Abstract
We have developed a highly sensitive and specific method for quantification of salivary 3-hydroxybutyrate (3HB), 3-hydroxyisobutyrate (3HIB), 3-hydroxy-3-methylbutyrate (3HMB) and 2-hydroxybutyrate (2HB), which could be new non-invasive biomarkers for catabolic pathways of fatty acids/ketogenic amino acids, valine, leucine, and methionine/threonine/α-ketobutyrate, respectively. The four hydroxybutyrates (3HB, 3HIB, 3HMB, and 2HB) were extracted from 5 µl of saliva, converted to 2-pyridylmethyl (2PM) ester derivatives, and measured by liquid chromatography–tandem mass spectrometry in positive electrospray ionization mode. [13C4]3HB was used as an internal standard. The detection limits for the 2PM esters were <1 pg (7.9–9.6 fmol) on-column (signal-to-noise ratio = 3). Reproducibilities and recoveries of the hydroxybutyrates were validated according to one-way layout and polynomial equation, respectively. The variances between sample preparations and between measurements were calculated to be 0.45–5.28 and 0.54–3.45 %, respectively. Experiments performed using 5 µl of saliva spiked with 3.8–154.4 pmol of the four hydroxybutyrates gave recoveries of 98.5 to 108.8 %, with a mean recovery of 104.1 %. In vitro experiments in hepatocytes or skeletal muscle cells showed that addition of palmitic acid, valine, leucine or α-ketobutyrate to culture medium markedly increased the targeted hydroxybutyrate concentrations. The salivary concentration of each targeted hydroxybutyrate was positively correlated with that in serum, and the salivary levels were elevated in patients with liver cirrhosis, which is characterized by upregulated catabolism of lipids and amino acids. The proposed method is useful for quantification of salivary 3HB, 3HIB, 3HMB, and 2HB for monitoring of catabolic activities of amino acids and fatty acids.Electronic supplementary materialThe online version of this article (doi:10.1186/s40064-015-1304-0) contains supplementary material, which is available to authorized users.
Highlights
Energy sources fluctuate among carbohydrates, lipids, and amino acids depending on nutritional and metabolic status, such as feeding, fasting, and exercise
Fatty acids are oxidized to acetyl-CoA in the mitochondrion (β-oxidation) and used in the TCA cycle, and excess acetyl-CoA in the liver is further metabolized to ketone bodies to supply energy to non-hepatic tissues, mainly brain and skeletal muscles (Fig. 1) (Laffel 1999)
selected reaction monitoring (SRM) chromatograms obtained for m/z 210 →
Summary
Energy sources fluctuate among carbohydrates, lipids, and amino acids depending on nutritional and metabolic status, such as feeding, fasting, and exercise. Fatty acids are oxidized to acetyl-CoA in the mitochondrion (β-oxidation) and used in the TCA cycle, and excess acetyl-CoA in the liver is further metabolized to ketone bodies to supply energy to non-hepatic tissues, mainly brain and skeletal muscles (Fig. 1) (Laffel 1999). Amino acids are an important energy source under conditions lacking glucose, and ketone bodies could be produced from ketogenic amino acids in the liver in such metabolic status (Fig. 1). In humans, branched-chain amino acids (BCAAs), valine (VAL), leucine (LEU) and isoleucine (ILE), are metabolized to succinyl-CoA or acetyl-CoA exclusively in skeletal muscle mitochondria and used in the TCA cycle (Shimomura et al 2006; Platell et al 2000; Rennie et al 2006; Kong et al 2012).
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