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Relationship between omega-3 fatty acids and plasma neuroactive steroids in alcoholism, depression and controls
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Relationship between omega-3 fatty acids and plasma neuroactive steroids in alcoholism, depression and controls L.R.G. Nieminena, K.K. Makinoa, N. Mehtaa, M. Virkkunenb, H.Y. Kima, J.R. Hibbelna, a
National Institutes of Health, National Institutes on Alcoholism and Alcohol Abuse, Laboratory of Membrane Biophysics and Biochemistry, Bethesda, MD 20814, USA b Department of Psychiatry, Helsinki University Central Hospital, Helsinki, Finland
Abstract Deficiency in the long-chain o-3 fatty acid, docosahexaenoic acid (DHA) has been associated with increased corticotropin releasing hormone and may contribute to hypothalamic pituitary axis (HPA) hyperactivity. Elevated levels of the neuroactive steroids, allopregnanolone (3a,5a-THP) and 3a,5a-tetrahydrodeoxycorticosterone (THDOC) appear to counter-regulate HPA hyperactivity. Plasma essential fatty acids and neurosteroids were assessed among 18 male healthy controls and among 34 male psychiatric patients with DSM-III alcoholism, depression, or both. Among all subjects, lower plasma DHA was correlated with higher plasma THDOC (r ¼ 0.3, Po0.05) and dihydroprogesterone (DHP) (r ¼ 0.52, Po0.05). Among psychiatric patients lower DHA was correlated with higher DHP (r ¼ 0.60, Po0.01), and among healthy controls lower plasma DHA was correlated with higher THDOC (r ¼ 0.83, Po0.01) and higher isopregnanolone (3b,5a-THP) (r ¼ 0.55, Po0.05). In this pilot observational study, lower long-chain o-3 essential fatty acid status was associated with higher neuroactive steroid concentrations, possibly indicating increased feedback inhibition of the HPA axis. r 2006 Elsevier Ltd. All rights reserved.
1. Introduction A growing body of clinical and epidemiological data suggests that the o-3 fatty acids docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) may be effective in the treatment and or prevention of depressive disorders [1]. Depressive disorders have been associated with excessive responses to stress and hyperactivity of the hypothalamic pituitary adrenal (HPA) axis [2]. However, few studies have examined a putative link between o-3 fatty acids, HPA hyperactivity and stress response. Hibbeln et al. [3] demonstrated that lower plasma levels of DHA predicted higher levels of corticotropin releasing hormone (CRH) in cerebrospinal fluid, suggesting that Corresponding author. NIAAA, 31 Center drive, 31/1B 58, Bethesda, MD 20892, USA. Tel.: +301 435 4028; fax: +301 402 0016. E-mail address: [email protected] (J.R. Hibbeln).
0952-3278/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.plefa.2006.07.012
deficiency in DHA may facilitate excessive stress responses and HPA hyperactivity. In support of this interpretation, DHA deficient rats had exaggerated distress behaviors compared to DHA adequate rats during administration of CRH and were normalized upon restoration of dietary DHA [4]. At least two double-blind placebo-controlled intervention trials with human subjects have demonstrated a stress protective benefit of supplementation with o-3’s. Among Japanese students, supplementation with DHA attenuated stress-induced increases in aggression and hostility [5]. Among stressed university staff, both o-3 and placebo significantly reduced perceived stress, however only the o-3 group was significantly better compared to the no-treatment controls. The latter study had several methodological limitations, including the use of olive oil as placebo, and therefore should be interpreted cautiously [6]. Excessive stress response and HPA hyper-activation may be reflected in concentrations of brain and plasma
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neuroactive steroids (NAS). NAS are produced de novo in the central nervous system from cholesterol and have the ability to rapidly (within seconds) alter neuronal excitability, in contrast to classic gonadal and adrenal steroid hormones acting over several hours to days [7]. 3a-hydroxy ring A-reduced pregnane neurosteroids 3a,5a-tetrahydroprogesterone (3a,5a-THP) and 3a,5atetrahydrodeoxycorticosterone (THDOC) behave similarly to benzodiazepines or barbiturates [8] acting as potent positive modulators of GABAergic neurotransmission. 3a,5a-THP and THDOC are metabolites of the 5a-pregnane steroids, 5a-dihydroprogesterone (DHP) and 5a-dihydrodeoxycorticosterone, which in turn, are metabolites of progesterone (PROG) and deoxycorticosterone, respectively [9]. PROG [10], but not DHP [11] nor stereoisomer 3b,5a-THP [12] have demonstrated the anxiolytic GABA enhancing potential of 3a,5a-THP and THDOC. Stress-induced CRH activation of the HPA axis resulted in elevated NAS concentrations [13] and NAS were elevated in both brain and plasma during stressinducing paradigms [14,15] and in panic disorder [16]. This stress-induced elevation of NAS, particularly 3a,5a-THP and THDOC, has been explained as a homeostatic mechanism to counter-regulate stress response via central and neuroendocrine pathways. 3a,5aTHP and THDOC potentiate GABAA receptor inhibition of the central nervous system [8] and have several anxiolytic properties [17,18] including: counteraction of the down regulated GABA receptor function observed in acute stress [19], decreased dopamine metabolism in prefrontal cortex [20], decreased gene expression of CRH in the hypothalamic paraventricular nucleus [21], and down-regulation of the HPA system [16,19,22]. In support of the proposed feedback inhibition paradigm, exogenous administration of THDOC and 3a,5a-THP attenuated the stress-induced release of CRH [23] and consequent anxiogenic effects [24]. We posit a model of interaction between o-3 essential fatty acid status, NAS and the HPA axis in which low DHA status predicts elevated CRH [3], elevated CRH induces HPA axis hyperactivity [25] and HPA axis hyperactivity results in higher levels of NAS [14]. Thus, in this observational study, we postulated that lower o-3 status would be correlated with higher plasma concentrations of 3a,5a-THP and THDOC.
