Matrix vesicles in osteomalacic hypophosphatasia bone contain apatite-like mineral crystals.

Hypophosphatasia, a heritable disease characterized by deficient activity of the tissue nonspecific isoenzyme of alkaline phosphatase (TNSALP), results in rickets and osteomalacia. Although identification of TNSALP gene defects in hypophosphatasia establishes a role of ALP in skeletal mineralization, the precise function remains unclear. The initial site of mineralization (primary mineralization) normally occurs within the lumen of TNSALP-rich matrix vesicles (MVs) of growth cartilage, bone, and dentin. We investigated whether defective calcification in hypophosphatasia is due to a paucity and/or a functional failure of MVs secondary to TNSALP deficiency. Nondecalcified autopsy bone and growth plate cartilage from five patients with perinatal (lethal) hypophosphatasia were studied by nondecalcified light and electron microscopy to assess MV numbers, size, shape, and ultrastructure and whether hypophosphatasia MVs contain apatite-like mineral, as would be the case if these MVs retained their ability to concentrate calcium and phosphate internally despite a paucity of TNSALP in their investing membranes. We found that hypophosphatasia MVs are present in approximately normal numbers and distribution and that they are capable of initiating internal mineralization. There is retarded extravesicular crystal propagation. Thus, in hypophosphatasia the failure of bones to calcify appears to involve a block of the vectorial spread of mineral from initial nuclei within MVs, outwards, into the matrix. We conclude that hypophosphatasia MVs can concentrate calcium and phosphate internally despite a deficiency of TNSALP activity.     

Phosphatase activities of rat intestinal enterocytes and their relation to diverse luminal pH, with special references to the possible localization of phytase along the brush border membrane

Phosphatase activities associated with the intestinal brush border membrane (BBM) of the rat were examined histochemically in relation to the characteristic environment of the intestine, where luminal pH fluctuates drastically between alkaline and acid pH ranges. Special attention was given to intestinal alkaline phosphatase (IALP) and phytase on the BBM. Whole body fresh-frozen sections of young rats and their rapidly frozen and freeze-substituted small intestines, embedded in Technovit 7100, were processed for the histochemical demonstration of phosphatase activity at three different pH values (9.2, 7.3, and 5.2), representing the deviation of luminal pH in vivo. Either an azo-dye method or lead-salt method was employed using naphthol AS-MX phosphate and ATP as substrate, respectively. With the azo-dye method, intense phosphatase reactions were demonstrated along the BBM at all three pH ranges. Phosphatase reactions of the BBM at pH 9.2 and 7.3 were abolished by L(+)-phenylalanine, heat pre-treatment, and EDTA chelation although some reaction remained at pH 7.3 after the treatment with EDTA or L(+)-phenylalanine. Phosphatase reactions of the BBM at pH 5.2 were resistant to L(+)-phenylalanine, L(+)-tartrate, PCMB and EDTA chelation, implying that the characteristics of the enzyme responsible for phosphohydrolysis at acid pH values differed from those at higher pH values. The lead-salt method in which ATP was used as substrate revealed intense reactions--which were dependent on Mg++ and stimulated by Ca++ and resistant to L(+)phenylalanine--to be localized along the BBM at alkaline and neutral pH values, but not at acid pH values. In vitro experiments showed progressive hydrolysis of naphthol AS-MX phosphate by purified phytase at pH 5.2, in a dose-dependent manner, and suggested the possible involvement of phytase in the phosphatase reactions of the BBM at acid pH. These data indicate that the phosphatase reactions at alkaline and neutral pH values, associated with the BBM of the rat intestine, represent IALP and Mg++/ Ca++-ATPase, while those at acid pH appear to correspond to phytase activity, something which has not been demonstrated by histochemical methods despite the availability of extensive data based on biochemical analyses.     

