Published January 8, 2016

Bixin uptake and antioxidative effect and role in immunoregulation in domestic cats1

J. S. Park, B. D. Mathison, B. M. Zawlocki, and B. P. Chew2

Department of Nutrition and Food Science, Texas A&M University, College Station 99164-6376

ABSTRACT: Bixin, a carotenoid found in the seed of
the Annatto plant, Bixa orellana, is a potent antioxi-
dant. Carotenoids are readily absorbed from the diet;
therefore, the purpose of this study was to examine
uptake of bixin by plasma, lipoproteins, and leukocytes
after dietary supplementation in domestic cats and to
assess effects on immune response. Female domestic
short hair cats (3 yr old; 4.79 0.13 kg BW) were fed
a single dose of 0, 1, 5, or 10 mg bixin, and blood was
taken at 0, 1, 2, 4 and 8 h after administration (n = 6/
treatment) to determine acute absorption rate. Then,
bixin was fed daily for 14 d to examine steady-state
plasma concentrations and subcellular distribution.
Following these preliminary experiments, cats (n =
8/treatment) were fed diets containing 0, 1, 5, or 10
mg bixin/d for 16 wk and blood was collected on wk
0, 6, 12, and 16 for analysis of leukocyte subpopula-
tions, cell-mediated responsiveness, and inflammatory
and oxidative biomarkers. Maximal uptake in plasma
occurred 1 h after a single oral dose of bixin, with a
maximal concentration of 0.119 M and elimination
half-life of 1.8 to 2.2 h. Daily feeding of bixin showed
a steady-state plasma concentration of 0.110 M at the
greatest doses. Bixin was primarily associated with the
high-density lipoprotein fraction of blood lipoproteins
and was primarily distributed in mitochondrial frac-
tions (5859%) of but also in microsomal and nuclear
fractions (3744%). Leukocyte subpopulations in
blood were variably affected by dietary bixin, with an
increase (P < 0.05) in total T cells but a concurrent
decrease (P < 0.05) in CD18+ and B cell subpopula-
tions. However, plasma IgG increased (P < 0.05) in the
10-mg treatment group by wk 6. Lymphoproliferation
was stimulated (P < 0.05) in the 5-mg bixin treatment
group by wk 16, and delayed-type hypersensitivity
response increased after nonspecific antigenic chal-
lenge. Conversely, when a specific challenge of
vaccine was assessed on wk 12 and 16, responsive-
ness decreased (P < 0.05) in the 10-mg bixin treatment
group. Bixin supplementation surprisingly caused an
increase (P < 0.05) in -acid glycoprotein but had no
effect on natural killer cell activity, other subpopula-
tions of leukocytes, or 8-oxo-2-deoxyguanosine, a
DNA damage biomarker. This experiment demon-
strated dose-dependent uptake of bixin in plasma
and blood lipoproteins and distribution in leukocyte
subcellular components and an impacted immune
response through cell-mediated and humoral actions.

Key words: bixin, feline, immunomodulation, oxidative biomarkers, uptake kinetics