2. Patients and methods This study population (n ¼ 52) consisted of male Finnish psychiatric patients with alcoholism, major depression, or both (n ¼ 34) and healthy controls without psychiatric disorders (n ¼ 18). As used in this manuscript, the term ‘psychiatric patients’ refers to those individuals with, at minimum, the diagnosis of
alcohol abuse or dependence, or depression, with most patients having both alcoholism and depression. DSMIII diagnosed Schizophrenia and other psychotic disorders were excluded, but comorbidity with other axis I or II disorders was not exclusionary. DSM-III diagnoses and plasma sample collections were preformed as previously described [26]. Subjects were studied under a human research protocol approved by the institutional review board of the National Institutes of Health, Bethesda, MD; the Office for Protection from Research Risks, Bethesda; the University of Helsinki Department of Psychiatry institutional review board, Helsinki, Finland; and the University of Helsinki Central Hospital institutional review board. All subjects provided written informed consent before participating in the study. Individual fatty acids in plasma were assessed [27] and are expressed as a weight percentage (wt%) of total fatty acids. Neurosteroid analysis of plasma was conducted using mass-spectroscopy methods for 3a,5a-THP (n ¼ 51) and THDOC (n ¼ 52), as well as metabolic precursors DHP (n ¼ 22) and PROG (n ¼ 19). We also assessed, d4dihydrotestosterone (DHT) (n ¼ 52) and isopregnanolone (3b,5a-THP) (n ¼ 47) as exploratory measures [28]. For identification and removal of outliers due to analytical error, a modified z-score was generated using the median of absolute deviation about the median (MAD) statistical technique [29]. Bivariate spearman’s rcorrelations were conducted using essential fatty acids and neurosteroids as variables among psychiatric patients (subjects with DSM-III alcohol dependence or abuse, major depression, or both alcoholism and depression), among healthy control subjects (subjects with no DSM-III diagnoses), and lastly among all subjects combined (SPSS release 11.0.1, SPSS Inc., Chicago, IL). In order to reduce errors due to multiple testing, the correlational analysis was limited to the essential fatty acids and background lipid profiles. Correlations between total fatty acids, total saturates, total polyunsaturates, total monounsaturates and neurosteroids were assessed in order to discern effects of individual fatty acids from overall lipid profiles. Independent samples t-tests and Levene’s test for equality of variances were conducted to compare the mean and variance, respectively, in concentrations of NAS and essential fatty acids between psychiatric patients and healthy controls. Logistic regression analyses were used to assess the possible confounding effects of DSM-III axis I or II comorbidity by comparing groups above or below the median split of neurosteroid measures.
3. Results Decreased plasma concentrations of long-chain o-3 fatty acids were correlated with increased plasma
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Plasma THDOC d0/d4
3.0
311
Table 1 Spearman’s r bivariate correlations (r-values and sample sizes shown) of plasma essential fatty acids (wt% total fatty acids) and plasma neurosteroids (ng/mL)
2.5
2.0
1.5
1.0 1.0
1.5
2.0
2.5 3.0 3.5 Plasma DHA (wt %)
4.0
4.5
Fig. 1. Lower plasma docosahexaenoic acid, DHA% (wt% total fatty acids) is correlated with higher plasma concentrations of 3a,5atetrahydrodeoxycorticosterone, THDOC (d0/d4, see methods reference [23]) among healthy controls (r ¼ 0.80, Po0.01) using spearman’s rregression analysis.
