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1.  Newborn screening and diagnosis of mucopolysaccharidoses: application of tandem mass spectrometry 
Mucopolysaccharidoses (MPS) are a group of lysosomal storage disorders caused by the deficiency of lysosomal enzymes. The enzymes are required to break down glycosaminoglycans (GAGs) that help build bone, cartilage, tendons, corneas, skin and connective tissue. In patients with MPS, a missing enzyme leads to the accumulation of GAGs in the cells, blood, connective tissues, and multiple organs. The consequence is permanent, with progressive cellular damage affecting patients’ appearance, physical abilities, organ and system function, and skeletal and mental development.
The measurement of each specific GAG in a variety of specimens is required to establish the correlation between GAGs and physiological status of patients and/or prognosis and pathogenesis of the disease and to separate the patients with MPS from the healthy controls.
We have developed a highly accurate, sensitive, and cost-effective liquid chromatography tandem mass spectrometry (LC-MS/MS) method for measurements of disaccharides derived from four specific GAGs [chondroitin sulfate (CS), dermatan sulfate (DS), heparan sulfate (HS), and keratan sulfate (KS)]. Disaccharides were produced by specific enzyme digestion of each GAG, and subsequently, quantified by negative ion mode of multiple reaction monitoring. Subclasses of GAGs with the same molecular weights can be separated by liquid chromatography.
We have also developed another GAG assay by high-throughput mass spectrometry (HT-MS/MS). The HT-MS/MS consists of an integrated solid phase extraction robot that binds and de-salts samples from assay plates and directly injects them into a MS/MS detector, reducing sample processing time to within ten seconds. HT-MS/MS consequently yields much faster throughput than conventional LC-MS/MS-based methods; however, the HT-MS/MS system does not use a chromatographic step, and therefore, cannot separate GAGs that have the same molecular weights. Both techniques can be applied to the analysis of dried blood spots, blood, and urine specimens.
In this review, we describe the assay methods for GAGs and the application to newborn screening and diagnosis of MPS.
PMCID: PMC4303184  PMID: 25620850
tandem mass spectrometry; glycosaminoglycans; diagnosis; mucopolysaccharidoses; newborn screening
2.  A rare case of spontaneous necrosis of primary renal cell carcinoma 
A 42-year-old woman was referred to our hospital with a chief complaint of asymptomatic gross hematuria. Computed tomography revealed a 4-cm tumour in the left kidney and radical nephrectomy was performed. Microscopically, the tumour was completely necrotic and consisted of nests of cells with abundant cytoplasm and large nuclei. Immunohistochemical analysis indicated complete infarction of the chromophobe renal cell carcinoma. Two years after surgery, the patient remained recurrence-free.
doi:10.5489/cuaj.2272
PMCID: PMC4301967  PMID: 25624965
3.  Newborn screening and diagnosis of mucopolysaccharidoses 
Mucopolysaccharidoses (MPS) are caused by deficiency of lysosomal enzyme activities needed to degrade glycosaminoglycans (GAGs), which are long unbranched polysaccharides consisting of repeating disaccharides. GAGs include: chondroitin sulfate (CS), dermatan sulfate (DS), heparan sulfate (HS), keratan sulfate (KS), and hyaluronan. Their catabolism may be blocked singly or in combination depending on the specific enzyme deficiency. There are 11 known enzyme deficiencies, resulting in seven distinct forms of MPS with a collective incidence of higher than 1 in 25,000 live births. Accumulation of undegraded metabolites in lysosomes gives rise to distinct clinical syndromes. Generally, the clinical conditions progress if untreated, leading to developmental delay, systemic skeletal deformities, and early death. MPS disorders are potentially treatable with enzyme replacement therapy or hematopoietic stem cell transplantation. For maximum benefit of available therapies, early detection and intervention are critical. We recently developed a novel high-throughput multiplex method to assay DS, HS, and KS simultaneously in blood samples by using high performance liquid chromatography/tandem mass spectrometry for MPS. The overall performance metrics of HS and DS values on MPS I, II, and VII patients vs. healthy controls at newborns were as follows using a given set of cut-off values: sensitivity, 100%; specificity, 98.5–99.4%; positive predictive value, 54.5–75%; false positive rate, 0.62–1.54%; and false negative rate, 0%. These findings show that the combined measurements of these three GAGs are sensitive and specific for detecting all types of MPS with acceptable false negative/positive rates. In addition, this method will also be used for monitoring therapeutic efficacy. We review the history of GAG assay and application to diagnosis for MPS.
