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 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.

Hypophosphatasia: Vitamin B6 status of affected children and adults.

Hypophosphatasia (HPP) is the heritable dento-osseous disease caused by loss-of-function mutation(s) of the gene ALPL that encodes the tissue-nonspecific isoenzyme of alkaline phosphatase (TNSALP). TNSALP is a cell-surface homodimeric phosphomonoester phosphohydrolase expressed in healthy people especially in the skeleton, liver, kidneys, and developing teeth. In HPP, diminished TNSALP activity leads to extracellular accumulation of its natural substrates including inorganic pyrophosphate (PPi), an inhibitor of mineralization, and pyridoxal 5'-phosphate (PLP), the principal circulating form of vitamin B6 (B6). Autosomal dominant and autosomal recessive inheritance involving >450 usually missense defects scattered throughout ALPL largely explains the remarkably broad-ranging severity of this inborn-error-of-metabolism. In 1985 when we identified elevated plasma PLP as a biochemical hallmark of HPP, all 14 investigated affected children and adults had markedly increased PLP levels. However, pyridoxal (PL), the dephosphorylated form of PLP that enters cells to cofactor many enzymatic reactions, was not low but often inexplicably elevated. Levels of pyridoxic acid (PA), the B6 degradation product quantified to assess B6 sufficiency, were unremarkable. Canonical signs or symptoms of B6 deficiency or toxicity were absent. B6-dependent seizures in infants with life-threatening HPP were later explained by their profound deficiency of TNSALP activity blocking PLP dephosphorylation to PL and diminishing gamma-aminobutyric acid synthesis in the brain. Now, there is speculation that altered B6 metabolism causes further clinical complications in HPP. Herein, we assessed the plasma PL and PA levels accompanying previously reported elevated plasma PLP concentrations in 150 children and adolescents with HPP. Their mean (SD) plasma PL level was nearly double the mean for our healthy pediatric controls: 66.7 (59.0) nM versus 37.1 (22.2) nM (P < 0.0001), respectively. Their PA levels were broader than our pediatric control range, but their mean value was normal; 40.2 (25.1) nM versus 39.3 (9.9) nM (P = 0.7793), respectively. In contrast, adults with HPP often had plasma PL and PA levels suggestive of dietary B6 insufficiency. We discuss why the B6 levels of our pediatric patients with HPP would not cause B6 toxicity or deficiency, whereas in affected adults dietary B6 insufficiency can develop.

Vitamin B6 deficiency with normal plasma levels of pyridoxal 5'-phosphate in perinatal hypophosphatasia.

Pyridoxal 5'-phosphate (PLP), the principal circulating form of vitamin B6 (B6), is elevated in the plasma of individuals with hypophosphatasia (HPP). HPP is the inborn-error-of-metabolism caused by loss-of-function mutation(s) of ALPL, the gene that encodes the "tissue-nonspecific" isoenzyme of alkaline phosphatase (TNSALP). PLP accumulates extracellularly in HPP because it is a natural substrate of this cell-surface phosphomonoester phosphohydrolase. Even individuals mildly affected by HPP manifest this biochemical hallmark, which is used for diagnosis. Herein, an exclusively breast-fed newborn boy with life-threatening perinatal HPP had uniquely normal instead of markedly elevated plasma PLP levels before beginning asfotase alfa (AA) TNSALP-replacement therapy. These abnormal PLP levels were explained by B6 deficiency, confirmed by his low plasma level of 4-pyridoxic acid (PA), the B6 degradation product. His mother, a presumed carrier of one of his two ALPL missense mutations, had serum ALP activity of 50 U/L (Nl 40-130) while her plasma PLP level was 9 µg/L (Nl 5-50) and PA was 3 µg/L (Nl 3-30). Her dietary history and breast milk pyridoxal (PL) level indicated she too was B6 deficient. With B6 supplementation using a breast milk fortifier, the patient's plasma PA level corrected, while his PLP level remained in the normal range but now in keeping with AA treatment. Our experience reveals that elevated levels of PLP in the circulation in HPP require some degree of B6 sufficiency, and that anticipated increases in HPP can be negated by hypovitaminosis B6.