concentrations of neurosteroids. Lower plasma concentrations of both DHA% (Fig. 1) and EPA% predicted higher levels of THDOC and the allopregnanolone stereoisomer, 3b,5a-THP among healthy controls. Among psychiatric patients lower plasma DHA% predicting higher plasma DHP was the only significant correlation detected. Among all subjects lower DHA% remained robust in predicting elevated concentrations of both THDOC and DHP (Table 1). Logistic regression analysis indicated that the relationship between THDOC and plasma DHA% remained significant (Wald ¼ 4.05, Po0.05) after adjustment for comorbidity of DSM-III axis I or axis II disorders. In contrast to the inverse correlations found between long-chain o-3 fatty acids and neurosteroids, 18:3n-3 linolenic acid (LNA) was positively correlated with both DHP among all subjects and THDOC among healthy controls. There were no significant relationships found between essential fatty acids and 3a5a-THP, PROG or DHT (data not shown). Total fatty acids, total saturates, total polyunsaturates and total monounsaturates did not predict neurosteroid concentrations (data not shown). Thus, correlational relationships between individual fatty acids and neurosteroids were not an artifact of overall background fatty acid profiles. Mean concentrations of essential fatty acids and neurosteroids did not significantly differ between psychiatric patients and healthy controls, as assessed by independent samples t-tests. The variance in THDOC and PROG concentrations were significantly higher among psychiatric patients compared to healthy controls, as assessed by Levene’s test for equality of variance (Table 2).
THDOCa
3b,5a-THPb
DHPb
All subjects 18:2n-6, LA1 20:4n-6, AA2 18:3n-3, LNA3 20:5n-3, EPA4 22:6n-3, DHA5
n ¼ 52 0.04 0.15 0.05 0.27 0.30
n ¼ 47 0.08 0.06 0.14 0.07 0.08
n ¼ 22 0.40 0.49 0.47 0.06 0.52
Psychiatric patients 18:2n-6, LA 20:4n-6, AA 18:3n-3, LNA 20:5n-3, EPA 22:6n-3, DHA
n ¼ 34 0.04 0.02 0.32 0.03 0.07
n ¼ 31 0.13 0.34 0.05 0.29 0.22
n ¼ 17 0.28 0.39 0.30 0.13 0.60
Healthy controls 18:2n-6, LA 20:4n-6, AA 18:3n-3, LNA 20:5n-3, EPA 22:6n-3, DHA
n ¼ 18 0.03 0.43 0.48 0.83 0.80
n ¼ 16 0.00 0.30 0.12 0.63 0.55
no10 NS NS NS NS NS
Bold text indicates significant correlations. 1 Linoleic acid. 2 Arachidonic acid. 3 Linolenic acid. 4 Eicosapentaenoic acid. 5 Docosahexaenoic acid. a These values were expressed as GC/MS response ratios d0/d4 (see methods reference [23]). b These values were expressed as ng/mL. Indicates Po.05. Indicates Po.01.
4. Discussion and conclusions To our knowledge, this is the first reported association between plasma essential fatty acid status and neurosteroid concentrations in humans or animals. In this pilot observational study, we found that lower plasma concentrations of long-chain o-3 fatty acids were associated with higher plasma concentrations of neurosteroids. Lower DHA% and EPA% were associated with higher concentrations of THDOC among healthy controls. DHA% remained a significant predictor of THDOC among all subjects, however neither DHA% nor EPA% were associated with THDOC among psychiatric patients. This may have been a result of the increased variability in THDOC concentrations among psychiatric patients as compared to healthy controls. The positive correlations between 18:3n-3 LNA and DHP among all subjects and THDOC among healthy controls are unexpected findings in light of the opposite relationship demonstrated between long chain o-3 fatty acids and neurosteroids in this study. This finding should be cautioned by the fact that LNA was present in
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Table 2 Means and standard deviations of essential fatty acids (expressed as wt% of total fatty acids) and neurosteroids (expressed as ng/mL) among psychiatric patients with alcoholism, depression, or both and healthy controls Mean (SD) Psychiatric patients Essential fatty acids (wt%) 18:2n-6, LA 20:4n-6, AA 18:3n-3, LNA 20:5n-3, EPA 22:6n-3, DHA Neurosteroids (ng/mL) 3b,5a-THP DHP 3a,5a-THP PROGa DHT THDOCb,c
Healthy controls
0.37 5.35 0.01 1.42 2.59
(0.19) (1.07) (0.002) (0.80) (0.94)
0.37 5.73 0.01 1.33 2.36
(0.21) (1.21) (0.003) (0.67) (0.76)
0.45 13.19 0.32 0.80 1.57 1.90
(0.21) (8.03) (0.09) (0.55) (0.85) (0.78)
0.46 17.42 0.35 0.80 1.57 1.76
(0.23) (7.86) (0.08) (0.16) (0.82) (0.50)
a Significant between groups difference in Levene’s test for equality of variance (F ¼ 5.079, Po 0.04). b Significant between groups difference in Levene’s test for equality of variance (F ¼ 5.163, Po0.03). c THDOC values expressed as d0/d4 (see methods reference [23]).
