Alkaline Phosphatase Isoenzymes Research Articles

P043 Fascinating phosphate: seek and you will find

Abstract Background/Aims A 62-year-old accountant was referred to the metabolic bone clinic with a 2 year history of thoracic back pain and a persistently raised ALP. There were no associated red flags for her back pain. She had a history of a gluteal lump, thought to be benign, for which she had declined excision. She has hypertension and had a previous navicular fracture. Medications included bendroflumethiazide and Adcal D3. She had restriction in neck movements and was tender to percussion in the thoracic spine. There was no proximal weakness or focal neurology. Systems and joint exam were unremarkable. Methods The case is discussed below. Results Salient abnormal results on presentation were a raised ALP of 207 and corrected calcium of 2.34. PTH was elevated at 8.2 (NR:1.6-6.9), Vitamin D 79 and a low phosphate of 0.34 (NR:0.8-1.50). Alkaline phosphatase isoenzymes showed the raised level came from bone. Protein and urine electrophoresis were normal. A bone density scan was normal, and a recent thoracic MRI showed only degenerative change. An isotope bone scan was requested and was reported to show increased activity in the nasal bone, maxilla and both orbits, raising the possibility of Paget’s disease. However, when reviewed in the Radiology meeting with a skull x-ray, the appearances were not felt to be in keeping with Paget’s. Despite physiotherapy, hydrotherapy, acupuncture and neuropathic medication the patient’s back pain continued. Her phosphate remained low and her calculated tubular reabsorption of phosphate from a 24h urine collection (TmP/GFR) was low at 0.42mmol/l (NR 0.80-1.35). She was started on phosphate replacement and calcitriol, and Adcal D3 was continued. The Fibroblast Growth Factor (FGF) 23 levels were sent and came back significantly elevated at 1380 (NR < 100). A 68Ga DOTA-TATE scan (whole body PET/CT scan), confirmed the right gluteal lump as the source of the FGF 23. The patient went on to have an excision biopsy and histology confirmed a mesenchymal tumour of the right buttock. Her phosphate replacement was gradually weaned, but on reducing the dose phosphate levels dropped and her symptoms returned. The repeat TmP/GFR was again low at 0.61, and FGF 23 levels were still raised at 204. A repeat 68Ga DOTA-TATE scan, 4 years after the first one, showed recurrence of the right gluteal lesion and a possible small lesion in the left gluteal muscle. She has been sent for further excision. Conclusion Tumour induced osteomalacia (TIO) is a rare condition and should be considered in cases of hypophosphataemia. Classical symptoms are proximal weakness and muscle and bone pain. They are typically associated with small benign tumours (most commonly mesenchymal tumours) which may be difficult to find. Excision is curative but if small amounts of tumour remain, relapses may occur. Disclosure J. Bakshi: None. C. Batten: None.

Hypophosphatasia screening in pediatric age based on records

Introduction: hypophosphatasia (HPP) is a congenital disease, characterized by a defect in bone and dentary mineralization, secondary to a deficiency in the biosynthesis of non-specific tissue isoenzyme of alkaline phosphatase (TNSALP), resulting in decreased levels of alkaline phosphatase (ALP) activity and the extracellular accumulation of its substrates. Our goal was to establish the interest of screening the hospital databases of pediatric patients with low levels of ALP for the diagnosis of HPP. Materials and methods: during the period from September 2016 to September 2017, 23231 patients were tested for ALP, 1752 of them showed low levels of ALP. Based on the clinic, basal disease and that in previous analyses they did not present ALT norm values, 14 cases were selected: 8 cases associated with early puberty, 1 case of low size, 1 case of liver disease and myasthenia and 1 case of teething problems. ALPL gene was studied in those cases. Results: the latter case was of an 8-year-old male with a pathological variant of HPP: c.343_348dupACCGCC (p.Thr115_Ala116dup) in exon 5 in dominant heterozygous, inherited from his mother. At the substrate level the levels of pyridoxal-5-phosphate were above 50μg/L. Conclusion: the diagnostic strategy with a high index of clinical suspicion of HPP should include the observation of low levels of serum ALP activity. Our study shows that HPP is an underdiagnosed disease.An appropriate protocol to detect HPP in a clinical setting in tertiary care hospitals is required.

Hypophosphatasia: is it an underdiagnosed disease even by expert physicians?

