Automated analysis of β-hydroxybutyric and citric acid in milk

 This article in Russian

Milk samples from each individual cow are tested on composition and quality by specialized laboratories. The quality test indicates whether the demands for hygiene during production and on-farm storage of the milk are met. The milk composition test is essential for e.g. feeding optimization and timely medical treatment. Reported data serve to take adequate dairy farm management decisions. Abnormal levels of β-Hydroxybutyric acid and Citric acid in milk give us important information about the cow’s health status.This information is essential for the farmer, as the milk production could be negatively influenced. Skalar offers a fully automatic and cost-effective continuous flow analyzer, San ++, for the enzymatic determination of β-Hydroxybutyric acid and Citric acid in milk.


Automated analysis in the San++



Samples can be offered to the system directly. The analyzer is equipped with a dialyzer, to remove protein, fat and larger molecules from the samples. Also a blank determination is measured prior to the addition of the enzyme as the sample can contain natural NADH or 340 nm interfering substances. The result is automatically subtracted by the data acquisition software from thetotal measurement.


Principle and Background enzymatic β-Hydroxybutyric acid analysis





In the presence of the enzyme β -hydroxybutyrate dehydrogenase (HBDH) the β-hydroxybutyric acid (BHB) is oxidized by nicotinamide-adenine dinucleotide (NAD) to acetoacetate and NADH. The amount of NADH formed in the reaction can be directly correlated with the amount of β-hydroxybutyric acid and is measured at 340 nm.


Energy deficiency in dairy cows can cause subclinical (without signs or symptoms) or clinical ketosis. In ketotic state the concentration of ketone bodies (acetone (AC), acetoacetic acid (ACAC) and β-hydroxybutyric acid) increases in the different body fluids. The early detection of elevated levels of ketone bodies – mainly during the subclinical stage of energy deficiency – is highly recommended, as these conditions are associated with decreased milk yield, reproductive performance and increased risk of clinical ketosis.


Principle and Background enzymatic Citric acid analysis




Citrate is converted into oxaloacetate and acetate, catalyzed by citrate lyase. Oxaloacetate decarboxylises easily into pyruvate. Both oxaloacetate and pyruvate are reduced by NADH to respectively 1-malate and 1-lactate, catalyzed by malate dehydrogenase and lactate dehydrogenase. The decrease of the amount of NADH is stoichiometric with the amount of citrate. The remaining NADH is measured at 340 nm.


Decreased levels of citric acid can be found in milk of cows, which have sub clinical or clinical mastitis. The impact of mastitis such as changes in milk composition reduces its quality. In addition, the antibiotic used in treating mastitis leaves residues in the milk, which interferes with the manufacturing process of many dairy products and can cause health problems to consumers. Therefore early detection of decreased citric acid levels during the subclinical stage is highly recommended.


The San++ continuous flow analyzer - for adequate dairy farm management



  • Fast sample through-put
  • High accuracy and reproducibility
  • Cost saving on reagents
  • Standardization of methods
  • Standardization of operation conditions
  • Inter-laboratory variations minimized
  • No transcription errors

Besides the β-hydroxybutyric and Citric acid methods, the San++ analyzer can handle also other relevant parameters for milk such as: Urea, Ammonia, Nitrate, Nitrite, Protein and Acetone




For more informationplease contact Skalar.