Dietary chlorophyll metabolites catalyze the photoreduction of plasma ubiquinone

Article abstract of DOI:10.1111/j.1751-1097.2012.01230.x

Ubiquinol is a plasma antioxidant. The mechanisms responsible for maintenance of plasma ubiquinol are poorly understood. Here, we show that metabolites of chlorophyll can be found in blood plasma of animals that are given a chlorophyll-rich diet. We also show that these metabolites catalyze the reduction of plasma ubiquinone to ubiquinol in the presence of ambient light, in vitro. We propose that dietary chlorophyll or its metabolites, together with light exposure, regulate plasma redox status through maintaining the ubiquinol pool. Blood vessels growing toward the light: can light protect blood plasma from oxidative damage?

Full text of DOI:10.1111/j.1751-1097.2012.01230.x

Photochemistry and Photobiology, 20**, **: *–*
Dietary Chlorophyll Metabolites Catalyze the Photoreduction of Plasma
Ubiquinone
1
2
2
3
,2
Jinfeng Qu , Li Ma , Junhua Zhang , Steffen Jockusch and Ilyas Washington*
1
Department of Ophthalmology of Peking University, Beijing, China
2
Department of Ophthalmology, Columbia University Medical Center, New York, NY
3
Department of Chemistry, Columbia University, New York, NY
Received 15 March 2012, accepted 6 August 2012, DOI: 10.1111/j.1751-1097.2012.01230.x
ABSTRACT
a chlorophyll-rich diet. Although chlorophyll is the most abun-
dant phytochemical found in green leafy vegetables, the benefi-
cial effects of such diets are mainly ascribed to ascorbic acid,
carotenoid, tocopherol and flavonoid antioxidants. We propose
that dietary chlorophyll is involved in plasma ubiquinol homeo-
stasis. This would provide a possible chemical mechanism for
the beneficial effects of diets rich in green leafy vegetables that
are high in chlorophyll.
Ubiquinol is a plasma antioxidant. The mechanisms responsi-
ble for maintenance of plasma ubiquinol are poorly under-
stood. Here, we show that metabolites of chlorophyll can be
found in blood plasma of animals that are given a chlorophyll-
rich diet. We also show that these metabolites catalyze the
reduction of plasma ubiquinone to ubiquinol in the presence of
ambient light, in vitro. We propose that dietary chlorophyll or
its metabolites, together with light exposure, regulate plasma
redox status through maintaining the ubiquinol pool.
MATERIALS AND METHODS
Screening. All chlorophyll derivatives and/or metabolites used were com-
mercially available (Frontier Scientific, Logan, UT), were all over 95% in
purity and used as received. We added bovine serum (700 uL), ascorbic
acid (400 mM), ubiquinone (20 mM) and the chlorophyll metabolite and/
INTRODUCTION
The observation that low levels of serum antioxidants correlate
with a high incidence of vasculature disease has led to the
hypothesis that the administration of lipid antioxidants may
decrease the incidence of diseases, including atherosclerosis, con-
gestive heart failure, hypertension (1–3) and general senescence
or derivative to a centrifuge tube and exposed the mixture to red light
À2
(
660 nm light-emitting diode [LED] at 1 W m ) for 2 h. After photoly-
sis, we isolated the quinones by precipitating the proteins with methanol
(1 mL), extracted the mixture with heptane (0.5 mL) and quantified
ubiquinol and ubiquinone using high-performance liquid chromatography
(
HPLC) according to the literature (17,18). To prevent the oxidation of
(
4–8). Ubiquinol–10 (also known as Coenzyme Q, shown in
the formed ubiquinol, the screening was run under an argon atmosphere.
