Alkaline Phosphatase Isoenzymes Research Articles

Serum alkaline phosphatase isoenzymes in SD rats detected by polyacrylamide-gel disk electrophoresis

Serum alkaline phosphatase (ALP) activity is frequently measured in toxicity studies. In the present study, we assessed the usefulness of a commercially available polyacrylamide-gel disk electrophoresis kit used in humans (AlkPhor System, Jokoh Co. Ltd., Tokyo, Japan) for identifying serum ALP isoenzymes in rats of the Sprague-Dawley strain (SD rats), which are commonly used in toxicity studies. We also examined age-related changes in serum ALP isoenzymes in SD rats. In order to identify the origin of each ALP isoenzyme, tissue ALP extracts from the liver, bone and small intestine (SI) and serum samples were treated with neuraminidase, antiintestinal ALP antibody, ALP inhibitor levamisole, and/or wheat germ agglutinin. It became clear that pretreatment of serum with neuraminidase is necessary for rat serum ALP isoenzyme analysis. The kit revealed that the main serum ALP isoenzymes in fasted 8-week-old intact rats were bone- and SI-derived and they tended to decrease with age. Serum liver-derived isoenzyme was slightly detected in both sexes of all ages examined, but it greatly increased in cholestasis model rats with bile-duct ligation, and rats of this model also had large molecular ALP detected in the stacking gel, suggesting hepatic damage. High-molecular intestinal ALP isoenzyme was slightly observed at the most cathodal side of the resolving gel. These results suggest that the present method is a useful tool for detecting serum ALP isoenzymes in SD rats and that concomitant levamisole inhibition with another gel is applicable for the evaluation of organ toxicity.

Relationship of non‐visualization of the fetal gallbladder and amniotic fluid digestive enzymes analysis to outcome

The aims of this study were evaluate the significance of non-visualization of fetal gallbladder at routine ultrasound scan in a series of 102 cases and to determine the contribution of amniotic fluid digestive enzyme (AF-DE) analysis towards the outcome. This is a multicenter retrospective study. Outcome of pregnancies, karyotype, and result of screening for CFTR gene mutations were known in all cases. Amniotic fluid gamma-glutamyl-transpeptidase and intestinal alkaline phosphatase isoenzyme were assayed. Non-visualization of the fetal gallbladder was associated with a severe disease in 25 cases (cystic fibrosis in ten, biliary duct atresia in eight, digestive tract anomalies in six, and chromosomal anomaly in one). In the remaining 77 cases, gallbladder agenesis was diagnosed in 22, and in 55, the gallbladder was subsequently demonstrated. Before 22 weeks of gestation (n=30), an abnormal AF-DE pattern had a 90% sensitivity and 80% specificity in detecting cystic fibrosis or biliary duct atresia. After 22 weeks, sensitivity fell to 53%. The AF-DE pattern was normal in 82% of gallbladder agenesis cases (benign) and in 91% of the cases where the gallbladder was subsequently detected. Non-visualization of the fetal gallbladder was associated with severe anomalies in 24% of cases. Prior to 22 weeks, determination of AF-DE contributes to the prediction of biliary atresia or the presence of cystic fibrosis.

Extremely high isolated maternal alkaline phosphatase serum concentration – 2 case reports and literature review

CasesWe report 2 cases of isolated elevated alkaline phosphatase (ALP) in pregnancy. Two healthy primigravida who are in their twenties presented with generally feeling unwell at approximately 36-37 weeks gestations....

