Abstract
An automated, spectro-photometric determination of blood acetoacetate and β-hydroxybutyrate was developed with a Gilford 3500 autoanalyzer. The stability of ketone bodies was studied in different conditions. An immediate precipitation with 0.6 M perchloric acid and cooling the sample effectively prevent the loss of acetoacetate from samples during transport to the laboratory (at 4°C a 6 % loss of acetoacetate was noted during 24 h). Freezing the sample makes it practically stable (less than 2 % loss of acetoacetate per week during a study lasting 2 months). At room temperature (20°C) the sample’s acetoacetate was instable and disappeared with a rate of 6 % per h. β-hydroxybutyrate was stable in precipitated samples. Because the precipitation also retains the sample’s glucose, 3 main parameters for the indication of ketosis could be analyzed automatically from the same sample with a total capacity of 40 samples in 2½ h.
Keywords: acetoacetate, bovine ketosis, Gilford 3500 analyzer, glucose, β-hydroxybutyrate
Sammanfattning
Man har utvecklat en automatisk spektrofotometrisk bestämningsraetod av acetoacetat och β-hydroxybutyrat i blod med Gilford 35øø autoanalyzer. Ketonkropparnas stabilitet har undersökts under olika förhållanden. Omedelbar precipitering med 0,6 M perklorsyra och nedkylning av provet förhindrar effektivt förlust av acetoacetat under transporten till laboratoriet (vid 4°C noterades en forlust av 6 % i acetoacetatkoncentrationen under ett dygn). Nedfrysningen av provet gör att det blir praktiskt taget stabilt (mindre än 2 % förlust av acetoacetat per vecka under en två månaders undersökning). Vid rumstemperatur (20°C) var acetoacetatet instabilt och koncentrationen minskade med 6 % i timmen.
β-hydroxybutyratet var stabilt i de precipiterade proven. Emedan precipiteringen också kvarhåller provets glukos, kan man ur samma prov automatiskt analysera tre huvudparametrar för indikering av ketos med en total kapacitet av 40 prov på 2,5 timmar.
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References
- Baird G. D., Hibbitt K. G., Hunter G. D., Lund P., Stubbs M., Krebs H. A. Biochemical aspects of bovine ketosis. Biochem. J. 1968;107:683–689. doi: 10.1042/bj1070683. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Baird G. D., Heitzman J. R., Hibbitt K. G. Effects of starvation on intermediary metabolism in the lactating cow. Biochem. J. 1972;128:1311–1318. doi: 10.1042/bj1281311. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Halse K., Tomasgard G., Benjaminsen E. Glykogen dannende stoffer til melkekyr I. (Glycogen forming substanceis to milking cows I) Norsk Vet.-T. 1976;88:83–89. [Google Scholar]
- Hansen J. L., Freier E. F. Direct assays of lactate, pyruvate, β-hydroxybutyrate, and acetoacetate with a centrifugal analyzer. Clin. Chem. 1978;24:475–479. doi: 10.1093/clinchem/24.3.475. [DOI] [PubMed] [Google Scholar]
- Kelly J. M. Changes in serum B-hydroxybutyrate concentrations in dairy cows kept under commercial farm conditions. Vet. Rec. 1977;101:499–502. [PubMed] [Google Scholar]
- Mellanby, J. & D. H. Williamson: Acetoacetate. In Methods of Enzymatic Analysis. H. U. Bergmeyer (ed.). Acad. Press, New York 1974, 1840–1843.
- Møller J. M., Halse K. Propylenglykol til kyr. Forebyggning av klinisk ketose. (Propyleneglycol to cows. Prevention of clinical ketosis). Norsk Vet.-T. 1078;90:145–156. [Google Scholar]
- Price C. P., Lloyd B., Alberti K. G. M. M. A kinetic spectrophotometric assay for rapid determination of acetoacetate in blood. Clin. Chem. 1977;23:1893–1897. doi: 10.1093/clinchem/23.10.1893. [DOI] [PubMed] [Google Scholar]
- Wildenhoff K. E. A micro-method for the enzymatic determination of acetoacetate and 3-hydroxybutyrate in blood and urine. Scand. J. clin. Lab. Invest. 1970;25:171–179. doi: 10.3109/00365517009049200. [DOI] [PubMed] [Google Scholar]
- Williamson D. H., Mellanby J., Krebs H. A. Enzymic determination of D(–)-β-hydroxybutyric acid and acetoacetic acid in blood. Biochem. J. 1962;82:90–96. doi: 10.1042/bj0820090. [DOI] [PMC free article] [PubMed] [Google Scholar]