Pyridoxine challenge reflects pediatric hypophosphatasia severity and thereby examines tissue-nonspecific alkaline phosphatase's role in vitamin B6 metabolism

Alkaline phosphatase (ALP) is detected in most human tissues. However, ALP activity is routinely assayed using high concentrations of artificial colorimetric substrates in phosphate-free laboratory buffers at lethal pH. Hypophosphatasia (HPP) is the inborn-error-of-metabolism caused by loss-of-function mutation(s) of the ALPL gene that encodes the ALP isoenzyme expressed in bone, liver, kidney, and elsewhere and is therefore designated "tissue-nonspecific" ALP (TNSALP). Consequently, HPP harbors clues concerning the biological function of this phosphohydrolase that is anchored onto the surface of cells. The biochemical signature of HPP features low serum ALP activity (hypophosphatasemia) together with elevated plasma levels of three natural substrates of TNSALP: i) phosphoethanolamine (PEA), a component of the linkage apparatus that binds ALPs and other proteins to the plasma membrane surface; ii) inorganic pyrophosphate (PPi), an inhibitor of bone and tooth mineralization; and iii) pyridoxal 5'-phosphate (PLP), the principal circulating vitameric form of vitamin B-6 (B-6). Autosomal dominant and autosomal recessive inheritance involving several hundred ALPL mutations underlies the remarkably broad-ranging expressivity of HPP featuring tooth loss often with muscle weakness and rickets or osteomalacia. Thus, HPP associates the "bone" isoform of TNSALP with biomineralization, whereas the physiological role of the "liver", "kidney", and other isoforms of TNSALP remains uncertain. Herein, to examine HPP's broad-ranging severity and the function of TNSALP, we administered an oral challenge of pyridoxine (PN) hydrochloride to 116 children with HPP. We assayed both pre- and post-challenge serum ALP activity and plasma levels of PLP, the B-6 degradation product pyridoxic acid (PA), and the B-6 vitamer pyridoxal (PL) that can enter cells. Responses were validated by PN challenge of 14 healthy adults and 19 children with metabolic bone diseases other than HPP. HPP severity was assessed using our HPP clinical nosology and patient height Z-scores. PN challenge of all study groups did not alter serum ALP activity in our clinical laboratory. In HPP, both the post-challenge PLP level and the PLP increment correlated (Ps < 0.0001) with the clinical nosology and height Z-scores (Rs = +0.6009 and + 0.4886, and Rs = -0.4846 and - 0.5002, respectively). In contrast, the plasma levels and increments of PA and PL from the PN challenge became less pronounced with HPP severity. We discuss how our findings suggest extraskeletal TNSALP primarily conditioned the PN challenge responses, and explain why they caution against overzealous B-6 supplementation of HPP.