Stress is an individual response to external and internal challenges ranging from behavioral to molecular adaptions, affecting the stress response network in the central nervous system and its crosstalk with peripheral organs via the endocrine and the autonomic nervous systems [1]. The hypothalamic-pituitary-adrenal axis (HPAA) plays a central role in such adaptation processes of the organism [2]. In response to acute stressors, the hypothalamus secretes corticotropin–releasing factor, which triggers the release of adrenocorticotropic hormone (ACTH) from the pituitary gland. ACTH then promotes the release of cortisol from the adrenal cortex. Cortisol affects numerous physiological functions, such as carbohydrate, lipid and protein metabolism, the immune system, and enhances the energy supply of the central nervous system. Chronic psychological stress has been shown to induce dysregulations of the HPAA, which are associated with impaired psychological and somatic well-being. Under such conditions, a biphasic response of the HPAA has been observed: prolonged psychological stress may first induce a hyper-activation of the HPAA, which may then be followed by a state of hypo-activation [3, 4]. While a hyper-activation of the HPAA has been shown to be associated with stress related disorders such as melancholic depression, anxiety disorders, and metabolic syndrome, a hypo-activation is rather linked to fatigue, pain, irritability and associated disorders such as fibromyalgia, arthritis, and chronic fatigue syndrome [5–7]. Thus, a hyper-activation of the HPAA seems to be associated with a hyper-reactivity to acute stress, while a reduced synthesis of cortisol may mainly account for a hypoactive state [6, 8–12].

Recent research showed that the intake of certain nutraceuticals reversed or normalized such dysregulations and improved stress symptomatology. For example, the intake of fertilized egg powder for the duration of four weeks normalized the psychological and physiological stress response in chronically stressed but otherwise healthy males [13]. Moreover, studies on different substances enriched with phospholipids have shown beneficial effects in chronically stressed subjects. It has been suggested that milk phospholipids may increase cortisol availability in subjects with high chronic stress [13–15].

Phosphatidic acid (PA) is one of the most important glycerophospholipids found in bio-membranes. PA has different roles in the cell: it is a precursor for other lipids such as phosphatidylserine (PS) or phosphatidylcholine via the conversion of PA to diacylglycerol [16]. Moreover, PA influences membrane curvature [17, 18] and acts as signaling lipid [19, 20]. Animal research suggests that PA may prevent or restore gastrointestinal disorders [21, 22] and may increase skeletal muscle mass [23]. PS plays a key role in neuronal cell structure and functioning and may improve memory, learning, mood and stress management [24–29]. PS has been shown to decrease ACTH and cortisol responses to acute physical and mental stress [30–35]. Additionally, the intake of PS has been associated with an improvement of psychiatric disorders, such as bipolar and major depressive disorders reviewed in [26, 36], as well as with the prevention of inflammatory neurodegenerative events [37].

Moreover, in combination with PS, PA has been shown to reduce cortisol levels and enhance well-being under acute social stress. In a previous study, we could show that the administration of PAS 400 per day over a period of 21 days resulted in both a significant decrease of the ACTH- and cortisol-response, and, in addition, a reduction of anxiety to the TSST [27]. The exact mechanisms on how PA and PS contribute to an adaptation of the stress response network are still unknown. Several stress-related disorders such as depression and mood disorders have been associated with fatty acid deficiencies [36, 38–41]. The intake of dietary phospholipids such as PA and PS may cause alterations and optimize the composition of cell membranes which in turn may modulate cellular function and activity of membrane [42].

The aim of the current study was to confirm the HPAA normalizing effects of a dose of PAS 400 found in a previous study. Here, we were especially interested whether there was a difference in effects between chronically stressed or non-stressed people. Chronic stress was assessed by using the screening scale for chronic stress of the Trier Inventory for Chronic Stress (TICS) [43]. Cut-off for low versus high chronic stress was defined as provided by a norm population of the TICS authors. In addition, we wanted to investigate whether these effects can also be observed with a lower dosage of PAS 200. In order to investigate PAS effects in a homogeneous study population, we included only male subjects and enlarged group size. We investigated the effects of two different doses of PAS (200 mg PA & 200 mg PS and 400 mg PA & 400 mg PS per day) over 42 days compared to placebo on psychological and physiological stress reactivity in subjects stratified by low and high chronic stress levels.

In the present study, we investigated the effects of a 42-day dose-dependent intake of PAS on biological and psychological responses to the TSST in both HCS and LCS. As expected, the TSST reliably induced robust biological and psychological stress responses. Perceived stress responses were similar in all treatment groups. The placebo group showed a slight tendency for higher anxiety (VAS) during the TSST as compared to the PAS groups. Compliance assessed by MEMS® (about 90%) and by counting remaining capsules (about 95%) was rather high. Differences between both might have been caused by subjects taking their daily dose along to work with only one opening of the track cap which then results in a lower number of total openings. In that case the number of openings as assessed by MEMS® does not reflect the number of intake occasions.

A pronounced stress dampening effect of PAS on HPAA responses could only be observed for the 400 mg treatment group in HCS. These data confirm our previous findings, and are in line with other studies showing that acute and chronic administration of PS and PAS can normalize cortisol responses to acute physical and mental stress [27, 30–32, 34, 35].

