Determination of lactate dehydrogenase (LDH) activity in milk by a fluorometric assay

Area of Science:

• Biochemistry
• Dairy Science
• Veterinary Diagnostics

Background:

• L-lactate dehydrogenase (LDH) in milk, originating from somatic cells, leukocytes, and microorganisms, is a potential indicator for mastitis detection.
• Current methods for measuring milk LDH activity require sample pretreatment and face methodological challenges, limiting their practical application.

Purpose of the Study:

• To develop and validate a fast, reliable, and pretreatment-free method for determining L-lactate dehydrogenase (LDH) activity in raw milk.
• To establish a fluorometric assay suitable for large-scale analyses and mastitis detection.

Main Methods:

• A fluorometric enzyme kinetic assay was developed for raw milk, measuring L-lactate conversion to pyruvate.
• The assay utilizes a linear response within 4-7 minutes, with an optimal substrate concentration of 35 mM.
• Specificity was confirmed using known LDH inhibitors (oxalic acid, oxamate, pyruvate).

Main Results:

• The developed method provides a fast and reliable determination of LDH activity in raw milk without pretreatment.
• The assay demonstrated good precision, with intra-plate CV of approximately 6% and inter-plate CV of approximately 10%.
• Analysis of reaction kinetics suggested variations in LDH isoenzyme composition between milk samples, potentially altered in high-activity samples.

Conclusions:

• A novel, rapid, and pretreatment-free fluorometric assay for milk LDH activity has been successfully established.
• This method offers improved reliability and suitability for large-scale screening, potentially enhancing mastitis diagnosis in dairy herds.
• Further investigation into LDH isoenzyme profiles may provide deeper insights into milk quality and udder health.