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Nutritional Neuroscience
An International Journal on Nutrition, Diet and Nervous System
ISSN: 1028-415X (Print) 1476-8305 (Online) Journal homepage: http://www.tandfonline.com/loi/ynns20
Supplementation with macular carotenoids
reduces psychological stress, serum cortisol, and
sub-optimal symptoms of physical and emotional
health in young adults
Nicole Tressa Stringham, Philip V. Holmes & James M. Stringham
To cite this article: Nicole Tressa Stringham, Philip V. Holmes & James M. Stringham (2017):
Supplementation with macular carotenoids reduces psychological stress, serum cortisol, and sub-
optimal symptoms of physical and emotional health in young adults, Nutritional Neuroscience, DOI:
10.1080/1028415X.2017.1286445
To link to this article: http://dx.doi.org/10.1080/1028415X.2017.1286445
Published online: 15 Feb 2017.
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Supplementation with macular carotenoids
reduces psychological stress, serum cortisol,
and sub-optimal symptoms of physical and
emotional health in young adults
Nicole Tressa Stringham 1,2, Philip V. Holmes1, 2, James M. Stringham 2
1
Interdisciplinary Neuroscience Program, Biomedical and Health Sciences Institute, University of Georgia,
Athens, GA 30602, USA,
2
Department of Psychology, University of Georgia, Athens, GA 30602, USA
Purpose: Oxidative stress and systemic inflammation are the root cause of several deleterious effects of
chronic psychological stress. We hypothesize that the antioxidant and anti-inflammatory capabilities of the
macular carotenoids (MCs) lutein, zeaxanthin, and meso-zeaxanthin could, via daily supplementation,
provide a dietary means of benefit.
Methods: A total of 59 young healthy subjects participated in a 12-month, double-blind, placebo-controlled
trial to evaluate the effects of MC supplementation on blood cortisol, psychological stress ratings, behavioural
measures of mood, and symptoms of sub-optimal health. Subjects were randomly assigned to one of three
groups: placebo, 13 mg, or 27 mg /day total MCs. All parameters were assessed at baseline, 6 months, and
12 months. Serum MCs were determined via HPLC, serum cortisol via ELISA, and macular pigment optical
density (MPOD) via customized heterochromatic flicker photometry. Behavioural data were obtained via
questionnaire.
Results: Significant baseline correlations were found between MPOD and Beck anxiety scores (r=−0.28;
P=0.032), MPOD and Brief Symptom Inventory scores (r=0.27; P=0.037), and serum cortisol and
psychological stress scores (r=0.46; P<0.001). Supplementation for 6 months improved psychological
stress, serum cortisol, and measures of emotional and physical health (P<0.05 for all), versus placebo.
These outcomes were either maintained or improved further at 12 months.
Conclusions: Supplementation with the MCs significantly reduces stress, cortisol, and symptoms of sub-
optimal emotional and physical health. Determining the basis for these effects, whether systemic or a
more central (i.e. brain) is a question that warrants further study.
Keywords: Lutein, Zeaxanthin, Macular pigment, Stress, Cortisol, Anxiety, Depression, Health
Introduction
The basis for all stress responses is the disruption of
some homeostatic set point, be it physical, physiologi-
cal, or psychological.
1
Physiologically, stress is associ-
ated with activation of autonomic and endocrine
systems,
2,3
which involves limbic, hypothalamic,
and brainstem circuits.
4
Chronic activation of these
systems may manifest as anxiety disorders
4,5
or other
stress-related disorders, such as depression.
6
Indeed,
the link between anxiety and chronic activation of
the limbic structures, such as the amygdaloid
complex, has been well established.
7
Psychologically,
the basis of anxiety and depression may involve
heightened vulnerability to stress or the inability to
cope with life stressors
8
and appears to be mediated
by dysregulation in cortico-limbic circuitry.
9
The convergence of physiological, psychological,
and neurological data in the case of stress, anxiety,
and depression is compelling. In fact, the basis for the
Depression Anxiety Stress Scales 21 (DASS-21)
10
is
the ‘tripartite model,’in which anxiety and depression
are both related to psychological stress: Anxiety arises
out of physiological hyperarousal, whereas depression
arises from low positive affectivity, both being impacted
by the negative affect brought on by stress. It appears,
therefore, that although anxiety and depression are
somewhat discrete phenomena, they both share a
common root in psychological stress .
5,11
This idea is
further supported by comorbidity rates exceeding
50%.
12
Susceptibility to stress could, therefore, be
Correspondence to: Nicole Tressa Stringham, Interdisciplinary
Neuroscience Program, Biomedical and Health Sciences Institute and
Department of Psychology, University of Georgia, Athens, GA 30602, USA
Email: ntwood@uga.edu
©2017InformaUKLimited,tradingasTaylor&FrancisGroup
DOI 10.1080/1028415X.2017.1286445 Nutritional Neuroscience 2017 1
considered to be a risk factor for those conditions, such
as anxiety and depression, that appear to result from
excessive psychological stress.
It has been suggested that dietary differences could
modulate susceptibility to stress.
13
Benton
14
noted
that effects of minor nutritional deficiencies would
manifest first as sub-clinical disruption of brain func-
tion, given the complexity and metabolic demands of
the brain. In general support of this idea, there have
been recent human and animal studies that report
stress-reducing effects of supplementation of specific
nutrients, such as curcumin,
15
alpha tocopherol,
16
and docosahexaenoic acid (DHA).
17
In each case, sup-
plementation appears to lead to reduced psychological
stress and physiological parameters of stress (e.g.
blood cortisol). Additionally, Long and Benton
18
con-
ducted a meta-analysis of studies on the effects of
vitamin and mineral supplementation on stress and
mood in sub-clinical populations, and found a
general trend towards stress reduction and improve-
ment in mood.
A recent report by El Ansari et al.
