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Measuring Corticotropin-Releasing Hormone (CRH): Insights and Methodological Challenges

Corticotropin-releasing hormone (CRH) is a 41-amino acid neuropeptide critical for stress responses and regulating the hypothalamic-pituitary-adrenal axis. Elevated CRH levels during pregnancy are linked to negative outcomes, making accurate measurement essential. Optimal sample collection involves using plasma in EDTA collection tubes and protease inhibitors, processed within 12 hours, refrigerated at 4°C, and stored at -80°C for less than two years. Assay selection requires consideration of cost, sensitivity, and ease of use, with ELISA preferred for rapid turnover and RIA for greater sensitivity.

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Measuring Corticotropin-Releasing Hormone (CRH): Insights and Methodological Challenges

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Presentation Transcript

  1. Lessons Learned/Challenges Corticotropin Releasing Hormone (CRH)

  2. Why Measure CRH? • 41 amino acid neuropeptide, plays a major role in the physiological response to stress • The principal hypothalamic regulator of the pituitary-adrenal axis • Higher levels of CRH associated with adverse pregnancy outcomes

  3. What Medium and Collection Method to Use? • Medium • Plasma • Collection Method • Collection tube • EDTA • Protease inhibitor • Time • Timing of sample collection – no circadian rhythm • Effects on plasma samples-process within 12 hrs • Timing during pregnancy • Temperature • Continuously chilled at 4°C

  4. Considerations when Processing • Sample Processing • Refrigerated Centrifugation • 3,000-4,500 rpm for 15 minutes • Aliquots siphoned and analyzed or stored • Storage • -80°C for < 2 years • Plasma extraction • Removes CRH from plasma • Methanol vs C18 Sep-Column • Immediately prior to assay

  5. Factors in Selection of Assay • Factors to consider • Cost • Ease • Reliability/sensitivity of tests • ELISA vs RIA • Elisa (easier, rapid turnover time) • RIA (well documented in literature, greater sensitivity)

  6. Lessons LearnedChallenges • Designing research study • Timing of collection in relation to EGA • Stratifying for ethnicity/race • Identify published protocol, consult with author/experts • Consider role of lab manager • Evaluate each plate result with consultant-assess standardized curve • Which method of assay?

  7. References • Glynn, L.M., DuneklSchetter, C., Chicz-DeMet, A., Hobel, C.J., & Sandman, C.A. (2006). Ethnic differences in adrenocorticotroic hormone, cortisol and corticotropin-releasing hormone during pregnancy. Peptides, 28, 1155-1161. • Latendress, G. & Ruiz, J. (2008). Bioassay research methodology: measuring CRH in pregnancy. Biological Research for Nursing, 10, 54-62. • Strong, E.F., Kleinman, K.P., Gillman, M.W. et al. (2006). Measuring corticotropin-releasting hormone in pregnant women-comparing blood collection protocols for epidemiological studies. Paediatric and Perinatal Epidemiology, 20, 67-71.

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