Stock solutions of ubiquinone and the chlorophyll metabolites and/or
derivatives were made in dimethyl sulfoxide. The light source was a
Fig. 1) is an endogenously synthesized lipid antioxidant that
scavenges free radicals and is involved in a-tocopherol homeo-
stasis (9–11). Ubiquinol is thought to be more effective in
preventing lipid oxidation than the dietary-derived antioxidants
a-tocopherol and L-ascorbic acid, and the carotenoids b-carotene
and lycopene (12–14). However, when ubiquinol is used as an
antioxidant, it is oxidized to ubiquinone. To act as an effective
antioxidant, the body must regenerate ubiquinol from ubiquinone
À2
660 nm LED light and luminous power density was 1.0 ± 0.2 W m as
measured using an LI–250A light meter (LI-COR Inc.).
Photoreduction of ubiquinone under ambient oxygen. We added
human serum, pheophorbide-a and a liposomal preparation of ubiquinone
that was used to increase solubility to a centrifuge tube. Both ubiquinone
and pheophorbide-a were added to give final concentrations of 2.5 lM
each. After vortexing for 30 s, we exposed the sample to red light
À2
(
1 W m ). We withdrew aliquots every 5 min for 15 min and quantified
(
Fig. 1). The efficacy with which the body regenerates ubiquinol
ubiquinol and ubiquinone using HPLC, as described above. For statistical
power, we repeated the reaction three times.
Preparation of liposomal ubiquinone. In a round-bottomed flask, we
dissolved ubiquinone (0.52 mg), egg phospholipid (45 mg), cholesterol
modulates the antioxidant ability of both the quinone and
a-tocopherol. In the sera of healthy individuals, more than 95%
of the quinone exists as ubiquinol. Although several enzymes
have been proposed to play a role in the maintenance of plasma
ubiquinol, the mechanisms for maintaining the plasma’s ubiqui-
nol reservoir are not entirely understood (15,16). Accordingly,
we sought to define mechanisms that may be involved in ubiqui-
nol homeostasis.
Herein, we show that metabolites and/or derivatives of
chlorophyll can catalyze the reduction of ubiquinone to generate
ubiquinol in plasma. We show that this catalysis does not cause
oxidative damage to plasma lipids and that the catalytic metabo-
lites of chlorophyll can be found in the plasma of animals given
(
7.2 mg) and Tween 80 (32 mg) in 2 mL of chloroform. Using a rot-
ovap, we evaporated the chloroform, then added 1 mL of phosphate
buffered saline, stirred the solution at 60°C for 30 min and probe soni-
cated the solution (using pulses of 10 s with 5 s rest in between) until it
turned from opaque to clear. To prevent lipid oxidation, all manipulations
were done under an argon atmosphere. An average liposome size of
6
0 nm was determined by dynamic light scattering upon dilution in
saline.
Lipid peroxidation assays. Reactions were carried out using pheophor-
bide-a, as described for the initial screening, under an atmosphere of 5%
oxygen and 95% nitrogen. Dark control mixtures were placed under the
LED lamp, but the lamp was covered with aluminum foil to block the
light. Immediately after the photoreduction, we analyzed for lipid peroxi-
dation using iodometric (19) and thiobarbituric acid (20) lipid peroxida-
tion assays. Both assays were conducted according to literature
procedures and are outlined below.
*
Corresponding author email: iw2101@columbia.edu (Ilyas Washington)
2012 Wiley Periodicals, Inc.
Photochemistry and Photobiology © 2012 The American Society of Photobiology 0031-8655/12
©
1

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Jinfeng Qu et al.
À2
strongly absorb. The intensity of red light used (1 ± 0.2 W m
)
was 30–60 times less than the level of red light that we measured
on a clear March afternoon in New York City and is less—after
attenuation—than capillaries, arteries and veins are exposed to in
vivo (26,27). Vitamin C was added as an oxidant, at levels that
are found in plasma in vivo. We used serum, free from red and
white blood cells because, in capillaries in vivo, plasma runs
along capillary walls whereas red and white blood cells concen-
trate and travel down the center of the lumen (28). We ran the
reactions under an atmosphere of argon to prevent the reverse
spontaneous air oxidation of ubiquinol to ubiquinone by oxygen,
and followed the reduction of ubiquinone by measuring the con-
centrations of ubiquinone and ubiquinol using HPLC (Fig. 2a).