19
on a large
(n=3706), generally healthy population of college-
aged adults examined, via survey, dietary patterns and
stress /depressive symptoms. They found a significant
relationship between consumption of healthy foods
(fresh fruits, salads, and cooked vegetables) and
reduced perceived psychological stress. Conversely,
consumption of ‘junk’food was associated with
increased perceived psychological stress. Although
these findings were correlational, there is a physiologi-
cal rationale to account for how healthy foods may
reduce psychological stress: consumption of antioxi-
dants. It has been shown that systemic oxidative stress
is induced by psychological stress (e.g. in medical stu-
dents
20
), and it appears that reduction of systemic oxi-
dative stress significantly reduces indicators of
psychological stress (via alpha-tocopherol adminis-
tration;
21
and by lutein
22
). Of particular relevance to
the present study, Yajima et al.
22
reported that lutein
(L) supplementation produced an anxiolytic-like
effect in mice exposed to constant illumination stress.
Taken together, these findings suggest a role for
dietary antioxidants in reducing psychological stress.
Based on the idea that any form of homeostatic upset
will produce a similar stress response in the body,
1
it is
perhaps the case that a situation, favouring oxidative
stress within the body, may produce an ‘alarm state’,
which may ultimately be interpreted psychologically
as uneasiness, or stress. Although the specific neurophy-
siological mechanisms are undoubtedly complex, we
believe this general idea to be plausible, based on
what is known in the literature on the matter.
Carotenoids, such as L, comprise a fairly large pro-
portion of dietary antioxidants for humans with a
reasonably healthy diet that includes daily
consumption of fruits and vegetables.
23
Along with
L, two other yellow-orange carotenoids, zeaxanthin
(Z), and meso-zeaxanthin (MZ) are deposited in rich
concentration in the central retina, where they form
the macular pigment (MP).
24
MP is most dense in
the metabolically intense central retina (fovea),
where its powerful antioxidant
25
and high-energy
short-wave light filtration properties
26
appear to
protect the macula from acute damage,
27
protect
against cumulative damage resulting in age-related
macular disease,
28
and maintain visual sensitivity
over a lifetime.
29
MP is strictly derived via diet, and
so a person’s level of MP is dependent upon his or
her consumption of foods that contain these caroten-
oids; for example, dark leafy-green vegetables, such
as kale and spinach, are excellent sources of L.
30
L
and Z also accumulate in the brain,
31,32
where they
may influence cognitive performance, especially in
aged individuals.
33–35
In a manner apparently similar
to the retina, L and Z cross the blood–brain barrier
and accumulate in the brain regions that maintain rela-
tively high metabolism (e.g. frontal and occipital
lobes, and hippocampus), and are therefore at higher
risk for oxidative stress and inflammation.
36
Importantly, MPOD has been shown to be signifi-
cantly correlated with brain levels of L and Z,
32
which suggests similar mechanisms of uptake, and
supports the idea that there is preferential deposition
of these powerful antioxidants /anti-inflammatories
in neural tissues that maintain high metabolism and
therefore concomitant oxygen tension and potential
for oxidative stress and inflammation.
There were two goals of the present study: (1) To
determine, in healthy young adults, the relationship
at baseline between MPOD and psychological stress
level, serum cortisol, and symptoms of sub-optimal
emotional and physical health, and (2) To determine
the effect of 12 months’L, Z, and MZ supplemen-
tation on the aforementioned parameters. Once depos-
ited in retinal tissue, L and Z (the two primary dietary
components of macular pigment
37
) are quite stable in
the absence of high oxidative stress, e.g. such as that
brought on by smoking
38
or diabetes.
39
Therefore, a
person’s macular pigment level is generally thought
to reflect his or her lifelong consumption of L and
Z. The baseline assessment of MPOD and psychologi-
cal stress, and physical /emotional health status
would thereby enable the analysis of potential cumu-
lative effects of diet on these outcome parameters. In
contrast, the 12-month supplementation trial enabled
the analysis of potential acute effects of MC
supplementation.
Methods
Fifty-nine subjects participated in this 12-month,
double-blind, randomized, placebo-controlled
Stringham et al. Macular carotenoids and stress
Nutritional Neuroscience 2017
2
supplementation trial. Subjects were generally healthy,
college-aged (18–25, mean =21.5 years; 27 males /32
female) non-smokers with a BMI <27. Subjects were
instructed to maintain their current diet; those who
were planning on changing their diet ( for whatever
reason) were excluded from consideration for the
trial. In consideration of macular pigment testing, all
subjects had uncorrected or contact lens-corrected
visual acuity of 20/20 or better in the test (right) eye,
and had no current or previous history of ocular path-
ology. Subjects were recruited from the population of
students at the University of Georgia in Athens,
Georgia. Informed consent was obtained from each
subject and the study adhered to the tenets of the
Declaration of Helsinki. The study was approved by
the Institutional Review Board of the University of
Georgia.
Several parameters were assessed over the course of
the study, including retinal status of MCs, serum cor-
tisol, serum lutein, serum zeaxanthin isomers.
Symptoms of sub-optimal health, psychological
health, and emotional health were assessed via ques-
tionnaire (see Table 1for a summary of questionnaires
used in the study and order of administration). All
measures were taken at baseline, 6 months, and 12
months. Laboratory visits included (in order): blood
draw, questionnaire completion, and vision testing.
Macular carotenoid supplementation
Subjects were randomly assigned to one of three
groups: placebo, n=10; 13 mg/day MC, n=24; or
27 mg/day MC. Pills were brown coloured, soft
gelatin capsules, with L, Z, and MZ suspended in saf-
flower oil. Independent analysis indicated that the
13 mg supplement contained 10.86 mg lutein /
2.27 mg zeaxanthin isomers, and the 27 mg sup-
plement contained 22.33 mg lutein /4.70 mg zeax-
anthin isomers. Placebos contained no L or Z
isomers, only safflower oil. Z and MZ were found in
roughly equal amounts in the active supplements. All
reported values were within ±5% variability.
Subjects were instructed to ingest one pill with a
meal ( preferably lunch or dinner) every day.
Compliance was ensured with weekly phone calls
and pill counts.
Measurement of macular pigment optical
density (MPOD)
The concentration of MCs in the central retina
(MPOD) was assessed with a non-invasive, perceptual
task called heterochromatic flicker photometry (HFP).
