Elevated plasma ceramide levels in post-menopausal women: a cross-sectional study

Circulating ceramide levels are abnormally elevated in age-dependent pathologies such as cardiovascular diseases, obesity and Alzheimer’s disease. Nevertheless, the potential impact of age on plasma ceramide levels has not yet been systematically examined. In the present study, we quantified a focused panel of plasma ceramides and dihydroceramides in a cohort of 164 subjects (84 women) 19 to 80 years of age. After adjusting for potential confounders, multivariable linear regression analysis revealed a positive association between age and ceramide (d18:1/24:0) (β (SE) = 5.67 (2.38); p = .0198) and ceramide (d18:1/24:1) (β (SE) = 2.88 (.61); p < .0001) in women, and between age and ceramide (d18:1/24:1) in men (β (SE) = 1.86 (.77); p = .0179). In women of all ages, but not men, plasma ceramide (d18:1/24:1) was negatively correlated with plasma estradiol (r = -0.294; p = .007). Finally, in vitro experiments in human cancer cells expressing estrogen receptors showed that incubation with estradiol (10 nM, 24 h) significantly decreased ceramide accumulation. Together, the results suggest that aging is associated with an increase in circulating ceramide levels, which in post-menopausal women is at least partially associated with lower estradiol levels.


INTRODUCTION
The ceramides are key lipid constituents of mammalian cells. They regulate the structural properties of the lipid bilayer [1] along with its interaction with cellular proteins [2], and control many signalling processes, including cell survival [3], growth and proliferation [4], differentiation [5], senescence [6] and apoptosis [7,8]. Dysfunctions in ceramide-mediated signalling may contribute to the initiation and progression of a variety of age-dependent diseases. Human studies have shown the existence of abnormal plasma levels of various ceramide species -including ceramide (d18:1/18:0), (d18:1/22:0), (d18:1/24:0) and (d18:1/24:1) -in several conditions such as obesity [9], type-2 diabetes [10], hypertension [11], atherosclerosis [12] and other cardiovascular diseases [13]. Furthermore, elevated serum levels of long-chain ceramides have been linked to the increased risk of memory deficits [14] and may be predictive of hippocampal volume loss and cognitive decline in patients affected by mild cognitive impairment [15]. Other studies have reported the existence of sex-dependent differences in circulating ceramides, albeit with apparently contrasting results [16][17][18]. For example, in a study of a large cohort of Mexican-Americans of median age 35.7 years, plasma ceramides were found to be higher in men than in women [17]. By contrast, in the Baltimore Longitudinal Study of Aging, whose participants were aged 55 or older, plasma ceramide concentrations were shown to be higher in women than in men [18]. These discrepancies may reflect across-study differences in design and participation.
Aging in rats and mice is associated with sexually dimorphic changes in the sphingolipid composition of several brain structures, including the hippocampus [19]. Age-dependent sphingolipid alterations have also been documented in peripheral rodent tissues [20]. In the present study, we hypothesized that aging in humans might be similarly associated with changes in ceramide levels. To test this idea, we profiled six ceramides and dihydroceramide species in lipid extracts of plasma from 164 subjects (84 women) of age 19 to 80 years, using liquid chromatography/mass spectrometry (LC-MS/MS).
In an additional analysis, we compared total ceramide levels in plasma of pre-and post-menopausal women with those measured in men of the same age group (Fig  3). The results show that pre-menopausal women had significantly lower levels of circulating ceramides (p < .05, 2-way ANOVA followed by Bonferroni post-hoc test) relative to men of similar age (Fig 3A). The difference disappeared after menopause (p > .05) ( Fig  3A).
Plasma ceramide levels are negatively correlated with estradiol in women, but not in men Next, we investigated a possible association between plasma levels of estradiol, which are known to fall significantly at menopause, and ceramides. Estradiol was measured with a competitive binding immunoassay. As expected, plasma estradiol was higher in pre-menopausal women (<55 years) compared to men in the same age group (p < .001, 2-way ANOVA followed by Bonferroni post-hoc test) (Fig 3B). After menopause, estradiol levels sharply decreased in both sexes ( Fig 3B). Fig 4 illustrates the results of Pearson's analyses of ceramide levels in female subjects of all ages. The results show a statistically significant negative correlation between estradiol and ceramide (d18:1/24:1) (r = -0.294; p = .007), a non-significant negative trend between estradiol and ceramide (d18:1/24:0) (r = -0.202; p = .066) and no correlations between estradiol and other ceramide species. By contrast, in men, no correlation was observed between estradiol and any ceramide species, including ceramide (d18:1/24:1) (r = -0.034; p = .763) (Fig 5), which was found to be correlated with aging (β (SE) = 1.86 (.77); p = .0179) (Table 2B).