Hypophosphatasia (HPP) is caused by mutations in the ALPL that encodes the tissue-nonspecific isoenzyme of alkaline phosphatase (ALP). Clinical manifestations range from extreme life-threatening lethal forms to no signs or symptoms at all. Consecutive 30,000 outpatients and inpatients with ALP data were screened retrospectively, out of which 1000 patients were found to have low levels of ALP more than once. Then, patients were evaluated for the symptoms and signs of HPP with further biochemical and genetic analyses. Thirty-seven patients who had severe musculoskeletal pain, recurrent fractures, and tooth anomalies were then screened with substrate and DNA sequencing analyses for HPP. It was determined that eight patients had variants in the ALPL gene. A total of eight different ALPL variants were identified in eight patients. The variants, namely c.244G > C (p.Gly82Arg), c.1444C > T (p.His482Tyr), c.1487A > G (p.Asn493Ser), and c.675_676insCA (p.Met226GlnfsTer52), had not been previously reported. Considering the wide spectrum of clinical signs and symptoms, HPP should be among the differential lists of bone, muscle, and tooth abnormalities at any age.

Intestinal alkaline phosphatase modulation by food components: predictive, preventive, and personalized strategies for novel treatment options in chronic kidney disease.

Alkaline phosphatase (AP) is a ubiquitous membrane-bound glycoprotein that catalyzes phosphate monoesters' hydrolysis from organic compounds, an essential process in cell signaling. Four AP isozymes have been described in humans, placental AP, germ cell AP, tissue nonspecific AP, and intestinal AP (IAP). IAP plays a crucial role in gut microbial homeostasis, nutrient uptake, and local and systemic inflammation, and its dysfunction is associated with persistent inflammatory disorders. AP is a strong predictor of mortality in the general population and patients with cardiovascular and chronic kidney disease (CKD). However, little is known about IAP modulation and its possible consequences in CKD, a disease characterized by gut microbiota imbalance and persistent low-grade inflammation. Mitigating inflammation and dysbiosis can prevent cardiovascular complications in patients with CKD, and monitoring factors such as IAP can be useful for predicting those complications. Here, we review IAP's role and the results of nutritional interventions targeting IAP in experimental models to prevent alterations in the gut microbiota, which could be a possible target of predictive, preventive, personalized medicine (PPPM) to avoid CKD complications. Microbiota and some nutrients may activate IAP, which seems to have a beneficial impact on health; however, data on CKD remains scarce.

Comparison of Heat Fractionation and Gel Electrophoresis Methods for the Quantitative Determination of Alkaline Phosphatase Isoenzymes

Abstract Introduction Alkaline phosphatase (ALP) is important in the diagnostic work-up for hepatobiliary and bone diseases. ALP isoenzymes are expressed in the bone, liver, kidney, placenta, and intestine, and vary in heat stability and electrophoretic mobility. Distinguishing the different ALP isoenzymes is clinically important for the diagnosis of pathologies associated with elevated ALP activity. Current modalities available to measure ALP isoenzymes utilize the heat stability, electrophoretic mobility, and immunochemical properties of the isoenzymes. The differences inherent in these methods allow for unique benefits of each method in identifying ALP isoenzymes. The objective of this study was to compare bone, liver, and placental ALP isoenzyme results determined by heat fractionation and gel electrophoresis and to characterize the heat-stable non-liver fraction (t1/2 >11 min), reported by heat fractionation, using gel electrophoresis. Methods A total of 72 de-identified serum samples that span a wide range of known ALP isoenzyme concentrations and disease states were used to measure ALP using gel electrophoresis and heat fractionation. Heat fractionation was achieved by selective inactivation of the isoenzymes at 56 °C in 10, 15, and 20-minute intervals. Log-percent activity of the total and heat-inactivated fractions at each time point was plotted against time in minutes. The linear activity decay between 10 and 20 minutes determined the relative amount of liver isoenzyme activity and the slope of the line determined the half-lives of ALP isoenzymes. Electrophoresis was performed according to the manufacturer’s protocol using the Hydragel ISO-PAL gel to resolve ALP isoenzymes based on their electrophoretic mobility and interaction with lectin. ALP isoenzymes were quantified by densitometry. Results Our results show a significant correlation coefficient (r) of 0.98, Deming regression slope of 1.1, and bias of -1.2% for the liver isoenzyme (n=43). However, liver fractions are not distinguishable by heat fractionation when heat-stable isoforms are present. The bone fraction (n=43) showed a coefficient of correlation of 0.86, slope of 0.55, and bias of -31%. Although, with a small sample size (n=6), the placental isoenzyme showed a significant agreement between the two methods: r = 0.999, slope = 0.98, and a -3.5% bias. Of the non-liver fractions reported by heat fractionation (n=13, ALP >100 U/L) eleven (85%) showed distinct qualitative bands in the intestinal lane on gel electrophoresis; however, quantitative values did not correlate between the two methods. Conclusion Our data support an agreement between the heat fractionation and gel electrophoresis methods for the quantitative determination of liver and placental alkaline phosphatase isoenzymes. Although there is an association between the two methods, the activity of the bone isoenzyme was underestimated by the gel electrophoresis method, likely due to saturation of the gel and densitometry scan because of elevated protein concentrations. The non-liver fractions were qualitatively identified as intestinal isoenzyme.