A densitometer (Macular Metrics Corp., Rehoboth,
MA) described by Wooten et al.
40
was used for this
purpose. The densitometer, detailed measurement pro-
cedures, and the principle of HFP have been fully
described in earlier publications.
41,42
Briefly, subjects
are presented with two superimposed lights that are
temporally alternated in square-wave counterphase.
This gives the subject an impression on a flickering
disc of light. The peak (550 nm) of the spectral compo-
sition of one of the lights is chosen to bypass the
absorption of MP, and the other (460 nm) is strongly
absorbed by MP. The subject’s task is to adjust the
relative radiance of the two lights until a percept of
no flicker is achieved. All other factors being equal,
a subject that requires more short-wave (i.e. 460 nm)
relative to middle-wave (i.e. 550 nm) light to achieve
null flicker has higher MPOD. This task is performed
for the locations of interest within the fovea, which
presumably contain MP, and for a reference location
in the parafovea that does not (about 7° eccentricity).
To obtain a measure of MPOD at a given test locus,
the logarithmic ratio of short- to middle-wave radiance
(for null flicker) at the reference location is subtracted
from the corresponding logarithmic ratio found at the
test locus.
Blood collection
Fasting blood was collected between 9 am and 11 am,
by a licensed phlebotomist, at baseline, 6-month, and
12-month visits. Subjects’whole blood was collected
into a serum separator vacutainer tube (SST) via veni-
puncture. Blood was allowed to clot for 30 minutes at
room temperature before centrifugation for 15 minutes
at 1000 ×g. Serum was then removed and stored in
microvials at −20° C until analysis.
High-performance liquid chromatography
(HPLC)
Sample extractions and analyses were completed
under yellow light. Serum proteins were precipitated
with an equal volume of ethanol (1% BHT), contain-
ing the internal standard, trans-β-apo-8′-carotenal.
After centrifugation, samples were extracted three
times with n-hexanes, mixing, and centrifugation.
Organic layers were pooled and evaporated to
dryness with nitrogen and re-suspended in the
mobile phase. An Agilent 1200 series HPLC system,
consisting of a quaternary pump with degasser, auto-
sampler, thermostated column compartment, UV–vis
diode array detection (DAD) with standard flow
cell, and 3D ChemStation software (Agilent
Technologies, Santa Clara, CA, USA), was employed
for the chromatography. A reversed-phase YMC C30
column (4.6 ×250 mm, 5-μm particle size) was uti-
lized. A stepwise elution consisting of mobile phase
A (95% methanol) and mobile phase B (methyl tert-
butyl ether) from 15 to 85% B over a 27-minute
period at a flow rate of 1 mL/min was employed. A
volume of 100 μL was injected for each of the serum
samples. Detection wavelengths were λ=447 nm (L)
and 450 nm (Z isomers).
Stringham et al. Macular carotenoids and stress
Nutritional Neuroscience 2017 3
Enzyme-linked immunosorbent assay (ELISA)
Serum was diluted and processed according to the
manufacturer’s instructions for the Parameter
Cortisol Human ELISA kit (KGE008, R&D
Systems, Minneapolis MN, USA). Wells were read at
450 nm (MiniReader MR590, Dynatech Instruments,
Inc, Santa Monica CA, USA), averaged across dupli-
cates, and a curve of best fit was used to calibrate to
standards. Cortisol concentration data are reported
as ng/mL. All coefficient of variability values were
under 10%.
Psychological stress measure
Subjects’psychological stress level was assessed via
questionnaire with the 9-item Psychological Stress
Measure (PSM-9).
43
Brief symptom inventory
Subjects’current psychological distress was assessed
with the Brief Symptom Inventory (BSI),
44
a 53-
item, self-report instrument developed from the
longer SCL-90-R.
Beck anxiety inventory
Subjects’symptoms of anxiety were assessed with the
Beck Anxiety Inventory (BAI),
45
a 21-item self-
report instrument that is validated for measuring the
severity of anxiety.
Beck depression inventory
Subjects’symptoms of depression were assessed with
the Beck Depression Inventory (BDI),
46
a 21-item
self-report instrument that is validated for measuring
the severity of depression.
General health status
The number of physical symptoms of sub-optimal
health was determined via self-report questionnaire,
using the 25-item Suboptimal Health Status
Questionnaire (SHSQ-25).
47
Statistical analysis
Graphs and statistical analysis, including descriptive
statistics, Pearson product-moment correlations,
dependent-samples t-tests, and Repeated-Measures
ANOVA were generated using Origin software
(Northampton, MA, USA). Statistical significance
was determined at the P=0.05 level. The number of
subjects required to detect effects (if present) was cal-
culated via power analysis, which was based on a
20% change in the composite outcome measure of
psychological stress /cortisol, and assumed a
placebo group with n=10.
Results
At baseline, significant correlations were determined
between MPOD and BAI scores (r=−0.28; P=
0.032 –see Table 2), MPOD and BSI scores
(r=−0.27; P=0.037 –see Table 2), and between
serum cortisol and PSM-9 scores (r=0.46; P<0.001
–see Fig. 1). Although not statistically significant,
marginal correlations were determined at baseline for
MPOD and serum cortisol (r=−0.202; P=0.124),
MPOD and psychological stress (r=−0.218; P=
0.10), and MPOD and symptoms of sub-optimal
health (r=−0.22; P=0.092). See Table 2for a
summary of baseline and supplementation effects for
all behavioural measures.
After 6 months of MC supplementation, repeated-
measures ANOVA revealed that there were no signifi-
cant beneficial changes from baseline in any parameter
for the placebo group. At 6 months, however, serum
cortisol was found to increase significantly from base-
line. This change did not persist, and returned to base-
line levels at 12 months (see Fig. 2). For the 13 mg/day
group however, we found that MPOD (P<0.001) was
significantly higher (see Fig. 2), and serum cortisol
(P<0.001 –see Fig. 2), BSI scores (P=0.005), and
number of sub-optimal health symptoms (P=
0.0012) were significantly lower compared to baseline.
