Hemamalini Ketha

Sclerostin alters serum vitamin D metabolite and fibroblast growth factor 23 concentrations and the urinary excretion of calcium

Inactivating mutations of the SOST (sclerostin) gene are associated with overgrowth and sclerosis of the skeleton. To determine mechanisms by which increased amounts of calcium and phosphorus are accreted to enable enhanced bone mineralization in the absence of sclerostin, we measured concentrations of calciotropic and phosphaturic hormones, and urine and serum calcium and inorganic phosphorus in mice in which the sclerostin (sost) gene was replaced by the β-D-galactosidase (lacZ) gene in the germ line. Knockout (KO) (sost−/−) mice had increased bone mineral density and content, increased cortical and trabecular bone thickness, and greater net bone formation as a result of increased osteoblast and decreased osteoclast surfaces compared with wild-type (WT) mice. β-Galactosidase activity was detected in osteocytes of sost KO mice but was undetectable in WT mice. Eight-week-old, male sost KO mice had increased serum 1α,25-dihydroxyvitamin D, decreased 24,25-dihydroxyvitamin D, decreased intact fibroblast growth factor 23, and elevated inorganic phosphorus concentrations compared with age-matched WT mice. 25-Hydroxyvitamin D 1α-hydroxylase cytochrome P450 (cyp27B1) mRNA was increased in kidneys of sost KO mice compared with WT mice. Treatment of cultured proximal tubule cells with mouse recombinant sclerostin decreased cyp27B1 mRNA transcripts. Urinary calcium and renal fractional excretion of calcium were decreased in sost KO mice compared with WT mice. Sost KO and WT mice had similar serum calcium and parathyroid hormone concentrations. The data show that sclerostin not only alters bone mineralization, but also influences mineral metabolism by altering concentrations of hormones that regulate mineral accretion.

Clinical applications of LC-MS sex steroid assays: evolution of methodologies in the 21st century.

Purpose of reviewThe purpose of this review is to summarize why and how liquid chromatography tandem mass spectrometry (LC-MS/MS) is increasingly replacing other methodologies for the measurement of sex steroids. Recent findingsMeasurement of sex steroids, particularly testosterone and estradiol, is important for diagnosis or management of a host of conditions (e.g. disorders of puberty, hypogonadism, polycystic ovary syndrome, amenorrhea, and tumors of ovary, testes, breast and prostate). Historically, metabolites of testosterone and estradiol were measured as ketosteroids in urine using colorimetric assays that lacked sensitivity and specificity due to endogenous and exogenous interferences. Extracted competitive manual radio-immunoassays provided improved, but still imperfect, specificity, and offered increased sensitivity. As testing demand increased, they were displaced by automated immunoassays. These offered better throughput and precision, but suffered worse specificity problems. Moreover, agreement between different immunoassays has often been poor and they are all compromised by a limited dynamic measurement range. To overcome these problems, LC-MS/MS methods have been developed and validated for quantitation of sex steroids. These methods reduce interferences, provide better specificity, improve dynamic range, and reduce between-method bias. SummaryEndocrine Society and Urology Society guidelines have highlighted the limitations of the immunoassays for sex steroids and have provided convincing evidence that mass spectrometric methods are preferable for measurement of sex steroid hormones. In this review, we describe LC-MS/MS methods for measurement of testosterone and estradiol.

Research Resource: Whole Transcriptome RNA Sequencing Detects Multiple 1α,25-Dihydroxyvitamin D3-Sensitive Metabolic Pathways in Developing Zebrafish