Peripartum changes in serum activities of three major alkaline phosphatase isoenzymes in Holstein dairy cows.

This study investigated changes in serum levels of hepatic, bone, and intestinal alkaline phosphatase (ALP) isoenzymes (ALP2, ALP3, and ALP5, respectively) in Holstein cows around parturition. Tartrate-resistant acid phosphatase 5b (TRAP5b) activity and calcium (Ca) concen-trations were also measured. We analyzed blood samples from 11 late-pregnant heifers (primipa-rous group) and 13 multiparous (2-4 lactations; multiparous group) cows at 3 weeks (18-24 days prepartum; -3 weeks), 2 weeks (17-11 days prepartum; -2 weeks), and 1 week (10-4 days prepar-tum; -1 weeks) before parturition; the day of calving (within 12 h post-calving; day 0); and 5 days postpartum (5 days). ALP3 activity was significantly higher in the primiparous group than in the multiparous group, whereas the activities decreased significantly in both groups after 5 days. ALP2 and ALP5 activities did not change, whereas ALP2 activity was significantly higher in the primiparous group than in the multiparous group. TRAP5b activity was significantly higher in the primiparous group than in the multiparous group and showed a transient significant increase at day 0. Ca concentration significantly decreased at day 0 in both groups; the Ca level at day 0 was significantly higher in the primiparous group than in the multiparous group. These data show that ALP3 activity in serum may indicate a change in osteoblastic bone forma-tion after calving, but further study is needed to determine the clinical application for measuring ALP isoenzymes in bovine medicine.

Single-Molecule Analysis Determines Isozymes of Human Alkaline Phosphatase in Serum.

Alkaline phosphatase (ALP) is an important biomarker, as high levels of ALP in blood can indicate liver disease or bone disorders. However, current clinical blood tests only measure the total concentration of ALP but are unable to distinguish enzyme isotypes. Here, we demonstrate a novel and rapid approach to profile various ALP isozymes in blood via a single-molecule-analysis platform. The microarray platform provides enzyme kinetics of hundreds of individual molecules at high throughput. Using these single molecule kinetics, we characterize the different activity profiles of ALP isotypes. By analyzing both healthy and disease samples, we found the single molecule activity distribution of ALP in serum reflects the health status of patients. This result demonstrates the potential utility of the method for improving the conventional ALP test, as well as for analyzing other enzymatic biomarkers, including enzyme isotypes.

An Explosive Workup for Diarrhea: The Cascade Effect From an Incidental Finding on a Laboratory Panel.

A 19-month-old boy with a medical history of 1 febrile seizure presented to the emergency department (ED) with 2 days of vomiting and 1 day of diarrhea. He was accompanied by his non–English-speaking mother, who reported several episodes of nonbloody, nonbilious emesis and dark brown, watery stools. The patient had decreased intake of solid foods, but he had been breastfeeding well. Because of his diarrhea, the patient’s mother was unsure how many voids he had had in the past day. He had not had fevers. The family history was significant for the patient’s father recently being diagnosed with hepatitis C. The patient’s mother brought him into the ED because of his symptoms and concern that he looked “yellow.” In the ED, the patient was afebrile with normal vital signs for his age (including heart rate of 146 beats per minute), and he was “well appearing and active” with moist mucous membranes and a capillary refill of <2 seconds. However, the ED provider additionally documented that the patient appeared “moderately dehydrated [and] slightly jaundiced,” and he ordered a comprehensive metabolic panel (CMP). All values were within normal limits, including total bilirubin, except for a low bicarbonate of 16 mmol/L (normal 21–30 mmol/L) and an elevated alkaline phosphatase (ALP) of 926 U/L (normal 129–291 U/L). The provider consulted endocrinology for the elevated ALP, who noted a broad differential for elevated ALP and that transient hyperphosphatasemia is a diagnosis of exclusion. They recommended checking 25-hydroxyvitamin D, phosphate, and parathyroid hormone to evaluate for underlying parathyroid, vitamin D, and bone disease. They also wrote that the team could check total and isoenzymes of ALP “if desiring further testing to help confirm [the] diagnosis of transient hyperphosphatasemia.” The ED provider obtained these recommended laboratories, including the ALP laboratories, as well as a gamma-glutamyl transferase, acute …

Agarose gel electrophoresis pattern of serum alkaline phosphatase isoenzymes in Holstein cows during lactation.