Persistent idiopathic hyperphosphatasemia from bone alkaline phosphatase in a healthy boy.

Alkaline phosphatase (ALP) in humans comprises a family of four cell-surface phosphomonoester phosphohydrolase isozymes. Three genes separately encode the "tissue-specific" ALPs whereas the fourth gene encodes ubiquitous homodimeric "tissue-nonspecific" ALP (TNSALP) richly expressed in bone, liver, kidney, and developing teeth. TNSALP monomers have five putative N-linked glycosylation sites where different post-translational modifications account for this isozyme's distinctive physicochemical properties in different organs. Three bone-derived TNSALP (BALP) isoforms (B/I, B1, and B2) are present in healthy serum, whereas a fourth BALP isoform (B1x) can circulate in chronic kidney disease. Herein, we report a healthy boy with persistent hyperphosphatasemia due to BALP levels two- to threefold higher than age-appropriate reference values. High-performance liquid chromatography, electrophoresis, heat inactivation, catalysis inhibition, and polyethylene glycol precipitation revealed increased serum B/I, B1, and B2 differing from patterns found in skeletal diseases. B/I was ~23-fold elevated. Absence of mental retardation and physical stigmata excluded Mabry syndrome, the ALP-anchoring disorder causing hyperphosphatasemia. Routine biochemical studies indicated intact mineral homeostasis. Serum N-terminal propeptide of type I procollagen (P1NP) level was normal, but C-terminal cross-linking telopeptide of type I collagen (CTX) level was elevated. However, radiological studies showed no evidence for a generalized skeletal disturbance. Circulating pyridoxal 5'-phosphate, a TNSALP natural substrate, was not low despite the laboratory hyperphosphatasemia, thereby suggesting BALP phosphohydrolase activity was not elevated endogenously. Mutation analysis of the ALPL gene encoding TNSALP revealed no defect. His non-consanguineous healthy parents had serum total ALP activity and BALP protein levels that were normal. Our patient's sporadic idiopathic hyperphosphatasemia could reflect altered post-translational modification together with increased expression and/or impaired degradation of BALP.

Hypophosphatasia: Biochemical hallmarks validate the expanded pediatric clinical nosology.

Hypophosphatasia (HPP) is the inborn-error-of-metabolism due to loss-of-function mutation(s) of the ALPL (TNSALP) gene that encodes the tissue non-specific isoenzyme of alkaline phosphatase (TNSALP). TNSALP represents a family of cell-surface phosphohydrolases differing by post-translational modification that is expressed especially in the skeleton, liver, kidney, and developing teeth. Thus, the natural substrates of TNSALP accumulate extracellularly in HPP including inorganic pyrophosphate (PPi), a potent inhibitor of mineralization, and pyridoxal 5'-phosphate (PLP), the principal circulating form of vitamin B6. The superabundance of extracellular PPi regularly causes tooth loss, and when sufficiently great can lead to rickets or osteomalacia. Sometimes diminished hydrolysis of PLP engenders vitamin B6-dependent seizures in profoundly affected babies. Autosomal dominant and autosomal recessive inheritance from among >340 ALPL mutations identified to date, typically missense and located throughout the gene, largely explains the remarkably wide-ranging severity of HPP, greatest of all skeletal diseases. In 2015, our demographic, clinical, and DXA findings acquired over 25 years from 173 children and adolescents with HPP validated and expanded the clinical nosology for pediatric patients to include according to increasing severity "odonto" HPP, "mild childhood" HPP, "severe childhood" HPP, "infantile" HPP, and "perinatal" HPP. Herein, we assessed this expanded nosology using biochemical hallmarks of HPP. We evaluated exclusively data from the 165 preteenage HPP patients in this cohort to exclude potential effects from physiological changes in TNSALP levels across puberty. All patients had subnormal serum total and bone-specific ALP and elevated plasma PLP, and nearly all had excessive urinary PPi excretion. Only the PLP levels were unchanged across puberty. Mean levels of all four biomarkers correlated with HPP severity ranked according to the HPP nosology, but the data overlapped among all four patient groups. Hence, these four biochemical hallmarks represent both a sensitive and reliable tool for diagnosing children with HPP. Furthermore, the hallmarks validate our expanded clinical nosology for pediatric HPP that, with limitations, is an improved framework for conceptualizing and working with this disorder's remarkably broad-ranging severity.