low amounts in this study, and secondly, because there are no previous studies we are aware of demonstrating opposing or competing effects for short and long chain o-3 fatty acids. Pisu and Serra [17], in their 2004 review, comment that ‘‘it remains to be determined y whether changes in the concentrations of NAS are a cause of, a risk factor for, or a consequence of mental disorders’’. In terms of the association presented here and its implication for the stress response system, it has been similarly challenging to interpret increased levels of anxiolytic NAS as either beneficial or indicative of HPA hyperactivity. Further, because plasma samples were collected at a single time point it is impractical to determine whether these levels are representative of elevated systemic concentrations or acute elevations as part of the proposed negative feedback mechanism. Thus, lower plasma DHA% associated with higher THDOC concentrations can be interpreted as indicating either stress protection or greater HPA activation. Both interpretations and corresponding evidence are presented below. Elevated NAS may indicate a counter-regulation of excessive hyperactivity of the HPA axis due to excessive stress or increased sensitivity. In support of this interpretation, THDOC was elevated in panic disorder [16] and major depression [30] and was normalized after 50 days of treatment with fluoxetine [31]. This decrease in THDOC may indicate attenuated need for neurosteroid down-regulation of HPA activation due to increased effectiveness of HPA axis negative feedback and
decreased CRH release [17,32]. Thus, the association between lower DHA% and greater concentrations of THDOC reported here may be interpreted as indicating greater HPA activation and homeostatic counter-regulation via elevated NAS levels. This interpretation is cautioned by the absence of other measures of HPA axis function, such as corticosterone and ACTH, and the absence of a significant relationship between essential fatty acid status and 3a,5a-THP. Alternatively, it is possible to postulate that higher THDOC and thus lower plasma DHA% is stress protective, since THDOC is a potent GABA agonist and has anxiolytic properties similar to benzodiazepines and barbiturates [8]. Consistent with this interpretation, social isolation induces anxiety-like behaviors along with marked decreases in plasma THDOC [33], and plasma THDOC was decreased in the early periods of ethanol withdrawal concomitant with increased anxiety and depression [34]. In addition, we note that there is a non-significant trend toward greater plasma DHA among the psychiatric patient group. This finding has high variability and could potentially be due to differences in smoking, exercise, medication use, metabolism or dietary habits. Providing additional support for a link between lowered o-3 status and increased neurosteroids, decreased plasma EPA% and DHA% were associated with higher 3b,5a-THP among healthy controls, and decreased DHA% was associated with increased DHP among psychiatric patients and across all subjects. DHP is the immediate metabolic precursor of 3a,5a-THP and reportedly lacks the GABA-enhancing anxiolytic potential of its metabolite [11], as does 3b,5a-THP [12]. Thus, little is known as to the clinical implications, if any, of altered DHP and 3b,5a-THP. The observation of lower DHA% predicting higher DHP and 3b,5a-THP should be interpreted cautiously because of the limited sample size available among these groups. These data suggest that there may be an association between readily modifiable o-3 fatty acid status and neurosteroids, of which the implications for HPA function and resulting stress response remain to be elucidated. Of the fatty acids assessed, o-3 fatty acids comprised all but one of the significant relationships with NAS (arachidonic acid, AA% and DHP; 0.42, Po0.05), suggesting a selective role. Decreased EPA% and DHA% predicted increased NAS among psychiatric patients with alcoholism, major depression or both, healthy controls, and among all subjects combined suggesting that this relationship was not specific to psychiatric populations. The finding that THDOC and DHP are negatively correlated with DHA% in plasma was consistent across groups with two exceptions, DHP among healthy controls and THDOC among psychiatric patients. We note that among healthy controls DHP has a sample size of less than 10, making comparisons in this
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group difficult to interpret. Among psychiatric patients it is interesting to speculate as to whether the increased variability in THDOC concentrations contributed to the lack of relationship between THDOC and DHA%. Future studies will be needed to determine if there are group differences in the interaction of o-3 fatty acids and neurosteroids. These preliminary correlational results do not demonstrate causality and should be interpreted with caution, especially in light of the possibility of hidden confounding variables and several limitations of this relatively small, male-only psychiatric population. Future studies should include large enough sample sizes to allow alcoholism and depression to be assessed as separate statistical analyses. We did not have complete and accurate data on the use, dosage or duration of antidepressant medications among psychiatric patients, and thus were unable to assess these variables as covariates. Because of the potentially confounding effects of multiple testing, a more conservative approach may be to assess significance of these results with an a ¼ 0.01, rather than 0.05. Further studies with larger sample sizes will be needed to confirm the associations reported here. Nonetheless, these pilot data, taken together with prior clinical and epidemiological studies, provide justification to conduct placebo-controlled intervention trials to determine if long chain o-3 fatty acids can reduce NAS concentrations while abating HPA hyperactivity.
Acknowledgments This study was funded by the Division of Intramural Clinical and Basic Science Research, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health.
