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Enzyme replacement therapy (ERT) is an effective treatment for several lysosomal storage disorders (LSDs). Intravenously infused enzymes are taken up by tissues through either the mannose 6-phosphate receptor (M6PR) or the mannose receptor (MR). It is generally believed that M6PR-mediated endocytosis is a key mechanism for ERT in treating LSDs that affect the non-macrophage cells of visceral organs. However, the therapeutic efficacy of MR-mediated delivery of mannose-terminated enzymes in these diseases has not been fully evaluated. We tested the effectiveness of a non-phosphorylated α-galactosidase A produced from moss (referred to as moss-aGal) in vitro and in a mouse model of Fabry disease. Endocytosis of moss-aGal was MR-dependent. Compared to agalsidase alfa, a phosphorylated form of α-galactosidase A, moss-aGal was more preferentially targeted to the kidney. Cellular localization of moss-aGal and agalsidase alfa in the heart and kidney was essentially identical. A single injection of moss-aGal led to clearance of accumulated substrate in the heart and kidney to an extent comparable to that achieved by agalsidase alfa. This study suggested that mannose-terminated enzymes may be sufficiently effective for some LSDs in which non-macrophage cells are affected, and that M6P residues may not always be a prerequisite for ERT as previously considered. Electronic supplementary material The online version of this article (doi:10.1007/s10545-015-9886-9) contains supplementary material, which is available to authorized users.

Fabry disease is caused by deficient activity of α-galactosidase A and subsequent accumulation of glycosphingolipids (mainly globotriaosylceramide, Gb3), leading to multisystem organ dysfunction. Oxidative stress and nitric oxide synthase (NOS) uncoupling are thought to contribute to Fabry cardiovascular diseases. We hypothesized that decreased tetrahydrobiopterin (BH4) plays a role in the pathogenesis of Fabry disease. We found that BH4 was decreased in the heart and kidney but not in the liver and aorta of Fabry mice. BH4 was also decreased in the plasma of female Fabry patients, which was not corrected by enzyme replacement therapy (ERT). Gb3 levels were inversely correlated with BH4 levels in animal tissues and cultured patient cells. To investigate the role of BH4 deficiency in disease phenotypes, 12-month-old Fabry mice were treated with gene transfer-mediated ERT or substrate reduction therapy (SRT) for 6 months. In the Fabry mice receiving SRT but not ERT, BH4 deficiency was restored, concomitant with ameliorated cardiac and renal hypertrophy. Additionally, glutathione levels were decreased in Fabry mouse tissues in a sex-dependent manner. Renal BH4 levels were closely correlated with glutathione levels and inversely correlated with cardiac and kidney weight. In conclusion, this study showed that BH4 deficiency occurs in Fabry disease and may contribute to the pathogenesis of the disease through oxidative stress associated with a reduced antioxidant capacity of cells and NOS uncoupling. This study also suggested dissimilar efficacy of ERT and SRT in correcting pre-existing pathologies in Fabry disease.

Odanacatib (ODN, MK-0822) is an investigational drug under development for the treatment of osteoporosis. A quantitative LC/MS–MS methodology was developed and validated to determine ODN concentrations in human plasma, with a linear calibration range from 0.500 to 500 ng/mL. Stable isotope 13 C 6 -labeled ODN was employed as the internal standard (IS). Sample preparation was based on liquid–liquid extraction of basified plasma with methyl t-butyl ether in a 96-well plate format. The extracted samples were analyzed on a liquid chromatography–tandem mass spectrometry system equipped with a turbo ion spray source. Chromatographic separation of the analyte and IS was achieved on a Phenomenex Luna C18 (50 mm × 2.0 mm, 5 μm) column. Ion pairs m / z 526 → 313 for the analyte and m / z 532 → 319 for the IS were monitored in positive ionization mode for MS detection. This methodology has been fully validated and proved to be rugged and reproducible. Intra- and inter-run variability was within 5.88%, with accuracy between 95.6 and 106% of the nominal concentrations. Analyte stability was evaluated under various sample preparation, analysis and storage conditions. This assay has been utilized to analyze human plasma samples obtained from phase I to III clinical trials.