Enzyme replacement prevents enamel defects in hypophosphatasia mice

Hypophosphatasia (HPP) is the inborn error of metabolism characterized by deficiency of alkaline phosphatase activity, leading to rickets or osteomalacia and to dental defects. HPP occurs from loss-of-function mutations within the gene that encodes the tissue-nonspecific isozyme of alkaline phosphatase (TNAP). TNAP knockout (Alpl(-/-), aka Akp2(-/-)) mice closely phenocopy infantile HPP, including the rickets, vitamin B6-responsive seizures, improper dentin mineralization, and lack of acellular cementum. Here, we report that lack of TNAP in Alpl(-/-) mice also causes severe enamel defects, which are preventable by enzyme replacement with mineral-targeted TNAP (ENB-0040). Immunohistochemistry was used to map the spatiotemporal expression of TNAP in the tissues of the developing enamel organ of healthy mouse molars and incisors. We found strong, stage-specific expression of TNAP in ameloblasts. In the Alpl(-/-) mice, histological, µCT, and scanning electron microscopy analysis showed reduced mineralization and disrupted organization of the rods and inter-rod structures in enamel of both the molars and incisors. All of these abnormalities were prevented in mice receiving from birth daily subcutaneous injections of mineral-targeting, human TNAP at 8.2 mg/kg/day for up to 44 days. These data reveal an important role for TNAP in enamel mineralization and demonstrate the efficacy of mineral-targeted TNAP to prevent enamel defects in HPP.

Enzyme-Replacement Therapy in Life-Threatening Hypophosphatasia

BackgroundHypophosphatasia results from mutations in the gene for the tissue-nonspecific isozyme of alkaline phosphatase (TNSALP). Inorganic pyrophosphate accumulates extracellularly, leading to rickets or osteomalacia. Severely affected babies often die from respiratory insufficiency due to progressive chest deformity or have persistent bone disease. There is no approved medical therapy. ENB-0040 is a bone-targeted, recombinant human TNSALP that prevents the manifestations of hypophosphatasia in Tnsalp knockout mice.MethodsWe enrolled infants and young children with life-threatening or debilitating perinatal or infantile hypophosphatasia in a multinational, open-label study of treatment with ENB-0040. The primary objective was the healing of rickets, as assessed by means of radiographic scales. Motor and cognitive development, respiratory function, and safety were evaluated, as well as the pharmacokinetics and pharmacodynamics of ENB-0040.ResultsOf the 11 patients recruited, 10 completed 6 months of therapy; 9 completed 1 year. Healing of rickets at 6 months in 9 patients was accompanied by improvement in developmental milestones and pulmonary function. Elevated plasma levels of the TNSALP substrates inorganic pyrophosphate and pyridoxal 5′-phosphate diminished. Increases in serum parathyroid hormone accompanied skeletal healing, often necessitating dietary calcium supplementation. There was no evidence of hypocalcemia, ectopic calcification, or definite drug-related serious adverse events. Low titers of anti–ENB-0040 antibodies developed in four patients, with no evident clinical, biochemical, or autoimmune abnormalities at 48 weeks of treatment.ConclusionsENB-0040, an enzyme-replacement therapy, was associated with improved findings on skeletal radiographs and improved pulmonary and physical function in infants and young children with life-threatening hypophosphatasia. (Funded by Enobia Pharma and Shriners Hospitals for Children; ClinicalTrials.gov number, NCT00744042.)

“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.

Polyoxometalates as potent and selective inhibitors of alkaline phosphatases with profound anticancer and amoebicidal activities

The biological significance of polyoxometalates is well renowned owing to their anticancer, antiviral and antibiotic properties. Here another therapeutic aspect of polyoxometalates has been explored as alkaline phosphatase inhibitors along with the remarked anticancer and amoebicidal properties. Synthesis and inhibitory studies of a set of seven polyoxotungstates against two major isozymes of alkaline phosphatase i.e. tissue specific and tissue non-specific alkaline phosphatase revealed their promising activity as alkaline phosphatase inhibitors. All compounds exhibited alkaline phosphatase inhibitory potency in nanomolar ranges. For tissue specific alkaline phosphatase, Na(10)[H(2)W(12)O(42)]·27H(2)O (A6) was found to be the most potent inhibitor (K(i) value 313 ± 7 nM), while for tissue non-specific alkaline phosphatase Na(33)[H(7)P(8)W(48)O(184)]·92H(2)O (A3) showed the highest inhibition potency (K(i) values 135 ± 10 nM). Moreover cytotoxicity evaluation of these compounds against lung carcinoma cells and immortalized human corneal epithelial cells demonstrated their anticancer potential with no cytotoxic effects on normal human cell lines. All anticancer drugs result in an impaired immune system and such immunocompromised persons become vulnerable to opportunistic infections specially Acanthamoeba which causes granulomatous amoebic encephalitis (GAE) which almost always results in death. The exclusive property of our tested polyoxotungstates is their strong amoebicidal activity against Acanthamoeba. Hence the study reveals a new window towards cancer therapy with the combined control of elevated levels of alkaline phosphatase and granulomatous amoebic encephalitis in cancer patients.

