Edwin A. Knecht

Stability of urinary female reproductive hormones stored under various conditions.

Urinary reproductive hormones afford specific and sensitive evaluation of female reproductive potential in epidemiologic and clinical settings. The goal of this study was to characterize the stability of urinary luteinizing hormone, follicle stimulating hormone, estrone 3-glucuronide, pregnanediol 3-glucuronide, and creatinine during storage as functions of time, temperature, and additives. After 2 weeks with no additives, activity of the four analytes, relative to initial concentrations, ranged from 91.9 to 102.8% at 4 degrees C, 35.1 to 89.6% at 25 degrees C, and 7.5 to 66.9% at 37 degrees C. Antimicrobial additives did not consistently improve stability. Analyte activity for samples stored with no additives for 24 weeks at -80 degrees C ranged from 69.0 to 101.2%. Glycerol and bovine serum albumin improved analyte stability; activity ranged from 91.1 to 106.3%. Other additives were ineffective. These results reveal conditions for storing reproductive hormone analytes in urine during epidemiologic field studies.

Validations of time-resolved fluoroimmunoassays for urinary estrone 3-glucuronide and pregnanediol 3-glucuronide

Competitive time-resolved fluoroimmunoassays (FIAs) were developed for measuring 1,3,5(10)-estratrien-3-ol-17-one glucosiduronate (estrone 3-glucuronide, E(1)3G) and 5 beta-Pregnane-3 alpha,20 alpha-diol 3-glucosiduronate (pregnanediol 3-glucuronide, Pd3G) in unextracted urine. The assays are specific, detect 0.98 ng E(1)3G/mL and 0.035 microgram Pd3G/mL, measure 102.8 +/- 2.0% of E(1)3G and 93.6 +/- 2.9% of Pd3G added, and exhibit between and within assay coefficients of variation, respectively, of 5.3% and 7.1% for E(1)3G and 6.8% and 7.8% for Pd3G. The urine matrix does not interfere with the assay. Urinary steroid glucuronide profiles measured by these FIAs conform to those of urinary steroid glucuronides and serum estradiol and progesterone measured by other established immunoassays. These FIAs afford the advantages of non-radioisotopic procedures and urine sample collection (convenience, non-invasiveness, integration of pulsatile secretion) to evaluate menstrual function in epidemiological, medical, and athletic populations.

Time-resolved immunofluorometric assays for urinary luteinizing hormone and follicle stimulating hormone

Abstract The goal of this effort was to develop and validate non-radioisotopic immunoassays for measuring luteinizing hormone and follicle stimulating hormone in unextracted urine. Towards this goal, commercial time-resolved immunofluorometric assays (IFMAs) were modified. Validation demonstrated that the resultant assays were specific, sensitive, accurate, and precise. Urine matrix was shown not to interfere with the assay. Gonadotropin profiles generated using these assays conformed to those measured in urine and serum by other established immunoassays. These IFMAs afford the collective advantages of non-radioisotopic procedures and urine sample collection (convenience, noninvasiveness, integration of pulsatile secretion), plus the superior sensitivity and specificity of IFMAs. Applications include epidemiology and medicine.

Feasibility issues in reproductive biomonitoring of female flight attendants and teachers.

Flight attendants (FAs) may be at risk of adverse reproductive outcomes. We investigated the feasibility of biomonitoring studies in this mobile workforce. Forty-five female FAs and 26 female teachers (referents) collected daily urine and saliva samples for one menstrual cycle, provided daily diary data for approximately three months, and wore a wrist monitor to measure sleep disruption. A transport system enabled FAs to store samples while traveling. Overall, participation rates were low (37%) but of those recruited, over 90% of FAs and teachers completed the biomonitoring cycle. Data collection and sample integrity were not diminished by travel. Study methods resulted in good compliance and high quality data. It is possible to conduct studies of menstrual cycle function, sleep disruption, and circadian rhythm disruption in a mobile workforce potentially exposed to reproductive hazards.