Characterization of phosphate binding by alkaline phosphatase in rat kidney brush border membrane

Phosphate binding by rat renal brush border membranes occurs on a single protein, as visualized by SDS polyacrylamide gel electrophoresis. The same protein can also be specifically labelled by gamma-32P ATP at 0 degree C or in the absence of magnesium. The phosphate binding protein co-migrates with monomers of two alkaline phosphatase activity bands previously localized on acrylamide gel. Measurement of binding by TCA precipitation, ion-exchange chromatography and dialysis gave an average of 31.1 +/- 5.7 pmol phosphate bound/mg protein. Alkaline phosphatase would then represent 0.23% of total brush border membrane protein. Maximal binding activity is obtained at pH 6.5, but when membranes are phosphorylated at pH 6.5 and the pH increased to 9.4, 50% of the bound radioactivity is released. The binding of phosphate to this protein presents two different apparent Km: one at 40 microM for low and one at 390 microM for high substrate concentrations. The membrane bound phosphate is readily exchangeable with phosphate in the medium. Phosphate binding and phosphate release are complete within 5 s. Alkaline phosphatase substrates and EDTA are potent inhibitors of phosphate binding and produce over 90% inhibition. Characteristics of phosphate binding for kidney membrane bound alkaline phosphatase seem very similar to the soluble form of the enzyme from various sources.     

Adult hypophosphatasia without apparent skeletal disease: “odontohypophosphatasia“ in four heterozygote members of a family

Summary Twenty members of a family with adult hypophosphatasia were examined clinically and biochemically. Severe caries causing early loss of permanent teeth was the only clinical symptom which could be attributed to hypophosphatasia. None of them had a history of defective bone mineralization, rachitic skeletal alterations, and recurrent pseudofractures or fractures. An iliac crest bone biopsy of the proposita showed a normal finding corresponding to the age of the patient.Four family members in two subsequent generations were affected, thus suggesting an autosomal dominant inheritance. Their serum and leukocyte alkaline phosphatases were reduced. The phosphoethanolamine (PEA) excretion in the urine was increased to a level which suggests a heterozygote state. The serum alkaline phosphatase activity could be ascribed to the liver isoenzyme fraction. This was shown by polyacrylamide electrophoresis, by inhibition studies with organ-specific inhibitors, heat inactivation, inhibition by antibodies, and treatment with neuraminidase.The proposita had an unexplained, diffuse fatty infiltration of the liver. Thus, not only alterations of bone but also of liver metabolism in hypophosphatasia should be considered.The variety of adult hypophosphatasia described in this paper is characterized by the lack of severe bone abnormalities, the apparently autosomal dominant inheritance, and the reduction of bone and intestinal isoenzyme in the serum. Our study suggests that hypophosphatasia is a heterogenous disorder which includes both severe and clinically mild forms.     

Nucleotide hydrolysis by solubilized membrane-bound enzymes of the brush border plasma membrane of Hymenolepis diminuta

Pyrophosphate, p-nitrophenyl phosphate and a variety of pyrimidine and purine nucleotides are hydrolyzed by the solubilized membrane-bound enzymes of the brush border plasma membrane of Hymenolepis diminuta. The pH optima (or ranges) for hydrolysis of substrates are 8.0 (pyrophosphate), 8.8 (p-nitrophenyl phosphate), 8.4-8.9 (nucleoside monophosphates), and 7.1-8.1 (nucleoside triphosphates); all substrates, with the exception of nucleoside triphosphates, have a higher affinity for the solubilized enzyme at pH 7.4 than at their optimal pH for hydrolysis. ATP is degraded completely by the enzyme preparation to adenosine and inorganic phosphate, but since neither ADP nor ATP accumulate in the incubation medium it is not known whether ATP hydrolysis involves the sequential hydrolysis of terminal phosphate groups. Isoelectric focusing and various chromatographic procedures (gel permeation, ion-exchange and hydrophobic interaction chromatography) fail to separate the alkaline phosphatase, phosphodiesterase, 5'-nucleotidase, adenosine triphosphatase and ribonuclease activities associated with the solubilized membrane preparation. Additionally, inhibitor studies indicate that only a single enzyme with low substrate specificity is involved in the hydrolysis of nucleotides, p-nitrophenyl phosphate, pyrophosphate and hexose phosphate esters. Purines and pyrimidines and their nucleosides interact with the active site, and in some instances activity of the enzyme is stimulated by an unknown mechanism.     