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Prostaglandins, Leukotrienes and Essential Fatty Acids 75 (2006) 309–314 www.elsevier.com/locate/plefa
Relationship between omega-3 fatty acids and plasma neuroactive steroids in alcoholism, depression and controls L.R.G. Nieminena, K.K. Makinoa, N. Mehtaa, M. Virkkunenb, H.Y. Kima, J.R. Hibbelna, a
National Institutes of Health, National Institutes on Alcoholism and Alcohol Abuse, Laboratory of Membrane Biophysics and Biochemistry, Bethesda, MD 20814, USA b Department of Psychiatry, Helsinki University Central Hospital, Helsinki, Finland
Abstract Deficiency in the long-chain o-3 fatty acid, docosahexaenoic acid (DHA) has been associated with increased corticotropin releasing hormone and may contribute to hypothalamic pituitary axis (HPA) hyperactivity. Elevated levels of the neuroactive steroids, allopregnanolone (3a,5a-THP) and 3a,5a-tetrahydrodeoxycorticosterone (THDOC) appear to counter-regulate HPA hyperactivity. Plasma essential fatty acids and neurosteroids were assessed among 18 male healthy controls and among 34 male psychiatric patients with DSM-III alcoholism, depression, or both. Among all subjects, lower plasma DHA was correlated with higher plasma THDOC (r ¼ 0.3, Po0.05) and dihydroprogesterone (DHP) (r ¼ 0.52, Po0.05). Among psychiatric patients lower DHA was correlated with higher DHP (r ¼ 0.60, Po0.01), and among healthy controls lower plasma DHA was correlated with higher THDOC (r ¼ 0.83, Po0.01) and higher isopregnanolone (3b,5a-THP) (r ¼ 0.55, Po0.05). In this pilot observational study, lower long-chain o-3 essential fatty acid status was associated with higher neuroactive steroid concentrations, possibly indicating increased feedback inhibition of the HPA axis. r 2006 Elsevier Ltd. All rights reserved.
1. Introduction A growing body of clinical and epidemiological data suggests that the o-3 fatty acids docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) may be effective in the treatment and or prevention of depressive disorders [1]. Depressive disorders have been associated with excessive responses to stress and hyperactivity of the hypothalamic pituitary adrenal (HPA) axis [2]. However, few studies have examined a putative link between o-3 fatty acids, HPA hyperactivity and stress response. Hibbeln et al. [3] demonstrated that lower plasma levels of DHA predicted higher levels of corticotropin releasing hormone (CRH) in cerebrospinal fluid, suggesting that Corresponding author. NIAAA, 31 Center drive, 31/1B 58, Bethesda, MD 20892, USA. Tel.: +301 435 4028; fax: +301 402 0016. E-mail address: [email protected] (J.R. Hibbeln).
0952-3278/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.plefa.2006.07.012
deficiency in DHA may facilitate excessive stress responses and HPA hyperactivity. In support of this interpretation, DHA deficient rats had exaggerated distress behaviors compared to DHA adequate rats during administration of CRH and were normalized upon restoration of dietary DHA [4]. At least two double-blind placebo-controlled intervention trials with human subjects have demonstrated a stress protective benefit of supplementation with o-3’s. Among Japanese students, supplementation with DHA attenuated stress-induced increases in aggression and hostility [5]. Among stressed university staff, both o-3 and placebo significantly reduced perceived stress, however only the o-3 group was significantly better compared to the no-treatment controls. The latter study had several methodological limitations, including the use of olive oil as placebo, and therefore should be interpreted cautiously [6]. Excessive stress response and HPA hyper-activation may be reflected in concentrations of brain and plasma
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neuroactive steroids (NAS). NAS are produced de novo in the central nervous system from cholesterol and have the ability to rapidly (within seconds) alter neuronal excitability, in contrast to classic gonadal and adrenal steroid hormones acting over several hours to days [7]. 3a-hydroxy ring A-reduced pregnane neurosteroids 3a,5a-tetrahydroprogesterone (3a,5a-THP) and 3a,5atetrahydrodeoxycorticosterone (THDOC) behave similarly to benzodiazepines or barbiturates [8] acting as potent positive modulators of GABAergic neurotransmission. 3a,5a-THP and THDOC are metabolites of the 5a-pregnane steroids, 5a-dihydroprogesterone (DHP) and 5a-dihydrodeoxycorticosterone, which in turn, are metabolites of progesterone (PROG) and deoxycorticosterone, respectively [9]. PROG [10], but not DHP [11] nor stereoisomer 3b,5a-THP [12] have demonstrated the anxiolytic GABA enhancing potential of 3a,5a-THP and THDOC. Stress-induced CRH activation of the HPA axis resulted in elevated NAS concentrations [13] and NAS were elevated in both brain and plasma during stressinducing paradigms [14,15] and in panic disorder [16]. This stress-induced elevation of NAS, particularly 3a,5a-THP and THDOC, has been explained as a homeostatic mechanism to counter-regulate stress response via central and neuroendocrine pathways. 3a,5aTHP and THDOC potentiate GABAA receptor inhibition of the central nervous system [8] and have several anxiolytic properties [17,18] including: counteraction of the down regulated GABA receptor function observed in acute stress [19], decreased dopamine metabolism in prefrontal cortex [20], decreased gene expression of CRH in the hypothalamic paraventricular nucleus [21], and down-regulation of the HPA system [16,19,22]. In support of the proposed feedback inhibition paradigm, exogenous administration of THDOC and 3a,5a-THP attenuated the stress-induced release of CRH [23] and consequent anxiogenic effects [24]. We posit a model of interaction between o-3 essential fatty acid status, NAS and the HPA axis in which low DHA status predicts elevated CRH [3], elevated CRH induces HPA axis hyperactivity [25] and HPA axis hyperactivity results in higher levels of NAS [14]. Thus, in this observational study, we postulated that lower o-3 status would be correlated with higher plasma concentrations of 3a,5a-THP and THDOC.
