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.

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.

Vitamin B-6 Metabolism and Interactions with TNAP.

Two observations stimulated the interest in vitamin B-6 and alkaline phosphatase in brain: the marked increase in plasma pyridoxal phosphate and the occurrence of pyridoxine responsive seizures in hypophosphatasia. The increase in plasma pyridoxal phosphate indicates the importance of tissue non-specific alkaline phosphatase (TNAP) in transferring vitamin B-6 into the tissues. Vitamin B-6 is involved in the biosynthesis of most of the neurotransmitters. Decreased gamma-aminobutyrate (GABA) appears to be most directly related to the development of seizures in vitamin B-6 deficiency. Cytosolic pyridoxal phosphatase/chronophin may interact with vitamin B-6 metabolism and neuronal development and function. Ethanolaminephosphate phospholyase interacts with phosphoethanolamine metabolism. Extracellular pyridoxal phosphate may interact with purinoceptors and calcium channels. In conclusion, TNAP clearly influences extracellular and intracellular metabolism of vitamin B-6 in brain, particularly during developmental stages. While effects on GABA metabolism appear to be the major contributor to seizures, multiple other intra- and extra-cellular metabolic systems may be affected directly and/or indirectly by altered vitamin B-6 hydrolysis and uptake resulting from variations in alkaline phosphatase activity.

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.

"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.

Chronic recurrent multifocal osteomyelitis mimicked in childhood hypophosphatasia.

Hypophosphatasia (HPP) is the inborn error of metabolism characterized by low serum alkaline phosphatase (ALP) activity caused by inactivating mutations within TNSALP, the gene that encodes the "tissue-nonspecific" isoenzyme of ALP (TNSALP). In HPP, extracellular accumulation of inorganic pyrophosphate, a TNSALP substrate, inhibits hydroxyapatite crystal growth leading to rickets or osteomalacia. Chronic recurrent multifocal osteomyelitis (CRMO) is the pediatric syndrome of periarticular pain and radiographic changes resembling infectious osteomyelitis but without lesional pathogens. Some consider CRMO to be an autoinflammatory disease. An unrelated boy and girl with the childhood form of HPP suffered chronic, multifocal, periarticular pain, and soft tissue swelling. To investigate this unusual complication, we evaluated their cumulative clinical, biochemical, radiological, and histopathological findings and performed mutation analysis of their TNSALP alleles. The earliest radiographic disturbances were typical of childhood HPP. Subsequently, changes consistent with CRMO developed at sites where there was pain, including lucencies, osteosclerosis, and marked expansion of the underlying metaphyses. Bone marrow edema was shown by MRI. Biopsies of affected bone showed nonspecific histopathological findings and no pathogens. The boy was heterozygous (c.1133A>T, p.D378V) and the girl compound heterozygous (c.350A>G, p.Y117C, c.400_401AC>CA, p.T134H) for different TNSALP missense mutations. Nonsteroidal anti-inflammatory drugs diminished their pain, which improved or resolved at maturity. HPP should be considered when CRMO is a diagnostic possibility. Metaphyseal radiographic changes and marrow edema associated with periarticular bone pain and soft tissue swelling suggestive of osteomyelitis can complicate childhood HPP.

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.

Infantile hypophosphatasia: transplantation therapy trial using bone fragments and cultured osteoblasts.

BACKGROUND: Hypophosphatasia (HPP) is a rare, heritable, metabolic bone disease due to deficient activity of the tissue-nonspecific isoenzyme of alkaline phosphatase. The infantile form features severe rickets often causing death in the first year of life from respiratory complications. There is no established medical treatment. In 1997, an 8-month-old girl with worsening and life-threatening infantile HPP improved considerably after marrow cell transplantation. OBJECTIVE: Our aim was to better understand and to advance these encouraging transplantation results. DESIGN: In 1999, based on emerging mouse transplantation models involving implanted donor bone fragments as well as osteoblast-like cells cultured from bone, we treated a 9-month-old girl suffering a similar course of infantile HPP. RESULTS: Four months later, radiographs demonstrated improved skeletal mineralization. Twenty months later, PCR analysis of adherent cells cultured from recipient bone suggested the presence of small amounts of paternal (donor) DNA despite the absence of hematopoietic engraftment. This patient, now 8 yr old (7 yr after transplantation), is active and growing, and has the clinical phenotype of the more mild, childhood form of HPP. CONCLUSIONS: Cumulative experience suggests that, after immune tolerance, donor bone fragments and marrow may provide precursor cells for distribution and engraftment in the skeletal microenvironment in HPP patients to form tissue-nonspecific isoenzyme of alkaline phosphatase-replete osteoblasts that can improve mineralization.