Histologic and ultrastructural studies on the mineralization process in hypophosphatasia

Chondroosseous tissue from six infants with infantile hypophosphatasia and six control infants were studied by light, transmission, and scanning electron microscopy. Alkaline phosphatase histochemical reaction of the growth plate was studied in two infants and was greatly reduced when compared to two control infants. Hypertrophic chondrocytes were increased in number with persisting cartilage islets in the metaphysis. In five of the six cases studied, chondrocytes and intercartilagenous intercellular chondroid matrix appeared ultrastructurally normal. Matrix vesicle distribution was similar to that of control subjects, but they were associated with few mineral crystals. In two infants, the matrix vesicles were alkaline phosphatase nonreactive. In the calcifying zone of the growth plate and in the newly formed metaphyseal trabecular bone, cartilagenous calcospherites often were small and the orientation of crystals was nonradial when compared to that of control infants. The mineralization of diaphyseal bone appeared normal. It seems that matrix vesicles are present in hypophosphatasia and that the impaired mineralization of cartilage is due primarily to the deficiency of alkaline phosphatase. In spite of the lack of alkaline phosphatase, secondary mineralization of bone which is not mediated by matrix vesicles was normal.     

Hypophosphatasia-study on two autopsy cases.

The autopsy findings of two cases of infantile hypophosphatasia are described and compared with those of 16 previously reported cases. Histochemical and biochemical tissue analysis for alkaline phosphatase showed a marked decrease in activity in liver, kidney, and bones. However, intestinal alkaline phosphatase possessed normal or slightly elevated activity. Nephrocalcinosis is a frequent complication and its development depends on hypercalcemia and length of survival of the patient. Electron microscopic findings are illustrated, and a mechanism for the development of nephrocalcinosis is proposed. For the first time, marked elevations of parathyroid hormone was detected. This finding, coupled with the extreme difficulty in locating the parathyroid glands in cases of hypophosphatasia, is enigmatic. Areas for furture investigation are suggested.     

Characterization of eleven novel mutations (M45L, R119H, 544delG, G145V, H154Y, C184Y, D289V, 862+5A, 1172delC, R411X, E459K) in the tissue-nonspecific alkaline phosphatase (TNSALP) gene in patients with severe hypophosphatasia. Mutations in brief no. 217. Online

Hypophosphatasia is a rare inherited disorder characterized by defective bone mineralization and deficiency of serum and tissue liver/ bone/kidney tissue alkaline phosphatase (L/B/K ALP) activity. We report the characterization of tissue-nonspecific alkaline phosphatase (TNSALP) gene mutations in a series of 9 families affected by severe hypophosphatasia. Fourteen distinct mutations were found, 3 of which were previously reported in the North American or Japanese populations. Seven of the 11 new mutations were missense mutations (M45L, R119H, G145V, C184Y and H154Y, D289V, E459K), the four others were 2 single nucleotide deletions (544delG and 1172delC), a mutation affecting donor splice site (862 + 5A) and a nonsense mutation (R411X).     

Severe hypophosphatasia: characterization of fifteen novel mutations in the ALPL gene

Hypophosphatasia is an inherited disorder characterized by defective bone mineralization and deficiency of serum and tissue liver/bone/kidney alkaline phosphatase (L/B/K ALP) activity. We report the characterization of ALPL gene mutations in a series of 11 families from various origins affected by perinatal and infantile hypophosphatasia. Sixteen distinct mutations were found, fifteen of them not previously reported: M45V, G46R, 388-391delGTAA, 389delT, T131I, G145S, D172E, 662delG, G203A, R255L, 876-881delAGGGGA, 962delG, E294K, E435K, and A451T. This confirms that severe hypophosphatasia is due to a large spectrum of mutations in Caucasian populations.     

