A novel assay for triglycerides using glycerol dehydrogenase and a water-soluble formazan dye, WST-8

Miki Kawano, Eisaku Hokazono, Susumu Osawa, Shouichi Sato, Takiko Tateishi, Masahiro Manabe, Hirotaka Matsui, Yuzo Kayamori

Research output: Contribution to journalArticle

Abstract

Background: The glycerol-3-phosphate (GPO)-peroxidase (POD) chromogenic method is one of the most widely used methods to assay triglycerides. However, it is well known that peroxidase is affected by reducing agents, and recently, it has been reported that some materials affect its activity. Moreover, there is a high possibility of non-specific reaction, as the method uses many enzymes. Against this background, we developed a simpler assay method for triglycerides without using peroxidase. Methods: Triglycerides were hydrolysed to glycerol and fatty acids by lipoprotein lipase followed by the oxidation of glycerol to dihydroxyacetone with simultaneous production of NADH by glycerol dehydrogenase. To overcome incomplete conversion of glycerol to dihydroxyacetone by glycerol dehydrogenase at equilibrium, we added 2-(2-methoxy-4-nitrophenyl)-3–(4-nitrophenyl)-5–(2,4-disulfophenyl)-2H-tetrazolium monosodium salt (WST-8) to the reaction mixture to remove NADH, allowing the reaction to complete while showing stoichiometric production of reduced WST-8. Results: The reaction was linear up to 6.4 mmol/L. The mean intra-assay (n = 20) and inter-assay (n = 20) imprecision, as determined by replicate analysis of three pooled human serum samples with different triglyceride concentrations, were 1.1–2.3% and 1.1–1.5% coefficient of variation (%CV), respectively. No interference by 2.5 g/L haemoglobin, 65 μmol/L free bilirubin and 359 μmol/L conjugated bilirubin was observed. The equation obtained in comparison with that by the GPO-POD method including endogenous glycerol-eliminating step was: y = 1.0002x + 0.0395 mmol/L; r = 0.999; Sy/x = 0.049 mmol/L; n = 97. Conclusion: Our method is an accurate, yet simpler and more sensitive for the quantitative analysis of triglycerides.

Original languageEnglish
Pages (from-to)442-449
Number of pages8
JournalAnnals of Clinical Biochemistry
Volume56
Issue number4
DOIs
Publication statusPublished - Jul 1 2019

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glycerol dehydrogenase
Formazans
Assays
Triglycerides
Coloring Agents
Glycerol
Peroxidase
Dihydroxyacetone
Water
Bilirubin
NAD
Chromogenics
Lipoprotein Lipase
Reducing Agents
NADH Dehydrogenase
Hemoglobins
Fatty Acids
2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H tetrazolium monosodium salt
Lipase
Oxidation

All Science Journal Classification (ASJC) codes

  • Clinical Biochemistry

Cite this

A novel assay for triglycerides using glycerol dehydrogenase and a water-soluble formazan dye, WST-8. / Kawano, Miki; Hokazono, Eisaku; Osawa, Susumu; Sato, Shouichi; Tateishi, Takiko; Manabe, Masahiro; Matsui, Hirotaka; Kayamori, Yuzo.

In: Annals of Clinical Biochemistry, Vol. 56, No. 4, 01.07.2019, p. 442-449.

Research output: Contribution to journalArticle

Kawano, Miki ; Hokazono, Eisaku ; Osawa, Susumu ; Sato, Shouichi ; Tateishi, Takiko ; Manabe, Masahiro ; Matsui, Hirotaka ; Kayamori, Yuzo. / A novel assay for triglycerides using glycerol dehydrogenase and a water-soluble formazan dye, WST-8. In: Annals of Clinical Biochemistry. 2019 ; Vol. 56, No. 4. pp. 442-449.
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abstract = "Background: The glycerol-3-phosphate (GPO)-peroxidase (POD) chromogenic method is one of the most widely used methods to assay triglycerides. However, it is well known that peroxidase is affected by reducing agents, and recently, it has been reported that some materials affect its activity. Moreover, there is a high possibility of non-specific reaction, as the method uses many enzymes. Against this background, we developed a simpler assay method for triglycerides without using peroxidase. Methods: Triglycerides were hydrolysed to glycerol and fatty acids by lipoprotein lipase followed by the oxidation of glycerol to dihydroxyacetone with simultaneous production of NADH by glycerol dehydrogenase. To overcome incomplete conversion of glycerol to dihydroxyacetone by glycerol dehydrogenase at equilibrium, we added 2-(2-methoxy-4-nitrophenyl)-3–(4-nitrophenyl)-5–(2,4-disulfophenyl)-2H-tetrazolium monosodium salt (WST-8) to the reaction mixture to remove NADH, allowing the reaction to complete while showing stoichiometric production of reduced WST-8. Results: The reaction was linear up to 6.4 mmol/L. The mean intra-assay (n = 20) and inter-assay (n = 20) imprecision, as determined by replicate analysis of three pooled human serum samples with different triglyceride concentrations, were 1.1–2.3{\%} and 1.1–1.5{\%} coefficient of variation ({\%}CV), respectively. No interference by 2.5 g/L haemoglobin, 65 μmol/L free bilirubin and 359 μmol/L conjugated bilirubin was observed. The equation obtained in comparison with that by the GPO-POD method including endogenous glycerol-eliminating step was: y = 1.0002x + 0.0395 mmol/L; r = 0.999; Sy/x = 0.049 mmol/L; n = 97. Conclusion: Our method is an accurate, yet simpler and more sensitive for the quantitative analysis of triglycerides.",
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T1 - A novel assay for triglycerides using glycerol dehydrogenase and a water-soluble formazan dye, WST-8