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.

Roscovitine targets, protein kinases and pyridoxal kinase.

(R)-Roscovitine (CYC202) is often referred to as a "selective inhibitor of cyclin-dependent kinases." Besides its use as a biological tool in cell cycle, neuronal functions, and apoptosis studies, it is currently evaluated as a potential drug to treat cancers, neurodegenerative diseases, viral infections, and glomerulonephritis. We have investigated the selectivity of (R)-roscovitine using three different methods: 1) testing on a wide panel of purified kinases that, along with previously published data, now reaches 151 kinases; 2) identifying roscovitine-binding proteins from various tissue and cell types following their affinity chromatography purification on immobilized roscovitine; 3) investigating the effects of roscovitine on cells deprived of one of its targets, CDK2. Altogether, the results show that (R)-roscovitine is rather selective for CDKs, in fact most kinases are not affected. However, it binds an unexpected, non-protein kinase target, pyridoxal kinase, the enzyme responsible for phosphorylation and activation of vitamin B6. These results could help in interpreting the cellular actions of (R)-roscovitine but also in guiding the synthesis of more selective roscovitine analogs.

Marrow cell transplantation for infantile hypophosphatasia.

An 8-month-old girl who seemed certain to die from the infantile form of hypophosphatasia, an inborn error of metabolism characterized by deficient activity of the tissue-nonspecific isoenzyme of alkaline phosphatase (TNSALP), underwent the first trial of bone marrow cell transplantation for this heritable type of rickets. After cytoreduction, she was given T-cell-depleted, haplo-identical marrow from her healthy sister. Chimerism in peripheral blood and bone marrow became 100% donor. Three months later, she was clinically improved, with considerable healing of rickets and generalized skeletal remineralization. However, 6 months post-transplantation, worsening skeletal disease recurred, with partial return of host hematopoiesis. At the age of 21 months, without additional chemotherapy or immunosuppressive treatment, she received a boost of donor marrow cells expanded ex vivo to enrich for stromal cells. Significant, prolonged clinical and radiographic improvement followed soon after. Nevertheless, biochemical features of hypophosphatasia have remained unchanged to date. Skeletal biopsy specimens were not performed. Now, at 6 years of age, she is intelligent and ambulatory but remains small. Among several hypotheses for our patient's survival and progress, the most plausible seems to be the transient and long-term engraftment of sufficient numbers of donor marrow mesenchymal cells, forming functional osteoblasts and perhaps chondrocytes, to ameliorate her skeletal disease.

Cutaneous metabolism of vitamin B-6.

Vitamin B-6 is important for skin development and maintenance. We examined vitamin B-6 metabolism in human and mouse skin collected at different phases of the hair cycle; in hamster melanomas; in normal and immortalized human keratinocytes (HaCaT) and several human melanoma cell lines. Pyridoxamine 5'-phosphate content was higher in mouse and hamster than in human skin. Activity of both pyridoxamine 5'-phosphate oxidase and pyridoxal 5'-phosphate hydrolase was significantly increased in rapidly growing melanomas compared to either normal skin or slower growing skin tumors. Reducing the pyridoxine content of the culture medium significantly increased the activity of pyridoxal kinase and pyridoxamine 5'-phosphate oxidase. Pyridoxal 5'-phosphate hydrolase has been proposed as a regulatory enzyme for vitamin B-6, but we found B-6 vitamer content to be significantly correlated only with kinase and oxidase activity and not with pyridoxal 5'-phosphate hydrolase activity. Although pyridoxal 5'-phosphate hydrolase activity is usually attributed to tissue-nonspecific alkaline phosphatase, tissue-nonspecific alkaline phosphatase knockout mice showed preservation of normal histology of the skin and adnexal structures. Furthermore, expression of tissue-nonspecific alkaline phosphatase mRNA was not detected in either HaCaT cells or human skin, both of which exhibited significant pyridoxal 5'-phosphate hydrolase activity. This suggests that an enzyme different from the classical tissue-nonspecific alkaline phosphatase may perform cutaneous pyridoxal 5'-phosphate hydrolase activity.