The biological role of vitamin D receptors (VDR), which are abundantly expressed in developing zebrafish (Danio rerio) as early as 48 h after fertilization, and before the development of a mineralized skeleton and mature intestine and kidney, is unknown. We probed the role of VDR in developing zebrafish biology by examining changes in expression of RNA by whole transcriptome shotgun sequencing (RNA-seq) in fish treated with picomolar concentrations of the VDR ligand and hormonal form of vitamin D(3), 1α,25-dihydroxyvitamin D(3) [1α,25(OH)(2)D(3))].We observed significant changes in RNAs of transcription factors, leptin, peptide hormones, and RNAs encoding proteins of fatty acid, amino acid, xenobiotic metabolism, receptor-activator of NFκB ligand (RANKL), and calcitonin-like ligand receptor pathways. Early highly restricted, and subsequent massive changes in more than 10% of expressed cellular RNA were observed. At days post fertilization (dpf) 2 [24 h 1α,25(OH)(2)D(3)-treatment], only four RNAs were differentially expressed (hormone vs. vehicle). On dpf 4 (72 h treatment), 77 RNAs; on dpf 6 (120 h treatment) 1039 RNAs; and on dpf 7 (144 h treatment), 2407 RNAs were differentially expressed in response to 1α,25(OH)(2)D(3). Fewer RNAs (n = 481) were altered in dpf 7 larvae treated for 24 h with 1α,25(OH)(2)D(3) vs. those treated with hormone for 144 h. At dpf 7, in 1α,25(OH)(2)D(3)-treated larvae, pharyngeal cartilage was larger and mineralization was greater. Changes in expression of RNAs for transcription factors, peptide hormones, and RNAs encoding proteins integral to fatty acid, amino acid, leptin, calcitonin-like ligand receptor, RANKL, and xenobiotic metabolism pathways, demonstrate heretofore unrecognized mechanisms by which 1α,25(OH)(2)D(3) functions in vivo in developing eukaryotes.

Iatrogenic vitamin D toxicity in an infant – a case report and review of literature

Public concern over vitamin D deficiency has led to widespread use of over the counter (OTC) vitamin D (-D3 or -D2) supplements, containing up to 10,000 IU/unit dose (400 IU=10μg). Overzealous use of such supplements can cause hypercalcemia due to vitamin D toxicity. Infants are particularly vulnerable to toxicity associated with vitamin D overdose. OTC supplements are not subject to stringent quality control regulations from FDA and high degree of variability in vitamin D content in OTC pills has been demonstrated. Other etiologies of vitamin D induced hypercalcemia include hyperparathyroidism, granulomatous malignancies like sarcoidosis and mutations in the CYP24A1 gene. The differential diagnosis of hypercalcemia should include iatrogenic and genetic etiologies. C24-hydroxylation and C3-epimerization are two important biochemical pathways via which 25-hydroxyvitamin D3 (25(OH)D3) is converted to its metabolites, 24,25-dihydroxyvitamin D3 (24,25(OH)2D3) or its C3 epimer, 3-epi-25-OH-D3 respectively. Mutations in the CYP24A1 gene cause reduced serum 24,25(OH)2D3 to 25(OH)D3 ratio (<0.02), elevated serum 1,25-dihydroxyvitamin D (1,25(OH)2D3), hypercalcemia, hypercalciuria and nephrolithiasis. Studies in infants have shown that 3-epi-25(OH)D3 can contribute 9-61.1% of the total 25(OH)D3. Therefore, measurements of parathyroid hormone (PTH) and vitamin D metabolites 25(OH)D3, 1,25(OH)2D3, 3-epi-25(OH)D3 and 24,25(OH)2D3 are useful to investigate whether the underlying cause of vitamin D toxicity is iatrogenic versus genetic. Here we report a case of vitamin D3 associated toxicity in a 4-month-old female who was exclusively breast-fed and received an oral liquid vitamin D3 supplement at a dose significantly higher than recommended on the label. The vitamin D3 content of the supplement was threefold higher (6000 IU of D/drop) than listed on the label (2000 IU). Due to overdosing and higher vitamin D3 content, the infant received ∼50,000 IU/day for two months resulting in severe hypercalcemia, hypercalciuria and nephrocalcinosis. We also review the relevant literature on vitamin D3 toxicity in this report.

Estradiol assays--The path ahead.

