There has been increased interest in the measurement of melatonin in plasma and saliva recently either as a marker of circadian phase or to understand the physiological role of melatonin. For both ...situations, there is a need for a specific assay for melatonin that is sensitive enough to detect low concentrations (<2 pg/mL). Since the mid‐1970s, there have been many assays developed to measure melatonin in blood and saliva. Radioimmunoassays and ELISA have predominated because of their relative simplicity and high throughput. In this review, I show that the early radioimmunoassays while providing valuable information about nocturnal melatonin levels in humans, generally produced inaccurate basal (daytime) levels. Mass spectrometry assays, however, have provided us with the target values that immunoassays need to achieve, that is, daytime plasma melatonin levels <1 pg/mL. There are now many contemporary commercial assays available utilising both RIA and ELISA technologies, but not all achieve the standards set by the mass spectrometry assays. The performance of these assays is reviewed. I conclude with recommendations on issues researchers need to consider when conducting melatonin studies, including the importance of time of day of collection, validation of assays, the potential causes of poor assay specificity at low levels, the advantages/disadvantages of using saliva vs plasma and extraction assays vs direct assays, kit manufacturers responsibilities and the reporting requirements when publishing melatonin studies.
The majority of equine progesterone research is over 30 years old and data is lacking regarding the use of current laboratory analysis methods. The research objective was to evaluate the effect of ...storage time on progesterone concentrations in refrigerated whole mare blood. Mares were ultrasounded twice weekly to confirm follicular development, ovulation, and cycling status before enrollment into the study. Twelve cycling, Quarter Horse mares were enrolled into the study from March to July, and blood samples were collected during multiple estrous cycles for an average of 2 cycles per mare. Mares were ultrasounded daily once a 35 mm follicle was detected on an ovary until ovulation, and then every other day to monitor corpus luteum formation. Open mares were reenrolled into the study on a subsequent estrous cycle, allowing for 28 total blood collection periods. Blood collections occurred 5 d after the ovulation of a previously recorded follicle and confirmation via ultrasonography of a corpus luteum on the same ovary. On each sample collection day, 6 cc of whole blood was collected from the mare into 5 vacutainer serum blood collection tubes from a single venipuncture, for a total of 30 cc of whole blood. The 5 blood samples were stored under refrigerated conditions at 4°C for 0, 6, 12, 24, and 48 h. The 0-h treatment was centrifuged once an adequate blood clot had formed or 30 min post-collection. Following the assigned storage time, each blood sample was centrifuged for 15 min and then serum was frozen at −22°C until analyzed for P4 using solid phase radioimmunoassay kit reagents validated for equine plasma. Statistical analysis of progesterone data was analyzed as a repeated measures design using the GLM procedure of SAS. Progesterone concentrations were similar among the refrigerated storage times at 0 (P = 0.278), 6 (P = 0.230), 12 (P = 0.198), 24 (P = 0.243), and 48 (P = 0.282) hours. In summary, refrigerated samples of whole blood from mares may be stored for up to 48 h without an effect on progesterone concentration analysis.
Research on the neurobiological and behavioral effects of oxytocin (OT), as well as on its possible therapeutic applications, has intensified in the past decade. Accurate determination of peripheral ...OT levels is essential to reach meaningful conclusions and to motivate, support and inform clinical interventions. Different, but concordant, methods for measuring plasma OT have been developed over the past four decades, but since 2004 several commercially available methods have been favored in research with humans. Evaluation of these methods reveals that they lack reliability when used on unextracted samples of human fluids, and that they tag molecules in addition to OT, yielding estimates that are wildly discrepant with an extensive body of earlier findings that were obtained using methods that are well validated, but more laborious. An accurate, specific, and readily available method for measuring OT that can be adopted as the standard in the field is urgently needed for advances in our understanding of OT's roles in cognition and behavior.
Current threshold values for primary aldosteronism (PA) diagnostic testing are based on measuring aldosterone (PAC) using immunoassays. Quantification of PAC by liquid chromatography-tandem mass ...spectrometry (LC-MS/MS) yields lower values.
To compare aldosterone measurement by radioimmunoassay (RIA) with LC-MS/MS and evaluate performances of proposed LC-MS/MS-specific cutoffs for PA screening and confirmatory testing.
Forty-one patients underwent aldosterone/renin ratio (ARR) testing to screen for, and fludrocortisone suppression testing (FST) to confirm or exclude, PA. Renin (DRC) was measured by chemiluminescent immunoassay.
Median serum PACLC-MS/MS was 27.8% lower (P < 0.05) than plasma PACRIA in 164 pairs of FST samples. A positive correlation (Spearman coefficient, 0.894, P < 0.01; Pearson r coefficient, 0.861, P < 0.01) was observed between the two assays. Thirty-seven patients showed consistent FST diagnoses (29 positive, 8 negative), whereas four showed inconsistent FSTs by the two assays. Good agreement (κ coefficient, 0.736; P < 0.01) was observed between the current FST diagnostic PACRIA cutoff of 165 pmol/L and the proposed PACLC-MS/MS cutoff of 133 pmol/L. Among 37 patients with consistent FST results, no differences were observed in sensitivity (89.7% vs 93.1%) or specificity (87.5% vs 87.5%) for PA screening between the current ARR cutoff of 70 pmol/mU (PACRIA/DRC) and the proposed cutoff of 55 pmol/mU (PACLC-MS/MS/DRC).
