Hypophosphatasia (HPP) is the dento-osseous disorder caused by deactivating mutation(s) of ALPL, the gene that encodes the \"tissue-nonspecific\" isoenzyme of alkaline phosphatase (TNSALP). In HPP, 3 natural substrates of cell-surface TNSALP accumulate extracellularly; phosphoethanolamine (PEA), inorganic pyrophosphate (PPi), and pyridoxal 5\'-phosphate (PLP). Hypophosphatasemia together with elevated plasma levels of PEA, PPi, and PLP comprise its biochemical signature. PPi can inhibit mineralization and in extracellular excess can impair bone and tooth hardening and perhaps explain weak muscle. Autosomal dominant or autosomal recessive inheritance from among more than 400 mutations of ALPL largely accounts for HPP\'s broad-ranging severity, greatest among all skeletal diseases. Pediatric HPP spans life-threatening perinatal and infantile forms, childhood forms, and odonto-HPP selectively featuring premature loss of deciduous teeth. ALPL gene testing and TNSALP supplementation therapy have bolstered familiarity with HPP, but there are new considerations for diagnosis. Herein, diagnosis of a boy\'s mild childhood HPP was delayed by missteps involving his medical and dental history, physical examination, radiographic findings, and clinical laboratory studies. We review how pediatric HPP is now identified. Prompt diagnosis while appreciating the broad-ranging severity of HPP underlies the safe and effective management of this inborn-error-of-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 B6 (B6). 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 B6 degradation product pyridoxic acid (PA), and the B6 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 B6 supplementation of HPP.

Pseudoxanthoma elasticum (PXE) is a heritable multisystem ectopic calcification disorder. The gene responsible for PXE, ABCC6, encodes ABCC6, a hepatic efflux transporter regulating extracellular inorganic pyrophosphate (PPi), a potent endogenous calcification inhibitor. Recent studies demonstrated that in addition to the deficiency of plasma PPi, the activated DDR/PARP signaling in calcified tissues provides an additional possible mechanism of ectopic calcification in PXE. This study examined the effects of etidronate (ETD), a stable PPi analog, and its combination with minocycline (Mino), a potent inhibitor of DDR/PARP, on ectopic calcification in an Abcc6-/- mouse model of PXE. Abcc6-/- mice, at 4 weeks of age, before the development of ectopic calcification, were treated with ETD, Mino, or both for 18 weeks. Micro-computed tomography, histopathologic examination, and quantification of the calcium content in Abcc6-/- mice treated with both ETD and Mino revealed further reduced calcification than either treatment alone. The effects were associated with reduced serum alkaline phosphatase activity without changes in plasma PPi concentrations. These results suggest that ETD and Mino combination therapy might provide an effective therapeutic approach for PXE, a currently intractable disease.

Inorganic pyrophosphate (PPi) is the endogenous inhibitor for vascular calcification (VC). The present study was to investigate the effects of adenosine disodium triphosphate (ADTP) and alendronate sodium (AL), two exogenous PPi sources, on the atheromatous calcification (AC) in Apolipoprotein E knockout (ApoE KO) mice. ApoE KO mice were randomly divided into five groups: ApoE KO group, ApoE KO + ADTP (Low) group, ApoE KO + ADTP (High) group, ApoE KO + AL (Low) group and ApoE KO + AL (High) group. The mice in ApoE KO + ADTP (Low) group and ApoE KO + ADTP (High) group were intraperitoneally injected with ADTP with dose of 0.5 and 1.0 mg/kg/day for 2 months respectively. The mice in ApoE KO + AL (Low) group and ApoE KO + AL (High) group were intraperitoneally injected with AL with dose of 0.6 and 1.2 mg/kg/day for 2 months respectively. The age matched C57 mice were used as control group. All ApoE KO and C57 mice were fed with normal chow throughout the experiment. The calcification was evaluated using von Kossa method. The contents of PPi, triglyceride (TG), total cholesterol (TC), high density lipoprotein (HDL) and low density lipoprotein (LDL), tumor necrosis factor α (TNF-α), interleukin-6 (IL-6), interferon-γ (IFN-γ) and interleukin-10 (IL-10) as well as the activity of alkaline phosphatase (ALP) in serum were measured. The results showed that compared with C57 mice, ApoE KO mice developed severe AC accompanied with high levels of TC, TG, LDL, IL-6, TNF-α and IFN-γ in serum and with low levels of PPi and IL-10 in serum. Both ADTP and AL dose-dependently reduced the AC in ApoE KO mice compared with that of ApoE mice, without affecting the contents of lipid profiles. In addition, ADTP and AL increased the contents of PPi and IL-10 while decreased the contents of TNF-α, IL-6 and IFN-γ in serum of ApoE KO mice, having no affection on ALP activity. The results suggested that ADTP and AL reduced AC in ApoE KO mice by increasing the PPi level and regulating the inflammation.

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

Pseudoxanthoma elasticum (PXE) is a rare ectopic calcification disorder affecting soft connective tissues that is caused by biallelic ABCC6 mutations. While the underlying pathomechanisms are incompletely understood, reduced circulatory levels of inorganic pyrophosphate (PPi)-a potent mineralization inhibitor-have been reported in PXE patients and were suggested to be useful as a disease biomarker. In this study, we explored the relation between PPi, the ABCC6 genotype and the PXE phenotype. For this, we optimized and validated a PPi measurement protocol with internal calibration that can be used in a clinical setting. An analysis of 78 PXE patients, 69 heterozygous carriers and 14 control samples revealed significant differences in the measured PPi levels between all three cohorts, although there was overlap between all groups. PXE patients had a ±50% reduction in PPi levels compared to controls. Similarly, we found a ±28% reduction in carriers. PPi levels were found to correlate with age in PXE patients and carriers, independent of the ABCC6 genotype. No correlations were found between PPi levels and the Phenodex scores. Our results suggest that other factors besides PPi are at play in ectopic mineralization, which limits the use of PPi as a predictive biomarker for severity and disease progression.

