Abstract
Because of the potential carcinogenic properties of hydrazine and because of other health hazards associated with its use in the laboratory, an enzymatic assay has been developed for d-3-hydroxybutyrate that is not dependent on hydrazine to drive the reaction toward completion. The use of a high concentration of NAD+ and a buffer at pH 9.5 resulted in a favorable conversion of d-3-hydroxybutyrate to acetoacetate by d-3-hydroxybutyrate dehydrogenase even though the reaction favors d-3-hydroxybutyrate formation under physiological conditions. The assay was also completed faster than previous assays using hydrazine so that the amount of enzyme used for the assay could be reduced. The recovery of d-3-hydroxybutyrate added to liver samples was 98 ± 1% (mean ± SEM, n = 6). The assay was found to be suitable for the measurement of d-3-hydroxybutyrate in samples such as perchloric acid extracts of isolated hepatocytes even when the acetoacetate to d-3-hydroxybutyrate ratio was 4 to 1. This assay presents a reliable alternative to the use of hydrazine and may be used for the assay of d-3-hydroxybutyrate in a variety of physiological and experimental samples.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 478-482 |
| Number of pages | 5 |
| Journal | Analytical Biochemistry |
| Volume | 131 |
| Issue number | 2 |
| DOIs | |
| State | Published - Jun 1983 |
| Externally published | Yes |
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Keywords
- d-3-hydroxybutyrate
- enzymatic assay
- hydrazine
- ketone bodies
ASJC Scopus subject areas
- Biochemistry
- Biophysics
- Molecular Biology
Cite this
A spectrophotometric, enzymatic assay for d-3-hydroxybutyrate that is not dependent on hydrazine. / Brashear, Allison; Cook, George A.
In: Analytical Biochemistry, Vol. 131, No. 2, 06.1983, p. 478-482.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - A spectrophotometric, enzymatic assay for d-3-hydroxybutyrate that is not dependent on hydrazine
AU - Brashear, Allison
AU - Cook, George A.
PY - 1983/6
Y1 - 1983/6
N2 - Because of the potential carcinogenic properties of hydrazine and because of other health hazards associated with its use in the laboratory, an enzymatic assay has been developed for d-3-hydroxybutyrate that is not dependent on hydrazine to drive the reaction toward completion. The use of a high concentration of NAD+ and a buffer at pH 9.5 resulted in a favorable conversion of d-3-hydroxybutyrate to acetoacetate by d-3-hydroxybutyrate dehydrogenase even though the reaction favors d-3-hydroxybutyrate formation under physiological conditions. The assay was also completed faster than previous assays using hydrazine so that the amount of enzyme used for the assay could be reduced. The recovery of d-3-hydroxybutyrate added to liver samples was 98 ± 1% (mean ± SEM, n = 6). The assay was found to be suitable for the measurement of d-3-hydroxybutyrate in samples such as perchloric acid extracts of isolated hepatocytes even when the acetoacetate to d-3-hydroxybutyrate ratio was 4 to 1. This assay presents a reliable alternative to the use of hydrazine and may be used for the assay of d-3-hydroxybutyrate in a variety of physiological and experimental samples.
AB - Because of the potential carcinogenic properties of hydrazine and because of other health hazards associated with its use in the laboratory, an enzymatic assay has been developed for d-3-hydroxybutyrate that is not dependent on hydrazine to drive the reaction toward completion. The use of a high concentration of NAD+ and a buffer at pH 9.5 resulted in a favorable conversion of d-3-hydroxybutyrate to acetoacetate by d-3-hydroxybutyrate dehydrogenase even though the reaction favors d-3-hydroxybutyrate formation under physiological conditions. The assay was also completed faster than previous assays using hydrazine so that the amount of enzyme used for the assay could be reduced. The recovery of d-3-hydroxybutyrate added to liver samples was 98 ± 1% (mean ± SEM, n = 6). The assay was found to be suitable for the measurement of d-3-hydroxybutyrate in samples such as perchloric acid extracts of isolated hepatocytes even when the acetoacetate to d-3-hydroxybutyrate ratio was 4 to 1. This assay presents a reliable alternative to the use of hydrazine and may be used for the assay of d-3-hydroxybutyrate in a variety of physiological and experimental samples.
KW - d-3-hydroxybutyrate
KW - enzymatic assay
KW - hydrazine
KW - ketone bodies
UR - http://www.scopus.com/inward/record.url?scp=0020586250&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0020586250&partnerID=8YFLogxK
U2 - 10.1016/0003-2697(83)90201-4
DO - 10.1016/0003-2697(83)90201-4
M3 - Article
C2 - 6614481
AN - SCOPUS:0020586250
VL - 131
SP - 478
EP - 482
JO - Analytical Biochemistry
JF - Analytical Biochemistry
SN - 0003-2697
IS - 2
ER -