The 27 mg/day group was found to significantly
increase in MPOD (P<0.001 –see Fig. 2), and
Table 1 Summary of self-report questionnaires used during the course of the study
Instrument Items Outcome measure
Range of
scores
Cronbach’s
α
Test/retest
reliability Published Order
PSM-9 Psychological Stress
Measure 9
9 Stress in general
population
9–72 0.95 0.68–8 Lemyre et al.
43
5
BSI Brief Symptom
Inventory
53 Current
psychological
distress
0–212 0.71-0.85 0.68–91 Derogatis and
Melisaratos
44
1
BAI Beck Anxiety
Inventory
21 Severity of anxiety 0–63 0.92 0.75 Beck et al.
45
2
BDI Beck Depression
Inventory
21 Severity of
depression
0–63 0.86 0.93 Beck et al.
46
3
SHSQ-25 Suboptimal Health
Status
Questionnaire
25 Suboptimal Health
Status
25–125 0.93 0.89–98 Yan et al.
47
4
Stringham et al. Macular carotenoids and stress
Nutritional Neuroscience 2017
4
Table 2 Relation of each self-report measure (PSM-9, BSI, BAI, and SHSQ-25) to MPOD at baseline, and descriptive statistics for each time point as a function of MC dose
Measure Psychological Stress Measure (PSM-9) Brief Symptom Inventory (BSI) Beck Anxiety Inventory (BAI)
Baseline relation to MPOD r=−0.218; P=0.10 r=−0.27; P=0.037 r=−0.28; P=0.032
Time point Baseline 6 months 12 months Baseline 6 months 12 months Baseline 6 months 12 months
Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD
0mg/day MC 26.7 6.43 29.1 5.07 30.1 7.39 18.1 14.22 17.2 13.27 17.5 14.18 8.7 5.89 8.4 6.2 8.6 6.08
13 mg/day MC 31.83 7.38 31.38 6.64 26.96
a
5.25 23 11.07 17.96
a
11.34 12.92
a,b
9.74 7.54 5.36 6.54 4.89 4.33
a,b
3.69
27 mg/day MC 31.44 10.24 27.76
a
6.73 27.4
a
7.67 27.4 18 20.12
a
14.07 13.44
a,b
12.22 6.48 5.87 4.32
a
4.29 2.96
a,b
3.42
Measure Beck Depression Inventory (BDI) Suboptimal Health Status Questionnaire (SHSQ-25)
Baseline relation to MPOD r=0.078; P0.671 r=0.22; P=0.092
Time point Baseline 6 months 12 months Baseline 6 months 12 months
Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD
0mg/day MC 5.3 8.01 5.2 11.77 5.2 13 30.3 7.93 29.9 4.89 30.4 7.82
13 mg/day MC 4.83 3.57 3.79 3.5 3.42 4.03 37.79 7.92 34
a
7.11 32.63
a,b
7.41
27 mg/day MC 4.2 4.03 3.36 3.32 2.4
a
3.18 37.76 10 34.64
a
9.23 33.28
a
7.49
a
P<0.05 compared to baseline.
b
P<0.05 compared to 6 months.
Stringham et al. Macular carotenoids and stress
Nutritional Neuroscience 2017 5
decrease significantly for the BSI (P=0.009 –see
Table 2), BAI scores (P<0.001 –see Table 2), psycho-
logical stress (P=0.05 –see Fig. 2), serum cortisol
(P=0.01 –see Fig. 2), and number of sub-optimal
health symptoms (P<0.001 –see Table 2).
There were no significant changes from baseline
determined for any measure in the placebo group at
12 months. For the 13 mg/day group, a significant
increase from 6 months to 12 months was found for
MPOD (P<0.001 –see Fig. 2), and significant
decreases were determined for BSI scores (P=0.002
–see Table 2), BAI scores (P=0.013 –see Table 2),
psychological stress (P=0.018 –see Fig. 2), serum
cortisol (P=0.0037 –see Fig. 2), and number of
sub-optimal health symptoms (P=0.007 –see
Table 2). Comparing 6- and 12-month measures, the
27 mg/day MC increased significantly in terms of
MPOD (P=0.0087 –see Fig. 2), and decreased sig-
nificantly for the BSI (P=0.013–see Table 2), the
BAI (P=0.038–see Table 2), serum cortisol (P=
0.037 –see Fig. 2), and symptoms of sub-optimal
health (P=0.041–see Table 2). A significant decrease
in scores on the PSM-9 and BDI was determined at 12
months for the 27 mg/day MC group when compared
to baseline (P=0.05 and 0.025; see Fig. 2and Table 2,
respectively).
Repeated-measures ANOVA determined that serum
L and Z isomers increased significantly after 6 months
of supplementation for both active supplement groups
(P<0.001; see Figs 3and 4, respectively) versus
placebo, and maintained an apparent steady-state
Figure 1 Baseline correlation between serum cortisol (ng/
mL) and PSM-9 scores. Dotted line least-squares fit to data.
Figure 2 MPOD, Serum cortisol, and PSM-9 scores for all groups, as a function of time in the study. Reported as percent change
from baseline. Means ±SEM plotted for baseline, 6- month, and 12- month measures.
Figure 3 Serum lutein concentration as a function of time in
the study, for all groups. Means ±SEM for baseline, 6-month,
and 12- month measures.
Stringham et al. Macular carotenoids and stress
Nutritional Neuroscience 2017
6
level at 12 months. As can be seen in Fig. 3, the steady-
state L serum level was found to be roughly 2.25 μg/
mL for the 13 mg/day MC group, and 3.25 μg/mL
for the 27 mg/day MC group. The placebo group
remained at a concentration of approximately
0.25 μg/mL throughout the 12-month study period.
The change in serum concentration of Z isomers was
also found to be significant at 6 months (P<0.001)
and, as in the case of L, maintained the 6-month
level through 12 months (see Fig. 4). From Fig. 4,it
can be seen that the Z isomer steady-state level for
the 13 mg/day MC group was 0.37 μg/mL, and
0.47 μg/mL for the 27 mg/day MC group. The
placebo group remained at a concentration of
roughly 0.10 μg/mL throughout the study.