A recent study found that an agarose gel electrophoresis (AGE) method yielded two distinct major bands corresponding to the hepatic and bone ALP isoenzymes (ALP2 and ALP3, respec-tively) in bovine serum treated with protease and neuraminidase (PN-treatment), although there were concerns that the intestinal ALP isoenzyme (ALP5) often overlapped with ALP3 in human serum treated with neuraminidase. Because ALP5 was separated from ALP3 in bovine serum treated with protease alone (P-treatment), we used a modified method employing both P- and PN-treated bovine sera to measure the activities of the three ALP isoenzymes in 53 lacta-ting Holstein cows: 24 primiparous and 29 multiparous. Upon electrophoresis, 51 of 53 samples (96.2%) subjected to P-treatment yielded a distinct fraction corresponding to ALP5, as did the control serum. All PN-treated sera yielded a definite ALP2 fraction. The ALP3 fraction was calculated as the remainder after excluding ALP2 and ALP5. The activities of total ALP (t-ALP) and ALP3 in primiparous cows were higher than those in multiparous cows (p ⟨ 0.001) at early-to-peak [10-110 days in milk (DIM)] and mid (111-220 DIM) lactation. In the multi-parous cows, the ALP3 activity at late lactation (221-477 DIM) was significantly higher than that at early-to-peak lactation. Thus, the modified AGE method described here is able to discrimi-nate three fractions of ALP isoenzymes in the sera of lactating cows. The AGE pattern of circu-lating ALP isoenzymes will contribute to the understanding of the physiological bone metabolism status in lactating cows.

Hypophosphatasia: Current Literature for Pathophysiology, Clinical Manifestations, Diagnosis, and Treatment.

Hypophosphatasia (HPP) is a rare inherited bone disorder identified by impaired bone mineralization. There are seven subtypes of HPP mainly characterized by their age of onset. These subtypes consist of perinatal (prenatal) benign, perinatal lethal, infantile, childhood, adult, odontohypophosphatasia, and pseudohypophosphatasia. Due to limited awareness of the condition, either misdiagnosis or delayed diagnosis is common. Furthermore, the condition is frequently treated with contraindicated drugs. This literature illustrates the most recent findings on the etiology, pathophysiology, clinical manifestations, diagnosing, and treatment for HPP and its subtypes. The etiology of the disease consists of loss-of-function mutations of the ALPL gene on chromosome one, which encodes for tissue nonspecific isoenzyme of alkaline phosphatase (TNAP).A decrease of TNAP reduces inorganic phosphate (Pi) for bone mineralization and allows for an increase in inorganic pyrophosphate (PPi) and phosphorylated osteopontin (p-OPN), which further reduces bone mineralization. The combination of these processes softens bone and mediates a clinical presentation similar to rickets/osteomalacia. HPP has an additional wide range of clinical features depending on its subtype.Although a concrete diagnostic guideline has not yet been established, many studies have supported a similar method of identifying HPP. Clinical features, radiological findings, and/or biomarker levels of the disorder should raise suspicion and encourage the inclusion of HPP as a differential diagnosis. Biomarkers, especially alkaline phosphatase (ALP), are major contributors to diagnosis. However, genetic testing is done for definitive diagnosis.The primary treatment for HPP is the reintroduction of TNAP as a recombinant enzyme called asfotase alfa. There are additional pharmaceutical treatments and in some cases, surgical intervention may be indicated. Pharmaceutical therapies such as bisphosphonates, denosumab, potent antiresorptive agents, and vitamin D are contraindicated in adults with HPP.We hope to raise awareness for HPP in order to prevent delayed diagnosis or misdiagnosis. We plan to encourage appropriate care and avoid treatments that may be contraindicating. We also encourage the development of a diagnostic guideline that will promote a consistently favorable patient prognosis.

Alkaline phosphatase: Structure, expression and its function in bone mineralization.