AU - Kawano, Miki

AU - Hokazono, Eisaku

AU - Osawa, Susumu

AU - Sato, Shouichi

AU - Tateishi, Takiko

AU - Manabe, Masahiro

AU - Matsui, Hirotaka

AU - Kayamori, Yuzo

PY - 2019/7/1

Y1 - 2019/7/1

N2 - Background: The glycerol-3-phosphate (GPO)-peroxidase (POD) chromogenic method is one of the most widely used methods to assay triglycerides. However, it is well known that peroxidase is affected by reducing agents, and recently, it has been reported that some materials affect its activity. Moreover, there is a high possibility of non-specific reaction, as the method uses many enzymes. Against this background, we developed a simpler assay method for triglycerides without using peroxidase. Methods: Triglycerides were hydrolysed to glycerol and fatty acids by lipoprotein lipase followed by the oxidation of glycerol to dihydroxyacetone with simultaneous production of NADH by glycerol dehydrogenase. To overcome incomplete conversion of glycerol to dihydroxyacetone by glycerol dehydrogenase at equilibrium, we added 2-(2-methoxy-4-nitrophenyl)-3–(4-nitrophenyl)-5–(2,4-disulfophenyl)-2H-tetrazolium monosodium salt (WST-8) to the reaction mixture to remove NADH, allowing the reaction to complete while showing stoichiometric production of reduced WST-8. Results: The reaction was linear up to 6.4 mmol/L. The mean intra-assay (n = 20) and inter-assay (n = 20) imprecision, as determined by replicate analysis of three pooled human serum samples with different triglyceride concentrations, were 1.1–2.3% and 1.1–1.5% coefficient of variation (%CV), respectively. No interference by 2.5 g/L haemoglobin, 65 μmol/L free bilirubin and 359 μmol/L conjugated bilirubin was observed. The equation obtained in comparison with that by the GPO-POD method including endogenous glycerol-eliminating step was: y = 1.0002x + 0.0395 mmol/L; r = 0.999; Sy/x = 0.049 mmol/L; n = 97. Conclusion: Our method is an accurate, yet simpler and more sensitive for the quantitative analysis of triglycerides.

AB - Background: The glycerol-3-phosphate (GPO)-peroxidase (POD) chromogenic method is one of the most widely used methods to assay triglycerides. However, it is well known that peroxidase is affected by reducing agents, and recently, it has been reported that some materials affect its activity. Moreover, there is a high possibility of non-specific reaction, as the method uses many enzymes. Against this background, we developed a simpler assay method for triglycerides without using peroxidase. Methods: Triglycerides were hydrolysed to glycerol and fatty acids by lipoprotein lipase followed by the oxidation of glycerol to dihydroxyacetone with simultaneous production of NADH by glycerol dehydrogenase. To overcome incomplete conversion of glycerol to dihydroxyacetone by glycerol dehydrogenase at equilibrium, we added 2-(2-methoxy-4-nitrophenyl)-3–(4-nitrophenyl)-5–(2,4-disulfophenyl)-2H-tetrazolium monosodium salt (WST-8) to the reaction mixture to remove NADH, allowing the reaction to complete while showing stoichiometric production of reduced WST-8. Results: The reaction was linear up to 6.4 mmol/L. The mean intra-assay (n = 20) and inter-assay (n = 20) imprecision, as determined by replicate analysis of three pooled human serum samples with different triglyceride concentrations, were 1.1–2.3% and 1.1–1.5% coefficient of variation (%CV), respectively. No interference by 2.5 g/L haemoglobin, 65 μmol/L free bilirubin and 359 μmol/L conjugated bilirubin was observed. The equation obtained in comparison with that by the GPO-POD method including endogenous glycerol-eliminating step was: y = 1.0002x + 0.0395 mmol/L; r = 0.999; Sy/x = 0.049 mmol/L; n = 97. Conclusion: Our method is an accurate, yet simpler and more sensitive for the quantitative analysis of triglycerides.

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