All chlorophyll metabolites and derivatives we investigated
showed an ability to catalyze the photoreduction of serum ubiqui-
none (Fig. 2b). In the absence of light or metabolites (Fig. 2b,
entries 9 and 10, respectively), ubiquinone was not reduced. Ubi-
quinone reduction was dependent on the amount of chlorophyll
metabolite added (Fig. 3a).
Figure 1. Oxidation and reduction cycle for the ubiquinol–ubiquinone
pair. In this work, we investigated the possible roles of dietarty chlro-
phyll metabolites and light in serum ubiqunol maintenance.
To show that the photoreduction could also take place under
atmospheric oxygen and in the absence of added vitamin C, we
exposed a mixture of human serum, ubiquinone and pheophor-
bide-a to red light and we quantified ubiquinol and ubiquinone
in the mixture using HPLC at various times after the onset of
light exposure (Fig. 3b). Ubiquinone was effectively reduced,
suggesting that oxygen in vivo, which is present at lower concen-
trations, would not sufficiently inhibit photoreduction.
For the iodometric assay: at the end of the reaction, plasma samples
were spiked with ethylenediaminetetraacetic acid and butylated hydroxytol-
uene (25 and 20 lM, final concentrations, respectively) to inhibit inadver-
tent lipid peroxidation during sample handling and during the assay
procedure. We then added 500 lL of the plasma to 1.5 mL of Merck
CHOD-iodide reagent (Merck, Darmstadt, Germany). The sample was
incubated for 30 min at room temperature in the dark, and the absorbance
was measured at 365 nm (Lamda 650; PerkinElmer, Waltham, MA).
For the thiobarbituric assay: 100 lL of plasma was added to a 1 mL
solution of 15% wt/vol trichloroacetic acid and 0.375% wt/vol thiobarbi-
turic acid in 0.25 N hydrochloric acid. The solution was prepared on the
day of its use. The mixture was heated at 90°C for 60 min, cooled and
then centrifuged at 3800 g for 5 min. The solution’s fluorescence (excita-
tion 515 nm; emission 553 nm) was measured using a fluorescence spec-
trometer (Fluromax-4; HORIBA Jobin Yvon, Edison, NJ).
To test for possible oxidative damage of plasma lipids during
the photoreduction, we used two widely used assays to measure
plasma lipid peroxidation: the thiobarbituric acid and the iodo-
metric assays (29,30). The iodometric assay takes advantage of
À
the ability of serum lipid peroxides to oxidize I to I
2
, which, in
À
À
turn, reacts with the excess of nonreacted I to form I3 . There
To check whether the assays responded linearly to increasing or
decreasing amounts of oxidized lipids, we varied the amount of plasma
used in the assays. Both assays gave increasing and/or decreasing values
for lipid peroxidation that were dependent on the amount of serum used.
The assays responded linearly within the concentrations of plasma used
in this work.
is a stoichiometric relationship between the amount of lipid
À
peroxides and the concentration of I
3
produced, which can be
measured using UV–Vis spectroscopy. A major end product of
Serum metabolites. Columbia University’s Institutional Animal Care
and Use Committee (IACUC) approved the animal protocols.
Three-month-old mice (Institute of Cancer Research, Charles River,
Wilmington, MA) were used. The chlorophyll-rich diet (Harlan Teklad,
Indianapolis, IN) contained 15% by weight spirulina (a food supplement
produced primarily from cyanobacteria, Arthrospira platensis and Arthro-
spira maxima [21,22]), giving ca 0.23% by weight chlorophyll, as a mix-
ture of mainly chlorophyll-a (86%) and chlorophyll-b (13%). The control
diet was a purified diet devoid of dietary chlorophylls (Harlan Teklad).
Five, 3-month-old, animals each were given each respective diet ad lib
for 2 weeks. Serum was collected (0.1 mL), vortexed in 2 mL ethanol,
clarified by centrifugation and analyzed using fluorescence spectroscopy
(
excitation = 410 nm; emission kmax = 665 nm). For a qualitative con-
centration determination, we used fluorescence spectroscopy and standard
curves generated from known solutions of the chlorophyll metabolite
pheophorbide-a.