2. Patients and methods This study population (n ¼ 52) consisted of male Finnish psychiatric patients with alcoholism, major depression, or both (n ¼ 34) and healthy controls without psychiatric disorders (n ¼ 18). As used in this manuscript, the term ‘psychiatric patients’ refers to those individuals with, at minimum, the diagnosis of
alcohol abuse or dependence, or depression, with most patients having both alcoholism and depression. DSMIII diagnosed Schizophrenia and other psychotic disorders were excluded, but comorbidity with other axis I or II disorders was not exclusionary. DSM-III diagnoses and plasma sample collections were preformed as previously described [26]. Subjects were studied under a human research protocol approved by the institutional review board of the National Institutes of Health, Bethesda, MD; the Office for Protection from Research Risks, Bethesda; the University of Helsinki Department of Psychiatry institutional review board, Helsinki, Finland; and the University of Helsinki Central Hospital institutional review board. All subjects provided written informed consent before participating in the study. Individual fatty acids in plasma were assessed [27] and are expressed as a weight percentage (wt%) of total fatty acids. Neurosteroid analysis of plasma was conducted using mass-spectroscopy methods for 3a,5a-THP (n ¼ 51) and THDOC (n ¼ 52), as well as metabolic precursors DHP (n ¼ 22) and PROG (n ¼ 19). We also assessed, d4dihydrotestosterone (DHT) (n ¼ 52) and isopregnanolone (3b,5a-THP) (n ¼ 47) as exploratory measures [28]. For identification and removal of outliers due to analytical error, a modified z-score was generated using the median of absolute deviation about the median (MAD) statistical technique [29]. Bivariate spearman’s rcorrelations were conducted using essential fatty acids and neurosteroids as variables among psychiatric patients (subjects with DSM-III alcohol dependence or abuse, major depression, or both alcoholism and depression), among healthy control subjects (subjects with no DSM-III diagnoses), and lastly among all subjects combined (SPSS release 11.0.1, SPSS Inc., Chicago, IL). In order to reduce errors due to multiple testing, the correlational analysis was limited to the essential fatty acids and background lipid profiles. Correlations between total fatty acids, total saturates, total polyunsaturates, total monounsaturates and neurosteroids were assessed in order to discern effects of individual fatty acids from overall lipid profiles. Independent samples t-tests and Levene’s test for equality of variances were conducted to compare the mean and variance, respectively, in concentrations of NAS and essential fatty acids between psychiatric patients and healthy controls. Logistic regression analyses were used to assess the possible confounding effects of DSM-III axis I or II comorbidity by comparing groups above or below the median split of neurosteroid measures.
3. Results Decreased plasma concentrations of long-chain o-3 fatty acids were correlated with increased plasma
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Plasma THDOC d0/d4
3.0
311
Table 1 Spearman’s r bivariate correlations (r-values and sample sizes shown) of plasma essential fatty acids (wt% total fatty acids) and plasma neurosteroids (ng/mL)
2.5
2.0
1.5
1.0 1.0
1.5
2.0
2.5 3.0 3.5 Plasma DHA (wt %)
4.0
4.5
Fig. 1. Lower plasma docosahexaenoic acid, DHA% (wt% total fatty acids) is correlated with higher plasma concentrations of 3a,5atetrahydrodeoxycorticosterone, THDOC (d0/d4, see methods reference [23]) among healthy controls (r ¼ 0.80, Po0.01) using spearman’s rregression analysis.