Hypophosphatasia: validation and expansion of the clinical nosology for children from 25 years experience with 173 pediatric patients.

Hypophosphatasia (HPP) is caused by loss-of-function mutation(s) within the gene TNSALP that encodes the "tissue-nonspecific" isoenzyme of alkaline phosphatase (TNSALP). In HPP, inorganic pyrophosphate, an inhibitor of mineralization and substrate for TNSALP, accumulates extracellularly often leading to rickets or osteomalacia and tooth loss, and sometimes to craniosynostosis and calcium crystal arthropathies. HPP's remarkably broad-ranging expressivity spans stillbirth from profound skeletal hypomineralization to adult-onset dental problems or arthropathies without bone disease, which is largely explained by autosomal recessive versus autosomal dominant transmission from among several hundred, usually missense, TNSALP mutations. For clinical purposes, this expressivity has been codified according to absence or presence of skeletal disease and then patient age at presentation and diagnosis. Pediatric patients are reported principally with "odonto", "childhood", "infantile", or "perinatal" HPP. However, this nosology has not been tested using a cohort of patients, and the ranges of the clinical and laboratory findings have not been defined and contrasted among these patient groups. To evaluate the extant nosology for HPP in children, we assessed our 25 years experience with 173 pediatric HPP patients. Data were exclusively from inpatient studies. The childhood form of HPP was further designated "mild" or "severe". Here, we focused on demographic, clinical, and dual-energy X-ray absorptiometry parameters compared to data from healthy American children. The 173-patient cohort comprised 64 individuals with odonto HPP, 38 with mild childhood HPP, 58 with severe childhood HPP, and 13 with infantile HPP. None was a survivor of perinatal HPP. TNSALP analysis revealed a mutation(s) in all 105 probands tested. Thirteen mutations were unique. Most patients represented autosomal dominant inheritance of HPP. Mutant allele dosage generally indicated the disorder's severity. Gender discordance was found for severe childhood HPP; 42 boys versus 16 girls (p=0.006), perhaps reflecting parental concern about stature and strength. Key disease parameters (e.g., height, weight, numbers of teeth lost prematurely, grip strength, spine and hip bone mineral density) were increasingly compromised as HPP was designated more severe. Although data overlapped successively between the four patient groups, body size (height and weight) differed significantly. Thus, our expanded nosology for HPP in children organizes the disorder's broad-ranging expressivity and should improve understanding of HPP presentation, natural history, complications, and prognosis.

Severe skeletal toxicity from protracted etidronate therapy for generalized arterial calcification of infancy.

Generalized arterial calcification (AC) of infancy (GACI) is an autosomal recessive disorder that features hydroxyapatite deposition within arterial elastic fibers. Untreated, approximately 85% of GACI patients die by 6 months of age from cardiac ischemia and congestive heart failure. The first-generation bisphosphonate etidronate (EHDP; ethane-1-hydroxy-1,1-diphosphonic acid, also known as 1-hydroxyethylidene-bisphosphonate) inhibits bone resorption and can mimic endogenous inorganic pyrophosphate by blocking mineralization. With EHDP therapy for GACI, AC may resolve without recurrence upon treatment cessation. Skeletal disease is not an early characteristic of GACI, but rickets can appear from acquired hypophosphatemia or prolonged EHDP therapy. We report a 7-year-old boy with GACI referred for profound, acquired, skeletal disease. AC was gone after 5 months of EHDP therapy during infancy, but GACI-related joint calcifications progressed. He was receiving EHDP, 200 mg/day orally, and had odynodysphagia, diffuse opioid-controlled pain, plagiocephaly, facial dysmorphism, joint calcifications, contractures, and was wheelchair bound. Biochemical parameters of mineral homeostasis were essentially normal. Serum osteocalcin was low and the brain isoform of creatine kinase and tartrate-resistant acid phosphatase 5b (TRAP-5b) were elevated as in osteopetrosis. Skeletal radiographic findings resembled pediatric hypophosphatasia with pancranial synostosis, long-bone bowing, widened physes, as well as metaphyseal osteosclerosis, cupping and fraying, and "tongues" of radiolucency. Radiographic features of osteopetrosis included osteosclerosis and femoral Erlenmeyer flask deformity. After stopping EHDP, he improved rapidly, including remarkable skeletal healing and decreased joint calcifications. Profound, but rapidly reversible, inhibition of skeletal mineralization with paradoxical calcifications near joints can occur in GACI from protracted EHDP therapy. Although EHDP treatment is lifesaving in GACI, surveillance for toxicity is crucial.