As addressed in the introduction, chronic stress can first induce a hyper- and then a subsequent hypo-cortisolemic state. Phospholipid effects on the HPAA can best be described in terms of a normalization of stress responsivity. If chronic stress results in a hyper-responsivity of the HPAA, as observed in this and other studies [15, 27], phospholipids can be expected to buffer the HPAA response to stress. On the other hand, phospholipids may restore normal ACTH- and cortisol responses to stress in subjects with a reduced HPAA response [13, 28]. Notably, relaxation and stress management techniques show similar effects [50–55]. These observations support the assumption that both phospholipids and relaxation can normalize the HPAA-responsivity to stress.

As in our previous PAS study, effects of PAS 400 could only be observed for endocrine but not for autonomic stress responses. If PAS would primarily affect the stress response network in the brain, one would expect common secondary effects on psychological, endocrine, and autonomic measures. This is unlikely, since in both studies psychological effects were inconsistent and autonomic effects could not be observed. This points to the possibility, that PAS primarily targets peripheral components of the HPAA. Chronic stress has been shown to reduce cortisol binding globulin (CBG) [56–58]. CBG is a glycoprotein synthesized in the liver and secreted in the blood where it binds with a high affinity but low capacity to glucocorticoid hormones, such as cortisol in humans and corticosterone in laboratory rodents. In mammals, 95% of circulating glucocorticoids are bound to either CBG (80%) or albumin (15%), and only the 5% free fraction is able to enter the brain [59]. During stress, the concentration of glucocorticoids rises significantly, and the free fraction increases even more, once CBG becomes saturated. However, glucocorticoids unbound to CBG are cleared from the blood more quickly. It is assumed that CBG plays an important role in the fast actions of glucocorticoids on behavior by maintaining a blood glucocorticoid pool that will be able to access the brain for the fast effects of glucocorticoids [60, 61].

The observed pronounced increase of ACTH and cortisol in the TSST can possibly be explained by a drop of CBG in chronically stressed subjects. If so, one may hypothesize that PAS 400 first causes a normalization of CBG levels under such conditions, which then would result in a normalization of the activity and reactivity of the HPAA.

In chronically stressed subjects, PAS 400 can be expected to buffer a hyper-responsivity of the HPAA to acute stressors by normalizing cortisol responses. In contrast, PAS 400 does not affect endocrine stress response in LCS who do not have elevated cortisol levels. Although sample sizes for subgroup analyses of chronic stress were rather small, the results of this study are intriguing.

The CBG-hypothesis, derived from this study, can now be tested to elucidate a possible relevant mechanism of the action of PAS. If this hypothesis holds true, an indication of PAS for stress related disorders is evident, particularly for disorders which are considered to occur in consequence of stress-elevated cortisol levels, such as cardiovascular disease, central obesity, and the metabolic syndrome [5, 62]. In sum, we conclude that PAS 400 (MemreePlus™) is a safe and effective supplement for the reduction of both physical and mental stress. Based on the previous study, it can be speculated that women also benefit from MemreePlus™ supplementation. This needs to be confirmed in further studies.

Participants and methods

Participant selection

Healthy, non-smoking male volunteers between age 20 and 45 years were recruited by newspaper advertisement and flyer distribution in the area of Trier, Germany. Subjects’ health was assessed in a structured medical interview by a study physician and by assessing clinical blood chemistry. Individuals were not eligible if any of the following criteria applied: known allergies to ingredients of the test substance, addiction to nicotine, drugs or alcohol, recent changes in nutritional habits (e.g. start of a weight loss diet), any serious general illness within the last 12 months, any febrile illness (longer than 24 hours) within seven days prior to assessment, any intake of antibiotics during the four weeks prior to study inclusion, diabetes mellitus, heart disease, hypertension, kidney disease, significant respiratory disease, or epilepsy, immunologic or infectious disease (e.g. hepatitis, tuberculosis, HIV or AIDS, lupus, rheumatoid arthritis) which could place the subject at risk or interfere with the accuracy of the study results, current or past participation in a TSST study, employee of the sponsor or CRO, medications that are likely to affect treatment response, or any conditions that may affect the ability of the individual to complete the study or the interpretation of the study results.A total number of 124 subjects were screened for study inclusion and exclusion criteria of which 75 male subjects met the eligibility criteria. These subjects were randomly assigned and stratified by chronic stress level to one of the three treatment groups (25 subjects per group: placebo, PAS 200, PAS 400). After study inclusion, one subject of the placebo group and two subjects of the PAS 200 group were lost to follow up, leaving 24 subjects of the placebo group, 23 subjects of the PAS 200 group, and 25 subjects of the PAS 400 group for analysis (total sample size of 72 subjects) (see Figure 4). The study protocol was approved by the ethical review board of the Medical Association of Rhineland-Palatinate (Mainz, Germany), and written informed consent was obtained from all participants. The study was performed in accordance with the ethical principles that have their origin in the Declaration of Helsinki and are consistent with International Conference on Harmonization/Good Clinical Practice (2008).