The Fentanyl Epidemic and Evolution of Fentanyl Analogs in the United States and the European Union.

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.

Effect of Diabetes Mellitus on Sickle Hemoglobin Quantitation in Sickle Cell Trait.

OBJECTIVES: We evaluated the effect of diabetes on sickle hemoglobin (HbS) measurement on two common clinical hemoglobin separation platforms. METHODS: We performed a method comparison between the Bio-Rad Variant II high-performance liquid chromatography (HPLC) and Sebia Capillarys 2 Flex Piercing capillary electrophoresis (CE) using clinical specimens from 38 patients without hemoglobin variants and 57 patients with sickle cell trait (AS) (HbA1c%, 4.1%-15.4%). We investigated the effect of intermethod Hb% differences on result interpretation by a panel of expert clinical observers. RESULTS: In diabetic specimens, HPLC undermeasured HbS% up to 7.4% vs CE, due to S1c eluting closely with A0 in HPLC. This increased concern for underlying α-thalassemia in diabetic patients with AS based on HPLC results. HPLC P2% was linearly related to HbA1c% and can be a screen for diabetic AS samples. CONCLUSIONS: Glycosylation can interfere with HbS% measurement by HPLC. Susceptible specimens should be identified and preferentially analyzed via CE.

10 years of 25-hydroxyvitamin-D testing by LC-MS/MS-trends in vitamin-D deficiency and sufficiency.

In early 2000's vitamin-D deficiency was shown to be prevalent in several countries including the United States (US). Studies exploring the role of vitamin-D metabolism in diverse disease pathways generated an increased demand for vitamin-D supplementation and an immense public interest in measurement of vitamin-D metabolite levels. In this report, we review the role of vitamin-D metabolism in disease processes, clinical utility of measuring vitamin-D metabolites including 25-hydroxyvitamin-D (25(OH)D), 1,25-dihydroxyvitamin-D and 24,25-dihydroxyvitamin-D and discuss vitamin-D assay methodologies including immunoassays and liquid chromatography mass spectrometry (LC-MS/MS) assays. We also provide examples of vitamin-D toxicity and insight into the trends in serum 25(OH)D levels in the US population based on 10 years of data from on serum 25(OH)D values from ~5,000,000 patients who were tested at the Mayo Medical Laboratories between February 2007-February 2017.

Comparison of the effect of daily versus bolus dose maternal vitamin D3 supplementation on the 24,25-dihydroxyvitamin D3 to 25-hydroxyvitamin D3 ratio.

OBJECTIVE: Supplementing lactating mothers with high doses of vitamin D3 can adequately meet vitamin D requirements of the breastfed infant. We compared the effect of bolus versus daily vitamin D3 dosing in lactating mothers on vitamin D3 catabolism. We hypothesized that catabolism of 25(OH)D3 to 24,25(OH)2D3 would be greater in the bolus than in the daily dose group. DESIGN, SETTING AND PATIENTS: Randomized controlled trial (clinicaltrials.govNCT01240265) in 40 lactating women. INTERVENTIONS: Subjects were randomized to receive vitamin D3 orally, either a single dose of 150,000IU or 5000IU daily for 28days. Vitamin D metabolites were measured in serum and breast milk at baseline, 1, 3, 7, 14 and 28days. MAIN OUTCOME MEASURE: Temporal changes in the serum 24,25(OH)2D3/25(OH)D3 ratio. RESULTS: The concentration of serum 24,25(OH)2D3 was directly related to that of 25(OH)D in both groups (r2=0.63; p<0.001). The mean (±SD) 24,25(OH)2D3/25(OH)D3 ratio remained lower at all time points than baseline values in the daily dose group (0.093±0.024, 0.084±0.025, 0.083±0.024, 0.080±0.020, 0.081±0.023, 0.083±0.018 at baseline, 1, 3, 7, 14, and 28days, respectively). In the single dose group, the increase in 24,25(OH)2D3 lagged behind that of 25(OH)D, but the 24,25(OH)2D3/25(OH)D3 values (0.098±0.032, 0.067±0.019, 0.081±0.017, 0.092±0.024, 0.103±0.020, 0.106±0.024, respectively) exceeded baseline values at 14 and 28days and were greater than the daily dose group at 14 and 28days (p=0.003). The 24,25(OH)2D3/25(OH)D3 ratio remained in the normal range with both dosing regimens. Greater breast milk vitamin D3 values in the single dose group were inversely associated with the 24,25(OH)2D3/25(OH)D3 ratio (r2=0.14, p<0.001), but not with daily dosing. CONCLUSIONS: After a 14-day lag, a single high dose of vitamin D led to greater production of 24,25(OH)2D3, presumably via induction of the 24-hydroxylase enzyme (CYP24A1), relative to the 25(OH)D3 value than did daily vitamin D supplementation, and this effect persisted for at least 28days after vitamin D administration. A daily dose of vitamin D may have more lasting effectiveness in increasing 25(OH)D3 with lesser diversion of 25(OH)D3 to 24,25(OH)2D3 than does larger bolus dosing.