Type 1 diabetes impairs vitamin B(6) metabolism at an early stage of women's adulthood.

Vitamin B(6) (pyridoxine) metabolism in diabetes has never been investigated except for a few reports on plasma pyridoxal 5'-phosphate (PLP). These studies indicated that this most active (coenzyme) vitamer can be reduced. The present clinical investigation aimed to measure all vitamers in blood and urine by high performance liquid chromatography as well as important related factors, in women during active reproductive years. Thirty-two insulin-treated type 1 diabetic (T1D) patients, without renal complication, and 27 well-matched healthy controls, aged 30 to 40 years old, were recruited using rigorous criteria. Both groups had normal hemoglobin and serum albumin levels. Plasma PLP and pyridoxal (PL) did not differ significantly in the T1D group but alkaline phosphatase (ALP) activity was greater (p < 0.01). This produced a shift in plasma PLP-PL profile, as evidenced by a lower plasma PLP/PL ratio (p < 0.05). Enhanced ALP activity meant more PLP being dephosphorylated to PL (the membrane transfer form), with more ending up in erythrocytes to be rephosphorylated in its active form, as suggested by the significant positive correlation (p < 0.001) between plasma PL and erythrocyte PLP. More PL into blood circulation also means more oxidation of this vitamer to 4'-pyridoxic acid in kidneys, as confirmed by the positive correlation between plasma PL and urinary 4'-pyridoxic acid (p < 0.001). The positive correlation (p < 0.001) between ALP activity and glycosylated hemoglobin indicated a direct effect of the disease. The T1D-induced alteration in vitamin B(6) metabolism, consecutive to enhanced ALP activity, may put patients at greater risk of vitamin B(6) deficiency and diabetic complications.

"Atypical femoral fractures" during bisphosphonate exposure in adult hypophosphatasia.

We report a 55-year-old woman who suffered atypical subtrochanteric femoral fractures (ASFFs) after 4 years of exposure to alendronate and then zolendronate given for "osteoporosis." Before alendronate treatment, she had low bone mineral density. After several months of therapy, metatarsal stress fractures began. Bisphosphonate (BP) administration was stopped following the ASFFs, and the adult form of hypophosphatasia (HPP) was diagnosed from low serum alkaline phosphatase (ALP) activity, high endogenous levels of two natural substrates for the "tissue-nonspecific" isoenzyme of ALP (TNSALP), and a heterozygous mutation within the gene that encodes this enzyme. Experience with other HPP families showed that her mutation (Arg71His) with a second defective TNSALP allele can cause severe HPP in infancy, and when heterozygous can cause mild HPP featuring premature loss of deciduous teeth in children. Because the skeletal disease of HPP results from extracellular accumulation of the TNSALP substrate inorganic pyrophosphate (PPi) and its inhibitory effect on mineralization, perhaps HPP patients or carriers will have adverse effects from BPs. BPs are analogues of PPi and can suppress bone turnover but also deactivate TNSALP. Our report is the first of BP exposure preceding ASFFs in adult HPP. To explore a potential role for TNSALP deactivation in ASFFs, mutation analysis of TNSALP should be studied in a cohort of these patients. Meanwhile, clinicians must suspect HPP when clinical or laboratory clues include premature loss of primary dentition, pseudofractures or recurrent poorly healing metatarsal stress fractures, a family history suggestive of HPP, or low serum ALP activity. If HPP is documented, BP treatment might be avoided. To establish the diagnosis of HPP, assays for two natural substrates for TNSALP and TNSALP mutation analysis are available in commercial laboratories. With positive findings, radiological or bone biopsy evidence of acquired osteomalacia would indicate the adult form of this inborn-error-of-metabolism.

