Beyond BHB: Metabolomic Signatures of Ketosis in Dairy Cows

β-hydroxybutyrate (BHB) is a key marker of ketosis, but the metabolic disturbance in ketotic dairy cows extends well beyond ketone body accumulation. Metabolomic studies using LC–MS, GC–MS, and ^1H-NMR have identified coordinated changes in lipid, amino acid, carbohydrate, and energy metabolism. Examining these changes across blood, urine, milk, rumen fluid, and tissues provides a broader view of the metabolic alterations associated with ketosis. 

Blood Metabolites Reflect Systemic Metabolic Disturbance 

Blood provides a direct snapshot of systemic metabolic status in ketotic cows. Along with increased BHB, acetoacetate, and acetone, ketosis is associated with increased non-esterified fatty acids (NEFAs) and long-chain fatty acids such as palmitic and stearic acids, reflecting increased adipose tissue mobilization^[1]. 

Metabolomic profiling also reveals changes beyond lipid metabolism. Glucogenic amino acids, including alanine, glutamate, glutamine, histidine, and phenylalanine, typically decline, consistent with increased hepatic demand for gluconeogenic substrates. Altered lysine metabolism has also been reflected by increased 2-piperidinecarboxylic acid^[2],[3]. 

Acylcarnitine profiles provide another metabolic signal, with alterations in short- and medium-chain acylcarnitines associated with impaired mitochondrial β-oxidation. GC–MS analysis has also identified increased cis-9-hexadecenoic acid as a potential biomarker candidate for clinical ketosis^[1]. 

Urine Offers a Noninvasive Metabolic Readout^[1] 

Urinary metabolites can complement blood-based assessment by reflecting downstream metabolic and excretory changes. Urinary BHB and acetone increase in ketotic cows, while pathway analyses identify alterations in phenylalanine metabolism and the alanine, aspartate, and glutamate pathways. 

In cows with displaced abomasum and concurrent ketosis, reductions in urinary hippuric acid and trimethylamine N-oxide (TMAO) have also been reported. These changes were associated with reduced feed intake and alterations in rumen microbial metabolism affecting gut-derived metabolites. 

Milk Metabolites Can Support Herd-Level Monitoring^[1] 

Milk provides another useful matrix because its composition reflects mammary gland metabolism and systemic energy status. Milk BHB shows a strong correlation with blood BHB and can serve as an indicator of negative energy balance. Changes in glycine and acetylcarnitine have also been associated with the severity of negative energy balance, reflecting altered fatty acid transport and utilization by the mammary gland. 

Ketotic cows may also show reduced milk concentrations of galactose-1-phosphate, glucose-1-phosphate, uridine, and orotate, indicating changes in purine and pyrimidine metabolism. Citrate and formate, which are involved in TCA-cycle-related metabolism, can also be altered. 

For larger-scale monitoring, mid-infrared (MIR/FTIR) spectroscopy can predict milk BHB and acetone and provide estimates of circulating NEFA and urea, offering a potentially cost-effective approach to metabolic risk screening. 

Tissue Metabolomics Shows Where Metabolic Dysfunction Occurs^[1] 

Tissue-level profiling provides insight into metabolic changes that are not fully captured by circulating biomarkers. In skeletal muscle from clinically ketotic cows, reduced concentrations of pyruvate, succinate, α-ketoglutarate, fumarate, malate, and citrate, together with increased local BHB, indicate suppressed TCA-cycle activity and impaired mitochondrial energy production. 

The liver shows metabolic changes associated with fatty liver, including lipid and cholesterol accumulation and reduced hepatic glucose concentrations. Increased glycocholate, tauroursodeoxycholate, and cholesteryl linoleate have also been identified as potential biomarkers associated with fatty liver disease. 

Adipose tissue provides another perspective. In subclinical ketosis, multi-omics analyses have identified disruption of sphingolipid metabolism, particularly accumulation of ceramides associated with oxidative stress and inflammation. 

Rumen Fluid Links Fermentation with Ketosis^[1] 

Rumen metabolomics highlights an important upstream component of ketosis. Studies report increased concentrations of the ketogenic volatile fatty acids (VFAs) butyrate and valerate, together with reduced propionate, the major gluconeogenic precursor in cattle. This altered VFA pattern can reduce the availability of glucose precursors to the liver and contribute to the metabolic conditions associated with ketosis. 

Changes in other rumen metabolites, including formate and succinate, further indicate alterations in microbial activity and fermentation patterns. 

Looking Beyond a Single Biomarker 

For practicing veterinarians, the key value of metabolomics is its ability to move assessment beyond BHB alone. Blood profiles capture systemic changes in ketone bodies, fatty acids and amino acids; milk and urine offer additional monitoring matrices; while liver, muscle, adipose tissue and rumen fluid reveal tissue-specific and fermentation-related disturbances. Together, these metabolic signatures provide a more comprehensive picture of ketosis and may help identify biomarkers for earlier detection and metabolic monitoring as metabolomic technologies continue to develop.

References:

1. Zhang Y, Wang C, Khan MZ, Ju Z, Huang J. Current Understanding of Bovine Ketosis: From Molecular Basis to Farm-Level Management. Animals. 2025 Dec 18;15(24):3644. https://www.mdpi.com/2076-2615/15/24/3644
2. Zhang H, Wu L, Xu C, Xia C, Sun L, Shu S. Plasma metabolomic profiling of dairy cows affected with ketosis using gas chromatography/mass spectrometry. BMC Veterinary Research. 2013 Sep 26;9(1):186. https://link.springer.com/content/pdf/10.1186/1746-6148-9-186.pdf
3. Li Y, Xu C, Xia C, Zhang HY, Sun LW, Gao Y. Plasma metabolic profiling of dairy cows affected with clinical ketosis using LC/MS technology. Veterinary Quarterly. 2014 Jul 3;34(3):152-8. https://www.tandfonline.com/doi/pdf/10.1080/01652176.2014.962116