Introduction
Understanding the nutritional cycles of dairy cows is very important for a number of reasons. For instance, to monitor:
- the health of the animal and its offspring
- production efficiency
- environmental footprint of dairying operations
- the quality of the resulting milk and affiliated products
Protein content of milk has long been documented to determine the nutritional value and requirements of dairy cows, and ultimately the value of the milk produced. The concentration and profile of free amino acids found in cow plasma can provide information on protein synthesis. For example, which amino acids are rate-limiting and facilitate the production of milk protein.
Increasing efficiency and minimising waste is an integral part of all food production. The dairy industry is no exception. The nutritional needs of dairy cows must be empirically understood in order to limit excess waste and encourage responsible resource management. Also, evaluating free amino acids can provide a measure of health; as concentrations of certain free amino acids may vary with metabolic disease.
Determining amino acid levels has bore witness to a number of quantifying methods; including ion exchange chromatography, reverse-phase chromatography and high-pressure liquid chromatography (HPLC). Whereby, subsequent amino acid derivatives are detected using ultraviolet (UV) or fluorescence spectrometry. The aforementioned study by Reinhardt et al devised a robust and accurate method to measure amino acid concentrations in bovine plasma using precolumn phenyl isothiocyanate (PITC) derivatisation; in addition to HPLC and positive electrospray ionization (ESI) single quadrupole mass spectrometry detection (HPLC/MS).
Materials and Products
The unlabelled 20 amino acid standards (MSK-A2-US-S) and the corresponding stable isotope labelled amino acids standards (MSK-CAA) were supplied by Cambridge Isotope Laboratories Inc. and consisted of the following:
- glycine (13C2; 15N)
- l-alanine (13C3; 15N)
- l-arginine·HCl (13C6; 15N4)
- l-asparagine·H2O (13C4; 15N2)
- l-aspartic acid (13C4; 15N)
- l-cystine (13C6; 15N2)
- l-glutamic acid (13C5; 15N)
- l-glutamine (13C5; 15N2)
- l-histidine·HCl·H2O (13C6; 15N3)
- l-isoleucine (13C6; 15N)
- l-leucine (13C6; 15N)
- l-lysine·2HCl (13C6; 15N2)
- l-methionine (13C5; 15N)
- l-phenylalanine (13C9; 15N)
- l-proline (13C5; 15N)
- l-serine (13C3; 15N)
- l-threonine (13C4; 15N)
- l-tryptophan (13C11; 15N2)
- l-tyrosine (13C9; 15N)
- l-valine (13C5; 15N)
Results and Discussion
In order to quantitate the endogenous amino acids in bovine plasma a robust LC/MS method was developed. LC/MS was selected over the alternatives due to the lack of complexity, inexpensiveness and all the analytes chosen to study are known. However, LC/MS is less sensitive for amino acids such as glycine that are not highly ionisable; therefore, precolumn derivatisation was necessary. Derivatising reagent PITC (Figure 1) was chosen due to its ease of use and removal of excess reagents by vacuum. Sufficient excess of PITC reagent was added to complete the reaction in just 20 minutes. Overall, the study contained over 1000 processed samples.
The use of a stable isotope labelled version of each analyte as Internal Standard (IS), especially those incorporating 13C and 15N isotope, added to each sample at the beginning of the procedure corrected for any losses, instrument variability and chemistry of derivatisation. Furthermore, Electrospray Ionisation (ESI) MS detection allowed overlapping peaks in the chromatogram to be quantified, thereby shortening the amount of time required for the chromatographic run.
Sufficient sample purification (via deproteination with methanol followed by binding and elution from cation exchange resin) reduced the potential for ionisation suppression and increased column lifetime. Consequently, this also decreased instrument maintenance, adding to the robust nature of this procedure.
This procedure was deemed successful and has since been implemented in further studies to determine plasmid amino acid concentrations in bovine subjects. Most notably in “Crude Protein Oscillation in Diets Adequate and Deficient in Metabolizable Protein: Effects on Nutrient Digestibility, Nitrogen Balance, Plasma Amino Acids, and Greenhouse Gas Emissions,” Journal of Dairy Science 107, no. 6 (2024): 3558–3572.
Full Article
Laurie A. Reinhardt, Levi Svaren, Ritvik Marathe, Geoffrey I. Zanton, Michael L. Sullivan