Alkaline phosphatase (ALP) is highly expressed in the cells of mineralized tissue and plays a critical function in the formation of hard tissue. The existing status of this critical enzyme should be reviewed periodically. ALP increases inorganic phosphate local rates and facilitates mineralization as well as reduces the extracellular pyrophosphate concentration, an inhibitor of mineral formation. Mineralization is the production, inside matrix vesicles, of hydroxyapatite crystals that bud from the outermembrane of hypertrophic osteoblasts and chondrocytes. The expansion of hydroxyapatite formsinto the extracellular matrix and its accumulation between collagen fibrils is observed. Among various isoforms, the tissue-nonspecific isozyme of ALP (TNAP) is strongly expressed in bone, liver and kidney and plays a key function in the calcification of bones. TNAP hydrolyzes pyrophosphate and supplies inorganic phosphate to enhance mineralization. The biochemical substrates of TNAP are believed to be inorganic pyrophosphate and pyridoxal phosphate. These substrates concentrate in TNAP deficient condition which results in hypophosphatasia. The increased level of ALP expression and development in this environment would undoubtedly provide new and essential information about the fundamental molecular mechanisms of bone formation, offer therapeutic possibilities for the management of bone-related diseases.

SUN-LB64 Urine Phosphoethanolamine Is an Underutilized Biomarker for Hypophosphastasia

Hypophosphatasia (HPP) is a rare disease caused by a loss-of-function mutation in the ALPL gene leading to a deficiency in the tissue-non-specific isoenzyme alkaline phosphatase (ALP) and excess of extracellular inorganic pyrophosphate (PPi) and pyridoxal 5’phosphate (PLP). Patients with HPP have a widely variable clinical phenotype, from neonatal seizures and hypomineralization to isolated dental or joint disease in adults. Patients present with ALP activity levels below their age-adjusted reference interval and elevations in PLP. This variable clinical phenotype leads to significant diagnostic challenges, particularly in those with minor disease manifestations. Biomarkers for diagnosing HPP and monitoring patients on enzyme replacement therapy (ERT) are limited. Low ALP activity is not specific for HPP; it can be low in other conditions including untreated hypothyroidism, and other skeletal dysplasias. Accurate PLP assessment is limited in patients on ERT due to hydrolysis of the substrate. Phosphoethanolamine (PEA) is a substrate hydrolyzed by TNSALP and elevated levels of PEA may be observed in HPP, supporting the diagnosis of HPP, but have been reported to be non-specific. We hypothesized that urine PEA levels could be used to diagnose HPP and as a surrogate marker for ERT compliance. We performed a retrospective analysis on 83 adult patients (63F: 20M) presenting for investigation or follow-up of HPP from 7/1/2014 to 1/7/2020. Urine was sent to the Children’s Hospital in Colorado for PEA testing. Patients were diagnosed with HPP if they had ALP levels persistently below the age-adjusted reference interval, were positive for an ALPL gene mutation/had a family history of HPP and had musculoskeletal/dental symptoms consistent with the disease. Patients were negative for HPP if ALP was not persistently low, vitamin B6 levels were normal without supplementation, negative ALPL genetic testing results (when available) and no musculoskeletal/dental symptoms. PEA levels were not considered for diagnosis. The following was collected from the EMR: PEA results, diagnosis, and ERT status. PEA levels were higher in patients with HPP not on ERT (median=117.0, N=39) vs. those negative for HPP (median=24.0, N=15, p<0.0001). The receiver operator curve for PEA in diagnosing HPP had an AUC of 0.94 (SE=0.03, p<0.0001). Specificity was 100% at PEA levels >53.5 ng/ml (sensitivity=79.5%) with an NPV of 65.2% and PPV of 100%. Initial PEA values in patients with HPP and not on ERT were higher (median 117.0, N=39) than patients on ERT (median 65.0, N=16, p=0.004). Patients who began ERT had a decline in PEA levels after treatment (mean decrease 68.1%). PEA is a specific diagnostic marker for HPP in patients undergoing investigation for HPP and may be used as a surrogate marker to monitor ERT compliance. Future studies are necessary to evaluate the association between PEA levels and functional performance.

O01 Hypophosphatasia in adults at a specialist centre in the UK: the spectrum of musculoskeletal disease

