Development and Validation of a LC-MS/MS Assay for Quantification of Parathyroid Hormone (PTH 1-34) in human Plasma

Background: Teriparatide [recombinant human PTH (1-34)] is an osteoanabolic agent for treatment of osteoporosis. The effect on bone decreases the risk of vertebral and non-vertebral fractures and increases bone mineral density (BMD) in post-menopausal women with osteoporosis. Measurement of PTH (1-34) is valuable in assessing treatment response and concordance with therapy.Aim: To develop and validate a method for quantification of PTH (1-34) using Liquid chromatography tandem mass spectrometry (LC-MS/MS) and to perform comparison with a commercial immunoassay.  Method: Sample extraction was developed using a Waters (Milford, MA, USA) Oasis® HLB µElution solid phase extraction. Quantification m/z transition 589>656 was used on Waters/Micromass® Quattro Ultima™ Pt mass spectrometer to measure PTH (1-34) in human plasma using rat PTH (1-34) as internal standard. Validation criteria were carried out against industry standards. PTH (1-34) results obtained from human subjects given Teriparatide (Fortsteo, Eli Lilly, IN, USA) (n=390) were compared against results obtained from an immunoassay (IDS; Boldon Tyne and Wear. UK).  Results and Discussion: LC-MS/MS produced a linear calibration curve from 10 to 2000 pg/mL (r2 >0.990). The LLoQ and LLoD for PTH (1-34) were 10 pg/mL and 2.1 pg/mL respectively. The inter- /intra-assay precision (CV%) of the method were 98.3% for four QCs (20, 100, 200, and 800 pg/mL). The mean recovery of PTH (1-34) was 107.2%. Method comparison between the LC-MS/MS and immunoassay using human EDTA plasma samples showed a high correlation (r2 = 0.950). A concentration-dependent, negative bias of 35.5% was observed across the range of 0 – 800 pg/mL. The immunoassay showed a 7% cross reactivity to human PTH (1-84) and 44% to rat PTH (1-34), no interference was observed in the LC-MS/MS method. Matrix effect and cross reactivity to human PTH (1-84) in the immunoassay were the likely contributing factors to the bias between the methods. The oxidised form of PTH (1-34) does not interfere with our LC-MS/MS method.  Conclusion: Our LC-MS/MS method demonstrated linearity over the calibration range, good precision and accuracy, excellent analyte recovery, and negligible matrix effects. The method was successfully used for measurements of PTH (1-34) in rat and human plasma

Flecainide Paradoxically Activates Cardiac Ryanodine Receptor Channels under Low Activity Conditions: A Potential Pro-Arrhythmic Action.

Cardiac ryanodine receptor (RyR2) mutations are implicated in the potentially fatal catecholaminergic polymorphic ventricular tachycardia (CPVT) and in atrial fibrillation. CPVT has been successfully treated with flecainide monotherapy, with occasional notable exceptions. Reported actions of flecainide on cardiac sodium currents from mice carrying the pro-arrhythmic homozygotic RyR2-P2328S mutation prompted our explorations of the effects of flecainide on their RyR2 channels. Lipid bilayer electrophysiology techniques demonstrated a novel, paradoxical increase in RyR2 activity. Preceding flecainide exposure, channels were mildly activated by 1 mM luminal Ca2+ and 1 µM cytoplasmic Ca2+, with open probabilities (Po) of 0.03 ± 0.01 (wild type, WT) or 0.096 ± 0.024 (P2328S). Open probability (Po) increased within 0.5 to 3 min of exposure to 0.5 to 5.0 µM cytoplasmic flecainide, then declined with higher concentrations of flecainide. There were no such increases in a subset of high Po channels with Po ≥ 0.08, although Po then declined with ≥5 µM (WT) or ≥50 µM flecainide (P2328S). On average, channels with Po < 0.08 were significantly activated by 0.5 to 10 µM of flecainide (WT) or 0.5 to 50 µM of flecainide (P2328S). These results suggest that flecainide can bind to separate activation and inhibition sites on RyR2, with activation dominating in lower activity channels and inhibition dominating in more active channels

Assessment of vitamin D status using MitraTM volumetric absorptive microsampling (VAMS) device