Estradiol suppresses ceramide accumulation in vitro
Sphingolipid-derived mediators such as ceramide, sphingosine, and sphingosine-1-phosphate regulate steroidogenesis [21], but it is still unknown whether estrogen hormones influence sphingolipid metabolism. To gain insight into the causality of the negative correlation observed between estradiol and ceramide in women, we asked whether the sex hormone might regulate ceramide mobilization (i.e. formation and/or degradation) in human MCF7 breast cancer cells, a cell line that expresses the estrogen receptor α (ERα) and β (ERβ) [22]. MCF7 cells were treated with estradiol (10 nM) for 24 h and ceramides quantified in lipid extracts by LC-MS/MS. Results indicate that the exposure to estradiol causes a substantial reduction in ceramide (d18:1/16:0) (p = .02, unpaired Student's t-test), (d18:1/24:0) (p = .0002) and (d18:1/24:1) (p = .0006) ( Table 3), thereby suggesting that estradiol causes a downregulation in the biosynthesis or/and an upregulation in the degradation of these substances.

DISCUSSION
In the present study, we investigated the age-and sexdependent trajectories of plasma ceramides in 80 men and 84 women aged 19-80 years. The results show that, in women, plasma levels of two ceramide species -(d18:1/24:0) and (d18:1/24:1) -increased with age, and that this change cannot be ascribed to confounding  AGING factors such as obesity, diabetes, and other health conditions. In men, the analysis revealed an association between age and ceramide (d18:1/24:1). Further analyses identified a significant negative correlation between circulating levels of estradiol and ceramide (d18:1/24:1) in women of all ages, but not in men. Finally, in vitro experiments showed that estradiol lowers ceramide levels in human cells expressing estradiol receptors. The findings suggest that aging is accompanied, in men and women, by an increase in the plasma concentrations of two ceramide species, (d18:1/24:0) and (d18:1/24:1), which are also known to be elevated in age-dependent pathologies such as atherosclerosis and cardiovascular disease [23,24]. The results also point to the intriguing possibility that estradiol might control circulating ceramide levels in a sexually dimorphic manner.
Previous studies have reported sex-dependent differences in plasma ceramides, but with somewhat contrasting results, which might be due to disparities in sex, fasting, age and body mass index across the studies. In a small investigation of blood serum samples from 10 Caucasian volunteers (5 males aged 27-33 years and 5 females aged 26-33 years), ceramide (42:1) was found to be higher in women compared to men [16]. Another study, performed on a much larger cohort of young Mexican Americans (1,076 individuals, 39.1% males, median age 35.7 years) [17], uncovered an association between plasma ceramides and sex after adjusting for    AGING age and body mass index: ceramide levels were lower in women than in men. These disparities were mostly driven by long-chain ceramide species, such as (d18:1/22:0), (d18:1/24:0) and (d18:1/24:1). Finally, in a multiethnic population sample of 366 women and 626 men aged over 55 years, enrolled in the Baltimore Longitudinal Study of Aging, plasma ceramide concentrations were found to be higher in women compared to men [18].
These studies did not focus on age as a variable. By contrast, in the present work we set out to address the impact of aging on ceramide levels and excluded subjects with disease conditions that had been previously shown to affect ceramides, such as diabetes [25], cancer [26], renal disease [27], cardiovascular disease [28] and obesity [29]. We did not exclude subjects with hypertension, however, because the association of this disease state with altered ceramides remains to be fully established [11,18]. In our sample, we were able to confirm the presence of age-and sexdependent differences in the plasma concentrations of certain ceramide species, but not others. A multivariable analysis showed that, in women, aging is accompanied by increased levels of ceramide (d18:1/24:0) and (d18:1/24:1). In men, the analysis also unmasked a statistically detectable association between age and ceramide (d18:1/24:1). These age-dependent changes could not be ascribed to obesity, diabetes, tobacco cigarette smoking and use of anti-hypercholesterol or contraceptive agents. Interestingly, secondary data analyses found that ceramide levels were significantly lower in female than male subjects aged 20-54, a difference that disappeared after menopause. In their 2015 study, Mielke and collaborators did not specifically include menopause as a variable, but suggested that menopause and estradiol may influence ceramide levels [18]. Two sets of results presented here support this prediction. First, we showed that, in women of all ages, plasma estradiol was negatively correlated with ceramide (d18:1/24:1) and displayed a trend toward correlation with ceramide (d18:1/24:0). No such relationship was detectable in men. Second, we found that incubation with exogenous estradiol (10 nM) lowered the intracellular levels of various ceramides, including ceramide (d18:1/24:0) and (d18:1/24:1), in human estrogen-sensitive MCF7 cells.