Estradiol quantitation is useful in the clinical assessment of diseases like hypogonadism, hirsutism, polycystic ovary syndrome (PCOS), amenorrhea, ovarian tumors and for monitoring response in women receiving aromatase inhibitor therapy. Physiologically relevant serum estradiol concentration in women can span across four orders of magnitude. For example, in women undergoing ovulation induction serum estradiol concentration can range between 250-2000 pg/mL whereas aromatase inhibitor therapy can decrease serum estradiol concentration to <5 pg/mL. While high-through-put automated un-extracted (direct) immunoassays accommodate the growing clinical need for estradiol quantitation, are amenable to implementation by most hospital clinical laboratories, they display a significant loss of specificity and accuracy at low concentrations. Most clinical scenarios (example: estradiol monitoring in fertility treatments) place a modest demand on accuracy and precision of the assay in use but accurate quantitation of estradiol in certain clinical scenarios (pediatric and male patients and for monitoring aromatase inhibitor therapy) can be challenging using currently available immunoassays since the direct immunoassays are prone to issues with sub-optimal accuracy and specificity due to cross reactivity with estradiol conjugates and metabolites. In this review we discuss the bases for the evolution of estradiol assays from extracted (indirect) radio-immunoassays to direct immunoassays to liquid-chromatography tandem mass spectrometry (LC-MS/MS) based assays, discuss technical factors relevant for development and optimization of a LC-MS/MS assay for estradiol and present the details and performance characteristics of an ultra-sensitive LC-MS/MS estradiol assay with a limit of quantitation of 0.2 pg/mL.

Altered Calcium and Vitamin D Homeostasis in First-Time Calcium Kidney Stone-Formers

Background Elevated serum 1,25-dihydroxyvitamin D (1,25(OH)2D) concentrations have been reported among cohorts of recurrent calcium (Ca) kidney stone-formers and implicated in the pathogenesis of hypercalciuria. Variations in Ca and vitamin D metabolism, and excretion of urinary solutes among first-time male and female Ca stone-formers in the community, however, have not been defined. Methods In a 4-year community-based study we measured serum Ca, phosphorus (P), 25-hydroxyvitamin D (25(OH)D), 1,25(OH)2D, 24,25-dihydroxyvitamin D (24,25(OH)2D), parathyroid hormone (PTH), and fibroblast growth factor-23 (FGF-23) concentrations in first-time Ca stone-formers and age- and gender frequency-matched controls. Results Serum Ca and 1,25(OH)2D were increased in Ca stone-formers compared to controls (P = 0.01 and P = 0.001). Stone-formers had a lower serum 24,25(OH)2D/25(OH)D ratio compared to controls (P = 0.008). Serum PTH and FGF-23 concentrations were similar in the groups. Urine Ca excretion was similar in the two groups (P = 0.82). In controls, positive associations between serum 25(OH)D and 24,25(OH)2D, FGF-23 and fractional phosphate excretion, and negative associations between serum Ca and PTH, and FGF-23 and 1,25(OH)2D were observed. In SF associations between FGF-23 and fractional phosphate excretion, and FGF-23 and 1,25(OH)2D, were not observed. 1,25(OH)2D concentrations associated more weakly with FGF-23 in SF compared with C (P <0.05). Conclusions Quantitative differences in serum Ca and 1,25(OH)2D and reductions in 24-hydroxylation of vitamin D metabolites are present in first-time SF and might contribute to first-time stone risk.

Clinical assays for quantitation of insulin-like-growth-factor-1 (IGF1).

Insulin-like growth factor 1 (IGF1), a 70 amino acid peptide hormone is the principal mediator of effects of growth hormone (GH). Since GH secretion is pulsatile in nature and is affected by many factors including sleep, feeding and exercise it is not a reliable marker for diagnosis of GH related disorders. On the other hand, IGF1 levels does not undergo short-term fluctuations in the manner that GH does making it the preferred IGF1 biomarker for the diagnosis of growth related disorders. There are several immunoassays available for IGF1 determination. Since majority (>90%) of IGF1 circulates as a ternary complex bound to its principal carrier/binding protein, IGF binding protein 3 (IGFBP3) and acid labile subunit (ALS), the assay methodology used to quantitate IGF1 has to dissociate IGF1 from IGFBPs prior to quantitation. IGFBPs are known to be a source of interference in immunoassays and many techniques have been employed to circumvent this issue. Immunoassays rely on antibody specificity towards IGF1 and differential cross reactivity towards IGFBPs. Mass spectrometry (MS) has also been employed for quantitation of IGF1. Liquid chromatography tandem mass spectrometry (LC-MS/MS) assays for IGF1 rely on generating tryptic peptides followed by selective reaction monitoring (SRM) while LC high resolution accurate-mass mass spectrometry (LC-HRAMS) approaches for intact IGF1 rely on mass accuracy for reliable, robust and accurate quantitation. This review article will focus on the clinical assays available and the clinical utility of quantitative assessment of IGF1. IGF1 quantitation using diverse assay platforms including immunoassay, LC-MS/MS and LC-HRAMS are discussed in detail.