Alkaline phosphatase activity of polymorphonuclear leucocytes and lymphocytes separated from normal human blood

Suspensions of polymorphonuclear leucocytes and lymphocytes were prepared from normal human blood by the glass bead column method of Rabinowitz (1964). The alkaline phosphatase activity of the separated cells was determined by biochemical and cytochemical techniques.Lymphocytes were found to contain an enzyme catalysing the hydrolysis of beta-glycerophosphate at alkaline pH but not of p-nitrophenylphosphate at the same pH and in the same buffer system. The alkaline phosphatase in polymorphonuclear leucocytes catalysed the hydrolysis of both substrates equally. The importance of this finding to the interpretation of clinical data is discussed.     

A novel missense mutation of the tissue-nonspecific alkaline phosphatase gene detected in a patient with hypophosphatasia

Hypophosphatasia is a rare heritable inborn error of metabolism characterized by abnormal bone mineralization associated with a deficiency of alkaline phosphatase. The clinical expression of hypophosphatasia is highly variable, ranging from death in utero to pathologic fractures first presenting in adulthood. We investigated the tissue-nonspecific alkaline phosphatase (TNSALP) gene from a Japanese female patient with hypophosphatasia. By a quantitative polymerase chain reaction (PCR) method, the amount of TNSALP mRNA appeared to be almost equal to that in normal individuals. Gene analysis clarified that the hypophosphatasia originated from a missense mutation and a nucleotide deletion. The missense mutation, a C ? T transition at position 1041 of cDNA, results in an amino acid change from Leu to Phe at codon 272, which has not yet been reported. The previously reported deletion of T at 1735 causes a frame shift mutation downstream from Leu at codon 503. Family analysis showed that the mutation 1041T and the deletion 1735T had been inherited from the proband's father and mother, respectively. An expression experiment revealed that the mutation 1041T halved the expression of alkaline phosphatase activity. Using homology analysis, the Leu-272 was confirmed to be highly conserved in other mammals.     

Changes in intralysosomal environment in the uterine epithelium during early pregnancy in the rat

Non-specific acid phosphatase activity was studied in the rat uterine luminal and glandular epithelium at oestrous, late dioestrous, and 6th d of pregnancy using the substrates beta-glycerophosphate and p-nitrophenyl phosphate, at both pH = 5.0 and pH = 6.0. At late dioestrous and 6th d of pregnancy, a significantly larger lysosomal population in the luminal and glandular epithelium was operating at pH = 5.0 rather than pH = 6.0. At oestrous, the epithelial lysosomes showed no preference for an operational pH = 5.0 or 6.0. Uniquely, the luminal epithelial lysosomes showed a significant preference for the hydrolysis of p-nitrophenyl phosphate over beta-glycerophosphate at 6th d of pregnancy, compared to no substrate preference at oestrous and late dioestrous. The activity of 5-Nucleotidase was also estimated semi-quantitatively at oestrous, late dioestrous, and 6th d of pregnancy in the luminal and glandular epithelium. 5-Nucleotidase activity was greatest in the luminal and glandular epithelium at 6th d of pregnancy. These findings are considered with respect to the possible mechanisms involved in implementing the changes and the relevance to blastocyst implantation.     