Hypophosphatasia: nonlethal disease despite skeletal presentation in utero (17 new cases and literature review).

Hypophosphatasia (HPP) is caused by deactivating mutation(s) within the gene that encodes the tissue-nonspecific isoenzyme of alkaline phosphatase (TNSALP). Patients manifest rickets or osteomalacia and dental disease ranging from absence of skeletal mineralization in utero to only loss of adult dentition. Until recently, HPP skeletal disease in utero was thought to always predict a lethal outcome. However, several reports beginning in 1999 emphasized a benign prenatal form of HPP (BP-HPP) where skeletal disease detected in utero had a mild postnatal course. Here we describe prenatal and postnatal findings of 17 additional BP-HPP patients among our 178 pediatric HPP patients. Their findings are compared with those of their siblings with HPP, carrier parents, and others with identical TNSALP mutations. New information concerning 7 previously published BP-HPP patients accompanies a review of the HPP literature. Among our 17 BP-HPP patients, prenatal ultrasound showed normal chest or abdominal circumferences where recorded. Sometimes, poor skeletal mineralization, fetal crowding, and third-trimester improvement were observed. Postnatally, extremity bowing further improved (13 patients). BP-HPP severity postnatally spanned the "infantile" to "odonto" HPP phenotypes, resembling our patients who harbored identical TNSALP mutation(s). Eight had autosomal dominant (AD) and 9 had autosomal recessive (AR) BP-HPP. Fourteen of our 15 mothers were HPP carriers or affected. Of the 41 cumulative BP-HPP patients (24 literature cases meriting a BP-HPP diagnosis since 1996 plus our 17 patients), 63% had AR BP-HPP. Maternally transmitted HPP involved 11 of the 13 total AD BP-HPP probands (p = 0.01), supporting a maternal in utero effect on the baby. Fetal crowding, normal fetal mineralization and chest size, and TNSALP heterozygosity seem to identify BP-HPP. However, bowed fetal long bones with AR HPP, specific TNSALP mutations, or poor skeletal mineralization before the third trimester do not reliably diagnose HPP lethality.

Bone metabolic abnormalities associated with well-controlled type 1 diabetes (IDDM) in young adult women: a disease complication often ignored or neglected.

OBJECTIVES: This investigation on a homogenous cohort of young adult Caucasian type 1 diabetic (IDDM) patients (1) aimed at studying the occurrence of low bone mineral density (BMD) at an early stage prior to menopause (i.e., during the first decade after peak bone mass) and (2) elucidating the possible mechanisms underlying IDDM-induced bone complication. METHODS: Twenty-seven female patients with insulin-treated and well-controlled diabetes, without renal complications, and 32 well-matched healthy controls, aged between 30 and 40 years and fulfilling rigorous inclusion criteria to minimize bone-confounding factors, were enrolled. Areal BMD was evaluated by dual energy X-ray absorptiometry at axial (lumbar spine) and appendicular (femur) sites, using diagnostic WHO reference (T-scores). Osteoblast functions, bone metabolism, related key minerals, and 2 osteoclast-stimulating calciotropic hormones regulating their serum levels were assessed biochemically. RESULTS: The number of cases with low BMD (T-score below -1.1 SD) was almost 2-fold greater (p < 0.01) in the IDDM group. BMD was significantly lower in this group for 3 lumbar sites (p < 0.01) and femur Ward's triangle (p < 0.05). Bone formation was reduced, as evidenced by the suppressions of osteocalcin (OC; p < 0.01) and IGF-I (p < 0.001). However, bone alkaline phosphatase (bALP) was induced (p < 0.01), in contrast to what is usually observed in cases of reduced bone formation. Correlated total ALP activity was also significantly increased. There was no change in the specific marker of bone resorption (urinary deoxypyridinoline). Serum calcium was significantly elevated, particularly after adjustment for albumin (p < 0.001), despite lower 1,25(OH)(2)D(3) (p < 0.001) and no elevation of PTH. All significant bone-related biochemical changes were significantly correlated with glycosylated hemoglobin, a clinical indicator of long-term glycemic control, indicating a direct effect of the disease. CONCLUSIONS: Bone loss in the IDDM group results from a decrease in bone formation rather than an increase of bone resorption. The induction of bALP is indicative of impaired osteoblast differentiation and maturation, which delayed (down-regulated) later stages of matrix mineralization, as evidenced by lower OC and BMD.

