Identification of hexose hydrolysis products in metabolic flux analytes: A case study of levulinic acid in plant protein hydrolysate

Biosynthetically directed fractional 13C labeling, a popular methodology of metabolic flux analysis, involves culture on a mixture of 13C and 12C substrates and preparation a ‘metabolic flux analyte’ (typically protein hydrolysate) from the biomass. Metabolic flux analytes prepared from complex eukaryotes may contain additional compounds than those prepared from microorganisms. We report the presence of such compounds (hexose hydrolysis products) in a plant metabolic flux analyte (acid hydrolyzed protein from soybean embryos). We designed NMR experiments to systematically identify these compounds, and found that they were levulinic acid (LVA; major) and hydroxyacetone (HyA; minor). These acid hydrolysis products of hexoses (glucose and mannose) were generated during acid hydrolysis of glycosylating sugars (glucosamine and mannose) associated with soybean embryo protein. Analysis of LVA by two-dimensional [13C, 1H] NMR and measurement of its J-coupling constants revealed long-range coupling between atoms C3 and C5, which enables LVA to provide more isotopomer information than its precursor hexose. Furthermore, we found that LVA and HyA preserve the isotopomeric composition of the metabolic hexose from which they are derived. An important consequence of these results is that comparison of LVA and HyA isotopomers from two separate metabolic flux analytes (protein hydrolysate and starch hydrolysate) from the same plant tissue can distinguish between parallel glycolysis and pentose phosphate pathways in different subcellular compartments.