Radioimmunoassay and tandem mass spectrometry measurement of bedtime salivary cortisol levels: a comparison of assays to establish hypercortisolism

CONTEXT: Although bedtime salivary cortisol measurement has been proposed as the optimal screening test for the diagnosis of Cushing's syndrome, its performance using commercially available assays has not been widely evaluated. OBJECTIVE: Our objective was to compare RIA and tandem mass spectrometry (LC-MS/MS) measurement of salivary cortisol in obese subjects and healthy volunteers. DESIGN AND SETTING: We conducted a cross-sectional prospective study of outpatients. SUBJECTS AND METHODS: We studied 261 obese subjects (186 female) with at least two additional features of Cushing's syndrome and 60 healthy volunteers (30 female). Subjects provided split bedtime salivary samples for cortisol measurement by commercially available RIA and LC-MS/MS. Results were considered normal or abnormal based on the laboratory reference range. Subjects with abnormal results underwent evaluation for Cushing's syndrome. RESULTS: In paired samples, RIA gave a lower specificity than LC-MS/MS in obese subjects (86 vs. 94%, P = 0.008) but not healthy volunteers (86 vs. 82%, P = 0.71). Among subjects with at least one abnormal result, both values were abnormal in 44% (confidence interval 26-62%) of obese and 75% (confidence interval 33-96%) of healthy volunteers. In obese subjects, salivary cortisol concentrations were less than 4.0 to 643 ng/dl (<0.11-17.7 nmol/liter; normal, < or =100 ng/dl, 2.80 nmol/liter) by LC-MS/MS and less than 50 to 2800 ng/dl (1.4-77.3 nmol/liter; normal, < or =170 ng/dl, 4.7 nmol/liter) by RIA. Cushing's syndrome was not diagnosed in any subject. CONCLUSION: Salivary cortisol levels should not be used as the sole test to diagnose Cushing's syndrome if laboratory-provided reference ranges are used for diagnostic interpretation.     

Mouse behavioral mutants have neuroimaging abnormalities

Impaired cognitive, memory, or motor performance is a distinguishing characteristic of neurological diseases. Although these symptoms are frequently the most evident in human patients, additional markers of disease are critical for proper diagnosis and staging. Noninvasive neuroimaging methods have become essential in this capacity and provide means of evaluating disease and tracking progression. These imaging methods are also becoming available to scientists in the research laboratory for assessment of animal models of neurological disease. Imaging in mouse models of neurological disease is of particular interest, owing to the availability of inbred strains and genetic manipulation tools that permit detailed investigation of the roles of various genes and gene products in disease pathogenesis. However, the relative prevalence of neuroimaging abnormalities in mice exhibiting neurological symptoms has not been reported. This prevalence has both theoretical and practical value because it is influenced by both the sensitivity of macroscopic anatomical measures to underlying genetic and disease processes and by the efficiency of neuroimaging in detecting and characterizing these effects. In this paper, we describe a meta-analysis of studies involving behavioral mouse mutants at our laboratory. In summary, we have evaluated 15 different mutant genotypes, of which 13 showed abnormal neuroimaging findings. This indicates a surprisingly high prevalence of neuroimaging abnormalities (87%) and suggests that disease processes affecting behavior generally alter neuroanatomy as well. As a consequence, neuroimaging provides a highly sensitive marker of neurological disease in mice exhibiting abnormal behavior.     

Effect of NSAID on muscle injury and oxidative stress

Indirect markers of muscle damage and delayed onset muscle soreness were examined and correlated to changes in oxidative stress, plasma antioxidant potential, and use or nonuse of non-steroidal anti-inflammatory drugs in 60 ultra-marathoners following the Western States Endurance Run. Blood was collected prior to and immediately following the race and analyzed for muscle damage by creatine phosphokinase and oxidative stress by F (2)-isoprostanes, protein carbonyls, and lipid hydroperoxides and antioxidant potential by the ferric reducing ability of plasma. Subjects recorded delayed onset muscle soreness during the week following the race. Lipid hydroperoxide concentrations were unchanged, but F (2)-isoprostanes, protein carbonyls, ferric reducing ability of plasma, creatine phosphokinase, and delayed onset muscle soreness increased significantly postrace. Protein carbonyls were significantly higher postrace in nonsteroidal anti-inflammatory drug users versus nonusers (p < 0.05). However, there was no difference between users and non-users for all other markers. Postrace creatine phosphokinase concentrations were not correlated with oxidative stress markers but were correlated with changes in delayed onset muscle soreness. Based upon these findings, caution should be used when consuming nonsteroidal anti-inflammatory drugs during ultra distance events.     

Chia seed does not promote weight loss or alter disease risk factors in overweight adults

The objective of this study was to assess the effectiveness of chia seed (Salvia hispanica L) in promoting weight loss and altering disease risk factors in overweight adults. The hypothesis was that the high dietary fiber and α-linolenic (ALA) contents of chia seed would induce a small but significant decrease in body weight and fat and improve disease risk factors. Subjects were randomized to chia seed (CS) and placebo (P) groups, and under single-blinded procedures, ingested 25 g CS or P supplements mixed in 0.25 L water twice daily before the first and last meal for 12 weeks. Ninety nondiseased, overweight/obese men and women between the ages of 20 and 70 years were recruited into the study, with 76 subjects (n = 39 CS, n = 37 P) completing all phases of the study. Pre- and poststudy measures included body mass and composition (dual energy x-ray absorptiometry), inflammation markers from fasting blood samples (C-reactive protein, interleukin 6, monocyte chemoattractant protein 1, and tumor necrosis factor α), oxidative stress markers (trolox equivalent antioxidant capacity and plasma nitrite), blood pressure, and a serum lipid profile. Plasma ALA increased 24.4% compared to a 2.8% decrease in CS and P, respectively (interaction effect, P = .012). No group differences were measured for changes in plasma eicosapentaenoic acid and docosahexaenoic acid (interaction effects, P = .420 and .980, respectively). Pre-to-post measures of body composition, inflammation, oxidative stress, blood pressure, and lipoproteins did not differ between CS and P for both sexes. In conclusion, ingestion of 50 g/d CS vs P for 12 weeks by overweight/obese men and women had no influence on body mass or composition, or various disease risk factor measures.     