Gas Chromatography Mass Spectrometry (GC-MS) for Identification of Designer Stimulants Including 2C Amines, NBOMe Compounds, and Cathinones in Urine.

Phenethylamine derivatives are being increasingly exploited for recreational use as "designer" stimulants designed to mimic psychostimulant properties of amphetamine or other illicit substances like 3,4-methylenedioxymethamphetamine (MDMA [ecstasy]). Clandestine operations meticulously design phenethylamines so the user can bypass legal action when detected, as many of these are yet to be regulated by government authorities. Substituted phenethylamines or 2C amines, N-methoxybenzyl derivatives of the corresponding 2C amines commonly known as NBOMe compounds, and cathinones are among the most commonly abused phenethylamines. Current FDA-approved assays used in screening for illicit drug use lack the sensitivity needed to detect designer stimulants making it challenging for toxicologists to accurately identify these compounds. Gas chromatography mass spectrometry (GC-MS) is a sensitive method for identifying designer stimulants. This unit describes and compares two qualitative GC-MS methods for identifying 2C amines, NBOMe compounds, and cathinones in urine. © 2017 by John Wiley & Sons, Inc.

Role of Mass Spectrometry in Clinical Endocrinology.

The advent of mass spectrometry into the clinical laboratory has led to an improvement in clinical management of several endocrine diseases. Liquid chromatography tandem mass spectrometry found some of its first clinical applications in the diagnosis of inborn errors of metabolism, in quantitative steroid analysis, and in drug analysis laboratories. Mass spectrometry assays offer analytical sensitivity and specificity that is superior to immunoassays for many analytes. This article highlights several areas of clinical endocrinology that have witnessed the use of liquid chromatography tandem mass spectrometry to improve clinical outcomes.

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.

LC-MS/MS for Identifying Patients with CYP24A1 Mutations.

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.

Quantitation of Albumin in Urine by Liquid Chromatography Tandem Mass Spectrometry.

Urinary excretion of human serum albumin (HSA), a 6.65 kDa monomeric protein, is a sensitive marker of renal damage associated with many diseases including diabetes mellitus. Albumin is synthesized by the liver and functions as a transport protein for fat-soluble hormones and drugs and for maintaining plasma colloid osmotic pressure and pH. Albumin is not filtered at the glomerulus and its presence in the urine at concentration above 30 mg/day is suggestive of glomerular damage. Early diagnosis of microalbuminuria (30-300 mg/24 h urine albumin excretion or 30-300 mg/g creatinine in random collections) has prognostic value for monitoring disease progression and early clinical management of diabetic nephropathy in prediabetic patients. Current methods for quantitation of urine albumin are based on immunoassays or size exclusion high-performance liquid chromatography coupled with UV detection (SEC-HPLC-UV). Studies have demonstrated discordance between the existing methods. It has been suggested that while immunoassays underestimate albumin in urine, SEC-HPLC-UV method overestimates albumin as it cannot separate co-eluting interferences. This chapter describes a liquid chromatography tandem mass spectrometry LC-MS/MS candidate reference method for albumin quantitation.