2016 American Society of Animal Science. All rights reserved. J. Anim. Sci. 2016.94:125134
INTRODUCTION
Formation of excess reactive oxygen species
(ROS) has been implicated in disease etiology, in-
1This work was supported by a grant from P&G Pet Care,
Lewisburg, OH, and the Agricultural Research Station, School of
Agriculture, Human and Natural Resource Sciences, Washington
State University, Pullman, WA.
2Corresponding author: boon.chew@tamu.edu
Received June 30, 2015.
Accepted October 16, 2015.
125
doi:10.2527/jas2015-9478
cluding cancer, cardiovascular diseases, and degen-
erative eye diseases, as well as in the aging process
(Day, 2010). Many carotenoids, including -carotene,
lutein, lycopene, zeaxanthin, and astaxanthin, have
been shown to be involved in modulating immune
response (Chew and Park, 2009) and metabolic dys-
function in humans (Esterbauer et al., 1989; Barona
et al., 2012) and rats (Hussein et al., 2007) as well as
cancer (Chew et al., 1999, 2003; Nakao et al., 2010).
The carotenoid bixin, found in the seed pericarp
of the Annatto plant, Bixa orellana, strongly quenches
singlet molecular oxygen (Di Mascio et al., 1990) and
126 Park et al.
inhibits lipid peroxidation formation (Zhang et al., 1991;
Canfield and Valenzuela, 1993) , thereby protecting cell
membranes from oxidation. Bioavailability of carot-
enoids is dependent on the amount consumed, the matrix
in which the carotenoid is incorporated, and linkage type.
Lutein diesters were shown to have greater bioavailability
than free lutein (Castenmiller and West, 1998). Therefore,
determining uptake and absorption of supplemental
bixin, which has 2 carboxylic groups, one of which is
a methyl ester (Levy et al., 1997), is an important pre-
liminary step to examining bioactivity. The carotenoids
lutein (Park et al., 1999), -carotene (Chew et al., 2000a),
and astaxanthin (Park et al., 2010b) are readily taken up
in cat plasma and absorbed in peripheral blood leukocyte
organelles (Park et al., 2010b), leading to enhanced cell-
mediated and humoral immune responses.
It is hypothesized that bixin will influence immune
regulation and have antioxidant effects after oral supple-
mentation in domestic cats. The objectives of this study
were to determine the uptake of bixin and norbixin in
plasma, blood lipids, and subcellular organelles and to
examine antioxidant and immune stimulating effects.
MATERIALS AND METHODS
The research protocol was approved by the
Washington State University Institutional Animal
Care and Use Committee (number 3229).
Animals and Experimental Design
Mature female domestic short hair cats (3 yr old;
4.79 0.13 kg BW; Liberty Research, Waverly, NY)
were fed a basal diet (P&G Pet Care, Lewisburg, OH)
containing 72 g/kg moisture, 310 g/kg protein, 52 g/
kg ash, 217 g/kg fat, 14 g/kg crude fiber, 11.5 g/kg Ca,
and 8.3 g/kg P. Cats were group housed in 2 by 2 m
pens (23 cats/pen) in light- (14 h light and 10 h dark)
and temperature-controlled (2022C) rooms and had
free access to food and water. In the initial trial, cats
(n= 8/treatment) were given a single dose by oral ga-
vage of 0, 1, 5, or 10 mg bixin and blood was taken
0, 1, 2, 4, and 8 h after feeding. To determine daily
uptake kinetics, oral doses of 0, 1, 5, or 10 mg bixin
were administered daily at 0800 h for 14 consecutive
days (n = 8/treatment) and blood was sampled 1 h after
each feeding, based on peak blood bixin concentra-
tions in the first experiment, on d 0, 1, 3, 6, 10, and 14.
In a consecutive experiment to examine immunomod-
ulatory effects, cats were randomly assigned (n = 14/
treatment) to receive 0, 1, 5, or 10 mg bixin daily for
16 wk. Blood was collected via jugular venipuncture
into heparin or EDTA vacutainers on wk 0, 6, 12, and
16 for assessment of immune function.
Cats were vaccinated on wk 12 and 14 with an
attenuated polyvalent vaccine containing feline her-
pesvirus-1, calicivirus, parvovirus, and Chlamydia
psittaci (Felocell; Pfizer Inc., New York City, NY),
and blood was collected on wk 16 to assess immune
response after antigenic challenge.
Bixin Preparation
A stock suspension of bixin in soybean oil was
prepared from annatto seed extract (17 g/L in soy-
bean oil containing 1.6% bixin and <0.3% norbixin;
INEXA, Quito, Ecuador) and warmed at 60 C for 15
min; then, bixin treatment solutions were diluted in
soybean oil for a final dosage volume of 0.5 mL.
Uptake HPLC Analysis
Blood was collected into heparinized vacutainers and
centrifuged at 400 g for 30 min at 4C, and plasma was
collected for HPLC analysis to evaluate plasma bixin and
norbixin concentrations. Plasma, lymphocyte homoge-
nate, and lymphocyte subcellular fractions were analyzed
for bixin content by HPLC (Alliance 2690; Waters Corp.,
Milford, MA) as previously described (Levy et al., 1997),
using an internal standard of 3.2 mol/L Sudan I (Sigma-
Aldrich, St. Louis, MO) in methanol. Sudan I (0.1 mL)
and plasma (0.3 mL) were vortexed for 1 min and then
incubated in the dark for 30 min. Chloroform (0.6 mL)
was added and samples were vortexed for 5 min and then
centrifuged 10 min at 1,000 g at 4C. The bottom lay-
er was collected and dried under nitrogen gas in a water
bath (40 C). The resulting residue was resuspended in
a mobile phase consisting of acetonitrile:2% acetic acid
in water (70:30, vol/vol); samples were eluted through
a 5-m silica C18 column (250 by 4.6 mm, Spherisorb;
Waters Corp.), and absorbance was monitored at 460 nm
(Photodiode Array Detector 996; Waters Corp.).
Buffy coat leukocytes collected on 14 d were en-
riched by underlaying the cell suspension with Percoll
(Histopaque-1077; Sigma-Aldrich) and centrifuging at
400 g for 30 min at room temperature (Chew et al.,
1993), and then the cell number was determined with