Isoenzima de fosfatasa alcalina placental-like, importancia clínica como marcador en tumores de tipo germinal

Background and purposeTo determine the clinical relevance of placental-like alkaline phosphatase isoenzyme (PLAP-like) as a marker for germ cell tumours (GCT). Patients and methodsWe report the behaviour of this isoenzyme in two patients; 1) A Child with an intracranial germinoma in the pineal region who showed 75% of PLAP-like activity at onset (normal value: 0%), with the rest of the tumour markers being negative, and 2) A girl with a stage I ovarian immature teratoma who had an elevated alpha-fetoprotein (AFP), 197ng/mL, PLAP-like 5.1%, at onset and 0.7% at tumour recurrence two years later. PLAP-like was determined by assessing the remaining activity after heat denaturation of the serum. Discussion and conclusionsPLAP-like constituted the only specific marker in the case of the pineal cell germ tumour, which justifies its use in association with neuroimaging studies in the diagnosis of cranial tumours. At follow-up analysis, PLAP-like was also critical since it confirmed the good response to treatment in the first case, while in the second one, it was the only marker to complement the assessment of tumour recurrence. We believe that this isoenzyme analysis is of relevance as a complementary marker in GCT.

Plasma Biochemistry Values of Recently Wild-Caught Purple Mouth Moray Eels (Gymnothorax vicinus)

The primary purpose of this study was to establish plasma biochemistry parameters for healthy recently wild-caught purple mouth moray eels (Gymnothorax vicinus) to provide a baseline of data for improved medical care in an aquarium or zoologic setting and for wild health assessments. Thirty-one clinically healthy purple mouth moray eels of unknown age and sex were caught from the wild, and were anesthetized 50 days following capture for blood collection from the ventral coccygeal vein. The median plasma biochemistry values were as follows: hematocrit = 21%, creatinine kinase = 2,100 U/L, lactate dehydrogenase = 97 U/L, aspartate aminotransferase = 88 U/L, alanine aminotransferase = 51 U/L, alkaline phosphatase 3,939 U/L, gamma-glutamyl transpeptidase = 1 U/L, amylase = 40 U/L, blood urea nitrogen = < 11 mg/dl, glucose = 21 mg/dl, calcium = 12.5 mg/dl, triglyceride = 206 mg/dl, creatinine = 0.1 mg/dl, cholesterol = 334 mg/dl, total bilirubin = < 0.1 mg/dl, phosphorus = 6.5 mg/dl, total protein = 4.2 g/dl, albumin = 1.5 g/dl, globulin = 2.7 g/dl, albumin/ globulin ratio = 0.6, sodium = 185 mmol/L, potassium = 3.7 mmol/L, and chloride = 175 mmol/L. Alkaline phosphatase isoenzyme results indicate that the majority of the plasma alkaline phosphatase is the liver isoenzyme. The data acquired in this study also provide baseline values for cholesterol and triglycerides in recently wild-caught moray eels to aid in monitoring elevations to these values in an aquarium setting over time so adjustments to the dietary regime may be utilized to prevent or improve conditions such as lipid keratopathy.

Bone mineral metabolism in patients with neurofibromatosis type 1 (von Recklingausen disease)

