# Serum Norepinephrine and Cholesterol Concentrations as Novel Diagnostic Biomarkers for Vitamin E Deficiency in Holstein Cows

## TL;DR

This study identifies norepinephrine and cholesterol as effective biomarkers for diagnosing vitamin E deficiency in dairy cows.

## Contribution

The study introduces serum norepinephrine and cholesterol as novel biomarkers for diagnosing vitamin E deficiency in Holstein cows.

## Key findings

- Vitamin E deficiency disrupts amino acid, lipid, and energy metabolism pathways in dairy cows.
- Norepinephrine and cholesterol show high diagnostic accuracy for vitamin E deficiency with sensitivities and specificities of 90% and 100%.
- Combining untargeted and targeted metabolomics reveals 31 differential metabolites associated with vitamin E deficiency.

## Abstract

This study investigated metabolic alterations in dairy cows having vitamin E deficiency (VED) using combined gas chromatography-time-of-flight mass spectrometry and liquid chromatography-mass spectrometry metabolomics approaches. There were significant disruptions in amino acid/lipid/energy metabolism pathways, with 31 differential metabolites identified between VED and healthy control groups. Notably, norepinephrine and cholesterol were identified as promising serum biomarkers for VED diagnosis, showing excellent diagnostic accuracy with high sensitivity and specificity.

Vitamin E deficiency (VED) represents a common micronutrient deficiency in dairy cows (DCs), leading to severe degenerative diseases, oxidative stress, immune dysfunction, and various health issues, ultimately causing significant economic losses for the global dairy sector. Accordingly, our objective was to explore the metabolic features of VED-afflicted cows by combining the untargeted gas chromatography-time-of-flight mass spectrometry (GC-TOF-MS) and targeted liquid chromatography-mass spectrometry (LC-MS) to identify effective serum VED biomarkers. Untargeted GC-TOF-MS analysis identified 31 differential metabolites (DMs): 20 were overexpressed and 11 were suppressed in the VED group compared to the healthy control group. These DMs were enriched in six major metabolic pathways: glycine, serine, and threonine; alanine, aspartate, and glutamate; cysteine and methionine; tyrosine; primary bile acid biosynthesis; and nitrogen metabolisms. These outcomes show that VED significantly disrupts amino acid/lipid/energy metabolism pathways in DCs. Further targeted LC-MS quantification revealed significant alterations in key metabolites, including increased levels of norepinephrine, glycine, cysteine, and L-glutamine, as well as a significant reduction in cholesterol concentrations. Binary logistic regression analysis identified norepinephrine and cholesterol as strong candidate biomarkers for VED. Receiver operating characteristic curve analysis established outstanding diagnostic accuracy for norepinephrine and cholesterol (for both p < 0.001, area under the curve = 0.980 and 0.990, correspondingly), with sensitivities and specificities of 90% and 100%, respectively. In conclusion, this study integrates untargeted and targeted metabolomics approaches to reveal VED-caused metabolic disruptions in DCs, particularly in amino acid/lipid/energy metabolism pathways. Norepinephrine and cholesterol were identified as highly accurate serum VED biomarkers with excellent diagnostic performance. Early detection and timely intervention using these biomarkers could promote disease treatment and cow health, as well as productivity, and decrease economic losses.