Pseudoxanthoma elasticum (PXE) is characterized by low levels of inorganic pyrophosphate (PPi) and a high activity of tissue-nonspecific alkaline phosphatase (TNAP). Lansoprazole is a partial inhibitor of TNAP. The aim was to investigate whether lansoprazole increases plasma PPi levels in subjects with PXE. We conducted a 2 × 2 randomized, double-blind, placebo-controlled crossover trial in patients with PXE. Patients were allocated 30 mg/day of lansoprazole or a placebo in two sequences of 8 weeks. The primary outcome was the differences in plasma PPi levels between the placebo and lansoprazole phases. 29 patients were included in the study. There were eight drop-outs due to the pandemic lockdown after the first visit and one due to gastric intolerance, so twenty patients completed the trial. A generalized linear mixed model was used to evaluate the effect of lansoprazole. Overall, lansoprazole increased plasma PPi levels from 0.34 ± 0.10 µM to 0.41 ± 0.16 µM (p = 0.0302), with no statistically significant changes in TNAP activity. There were no important adverse events. 30 mg/day of lansoprazole was able to significantly increase plasma PPi in patients with PXE; despite this, the study should be replicated with a large number of participants in a multicenter trial, with a clinical end point as the primary outcome.

Ectopic calcification is characterized by inappropriate deposition of calcium mineral in nonskeletal connective tissues and can cause significant morbidity and mortality, particularly when it affects the cardiovascular system. Identification of the metabolic and genetic determinants of ectopic calcification could help distinguish individuals at the greatest risk of developing these pathological calcifications and could guide development of medical interventions. Inorganic pyrophosphate (PPi ) has long been recognized as the most potent endogenous inhibitor of biomineralization. It has been intensively studied as both a marker and a potential therapeutic for ectopic calcification. Decreased extracellular concentrations of PPi have been proposed to be a unifying pathophysiological mechanism for disorders of ectopic calcification, both genetic and acquired. However, are reduced plasma concentrations of PPi a reliable predictor of ectopic calcification? This perspective article evaluates the literature in favor and against a pathophysiological role of plasma versus tissue PPi dysregulation as a determinant of, and as a biomarker for, ectopic calcification. © 2023 American Society for Bone and Mineral Research (ASBMR).

The coexistence of calcium pyrophosphate dihydrate crystal deposition (CPP) and synovial chondromatosis (SC) in the temporomandibular joint (TMJ) is rarely reported. CPP disease (CPPD) is complex arthritis synonymous with excessive pyrophosphate production and variable aberrations in mineral and organic phase metabolism of the joint cartilage, leading to local inundated CPP and crystal deposition of partially deciphered predispositions. Meanwhile, SC is a rare benign synovial joint proliferative disease of unclear etiology and has a low risk of malignant transformation. However, SC manifests severe joint disability and dysfunction because of connective tissue metaplasia of the synovial membrane, which forms cartilaginous nodules with or without calcifications or ossifications. These nodules often detach and form intra-articular loose bodies and very rarely within extraarticular spaces.
We report the case of a 61-year-old man to expand the body of literature on these unusual coexisting arthropathies of the TMJ. The patient presented to our hospital in 2020 with complaints of pain in the right TMJ and trismus for over 6 months. Radiographic assessments of the TMJ provided a preoperative provisional diagnosis of SC. However, the histopathology of the open biopsy revealed tumor-like lesions comprising several deposits of rhomboid and rod-shaped crystals that displayed positive birefringence in polarized light, confirming a coexistence of CPPD. A second-stage operation was performed for the complete removal of the loose bodies and chalk-like lesions including synovectomy. No evidence of recurrence was recorded after a follow-up of nearly 1.5 years.
Isolated CPPD and SC of the TMJ are prevalent in the literature however, monoarticular coexistence of these diseases is rare, due to the lack of consistency in the diagnostic criteria in clinical practice. Moreover, optimal treatment depends on several considerations. This report delineated the molecular etiopathology and underscored the need for continued deciphering of the causal mechanisms of coexisting CPPD and SC of the TMJ. In addition, the importance of confirmatory testing for accurate diagnosis, and appropriate management of these diseases were discussed.

Generalised arterial calcification of infancy (GACI) is an ultra-rare life-threatening genetic disorder. Arterial calcification is identified during foetal ultrasound scan (USS) as increased cardiac and/or vascular echogenicity. Inorganic pyrophosphate (PPi) is the main inhibitor of arterial calcification. Pathogenic variants in ENPP1, ABCC6 and NT5E causing low PPi lead to ectopic calcifications. Rheumatoid arthritis (RA) is an acquired condition that can also lead to arterial calcification in adults. We present an extremely rare case of a transient GACI-like condition identified during foetal echocardiogram of an infant born to a mother diagnosed with RA, which spontaneously resolved postnatally. This case highlights that foetal ultrasound scans of pregnant women with RA should be carefully evaluated for cardiovascular calcifications.