The neurofibromatosis type 1 (NF1) is characterized by specific cutaneous features (neurofibromas, "café-au-lait" spots of the skin) and alterations of several tissue (nervous, vascular) and bone deformities, such as scoliosis, congenital pseudoarthrosis and bone dysplasia of tibia. Moreover, several studies have shown systemic involvement of bone tissue in NF1 patients, leading to reduced bone mass. The aim of our study was to evaluate some bone mineral metabolism parameters before and after calcium and vitamin D supplementation in NF1 patients. We evaluated in 70 NF1 consecutive patients the mineral metabolism and bone mineral density compared with 40 normal subjects. We showed bone alterations in 35% of patients and the increase of bone formation markers, such as bone isoenzyme of alkaline phosphatase (41.2±15.5 vs. 25.6±8.7UI; P<0.05, respectively) and osteocalcin (18.1±5.6 vs. 7.6±1.9ng/ml; P<0.05) and reduction of circulating levels of (25OH)-vitamin D (21.8±12.3ng/ml) with an high percentage of hypovitaminosys D (>60%). Moreover, we revealed a significant reduction of bone mass density at spine (L1-L4) (0.935±0.13 vs. 1.110±0.17g/cm(2); P<0.001) and femoral neck side (0.765±0.09 vs. 0.839±0.12g/cm(2); P<0.02), with high prevalence of osteopenia (44%) and osteoporosis (18%). After 12months of calcium (1,200mg/die) and cholecalciferol (800UI/die) supplementation, we found a significant increase of (25) OH-vitamin D level (21.8±12.3 vs. 35±13ng/ml; P<0.01), without changes in bone mass density. In conclusion, NF1 patients may present a mineral bone involvement, with vitamin D deficiency; calcium and vitamin D supplementation is necessary to restore these bone mineral metabolic alterations.

Transition of Serum Alkaline Phosphatase Isoenzymes during Liver Regeneration in Humans

Serum alkaline phosphatase (ALP) levels tend to increase after hepatectomy. However, no previous examinations have yet focused on the relationship between liver regeneration and the individual ALP isoenzymes levels. Forty living liver transplantation donors who underwent hemi-hepatectomy were herein investigated. We evaluated the serum ALP levels and ALP isoenzyme levels preoperatively and postoperatively. The liver regeneration rate (LRR) was calculated using volumetry. According to the LRR, we divided the donors into two groups, consisting of a high regeneration group (HG) and a low regeneration group (LG). The total serum ALP levels increased gradually after hepatectomy and peaked on postoperative day (POD) 14. ALP-1 was not detected in any donor preoperatively. However, it was detected after hepatectomy, peaking on POD 7. The serum ALP-2 level increased after hepatectomy, reaching a peak level on POD 14. The ALP-2 levels gradually increased after hepatectomy and reached peak levels on POD 14 in both groups. However, the ALP-2 level on POD 14 was significantly higher in HG than LG. The serum ALP-2 levels after POD 14 might therefore be a useful indicator of favorable liver regeneration following hepatectomy, especially in patients who have a normal liver function.

A novel characterization of alkaline phosphatase enzyme in acute lymphoblastic leukemias patients serum

Introduction: Alkaline phosphatase (ALP) is a member of a group of phosphomonoesterase enzymes generating an organic radical and inorganic phosphate by catalyzing the hydrolysis of phosphate esters in an alkaline environment. The aim of the present study was to investigate the clinical value of alkaline phosphatase isoenzymes, as metastases markers. Materials and Methods: The alkaline phosphatase activity of the human serum has been partially mesured in patients with lymphoblast leukemia's acute diseases. Then the in vitro‘ effect of heavy metals on enzymatic activity has been analyzed. Results: The optimal pH was 10.2 for alkaline phosphatase activity. The effect of heavy metals is dependent on the serum of patients analyzed. Zn showed the highest inhibition for normal serum enzyme activity but Mn + 2 showed the highest inhibition for lymphoblast leukemia's acute cancer serum enzyme.The parameter Km For normal serum and cancer serum was 18 and 20, respectively. Conclusion: This is a different and unique isoenzyme which may use as a diagnostic marker in lymphoblast leukemia's acute patients due to increase in its activity, different km value, different pH optima, different effect of Zn and Mg comparing to another members of phosphomonoesterase group.

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.

