Plasma amino acid profile as a biomarker of malate-aspartate shuttle overload in a hepatic glycolytic defect (PFKFB1): a pre/post supplementation case study

Abstract

Background: it has been proposed that the hepatic glycolytic blockade associated with a hemizygous PFKFB1 variant (p.Ser159Thr) causes cytosolic NADH accumulation, with a secondary increase in demand on the malate-aspartate shuttle (MAS) — the principal mechanism for transferring reducing equivalents from the cytosol to the mitochondria in the hepatocyte. In the index case (companion report cited above), a selective deficit of aspartate and glutamine was documented, with the remainder of the amino acid profile within range, in the context of adequate protein intake — a pattern biochemically inconsistent with a nutritional deficiency and compatible with MAS overload. Objective: to describe the evolution of this participant's plasma amino acid profile before and after a 40-day intervention that included targeted supplementation with magnesium aspartate (200 mg/day), in the context of the naturopathic and dietary optimization of the ketogenic bypass described in the companion report. Methods: pre/post comparison of serum/plasma amino acid profiles: baseline (May 2026, prior to starting magnesium aspartate supplementation) and post-intervention (10 July 2026, four days after the end of the 40-day phase). Single-case study, no control group, exploratory and hypothesis-generating in nature. Results: aspartate rose from <1 µmol/L (below the lower reference limit, 2–21) to 4.8 µmol/L (within range). Glutamine rose from 351 µmol/L (below the lower limit, 361–856) to 376 µmol/L (within range, close to the lower limit). The rest of the amino acid profile remained largely within range, with some elevations (branched-chain amino acids, glycine, taurine, ornithine) discussed with caution given a change of laboratory and reference ranges between the two determinations. Conclusions: normalization of aspartate and glutamine following targeted supplementation is consistent with the hypothesis of MAS overload secondary to the PFKFB1 glycolytic blockade, and with experimental evidence that aspartate supplementation can restore the NAD+/NADH ratio in cellular models with MAS defects. However, a single post-intervention timepoint, the change of laboratory, and the impossibility of isolating the effect of magnesium aspartate from the rest of the Phase II intervention preclude establishing causality. These findings are hypothesis-generating and require confirmation through targeted metabolomics and controlled designs