Stability of analytes related to clinical chemistry and bone metabolism in blood specimens after delayed processing.

OBJECTIVES: We investigated the stability of 36 analytes related to clinical chemistry in a controlled storage study. DESIGN AND METHODS: Blood was collected from 11 subjects and was maintained for 45 min, 2.5 h, 5 h, or 24 h after phlebotomy before centrifugation. RESULTS: Statistically significant changes were observed only for parathyroid hormone, osteocalcin, zinc, pyridoxal 5'-phosphate, and homocysteine. CONCLUSIONS: These studies indicate that many analytes in clinical chemistry are stable for 24 h before centrifugation.

Autosomal recessive hypophosphatasia manifesting in utero with long bone deformity but showing spontaneous postnatal improvement.

CONTEXT: Hypophosphatasia (HPP) is a heritable metabolic disorder of the skeleton that includes variable expressivity conditioned by gene dosage effect and the variety of mutations in the tissue nonspecific alkaline phosphatase (TNSALP) gene. Patient age when skeletal problems first manifest generally predicts the clinical course, with perinatal HPP causing bone disease in utero with postnatal lethality. OBJECTIVE: Our objective was to identify TNSALP mutations and characterize the inheritance pattern of a family with clinically variable HPP with one child manifesting in utero with long bone deformity but showing spontaneous prenatal and postnatal improvement. DESIGN: TNSALP enzyme and substrate analysis and TNSALP mutation analysis were performed on all family members. PATIENTS: A boy with HPP showing long bone deformity that spontaneously improved in utero and after birth is described. His older brother has the childhood form of HPP without findings until after infancy. His parents and twin sister are clinically unaffected. RESULTS: Both boys are compound heterozygotes for the same missense mutations in TNSALP, documenting autosomal recessive inheritance for their HPP. The parents each carry one defective allele. CONCLUSIONS: The patient is an autosomal recessive case of HPP with prenatal long bone deformity but with spontaneous prenatal and postnatal improvement. Thus, prenatal detection by sonography of bowing of long bones from HPP, even with autosomal recessive inheritance, does not necessarily predict lethality but can represent variable expressivity or the effects of modifiers on the TNSALP defect(s).

N-methylpyridoxamine: novel canine vitamin B6 urine metabolite.

Cation-exchange HPLC analysis of urine from dogs given large daily doses of pyridoxamine revealed an unidentified metabolite hypothesized to be N-methylpyridoxamine. Identity was established by N-methylpyridoxamine synthesis and HPLC comparison to the canine metabolite. Compound synthesis was confirmed by IR, NMR, UV-vis and emission spectroscopy. It seems to have less fluorescent character than other routinely-measured vitamin B(6) metabolites. Upon administration of substantial pyridoxamine doses, N-methylpyridoxamine appears to be a quantifiable canine urine metabolite, although, at either pharmacological or dietary pyridoxamine intakes, its relevance to vitamin B(6) metabolism in other species, including humans, is not yet determined.

Pyridoxine-responsive seizures as the first symptom of infantile hypophosphatasia caused by two novel missense mutations (c.677T>C, p.M226T; c.1112C>T, p.T371I) of the tissue-nonspecific alkaline phosphatase gene.