RESULTS AND DISCUSSION
Figure 2. (a) Representative HPLC traces of extracted quinones before
bottom) and after light exposure (top). We mixed bovine serum, ubiqui-
(
To demonstrate that dietary chlorophyll metabolites could cata-
lyze the reduction of ubiquinone, we incubated bovine serum with
ubiquinone, vitamin C and the several chlorophyll metabolites
and derivatives near concentrations that have been shown to be
present in plasma in vivo (23–25). We exposed the solution to red
light (kmax = 660 nm), to which biological tissues are relatively
transparent, but that chlorophyll metabolites and derivatives
none, the chlorophyll metabolite pheophorbide-a and vitamin C and
À2
exposed the mixture to 660 nm light of 1.0 W m for 2 h. Before light
exposure, ubiquinol was not detected in the mixture; however, after light
exposure, ubiquinol was detected. (b) We repeated the reaction described
in panel (a) with the listed chlorophyll metabolites and/or derivatives. All
chlorophyll metabolites and/or derivatives catalyzed the photoreduction
of ubiquinone to ubiquinol. The hydrophobic and copper derivatives
(
entries 4 and 5 in panel b, respectively) were slightly less reactive.

Photochemistry and Photobiology
3
Figure 3. (a) Dose response curve for pheophorbide-a (entry 8 in
Fig. 2b). We repeated the reaction described in Fig. 2 with various con-
centrations of pheophorbide-a. The photoreduction of ubiquinone was
dependent on pheophorbide-a dose. (b) Photoreduction of ubiquinone cat-
alyzed by pheophorbide-a in the presence of atmospheric oxygen. Con-
centrations of ubiquinol and ubiquinone at time zero are normalized to 1.
The reaction was run in triplicate. Means and standard deviations are
shown. Some standard deviations were smaller than the mark and thus
cannot be seen in the figure. We mixed human serum, pheophorbide-a
and liposomal ubiquinone, both at a final concentration of 2.5 lM. We
Figure 4. Assessment of lipid oxidative damage during the photoreduc-
tion. We repeated the reaction described in Fig. 2a using pheophorbide-a
under an atmosphere of 5% oxygen and 95% nitrogen. Control samples
were kept in the dark (Treated Dark) or were not treated at all (Before
Treatment). Immediately, after the reaction, we measured lipid oxidation
using iodometric and thiobarbituric acid lipid peroxidation assays. Reac-
tions were run in quadruplicate. Means and standard deviations are
shown. The higher the absorbance or fluorescence intensities, the greater
the amount of oxidized lipid. Comparisons between groups were made
by two-tailed, unpaired t-test. A P value less than 0.05 was considered
significant. Both assays were in agreement and showed statistically negli-
gible oxidative damage.
À2
then exposed the sample to red light (1.0 W m ) and, at the specified
times, withdrew aliquots and quantified ubiquinol and ubiquinone by
HPLC.
plasma lipid oxidation is malondialdehyde, which is formed from
the [2 + 2] cycloadduct of singlet oxygen to plasma polyunsatu-
rated acids. Thiobarbituric acid forms a 2:1 adduct with malondi-
aldehyde. Concentration changes in malondialdehyde in the
nanomolar range are readily detected by the use of fluorescence
spectroscopy for adduct quantification. Although absolute values
of lipid oxidative damage are difficult to obtain via the above
assays, together, these assays give a good metric of relative oxi-
dative damage of similarly treated samples. For assessment of
oxidative damage during photoreduction, reactions were con-
ducted employing pheophorbide-a under an atmosphere of 5%
oxygen and 95% nitrogen to simulate oxygen tensions in blood
serum in vivo (31). As controls, we compared the amount of oxi-
dized lipids in samples treated similarly but kept in the dark and
before any treatment. We did not detect a statistically significant
difference in lipid oxidation as measured by both assays relative
to control samples (Fig. 4).