Adjustment of the current cutoffs for PA diagnostic testing is necessary if PAC is measured by LC-MS/MS. Our preliminary results suggest that the proposed LC-MS/MS cutoffs for ARR and FST perform as well as current RIA cutoffs.
Serum progesterone (sP4) measurement is commonly used to determine the optimal time for mating in bitches, and to diagnose reproduction-related abnormalities. Radioimmunoassay (RIA) is the gold ...standard assay, but is becoming less available, and has several practical disadvantages. Chemiluminescence immunoassays (CLIA) are commonly used in human medicine for sP4 measurement, and are becoming more available in veterinary medicine. Our objective was to compare the sP4 results obtained by RIA and two CLIA systems (Immulite-Siemens IS-CLIA and Elecsys-Roche ER-CLIA) in the same sera in 60 client-owned healthy bitches at different estrous cycle stages. The agreement between the two CLIAs and RIA was examined using the Passing-Bablok regression and Bland Altman plots. Comparing sP4 concentrations measured by the IS-CLIA to the RIA yielded an intercept of 0.16 ng/mL (95% confidence interval 95%CI, 0.03–0.25) with a slope of 0.45 (95%CI, 0.44–0.47) and a median difference of −2.10 ng/mL (P < 0.0001) that was strongly correlated to the average of measurements (r = −0.97; P < 0.0001). Comparing sP4 concentrations measured by the ER-CLIA to the RIA yielded an intercept of −0.23 ng/mL (95%CI, −0.56 to −0.09) with a slope of 1.06 (95%CI, 1.00–1.12) and a median difference of −0.09 ng/mL (P = 0.9), that was weakly correlated to the average of measurements (r = 0.34; P = 0.018). The performance of the ER-CLIA was similar to the RIA, while the IS-CLIA showed significantly different results compared to the RIA. Our study supports the conclusion that sP4 results generated by the ER-CLIA can be used interchangeably with RIA results for clinical purposes, while IS-CLIA results require adjustment to RIA results for clinical practice.
•Female dog serum progesterone was measured using three immunoassays.•Radioimmunoassay compared with Immulite and Elecsys Chemiluminscence immunoassay.•Immulite underestimates radioimmunoassay for serum progesterone measurements.•Elecsys consistently closer to radioimmunoassay for serum progesterone measurements.
The first issue of Hormones and Behavior was published 50 years ago in 1969, a time when most of the techniques we currently use in Behavioral Endocrinology were not available. Researchers have ...during the last 5 decades developed techniques that allow measuring hormones in small volumes of biological samples, identify the sites where steroids act in the brain to activate sexual behavior, characterize and quantify gene expression correlated with behavior expression, modify this expression in a specific manner, and manipulate the activity of selected neuronal populations by chemogenetic and optogenetic techniques. This technical progress has considerably transformed the field and has been very beneficial for our understanding of the endocrine controls of behavior in general, but it did also come with some caveats. The facilitation of scientific investigations came with some relaxation of methodological exigency. Some critical controls are no longer performed on a regular basis and complex techniques supplied as ready to use kits are implemented without precise knowledge of their limitations. We present here a selective review of the most important of these new techniques, their potential problems and how they changed our view of the hormonal control of behavior. Fortunately, the scientific endeavor is a self-correcting process. The problems have been identified and corrections have been proposed. The next decades will obviously be filled with exciting discoveries in behavioral neuroendocrinology.
•Most of the current techniques in behavioral endocrinology did not exist in 1969.•Hormones and gene expression can now be quantified in small biological samples.•The activity of specific neurons can also be manipulated at various time scales.•-These new techniques are briefly presented together with their potential limitations.•These techniques should still lead to exciting discoveries in neuroendocrinology.
The dodecapeptide angiotensin-(1–12) Ang-(1–12) functions as an intracrine/paracrine substrate for local production of angiotensin II. We developed a reliable and specific radioimmunoassay (RIA) ...method for the measurement of Ang-(1–12) in human plasma and urine using an affinity purified antibody fraction directed towards the C-terminus of the human Ang-(1–12) sequence. The RIA method was applied to quantify the Ang-(1–12) in plasma and urine collected from thirty-four human subjects (29 treated with antihypertensive medicines and 5 untreated patients). Plasma Ang-(1–12) level was significantly higher (P < 0.05) in patients with systolic blood pressure ≥140 mm Hg (n = 10) compared to the group with systolic blood pressure <140 mm Hg (n = 24). No significant difference (P = 0.22) was found in spot urine between the groups. Our study also shows that the polyclonal antibody neutralizes the cleavage sites of the human Ang-(1–12) from recombinant human chymase (rhChymase) and serum angiotensin converting enzyme (ACE) mediated Ang II generating hydrolysis. Overall, this newly developed RIA method is reliable and applicable to accurately quantify the Ang-(1–12) level in clinical samples (plasma and urine). Further, our in vitro neutralization study suggests that the anti-Ang-(1–12)-antibody might be used as an in vivo therapeutic agent for preventing Ang-(1–12)/Ang II-mediated hypertension and organ damage.
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