Magnetic resonance thermometry of flowing blood

Blood temperature is a key determinant of tissue temperature and can be altered under normal physiological states, such as exercise, in diseases such as stroke or iatrogenically in therapies which modulate tissue temperature, such as therapeutic hypothermia. Currently available methods for the measurement of arterial and venous temperatures are invasive and, for small animal models, are impractical. Here, we present a methodology for the measurement of intravascular and tissue temperature by magnetic resonance imaging (MRI) using the lanthanide agent TmDOTMA^? (DOTMA, tetramethyl‐1,4,7,10‐tetraazacyclododecane‐1,4,7,10‐tetraacetic acid; Tm, thulium). The approach makes use of phase‐sensitive imaging measurements, combined with spectrally selective excitation, to monitor the temperature‐dependent shift in the resonance of proton nuclei associated with water and with methyl groups of TmDOTMA^?. Measurements were first made in a flow phantom modelling diastolic blood flow in the mouse aorta or inferior vena cava (IVC) and imaged using 7‐T preclinical MRI with a custom‐built surface coil. Flowing and static fluid temperatures agreed to within 0.12°C for these experiments. Proof‐of‐concept experiments were also performed on three healthy adult mice, demonstrating temperature measurements in the aorta, IVC and kidney following a bolus injection of contrast agent. A small (0.7–1°C), but statistically significant, higher kidney temperature compared with the aorta (p  = 0.002–0.007) and IVC (p  = 0.003–0.03) was shown in all animals. These findings demonstrate the feasibility of the technique for in vivo applications and illustrate how the technique could be used to explore the relationship between blood and tissue temperature for a wide range of applications.     

Recruitment and retention of women for clinical leiomyoma trials

BackgroundSubject recruitment and retention in clinical leiomyoma trials is challenging. We evaluated strategies to increase patient enrollment and completion in leiomyoma trials.Materials and methodsRandomized trials for treatment of symptomatic leiomyoma published from 2000 through 2008 were evaluated and thirteen trials were selected. Subject enrollment and completion rates, recruitment methods and reasons for patient drop-out were assessed.ResultsRecruitment by study personnel or clinic staff during evaluation for symptomatic leiomyoma was the most common strategy for enrollment. Additional methods included local media, internet postings and physician referrals. Seven to 85% of patients enrolled after screening, with a median enrollment of 70%. Sixty-five to 100% of patients completed the study after enrollment with a median completion rate of 89%. Reasons for drop-out at the screening stage included failure to meet inclusion criteria, patient refusal and patient preference for specific treatment. Commonly reported reasons for drop-out after enrollment were refusal of treatment following randomization, adverse reaction to study intervention and non-compliance with study protocol or follow-up visits.ConclusionWomen with symptomatic uterine leiomyomas may be attracted to participate in leiomyoma trials, however desire for specific treatment and persistent symptoms following intervention may hinder their participation. Randomization to placebo treatment and stringent inclusion criteria appear to adversely impact accrual. A wide range of recruiting tactics is needed and media sources or direct mailings may prove particularly effective to improve subject recruitment and retention in clinical leiomyoma trials.     

Toward Computational Identification of Multiscale “Tipping Points” in Acute Inflammation and Multiple Organ Failure

Sepsis accounts annually for nearly 10% of total U.S. deaths, costing nearly $17?billion/year. Sepsis is a manifestation of disordered systemic inflammation. Properly regulated inflammation allows for timely recognition and effective reaction to injury or infection, but inadequate or overly robust inflammation can lead to Multiple Organ Dysfunction Syndrome (MODS). There is an incongruity between the systemic nature of disordered inflammation (as the target of inflammation-modulating therapies), and the regional manifestation of organ-specific failure (as the subject of organ support), that presents a therapeutic dilemma: systemic interventions can interfere with an individual organ system’s appropriate response, yet organ-specific interventions may not help the overall system reorient itself. Based on a decade of systems and computational approaches to deciphering acute inflammation, along with translationally-motivated experimental studies in both small and large animals, we propose that MODS evolves due to the feed-forward cycle of inflammation?→?damage?→?inflammation. We hypothesize that inflammation proceeds at a given, “nested” level or scale until positive feedback exceeds a “tipping point.” Below this tipping point, inflammation is contained and manageable; when this threshold is crossed, inflammation becomes disordered, and dysfunction propagates to a higher biological scale (e.g., progressing from cellular, to tissue/organ, to multiple organs, to the organism). Finally, we suggest that a combination of computational biology approaches involving data-driven and mechanistic mathematical modeling, in close association with studies in clinically relevant paradigms of sepsis/MODS, are necessary in order to define scale-specific “tipping points” and to suggest novel therapies for sepsis.