Abstract Background Hypophosphatasia (HPP) is a condition arising due to mutations in the gene encoding the tissue-non-specific alkaline phosphatase (TNSALP) isoenzyme (ALPL), leading to deficient activity of TNSALP. It can be inherited as an autosomal dominant or recessive disorder. There are over 300 known mutations in the ALPL gene associated with HPP, leading to varying degrees of deficiency in the activity of TNSALP. There are six clinical forms of HPP, with a great degree of heterogeneity in clinical presentation. The adult form is generally milder than perinatal and infantile forms, which may be fatal. Estimates of the prevalence of HPP range from 0.3/100,000 (more severe forms), to 1/6370 (less severe forms). Diagnosis is based on clinical features, biochemical abnormalities (low serum alkaline phosphatase (ALP), raised serum pyridoxal-5’-phosphate and raised urinary phosphoethanolamine), and genetic testing. The dentition and musculoskeletal system are often affected. Low trauma, metatarsal and atypical subtrochanteric femoral fractures may be seen, often with poor healing. However in some cases, individuals may present with calcium pyrophosphate deposition disease (CPPD) or non-specific musculoskeletal features. Under-recognition of HPP and diagnostic delay are therefore common. This retrospective observational study, performed at a specialist centre, aimed to characterise the spectrum of musculoskeletal manifestations in adults with HPP. Methods Patient records of adults with a confirmed diagnosis of HPP seen in the Rheumatology department at St George’s Hospital, London, were consulted. Clinical features at presentation and those developed subsequently were recorded, with a focus on musculoskeletal manifestations. Biochemical, genetic and radiographic data were also obtained. Results Fifteen individuals (two male, thirteen female) with a confirmed diagnosis of HPP were identified. Average delay to diagnosis was 21 years. The commonest presenting feature for the whole cohort was joint pain or swelling, followed by dental caries. The commonest presenting musculoskeletal feature in childhood was fracture (often low trauma and recurrent); in adults it was joint pain followed by poor mobility. There were eight diagnoses of osteoarthritis, five cases of tendinopathy and three of bursitis. Four patients had previously been diagnosed with psoriatic arthritis and three with pseudogout. Other musculoskeletal diagnoses included patellofemoral dysfunction, hypermobility and musculoskeletal tumours (two individuals). The upper limb joints were most commonly affected; some individuals reported back and knee pain. Musculoskeletal morbidity was high: eight individuals had poor mobility or abnormal gait, and three were unable to work through ill health. Conclusion There is a long delay to diagnosis of HPP. The condition may present in adults with non-specific musculoskeletal or dental features. It is therefore important to consider the diagnosis in individuals with a low ALP and musculoskeletal features which may suggest HPP, such as fractures or CPPD. Earlier recognition may avoid potential harm through inappropriate bisphosphonate or high-dose vitamin D therapy. Disclosures J. Kotecha None. S. Mansour None. C. Hall None. K.E. Moss None.

Healing of vitamin D deficiency rickets complicating hypophosphatasia suggests a role beyond circulating mineral sufficiency for vitamin D in musculoskeletal health.

Hypophosphatasia (HPP) is the metabolic bone disease caused by loss-of-function mutation(s) of the ALPL gene that encodes the cell-surface tissue-nonspecific isoenzyme of alkaline phosphatase (TNSALP). In HPP, extracellular accumulation of inorganic pyrophosphate (PPi), a TNSALP natural substrate and inhibitor of biomineralization, often leads to rickets or osteomalacia despite normal or sometimes elevated circulating levels of calcium (Ca) and inorganic phosphate (Pi). We report an infant girl with vitamin D deficiency rickets subsequently healed by cholecalciferol administration alone before receiving TNSALP-replacement therapy for accompanying HPP. Throughout her clinical course, circulating Ca and Pi levels were normal or elevated. At presentation with failure-to-thrive at age six months, radiographs revealed severe rickets and serum 25(OH)D was 8 ng/mL (Nl, 30-100), yet low ALP activity 55 U/L (Nl, 124-341), normal Ca 9.3 mg/dL (Nl, 8.5-10.1) and Pi 6.4 mg/dL (Nl, 3.5-7.0), and low-normal parathyroid hormone 21 pg/mL (Nl, 14-72) were instead consistent with HPP. At age nine months, after 1000 IU of cholecalciferol orally each day for six weeks, serum 25(OH)D was 86 ng/mL, strength markedly better, and radiographs documented significant improvement of rickets. At age 18 months, with fully healed vitamin D deficiency rickets, findings of underlying HPP included a waddling gait and Gower sign, metaphyseal "tongues" of radiolucency, elevated serum pyridoxal 5'-phosphate 121 ng/mL (Nl, 2-33), and bi-allelic ALPL missense mutations. Then, nearly complete restoration of strength and radiographic healing of her remaining skeletal disease from HPP occurred during asfotase alfa enzyme replacement treatment. At no time, including presentation, were circulating Ca or Pi levels compromised. Instead, and in keeping with HPP, high-normal or elevated serum Ca and Pi concentrations were consistently documented. Thus, our findings suggest some role for vitamin D in musculoskeletal health beyond assuring circulating mineral sufficiency.

Recent advances with alkaline phosphatase isoenzymes and their inhibitors.