a Coulter counter (Coulter Electronics, Hialeah, FL).
Cells were resuspended in PBS containing 30 g/L sodi-
um ascorbate (Sigma-Aldrich) to prevent oxidation and
disrupted by sonication (30 s). Total whole lymphocyte
bixin concentration was determined by HPLC after ex-
traction from an aliquot of the lymphocyte homogenate.
Subcellular fractions were also prepared from the cell ho-
mogenates as previously described (Chew et al., 1993).
Briefly, homogenates were differentially centrifuged to
obtain nuclear (600 g for 10 min at 4C), mitochondrial
(17,300 g for 20 min at 4C), microsomal (102,000 g
 Bixin uptake and immunomodulation in cats
for 60 min at 4C; pellet) and cytosolic (102,000 g for
60 min at 4C; supernatant). All samples were stored at
80C before HPLC analysis.
Lipoprotein Uptake and Cholesterol Analysis
Blood was collected into vacutainers containing no
anticoagulants and centrifuged at 400 g for 30 min at
4C, and serum was collected and stored at 80C until
analysis. Lipoproteins were isolated from serum using
density gradient ultracentrifugation (Terpstra et al., 1981;
Chew et al., 1993). Briefly, solid KBr (0.114 g), sucrose
(0.025g), serum (1mL), and Sudan black (2.19 mol/L
in ethylene glycol; Sigma-Aldrich) were combined in a
polycarbonate centrifuge tube and then overlayed with
2.4 mL salt solution (0.195 mol/L NaCl, 0.638 mol/L
KBr, and 0.2 mmol/L disodium EDTA; density 1.06 g/
mL) followed by 2.4mL of 0.2 mmol/L disodium EDTA
in distilled water and then centrifuged at 232,000 g for
7 h at 20C (Sorvall T-865.1 fixed angle rotor, Sorvall
OTD65B; Thermo Scientific, Waltham, MA). The li-
poprotein fractions were analyzed for bixin by HPLC
and the cholesterol content was determined (Chew et al.,
1993). The cholesterol reagent was prepared by dissolv-
ing 520 mg ferric perchlorate in 600 mL ethyl acetate,
cooling, and then adding 400 mL cold concentrated sul-
furic acid. The cholesterol reagent (25mL) was added to
25 L of sample or standard (2 mg cholesterol/L in gla-
cial acetic acid), allowed to react for 1.5 min in a 100C
water bath, and then cooled to less than 20C. Optical
density (OD) was read at 600 nm.
Hematology and Phenotyping of Leukocyte Subsets
Whole blood collected into EDTA vacutainers was
analyzed for complete blood count (white blood cell,
red blood cell [RBC], and platelet counts; lymphocyte,
monocyte, and granulocyte differential counts; he-
matocrit, hemoglobin, and mean corpuscular volume;
hemoglobin and hemoglobin concentration; and plate-
let volume) using a hematology analyzer (Vet ABC-
Hematology Analyzer; Heska Corp., Fort Collins, CO).
Whole blood collected into heparinized vacutainers
was used for leukocyte subpopulation determination. Red
blood cells were lysed using hypotonic shock followed
by centrifugation at 300 g for 5 min at 20C. Cells
were resuspended in PBS, pH 7.3, containing 10% fetal
bovine serum and 0.02% sodium azide and then stained
as previously described (Kim et al., 2000). Monoclonal
antibodies (Leukocyte Antigen Biology Laboratory,
Davis, CA) against specific subpopulations included
CD5+ (FE1.1B11, total T cells), CD4+ (FE1.7B12, help-
er T cells [Th]), CD8+ (FE1.10E9, cytotoxic T cells),
MHCII+ (42.3), and CD21+ (CA2.ID6, mature B cells).
127
Flow cytometry (FACSCaliber; BD Biosciences, San
Jose, CA) was used to quantitate changes in subpopula-
tions of cells on wk 0, 6, 12, and 16.
Plasma Immunoglobulins
Concentrations of IgG, IgM, and IgA in plasma
were measured using commercially available ELISA
kits (Cat IgG ELISA Quantitation Kit, Cat IgM ELISA
Quantitation Kit, and Cat IgA ELISA Quantitation Kit;
Bethyl Laboratories, Montgomery, TX). Lower limits
of detection were 7.8, 15.6, and 15.6 ng/mL for IgG,
IgM, and IgA, respectively.
Lymphoproliferation
Peripheral blood mononuclear cells were assessed for
ability to respond to the T cell mitogens (Sigma-Aldrich),
phytohemagglutinin (PHA; 0.25 and 1.25 mg/L final
concentration), and concanavalin A (ConA; 0.5 and 2.5
mg/L) as well as the T/B cell mitogen derived from poke-
weed (0.025 and 0.125 mg/L) were assessed (Kim et al.,
2000). Heparinized blood collected on wk 0, 6, 12, and
16 was diluted 1:8 in RPMI-1640 (developed at Roswell
Park Memorial Institute, Buffalo, NY) supplemented
with 10% newborn calf serum and penicillinstreptomy-
cin (Sigma-Aldrich). Whole blood was cultured in order
to mimic in vivo conditions. Diluted blood and mitogens
were incubated for 72 h at 37 C in a humidified, 5%
CO2 atmosphere. At 68 h of the incubation period, [3H]-
thymidine (thymidine 5-triphosphate [methyl-3H]; MP
Biomedicals, Santa Ana, CA) at 1 Ci/well was added
and allowed to incubate for an additional 4 h. Uptake was
measured by liquid scintillation, and the results were ex-
pressed as a stimulation index (SI), calculated as the per-
cent proliferation compared with unstimulated controls.
Natural Killer Cell Cytotoxic Activity
Natural killer cell cytotoxicity against tumor cells
was assessed using a modified bioassay (Gondolf et al.,
1996). The Crandell feline kidney fibroblast cell (CrFK)
line (ATCC CRL-9761; ATCC, Manassas, VA) was used
as the target. Cells were plated in 96-well flat-bottom cul-
ture plates at 2 104 cells/well in Dulbeccos modified
Eagles medium supplemented with 10% fetal bovine
serum and antibiotics and incubated at 37 C for 8 h in
a humidified, 5% CO2 atmosphere. Ficoll-separated pe-
ripheral blood mononuclear cells, which acted as effector
cells, were added to the CrFK target cells at 1 105 or 2
105/well to provide effector:target cell ratios of 5:1 and
10:1. After incubating for 8 h, 20 L rose bengal (0.25%
in PBS; Sigma-Aldrich Chemical, St. Louis, MO) was
added and the mixture was incubated an additional 3 min
128 Park et al.

