β-Hydroxybutyrate (β-HB; 3-hydroxybutyric acid) is a “ketone body” which is produced in the liver, mainly from the oxidation of fatty acids, and is exported to peripheral tissues for use as an energy source. The term ‘ketone body’ refers to three molecules, acetoacetate, β-HB, and acetone. β-HB and acetoacetate transport energy from the liver to the other tissues and acetone is generated by spontaneous decarboxylation of acetoacetate.¹ The presence of ketosis may be normal or pathologic. Normally ketosis can indicate that lipid metabolism has been activated and the pathway of lipid degradation is intact. Normal ketosis is prevalent in many circumstances such as during fasting, after prolonged exercise or after a high fat diet. Pathological causes of ketosis include multiple organ failure, diabetes, childhood hypoglycemia, corticosteroid or growth hormone deficiency, intoxication with alcohol or salicylates and several inborn errors of metabolism.² In acutely ill patients, these ketone bodies can accumulate in the body to cause ketoacidosis, which leads to the potentially life threatening condition known as metabolic acidosis.³ The presence and degree of ketosis can be determined by measuring blood levels of β-HB. Ordinarily, β-HB accounts for approximately 75% of the ketone bodies in serum.^4,5,6 Measurement of β-HB provides a reliable index of the level of ketoacidosis, including the detection of subclinical ketosis.^7,8,9 In diabetics, β-HB measurements (and blood glucose) can be used for the assessment of the severity of diabetic coma and is essential for the exclusion of hyperosmolar non-ketotic diabetic coma. The measurement of β-HB is also used to monitor insulin requirements, based on existing hyperketonemia.¹⁰ β-HB has more recently been evaluated for use in neurodegenerative diseases and inhibition of adipocyte lipolysis.^{11,12,13,14,15} Cayman’s β-HB (Ketone Body) Assay Kit provides a simple, reproducible, and sensitive tool for measuring β-HB levels in plasma, serum, or urine. The method for β-HB determination is based upon the oxidation of D-3-Hydroxybutyrate to acetoacetate by the enzyme 3-hydroxybutyrate dehydrogenase.¹⁶ Concomitant with this oxidation, the cofactor NAD⁺ is reduced to NADH. In the presence of diaphorase, NADH reacts with the colorimetric detector WST-1 to produce a formazan dye with an absorbance maximum at 445-455 nm. The absorbance of the dye is directly proportional to the β-HB concentration.

1 Guthrie, J.P., and Jordan, F. Amine-catalyzed decarboxylation of acetoacetic acid. The rate constant for decarboxylation of a β-imino acid. J Am Chem Soc 94(26) 9136-9141 (1972).

2 Galán, A., Hernández, J.M., and Jimenez, O. Measurement of blood acetoacetate and β-hydroxybutyrate in an automatic analyser. J Autom Methods Manag Chem 23(3) 69-76 (2001).

3 Foster, D.W., and McGarry, J.D. The metabolic derangements and treatment of diabetic ketoacidosis. N Engl J Med 309(3) 159-169 (1983).

4 Persson, B. Determination of plasma acetoacetate and D-b-hydroxy-butyrate in new-born infants by an enzymatic fluorometric micro-method. Scand J Clin Lab Invest 25(1) 9-18 (1970).

5 Wildenhoff, K.E. A micro-method for the enzymatic determination of acetoacetate and 3-hydroxybutyrate in blood and urine. Scand J Clin Lab Invest 25(2) 171-179 (1970).

6 Koch, D.D., and Feidbruegge, H. Optimized kinetic method for automated determination of β-hydroxybutyrate. Clin Chem 33(10) 1761-1766 (1987).

7 Li, P.K., Lee, J.T., MacGillivray, M.H., et al. Direct, fixed-time kinetic assays for β-hydroxybutyrate and acetoacetate with a centrifugal analyzer or a computer-backed spectrophotometer. Clin Chem 26(12) 1713-1717 (1980).

8 Harano, Y., Kosugi, K., Hyosu, T., et al. Sensitive and simplified method for the differential determination of serum levels of ketone bodies. Clin Chim Acta 134 327-336 (1983).

9 MacGillivray, M.H., Li, P.K., Lee, J.T., et al. Elevated plasma b-hydroxybutyrate concentrations without ketonuria in healthy insulin-dependent diabetic patients. J Clin Endocrinol Metab 54(3) 665-668 (1982).

10 Alberti, K.G.M.M., and Hockaday, T.D.R. Rapid blood ketone body estimation in the diagnosis of diabetic ketoacidosis. Br Med J 2 565-568 (1972).

11 Kashiwaya, Y., Takeshima, T., Mori, N., et al. D-β-hydroxybutyrate protects neurons in models of Alzheimer’s and Parkinson’s disease. Proc Natl Acad Sci USA 97(10) 5440-5444 (2000).