As noted above, MPOD increased significantly
from baseline at 6 months, and from 6 months to 12
months in both 13- and 27- mg/day MC groups (see
Fig. 2). Despite double the amount of carotenoid in
the 27 mg/day MC group’s supplement (27 mg vs.
13 mg), retinal response across the study period was
virtually identical for both groups.
In terms of change in measures over the 12-month
study period, the relationship between increases in
MPOD and decreases in serum cortisol was found to
be significant (r=−0.454; P<0.001; see Fig. 5).
This same relationship was found for psychological
stress, where increases in MPOD were significantly
related to reduced PSM-9 scores (r=0.398; P=
0.002 –see Fig. 6). This kind of relationship with
change in MPOD was not found for the other behav-
ioural measures. There were, however, nearly signifi-
cant relationships determined between the change in
symptoms of sub-optimal health and psychological
stress (P=0.08), and cortisol (P=0.07), respectively.
The finding of a relationship between cortisol and
sub-optimal health symptoms was also determined
by Yan et al.,
48
using the same scale (SHSQ-25) as
the present study.
Discussion
Given the results of this study, it appears that there is a
significant role for diet, specifically the MCs, in redu-
cing stress and improving symptoms of both physical
and emotional health. Although similar improvements
were determined for all outcome measures over the
course of the study in both active supplement groups,
measures of stress (serum cortisol and PSM-9) were
the only measures that were related directly to increases
in MPOD. The mechanism for the stress reduction
effects appears, therefore, to be related to the accumu-
lation of the MCs in the retina (and presumably the
brain). Given the biochemical properties of the MCs,
a plausible mechanism for this finding may involve
the direct antioxidant and anti-inflammatory action
within specific neural tissues that ultimately leads to
production of stress-related hormones. Additionally
(as suggested in the Introduction section), it could be
that the presumed reduction of systemic or local
neural oxidative stress via L, Z, and MZ supplemen-
tation effectively produced lower physiological stress,
which led to reduced psychological stress. As for the
measures related to mood (BAI, BDI, BSI) and physical
health (SHSQ-25), there was a clear benefit of sup-
plementation with the MCs, but the improvements
were not directly related to the change in MPOD. If
retinal /brain deposition of L, Z, and MZ, does not
account for the improvements in physical /emotional
health symptoms, then it would seem plausible that
changes in systemic (i.e. serum) carotenoid levels
could explain the effects. But changes in serum caroten-
oid levels were not directly related to changes in
physical /emotional health symptoms. There are
several possible reasons for this. It may be that differ-
ences in systemic oxidative stress and inflammation
among participants served to modify serum carotenoid
levels in such a way as to mask any relationship
between mood /health scales and serum carotenoid
Figure 4 Serum zeaxanthin concentration as a function of
time in the study, for all groups. Means ±SEM for baseline,
6-month, and 12- month measures
Figure 5 Change in serum cortisol over the 12-month study
period as a function of change in MPOD over the same time
period. Dotted line least-squares fit to data.
Stringham et al. Macular carotenoids and stress
Nutritional Neuroscience 2017 7
concentration. Alternatively, retinal and brain caroten-
oid transport efficiency differs substantially between
individuals,
24
and this may have impacted serum
levels in a non-systematic way. Additionally, serum
carotenoid concentrations for a supplementation trial,
such as the present study, tend to saturate by about 12
weeks of daily supplementation.
49,50
Therefore, corre-
lations involving analysis of change would be limited,
due to the fact that our subjects probably reached
serum saturation long before their second measure (6
months). Lastly, and perhaps most parsimoniously,
the reduced psychological stress levels seen in our treat-
ment groups may have served to reduce symptoms of
anxiety, depression, and sub-optimal health symptoms
in a manner that is not related to our serum measures
of either cortisol or carotenoids. Whatever the case,
the effects found in our study are consistent with
either systemic or neural tissue elevation of MCs.
Based on our supplementation data for serum and
MPOD, it appears that deposition in neural tissues
requires a consistent, relatively elevated serum concen-
tration of L, Z, and MZ –that the placebo group
(which did not exhibit improvements in any outcome
parameter) did not increase in either serum or MPOD
speaks convincingly to this point.
MPOD response to supplementation in both active
supplement groups was very similar, despite the
higher dose supplement containing roughly double
the amount of carotenoids. Serum response was
about 30% higher for the 27 mg/day MC group,
which indicates that the additional carotenoids either
remained higher in serum, or were deposited in other
tissues, such as skin or adipose tissue. Another possi-
bility is that, despite random assignment, participants
assigned to 13 mg/day MC group tended to (overall)
respond more favourably in the retina, compared to
those in 27 mg/day MC group. Variability in retinal
response to supplementation with retinal carotenoids
has been shown previously.
24
Moreover, retinal
response typically is found to increase somewhat line-
arly with increased dose.
24,50
Although the results of
these previous studies are difficult to reconcile with
the present results, retinal response was nevertheless
robust in both active supplement groups.
Taken together, the cross-sectional and supplemen-
tation-trial data make a strong case for the involve-
ment of L, Z, and MZ in psychological stress levels
and physical /emotional health symptoms. In terms
of stress and physical health, given the well-established
relationship between psychological stress and compro-
mised immune function, it is quite possible that the
reduction in sub-optimal health symptoms over the
period of the study is an effect subsequent to the
reduction in stress seen with supplementation.
Support for this possibility is provided by the baseline
relationship between psychological stress level and
number of sub-optimal health symptoms (r=0.415;
P=0.0011 –see Fig. 7). The nearly significant
relationships between change in symptoms of sub-
optimal health and change in both serum cortisol
(r=0.24; P=0.07), and change in psychological
stress (P=0.08) over the study period is further evi-
dence for this idea.
Cortisol is the effector hormone of the hypothala-
mic-pituitary-adrenal (HPA) axis (the ‘stress’axis),
and widely considered to be an excellent physiological
marker for psychological stress.