doi:10.1016/j.ymgme.2013.06.007
PMCID: PMC4047214  PMID: 23860310
Mucopolysaccharidoses; Tandem mass spectrometry; Newborn screening; Glycosaminoglycans; Monitoring
4.  Establishment of Glycosaminoglycan Assays for Mucopolysaccharidoses 
Metabolites  2014;4(3):655-679.
Mucopolysaccharidoses (MPS) are a group of lysosomal storage disorders caused by deficiency of the lysosomal enzymes essential for catabolism of glycosaminoglycans (GAGs). Accumulation of undegraded GAGs results in dysfunction of multiple organs, resulting in distinct clinical manifestations. A range of methods have been developed to measure specific GAGs in various human samples to investigate diagnosis, prognosis, pathogenesis, GAG interaction with other molecules, and monitoring therapeutic efficacy. We established ELISA, liquid chromatography tandem mass spectrometry (LC-MS/MS), and an automated high-throughput mass spectrometry (HT-MS/MS) system (RapidFire) to identify epitopes (ELISA) or disaccharides (MS/MS) derived from different GAGs (dermatan sulfate, heparan sulfate, keratan sulfate, and/or chondroitin sulfate). These methods have a high sensitivity and specificity in GAG analysis, applicable to the analysis of blood, urine, tissues, and cells. ELISA is feasible, sensitive, and reproducible with the standard equipment. HT-MS/MS yields higher throughput than conventional LC-MS/MS-based methods while the HT-MS/MS system does not have a chromatographic step and cannot distinguish GAGs with identical molecular weights, leading to a limitation of measurements for some specific GAGs. Here we review the advantages and disadvantages of these methods for measuring GAG levels in biological specimens. We also describe an unexpected secondary elevation of keratan sulfate in patients with MPS that is an indirect consequence of disruption of catabolism of other GAGs.
doi:10.3390/metabo4030655
PMCID: PMC4192686  PMID: 25116756
mucopolysaccharidoses; ELISA; tandem mass spectrometry; glycosaminoglycan; chondroitin sulfate; dermatan sulfate; heparan sulfate; keratan sulfate
5.  Assay for Glycosaminoglycans by Tandem Mass Spectrometry and its Applications 
Glycosaminoglycans (GAGs) are distributed in the whole body and play a variety of important physiological roles associated with inflammation, growth, coagulation, fibrinolysis, lipolysis, and cell-matrix biology. Accumulation of undegraded GAGs in lysosomes gives rise to a distinct clinical syndrome, mucopolysaccharidoses. Measurement of each specific GAG in a variety of specimens is urgently required to understand GAG interaction with other molecules, physiological status of patients, and prognosis and pathogenesis of the disease.
We established a highly sensitive and accurate tandem mass spectrometry (LC-MS/MS) method for measurements of disaccharides derived from four specific GAGs [dermatan sulfate (DS), heparan sulfate (HS), keratan sulfate (KS), and chondroitin sulfate (CS)]. Disaccharides were produced by specific enzyme digestion of each GAG, and quantified by negative ion mode of multiple reaction monitoring. Subclasses of HS and GAGs with identical molecular weights can be separated using a Hypercarbcolumn (2.0 mm×50 mm, 5 μm) with an aectonitrile gradient in ammonium acetate (pH 11.0).