Introduction: The use of dried blood spot (DBS) sampling for general wellness assessment and in clinical diagnostics has gained popularity as a convenient and less invasive alternative to venous sampling. Collection of blood samples from a finger/heel prick using conventional filter paper suffers from variability in sample volume and spot sizes which undermine the quality of results. We describe the use of a volumetric absorptive microsampler (VAMS), called MitraTM (Torrance, CA, USA) for measurement of 25OHD3 and interpretation of vitamin D status according to current international guidelines.  Method: A liquid-chromatography mass spectrometry (LC-MS/MS) method was used for measurement of 25OHD3 (Tang et al. ASBMR 2015, LB-MO0026). We compared results from patient samples (n=97) collected by VAMS and Whatman® 903 cards extracted as whole spot (wDBS) and sub-punches (spDBS) against plasma 25OHD3 concentration. We investigated the volume displacement effects of haematocrit (Hct) on DBS 25OHD3 measurements and described the use of DBS-to-plasma equivalence value (PEV) to allow accurate interpretation of vitamin D status.  Results: VAMS showed the best assay precision CV (<8.2%) compared to wDBS (<16.6%) and spDBS (<15.1%) across the assay range of 0.1-125 nmol/L, the least variability in recovery and lowest LLoQ (Figure 1). We observed a decrease in DBS 25OHD3 concentration in proportion to the reduction in plasma volume and increase in packed cell volume. The displacement effect of Hct resulted in a strong but negatively biased correlation (r2=0.893, -39.3%) between raw DBS values and plasma concentrations, that was dependent upon the level of Hct present in sample. We demonstrated the use of simple linear regression model to transform raw DBS values into PEVs. In a subsequent cohort of patient samples (n=70), PEVVAMS produced the most accurate interpretation of vitamin D status compared to PEVwDBS and PEVspDBS.  Discussion: We present data supporting the use of VAMS for measurement of 25(OH)D3, particularly in circumstances where venesection may be impossible or difficult and where sample volume may be limited. Although the recovery of analyte remains Hct-dependent, the use of DBS-to-plasma equivalence values improves the clinical applicability and broadens the utility of DBS as a sampling technique

Stress response during early sedation with dexmedetomidine compared with usual-care in ventilated critically ill patients

Background: Sedative agents may variably impact the stress response. Dexmedetomidine is a sympatholytic alpha2-adrenergic agonist mainly used as a second-line sedative agent in mechanically ventilated patients. We hypothesised that early sedation with dexmedetomidine as the primary agent would result in a reduced stress response compared to usual sedatives in critically ill ventilated adults. Methods: This was a prospective sub-study nested within a multi-centre randomised controlled trial of early sedation with dexmedetomidine versus usual care. The primary outcome was the mean group differences in plasma levels of stress response biomarkers measured over 5 days following randomisation. Other hormonal, biological and physiological parameters were collected. Subgroup analyses were planned for patients with proven or suspected sepsis. Results: One hundred and three patients were included in the final analysis. Baseline illness severity (APACHE II score), the proportion of patients receiving propofol and the median dose of propofol received were comparable between groups. More of the usual-care patients received midazolam (57.7% vs 33.3%; p = 0.01) and at higher dose (median (95% interquartile range) 0.46 [0.20–0.93] vs 0.14 [0.08–0.38] mg/kg/day; p < 0.01). The geometric mean (95% CI) plasma level of the stress hormones, adrenaline (0.32 [0.26–0.4] vs 0.38 [0.31–0.48]), noradrenaline (4.27 [3.12–5.85] vs 6.2 [4.6–8.5]), adrenocorticotropic hormone (17.1 [15.1–19.5] vs 18.1 [15.9–20.5]) and cortisol (515 [409–648] vs 618 [491–776)] did not differ between dexmedetomidine and usual-care groups, respectively. There were no significant differences in any other assayed biomarkers or physiological parameters Sensitivity analyses showed no effect of age or sepsis. Conclusions: Early sedation with dexmedetomidine as the primary sedative agent in mechanically ventilated critically ill adults resulted in comparable changes in physiological and blood-borne parameters associated with the stress-response as with usual-care sedation

Effects of ACTH, dexamethasone, and adrenalectomy on 11β-hydroxylase (CYP11B1) and aldosterone synthase (CYP11B2) gene expression in the rat central nervous system

