Abstract
Detection and quantification of lysine degradation metabolites in plasma is necessary for the diagnosis and follow-up of diseases such as pyridoxine-dependent epilepsy. The principal metabolites involved in the disease are related to the first steps of lysine oxidation, either through the saccharopine or the pipecolate pathways. Currently, there are three different analytical methods used to assess the content of these metabolites in urine and plasma, but they require different sample preparations and analytical equipment. Here, we describe a protocol that calls for a simple sample preparation and uses liquid chromatography tandem mass spectrometry (LC–MS/MS) that allows simultaneous detection and quantification of underivatized l-saccharopine, l-aminoadipic acid, l-pipecolic acid, piperideine-6-carboxylate, l-glutamic acid, and pyridoxal-5-phosphate in plasma samples. To validate the method we analyzed the time course degradation after intraperitoneal injection of l-lysine in C57BL/6/J mice. We observed that the degradation of lysine through the saccharopine pathway reached a maximum within the first 2 h. At this time point there was an increase in the levels of the metabolites saccharopine, aminoadipic acid, and pipecolic acid by 3-, 24- and 3.4-fold, respectively, compared to time zero levels. These metabolites returned to basal levels after 4–6 h. In conclusion, we have developed a LC–MS/MS approach, which allows simultaneous analysis of lysine degradation metabolites without the need for derivatization.Electronic supplementary materialThe online version of this article (doi:10.1186/s40064-016-1809-1) contains supplementary material, which is available to authorized users.
Highlights
Lysine oxidation in mammals is essential for regulating the free levels of this amino acid, for the balance of nitrogen and conversion to ketone bodies
The principal metabolite of lysine degradation detected in the mice plasma was aminoadipic acid (AAA), which reached a peak of approximately 70 μM in the plasma 1 h after IP lysine injection (Fig. 3a). This value represents a 24-fold increase compared to the basal AAA values seen at zero time point that was estimated at approximately 3 μM. This value is similar to the 4 μM basal AAA concentration found in normal mouse plasma, as reported in the mouse multiple tissue metabolome database (MMMDB) (Sugimoto et al 2012) and in the human metabolome database (HMDB 2.0) (Vallat et al 1996; Wishart et al 2013) These results suggest that lysine is rapidly oxidized to AAA reaching a maximum within the first 2 h after lysine administration
The method did not detect AASA but since this compound is in chemical equilibrium with its cyclic form P6C, the detection of the latter is highly correlated to AASA levels (Struys et al 2012)
Summary
Lysine oxidation in mammals is essential for regulating the free levels of this amino acid, for the balance of nitrogen and conversion to ketone bodies. The first step of this pathway is catalyzed by lysine-ketoglutarate reductase (LKR), which condenses lysine and α-ketoglutaric acid to form saccharopine (SAC) (Arruda et al 2000). PDE is caused by a mutation in the aldh7a1 gene that abolishes the AASADH activity and patients accumulate high levels of AASA/P6C and pipecolic acid (PIP) (Mills et al 2006). Most of the experiments conducted to verify the levels of saccharopine or the absence of AASS activity in cerebral tissues were performed in mice without lysine supplementation and/or after several hours following intraperitonial lysine injection (Sauer et al 2011; Posset et al 2015). We propose a LC–MS/MS approach capable of detecting and quantifying all of these metabolites in a single method without derivatization. It was our primary interest to validate the method using a lysine-injected mouse model by monitoring the time course of lysine-derived metabolite levels in peripheral blood
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