The findings outlined above suggest that age-dependent changes in estradiol may affect ceramide metabolism and/or transport in microsomal triglyceride transporter protein [30] differentially in men and women. The correlation between estradiol and ceramide raises the possibility that changes in the availability of certain ceramide species [e.g. ceramide (d18:1/24:1)] might be implicated in the reported cardioprotective, antihypertensive and neuroprotective effects of estradiol [31][32][33]. In this context, it is important to point out that increased plasma ceramide levels have been consistently linked to heightened risk of myocardial infarction and stroke [34]. Moreover, recent evidence indicates that alterations of ceramide levels may mirror, indirectly, ongoing neurodegenerative processes and might be used as a biomarker for Alzheimer's disease development and progression [35]. Interestingly, the close association between alterations in ceramide levels and the likelihood of developing cognitive impairment and Alzheimer-related pathology may be particularly AGING strong in women, thereby indicating that sex-related mechanisms might participate in shaping the Alzheimer phenotype. Of note, elderly women show changes in plasma ceramide levels at the onset of their memory impairment [15]. In the past few years, the neurobiology of memory disorders has received increasing attention [36]. Memory deficits are often reported by women in temporal proximity of menopause [37], and recent findings indicate distinct changes in memory processing that appear to be linked more to the post-menopausal status than to chronological aging: in peri-menopausal women, the onset and progression of cognitive decline are often associated with a menopause-related decrease in estradiol levels [38]. Thus, the interplay between estradiol and ceramides, described here, may potentially be of broad significance for a variety of age-dependent disorders, including cardiovascular disease and cognitive impairment or any intersection of the two conditions [39].
The present study has several limitations. First, even though we excluded persons affected by obesity and diabetes, we did not collect information on metabolic factors that could potentially impact ceramide mobilization -such as adiposity, physical activity and levels of cholesterol, triacylglycerol and glucose in blood [18,[40][41][42]. Second, our study was focused on a specific subset of ceramides that we had previously found to be altered in the hippocampus of aged male and female mice [19]. This group of ceramides has been proposed as potential biomarker for cardiovascular risk [43], but is still only a small fraction of the vast number of ceramides produced by the body. Third, we measured total circulating levels of ceramides and did not attempt to separate ceramide pools bound to specific plasma lipoproteins [44]. It is possible that aging might exert an effect similar to obesity and diabetes, which have been shown to change the distribution of ceramides among lipoproteins [41].
Our findings also raise a number of relevant questions, which should be addressed in future work. First, even though the results suggest that estradiol modulates ceramide mobilization in women, the precise mechanism and functional significance of this effect remain to be determined. One possibility is that activation of estrogen receptors results in the downregulation of de novo ceramide biosynthesis, for example by suppressing the expression of key enzymes such as serine-palmitoyltransferase and ceramide synthase [45]. Other possibilities are that estradiol might stimulate the expression of ceramide-hydrolyzing enzymes such as acid or neutral ceramidase or of ceramide-transporting proteins [30]. Probing the link between estradiol and ceramides will require additional experimentation, which may include measuring circulating ceramide levels in women throughout the menstrual cycle or assessing the impact of endogenous and exogenous estradiol on sphingolipid metabolism in female animals. At the functional level, studies are needed to correlate ceramide and estrogen levels to a broad panel of biomarkers (e.g., lipoprotein profile, Creactive protein) and clinical outcome measures (e.g., future adverse cardiovascular events and cognitive impairment) [43]. Without such information, the clinical significance of our findings remains speculative. Second, the lack of correlation between estradiol and ceramide (d18:1/18:0) and lack of association with age implies that this ceramide species, though elevated after menopause, may be subjected to a different regulation than ceramide (d18:1/24:1), whose levels are correlated with estradiol and are statistically associated with age. Third, we observed a substantial age-dependent decrease in plasma dihydroceramide (d18:0/24:0) in men aged 54-80 years. The significance of this finding is presently unclear, but warrants further attention. Fourth, we unexpectedly uncovered an association between hypertension in post-menopausal women and elevated levels of ceramide (d18:1/16:0) and (d18:1/18:0). While this result is consistent with previous reports [11], caution is warranted until studies with a larger cohort of pre-and post-menopausal women are performed. Finally, as mentioned above, the relation between ceramide levels and premorbid changes in cardiovascular, metabolic or cognitive function was not investigated in our study and deserves further investigations. Despite these unanswered questions, our results reveal the existence of a link between age, estradiol and ceramides, which might contribute to age-dependent pathologies in postmenopausal women.

