Endocrine Abstracts

Background: Melatonin and cortisol production demonstrate circadian rhythms; disruption of these rhythms feature in endocrine and neurodegenerative disorders, such as Addison’s disease, Huntington’s disease (HD) and Alzheimer’s disease (AD). Measuring serum and saliva concentrations at nadir is challenging using immunoassays. We have developed LC-MSMS methods for measuring serum and saliva melatonin, cortisol and cortisone to analyse 24hr profile samples obtained from sleep studies.

Methods: 4 hourly serum samples (n=231) collected over a 24hr period were obtained with consent from 28 HD patients and 14 controls. Hourly saliva samples (n=523) from 30 individuals with APOE- ε ^3/3 and APOE- ε ^3/4 genotypes at higher risk of developing AD having undergone partial sleep deprivation (sleep restricted to 4hrs) or multi-nap cycles (4hrs 160min sleep,80min wake). LC-MSMS methods were developed using a Waters Xevo TQ-XS LC-MSMS. The method was compared against commercial ELISA (IBL, Germany).

Results: The LC-MSMS method showed intra/inter-assay precision (CV%) of <8.1% and <9.6% across the assay range for melatonin(0.1-430 pmo/l), cortisol and cortisone (saliva/serum) of (1.3-165 nmol/l/28.5-806 nmol/l), (2.76-259/2.87-108 nmol/l), respectively. LLoQ and LLoD for melatonin were 2.7 pmo/l and 0.3 nmol/l for cortisol and cortisone. Comparison of LC-MS/MS melatonin values against ELISA showed a negatively biased correlation in serum (y=0.5738x–10.667, r²=0.7265); no correlation was observed in saliva (y=0.2273x+8.719, r²=0.1927). The sleep studies showed significant rhythm observed in serum melatonin (time, conc) peak (3:30am, 162.1 pmo/l), nadir (2:45pm, 0.8 pmo/l); cortisol peak (7:00am, 456.6 nmol/l), nadir (11:00pm, 99.3 nmol/l). HD patients exhibited lower concentrations of melatonin and cortisol across all time points compared to the controls. Saliva in APOE carriers showed circadian rhythm in the sleep-deprived group, but not in the multi-nap group.

Conclusion: Our LC-MSMS methods were able to detect circadian rhythms of melatonin, cortisol and cortisone in serum and saliva samples. Salivary method advancement has clinical applications and offers an alternative to venous samples in research studies.