Alkaline phosphatases are found in different living species and play crucial roles in various significant functions, such as hydrolyzing a variable spectrum of phosphate-containing physiological compounds, contributing to DNA synthesis, bone calcification, and attenuation of inflammation. They are homodimeric enzymes; each subunit contains one magnesium ion and two zinc ions crucial for the catalytic activity of the enzyme. Alkaline phosphatases exist in four distinct isoenzymes (placental, intestinal, germ cell, and tissue nonspecific alkaline phosphatases), which are expressed by four different genes; each one of them has distinguished functions. Any disturbance in the gene expression of alkaline phosphatase eventually induces serious disease conditions. Thus, the need to explore new lead inhibitors has increased recently. In this literature review, we aim to investigate the role of alkaline phosphatase in different diseases and physiological conditions and to study the structure-activity relationships of recently reported inhibitors. We focused on the lead compounds reported in the last 5 years (between 2015 and 2019).

Hyperphosphatemia with low FGF7 and normal FGF23 and sFRP4 levels in the circulation characterizes pediatric hypophosphatasia.

Hypophosphatasia (HPP) is the inborn-error-of-metabolism caused by loss-of-function mutation(s) of the ALPL gene that encodes the tissue-nonspecific isoenzyme of alkaline phosphatase (TNSALP). TNSALP in healthy individuals is on cell surfaces richly in bone, liver, and kidney. Thus, TNSALP natural substrates accumulate extracellularly in HPP, including inorganic pyrophosphate (PPi), a potent inhibitor of hydroxyapatite crystal formation and growth. Superabundance of extracellular PPi (ePPi) in HPP impairs mineralization of bones and teeth, often leading to rickets during childhood and osteomalacia in adult life and to tooth loss at any age. HPP's remarkably broad-ranging severity is largely explained by nearly four hundred typically missense mutations throughout the ALPL gene that are transmitted as an autosomal dominant or autosomal recessive trait. In the clinical laboratory, the biochemical hallmark of HPP is low serum ALP activity (hypophosphatasemia). However, our experience indicates that hyperphosphatemia from increased renal reclamation of filtered inorganic phosphate (Pi) is also common. Herein, from our prospective single-center study, we document throughout the clinical spectrum of non-lethal pediatric HPP that hyperphosphatemia reflects increased renal tubular threshold maximum for phosphorus adjusted for the glomerular filtration rate (TmP/GFR). To explore its pathogenesis, we studied mineral metabolism and quantitated circulating levels of three phosphatonins [fibroblast growth factor 23 (FGF23), secreted frizzled-related protein 4 (sFRP4), and fibroblast growth factor 7 (FGF7)] in 41 pediatric patients with HPP, 73 with X-linked hypophosphatemia (XLH), and 15 healthy pediatric control (CTR) subjects. The HPP and XLH cohorts had normal serum total and ionized calcium and parathyroid hormone levels (Ps > 0.10) and uncompromised glomerular filtration. In XLH, serum FGF23 was characteristically elevated (P < 0.0001) and despite hypophosphatemia sFRP4 was normal (P > 0.4) while FGF7 was low (P < 0.0001). In HPP, despite hyperphosphatemia serum FGF23 and sFRP4 were normal (Ps > 0.8) while FGF7 was low (P < 0.0001). Subsequently, in rats, we confirmed that FGF7 is phosphaturic. Thus, hyperphosphatemia in non-lethal pediatric HPP is associated with phosphatonin insufficiency together with, as we discuss, ePPi excess and diminished renal TNSALP activity.

Infantile Hypophosphatasia: Clinical Case

Background . Hypophosphatasia is rare hereditary disease caused by deficiency of the tissue-nonspecific alkaline phosphatase isozyme. It manifests with bone and teeth mineralisation defects, electrolyte imbalance, respiratory disorders, convulsive syndrome, physical developmental delay, nephrocalcinosis. The rarity of this disease, clinical polymorphism, non-specificity of complains and signs are the major reasons of hypophosphatasia late diagnosis. The enzyme replacement therapy with recombinant alkaline phosphatase (asfotase alfa) can be used for treatment of severe forms of disease. Clinical Case Description . The girl (1 y 4 m) was routinely admitted with complains of physical developmental delay and psychomotor retardation, deformation of lower limbs, chest, gait abnormality, teeth loss and with diagnosis «protein-calorie malnutrition». Rickets-like changes in skeleton, myopathic syndrome, early normal teeth loss, hepatomegaly were revealed. Reduced activity of alkaline phosphatase in blood serum (33 u/l; reference range 156–369 u/l) was revealed. The infantile hypophosphatasia has been diagnosed. Due to molecular genetic testing of ALPL gene we revealed pathogenic variants c.526G>A (p.Ala176Thr) and c.1375G>A (p.Val459Met) in compound heterozygous state. The asphotase alpha therapy was initiated at the age of 1 y 10 m, the dose was 2 mg/kg subcutaneously 3 times a week. The results of 6 months of the therapy are the following: significant increase in the activity of alkaline phosphatase (maximum 4400 u/l), body weight (+ 2 kg), growth (+ 6 cm), reduction of bone deformation, normalisation of muscle tone and gait, exercise tolerance. The patient tolerated the drug administration well. Rarely there were hyperemia zones up to 4 cm in diameter with moderate induration at the injection site but they spontaneous disappeared in 2–3 days though. Conclusion. Patients with rickets-like diseases and low alkaline phosphatase activity requires performing of molecular genetic testing to confirm hypophosphatasia. Timely diagnosis and early initiation of enzyme replacement therapy can significantly improve the quality of life of children with hypophosphatasia.