2
We determined a
marginally significant relationship between MPOD
and serum cortisol at baseline (P=0.124), and a
strongly significant relationship between change in
MPOD and change in serum cortisol for all subjects
over the study period (r=0.454; P<0.001). As
noted above, however, the effect of cortisol reduction
was not related to serum carotenoid response. In
other words, a subject’s blood response was somewhat
Figure 6 Change in PSM-9 score over the 12-month study
period, as a function of change in MPOD over the same time
period. Dotted line least-squares fit to data.
Figure 7 Baseline SHSQ-25 scores (higher scores=greater
number of sub-optimal health symptoms), as a function of
baseline PSM-9 scores. Dotted line least-squares fit to data.
Stringham et al. Macular carotenoids and stress
Nutritional Neuroscience 2017
8
independent of cortisol reduction over the study
period. This apparent discrepancy could be explained
by the fact that the effect of stress reduction is driven
by the neural (presumably brain) deposition of these
carotenoids, and that this deposition may lead to
modulation of the HPA axis. The mechanism for this
could involve a local reduction of inflammation,
which has been previously shown to be closely linked
with stress.
51
Additionally, corticosteroids generated
from the stress response decrease the effectiveness of
endogenous antioxidant systems.
52
Because dietary
antioxidants, such as L and Z, supplement endogen-
ous antioxidant systems, such as glutathione and
superoxide dismutase,
53
it could be that local
reduction of both oxidation and inflammation (via L
and Z) plays a role in the cortisol and stress reduction
effects found in our study.
Serum cortisol concentration increased signifi-
cantly from baseline in the placebo group at
6months,andthenreturnedtobaselinelevelsat
12 months (see Fig. 2). This may be due to a seasonal
effect of variation in serum cortisol,
54
as the 6-month
measure fell within the months when cortisol is
reportedly elevated in healthy subjects. Nevertheless,
both supplementation groups’serum cortisol
decreased at both 6 and 12 months, suggesting an
overall long-term effect of L, Z, and MZ supplemen-
tation on serum cortisol.
That such specific nutrients are able to confer sub-
stantial and meaningful effects over a relatively short
time period could be interpreted in several ways.
First, it could be that human beings were meant to
consume significantly more foods (e.g. leafy-green veg-
etables) that contain these carotenoids than is cur-
rently the case.
55
Our serum data from the baseline
measure of our entire sample are indicative of low
intake (overall) of L, Z, and MZ. Indeed, data from
the National Health and Nutrition Examination
Survey (NHANES, 2003, as cited in Johnson
et al.
55
) indicate that Americans in the age range
(19–30 years.) corresponding to our subjects’general
age range consume a paltry 1.5 mg of L and Z daily.
At such low levels of consumption, the body may
use any available carotenoid for more immediate, sys-
temic purposes (e.g. inflammation, or oxidative stress)
rather than depositing it in tissues such as the retina or
brain (where our data suggest stress-reducing effects).
Perhaps, our intervention simply brought serum
MCs, MPOD (and brain carotenoid) levels up to a
point that facilitated relatively ‘normal’function. In
terms of psychological stress and cortisol, this point
can be argued not only from the standpoint of the
intervention but also from the cross-sectional analysis,
where subjects with higher levels of MPOD were found
to have marginally significantly lower psychological
stress levels and serum cortisol (see Table 2).
In addition to low baseline dietary intake of L, Z,
and MZ, another consideration for our findings is
the level of stress experienced by the study partici-
pants. Our subjects were young and healthy, but never-
theless experienced relatively high levels of
psychological stress, and reported a fair number of
sub-optimal physical and emotional health symptoms.
Stressful situations most often noted by subjects were
struggles with coursework (e.g. worrying about
grades), relationship problems, and worrying about
money. It may be the case that college students experi-
ence higher-than-average stress (and subsequent nega-
tive health symptoms) than the overall population. If
the MCs serve a function of reducing serum cortisol
and stress, then it follows logically that supplemen-
tation in individuals experiencing relatively high
levels of stress would produce acute benefits.
As with any study, caution should be exercised
before extending these results to other populations.
Although there are advantages in terms of experimen-
tal control to studying a fairly homogeneous group, it
can limit external validity. Our subjects were similar
along many dimensions, including age, BMI, edu-
cation level, and current life status (i.e. college
student); our findings may therefore hold true for
this group, but may not extend to others.
Additionally, it may be tempting to interpret the be-
havioural data (BAI, BDI, and BSI) as evidence for
the ability of MC supplementation to reduce anxiety
or depression. None of our subjects were diagnosed
with depression or an anxiety disorder. Our results
simply suggest that supplementation with the MCs
can reduce symptoms (however few) of anxiety and /
or depression. In order to address other populations
(e.g. clinically anxious or depressed individuals),
additional studies would need to be conducted. In
the future, we hope to investigate the effects character-
ized in the present study in subjects with different life-
style and dietary habits, in different age groups, and
different socioeconomic backgrounds.
Disclaimer statement
Contributors Author N.T.S. contributed to experimen-
tal design, data collection, blood collection and analy-
sis, data analysis, and writing of the manuscript.
Author P.V.H. contributed to experimental design
and manuscript writing. Author J.M.S. contributed
to experimental design, data collection, data analysis,
and writing of the manuscript.
Funding Omniactive Health Technologies, Inc.
Conflict of interest None.
Ethics approval The study was approved by the
Institutional Review Board of the University of
Georgia.
Stringham et al. Macular carotenoids and stress
Nutritional Neuroscience 2017 9
ORCID
Nicole Tressa Stringham http://orcid.org/0000-
0003-1721-7882
James M. Stringham http:// orcid.org/0000-0002-
1476-1084
References
1 McEwen BS, Wingfield JC. What’s in a name? Integrating
homeostasis, allostasis and stress. Horm Behav 2010;57(2):
105–11.
2 Chrousos GP. Stress, chronic inflammation, and emotional and
physical well-being: concurrent effects and chronic sequelae. J
Allergy Clin Immunol 2000;106(5 Suppl):S275–91.
3Al’absi M, Arnett DK. Adrenocortical responses to psychologi-
cal stress and risk for hypertension. Biomed Pharmacother.