Using a highly sensitive quantitative RT-PCR method for the measurement of CYP11B1 (11β-hydroxylase) and CYP11B2 (aldosterone synthase) mRNAs, we previously demonstrated that CYP11B2 expression in the central nervous system (CNS) is subject to regulation by dietary sodium. We have now quantified the expression of these genes in the CNS of male Wistar Kyoto (WKY) rats in response to systemic ACTH infusion, dexamethasone infusion, and to adrenalectomy. CYP11B1 and CYP11B2 mRNA levels were measured in total RNA isolated from the adrenal gland and discrete brain regions using real-time quantitative RT-PCR. ACTH infusion (40 ng/day for 7 days, N=8) significantly increased CYP11B1 mRNA in the adrenal gland, hypothalamus, and cerebral cortex compared with animals infused with vehicle only. ACTH infusion decreased adrenal CYP11B2 expression but increased expression in all of the CNS regions except the cortex. Dexamethasone (10 μg/day for 7 days, N=8) reduced adrenal CYP11B1 mRNA compared with control animals but had no significant effect on either gene's expression in the CNS. Adrenalectomy (N=6 per group) significantly increased CYP11B1 expression in the hippocampus and hypothalamus and raised CYP11B2 expression in the cerebellum relative to sham-operated animals. This study confirms the transcription of CYP11B1 and CYP11B2 throughout the CNS and demonstrates that gene transcription is subject to differential regulation by ACTH and circulating corticosteroid levels

Autonomic Dysregulation in Adolescent Concussion Is Sex- and Posture-Dependent

Objective: To study autonomic responses to postural changes in concussed adolescents. The influence of sex was also studied. Design: Longitudinal cohort observational study. Participants: Concussed adolescents (CONC; n = 65; 26 male adolescents; age 15 ± 1 years, range = 12-18 years) and a control (CTRL) group of nonconcussed adolescents of similar age and sport (CTRL; n = 54; 29 male adolescents; age 14 ± 1 years, range = 12-18 years). Interventions: Concussed participants were monitored through 6 weekly visits throughout usual physician care. Control participants underwent 2 visits separated by at least 1 week to account for intrapersonal variation in testing measures. Main Outcome Measures: Heart rate variability as the root mean square of successive differences in R–R intervals (RMSSD), heart rate (HR), and blood pressure [mean arterial pressure (MAP) and diastolic blood pressure (DBP)] were measured in supine, sitting, and standing postures. Results: A mixed analysis of variance revealed a group 3 sex 3 posture interaction (P = 0.04) where seated values of RMSSD were less in concussed female participants versus control female participants (42 ± 4 vs 61 ± 7 ms; P = 0.01; Mann–Whitney rank test). Compared with CTRL, CONC exhibited increased pretesting seated DBP (69 ± 1 vs 74 ± 1 mm Hg; P\u3c 0.01), MAP (83 ± 1 vs 86 ± 1 mm Hg; P = 0.02), and baseline seated HR (72 ± 1 vs 77 ± 2 bpm; P = 0.03). Values of DBP (P = 0.03) and MAP (P, 0.01) improved at clinical discharge, whereas the RMSSD in female participants did not (P \u3e 0.5). Data are mean ± SEM. Conclusions: A modest reduction in female cardiac autonomic regulation was observed during seated postures. Alterations in seated concussed DBP and MAP, but not RMSSD, resolved at clinical discharge (median = 37 days). The results indicate that, in adolescents, concussion may impair cardiovagal function in a sex- and posture-dependent manner. The findings also suggest that BP metrics, but not RMSSD, are associated with clinical concussion recovery

Reference intervals for serum 24,25-Dihydroxyvitamin D and the ratio with 25-Hydroxyvitamin established using a newly developed LC-MS/MS method

24,25(OH)2D is the product of 25(OH)D catabolism by CYP24A1.The measurement of serum 24,25(OH)2D concentration may serve as an indicator of vitamin D catabolic status and the relative ratio with 25(OH)D can be used to identify patients with inactivating mutations in CYP24A1. We describe a LC-MS/MS method to determine: 1) the relationships between serum 24,25(OH)2D and 25(OH)D; 2) serum reference intervals in healthy individuals; 3) the diagnostic accuracy of 24,25(OH)2D measurement as an indicator for vitamin D status; 4) 24,25(OH)2D cut-off value for clinically significant change between inadequate and sufficient 25(OH)D status. Serum samples of healthy participants (n=1996) from Army recruits and patients (n=294) were analysed. The LC-MS/MS assay satisfied industry standards for method validation. We found a positive, concentration-dependent relationship between serum 24,25(OH)2D and 25(OH)2D concentrations. The 25(OH)D:24,25(OH)2D ratio was significantly higher (p4.2 nmol/L was identified as a diagnostic cut-off for 25(OH)D replete status. One patient sample with an elevated 25(OH)D:24,25(OH)2D ratio of 32 and hypercalcaemia who on genetic testing confirmed to have a biallelic mutation of CYP24A1. Our study demonstrated the feasibility of a combined 24,25(OH)2D and 25(OH)D assessment profile. Our established cut-off value for 24,25(OH)2D and ratio reference ranges can be useful to clinicians in the investigation of patients with an impaired calcium/phosphate metabolism and may point towards the existence of CYP24A1 gene abnormalities