The aim of our study was to measure globotriaosylceramide (Gb(3)) and lyso-Gb(3) levels by tandem mass spectrometry in the urine and kidney in Fabry (gla knockout) mice and wild-type controls. We found that urine Gb(3) of male and female Fabry mice was higher than wild-type mice of the same sex but also significantly higher in male mice compared with females of the same genotype. In kidney tissue, sex and genotype-dependent differences in Gb(3) levels paralleled those in the urine. Isoforms C16, C22:1, and C24OHA were particularly higher in males compared with females in both wild-type and Fabry mice. Similarly, kidney lyso-Gb(3) concentrations were significantly higher in 12-month-old male Fabry mice than in their homozygous female counterparts. However, lyso-Gb(3) was undetectable in wild-type mice of both sexes. α-Galactosidase A activity and mRNA levels in kidney were significantly lower in male wild-type mice compared with female mice. This study shows the sex differences in kidney and urine Gb(3) and kidney lyso-Gb(3) levels in both wild-type and Fabry mice, and it suggests that these male-female differences should be taken into consideration when using murine models for Fabry disease.

Background Flow-injection MS/MS methods for elevated acylcarnitines are routinely performed in most newborn screening and biochemical genetics laboratories; however this technique cannot distinguish between isobaric compounds; therefore, chromatographic separation is required to quantitate isomers for differential diagnosis of some inborn errors of metabolism. Methods A UPLC–MS/MS method has been developed for the simultaneous quantitation of isobutyrylcarnitine and butyrylcarnitine, and a second UPLC–MS/MS method for the quantitation of isovalerylcarnitine, ( S ) and ( R ) 2-methylbutyrylcarnitine, pivaloylcarnitine and valerylcarnitine. Plasma and dried blood spots samples are extracted with methanol and derivatized with butanolic HCl. Deuterium labeled internal standards are used for quantitation. Separation is obtained using a methanol/water gradient with a C18 BEH, 1×100mm, 1.7μm UPLC column, at 60°C; run time is less than 10min. The isomers are detected with a Quattro Premier triple quadrupole, with electrospray ionization in positive ion mode. Results Intra-day precision in plasma and dried blood spots ranged from 1.4% to 14% and accuracy from 88% to 114% respectively for butyrylcarnitine and isobutyrylcarnitine. Precision for the isomers of C5-acylcarnitine ranged from 1.3% to 15% and accuracy 87% to 119%, respectively in plasma or dried blood spots. Inter-day precision was within 20% at each concentration of isobutyrylcarnitine and butyrylcarnitine. Precision for 2-methylbutyrylcarnitine and isovalerylcarnitine at concentrations above the normal range was within 24%. Conclusions Two diagnostic tests based on the separation of C4-acylcarnitine and C5-acylcarnitine isomers by UPLC–MS/MS provide fast differential diagnosis of SCAD deficiency versus IBCD deficiency and IVA versus 2-MBCD deficiency. The separation of C5-acylcarnitines can reveal false elevation due to pivalic acid-containing antibiotics. Abnormal newborn screen results due to pivalate-generating prodrug antibiotics of maternal origin were confirmed. This separation of isomers can resolve multiple diagnostic challenges in both newborn screening and in cases with ambiguous metabolic test results.

Fabry disease is caused by deficient activity of lysosomal enzyme α-galactosidase A. The enzyme deficiency results in intracellular accumulation of glycosphingolipids, leading to a variety of clinical manifestations including hypertrophic cardiomyopathy and renal insufficiency. The mechanism through which glycosphingolipid accumulation causes these manifestations remains unclear. Current treatment, especially when initiated at later stage of the disease, does not produce completely satisfactory results. Elucidation of the pathogenesis of Fabry disease is therefore crucial to developing new treatments. We found increased activity of androgen receptor (AR) signaling in Fabry disease. We subsequently also found that blockade of AR signaling either through castration or AR-antagonist prevented and reversed cardiac and kidney hypertrophic phenotype in a mouse model of Fabry disease. Our findings implicate abnormal AR pathway in the pathogenesis of Fabry disease and suggest blocking AR signaling as a novel therapeutic approach.

