slightly decreased (by about 6.7%) during the first 2 h of delayed deproteinization, followed by a slight increase in the 3rd through 6th hours and overnight. The magnitude of the increases ranged from 13.3% to 4.4%, with the highest at the 3rd hour. However, the pyruvate levels at each hour of delayed deproteinization were not statistically significantly different compared to the result after immediate deproteinization. In samples kept at 4°C before deproteinization, pyruvate levels were markedly reduced, ranging from 37.8% to 62.2% lower than those in samples maintained at RT, and a paired t test showed significant differences between the results obtained from samples stored at RT and those stored at 4°C (P b 0.001) (Fig. 1).
Hemolyzed sample preparation A total of 10 blood samples (about 5 mL for each sample) were used in this part of the study. Hemolyzed samples were prepared by using the cell lysis method reported by Yulcel and Dalva [11]. Blood samples (2 mL) were hemolyzed by stirring with a metal bar for 1 or 2 min. The degree of hemolysis was determined by automated plasma hemoglobin measurement (Gen. S System, Beckman-Coulter, Fullerton, CA, USA). The remaining 3 mL from each blood sample without hemolysis was used for control. The samples were then deproteinizied as described above. Assay procedure A commercial pyruvate kit was purchased from Instruchemie (Delfzijl, The Netherlands) that contained pyruvate standard, control, Tris-buffer reagent (stabilizers), β-NADH, and LDH reagent. The COBAS-MIRA centrifugal analyzer was from
Fig. 1. Comparisons of pyruvate levels in whole blood stored at RT or in an ice bath, and supernatants stored at RT or at 4°C immediately after the blood was drawn (0 h), at 1 h intervals after drawing for 6 h, and finally on samples stored overnight (paired t test showed a significant mean difference between whole blood stored at RT or in an ice bath (P b 0.001), as well as between whole blood and supernatant stored at RT (P b 0.005); no significant mean difference was found between supernatants stored at RT or at 4°C).
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C.-K. Chuang et al. / Clinical Biochemistry 39 (2006) 74–77
In the supernatant stored at RT and 4°C for varying intervals after immediate deproteinization, the pyruvate was slightly reduced at 1 h and then remained constant from the 2nd hour to the end. The supernatant stored at RT was reduced by 15.6% and that at 4°C by 11.1%, with no statistically significant difference between them (Fig. 1). There was a significant difference by a paired t test between the results for whole blood and for supernatant stored at RT (P b 0.005). For overall comparison of the results after delayed deproteinization with those after immediate deproteinization, there was a significant correlation only in the supernatants stored in RT (r = 0.753 to 0.871), and the whole blood sample stored at RT at 1 h after delayed deproteinization (r = 0.697). The averaged pyruvate levels on non-hemolytic and hemolytic samples were 0.424 (±0.088) and 0.567 (±0.127) mg/dL, respectively. The mean degree of hemolysis was 0.9 g Hb per deciliter (0.3–1.4 g/dL). Hemolysis resulted in a mean increase in the pyruvate level of 29.7% (±15.8). The magnitude of increase was equivalent to 0.1782 mg pyruvate per gram per deciliter of hemoglobin. In an overall comparison with the original data, the correlation was significant (P = 0.011; r = 0.728). There was a significant difference in a paired t test between the hemolyzed and non-hemolyzed samples (P b 0.001). Discussion Lactate and pyruvate are physiological blood components, the levels of which reflect the equilibrium between their cytoplasmic production from glycolysis and their consumption by various tissues. That is, the blood lactate and pyruvate concentrations are affected by the rate of production as well as the rate of metabolism. These substances indicate the severity of circulatory failure and the state of tissue oxidation. Pyruvate in the blood is extremely unstable, so careful attention must be paid to blood sample preparation and storage to avoid errors in the results. According to the literature, if blood samples are not treated immediately, lactate will increase rapidly as a result of glycolysis, whereas the blood pyruvate will be significantly reduced [12,13]. This explains why we wanted to investigate the influence of delayed deproteinization on the results of a blood pyruvate enzymatic assay. Delays in processing are frequent because of problems in transporting the samples to the laboratory or from hemolysis resulting from improper venipuncture technique or handling, particularly