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• The contents of plasma CORT, ALD and 11-DHC were unimodally fluctuated and showed the similar age-associated patterns. • Plasma gonadal steroids were unimodally altered except A4, showing the maximum at 2.7-month-old. • The contents of both E1 and E2 in plasma were declined with age. • The hair levels of endogenous compounds and their ratios were varied with age similar to plasma biomarkers. Age-related level changes of hormones, endocannabinoids and their ratios are of pathophysiological significance for understanding functions, activities and interactions of the endocrine systems, including the hypothalamic–pituitaryadrenal (HPA), hypothalamic-pituitary–gonadal (HPG) axes and endogenous cannabinoid system (ECS). The present study aimed to investigate the age-dependent fluctuations of glucocorticoids, gonadal steroids, endocannabinoids and their ratios from 21 days to 10 months in both plasma and hair from the male C57BL/6 mice. A novel framework based on the liquid chromatography-tandem mass spectrometry was developed to simultaneously determine ten hormones and two endocannabinoids in plasma and hair. Results showed that glucocorticoids, corticosterone (CORT), aldosterone (ALD), 11-dehydrocorticosterone (11-DHC), gonadal steroids, progesterone (P), dehydroepiandrosterone (DHEA), testosterone (T) and dihydrotestosterone (DHT) in plasma were unimodally fluctuated (p s < 0.001) along age with the maximum value at 2.7-month-old. In contrast, the other two gonadal steroids, estrone (E1) and estradiol (E2) were declined with age (p s < 0.001). Differently, endocannabinoids, N-arachidonoyl-ethanolamine (AEA) and 1-arachydonoyl glycerol (1-AG) showed nadir and zenith values at 2.7-month-old and 3.4-month-old, respectively (p s < 0.001). Additionally, the ratios of CORT to 11-DHC and ALD in plasma were dropped similarly with age (p s < 0.001). The ratios of 1-AG to AEA, and of T to A4 and DHT, and of DHEA to A4 were unimodally changed (p s < 0.001) along age with maximum value at 2.7- or 3.4-month-old. In contrast, the ratios of E2 to T and E1 to A4 were decreased with age (p s < 0.05). The rest six ratios that reflected the interactions among the three endocrine systems, were similar age-dependent and showed nadir and zenith values at 2.7-month-old and 3.4-month-old, respectively (p s < 0.05). Most importantly, these findings in light of age-related changing patterns in plasma were repeated in hair, suggesting that the fi41-ndings in the two matrices were mutually validated. However, it was worth noting that their magnitude of levels in the two bio-matrices were markedly different. The current findings could provide reliable hormone and endocannabinoid signatures with age on neuroendocrine profiles as well as their ratios for the male C57BL/6 mice. [ABSTRACT FROM AUTHOR]

• LC-APCI-MS/MS was developed for quantitation of 12 compounds in rodent's plasma and hair. • The mice having more-diversity gut microbiota had higher hair levels of CORT, 11-DHC and DHEA. • Gut microbiota possibly influence long-term activity of the HPA and HPG axes and ECS. • The mice with less-diversity gut microbiota show sex-specific pattern in plasma hormones. The effect of gut microbiota on the activity of the HPA and HPG axes and ECS is a short-term or long-lasting process remains unclear in rodents. However, the extant studies focused only on its short-term effect on the HPA activity because there is lack of reliable biomarkers characterizing short-term activity of the HPG axis and ECS and long-term activities of the three endocrine systems. The endogenous levels of aldosterone (ALD), 11-dehydrocorticosterone (11-DHC), estradiol (E2), estrone (E1), androstenedione (A4), dihydrotestosterone (DHT), corticosterone (CORT), dehydroepiandrosterone (DHEA), testosterone (T), progesterone (P), N-arachidonoyl ethanoamide (AEA) and 1-arachydonoyl glycerol (1-AG) in hair and plasma are the potential long-term and short-term biomarkers of the three systems. This study aimed to develop the sensitive and selective methods for simultaneous quantitation of the twelve compounds in rodent's hair and plasma. Then the methods were used to explore the differences in the hair levels of the twelve compounds between the mice in XZ group possibly having gut microbiota with more diversity and SPF group possibly having gut microbiota with less diversity and the inter-group differences in the plasma levels in the response to 1-h restraint stress. The methods were based on high performance liquid chromatography tandem mass spectrometry with atmospheric pressure chemical ionization in positive mode. The methods were adopted for 20 mg hair and 100 μL plasma, respectively. Hair samples were incubated in methanol at 40 ℃ for 24 h, and were performed by solid phase extraction. Plasma samples were implemented by liquid-liquid extraction with ethyl acetate. The methods showed limit of quantification at 0.06–1.3 pg/mg and 0.03-0.6 ng/mL and recovery ranging between 87.7–115.1 % and 86.1–114.6 % for all compounds in rodent's hair and plasma, and the intra-day and inter-day coefficients of variation less than 15 %, and good freeze/thaw and short-term stability. The present methods also had good reliability as demonstrated by the sex difference in the testosterone levels in hair and plasma. The inter-group comparison revealed that the mice in XZ group showed significantly higher hair levels than those in SPF group for 1-AG and most of hormones except for T and P. The non-stressed female mice in SPF showed significantly higher plasma levels than those in XZ for AEA and most of hormones except for E2, A4, DHT, T and 1-AG, but there were no inter-group differences for the stressed mice except for DHEA and 11-DHC and for the non-stressed male mice. Additionally, the stressed mice showed significantly higher corticosterone level in plasma than controls for male and female mice in XZ and male mice in SPF, but it was not true for female mice in SPF. [ABSTRACT FROM AUTHOR]

• Twenty-one biomarkers were screened to assess the effect of exogenous testosterone (T). • Salivary levels of the biomarkers were determined using a developed LC-MS/MS method. • Thirteen biomarkers showed intergroup differences after the T administration. • Fifteen biomarkers showed time-dependent changes after the T administration. • Ten biomarkers showed moderate and over relative stability after the T administration. The contents of steroids and endocannabinoids along with the ratios between them would be candidate biomarkers for sensitively and comprehensively assessing the role of testosterone in regulating the activities of the hypothalamus-pituitary–gonadal (HPG) axis, the hypothalamus–pituitaryadrenal (HPA) axis and endogenous cannabinoid system (ECS). However, previous studies mostly used the contents rather than the ratios as biomarkers. This study aimed to systematically screen and identify sensitive biomarkers from 21 candidates including both the contents of nine steroids and one endocannabinoid and their ratios in saliva. Three screening criteria were whether there were intergroup differences, time-dependent changes and considerable relative stability during a 4-h period after exogenous testosterone administration. This study used LC-APCI+-MS/MS to determine the salivary levels of the candidate biomarkers on 62 male healthy undergraduates who were divided into testosterone administration and placebo control groups. The results revealed that salivary testosterone, androstenedione, DHEA and the ratios of testosterone to estradiol and AEA, and of cortisol to testosterone and DHEA were sensitive biomarkers for assessing the effects of testosterone administration on the three neuroendocrine systems because they all showed significant intergroup differences and time-dependent changes and good relative stability. Salivary cortisol, cortisone and the ratios of testosterone to androstenedione and DHEA and of androstenedione to estrone, and of cortisol to cortisone, androstenedione and AEA might be suitable biomarkers because they met only two of the three criteria, but needed to be validated in the future. The rest biomarkers were unsuitable because they mostly showed no significant intergroup differences, blunt time-dependent changes and poor relative stability. [ABSTRACT FROM AUTHOR]