Background Elevated urinary globotriaosylceramide (Gb 3 ) has been considered a hallmark of F abry disease, an X‐linked lysosomal disorder that is a risk factor for most types of heart disease. Methods and Results We screened 1421 consecutive patients with common forms of heart disease for F abry disease by measuring urinary Gb 3 in whole urine using tandem mass spectrometry, α‐galactosidase A activity in dried blood spots, and we looked for GLA mutations by parallel sequencing of the whole gene (exons and introns) in pooled genomic DNA samples followed by Sanger sequencing verification. GLA variants were found in 13 patients. In the 1408 patients without GLA mutations, urinary Gb 3 levels were significantly higher in heart disease patients compared to 116 apparently healthy controls (median difference=10.0 ng/mL and P F abry disease (n=7) and heart disease patients with elevated urinary Gb 3 (n=6). Sphingomyelin and Gb 3 levels were abnormal in the left ventricular wall of patients with ischemic heart failure. Elevated levels of urinary Gb 3 were independently associated with increased risk of death in the average follow‐up of 17 months (hazard ratio=1.59 for increase in Gb 3 of 200, 95% CI =1.36 and 1.87, and P Conclusions In heart disease patients who do not have F abry disease or GLA gene mutations, a higher level of urinary Gb 3 is positively associated with near‐term mortality. The elevation of urinary Gb 3 and that of other lipids suggests that heart disease is associated with multiorgan lipid abnormalities. Clinical Trial Registration URL: clinicaltrials.gov. Unique Identifier: NCT 01019629.

Reply: We appreciate the interest in our report on newborn screening for succinic semialdehyde dehydrogenase (SSADH) deficiency by Drs. Stove, Ingels, and Lambert [1]. We are also grateful for their highlighting of excellent GCMS methods for determining gamma-hydroxybutyric acid (GHB) in dried blood spots (DBS) for use in toxicology or forensics [2,3]. Our method was developed to screen DBS of newborns for the very high elevations of GHB found in the blood of patients with the inherited disorder, SSADH deficiency [4]. It utilized LC-MS/MS which is currently available in most newborn screening laboratories. Our method was not intended for toxicology use, or for the accurate determination of low endogenous levels in DBS of newborns not affected with SSADH deficiency. Nevertheless it does permit establishing a screening cut-off for GHB above which a deficiency of SSADH is likely. As with most newborn screening tests, elevations of GHB in DBS above the cut-off would need to be followed up with diagnostic tests to determine the final diagnosis, which could include accurate tests for GHB levels in blood or urine, urine organic acid analysis for GHB levels in biochemical genetics laboratories, or sequencing DNA for mutations in the ALDH5A1 gene. With this report our goal was to demonstrate the feasibility of this approach, the manuscript was not intended as a detailed method development and/or validation paper; accordingly, only a brief description of the validation was summarized in the results. The letter to the editor correctly points out that the GHB concentrations were μM, which was an oversight on our part. We believe our report is an important contribution to the prospect of widespread newborn screening for SSADH deficiency, for which potential specific therapy is currently in clinical trials.

Objective SSADH deficiency, the most prevalent autosomal recessive disorder of GABA degradation, is characterized by elevated gamma-hydroxybutyric acid (GHB). Neurological outcomes may be improved with early intervention and anticipatory guidance. Morbidity has been compounded by complications, e.g. hypotonia, in undiagnosed infants with otherwise routine childhood illnesses. We report pilot methodology on the feasibility of newborn screening for SSADH deficiency. Method Dried blood spot (DBS) cards from patients affected with SSADH deficiency were compared with 2831 archival DBS cards for gamma-hydroxybutyric acid content. Following extraction with methanol, GHB in DBS was separated and analyzed using ultra high-performance liquid chromatography tandem mass spectrometry. Results Methodology was validated to meet satisfactory accuracy and reproducibility criteria, including intra-day and inter-day validation. Archival refrigerated dried blood spot samples of babies, infants and children (N = 2831) were screened for GHB, yielding a mean +/− S.D. of 8 ± 5 nM (99.9%-tile 63 nM) (Min 0.0 Max 78 nM). The measured mean and median concentrations in blood spots derived from seven SSADH deficient patients were 1182 nM and 699 nM respectively (Min 124, Max 4851 nM). Conclusions GHB concentration in all 2831 dried blood spot cards was well below the lowest concentration of affected children. These data provide proof-of-principle for screening methodology to detect SSADH deficiency with applicability to newborn screening and earlier diagnosis.

and C17-Gb3 as internal standards. Ten microliters are injected into a UPLC-MS/MS for the simultaneous determination of creatinine and Gb3. Urine of 100 healthy subjects from newborns to 65 years old was analyzed. The concentration of creatinine in urine varied widely due to diet or age: from less then 1 lmol/mL in infants and some adults to higher than 30 lmol/mL in adults. Gb3 in urine is usually expressed as Gb3/creatinine concentration because creatinine is believed to correct for the excretion of more or less concentrated urine [4]. Low creatinine (