Background:​ Inorganic pyrophosphate (PP_i) is highly regulated as it plays a critical role in the regulation of physiological mineralization. Dysregulation of plasma PP_i is associated with skeletal hypomineralization and pathogenic mineralization in soft connective tissue, arteries, and heart valves. There is no standard approach to measuring PP_i, making it difficult to establish PP_i as a biomarker of mineralization disorders. This study aims to determine the impact of time of day, meals, or exercise on plasma PP_i homeostasis using a highly sensitive PP_i 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 PP_i 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 PP_i) was also measured to determine whether these conditions influence PP_i levels through ENPP1 activity.

Results:​ There is a circadian increase in mean PP_i 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 PP_i levels when compared to the same individual fasting. PP_i levels and ENPP1 activity exhibited a short-term increase after intense exercise. We found PP_i ranges from 1465 nM to 2969 nM (mean 2164 nM) after fasting overnight. Within this range, there was lower intra-subject variability in PP_i, suggesting that each individual has a uniquely regulated normal PP_i range.

Conclusion:​ Plasma levels of PP_i can be reliably measured after an overnight fast and show promise as a biomarker of mineralization disorders.