concentrations of neurosteroids. Lower plasma concentrations of both DHA% (Fig. 1) and EPA% predicted higher levels of THDOC and the allopregnanolone stereoisomer, 3b,5a-THP among healthy controls. Among psychiatric patients lower plasma DHA% predicting higher plasma DHP was the only significant correlation detected. Among all subjects lower DHA% remained robust in predicting elevated concentrations of both THDOC and DHP (Table 1). Logistic regression analysis indicated that the relationship between THDOC and plasma DHA% remained significant (Wald ¼ 4.05, Po0.05) after adjustment for comorbidity of DSM-III axis I or axis II disorders. In contrast to the inverse correlations found between long-chain o-3 fatty acids and neurosteroids, 18:3n-3 linolenic acid (LNA) was positively correlated with both DHP among all subjects and THDOC among healthy controls. There were no significant relationships found between essential fatty acids and 3a5a-THP, PROG or DHT (data not shown). Total fatty acids, total saturates, total polyunsaturates and total monounsaturates did not predict neurosteroid concentrations (data not shown). Thus, correlational relationships between individual fatty acids and neurosteroids were not an artifact of overall background fatty acid profiles. Mean concentrations of essential fatty acids and neurosteroids did not significantly differ between psychiatric patients and healthy controls, as assessed by independent samples t-tests. The variance in THDOC and PROG concentrations were significantly higher among psychiatric patients compared to healthy controls, as assessed by Levene’s test for equality of variance (Table 2).
THDOCa
3b,5a-THPb
DHPb
All subjects 18:2n-6, LA1 20:4n-6, AA2 18:3n-3, LNA3 20:5n-3, EPA4 22:6n-3, DHA5
n ¼ 52 0.04 0.15 0.05 0.27 0.30
n ¼ 47 0.08 0.06 0.14 0.07 0.08
n ¼ 22 0.40 0.49 0.47 0.06 0.52
Psychiatric patients 18:2n-6, LA 20:4n-6, AA 18:3n-3, LNA 20:5n-3, EPA 22:6n-3, DHA
n ¼ 34 0.04 0.02 0.32 0.03 0.07
n ¼ 31 0.13 0.34 0.05 0.29 0.22
n ¼ 17 0.28 0.39 0.30 0.13 0.60
Healthy controls 18:2n-6, LA 20:4n-6, AA 18:3n-3, LNA 20:5n-3, EPA 22:6n-3, DHA
n ¼ 18 0.03 0.43 0.48 0.83 0.80
n ¼ 16 0.00 0.30 0.12 0.63 0.55
no10 NS NS NS NS NS
Bold text indicates significant correlations. 1 Linoleic acid. 2 Arachidonic acid. 3 Linolenic acid. 4 Eicosapentaenoic acid. 5 Docosahexaenoic acid. a These values were expressed as GC/MS response ratios d0/d4 (see methods reference [23]). b These values were expressed as ng/mL. Indicates Po.05. Indicates Po.01.
4. Discussion and conclusions To our knowledge, this is the first reported association between plasma essential fatty acid status and neurosteroid concentrations in humans or animals. In this pilot observational study, we found that lower plasma concentrations of long-chain o-3 fatty acids were associated with higher plasma concentrations of neurosteroids. Lower DHA% and EPA% were associated with higher concentrations of THDOC among healthy controls. DHA% remained a significant predictor of THDOC among all subjects, however neither DHA% nor EPA% were associated with THDOC among psychiatric patients. This may have been a result of the increased variability in THDOC concentrations among psychiatric patients as compared to healthy controls. The positive correlations between 18:3n-3 LNA and DHP among all subjects and THDOC among healthy controls are unexpected findings in light of the opposite relationship demonstrated between long chain o-3 fatty acids and neurosteroids in this study. This finding should be cautioned by the fact that LNA was present in
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Table 2 Means and standard deviations of essential fatty acids (expressed as wt% of total fatty acids) and neurosteroids (expressed as ng/mL) among psychiatric patients with alcoholism, depression, or both and healthy controls Mean (SD) Psychiatric patients Essential fatty acids (wt%) 18:2n-6, LA 20:4n-6, AA 18:3n-3, LNA 20:5n-3, EPA 22:6n-3, DHA Neurosteroids (ng/mL) 3b,5a-THP DHP 3a,5a-THP PROGa DHT THDOCb,c
Healthy controls
0.37 5.35 0.01 1.42 2.59
(0.19) (1.07) (0.002) (0.80) (0.94)
0.37 5.73 0.01 1.33 2.36
(0.21) (1.21) (0.003) (0.67) (0.76)
0.45 13.19 0.32 0.80 1.57 1.90
(0.21) (8.03) (0.09) (0.55) (0.85) (0.78)
0.46 17.42 0.35 0.80 1.57 1.76
(0.23) (7.86) (0.08) (0.16) (0.82) (0.50)
a Significant between groups difference in Levene’s test for equality of variance (F ¼ 5.079, Po 0.04). b Significant between groups difference in Levene’s test for equality of variance (F ¼ 5.163, Po0.03). c THDOC values expressed as d0/d4 (see methods reference [23]).
