Ketha, Hemamalini

Abstract BACKGROUND Since 2013, an unprecedented surge in fentanyl overdose deaths has been caused by heroin laced with illicitly produced fentanyl and/or fentanyl analogs (FAs) sold as heroin. The US Drug Enforcement Agency's National Forensic Laboratory Information System reported a >300% increase in fentanyl encounters from 4697 in 2014 to 14440 in 2015. In 2015, the CDC reported 9580 deaths caused by synthetic opioids, primarily fentanyl, a 72% increase from 2014. The European Monitoring Centre for Drugs and Drug Addiction has also encountered several new FAs in the heroin supply. Counterfeit pharmaceuticals containing mixtures of fentanyl and FAs continue to be a poorly recognized worldwide problem despite the WHO classifying several FAs as a serious threat to public health. CONTENT This review covers the epidemiology of fentanyl abuse and discusses the clinical practice implications of widespread fentanyl abuse. It includes a historical perspective on the illicit FAs that have appeared in the US and European Union and reviews the methods available to identify FAs and emerging technologies useful for identifying previously undescribed analogs. A compilation of structural and mass spectral data on FAs reported thus far is provided. SUMMARY Fentanyl and FAs have evolved into a global public health threat. It is important to understand the analytical, clinical, and regulatory efforts underway to assist communities affected by the current fentanyl epidemic.

Abstract BACKGROUND Patients have been described with loss-of-function CYP24A1 (cytochrome P450, family 24, subfamily A, polypeptide 1) mutations that cause a high ratio of 25-hydroxyvitamin D to 24,25-dihydroxyvitamin D [25(OH)D/24,25(OH)2D], increased serum 1,25-dihydroxyvitamin D, and resulting hypercalcemia, hypercalciuria and nephrolithiasis. A 25(OH)D/24,25(OH)2D ratio that can identify patients who are candidates for confirmatory CYP24A1 genetic testing would be valuable. We validated an LC-MS/MS assay for 24,25(OH)2D (D3 and D2) and determined a 25(OH)D/24,25(OH)2D cutoff to identify candidates for confirmatory genetic testing. METHODS After addition of isotope-labeled internal standard, serum samples were extracted by solid-phase extraction, derivatized with 4-phenyl-1,2,4,-triazoline-3,5-dione, and quantified by LC-MS/MS. We measured 25(OH)D/24,25(OH)2D in 91 healthy patients and 34 patients with clinically suspected CYP24A1-mediated hypercalcemia. RESULTS The limits of detection and quantification were 0.03 (0.2) and 0.1 (0.24) nmol/L, respectively, for 24,25(OH)2D3, and 0.1 (0.23) and 0.5 (1.16) nmol/L for 24,25(OH)2D2. Intra- and interassay imprecision was 4%–15% across the analytical measurement range of 0.1–25 ng/mL (0.2–60 nmol/L). No interference was observed with 25(OH)D and 1,25(OH)2D. 25(OH)D/24,25(OH)2D of 7–35 was observed in healthy patients, whereas in 2 patients with CYP24A1 mutations, 25(OH)D/24,25(OH)2D was significantly increased (99–467; P < 0.001). A 25(OH)D/24,25(OH)2D ratio ≥99 identified patients who were candidates for CYP24A1 genetic testing. CONCLUSIONS Increased 25(OH)D/24,25(OH)2D supports the diagnosis of reduced CYP24A1 activity due to mutations in CYP24A1. Measurement of 25(OH)D/24,25(OH)2D should be considered a part of the clinical workup in patients with hypercalcemia of otherwise unknown etiology.

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.

AbstractThe 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 D3, 1α,25-dihydroxyvitamin D3 [1α,25(OH)2D3)].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)2D3-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)2D3. Fewer RNAs (n = 481) were altered in dpf 7 larvae treated for 24 h with 1α,25(OH)2D3vs. those treated with hormone for 144 h. At dpf 7, in 1α,25(OH)2D3-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)2D3 functions in vivo in developing eukaryotes.