Vitamin B6 status of children with sickle cell disease.

PURPOSE: In vitro, vitamin B(6) has antisickling properties, but the effect of vitamin B status on the health of children with sickle cell disease-SS (SCD-SS) is not well described. The purpose of this study was to assess vitamin B(6) status of children with SCD-SS ages 3 to 20 years and determine its relationship to growth, dietary intake, and disease severity. PATIENTS AND METHODS: Vitamin B(6) status was assessed by serum pyridoxal 5-phosphate (PLP) concentration in subjects with SCD-SS and by urinary 4-pyridoxic acid (4-PA) concentration in other subjects with SCD-SS and healthy control children. Concentration of PLP was compared with anthropometric measures of growth and nutritional status, dietary intake, hematologic indices, and frequency of SCD-related illness. RESULTS: The PLP concentration of subjects with SCD-SS was 15.6 +/- 15.2 nmol/L. Seventy-seven percent had a PLP concentration below the deficiency criterion (20 nmol/L) suggested by the Dietary Reference Intakes (1998). Controlling for alkaline phosphatase, age, and gender, PLP concentration was associated positively with weight, body mass index, and arm circumference -scores and negatively with reticulocyte count. Urinary 4-PA was lower in children with SCD-SS versus controls, although 4-PA/creatinine values did not differ between groups. CONCLUSIONS: Children with SCD-SS had apparently low serum PLP concentrations in the absence of excess vitamin B(6) excretion, suggesting low vitamin B(6) status. Low serum PLP concentration was associated with indicators of poor nutritional status and may be related to increased hemolysis in children with SCD-SS.

Vitamin B6 metabolism and homocysteine in end-stage renal disease and chronic renal insufficiency.

Homocysteine (tHcy) is a risk factor for atherosclerosis in patients with end-stage renal disease and chronic renal insufficiency (CRI). Vitamin B6 deficiency may result in high tHcy levels, especially after a methionine load (PML). Therefore, we evaluated vitamin B6 metabolism and tHcy (fasting and PML) levels in patients with CRI and those on hemodialysis (HD) therapy before and during high-dose sequential vitamin B6 and folic acid supplementation in male patients (27 patients, HD, 17 patients, CRI) and 19 age-matched healthy controls. Vitamin B6 doses were 100 mg/d in patients with CRI and 200 mg/d in HD patients, plus folic acid (5 mg/d), for more than 3 months in each period. We analyzed vitamin B6 metabolites by high-performance liquid chromatography in plasma and red blood cells (RBCs) and fasting tHcy in all cases and PML in subgroups of 11 HD patients and 14 patients with CRI. We found vitamin B6 deficiency and high tHcy (fasting and PML) levels in all patients. Plasma and RBC levels of pyridoxal and pyridoxal phosphate were abnormally low, whereas levels of pyridoxic acid (PA), an end product of vitamin B6 metabolism, were extremely high in both groups. Fasting and PML tHcy levels were partially resistant to vitamin B6 supplements, with different response patterns in HD patients and those with CRI. Thus, the PML defect was more responsive to folic acid in HD patients, whereas vitamin B6 partially reduced PML tHcy levels in patients with CRI. Resistance of tHcy to vitamin B6 treatment in patients with CRI and HD patients is not caused by poor absorption or low tissue stores. Rather, nonvitamin factors or potentially toxic PA levels may be implicated in abnormal vitamin B6 and/or tHcy metabolism during renal insufficiency.

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.