Calcium and phosphate supplementation promotes bone cell mineralization: implications for hydroxyapatite (HA)-enhanced bone formation

Organic phosphate, in particular beta-glycerophosphate (beta-GP), has been used to induce mineralization in cell culture systems. It serves as a source of inorganic phosphate when hydrolyzed by alkaline phosphatase. This study examined the effect of supplemental calcium and phosphate as well as the influence of various metabolic inhibitors on mineralization in a rat osteoblast-like cell-culture system. Mineralization was induced by supplementation of 1.8 mM of Ca(+2) and 5 mM of beta-GP or Pi. Mineral deposits associated with in vitro mineralization were revealed under SEM and TEM. Levamisole (10-100 microM) inhibited alkaline phosphatase activity and effectively reduced mineral formation. Actinomycin (500 ng/mL) and cycloheximide (50 microg/mL) also reduced mineral depositions by blocking RNA synthesis and protein synthesis, respectively. Levamisole and beta-GP did not appear to influence DNA synthesis. Spontaneous precipitation of calcium phosphate mineral was not detected in the culture medium with calcium and phosphate supplements in the absence of cell culture. The findings suggest that an elevated concentration of calcium and phosphate is crucial for in vitro mineralization. Furthermore, the mineralization process is associated with biologic events rather than with a spontaneous precipitation of calcium phosphate mineral. In view of the degradation potential of hydroxyapatite (HA)-coated implants, these results may be a viable indication that HA enhances bone formation through a similar mechanism.     

Hypophosphatasia: the mutations in the tissue-nonspecific alkaline phosphatase gene

Hypophosphatasia is an inborn error of metabolism caused by a deficiency of liver-, bone- or kidney-type alkaline phosphatase due to mutations in the tissue-nonspecific alkaline phosphatase (ALPL) gene. Most of the 65 distinct mutations described to date are missense mutations, a result which must be correlated with the great variability of clinical expression ranging from stillbirth without mineralized bone to pathologic fractures developing only late in adulthood. Correlations of genotype and phenotype have been established on the basis of clinical data exhibited by the patients, transfection studies, computer-assisted modeling, and examination of biochemical properties of ALP in cultured fibroblasts of patients. Screening for mutations in the TNSALP gene allows genetic counseling and prenatal diagnosis of the disease in families with severe forms of hypophosphatasia, and screening may also be helpful in confirming diagnosis of hypophosphatasia when biochemical and clinical data are not clear. Screening is also the necessary first step in further studies to elucidate dominant transmission of the disease and of liver-, bone- and kidney-type alkaline phosphatase activity mechanism.     

Origins of serum alkaline phosphatase

A very rapid method of agar gel electrophoresis on glass slides, together with a superior visualization technique employing simultaneous coupling of a hydrolysed naphthol substrate, have been developed for the identification of the tissues of origin of serum alkaline phosphatase. Combined with L-phenylalanine inhibition, specific for the intestinal enzyme, and heat inactivation, specific for the placental enzyme, the heterogeneity of serum alkaline phosphatase has been demonstrated. Normal adult serum contains predominantly liver-type alkaline phosphatase with a small but variable quantity of intestinal enzyme, and little or no bone enzyme.     

Distribution of alkaline phosphatase in the serum proteins in hypophosphatasia

A patient with hypophosphatasia and her relatives, including an affected brother, were studied with regard to serum alkaline phosphatase levels, phosphoethanolamine excretion, leucocyte alkaline phosphatase, blood group genes, and alkaline phosphatase distribution in the serum protein fractions.     

Hypophosphatasia: screening and family investigations in an endogamous Hungarian village

A screening programme for hypophosphatasia was performed in a Hungarian village where a family with the disease had previously been observed. Consanguinity was known to be common in this area.
198 schoolchildren were tested. As to physical signs, low serum alkaline phosphatase activity, and phospho-ethanolamine excretion, 3 homozygotes and 12 heterozygotes of the juvenile type of hypophosphatasia were discovered among them. The families of the positive children were investigated; thus altogether 34 subjects were found to be affected by the disease.
Tubular reabsorption of phosphates proved to be decreased in the homozygotes and in about half the heterozygotes.
In 4 families a transition between hypophosphatasia and pseudohypophosphatasia, i.e. the typical clinical and biochemical picture without a low serum alkaline phosphatase level, was observed. This suggests that the two conditions belong to the same entity.     