for pediatric patients. It may be that correction factors can be derived to help reduce the magnitude of laboratory error. In our study, immediate deproteinization did not appear to be necessary, as delay resulted in only slight decreases in the measured pyruvate level. Maintaining a blood sample at RT also resulted in only insignificant differences. It appears that whole blood samples should not be iced before deproteinization. If the sample is immediately deproteinized, however, the supernatant should be kept at 4°C rather than at RT. According to another report, pyruvate in metaphosphoric acid filtrates of blood remains stable for 6 days at RT and for 8 days at 4°C [14]. Hemolysis influences most biochemical test values, either because it dilutes the plasma or releases intracellular constitu-
ents into plasma from erythrocytes. The errors can be slight or severe depending on the relative concentrations of erythrocyte constituents [11,15]. The measuring error can be avoided if the effect of hemolysis on a particular test is known. Red blood cells use anaerobic glycolysis for energy delivery and contain large amounts of LDH [11,16]. Hemolysis causes a false increase in the pyruvate concentration due to LDH release, thus accelerating the reaction between pyruvate and lactate. The optimum concentrations of LDH and its effects on blood lactate and pyruvate enzymatic measurement have been investigated by Marbach and Weil [3]. The magnitude of the change in pyruvate is not directly proportional to the degree of hemolysis. The reference interval for whole blood pyruvate concentration ranges from 0.3 to 0.7 mg/dL [7,14]. Given this relatively small amount in the blood, it seems likely that hemolysis could introduce a substantial error in measurement. The cell lysis method we used was intended to mimic the mechanical disruption of erythrocytes which typically happens when venipuncture is difficult or the sample is improperly handled. Improper handling or storage of blood samples and sample hemolysis will affect the results of a blood pyruvate enzymatic assay. In conclusion, whole blood samples kept at room temperature are acceptable, as long as the sample is immediately deproteinized; whole blood sample should not be stored in an ice bath for transport prior to deproteinization; and hemolyzed samples are not acceptable for a blood pyruvate enzymatic assay.
Acknowledgments We are grateful to the Mackay Memorial Hospital (MMH # 9330) and the National Science Council, Taiwan (NSC-922314-B-195-032) for supporting research grants and necessary instrumentations for this study. In addition, we would like to express our sincere thanks to Dr. Mary Jeanne Buttrey for her revision of this article. References [1] Huckabee WE. Relationships of pyruvate and lactate during anaerobic metabolism: I. Effects of infusion of pyruvate or glucose and of hyperventilation. J Clin Invest 1958;37(2):244–54. [2] Huckabee WE. Relationships of pyruvate and lactate during anaerobic metabolism: II. Exercise and formation of O-debt. J Clin Invest 1958;37 (2):255–63. [3] Marbach EP, Weil MH. Rapid enzymatic measurement of blood lactate and pyruvate. Use and significance of metaphosphoric acid as a common precipitant. Clin Chem 1967;13(4):314–25. [4] Neville Jr JF, Gelder RL. Modified enzymatic methods for the determination of L-(+)-lactic and pyruvic acids in blood. Am J Clin Pathol 1971;55(2):152–8. [5] Artuch R, Vilaseca MA, Farre C, Ramon F. Determination of lactate, pyruvate, beta-hydroxybutyrate and acetoacetate with a centrifugal analyser. Eur J Clin Chem Clin Biochem 1995;33(8):529–33. [6] Bonora R, Pagani F, Panteghini M. Pyruvate measured in whole blood with the Cobas Bio analyzer. Clin Chem 1989;35(2):325. [7] Hansen JL, Freier EF. Direct assays of lactate, pyruvate, beta-hydroxybutyrate, and acetoacetate with a centrifugal analyzer. Clin Chem 1978; 24(3):475–9.
C.-K. Chuang et al. / Clinical Biochemistry 39 (2006) 74–77 [8] Panteghini M, Pagani F. Biological variation of lactate and pyruvate in blood. Clin Chem 1993;39(5):908. [9] Chariot P, Ratiney R, Ammi-Said M, Herigault R, Adnot S, Gherardi R. Optimal handling of blood samples for routine measurement of lactate and pyruvate. Arch Pathol Lab Med 1994;118(7):695–7. [10] Segal S, Blair AE, Wyngaarden JB. An enzymatic spectrophotometric method for the determination of pyruvic acid in blood. J Lab Clin Med 1956;48(1):137–43. [11] Yucel D, Dalva K. Effect of in vitro hemolysis on 25 common biochemical tests. Clin Chem 1992;38(4):575–7. [12] Alderman JA, Cross RE. Adaptation to the centrifugal analyzer of an
[13]
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