Ethics statement
Investigation has been conducted in accordance with the ethical standards and according to the Declaration of Helsinki and according to national and international guidelines and has been approved by the authors' institutional review board.

Study subjects
We recruited 164 Italian subjects (84 women) from 19 to 80 years of age (Table 1). A primary criterion for subject selection was the absence of major medical illnesses and, particularly, conditions that had been previously linked to ceramide alterations. A subdivision of the subjects by age and menopause status is reported in Table S1. There were no significant differences between male and female subjects with regard to age, AGING years of education, ethnic background, cognitive status, as assessed by the Mini-Mental State Examination (MMSE), obesity and diabetes, hypertension and use of anti-hypercholesterol drugs. By contrast, there was difference in smoking status (20.23% women versus 7.5% men). Moreover, 6 of 44 pre-menopause women used contraceptives at time of enrollment. None of the post-menopause women was under hormone replacement therapy (HRT).
Exclusion criteria were: (i) suspicion of cognitive impairment or dementia based on MMSE [46] (score ≤ 26, consistent with normative data collected in the Italian population) and confirmed by a detailed neuropsychological evaluation using the mental deterioration battery [47] and clinical criteria for Alzheimer's dementia [48] or mild cognitive impairment [49] Blood collection and analyses were approved by the Santa Lucia Foundation Ethics Committee. A written consent form was signed by all participants after they received a full explanation of the study procedures. Informed consent has been obtained.