followed by washing with PBS to remove excess dye.

Phosphate-buffered saline:ethanol (50%, vol/vol) was
added and the OD was measured at 570 nm with a refer-
ence OD at 630 nm. The percent of specific cytotoxicity
was calculated by subtracting the effector OD from the
effector + target OD and dividing by the target OD.

Skin Hypersensitivity Response

Skin induration response was assessed using a

delayed-type hypersensitivity (DTH) assay on wk 12
and 16 after intradermal injection of 100 L of physi-
ologic saline (negative control); an attenuated poly-
valent vaccine containing feline herpesvirus-1, cali-
civirus, parvovirus, and Chlamydia psittaci (Felocell;
Pfizer Inc.); and ConA (0.5 g/L saline), as previously
described (Kim et al., 2000). Skin thickness was mea-
sured at 0, 24, 48, and 72 h after injection, and results
were expressed as the percentage of increase in skin
thickness compared with 0 h.

Deoxyribonucleic Acid Oxidative Damage

Oxidative damage was assessed in plasma by

measuring 8-hydroxy-2-deoxyguanosine (8-OHdG)
using a competitive ELISA (BIOXYTECH 8-OHdG-
EIA; Percipio Biosciences, Burlingame, CA), with a
lower limit of detection of 0.5 ng/mL.