Urinary reproductive hormone level differences between African American and Caucasian women of reproductive age

OBJECTIVE To compare urinary levels of reproductive hormones in African American and Caucasian women. DESIGN Cross-sectional study. SETTING Ten United States Air Force (USAF) bases. PATIENT(S) African American (n = 33) and Caucasian (n = 65) women of reproductive age from a larger study of USAF women (n = 170). INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) Urinary endocrine end points: follicular luteinizing hormone (LH), preovulatory LH, level of LH surge peak, early follicular follicle stimulating hormone (FSH), follicular LH:FSH ratio, midluteal FSH, FSH rise before menses, early follicular estrone 3-glucuronide (E(1)3G), midfollicular E(1)3G, periovulatory E(1)3G peak, midluteal E(1)3G, early follicular pregnanediol 3-glucuronide (Pd3G), follicular Pd3G, rate of periovulatory Pd3G increase, E(1)3G:Pd3G on the day of luteal transition, slope of E(1)3G:Pd3G, and midluteal Pd3G. RESULT(S) Relative to Caucasians, African American women had significantly lower follicular phase LH:FSH ratios (mean +/- SD: 0.7 +/- 0.4 vs. 1.0 +/- 0.6), lower follicular phase Pd3G levels (1.0 +/- 0.5 vs. 1.2 +/- 0.8 microg/mg creatinine), and lower rates of periovulatory Pd3G increase (0.5 +/- 0.7 vs. 1.0 +/- 1.2 microg/mg creatinine). CONCLUSION(S) Findings of this analysis should be considered preliminary evidence of racial differences in hormone levels. Future studies are needed to determine whether these differences have clinical significance.

Immune responses of cynomolgus monkeys to phthalic anhydride

Four groups of four Macaca fascicularis monkeys were administered 10 consecutive weekly subcutaneous injections of 2 mg aluminum hydroxide plus one of the following: 200 micrograms of phthalic anhydride (PA)-monkey serum albumin (PA-MSA, group 1); 200 micrograms of PA dissolved in ethanol-saline (EtOH-sal, group 2); 200 micrograms of MSA (group 3); or EtOH-sal alone (group 4). Direct intracutaneous tests to PA-MSA, PA-EtOH-sal, MSA, and EtOH-sal were applied at biweekly intervals throughout the course of the immunization. Serum-specific IgG to PA-MSA and specific IgE to PA-MSA were determined at 2-week intervals according to the ELISA and RAST methods, respectively. The prevalence of cutaneous sensitivity to PA-MSA in the PA-MSA-immunized group (group 1) was significantly greater after 4 and 6 (p less than 0.01) and 8 and 10 (p less than 0.05) weeks, compared with the other treatment groups. Significantly elevated (p less than 0.01) PA-MSA-specific IgG was also observed in monkeys in group 1 compared with the other treatment groups. No significant changes in PA-MSA RAST or total IgE were observed in any group during the study. These results indicate that parenteral sensitization to PA in subhuman primates requires the presence of new antigenic determinants formed by PA on protein carriers.

Acute airway narrowing in monkeys from challenge with 2.5 PPM formaldehyde generated from formalin

Nine adult male Cynomolgus monkeys (Macaca fascicularis) were exposed for 10 min to 2.55 +/- 0.03 ppm formaldehyde (HCHO; mean +/- standard error of the mean, SEM) generated from formalin with a newly developed HCHO challenge system. The generation system was capable of producing highly stable HCHO vapor concentrations with fluctuations of HCHO concentrations of less than +/- 5%. The experimental design included pre-exposure methacholine challenge to determine if responses to HCHO were associated with pre-existing bronchial hyperreactivity. Significant changes in average pulmonary flow resistance (RL) were observed (compared to control RL values) at 2 (p less than 0.01), 5 (p less than 0.01), and 10 min (p less than 0.005) post-HCHO challenge. Pre-challenge RL values (mean +/- SEM) were 11.3 +/- 1.4 cm H2O.l/s, while at 2, 5, and 10 min after HCHO challenge, values were 16.1 +/- 2.1, 16.9 +/- 2.8 and 20.0 +/- 3.4 cm H2O.l/s, respectively. Methacholine challenge data suggest that reactions to HCHO tend to be greater in monkeys hyperreactive to methacholine, but the relationship does not reach statistical significance in this small series of animals. These data indicate that significant pulmonary function deficits occur immediately after challenge with 2.55 ppm HCHO vapor in monkeys.

An algorithm for detecting features of the hormone profiles of the human menstrual cycle

An algorithm for detecting features of the cycles of the gonadotropic and ovarian hormones in women is described. The algorithm can detect hormone peaks and normal cycles defined in terms of the peaks in sequences of measurements that have an arbitrary starting point in the menstrual cycle and are of arbitrary length. The algorithm makes use of fuzzy set theory and is optimized using signal detection theory.