Determination of 3-HydroxyButyric Acid and Acetone in Milk

In lactating diary cows, ketosis could occur because of a deficiency in energy rich food. In case of ketosis, abnormal levels of Acetone, Aceto-Acetate and β-HydroxyButyric Acid (3-HBA) can be found. The cow itself does not show illness in this stage. It is very important to diagnose a ketosis as the milk production could be negatively influenced by this disease. In order to check for this disease by the normal NIR analysis of the milk, a suitable calibration for the NIR system has to be performed. Therefore Skalar developed in cooperation with the Milk Control Station Nederland in Zutphen, The Netherlands, a suitable analyzer for the analyses of Acetone and 3-HBA in milk.

Reaction mechanism for 3-HBA

In cows, the following reaction can take place:

3-Hydroxybutyric acid Aceto-Acetate + 2H+ CO2 + Acetone

But the equation is in favor of 3-HBA: about 75% of the ketone bodies will be in the 3-HBA form. The remainder will be as Aceto-Acetate and a small percentage as Acetone. So it is important to measure the 3-HBA concentration.

For the determination of 3-HBA an enzymatic reaction is used:


3-Hydroxybutyric acid + NAD AcetoAcetate + NADH

The changes in NAD/NADH ratio is measured at 340 nm. A dialyzer is used in order to remove protein, fat and larger molecules from the samples. Also a blanc determination is measured prior to the addition of the enzyme as the sample can contain natural NADH or 340 nm interfering substances. After subtraction of the blanc, the photometer signals are automatically processed and the concentration calculated.

Without any pretreatment, about 40 samples per hour can be measured simultaneously for 3-HBA and Acetone. If Aceto-Acetate has to be measured, the milk sample has to be pretreated by heating it up to 100°C prior to analyses. All Aceto-Acetate is then converted to 3-HBA and subsequently measured.

For more information on the Skalar Analyzers or other Skalar products, please contact Skalar Analytical BV