2000;54(5):234–44.
4 Mitra R, Sapolsky RM. Acute corticosterone treatment is suffi-
cient to induce anxiety and amygdaloid dendritic hypertrophy.
Proc Natl Acad Sci USA 2008;105(14):5573–8.
5 Mitra R, Vyas A, Chatterjee G, Chattarji S. Chronic-stress
induced modulation of different states of anxiety-like behavior
in female rats. Neurosci Lett 2005;383(3):278–83.
6 Rainnie DG, Bergeron R, Sajdyk TJ, Patil M, Gehlert DR,
Shekhar A. Corticotrophin releasing factor-induced synaptic
plasticity in the amygdala translates stress into emotional dis-
orders. J Neurosci 2004;24(14):3471–9.
7 Adamec RE, Blundell J, Burton P. Neural circuit changes med-
iating lasting brain and behavioral response to predator stress.
Neurosci Biobehav Rev 2005;29(8):1225–41.
8 Cole DA, Peeke LG, Martin JM, Truglio R, Seroczynski AD. A
longitudinal look at the relation between depression and anxiety
in children and adolescents. J Consult Clin Psychol 1998;66(3):
451–60.
9 Duman RS, Monteggia LM. A neurotrophic model for stress-
related mood disorders. Biol Psychiatry 2006;59(12):1116–27.
10 Henry JD, Crawford JR. The short-form version of the
depression anxiety stress scales (DASS-21): construct validity
and normative data in a large non-clinical sample. Br J Clin
Psychol 2005;44:227–39.
11 Pittenger C, Duman RS. Stress, depression, and neuroplasticy: a
convergence of mechanisms. Neuropsychopharmacology
2008;33(1):88–109.
12 Kessler RC, Nelson C, McGonagle KA, Liu J, Swartz M, Blazer
DG. Comorbidity of DSM-III-R major depressive disorder in
the general population: results from the US National
Comorbidity Survey. Br J Psychiatry 1996;168(suppl 30):17–30.
13 Tannenbaum BM, Tannenbaum GS, Anisman H. Impact of life-
long macronutrient choice on neuroendocrine and cognitive
functioning in aged mice: differential effects in stressor-reactive
and stressor-resilient mouse strains. Brain Res 2003;985(2):
187–97.
14 Benton D. To establish the parameters of optimal nutrition do
we need to consider psychological in addition to physiological
parameters? Mol Nutr Food Res 2013;57(1):6–19.
15 Sciberras JN, Galloway SD, Fenech A, Grech G, Farrugia C,
Duca D, et al. Theeffectofturmeric(Curcumin)supplemen-
tation on cytokine and inflammatory marker responses following
2 hours of endurance cycling. J Int Soc Sports Nutr. 2015;12(1):
5–14.
16 Lodhi GM, Latif R, Hussain MM, Naveed AK, Aslam M.
Effect of ascorbic acid and alpha tocopherol supplementation
on acute restraint stress induced changes in testosterone, corticos-
terone and nor epinephrine levels in male Sprague Dawley rats. J
Ayub Med Coll Abbottabad 2014;26(1):7–11.
17 Keenan K, Hipwell AE, Bortner J, Hoffmann A, McAloon R.
Association between fatty acid supplementation and prenatal
stress in African Americans: a randomized controlled trial.
Obstet Gynecol 2014;124(6):1080–7.
18 Long SJ, Benton D. A double-blind trial of the effect of docosa-
hexaenoic acid and vitamin and mineral supplementation on
aggression, impulsivity, and stress. Hum Psychopharmacol
2013;28(3):238–47.
19 El Ansari W, Adetunji H, Oskrochi R. Food and mental health:
relationship between food and perceived stress and depressive
symptoms among university students in the United Kingdom.
Cent Eur J Public Health 2014;22(2):90–7.
20 Srivastava R, Batra J. Oxidative stress and psychological func-
tioning among medical students. Ind Psychiatry J 2014;23(2):
127–33.
21 Smitha KK, Mukkadan JK. Effect of different forms of acute
stress in the generation of reactive oxygen species in albino
Wistar rats. Indian J Physiol Pharmacol. 2014;58(3):229–32.
22 Yajima M, Matsumoto M, Harada M, Hara H, Yajima T.
Effects of constant light during perinatal periods on the behav-
ioral and neuronal development of mice with or without
dietary lutein. Biomed Res 2013;34(4):197–204.
23 Reboul E, Thap S, Perrot E, Amiot MJ, Lairon D, Borel P. Effect
of the main dietary antioxidants (carotenoids, gamma-toco-
pherol, polyphenols, and vitamin C) on alpha-tocopherol
absorption. Eur J Clin Nutr 2007;61(10):1167–73.
24 Bone RA, Landrum JT. Dose-dependent response of serum
lutein and macular pigment optical density to supplementation
with lutein esters. Arch Biochem Biophys 2010;504(1):50–55.
25 Krinsky NI, Landrum JT, Bone RA. Biologic mechanisms of the
protective role of lutein and zeaxanthin in the eye. Annu Rev
Nutr 2003;23:171–201.
26 Snodderly DM, Brown PK, Delori FC, Auran JD. The macular
pigment. I. Absorbance spectra, localization, and discrimination
from other yellow pigments in primate retinas. Invest
Ophthalmol Vis Sci 1984;25(6):660–73.
27 Ham WT Jr, Mueller HA, Sliney DH. Retinal sensitivity to
damage from short wavelength light. Nature 1976;260(5547):
153–5.
28 Snodderly DM. Evidence for protection against age-related
macular degeneration by carotenoids and antioxidant vitamins.
Am J Clin Nutr 1995;62(6 Suppl):1448S–61S.
29 Hammond BR Jr, Wooten BR, Snodderly DM. Preservation of
visual sensitivity of older subjects: association with macular
pigment density. Invest Ophthalmol Vis Sci 1998;39(2):
397–406.
30 Humphries JM, Khachik F. Distribution of lutein, zeaxanthin,
and related geometrical isomers in fruit, vegetables, wheat, and
pasta products. J Agric Food Chem 2003;51(5):1322–7.