low amounts in this study, and secondly, because there are no previous studies we are aware of demonstrating opposing or competing effects for short and long chain o-3 fatty acids. Pisu and Serra [17], in their 2004 review, comment that ‘‘it remains to be determined y whether changes in the concentrations of NAS are a cause of, a risk factor for, or a consequence of mental disorders’’. In terms of the association presented here and its implication for the stress response system, it has been similarly challenging to interpret increased levels of anxiolytic NAS as either beneficial or indicative of HPA hyperactivity. Further, because plasma samples were collected at a single time point it is impractical to determine whether these levels are representative of elevated systemic concentrations or acute elevations as part of the proposed negative feedback mechanism. Thus, lower plasma DHA% associated with higher THDOC concentrations can be interpreted as indicating either stress protection or greater HPA activation. Both interpretations and corresponding evidence are presented below. Elevated NAS may indicate a counter-regulation of excessive hyperactivity of the HPA axis due to excessive stress or increased sensitivity. In support of this interpretation, THDOC was elevated in panic disorder [16] and major depression [30] and was normalized after 50 days of treatment with fluoxetine [31]. This decrease in THDOC may indicate attenuated need for neurosteroid down-regulation of HPA activation due to increased effectiveness of HPA axis negative feedback and
decreased CRH release [17,32]. Thus, the association between lower DHA% and greater concentrations of THDOC reported here may be interpreted as indicating greater HPA activation and homeostatic counter-regulation via elevated NAS levels. This interpretation is cautioned by the absence of other measures of HPA axis function, such as corticosterone and ACTH, and the absence of a significant relationship between essential fatty acid status and 3a,5a-THP. Alternatively, it is possible to postulate that higher THDOC and thus lower plasma DHA% is stress protective, since THDOC is a potent GABA agonist and has anxiolytic properties similar to benzodiazepines and barbiturates [8]. Consistent with this interpretation, social isolation induces anxiety-like behaviors along with marked decreases in plasma THDOC [33], and plasma THDOC was decreased in the early periods of ethanol withdrawal concomitant with increased anxiety and depression [34]. In addition, we note that there is a non-significant trend toward greater plasma DHA among the psychiatric patient group. This finding has high variability and could potentially be due to differences in smoking, exercise, medication use, metabolism or dietary habits. Providing additional support for a link between lowered o-3 status and increased neurosteroids, decreased plasma EPA% and DHA% were associated with higher 3b,5a-THP among healthy controls, and decreased DHA% was associated with increased DHP among psychiatric patients and across all subjects. DHP is the immediate metabolic precursor of 3a,5a-THP and reportedly lacks the GABA-enhancing anxiolytic potential of its metabolite [11], as does 3b,5a-THP [12]. Thus, little is known as to the clinical implications, if any, of altered DHP and 3b,5a-THP. The observation of lower DHA% predicting higher DHP and 3b,5a-THP should be interpreted cautiously because of the limited sample size available among these groups. These data suggest that there may be an association between readily modifiable o-3 fatty acid status and neurosteroids, of which the implications for HPA function and resulting stress response remain to be elucidated. Of the fatty acids assessed, o-3 fatty acids comprised all but one of the significant relationships with NAS (arachidonic acid, AA% and DHP; 0.42, Po0.05), suggesting a selective role. Decreased EPA% and DHA% predicted increased NAS among psychiatric patients with alcoholism, major depression or both, healthy controls, and among all subjects combined suggesting that this relationship was not specific to psychiatric populations. The finding that THDOC and DHP are negatively correlated with DHA% in plasma was consistent across groups with two exceptions, DHP among healthy controls and THDOC among psychiatric patients. We note that among healthy controls DHP has a sample size of less than 10, making comparisons in this
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group difficult to interpret. Among psychiatric patients it is interesting to speculate as to whether the increased variability in THDOC concentrations contributed to the lack of relationship between THDOC and DHA%. Future studies will be needed to determine if there are group differences in the interaction of o-3 fatty acids and neurosteroids. These preliminary correlational results do not demonstrate causality and should be interpreted with caution, especially in light of the possibility of hidden confounding variables and several limitations of this relatively small, male-only psychiatric population. Future studies should include large enough sample sizes to allow alcoholism and depression to be assessed as separate statistical analyses. We did not have complete and accurate data on the use, dosage or duration of antidepressant medications among psychiatric patients, and thus were unable to assess these variables as covariates. Because of the potentially confounding effects of multiple testing, a more conservative approach may be to assess significance of these results with an a ¼ 0.01, rather than 0.05. Further studies with larger sample sizes will be needed to confirm the associations reported here. Nonetheless, these pilot data, taken together with prior clinical and epidemiological studies, provide justification to conduct placebo-controlled intervention trials to determine if long chain o-3 fatty acids can reduce NAS concentrations while abating HPA hyperactivity.
Acknowledgments This study was funded by the Division of Intramural Clinical and Basic Science Research, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health.
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