Alpha-1-Acid Glycoprotein

The acute phase protein -1-acid glycoprotein
(AGP) was measured in plasma using a commer-
cially available single radial immunodiffusion assay
(Feline Alpha 1 Acid Glycoprotein Assay; Tri Delta
Diagnostics, Morris Plains, NJ). The lower limit of de-
tection was 100 g AGP/mL plasma.
Statistics
Data were analyzed by repeated measures ANOVA
using the General Linear Model of SAS (SAS Inst.,
Inc., Cary, NC). The statistical model was Yijk = +
Treatmenti + Animalj (Treatmentj) + Sampling peri-
odj+ Treatmenti Periodj + eijk. Differences among
treatment means were compared by a protected LSD
test and considered different at P < 0.05.
RESULTS
Uptake in Cats
Bixin was not detectable in unsupplemented cat
plasma but was taken up in a dose-dependent manner
Figure 1. Concentrations of plasma bixin and subcellular uptake in cats
administered daily doses of 0, 1, 5, or 10 mg bixin for 14 d. Values are means
SEM (n = 8) as analyzed by repeated measures ANOVA. nd = non-detectable.
in all treatment groups after a single dose. Plasma bixin
peaked at 1 h after administration, with concentrations
in the 10-mg group (119 nM [SEM 18]) almost 13 times
greater (P < 0.05) than the 1-mg group (9 nM [SEM 2])
and nearly 3 times greater (P < 0.05) than the 5-mg group
(45 nM [SEM 7]) and with all treatments approaching
baseline levels by 8 h after administration. The half-life
of bixin in plasma was 2.2 h for the 1-mg group and 1.8 h
for the 5- and 10-mg groups. Norbixin uptake showed a
similar trend in uptake, although peak plasma concentra-
tions were relatively quite low, the greatest being <0.005
M in the 10-mg group. Concentrations of plasma
-tocopherol and retinol were not influenced by astax-
anthin supplementation and averaged 0.801 M (SEM
0.116) and 0.0.160 M (SEM 0.005), respectively.
Daily doses of bixin administered for 14 d also re-
sulted in a dose-dependent increase (P < 0.05) in plasma
concentrations (Fig. 1). Concentrations reached steady-
state levels of 0.010, 0.051, and 0.110 M for the 1-, 5-,
 Bixin uptake and immunomodulation in cats
Figure 2. Concentrations of bixin in whole lymphocytes and lipoprotein
fraction in cats administered daily doses of 0, 1, 5, or 10 mg bixin for 14 d.
Values are means SEM (n = 8) as analyzed by repeated measures ANOVA.
a,bMeans with different superscripts within a sampling period are significantly
different (P < 0.05). nd = non-detectable; VLDL = very-low-density lipopro-
tein; LDL = low-density lipoprotein; HDL = high-density lipoprotein.
and 10-mg groups, respectively, by the first day after
administration of treatments. Plasma concentrations in
cats fed 5 and 10 mg were 5 and 11 times greater (P<
0.05), respectively, than those fed 1 mg. Feeding of
bixin for 14 d did not significantly alter concentrations
of plasma -tocopherol and retinol (0.453 M [SEM
0.062] and 0.158 M [SEM 0.002], respectively).
Bixin uptake by d 14 in whole lymphocytes of cats
increased in a dose-dependent manner after daily treat-
ment (Fig. 1), with detectable uptake in even the low-
est (1 mg) treatment group but not in control cat cells.
Distribution of bixin in the subcellular fractions was
indeed dose-dependent, with the majority of the uptake
by mitochondria, 59 and 57%, in both the 5- and 10-mg
groups, respectively. There was also significant uptake of
bixin in both the nuclear (19%) and microsomal (22%)
fractions for both the 5- and 10-mg treatment groups.
The predominant (P < 0.05) lipoprotein fraction
with bixin in the blood of cats on d 6 and 14 was as-
sociated with the high-density lipoprotein (HDL)
fractions in both the 5- and 10-mg treatment groups
129
(Fig. 2). Bixin incorporation into HDL was dose de-
pendent (P < 0.05) for both of these treatment groups,
and although there was no further increase in bixin up-
take on d 14 compared with d 6, the HDL fraction was
always greater (P< 0.05) than the low-density lipopro-
tein (LDL) and very-low-density lipoprotein (VLDL)
fractions in both the 5- and 10-mg treatment groups.
Bixin was not detectable in any lipoprotein fraction
from unsupplemented control cats.
Hematology and Leukocyte Subset Phenotyping
Hematology results after 0, 6, 12, and 16 wk of bix-
in treatment are seen in Table 1. Bixin increased (P<
0.05) lymphocyte percentage and mean platelet volume
by 6 wk in the 1-mg treatment group and remained
elevated through wk 16, compared with the control.
Additionally, 5 mg bixin increased lymphocyte percent-
age from wk 6 through 16 and also increased (P < 0.05)
monocyte percentage by wk 6. An increase in hemoglo-
bin concentration (P < 0.05) was seen at wk 16 and in
red cell distribution width at wk 12 in the 10-mg treat-
ment group. Conversely, granulocyte percentage was
decreased (P< 0.05) in both the 1- and the 5-mg treat-
ment group by wk 6 and remained lower throughout
wk 16, when compared with the 0-mg treatment group.
Overall mean (SEM) for white blood cell ( 103/mm3),
RBC ( 106/mm3), mean cell hemoglobin (pg/mL),
mean cell hemoglobin concentration (hemoglobin/cell),
and total platelet count ( 103/mm3) were 4.1 (0.2), 8.6
(0.1), 14.8 (0.1), 34.8 (0.1), and 222 (9), respectively.
These parameters, as well as hematocrit and mean RBC
cell volume, fell within normal ranges.
CD4+ Th cells increased (P < 0.05) at wk 6 in the
5-mg treatment group (Table 1), when compared with the
control, with an analogous trend in increasing CD5+ pan
T lymphocytes (Tpan) lymphocytes. This correlates with
the increase in percent of lymphocytes seen in the he-
matology results. In addition, there was a decrease (P <
0.05) in the percentage of CD21+ B cell subpopulations
at wk 6, when compared with the control. Although there
were no other significant changes in subpopulations in
the cat blood after dietary bixin for 16 wk, there was a
tendency for T cell subpopulations to increase across the
16-wk time period, with the 5-mg group approaching
significance (P = 0.06) at wk 16, when compared with
the control. There was also a trend for the CD18+ sub-
population to decrease over the course of the intervention
in the 1- and 5-mg treatment groups, with the decrease
approaching significance (P= 0.06) on wk 12 again in
the 5-mg treatment group, when compared with the con-
trol. Major histocompatibility complex class II molecule-
positive subpopulations were unchanged throughout the
study with an overall mean of 87.2% (SEM 0.9).
130 Park et al.

