Effects of food, fasting, and exercise on plasma pyrophosphate levels and ENPP1 activity in healthy adults

Background: Inorganic pyrophosphate (PPi) is highly regulated as it plays a critical role in the regulation of physiological mineralization. Dysregulation of plasma PPi is associated with skeletal hypomineralization and pathogenic mineralization in soft connective tissue, arteries, and heart valves. There is no standard approach to measuring PPi, making it difficult to establish PPi as a biomarker of mineralization disorders. This study aims to determine the impact of time of day, meals, or exercise on plasma PPi homeostasis using a highly sensitive PPi assay.Methods: In this single-center trial, a clinical laboratory improvement amendment (CLIA) validated modified sulfurylase-based adenosine 5-triphosphate (ATP) assay was used to measure PPi levels throughout the day in 10 healthy adults under 3 conditions; normal diet (non-fasting), fasting, and normal diet with exercise. Serum ectonucleotide pyrophosphatase/phosphodiesterase 1 activity (ENPP1; an enzyme that produces PPi) was also measured to determine whether these conditions influence PPi levels through ENPP1 activity.Results: There is a circadian increase in mean PPi levels under fasting and non-fasting conditions between 8 am and 6 pm, followed by a rapid return to baseline overnight. A circadian increase in ENPP1 activity was also measured under fasting but was lost under non-fasting conditions. Meals increased the individual variability of PPi levels when compared to the same individual fasting. PPi levels and ENPP1 activity exhibited a short-term increase after intense exercise. We found PPi ranges from 1465 nM to 2969 nM (mean 2164 nM) after fasting overnight. Within this range, there was lower intra-subject variability in PPi, suggesting that each individual has a uniquely regulated normal PPi range. Conclusion: Plasma levels of PPi can be reliably measured after an overnight fast and show promise as a biomarker of mineralization disorders.