TWO SIBLINGS WITH HYPOPHOSPHATASIA

Two siblings with hypophosphatasia, one of whom was autopsied, were reported. The first case which was a product of a 26-year-old mother complicated by hydroamnios represented poor mineralization of the entire bones on X-ray examination and died shortly after birth. The second case weighing 1850 g delivered from the same mother had a rhizomelic micromelia and poor visualization of the skull, long bones and vertebral bones on X-ray at postmortem. The autopsy on the second case showed small thoracic cage with rachitic rosaries of ribs, membranous skull and poorly ossified vertebral and long bones. Microscopically, there was a marked disturbance of both enchondral and membranous ossifications similar to the histology of rachitis. A biochemical examination showed low alkaline phosphatase, high calcium and normal PTH in the serum. Further examination of their family revealed relatively low level of alkaline phosphatase of the parents and one of their brothers which suggested they were carriers of hypophosphatasia. Previous reports on hypophosphatasia were reviewed and differential diagnosis of hypophosphatasia from the other congential dwarfisms was discussed.     

High-molecular-weight alkaline phosphatase in serum has properties similar to the enzyme in plasma membranes of the liver.

Partially purified high-molecular-weight alkaline phosphatase from serum was compared with two other forms of the enzyme from the human liver, enzyme in native plasma membranes and purified alkaline phosphatase as a hydrophilic dimer. In a high-molecular-weight form from serum and plasma membranes, and when treated with 1% (v/v) Triton X-100, alkaline phosphatase showed a major band on gradient gel electrophoresis with a mobility equivalent to 400 kD. Nondetergent-treated material from both sources did not enter the gel and was in the voided volume of a gel permeation column. Stimulation of catalytic activity by four different phospholipids and by albumin yielded similar results for high-molecular-weight alkaline phosphatase and for the enzyme in plasma membranes, but these were different from the hydrophilic form. Inhibitors of alkaline phosphatase had similar effects on all forms. Of the three forms of the enzyme, only the hydrophilic dimer did not become incorporated into liposomes or adsorb to octyl-Sepharose after solubilization with Triton X-100 and removal of the detergent. Km (substrate concentration to give half maximal velocity) values with p-nitrophenylphosphate and heat and sodium dodecyl sulfate stabilities were similar for all forms. In the high-molecular-weight form from serum and in plasma membranes, alkaline phosphatase and 5'-nucleotidase showed similar rates of release by phosphatidylinositol phospholipase C. Three preparations of phospholipase D failed to release alkaline phosphatase from either the high-molecular-weight form or from plasma membranes. Based on these similarities, it is probable that the complex of high-molecular-weight alkaline phosphatase in serum most often originates from fragments of hepatic plasma membranes.     

Anion-stimulated phosphohydrolase activity of intestinal alkaline phosphatase

Phosphohydrolase activity of a highly enriched commercial preparation of calf intestinal alkaline phosphatase was stimulated in the presence of HCO3. SO4, Cl, SCN, and acetate did not stimulate hydrolysis, whereas SO3 exhibited a bimodal effect, stimulating at low (25mM) concentration but inhibiting at high (100 mM) concentration. The pH optimum of this stimulation by HCO3 or SO3 was 8.5--9.0 and was maximal at a Mg concentration of 0.5 mM. HCO3 increased the Vmax of the reaction without changing the Km for ATP. ATP, GTP, UTP, and xanthosine triphosphate were equally effective as substrates, whereas AMP and p-nitrophenyl phosphate were much less effective. Alkaline phosphatase activity was inhibited by L-cysteine and L-phenylalanine, compounds that also inhibited the HCO3-ATPase activity of the preparation. Passage of the commercial preparation through an anion-exchange column yielded a fraction with enriched alkaline phosphatase and HCO3-ATPase activities; this fraction proved to be a single protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, on isoelectric focusing, and by immunologic techniques. These studies strongly suggest that alkaline phosphatase and anion-stimulated phosphohydrolase activities are properties of the same protein in small intestine. It is possible that alkaline phosphatase may function as a HCO3-ATPase involved in intestinal absorption and secretion.