Table 1. Absolute percentage of leukocytes and rela-

tive percentage of leukocyte subpopulations in blood
from cats fed 0, 1, 5, or 10 mg bixin for 16 wk. Values
are means (SEM; n = 14)
Dietary bixin, mg/d
Variable Week 0 1 5 10
Lymphocyte, 6 27.9 (2.9)b 33.9 (2.0)a 34.8 (2.0)a 29.6 (2.2)ab
% 12 26.2 (1.8) 27.9 (2.6) 32.9 (0.4) 26.7 (1.2)
16 25.1 (2.1)b 31.9 (2.0)a 31.2 (2.4)a 25.0 (2.0)b
Monocyte, 6 6.8 (0.4)c 9.0 (0.5)ab 9.5 (0.7)a 7.8 (0.4)bc
12 8.9 (0.5) 8.1 (0.5) 12.2 (0.4) 7.8 (0.2)
% 16 7.0 (0.3) 8.0 (0.4) 7.5 (0.6) 6.8 (0.4)
Granulocyte, 6 66.0 (3.0)a 57.1 (2.1)b 55.7 (2.4)b 62.6 (2.3)ab
% 12 64.9 (2.2) 64.0 (3.0) 54.9 (0.2) 65.6 (1.2)
16 67.9 (2.3)a 60.1 (2.0)b 61.3 (2.5)b 68.2 (2.2)a
Relative percent
CD4+ T 6 36.0 (2.1)b 39.6 (2.7)ab 46.0 (4.1)a 41.0 (2.4)ab
helper 12 44.4 (3.2) 42.7 (2.4) 46.3 (3.4) 46.5 (3.0)
16 40.9 (2.9) 44.3 (3.1) 45.6 (3.1) 45.9 (2.5)
CD8+ T 6 24.9 (0.9) 25.7 (0.6) 26.0 (0.8) 26.4 (1.2)
cytotoxic 12 25.6 (0.8) 24.8 (0.5) 26.7 (1.1) 26.4 (1.1)
16 27.7 (1.0) 27.3 (0.7) 29.0 (1.0) 29.2 (1.2)
CD21+ B 6 34.0 (2.6)a 30.5 (3.3)ab 23.0 (3.6)b 30.4 (2.9)ab
12 31.4 (3.0) 30.9 (2.3) 27.7 (4.3) 26.8 (3.8)
16 31.5 (3.3) 28.9 (3.1) 24.4 (3.6) 24.0 (3.4)
acDifferences within variables between treatments (P < 0.05).

Plasma Immunoglobulins Figure 3. Delayed-type hypersensitivity skin induration response af-