We also developed a GAG assay by RapidFire with tandem mass spectrometry (RF-MS/MS). The RF system consists of an integrated solid phase extraction robot that binds and de-salts samples from assay plates and directly injects them into a MS/MS detector, reducing sample processing time to ten seconds. RF-MS/MS consequently yields much faster throughput than conventional LC-MS/MS-based methods.
However, the RF system does not have a chromatographic step, and therefore, cannot distinguish GAGs that have identical molecular weights. Both methods can be applied to analysis of dried blood spots, blood, and urine specimens.
In this article, we compare the assay methods for GAGs and describe their potential applications.
doi:10.4172/2155-9872.S2-006
PMCID: PMC4109812  PMID: 25068074
Tandem mass spectrometry; Glycosaminoglycans; Mucopolysaccharidoses; Chromatogram; Newborn screening
6.  Chondroitin 6-Sulfate as a Novel Biomarker for Mucopolysaccharidosis IVA and VII 
JIMD Reports  2014;16:15-24.
Chondroitin 6-sulfate (C6S), a glycosaminoglycan (GAG), is distributed mainly in the growth plates, aorta, and cornea; however, the physiological function of C6S is not fully understood. One of the limitations is that no rapid, accurate quantitative method to measure C6S has been established. Mucopolysaccharidosis IVA and VII (MPS IVA and VII) are caused by the deficiency of N-acetylgalactosamine-6-sulfate sulfatase and β-d-glucuronidase, respectively, resulting in accumulation of C6S and other GAG(s). While levels of keratan sulfate (KS), heparan sulfate, and dermatan sulfate in samples from MPS patients are well described, this is the first report of quantitative analysis of C6S levels in samples from MPS IVA and VII patients.
We developed a method to digest polymeric C6S and measure resultant disaccharides using liquid chromatography–tandem mass spectrometry (LC-MS/MS). C6S levels were measured in the blood from control subjects and patients with MPS IVA and VII aged from 0 to 58 years of age. We also assayed KS levels in the same samples for comparison with C6S.
Levels of C6S in the blood decreased with age and were significantly elevated in patients with MPS IVA and VII, compared with age-matched controls. Levels of KS in patients with MPS IVA were also higher than those in age-matched controls, although differences were less pronounced than with C6S. Combining KS and C6S data, discriminated patients with MPS IVA from age-matched control subjects were better than either C6S or KS levels alone.
In conclusion, this first report showing that blood levels of C6S are quantitatively evaluated in patients with MPS IVA and VII indicates that C6S could be a useful biomarker for these metabolic disorders.
doi:10.1007/8904_2014_311
PMCID: PMC4221613  PMID: 24850234
7.  Osteoinduction on Acid and Heat Treated Porous Ti Metal Samples in Canine Muscle 
PLoS ONE  2014;9(2):e88366.
Samples of porous Ti metal were subjected to different acid and heat treatments. Ectopic bone formation on specimens embedded in dog muscle was compared with the surface characteristics of the specimen. Treatment of the specimens by H2SO4/HCl and heating at 600°C produced micrometer-scale roughness with surface layers composed of rutile phase of titanium dioxide. The acid- and heat-treated specimens induced ectopic bone formation within 6 months of implantation. A specimen treated using NaOH followed by HCl acid and then heat treatment produced nanometer-scale surface roughness with a surface layer composed of both rutile and anatase phases of titanium dioxide. These specimens also induced bone formation after 6 months of implantation. Both these specimens featured positive surface charge and good apatite-forming abilities in a simulated body fluid. The amount of the bone induced in the porous structure increased with apatite-forming ability and higher positive surface charge. Untreated porous Ti metal samples showed no bone formation even after 12 months. Specimens that were only heat treated featured a smooth surface composed of rutile. A mixed acid treatment produced specimens with micrometer-scale rough surfaces composed of titanium hydride. Both of them also showed no bone formation after 12 months. The specimens that showed no bone formation also featured almost zero surface charge and no apatite-forming ability. These results indicate that osteoinduction of these porous Ti metal samples is directly related to positive surface charge that facilitates formation of apatite on the metal surfaces in vitro.
doi:10.1371/journal.pone.0088366
PMCID: PMC3919776  PMID: 24520375
8.  Detection of Novel Visible-Light Region Absorbance Peaks in the Urine after Alkalization in Patients with Alkaptonuria 
PLoS ONE  2014;9(1):e86606.