Consecutive changes in serum alkaline phosphatase isoenzyme 3 activities in Holstein heifers during the first 18 months of life.

This study investigated consecutive fluctuations in serum activities of bone-specific alkaline phosphatase (ALP) isoenzyme 3 (ALP3) in 11 clinically healthy Holstein heifers during the first 18 months of life. ALP3 activities at the first sampling time point after weaning (3 months) were significantly lower than those at multiple time points during the pre-weaning period. Those activities increased from a minimum at 3 months to a peak at 6 months during the post-weaning period. In the anthropometric data, daily body weight and wither height gains appeared to be below the public data at 4 months and 4–5 months, respectively. The data suggested that serum ALP3 activity can be used to monitor skeletal growth of heifers at weaning.

Regression formula to predict the International Federation of Clinical Chemistry and Laboratory Medicine measure of alkaline phosphatase activity in canine blood based on the Japan Society of Clinical Chemistry reference method.

The Japan Society of Clinical Chemistry reference method (JSCC method) is used to measure alkaline phosphatase (ALP) activity only in Japan. Other countries use the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) reference method to measure ALP activity. Since April 2020, human medical institutions in Japan have been gradually switching to the IFCC method. However, it is unclear whether the supply of reagents required for the JSCC method will be steady in the future. Additionally, the comparison of the performances and accuracies of these two methods for measuring ALP values remains uncertain in several animal species. In this investigation, we measured canine ALP activity using both methods and developed a formula to interconvert the two resulting values. The regression formula for ALP values measured using the modified JSCC (x) and IFCC (y) methods was determined as log10 y=0.960 log10 x−0.395 (r=0.997). However, the correlation between values based on JSCC and IFCC methods can change depending on the composition of ALP isozymes. Therefore, the developed formula can currently serve as a provisional strategy in calculating ALP levels. Nevertheless, this formula might avoid confusion in the clinical field during the transition from the JSCC to the IFCC method when both measurement values co-exist.

Determination of plasma bone-specific alkaline phosphatase isoenzyme activity in Holstein calves using a commercial agarose gel electrophoresis kit.

We measured the bone-specific alkaline phosphatase (ALP) isoenzyme activity in 67 plasma samples from 14 newborn Holstein calves using both a conventional method (featuring heat inactivation) and a commercial agarose gel electrophoresis (AGE) kit; the relevant isoenzymes were termed bone-specific ALP (BAP) and ALP isoenzyme 3 (ALP3). We explored whether the AGE kit afforded reliable data when used to analyze samples from Holstein calves. The blood was collected from the jugular vein of each calf immediately prior to the first colostrum feeding (pre-feeding), 20 and 40 h after pre-feeding, and on days 4 and 7; whereas three samples (from three calves) were not obtained. The total plasma ALP activity varied widely, exceeding the ranges of reference values. On electrophoresis, 52 of 67 plasma samples (77.6 %) clearly contained both ALP isoenzyme 2 and ALP3, as did control human serum. The total ALP activity of the 52 samples ranged from 166-1989 U/L (median: 1013 U/L), whereas the values for the other 15 samples (22.4%) exhibiting abnormal isoenzyme fractionation ranged from 1014-5118 U/L (median: 1780 U/L). In the 52 plasma samples exhibiting clearly separated isoenzymes, ALP3 and BAP activities were strongly positively correlated as revealed by Deming regression (y = 0.93x + 22.6, p⟨0.0001) and Bland-Altman analysis (ALP3/BAP activities limit of agreement: -5.1%). Thus, the AGE kit yields useful information on newborn calves, and can replace the conventional method when the total plasma ALP activity is less than approximately 1000 U/L.