lites of chlorophyll, such as chlorophyllide a, pheophytin-a, pheo-
phorbide-a, methyl pheophorbide-a, 10-OH-pheophorbide-a, 10-
OH-methyl pheophorbide-a, pyro pheophorbide-a and methyl pyr-
opheophorbide may theoretically be present in the blood. All of
the above metabolites can be formed from chlorophyll by chemis-
try that normally takes place in the body. Results in Fig. 2b sug-
gest that a variety of chlorin-type molecules can catalyze the
photoreduction. Therefore, any one of the above-named metabo-
lites, if present, might catalyze the photoreduction of ubiquinone.
To better follow ubiquinone photoreduction, we added coen-
zyme Q at higher concentrations than naturally found in plasma.
In this work, we used plasma containing as little as 2.5 lM of
added ubiquinone; conversely the sera of healthy adults have
been reported to contain an average of 1.4 lM coenzyme Q
mostly in the form of ubiquinol (33). However, for those taking
coenzyme Q supplements, levels of up to 2.9 lM can be reached
To show that dietary chlorophyll metabolites are present in
plasma, we fed mice a chlorophyll-rich diet. Extracts of plasma
from these mice exhibited red fluorescence at 675 nm when
excited with 410 nm light, typical of chlorophyll and its metabo-
lites (Fig. 5). The excitation spectrum of this 675 nm peak was
also similar to that of chlorophyll metabolites. This red fluores-
cence was absent when animals were given a chlorophyll-free
diet for 2 weeks. Using fluorescence spectroscopy and assuming
that the fluorescence quantum yield of the unknown pigment is
similar to a typical chlorophyll metabolite with an intact chlorin
(
33). It is of interest to employ a more sensitive HPLC detection
system, such as electrochemical detection, to be able to follow
the oxidation state of coenzyme Q when it is present at lower
concentrations closer to the in vivo situation. However, the likeli-
ring (i.e. pheophorbide-a, Φ = 0.28 [32]), we estimated a chlo-
f
rophyll metabolite concentration of ca 0.16 ± 0.03 lM in the
blood plasma for mice fed with chlorophyll-rich diet. These data
are consistent with literature data, which also suggest that dietary
chlorophyll metabolites are in circulation (23–25). For example,
chlorophyll metabolites (up to 2.5 lM) have been found in the
plasma of people taking certain chlorophyll-containing dietary
supplements (23).
Further research is needed to identify the red fluorescence
pigment(s) in the blood and to evaluate its (their) ability to
catalyze the photoreduction of ubiquinone. A variety of metabo-
Figure 5. Representative fluorescence spectra of ethanol extracts of
blood from a mouse fed a chlorophyll-rich (Treated) diet and a mouse
fed a chlorophyll poor (Control) diet (excitation = 410 nm).

4
Jinfeng Qu et al.
hood of such a photoreduction taking place in vivo is perhaps
more largely dependent on where blood chlorins localize with
respect to ubiquinone in serum. Proximity of the two reactants
would presumably increase photoreduction efficacy. Several
chlorophyll-derived chlorins have been shown to selectively
incorporate into the low-density lipoprotein and high-density
lipoprotein fractions of plasma, where most of the coenzyme Q
is concentrated (34–37).
Both light (26) and light-absorbing chlorophyll metabolites
can be present in capillaries, arteries and veins of several animals
including humans. If chlorophyll metabolites catalyze the photo-
reduction of plasma ubiquinol in vivo, it would be a novel mech-
anism to maintain high levels of plasma ubiquinol. Such a
mechanism would provide a chemical mechanism for the benefi-
cial effects of diets rich in green leafy vegetables that are high in
chlorophyll. Further study is needed to determine the extent to
which this mechanism may function.
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Acknowledgements—This work was supported by the Office of Naval
Research (N00014–08–10150), Nanoscale Science and Engineering
Initiative of the National Science Foundation under (NSF CHE–0117752
and CHE–0641523) and by NYSTAR and the Mebane Charitable
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