31 Craft NE, Haitema TB, Garnett KM, Fitch KA, Dorey CK.
Carotenoid, tocopherol, and retinol concentrations in elderly
human brain. J Nutr Health Aging 2004;8(3):156–62.
32 Vishwanathan R, Neuringer M, Snodderly DM, Schalch W,
Johnson EJ. Macular lutein and zeaxanthin are related to
brain lutein and zeaxanthin in primates. Nutr Neurosci
2013;16(1):21–29.
33 Johnson EJ, McDonald K, Caldarella SM, Chung HY, Troen
AM, Snodderly DM. Cognitive findings of an exploratory trial
of docosahexaenoic acid and lutein supplementation in older
women. Nutr Neurosci 2008;11(2):75–83.
34 Feeney J, Finucane C, Savva GM, Cronin H, Beatty S, Nolan
JM, Kenny RA. Low macular pigment optical density is associ-
ated with lower cognitive performance in a large population-
based sample of older adults. Neurobiol Aging 2013;34(11):
2449–56.
35 Vishwanathan R, Iannaccone A, Scott TM, Kritchevsky SB,
Jennings BJ, Carboni G, et al. Macular pigment optical
density is related to cognitive function in older people. Age
Ageing 2014;43(2):271–5.
36 Gemma C, Vila J, Bachstetter A, Bickford PC. Oxidative stress
and the aging brain: from theory to prevention (chapter 15).
Riddle DR, editor. Boca Raton (FL): CRC Press/Taylor &
Francis; 2007.
37 Bone RA, Landrum JT, Fernandez L, Tarsis SL. Analysis of the
macular pigment by HPLC: retinal distribution and age study.
Invest Ophthalmol Vis Sci 1988;29(6):843–9.
38 Hammond BR Jr, Wooten BR, Snodderly DM. Cigarette
smoking and retinal carotenoids: implications for age-related
macular degeneration. Vision Res 1996;36(18):3003–9.
39 Scanlon G, Connell P, Ratzlaff M, Foerg B, McCartney D,
Murphy A, et al. Macular pigment optical density is lower
in type 2 diabetes, compared with type 1 diabetes and normal
controls. Retina 2015;35(9):1808–16.
40 Wooten BR, Hammond BR, Land R, & Snodderly DM. A prac-
tical method of measuring macular pigment optical density.
Invest Ophthalmol Vis Sci 1999;40:2481–9.
41 Wooten BR, Hammond BR, and Smollon B. Assessment of the
validity of heterochromatic flicker photometry for measuring
macular pigment optical density in normal subjects. Optom
Vis Sci 2005;82:378–86.
42 Stringham JM, Hammond BR, Nolan JM, Wooten BR,
Mammen A, Smollon W, Snodderly DM. The utility of using
Stringham et al. Macular carotenoids and stress
Nutritional Neuroscience 2017
10
customized heterochromatic flicker photometry (cHFP) to
measure macular pigment in patients with age-related macular
degeneration. Exp Eye Res 2008;87(5):445–53.
43 Lemyre L, Chair M, Lalande-Markon MP. Psychological
Stress Measure (PSM-9): integration of evidence-based approach
to assessment, monitoring, and evaluation of stress in physical
therapy practice. Physiother Theory Pract 2009;25(5–6):453–62.
44 Derogatis LR, Melisaratos N. The brief symptom inventory: an
introductory report. Psychol Med. 1983;13(3):595–605.
45 Beck AT, Epstein N, Brown G, Steer RA. An inventory for
measuring clinical anxiety: psychometric properties. J Consult
Clin Psychol. 1988;56(6):893–97.
46 Beck AT, Ward CH, Mendelson M, Mock J, Erbaugh J. An
inventory for measuring depression. Arch Gen Psychiatry.
1961;4:561–71.
47 Yan YX, Liu YQ, Li M, Hu PF, Guo AM, Yang XH, et al.
Development and evaluation of a questionnaire for measuring
suboptimal health status in urban Chinese. J Epidemiol
2009;19(6):333–41.
48 Yan YX, Dong J, Liu YQ, Zhang J, Song MS, He Y, Wang W.
Association of suboptimal health status with psychosocial stress,
plasma cortisol and mRNA expression of glucocorticoid recep-
tor α/βin lymphocyte. Stress. 2015;18(1):29–34.
49 Meagher KA, Thurnham DI, Beatty S, Howard AN,
Connolly E, Cummins W, Nolan JM. Serum response to
supplemental macular carotenoids in subjects with and without
age-related macular degeneration. Br J Nutr 2013;110(2):
289–300.
50 Stringham JM, Stringham NT. Serum and retinal responses to
three different doses of macular carotenoids over 12 weeks of
supplementation. Exp Eye Res 2016;151:1–8.
51 Marsland AL, Walsh C, Lockwood K, John-Henderson NA.
The effects of acute psychological stress on circulating and
stimulated inflammatory markers: A systemic review and meta-
analysis. Brain Behav Immun 2017;S0889-1591(17)30011-9.
doi:10.1016/j.bbi.2017.01.013.
52 McIntosh LJ, Hong KE, Sapolsky RM. Glucocorticoids may
alter antioxidant enzyme capacity in the brain: baseline studies.
Brain Res. 1998;791(1–2):209–14.
53 Valko M, Leibfritz D, Moncol J, Cronin MT, Mazur M, Telser J.
Free radicals and antioxidants in normal physiological
functions and human disease. Int J Biochem Cell Biol 2007;39:
44–84.
54 Persson R, Garde AH, Hansen AM, Osterberg K, Larsson B,
Orbaek P, Karlson B. Seasonal variation in human
salivary cortisol concentration. Chronobiol Int 2008;25(6):
923–37.
55 Johnson EJ, Maras JE, Rassmussen HM, Tucker KL. Intake of
lutein and zeaxanthin differ with age, sex, and ethnicity. J Am
Diet Assoc 2010;110(9):1357–62.
Stringham et al. Macular carotenoids and stress
Nutritional Neuroscience 2017 11