Variables examined in relation to ceramide levels
All variables were assessed for each patient using the same methods. Demographic variables considered in linear regression analysis included age and sex. Medical history covariates included hypertension, use of antihypercholesterol agents and use of contraceptives. Current and former smoking status was ascertained by an oral interview.

Chemicals
Solvents and chemicals were purchased from Sigma Aldrich (Milan, Italy). Ceramide standards were from Avanti Polar Lipids (Alabaster, AL, USA).

Blood collection
Blood was drawn by venipuncture in the morning after an overnight fast, and collected into 10-ml tubes containing spray-coated EDTA (EDTA Vacutainer, BD Biosciences, San Diego, CA, USA). Plasma was obtained by blood centrifugation at 400 × g at 4 °C for 15 min. The plasma divided into aliquots was stored at −80 °C until analyses.

Lipid extraction
Lipids were extracted using a modified Bligh and Dyer method [54]. Briefly, plasma samples (50 µL) or cell pellets were transferred to glass vials and liquid-liquid extraction was carried out using 2 mL of a methanol/chloroform mixture (2:1 vol/vol) containing the odd-chain saturated ceramide (d18:1/17:0) as an internal standard. After mixing for 30 s, lipids were extracted with chloroform (0.5 mL), and extracts were washed with liquid chromatography-grade water (0.5 mL), mixing after each addition. The samples were centrifuged for 15 min at 3500 x g at room temperature. After centrifugation, the organic phases were collected and transferred to a new set of glass vials. To increase overall recovery, the aqueous fractions were extracted again with chloroform (1 mL). The two organic phases were pooled, dried under a stream of N 2 and residues were dissolved in methanol/chloroform (9:1 vol/vol, 0.07 mL). After mixing (30 s) and centrifugation (10 min, 5000 x g, room temperature) the samples were transferred to glass vials for analyses.

Estradiol quantification
Plasma 17-β-estradiol (E2) levels were quantified using a competitive binding immunoassay kit (Human E2 ELISA kit, Invitrogen, Milan, Italy) following manufacturer's instructions. Briefly, plasma samples, controls and standard curve samples (50 µL) were incubated with E2-horseradish peroxidase conjugate (50 µL) and anti-estradiol antibody (50 µL) in a 96-well plate for 2 h, at room temperature, on a shaker set at 700 ± 100 rpm. Washing was carried out by completely aspirating the liquid, filling the wells with diluted wash buffer (0.4 mL) provided in the kit and then aspirating again. After repeating this procedure 4 times, chromogen solution (200 µL) was added to each well; reactions were run for 15 min and stopped adding 50 µL of the stop solution provided in the kit. Absorbance was measured at 450 nm and estradiol concentrations were calculated by interpolation from the reference curve.

Statistical analyses
Results are expressed as mean ± SEM (standard error of the mean). Sex differences in baseline demographic and health-related characteristics were examined using Fisher's exact test for categorical variables and unpaired Student's t-test for continuous variables. Data were analyzed by unpaired Student's t-test or 2-way ANOVA followed by Bonferroni post-hoc test. Pearson's correlation coefficient assessed the pairwise correlation between estradiol and ceramide levels. Significant outliers were excluded using the Grubbs' test. Multivariable linear regression method was used to assess the association between ceramides and patients' characteristics. Differences between groups were considered statistically significant at values of p < .05. The GraphPad Prism software (GraphPad Software, Inc., La Jolla, CA, USA) and SAS 9.4 (SAS Institute, Cary, NC, USA) were used for statistical analyses.

CONFLICTS OF INTEREST
The authors have no conflicts of interest to disclose.

FUNDING
The study received support from the Italian Ministry of Health (grant RF-2013-02359074) and from grant UL1 TR001414 from the National Center for Advancing Translational Sciences, National Institutes of Health (NIH), through the Biostatistics, Epidemiology and Research Design Unit. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.
AGING SUPPLEMENTARY MATERIAL Table S1. Clinical characteristics of men and women included in the study divided by age or menopause status.