Quantitation of Insulin-Like Growth Factor 1 in Serum by Liquid Chromatography High Resolution Accurate-Mass Mass Spectrometry.

Insulin-like growth factor 1 (IGF-1) is a 70 amino acid peptide hormone which acts as the principal mediator of the effects of growth hormone (GH). Due to a wide variability in circulating concentration of GH, IGF-1 quantitation is the first step in the diagnosis of GH excess or deficiency. Majority (>95 %) of IGF-1 circulates as a ternary complex along with its principle binding protein insulin-like growth factor 1 binding protein 3 (IGFBP-3) and acid labile subunit. The assay design approach for IGF-1 quantitation has to include a step to dissociate IGF-1 from its ternary complex. Several commercial assays employ a buffer containing acidified ethanol to achieve this. Despite several modifications, commercially available immunoassays have been shown to have challenges with interference from IGFBP-3. Additionally, inter-method comparison between IGF-1 immunoassays has been shown to be suboptimal. Mass spectrometry has been utilized for quantitation of IGF-1. In this chapter a liquid chromatography high resolution accurate-mass mass spectrometry (LC-HRAMS) based method for IGF-1 quantitation has been described.

Quantitation of Parathyroid Hormone in Serum or Plasma by Liquid Chromatography-Tandem Mass Spectrometry.

Parathyroid hormone (PTH), an 84 amino acid peptide hormone, is an important regulator of calcium homeostasis. Quantitation of PTH in serum is useful for the diagnosis of primary hyperparathyroidism, hypoparathyroidism, and for monitoring osteodystrophy in patients with renal failure. The biological activity of PTH arises from binding of PTH (N terminus) to its target receptor (D'Amour et al., Kidney Int 68: 998-1007, 2005). Several C-terminal and N-terminal fragments circulate in normal subjects. Recent studies have demonstrated that accurate quantitation of PTH fragments may be of clinical value. In this chapter a mass spectrometry based method for quantitation of PTH(1-84) is described. This method involves immunoaffinity capture of PTH followed by trypsinization and quantitation of PTH-specific tryptic peptides by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The N-terminal tryptic peptide, PTH(1-13) as surrogate of 1-84 PTH, is used for quantitation.

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.

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.

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

PURPOSE OF REVIEW: The 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 FINDINGS: Measurement 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. SUMMARY: Endocrine 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.

Fit of fluxes of sunscreens and other compounds from propylene glycol:water (30:70) through human skin and silicone membrane to the Roberts-Sloan equation: the effect of polar vehicle (or water) solubility.

It would be useful to develop a surrogate for animal skin, which could be use to predict flux through human skin. The fluxes (and physicochemical properties) of sunscreens and other compounds from propylene glycol (PG):water (AQ), 30:70, through human skin have previously been reported. We measured the fluxes of several of those sunscreens and other compounds from PG:AQ, 30:70, through silicone membrane and fit both sets of data to the Roberts-Sloan (RS) equation to determine any similarities. For both sets of data, the fluxes were directly dependent on their solubilities in a lipid solvent [octanol (OCT), in this case] and in a polar solvent (PG:AQ, 30:70, or AQ in this case) and inversely on their molecular weights. The fit of the experimental (EXP) fluxes through human skin in vivo to RS was excellent: r² = 0.92 if the vehicle (VEH) PG:AQ, 30:70 was the polar solvent (RS¹) or r² = 0.97 if water was the polar solvent (RS²). The fit of the EXP fluxes through silicone membrane to RS was good: r² = 0.80 if the VEH PG:AQ, 30:70, was the polar solvent (RS¹) or r² = 0.81 if water was the polar solvent (RS²). The correlations between their EXP fluxes through human skin in vivo and their EXP fluxes through silicone membrane were good (r² = 0.85). In addition, the correlation between EXP fluxes from PG:AQ, 30:70, through human skin in vivo and their fluxes calculated from the coefficients of the fit of solubilities, molecular weights and fluxes from water through silicone membranes from a previous n = 22 database to RS was even better (r² = 0.94). These results suggest that flux through human skin can be calculated from flux through a silicone membrane.

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 ß-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. ß-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.