Concentrations of plasma IgM (overall mean of
4.19 mg/mL [SEM 0.19]) and IgA (overall mean of
0.42 mg/mL [SEM 0.02]) did not show changes in
concentrations throughout 16 wk of treatment with di-
etary bixin. Plasma IgG, on the other hand, increased
(P < 0.05) at wk 6 in the 10-mg treatment group, com-
pared with the 0-mg group.
Lymphoproliferation
Stimulation of lymphocytes with mitogens was
dose dependent with greater response in the presence
of greater concentrations of mitogens. There were no
treatment differences with stimulation by PHA at wk
6, 12, or 16, with overall means for low (0.25 g/mL)
and high PHA (1.25 g/mL) of 1.34 SI (SEM 0.05)
and 1.99 SI (SEM 0.1), respectively. There were also
no treatment differences with stimulation by poke-
weed mitogen, with overall means for low (0.025 g/
mL) and high (0.125 g/mL) of 1.93 SI (SEM 0.09)
and 4.53 SI (SEM 0.24), respectively. Concanavalin A
increased SI , with a greater (P < 0.01) response in the
5-mg bixin treatment group to the high concentration
(2.5 g/mL) of ConA as well as an increased (P < 0.01)
response in the 5-mg bixin treatment group to the lower
concentration of ConA (0.5 g/mL) at wk 16.
ter intradermal challenge with concanavalin A or vaccine seen in cats (n =
14) fed 0, 1, 5, and 10 mg bixin/d for 16 wk. Results are percent increase in
skin thickness (mean SEM). a,bDifferent superscripts within a sampling
period are significantly different (P < 0.05).
Natural Killer Cell Cytotoxic Activity
Natural killer cell cytotoxicity showed no significant
differences in natural killer cell killing efficiency between
treatments at any sampling period with an effector:target
ratio of 5:1. Overall mean was 47.4% (SEM 0.9) de-
creased viability. When an effector:target ratio of 10:1
was used in the cytotoxicity assay, there was decreased
(P< 0.05) killing efficiency compared with the control
treatment not only in the 10-mg bixin treatment group
but also at the lowest bixin treatment (1 mg).
Delayed-Type Hypersensitivity
Bixin had no effect on the DTH induration response
to ConA at wk 12 but did stimulate DTH response (P<
0.05) in the 10-mg treatment group (Fig. 3) at 24 h after
intradermal challenge at wk 16. Increase in skin thick-
ness with the 1- and 5-mg treatment groups also tended
to increase in a dose-dependent manner.
There was a slight (P < 0.08) stimulation of DTH
response at 12 wk of dietary bixin in the 1-mg treat-
ment group after challenge with vaccine. Conversely,
 Bixin uptake and immunomodulation in cats
Figure 4. Concentrations of plasma 8-hydroxy-2-deoxyguanosine,
an oxidative biomarker, and acid alpha glycoprotein, an inflammatory bio-
marker, seen in cats (n = 14) fed 0, 1, 5, and 10 mg bixin/d for 16 wk.
Results are mean SEM. acDifferent superscripts within a sampling pe-
riod are significantly different (P < 0.05).
at 72 h, there was a decrease (P < 0.05) in response
in the 10-mg treatment (Fig. 3), with a dose-related
trend in decreasing response with 1 and 5 mg dietary
bixin. In addition, at 16 wk, the 10-mg treatment
group showed the same depressed response (P < 0.05)
to vaccine challenge at 72 h. There was no effect of
dietary bixin on induration response to histamine chal-
lenge at 16 wk, with overall means of 28.2 (SEM 2.2),
28.0 (SEM 2.0), 35.7 (SEM 3.5), and 31.0 (SEM 2.0)
for 0, 1, 5, and 10 mg dietary bixin, respectively.
Acute Phase Protein
Plasma AGP concentrations were greater (P <
0.05) at both wk 12 and 16 in both the 5- and 10-mg
treatment groups when compared with the control. In
addition (Fig. 4), there was a tendency for elevated
AGP in the 1-mg treatment group at wk 12.
Deoxyribonucleic Acid Oxidative Damage
There were no significant changes in plasma
8-OHdG after 12 wk. However, after 1 wk of dietary
131
bixin treatment, plasma 8-OHdG levels were dramati-
cally suppressed by the 1- and 5-mg treatment levels
(Fig. 4) compared with the 0-mg control treatment.
DISCUSSION
Cellular oxidative damage by ROS has been
shown to be a factor in chronic disease, through de-
struction of cellular membranes, proteins, and nucleic
acids. Immune cells are very susceptible to peroxida-
tion, due to the high lipid content in the cell mem-
branes (Meydani et al., 1993). Aging compromises the
immune system, with changes in the balance of Th1
and Th2 activity, impaired functionality of cell-me-
diated immunity, and an increase in proinflammatory
cytokines (Day, 2010). Many carotenoids, including
-carotene, lutein, lycopene, zeaxanthin, and astaxan-
thin, have been shown to be involved in modulating
immune response (Chew and Park, 2009) as well as
cancer (Chew et al., 1999, 2003; Nakao et al., 2010).
Supplementation with carotenoids has also been
shown to mediate factors involved in metabolic dys-
function in humans (Esterbauer et al., 1989; Barona et
al., 2012) and rats (Hussein et al., 2007).
Bioavailability of carotenoids with oral supple-
mentation is an important factor in efficacy, whether
through immunomodulation or disease prevention.
Uptake and intracellular distribution varies greatly
between carotenoids, dependent on the nature of the
compounds (Yeum and Russell, 2002), as well the
matrix the carotenoid is presented in (Moelants et al.,
2012). The effects of absorption and bioconversion
will influence bioavailability (Castenmiller and West,
1998), and little is known about these parameters in
cats administered oral bixin. Although bixin is the
predominant carotenoid extracted from the annatto
seed, making up 70 to 80% of the pigment present,
norbixin is also present in small amounts and is the
most abundant water-soluble derivative after hydroly-
sis of bixin. Levy demonstrated measurable levels of
bixin and the water-soluble derivative, norbixin, in the
human blood after a single dose of Annatto food color
(Levy et al., 1997). In this study, bixin was rapidly
absorbed in cat plasma, with peak absorption occur-
ring within 1 h. Although this is more rapid than that
seen in humans given bixin (Levy et al., 1997) or dogs
and cats given oral astaxanthin (Park et al., 2010b)
or -carotene (Chew et al., 2000b,a), the elimination