12 Tieu, K., Perier, C., Caspersen, C., et al. D-β-hydroxybutyrate rescues mitochondrial respiration and mitigates features of Parkinson disease. J Clin Invest 112(6) 892-901 (2003).

13 Reger, M.A., Henderson, S.T., Hale, C., et al. Effects of β-hydroxybutyrate on cognition in memory-impaired adults. Neurobiol Aging 25 311-314 (2004).

14 Cheng, B., Yang, X., Hou, Z., et al. D-β-hydroxybutyrate inhibits the apoptosis of PC12 cells induced by 6-OHDA in relation to up-regulating the ratio of Bcl-2/Bax mRNA. Auton Neurosci 134 38-44 (2007).

15 Taggart, A.K.P., Kero, J., Gan, X., et al. (D)-β-hydroxybutyrate inhibits adipocyte lipolysis via the nicotinic acid receptor PUMA-G. J Biol Chem 280(29) 26649-26652 (2005).

16 McMurray, C.H., Blanchflower, W.J., and Rice, D.A. Automated kinetic method for D-3-hydroxybutyrate in plasma or serum. Clin Chem 30(3) 421-425 (1984).

Harano, Y., Kosugi, K., Hyosu, T., et al. Sensitive and simplified method for the differential determination of serum levels of ketone bodies. Clin Chim Acta 134 327-336 (1983).

MacGillivray, M.H., Li, P.K., Lee, J.T., et al. Elevated plasma b-hydroxybutyrate concentrations without ketonuria in healthy insulin-dependent diabetic patients. J Clin Endocrinol Metab 54(3) 665-668 (1982).

Alberti, K.G.M.M., and Hockaday, T.D.R. Rapid blood ketone body estimation in the diagnosis of diabetic ketoacidosis. Br Med J 2 565-568 (1972).

Reger, M.A., Henderson, S.T., Hale, C., et al. Effects of β-hydroxybutyrate on cognition in memory-impaired adults. Neurobiol Aging 25 311-314 (2004).

Cheng, B., Yang, X., Hou, Z., et al. D-β-hydroxybutyrate inhibits the apoptosis of PC12 cells induced by 6-OHDA in relation to up-regulating the ratio of Bcl-2/Bax mRNA. Auton Neurosci 134 38-44 (2007).

Taggart, A.K.P., Kero, J., Gan, X., et al. (D)-β-hydroxybutyrate inhibits adipocyte lipolysis via the nicotinic acid receptor PUMA-G. J Biol Chem 280(29) 26649-26652 (2005).

McMurray, C.H., Blanchflower, W.J., and Rice, D.A. Automated kinetic method for D-3-hydroxybutyrate in plasma or serum. Clin Chem 30(3) 421-425 (1984).

Hansen, J.L., and Freier, E.F. Direct assays of lactate, pyruvate, β-hydroxybutyrate, and acetoacetate with a centrifugal analyzer. Clin Chem 24(3) 475-479 (1978).

Feliz, B., Witt, D.R., and Harris, B.T. A neuropathology case report and review of prior cases. Arch Pathol Lab Med 127 325-328 (2003).

Tieu, K., Perier, C., Caspersen, C., et al. D-β-hydroxybutyrate rescues mitochondrial respiration and mitigates features of Parkinson disease. J Clin Invest 112(6) 892-901 (2003).

Galán, A., Hernández, J.M., and Jimenez, O. Measurement of blood acetoacetate and β-hydroxybutyrate in an automatic analyser. J Autom Methods Manag Chem 23(3) 69-76 (2001).

Foster, D.W., and McGarry, J.D. The metabolic derangements and treatment of diabetic ketoacidosis. N Engl J Med 309(3) 159-169 (1983).

Persson, B. Determination of plasma acetoacetate and D-b-hydroxy-butyrate in new-born infants by an enzymatic fluorometric micro-method. Scand J Clin Lab Invest 25(1) 9-18 (1970).

Wildenhoff, K.E. A micro-method for the enzymatic determination of acetoacetate and 3-hydroxybutyrate in blood and urine. Scand J Clin Lab Invest 25(2) 171-179 (1970).

Koch, D.D., and Feidbruegge, H. Optimized kinetic method for automated determination of β-hydroxybutyrate. Clin Chem 33(10) 1761-1766 (1987).

Li, P.K., Lee, J.T., MacGillivray, M.H., et al. Direct, fixed-time kinetic assays for β-hydroxybutyrate and acetoacetate with a centrifugal analyzer or a computer-backed spectrophotometer. Clin Chem 26(12) 1713-1717 (1980).

Kashiwaya, Y., Takeshima, T., Mori, N., et al. D-β-hydroxybutyrate protects neurons in models of Alzheimer’s and Parkinson’s disease. Proc Natl Acad Sci USA 97(10) 5440-5444 (2000).

Guthrie, J.P., and Jordan, F. Amine-catalyzed decarboxylation of acetoacetic acid. The rate constant for decarboxylation of a β-imino acid. J Am Chem Soc 94(26) 9136-9141 (1972).

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