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*
Department of Animal Sciences, Washington State University, Pullman, Washington and
The Iams Company, Lewisburg, Ohio, 45338
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: • ß-carotene • uptake • canine • plasma • leukocytes
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Others (Baker et al. 1986
,
Frohring 1935
, Steenbock et al. 1921
,
Turner 1934
) have reported low to moderate
concentrations of ß-carotene in the blood of the domestic canine. To
date, no systematic studies are available to address the biokinetic
uptake of dietary ß-carotene in the domestic dog. Interest in the
role of ß-carotene and other carotenoids on improving immunity and
health have increased in recent years. Indeed, ß-carotene has been
shown to enhance immunity (Chew 1995a
), to increase
reproduction (Chew et al. 1995b
) and to prevent cancer
(Weisburger 1991
) and atherosclerosis (Esterbauer et al. 1989
). For example, ß-carotene increased the number of
T helper cells (Alexander et al. 1985
), increased the
expression of IL-2 receptors on natural killer cells (Prabhala et al. 1989
) in humans and enhanced the proliferation and
induction of cytotoxic T cells in mice (Seifter et al. 1982
). Dietary ß-carotene similarly enhanced both humoral and
cell-mediated immune responses in the dog (Chew et al. 1998
). Also, there is a continuous effort to identify animal
species that will be appropriate models for studying carotenoid
absorption and metabolism in the human. So far, the use of the
preruminant calves (Hoppe et al. 1996
, Poor et al. 1992
), ferrets (Gugger et al. 1992
,
Ribaya-Mercado et al. 1989
) and rhesus monkeys
(Krinsky et al. 1990
) has been proposed. Our objectives are to study the uptake of dietary ß-carotene by blood plasma and leukocytes in the domestic canine, with the long-term objective of understanding the possible role of ß-carotene on health, and to investigate the suitability of domestic dogs as a model for the study of ß-carotene absorption and metabolism in humans.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Female beagle dogs (18–19 mo old, 7–9 kg body weight; Marshall Farms
USA, North Rose, NY) were used in all experiments and were fed a basal
diet (The Iams, Lewisburg, OH) that met or exceeded the requirement for
all essential nutrients (NRC 1985
). The diet composition
was as follows (g/kg): 66.2 moisture, 262 protein, 74.5 ash, 160 fat,
14.8 Ca, 10.3 P and 437.3 nitrogen-free extract. The vaccination
history for the dogs included commercial vaccines to parvovirus,
distemper, papilloma, parainfluenza, leptospirosis, adenovirus and
rabies. During the study, all dogs were housed in 2 x 2 m
pens (2 dogs/pen) in a temperature- (20–22°C) and light- (14 h
light) controlled facility. The study was approved by the Washington
State University Institutional Animal Care and Use Committee. Three
experiments were conducted to study the biokinetic uptake of
ß-carotene by blood plasma and leukocytes after a single dose or
multiple doses of ß-carotene.
Experiment 1.
To study the plasma uptake of ß-carotene in dogs given a single oral
dose of ß-carotene, dogs (n = 6) were given a single perorally
0, 50, 100 or 200 mg of ß-carotene (10% cold water dissolvable;
BASF, Ludwigshafen, Germany). Immediately prior to dosing, ß-carotene
was dissolved in 5 mL of water and fed orally by using a feeding
syringe. Blood samples were taken from the jugular vein at 0
(immediately prior to ß-carotene feeding), 1.5, 3, 6, 10, 18 and
24 h. These times were selected based on preliminary study using
three dogs. Plasma was stored at -80°C and the contents of
ß-carotene, retinol and 
-tocopherol were subsequently analyzed
using HPLC (described later).
Experiment 2.
To study ß-carotene uptake from repeated doses, dogs (n = 6) were fed daily at 0800 h for 7 d with 0, 12.5, 25, 50 or 100 mg ß-carotene. These doses were selected based on results obtained from expt. 1. To study ß-carotene uptake in dogs that were fed for a longer period, ß-carotene was similarly given for 30 d to dogs in the 50 and 100 mg groups with blood taken on d 10, 20 and 30 of feeding. Blood samples were taken from the jugular vein at 6 h after each dose because results from expt. 1 showed peak concentrations of ß-carotene at this time.