Background
Alkaptonuria, caused by a deficiency of homogentisate 1,2-dioxygenase, results in the accumulation of homogentisic acid (2,5-dihydroxyphenylacetic acid, HGA) in the urine. Alkaptonuria is suspected when the urine changes color after it is left to stand at room temperature for several hours to days; oxidation of homogentisic acid to benzoquinone acetic acid underlies this color change, which is accelerated by the addition of alkali. In an attempt to develop a facile screening test for alkaptonuria, we added alkali to urine samples obtained from patients with alkaptonuria and measured the absorbance spectra in the visible light region.
Methods
We evaluated the characteristics of the absorption spectra of urine samples obtained from patients with alkaptonuria (n = 2) and compared them with those of urine specimens obtained from healthy volunteers (n = 5) and patients with phenylketonuria (n = 3), and also of synthetic homogentisic acid solution after alkalization. Alkalization of the urine samples and HGA solution was carried out by the addition of NaOH, KOH or NH4OH. The sample solutions were incubated at room temperature for 1 min, followed by measurement of the absorption spectra.
Results
Addition of alkali to alkaptonuric urine yielded characteristic absorption peaks at 406 nm and 430 nm; an identical result was obtained from HGA solution after alkalization. The absorbance values at both 406 nm and 430 nm increased in a time-dependent manner. In addition, the absorbance values at these peaks were greater in strongly alkaline samples (NaOH- KOH-added) as compared with those in weakly alkaline samples (NH4OH-added). In addition, the peaks disappeared following the addition of ascorbic acid to the samples.
Conclusions
We found two characteristic peaks at 406 nm and 430 nm in both alkaptonuric urine and HGA solution after alkalization. This new quick and easy method may pave the way for the development of an easy method for the diagnosis of alkaptonuria.
doi:10.1371/journal.pone.0086606
PMCID: PMC3900575  PMID: 24466168
9.  Apatite-forming ability of titanium in terms of pH of the exposed solution 
In order to elucidate the main factor governing the capacity for apatite formation of titanium (Ti), Ti was exposed to HCl or NaOH solutions with different pH values ranging from approximately 0 to 14 and then heat-treated at 600°C. Apatite formed on the metal surface in a simulated body fluid, when Ti was exposed to solutions with a pH less than 1.1 or higher than 13.6, while no apatite formed upon exposure to solutions with an intermediate pH value. The apatite formation on Ti exposed to strongly acidic or alkaline solutions is attributed to the magnitude of the positive or negative surface charge, respectively, while the absence of apatite formation at an intermediate pH is attributed to its neutral surface charge. The positive or negative surface charge was produced by the effect of either the acidic or alkaline ions on Ti, respectively. It is predicted from the present results that the bone bonding of Ti depends upon the pH of the solution to which it is exposed, i.e. Ti forms a bone-like apatite on its surface in the living body and bonds to living bone through the apatite layer upon heat treatment after exposure to a strongly acidic or alkaline solution.
doi:10.1098/rsif.2012.0107
PMCID: PMC3405753  PMID: 22417910
titanium; NaOH or HCl treatments; apatite-forming ability; simulated body fluid; zeta potential; X-ray photoelectron spectroscopy
10.  Novel 5′TOPmRNAs Regulated by Ribosomal S6 Kinase Are Important for Cardiomyocyte Development: S6 Kinase Suppression Limits Cardiac Differentiation and Promotes Pluripotent Cells Toward a Neural Lineage 
Stem Cells and Development  2011;21(9):1538-1548.