Novel heterozygous tissue-nonspecific alkaline phosphatase (TNAP) gene mutations causing lethal perinatal hypophosphatasia

Hypophosphatasia is a rare inherited disorder characterized by poor bone mineralization and deficiency of alkaline phosphatase activity. It is caused by mutations in the liver/bone/kidney alkaline phosphatase gene encoding the tissue-nonspecific isoenzyme of alkaline phosphatase (TNAP), which displays many allelic heterogeneities, leading to different clinical phenotypes. This study reports the case of a patient diagnosed with lethal perinatal hypophosphatasia. His gene analysis showed compound heterozygocity of two novel mutations: c.650delTinsCTAA and c.984_986delCTT, which led to p.217delVinsAK and p.328delF, respectively. The two mutations originated separately from his parents, consistent with autosomal recessive perinatal hypophosphatasia. For these two novel mutations, we analyzed their functions through three-dimensional structural analysis. This revealed that V217 is located in the β-sheet area, V217 is deleted, and insertion of alanine and lysine alter the secondary structure, causing instability in the hydrophobic region, which may influence the metal-binding vicinity. This mutant structure loses its catalytic activity. Deletion of 328F also results in protein structural alteration and affects TNAP functions. These results may provide an explanation of the two novel mutated alleles correlating with the lethal phenotype of our patient. In conclusion, we demonstrated the case of a patient with lethal perinatal hypophosphatasia caused by two novel heterozygous mutations.

Novel methodology for the determination of alkaline phosphatase isoenzymes in plasma circulating

dp er os+1.5μg/kg/w sc. Eight rats were sham operated. Histomorphometric and histopathological studies were performed on tibias obtained after 30 days of treatment. OVX decreased trabecular bone (%BV/TV), and Z prevented and raised it (Sham: 35.3±4.7, control: 21.5±1.5*, MFP: 17.1±1.0*, Z: 64.3±3.5*, MFP±Z: 52.2±3.2*, * p<0.05 vs sham, mean±SE, ANOVA). Histological examination showed, in the sham group, an adequate bone-hemopoietic (HT) tissue relationship and mature bone trabeculae (Tb). The control group showed higher HT, immature Tb, fewer osteoblasts (Ob) and higher osteoclasts (Oc) compared with the sham group. The MFP group showed abundant HT and thin Tb with abundant lining cells (LC) and Ob. In Z, anastomosed and immature Tb with invasive tendency on the marrow space were observed. A decrease in Ob and HT was also observed. In contrast, the MFP+Z group showed mature Tb with well defined marrow spaces. The trabecular edge was covered by LC and Ob. We conclude that Z treatment, alone or combined with MFP, prevented the bone loss caused by OVX. Its combination with MFP normalized the histological features of bone. The MFP, by stimulating osteoblast activity, could modulate bone remodeling by producing a better quality bone. This article is part of a Special Issue entitled AAOMM 2010 Abstracts.

A case report of a rare type of skeletal dysplasia diagnosed by a common blood test

We report a rare case of enzyme defect affecting the skeleton. Antenatal scans gave suspicion of skeletal dysplasia (micromelia affecting all four limbs with bone echogenicity). This baby girl was...

Serum alkaline phosphatase isoenzymes in laboratory beagle dogs detected by polyacrylamide-gel disk electrophoresis

Serum alkaline phosphatase (ALP) activity is frequently measured in toxicity studies. Itoh et al. (2002) reported that a commercially available polyacrylamide-gel (PAG) disk electrophoresis kit used in humans (AlkPhor System, Jokoh Co., Ltd., Tokyo, Japan) for identifying serum ALP isoenzymes was useful for veterinary clinicopathological diagnosis in mongrel dogs. In the present study, based on the report of Itoh et al. (2002), we tried to expand the application range of this kit to laboratory beagle dogs which are commonly used in toxicity studies. In order to identify the origin of each ALP isoenzyme, tissue ALP extracts from the liver, bone and small intestine and serum samples were treated with neuraminidase, anti-small intestinal ALP antibody, ALP inhibitor levamisole and/or wheat germ agglutinin (WGA). The main serum ALP isoenzymes in 5-month-old intact beagle dogs were bone-derived (bone and atypical ALP: corresponding to human variant bone ALP) and they tended to decrease with age. However, liver-derived ALP isoenzyme greatly increased in the serum of cholestasis model dogs. The cholestasis model dogs also had a large molecular ALP detected in the resolving gel. This ALP could be originated from intestinal ALP or corticosteroid-induced ALP (CALP), because the activity remained even after levamisole inhibition. CALP was observed in intact laboratory beagle dogs with individual differences. These results suggest that the present method is a useful tool for detecting serum ALP isoenzymes in laboratory beagle dogs and concomitant levamisole inhibition with another gel is applicable for the evaluation of organ toxicity.