half-life was also much shorter for both bixin (1.82.2
h) and norbixin (1.82.8 h) compared with that seen in
human plasma, where baseline concentrations of bixin
were reached by 6 h after consumption. Astaxanthin
concentrations did not reach baseline until 21 h after
consumption (Park et al., 2010b). Daily administra-
132 Park et al.
tion of bixin resulted in a dose-dependent increase in
plasma bixin, which reached steady-state concentra-
tions within 24 h. This is remarkably different when
compared with the 12 wk for astaxanthin (Chew et al.,
2000a) and lutein (Park et al., 1999) to plateau.
In addition to differences in uptake kinetics be-
tween different carotenoids, there were also differ-
ences in distribution in lipoproteins and within leu-
kocytes. In humans, carotenoids such as -carotene,
lycopene, and -cryptoxanthin are predominantly as-
sociated with the LDL in plasma (Romanchik et al.,
1994), whereas the carotenoids lutein and zeaxanthin
are primarily transported by HDL (Wang et al., 2007).
High-density lipoproteins are the major lipoproteins
in the blood of dogs and cats, so it is not surprising
that the only lipoprotein with measurable astaxanthin
in both dogs and cats was the HDL fraction (Park et al.,
2010b). Bixin was also primarily associated with the
HDL fraction (84%) in cat blood, although there was
measurable bixin in both the LDL (14%) and VLDL
(2%) fractions on d 6. By d 14, there was a slight shift
in distribution between cat blood lipoprotein fractions
with 14% VLDL and 18% LDL, but the majority were
still associated with HDL (68%).
The dose-dependent uptake of bixin in blood
leukocytes on d 14 demonstrated that mitochondrial
bixin accounted for 61 to 82% of total bixin uptake,
dependent on oral dose, with additional uptake into
the microsomes and nuclei of up to 18 to 38% of to-
tal bixin but negligible uptake in the cytosolic frac-
tion. Mitochondria are a significant source of ROS
(Shigenaga et al., 1994), which may increase the
importance of bixin in preventing damage to mito-
chondrial membranes. Carotenoids reduce free radical
formation in lipid membranes (Machlin and Bendich,
1987). In fact, bixin may be more effective in main-
taining normal mitochondrial function and membrane
fluidity than astaxanthin and -carotene, because
compared with bixin, mitochondrial concentrations of
astaxanthin (42%) and -carotene (47%) were lower
(Chew et al., 2000a).
The risk of metabolic as well as cardiovascular dis-
eases increase with aging, which is frequently linked
with increased lipoperoxidation, inflammation, and pro-
duction of ROS (Veronica and Esther, 2012). The anti-
oxidant properties of bixin have been demonstrated in
cell-free in vitro models (Chist et al., 2011) and in im-
mune cell culture models and in retinal ganglion cells
(Rossoni-Jnior et al., 2012; Tsuruma et al., 2012). In
addition, aging induces immunosenescence, manifested
in impaired cell-mediated function, decreased Th sub-
populations, and decreased DTH response and ability
of lymphocytes to respond to mitogens (Day, 2010). In
this study, bixin appeared to influence immune param-
eters in a number of different ways. The DTH, which
was used to evaluate in vivo T lymphocyte function,
gave varied results in response to vaccine and ConA at
wk 12 and 16. It was previously shown that DTH re-
sponses after subdural injection with vaccine and mito-
gens were enhanced in cats receiving supplementation
with astaxanthin (Park et al., 2011) and -carotene (Park
et al., 2004). However, although DTH response to vac-
cine was also enhanced by lutein, there was no change
in response to mitogens (Kim et al., 2000). This is the
reverse of DTH results seen with bixin supplementation
after 16 wk, where response to mitogens was increased
24 h after subdural injection whereas response to vac-
cine was actually decreased 72 h after subdural injec-
tion, which again illustrates carotenoid-specific effects
on immunoregulation. Although the only significant
increase seen in T cell (Tpan and Th) percent was at wk
6, there was a tendency for increased Tpan as well as Th
at wk 12 and 16, which is consistent with an increased
DTH response. This increase in T cells has been ob-
served for astaxanthin, -carotene, and lutein (Kim et
al., 2000; Park et al., 2004, 2011). In addition, there was
a similar response to bixin seen with the other carot-
enoids in terms of increased production of plasma IgG.
Conversely, there was a concurrent decrease in B cell
subpopulations, which was not observed in response
to the other carotenoids. The acute phase protein AGP
also showed a significant increase in the high treat-
ment groups after 12 wk of supplementation. -1-Acid
glycoprotein has been shown to play an anti-inflamma-
tory and immunomodulatory role in immune function
(Hochepied et al., 2003) but inhibited ConA- and PHA-
induced proliferation response in vitro in humans. It is
therefore surprising that cats had an elevated lymphop-
roliferation response to ConA by wk 16.
Lastly, bixin had a significant impact on decreasing
DNA damage by ROS. Many researchers have shown
bixin to be an effective antioxidant in rats (Barcelos et
al., 2012; dos Santos et al., 2012). This is similar to the
inhibition of DNA oxidation seen in humans supple-
mented with astaxanthin (Park et al., 2010a).
Dog and cats have age-driven immunosenescent
changes similar to those seen in humans including
impaired cell-mediated function, decreased ability to
respond to mitogens, and decreased DTH response.
Aging is a response to not only programmed endog-
enous factors but a result of exogenous factors as well.
Dietary modification can slow or even reverse age-
related changes in immunocompetency (Day, 2010),
and the addition of a carotenoids, fatty acids, and pro-
biotics can facilitate this modification in aging effects
(Cupp et al., 2006; Day, 2010). This study provides
supporting evidence for the positive immunomodula-
tory and antioxidant effects of bixin in cats.
 Bixin uptake and immunomodulation in cats
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