Moving stem cells from bench to bedside has been a challenging task. Undermining this task is comprehending and optimizing the underlying regulatory mechanisms that drive differentiation of stem cells into desired cell and tissue types. Here we present evidence that ribosomal S6 kinase (S6K) is among the proteins upregulated as embryonic stem cells (ESCs) and human induced pluripotent stem cells differentiate into beating cardiomyocytes. We hypothesized that S6K plays a pivotal role in cardiomyogenesis, primarily because it regulates the translation of 3 cardiac-involved genes recently shown to have 5′ terminal oligopyrimidine (5′TOP) sequences: connexin 43 (Cx43), desmoplakin (Dsp), and phosphatase and tensin homolog (PTEN). Along with another independent laboratory, we confirmed that S6K is indeed upregulated in beating ESC-derived cardiomyocytes compared to the surrounding nonbeating, differentiated cells. S6K short interfering RNA-transfected stem cell cultures indicate that inhibition of S6K strongly hinders development of cardiomyocyte beating and translation of Cx43, Dsp, and PTEN; these cardiac 5′TOP mRNAs were only properly translated in cells with S6K, supporting our hypothesis. An unexpected discovery took the role of S6K one step further: S6K-knockdown stem cell cultures developed significantly more neurons than seen in embryoid bodies subjected to a typical cardiac differentiation protocol. These results introduced the novel idea that in addition to its critical cardiac roles, S6K may be a significant factor that prevents stem cells from pursuing a neuronal pathway. Overall, results have indicated the necessity of S6K for normal stem cell cardiomyogenesis, as well as lowered S6K expression for stem cell neurogenesis.
doi:10.1089/scd.2011.0582
PMCID: PMC3359640  PMID: 22165977
11.  Clinical characteristics and risk factors for septic shock in patients receiving emergency drainage for acute pyelonephritis with upper urinary tract calculi 
BMC Urology  2012;12:4.
Background
Acute pyelonephritis (APN) is a common complication of ureteral obstruction caused by urolithiasis, and it can be lethal if it progresses to septic shock. We investigated the clinical characteristics of patients undergoing emergency drainage and assessed risk factors for septic shock.
Methods
A retrospective study was performed of 98 patients (101 events) requiring emergency drainage at our urology department for obstructive APN associated with upper urinary tract calculi from January 2003 to January 2011. Clinical characteristics were summarized, and risk factors for septic shock were assessed by logistic regression analysis.
Results
Objective evidence of sepsis was found in 64 (63.4%) events, and 21 events (20.8%) were categorized as septic shock. Ninety-six patients recovered, but 2 patients died of septic shock. Multivariate analysis revealed that age and the presence of paralysis were independent risk factors for septic shock.
Conclusions
APN associated with upper urinary tract calculi is a severe disease that should be treated with caution, particularly when risk factors are present.
doi:10.1186/1471-2490-12-4
PMCID: PMC3353222  PMID: 22413829
12.  Positively charged bioactive Ti metal prepared by simple chemical and heat treatments 
Journal of the Royal Society Interface  2010;7(Suppl 5):S503-S513.
A highly bioactive bone-bonding Ti metal was obtained when Ti metal was simply heat-treated after a common acid treatment. This bone-bonding property was ascribed to the formation of apatite on the Ti metal in a body environment. The formation of apatite on the Ti metal was induced neither by its surface roughness nor by the rutile phase precipitated on its surface, but by its positively charged surface. The surface of the Ti metal was positively charged because acid groups were adsorbed on titanium hydride formed on the Ti metal by the acid treatment, and remained even after the titanium hydride was transformed into titanium oxide by the subsequent heat treatment. These results provide a new principle based on a positively charged surface for obtaining bioactive materials.
doi:10.1098/rsif.2010.0129.focus
PMCID: PMC3024574  PMID: 20444711
bioactive Ti metal; apatite formation; surface charge; chemical treatment; dental implant; orthopaedic implant
13.  Three Japanese Patients with Beta-Ketothiolase Deficiency Who Share a Mutation, c.431A>C (H144P) in ACAT1 
JIMD Reports  2011;3:107-115.
