Our results revealed sex differences in RAS serum enzymatic activities. Sex differences in soluble aminopeptidases, such as APA, have been described by other authors [19]. Moreover, it has been suggested that sex differences in blood pressure could be due to the effects of sex hormones on some of these peptidases, which are critical for the regulation of RAS [20]. In the present work, a critical range of age was selected for the inclusion of participants, all of whom were healthy middle-aged individuals, thus leading to a methodological improvement with respect to previous studies on this topic.

It has been proposed that sex differences in the regulation of arterial pressure by RAS could be due in part to a decrease in ACE serum activity in males, contrasting with a predominant ACE2 activity in females that could be lost in the post-menopausal period [16]. Sex differences in the incidence and evolution of cardiovascular disease and hypertension are also suggested to be due to the differential effects of sex steroids on critical components of RAS, which could possibly explain sex differences in blood pressure levels [21].

To detect specific physiological sex differences in the serum activities of RAS peptidases, we selected participants ranging from 41 to 70 years old, representing a period of time in which the onset of several cardiovascular and proliferative disorders and early stages of neurodegenerative diseases is most prevalent [11–13], who did not have any chronic pathology, and were not under any pharmacological treatment. Consequently, this sample was intended to constitute a specific ideal model, to avoid bias caused by concomitant pathologies and/or medications.

Estrogen decrease, whereas testosterone increases, ACE activity, and consequently Ang II levels [20]. Accordingly, in our study, men aged ≥55 years showed a significantly lower ACE activity with respect to women. Further, among men, a significant reduction in ACE activity was observed in older participants ( > 55 years), with respect to younger ones (<55 years). These results suggest a greater influence on ACE activity by testosterone, compared to estrogens.

Post-menopausal changes in estrogen levels are well known [22], as a consequence of the physiological decline in ovarian follicle numbers [23], leading to sleep and body temperature disturbances in older women [24]. Although andropausia has been described as a controversial concept [25], it has been suggested that androgen deficiency in older men has been overlooked in clinical settings [26]. In 2006, the Massachusetts Male Aging Study (MMAS) described a reduction in testosterone levels in 21% of men aged 55–59 years, 26% of those aged 60–69 years, and 31% of those aged 70–86 years [27]. In fact, late-onset hypogonadism (age-related testosterone deficiency) has been recently recognized as a clinical syndrome associated with low testosterone levels and alteration of health status [28, 29], and the possibility that subclinical hypogonadism in andropausal men could be a physiologic response of the hypothalamus has been proposed [30]. The breakpoint of 55 years selected for comparisons between groups of age in our study is relevant to these findings.

Pre-menopausal women have lower blood pressure with respect to post-menopausal women [31] and pre-andropausal men [32], indicating that a difference in cardiovascular risk could be mediated by RAS. In fact, androgens can stimulate RAS [33], and the sexually dimorphic pattern of hypertension in the spontaneously hypertensive rat is androgen-dependent, rather than estrogen-dependent [34]. Accordingly, serum peptidases, such as ACE, ACE2, NEP, APN, and APA, which are important elements of the RAS, were studied here because dysregulation of these enzymes has been associated with hypertension and cardiovascular risk [35]. Our results revealed sexual differences in these activities, with a different pattern in pre- and post-andropausal/post-menopausal subjects.

Sex differences in APA activity were observed in the selected subjects. More precisely, a significantly lower serum activity of this enzyme was found in men than in women. These results suggest lower production of angiotensin III in men, which exerts an important physiological regulatory action on cerebral circulation [36]. However, a different pattern of sexual differences was observed in participants ≥55 years of age. In this group, significantly lower APA and ACE serum activities were observed in men compared to women. Considering that angiotensin III promotes increased blood pressure when injected intracerebroventricularly [37], and taking into account that it has been reported that inhibitors of APA, the enzyme that produces angiotensin III, can act as antihypertensive drugs [38, 39]; these results could help explain the smaller changes in blood pressure usually observed in post-andropausal men, with respect to that observed in post-menopausal women.

The reduction in APA activity may be linked to a decrease in the production of angiotensin III, leading to a reduction in cerebral vasoconstriction. This could prevent the elevation of cerebral blood pressure, thus contributing to the decrease in cerebrovascular risk found in post-andropausal men compared to post-menopausal women. However, in participants <55 years old, although significantly higher NEP and APN serum activities were observed, a lack of changes in ACE, ACE2, and APA serum activities was observed in men, with respect to women. Notably, reduced APA could be linked to higher levels of angiotensin II, thus tending to increase blood pressure. Further, elevated NEP in younger men could increase angiotensin 1-7, reducing this parameter.

With respect to aging, these serum enzymatic activities presented sex differences in the activity patterns when it was observed between younger (<55 years) and older (≥55 years) participants. Thus, higher ACE2 activity was found in older women, whereas lower ACE and APA activities were observed in older men. Due to the fact that ACE2 activity can increase production of angiotensin 1-7, which leads to vasodilatatory, antiproliferative, and antifibrotic effects [40], and considering that antithrombotic effects have been recently proposed for the ACE2-angiotensin (1-7)-Mas receptor axis [41], the increase in ACE2 activity in the post-menopausal period in women could lead to a differential cardiovascular risk in men and women [42].

Although post-menopausal women are known to have an increased cardiovascular risk, this fact does not necessary contradict the beneficial cardiovascular effects of the increase in ACE2 activity in older women. Thus, it cannot be discarded that the ACE2-ang1-7-Mas axis could counterbalance negative cardiovascular effects of other age-related mechanisms. Further studies are needed to detect angiotensin peptide changes induced by aging in both sexes.

A limitation of the study could be that we lack data regarding alcohol consumption, cigarette smoking status, or physical activity habits, additional information that could influence RAS activity. However, we must emphasize that in meeting our inclusion criteria, the participants did not have any chronic pathology and were not under any pharmacological treatment, making this a reasonably good sample for this study.