Pyridoxine-responsive seizures (PRS) and the role of pyridoxine (PN, vitamin B(6)) in hypophosphatasia (HPP) are incompletely understood. Typically, PRS and HPP are rare, independent, metabolic disorders. In PRS, seizures resist standard anticonvulsants apart from PN, yet have a good prognosis. In HPP, inactivation of the tissue nonspecific isoenzyme of alkaline phosphatase (TNSALP) impairs skeletal mineralization and causes rickets in infants that can be fatal. Here, we report a 7-month-old girl, newly diagnosed with infantile HPP, who presented as a neonate with PRS but without bony abnormalities. Analysis of biogenic amines in cerebrospinal fluid (CSF) suggested brain pyridoxal 5'-phosphate (PLP) deficiency, although PLP in CSF was not decreased. She had normal cognitive milestones but failure to thrive and rickets. Nearly undetectable serum ALP activity, elevated plasma PLP and urinary phosphoethanolamine (PEA) and inorganic pyrophosphate (PPi) levels, hypercalcemia, hypercalciuria and nephrocalcinosis were consistent with infantile HPP. Only prednisolone reduced serum calcium levels. Despite improved growth and weight gain, she developed rib fractures and died from respiratory failure at age 9 months. Sequence analysis of the TNSALP gene revealed novel missense mutations in exon 7 (c.677T>C, p.M226T) and exon 10 (c.1112C>T, p.T371I). Our patient demonstrated that PRS in neonates may not necessarily be "idiopathic"; instead, such seizures can be caused by severe HPP that becomes clinically apparent later in infancy. The pathophysiology of PRS in HPP differs from the three other genetic defects known to cause PRS, but all may lead to brain PLP deficiency reducing seizure thresholds. All reported HPP patients with neonatal seizures died within 18 months of birth, suggesting that PRS is an indicator of HPP severity and lethal prognosis. We recommend that assessment of any neonate with PRS should include measurement of serum ALP activity.

Use of chlorite to improve HPLC detection of pyridoxal 5'-phosphate.

The sensitivity of fluorescent detection of the biologically active form of Vitamin B-6, pyridoxal 5'-phosphate (PLP), in biological samples has been improved approximately four-fold by adopting chlorite as a post-column derivatization reagent (instead of bisulfite) in high-performance liquid chromatography (HPLC) separation. Chlorite oxidizes PLP to the more fluorescent 4-pyridoxic acid 5'-phosphate, and avoids the toxicity and heating of the cyanide procedure. Detection of another major metabolite, 4-pyridoxic acid (4-PA), is not effected. Detection of pyridoxal (PL) is slightly lowered due to eluting at a lower pH.

Elevated plasma 4-pyridoxic acid in renal insufficiency.

BACKGROUND: Renal insufficiency is associated with altered vitamin B-6 metabolism. We have observed high concentrations of 4-pyridoxic acid, the major catabolite of vitamin B-6 metabolism, in plasma during renal insufficiency. OBJECTIVE: The objective was to evaluate the renal handling of 4-pyridoxic acid and the effects of renal dysfunction on vitamin B-6 metabolism. DESIGN: We measured the renal clearance of 4-pyridoxic acid and creatinine in 17 nonpregnant, 17 pregnant, and 16 lactating women. We then examined the influence of vitamin B-6 or alkaline phosphatase activity on the ratio of 4-pyridoxic acid to pyridoxal (PA:PL) in plasma in 10 men receiving a low (0.4 mg pyridoxine.HCl/d) or high (200 mg pyridoxine.HCl/d) vitamin B-6 intake for 6 wk, in 10 healthy subjects during a 21-d fast, in 1235 plasma samples from 799 people screened for hypophosphatasia, and in 67 subjects with a range of serum creatinine concentrations. RESULTS: Renal clearance of 4-pyridoxic acid was 232 +/- 94 mL/min in nonpregnant women, 337 +/- 140 mL/min in pregnant women, and 215 +/- 103 mL/min in lactating healthy women. These values were approximately twice the creatinine clearance, indicating that 4-pyridoxic acid is at least partially eliminated by tubular secretion. Elevated plasma creatinine concentrations were associated with marked elevations in 4-pyridoxic acid and PA:PL. PA:PL was not affected by wide variations in vitamin B-6 intake or by the wide range of pyridoxal-P concentrations encountered while screening for hypophosphatasia. CONCLUSIONS: Plasma 4-pyridoxic acid concentrations are markedly elevated in renal insufficiency. Plasma PA:PL can distinguish between increases in 4-pyridoxic acid concentrations due to increased dietary intake and those due to renal insufficiency.