Mitochondrial acetoacetyl-CoA thiolase (T2) deficiency affects both isoleucine catabolism and ketone body metabolism. The disorder is characterized by intermittent ketoacidotic episodes. We report three Japanese patients. One patient (GK69) experienced two ketoacidotic episodes at the age of 9 months and 3 years, and no further episodes until the age of 25 years. She had two uncomplicated pregnancies. GK69 was a compound heterozygote of the c.431A>C (H144P) and c.1168T>C (S390P) mutations in T2 (ACAT1) gene. She was not suspected of having T2 deficiency during her childhood, but she was diagnosed as T2 deficient at the age of 25 years by enzyme assay using fibroblasts. The other two patients were identical twin siblings who presented their first ketoacidotic crisis simultaneously at the age of 3 years 4 months. One of them (GK77b) died during the first crisis and the other (GK77) survived. Even during severe crises, C5-OH and C5:1 were within normal ranges in their blood acylcarnitine profiles and trace amounts of tiglylglycine and small amounts of 2-methyl-3-hydroxybutyrate were detected in their urinary organic acid profiles. They were H144P homozygotes. This H144P mutation has retained the highest residual T2 activity in the transient expression analysis of mutant cDNA thus far, while the S390P mutation did not retain any residual T2 activity. The “mild” H144P mutation may result in subtle profiles in blood acylcarnitine and urinary organic acid analyses. T2-deficient patients with “mild” mutations have severe ketoacidotic crises but their chemical phenotypes may be subtle even during acute crises.
doi:10.1007/8904_2011_72
PMCID: PMC3509868  PMID: 23430882
14.  Comparison of Liquid Chromatography–Tandem Mass Spectrometry and Sandwich ELISA for Determination of Keratan Sulfate in Plasma and Urine 
Biomarker Insights  2011;6:69-78.
Background and aim:
Mucopolysaccharidosis IVA (MPS IVA) leads to skeletal dysplasia through excessive storage of chondroitin-6-sulfate and keratan sulfate (KS). KS is synthesized mainly in cartilage and released into circulation, making it a critical biomarker for MPS IVA to evaluate clinical course and effectiveness of therapies. Therefore, an accurate and sensitive method is required to measure KS levels.
Material and methods:
Using sandwich ELISA and liquid chromatography tandem mass spectrometry (LC/MS/MS) assays, we measured KS levels in blood and urine from MPS IVA patients and healthy controls to evaluate comparability of results. Blood (patients, n = 110; controls, n = 364) and urine (patients, n = 103; controls, n = 326) specimens were obtained.
Results:
Plasma and urine KS measurements in patients were age-dependent and higher than age-matched controls. We observed a moderate correlation (r = 0.666; P < 0.001) between urine KS measurements and a weak correlation (r = 0.333; P = 0.002) between plasma KS measurements by ELISA and LC/MS/MS methods in patients. No correlation was found between plasma KS measurements in controls. The difference between KS measurements assayed by LC/MS/MS and ELISA was greater in controls than in patients. A moderate correlation between blood and urine KS measurements in the same individual was observed.
Conclusion:
These findings indicate that both methods to measure blood and urine KS are suitable for diagnosis, monitoring therapies, and longitudinal assessment of the disease course in MPS IVA, but the LC/MS/MS method measures over 10 times more KS present in body fluids.
doi:10.4137/BMI.S7451
PMCID: PMC3140273  PMID: 21792275
biomarker; clinical severity; correlation; monitor therapy; MPS IVA; Mucopolysaccharidosis IVA
15.  Survivin Selectively Modulates Genes Deregulated in Human Leukemia Stem Cells 
Journal of Oncology  2010;2011:946936.