Alkaline phosphatase isoenzymes in mouse plasma detected by polyacrylamide-gel disk electrophoresis

Plasma alkaline phosphatase (ALP) activity is frequently measured in toxicity studies. In the present study, we assessed the usefulness of a commercially available polyacrylamide-gel (PAG) disk electrophoresis kit used in humans (AlkPhor System, Jokoh Co., Ltd., Tokyo, Japan) for identifying plasma ALP isoenzymes in mice of the Crlj:CD1 strain (ICR mice), which are commonly used in toxicity studies. We also examined age-related changes in plasma ALP isoenzymes in ICR mice. Electrophoresis was performed according to the manufacturer's instructions. In order to identify the origin of each ALP isoenzyme, in addition to plasma samples, tissue ALP extracts from the liver, bone and small intestine were treated with neuraminidase, anti-small intestinal ALP antibody, ALP inhibitor levamisole and/or wheat germ agglutinin (WGA). The kit revealed that main plasma ALP isoenzyme in intact ICR mice was bone-derived one, and it tended to decrease with age. On the other hand, liver-derived ALP isoenzyme greatly increased in plasma of cholestasis model mice induced by bile duct ligation. This model mouse had also a large molecular ALP detected in the stacking gel. This ALP was thought to be of intestinal origin because its activity remained even after levamisole inhibition. In addition, a minimum sample volume for sufficient resolution of plasma ALP isoenzymes was only 14µl. The results of this study suggest that the present method is a useful tool for detecting plasma ALP isoenzymes in mice and that pre-treatment of plasma with neuraminidase and concomitant levamisole inhibition with another gel is applicable for the evaluation of organ toxicity.

Prevalence of c.1559delT in ALPL, a common mutation resulting in the perinatal (lethal) form of hypophosphatasia in Japanese and effects of the mutation on heterozygous carriers

Hypophosphatasia (HPP) is an inherited disorder caused by mutations in ALPL that encodes an isozyme of alkaline phosphatase (ALP), TNSALP. One of the most frequent ALPL mutations is c.1559delT, which causes the most severe HPP, the perinatal (lethal) form (pl-HPP). c.1559delT has been found only in Japanese and its prevalence is suspected to be high; however, the allele frequency of c.1559delT in Japanese remains unknown. We designed a screening system for the mutation based on high-resolution melting curve analysis, and examined the frequency of c.1559delT. We found that the c.1559delT carrier frequency is 1/480 (95% confidence interval, 1/1562-1/284). This indicates that ∼1 in 900 000 individuals to have pl-HPP caused by a homozygous c.1559delT mutation. In our analysis, the majority of c.1559delT carriers had normal values of HPP biochemical markers, such as serum ALP and urine phosphoethanolamine. Our results indicate that the only way to reliably detect whether individuals are pl-HPP carriers is to perform the ALPL mutation analysis.

Prolonged survival and phenotypic correction of Akp2−/− hypophosphatasia mice by lentiviral gene therapy

Hypophosphatasia (HPP) is an inherited systemic skeletal disease caused by mutations in the gene encoding the tissue-nonspecific alkaline phosphatase (TNALP) isozyme. The clinical severity of HPP varies widely, with symptoms including rickets and osteomalacia. TNALP knockout (Akp2−/−) mice phenotypically mimic the severe infantile form of HPP; that is, TNALP-deficient mice are born with a normal appearance but die by 20 days of age owing to growth failure, hypomineralization, and epileptic seizures. In this study, a lentiviral vector expressing a bone-targeted form of TNALP was injected into the jugular vein of newborn Akp2−/− mice. We found that alkaline phosphatase activity in the plasma of treated Akp2−/− mice increased and remained at high levels throughout the life of the animals. The treated Akp2−/− mice survived for more than 10 months and demonstrated normal physical activity and a healthy appearance. Epileptic seizures were completely inhibited in the treated Akp2−/− mice, and X-ray examination of the skeleton showed that mineralization was significantly improved by the gene therapy. These results show that severe infantile HPP in TNALP knockout mice can be treated with a single injection of lentiviral vector during the neonatal period. © 2011 American Society for Bone and Mineral Research.