A sensitive and rapid mass spectrometric method for the simultaneous measurement of eight steroid hormones and CALIPER pediatric reference intervals.

OBJECTIVES: To develop an accurate assay and establish the normal reference intervals for serum cortisol, corticosterone, 11-deoxycortisol, androstenedione, 21-hydroxyprogesterone, testosterone, 17-hydroxyprogesterone, and progesterone. These steroids are commonly used as biomarkers for the diagnosis and monitoring of endocrine diseases such as congenital adrenal hyperplasia. Appropriate age- and gender-stratified reference intervals are essential in accurate interpretation of steroid hormone levels. DESIGN AND METHODS: The samples analyzed in this study were collected from healthy, ethnically diverse children in the Greater Toronto Area as part of the CALIPER program. A total of 337 serum samples from children between the ages of 0 and 18years were analyzed. The concentrations were measured by using an LC-MS/MS method. The data were analyzed for outliers and age- and gender-specific partitions were established prior to establishing the 2.5th and 97.5th percentiles for the reference intervals. RESULTS: Reference intervals for all hormones required significant age-dependent stratification while testosterone and progesterone required additional sex-dependent stratification. CONCLUSIONS: We report a sensitive, accurate and relatively fast LC-MS/MS method for the simultaneous measurement of eight steroid hormones. Detailed reference intervals partitioned based on both age and gender were also established for all eight steroid hormones.

Influence of fasting and sample collection time on 38 biochemical markers in healthy children: a CALIPER substudy.

OBJECTIVES: Fasting samples can be difficult to obtain in the pediatric setting, particularly in neonates. As part of the Canadian Laboratory Initiative on Pediatric Reference Intervals (CALIPER), we aimed to determine if there are differences in serum concentrations of pediatric biochemical markers measured at fasting, postprandial, and random time points throughout the day. DESIGN AND METHODS: Blood was drawn from 27 healthy children and adolescents (aged 4-18) with informed consent at 4 time points: after overnight fast, mid-morning after breakfast, within 2h after lunch, and late afternoon. The effect of fasting on 38 chemistries was evaluated by paired, two-tailed student'st-tests. Analysis of the effect of time of day was done using paired, repeated-measures ANOVA. RESULTS: Fasting significantly affected 22 analytes, with HDL cholesterol being the most highly affected. Values tended to decrease postprandially, except for five analytes, including triglycerides, which increased. By ANOVA, 28 chemistries significantly differed across times of day tested. CONCLUSIONS: Fasting is necessary for analysis of certain chemistries in pediatric subjects. Pediatricians should consider diurnal factors when ordering non-fasting tests and interpreting test results.

A novel mechanism for myocardial stunning involving impaired Ca(2+) handling.

The mechanism of myocardial stunning has been studied extensively in rodents and is thought to involve a decrease in Ca(2+) responsiveness of the myofilaments, degradation of Troponin I (TnI), and no change in Ca(2+) handling. We studied the mechanism of stunning in isolated myocytes from chronically instrumented pigs. Myocytes were isolated from the ischemic (stunned) and nonischemic (normal) regions after 90-minute coronary stenosis followed by 60-minute reperfusion. Baseline myocyte contraction was reduced, P<0.01, in stunned myocytes (6.3+/-0.4%) compared with normal myocytes (8.8+/-0.4%). The time for 70% relaxation was prolonged, P<0.01, in stunned myocytes (131+/-8 ms) compared with normal myocytes (105+/-5 ms). The impaired contractile function was associated with decreased Ca(2+) transients (stunned, 0.33+/-0.04 versus normal, 0.49+/-0.05, P<0.01). Action potential measurements in stunned myocytes demonstrated a decrease in plateau potential without a change in resting membrane potential. These changes were associated with decreased L-type Ca(2+)-current density (stunned, -4.8+/-0.4 versus normal, -6.6+/-0.4 pA/pF, P<0.01). There were no differences in TnI, sarcoplasmic reticulum Ca(2+) ATPase (SERCA2a), and phospholamban protein quantities. However, the fraction of phosphorylated phospholamban monomer was reduced in stunned myocardium. In rats, stunned myocytes demonstrated reduced systolic contraction but actually accelerated relaxation and no change in Ca(2+) transients. Thus, mechanisms of stunning in the pig are radically different from the widely held concepts derived from studies in rodents and involve impaired Ca(2+) handling and dephosphorylation of phospholamban, but not TnI degradation.