ITD-Flt3 mutations are detected in leukemia stem cells (LSCs) in acute myeloid leukemia (AML) patients. While antagonizing Survivin normalizes ITD-Flt3-induced acute leukemia, it also impairs hematopoietic stem cell (HSC) function, indicating that identification of differences in signaling pathways downstream of Survivin between LSC and HSC are crucial to develop selective Survivin-based therapeutic strategies for AML. Using a Survivin-deletion model, we identified 1,096 genes regulated by Survivin in ITD-Flt3-transformed c-kit+, Sca-1+, and lineageneg (KSL) cells, of which 137 are deregulated in human LSC. Of the 137, 124 genes were regulated by Survivin exclusively in ITD-Flt3+ KSL cells but not in normal CD34neg KSL cells. Survivin-regulated genes in LSC connect through a network associated with the epidermal growth factor receptor signaling pathway and falls into various functional categories independent of effects on apoptosis. Pathways downstream of Survivin in LSC that are distinct from HSC can be potentially targeted for selective anti-LSC therapy.
doi:10.1155/2011/946936
PMCID: PMC3021862  PMID: 21253548
16.  Acute osteomyelitis of the acetabulum induced by Staphylococcus capitis in a young athlete 
Pediatric Reports  2010;2(1):e2.
Acute hematogenous osteomyelitis (AHOM) of the acetabulum is a rare condition in children and usually caused by Staphylococcus aureus. We present an 11-year-old soccer athlete who suffered from acute osteomyelitis involving the acetabulum caused by S. capitis, a normal flora of the human skin but never reported in this condition. The disease was associated with repetitive skin injuries of the knee and potential osseous microtrauma of the hip joint by frequent rigorous exercise. This unusual case suggests that osseous microtrauma of the acetabulum, in addition to repetitive skin injuries, allowed normal skin flora to colonize to the ipsilateral acetabulum, which served as a favorable niche and subsequently led to AHOM.
doi:10.4081/pr.2010.e2
PMCID: PMC3094007  PMID: 21589838
acute osteomyelitis; acetabulum; osseous microtrauma; pre-existing trauma; Staphylococcus capitis.
17.  Enhancement of drug delivery to bone: Characterization of human tissue-nonspecific alkaline phosphatase tagged with an acidic oligopeptide 
Molecular genetics and metabolism  2006;88(3):244-255.
Hypophosphatasia is caused by deficiency of activity of the tissue-nonspecific alkaline phosphatase (TNSALP), resulting in a defect of bone mineralization. Enzyme replacement therapy (ERT) with partially purified plasma enzyme was attempted but with little clinical improvement. Attaining clinical effectiveness with ERT for hypophosphatasia may require delivering functional TNSALP enzyme to bone. We tagged the C-terminal-anchorless TNSALP enzyme with an acidic oligopeptide (a six or eight residue stretch of L-Asp), and compared the biochemical properties of the purified tagged and untagged enzymes derived from Chinese hamster ovary cell lines. The specific activities of the purified enzymes tagged with the acidic oligopeptide were the same as the untagged enzyme. In vitro affinity experiments showed the tagged enzymes had 30-fold higher affinity for hydroxyapatite than the untagged enzyme. Lectin affinity chromatography for carbohydrate structure showed little difference among the three enzymes. Biodistribution pattern from single infusion of the fluorescence-labeled enzymes into mice showed delayed clearance from the plasma up to 18h post infusion and the amount of tagged enzyme retained in bone was 4-fold greater than that of the untagged enzyme. In vitro mineralization assays with the bone marrow from a hypophosphatasia patient using each of the three enzymes in the presence of high concentrations of pyrophosphate provided evidence of bone mineralization. These results show the anchorless enzymes tagged with an acidic oligopeptide are delivered efficiently to bone and function bioactively in bone mineralization, at least in vitro. They suggest potential advantages for use of these tagged enzymes in ERT for hypophosphatasia, which should be explored.
doi:10.1016/j.ymgme.2006.02.012
PMCID: PMC2587042  PMID: 16616566
Hypophosphatasia; Drug delivery system; Alkaline phosphatase

Results 1-17 (17)