Detection of IGF-1 Protein Variants by Use of LC-MS with High-Resolution Accurate Mass in Routine Clinical Analysis

To the Editor: Low or increased serum insulin-like growth factor 1 (IGF-1)1 concentrations might indicate growth hormone (GH) deficiency or overproduction, respectively. Currently, IGF-1 is mostly measured by automated immunometric assays. However, reagent availability issues prompted us to evaluate LC-MS with high-resolution accurate mass measurement (HRAM) as an alternative (1). We developed and validated an IGF-1 LC-MS HRAM method on a Q Exactive mass spectrometer (Thermo Scientific) using 1:70 000 resolution (at m / z 200) with a mass accuracy of 5 ppm. A similar method has been published for a qTOF instrument (Agilent) (2). We compared our LC-MS-HRAM method initially in 459 patient samples with the iSYS IGF-1 automated immunoassay (IDS). This yielded a least square linear fit of LC-MS HRAM = 0.84 × iSYS + 3.5, with an r 2 of 0.966. During further studies involving 1720 samples to generate reference ranges, the 2 methods continued to be highly correlated, with a reproducible systematic bias. However, we also identified 16 outliers for which the LC-MS HRAM method gave dramatically lower results than the iSYS immunoassay …

Analytical and clinical validation of parathyroid hormone (PTH) measurement in fine-needle aspiration biopsy (FNAB) washings

BACKGROUND Parathyroid hormone (PTH) quantitation in fine needle aspirate biopsy (FNAB) saline washings complements current modalities for parathyroid tissue localization. OBJECTIVES To establish the performance characteristics of the Roche Elecsys intact PTH immunoassay in FNAB needle washings and its diagnostic performance for the identification of parathyroid tissue. DESIGN AND METHODS Accuracy, precision, reportable range, and analytical specificity and sensitivity for the intact PTH immunoassay in FNAB needle washings were established. For clinical validation, 93 specimens from 79 patients were evaluated. Diagnostic cut-offs were established via receiver operator characteristic (ROC) curve analysis. Performance of PTH in FNAB needle washings was compared to cytology. RESULTS Measurement of the PTH in FNAB needle washings demonstrated a matrix interference that was overcome by supplementation of the samples with a protein based matrix prior to analysis. ROC area under the curve (AUC) was 0.96 for PTH in FNAB needle washings. A PTH concentration ≥100pg/mL showed 100% specificity and 82% sensitivity for identifying parathyroid tissue. On histology-confirmed parathyroid specimens, 21/38 (55%) were correctly identified by cytology; whereas 31/38 (82%) were identified by PTH. CONCLUSIONS Measurement of PTH in FNAB washings complements cytology for identification of parathyroid tissue. Analytical validation to exclude interference in the PTH immunoassay and proper localization of the parathyroid tissue by ultrasound is necessary to ensure the robustness of the method.

To Monitor Dabigatran or Not: A Matter of Patient Safety

In October 2010, the US Food and Drug Administration (FDA)2 approved dabigatran etexilate (Pradaxa), a new oral, direct thrombin inhibitor, for prevention of stroke and thrombosis in patients with nonvalvular atrial fibrillation (AF). This marked a new era in the development of fixed-dose novel oral anticoagulants (NOACs) with the hope of achieving improved safety and clinical outcomes compared with warfarin. The concept of a fixed-dose anticoagulant that requires no monitoring quickly made the transition from concept to practice, helped partially by the FDAs new approach to innovative therapies. This new approach gave the manufacturer, Boehringer Ingelheim, access to priority reviews and a shorter approval process for its blockbuster drug, dabigatran. Rather than the conventional requirement of at least 2 pivotal trials, only a single large clinical trial was necessary for approval. Fixed-dose dabigatran demonstrated noninferior performance compared with dose-optimized warfarin therapy in the Randomized Evaluation of Long-Term Anticoagulation Therapy (RE-LY) trial for prevention of stroke and thrombosis in patients with nonvalvular AF, thus providing the bulk of support for FDA approval. After approval by the FDA in 2010 and the European Medicines Agency (EMA) a year later, dabigatrans popularity grew quickly, and by the end of 2013 sales approached