Experiment 3.
This experiment was designed to study the uptake of ß-carotene by
blood lymphocytes and neutrophils and their subcellular fractions. Dogs
(n = 8) were fed 0, 50 or 100 mg of ß-carotene daily for 30 d. Blood was taken from the jugular vein on d 0, 10, 20 and 30 for HPLC
analysis of ß-carotene concentrations in the plasma, lymphocytes and
neutrophils. Blood lymphocytes and neutrophils were separated by
density gradient centrifugation (Chew et al. 1993
) and
cell purity assessed by using a Wright stain smear. Cell numbers were
enumerated using a particle counter (Coulter Electronics, Hialeah, FL),
and cells were resuspended in PBS containing 30 g/L sodium ascorbate
(Sigma Chemical, St. Louis, MO) as an antioxidant. Prior to HPLC
analysis, cells were disrupted by sonication (30 s), and the leukocyte
homogenates were extracted for the HPLC analysis of ß-carotene
content in whole cells. An aliquot of leukocyte homogenate from d 30
was used to quantitate ß-carotene content in subcellular fractions as
previously described (Chew et al. 1993
). Briefly, cells
were disrupted in 0.25 m sucrose by sonication and the homogenates were
centrifuged to obtain the nuclear (600 x g for 10
min at 4°C), mitochondrial (17,300 x g for 20
min at 4°C), microsomal (102,000 x g for 60 min
at 4°C) and cytosolic fractions. Each subcellular fraction was
analyzed by HPLC. All HPLC (Alliance 2690 Waters HPLC system fitted
with a photodiode array detector, Waters, Milford, MA) procedures were
as previously described (Park et al. 1998
).
trans-ß-apo-8'carotenal (Sigma Chemical, St. Louis,
MO) was used as the internal standard.
Data were analyzed by repeated sampling analysis of variance using the
General Linear Models Procedure of SAS (1991)
.
The statistical model was Yijk = µ +
Treatmenti +
Dogj(Treatmenti) (error A
used to test the effects of treatment) + Sampling
periodk + Treatmenti
· Periodj + eijk
(error B). Differences among treatment means within a sampling period
were compared by a protected least significant difference test.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Plasma ß-carotene was undetectable in unsupplemented dogs at all time
periods and in all three experiments. In contrast, there was a
dose-dependent increase (P < 0.01) in plasma
ß-carotene levels in dogs given a single oral dose of ß-carotene
(Fig. 1
). Peak concentrations were observed at 6 h postdosing and were
consistent in all treatment groups. Thereafter, there was a rapid
decrease (P < 0.01) in ß-carotene concentrations in
all ß-carotene supplemented dogs. In general, plasma ß-carotene
concentrations were similar in dogs fed 50 and 100 mg ß-carotene.
However, dogs fed 200 mg ß-carotene had higher (P < 0.05) plasma ß-carotene levels at 3, 6 and 10 h than those fed
50 or 100 mg ß-carotene. The half-life of plasma ß-carotene was
~3 h (50 and 100 mg) to 4 h (200 mg). Concentrations in all
treatment groups had returned to baseline values at 24 h
postdosing. No dogs could be considered as nonresponders to dietary
ß-carotene.
|
). The concentration (0.033 µmol/L; Fig. 2
) of plasma ß-carotene on
d 1 in dogs fed 100 mg ß-carotene was similar to that observed in
dogs fed the same amount of ß-carotene in expt. 1 (Fig. 1)
. Dogs fed
12.5 or 25 mg ß-carotene showed similar plasma ß-carotene
concentrations during the study, and plasma concentrations were ~0.02
µmol/L on d 7. Plasma ß-carotene levels continued to increase
through d 7 in dogs given 50 or 100 mg ß-carotene. Concentrations
after the last dose were approximately twofold higher than those
observed after the first dose. Plasma ß-carotene concentrations
continued to increase through d 30 of supplementation in dogs fed 50 or
100 mg ß-carotene (Table 1
).
|
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-tocopherol in all experiments. Plasma
concentrations of retinol and -tocopherol averaged 3.2 ± 0.12
and 43.6 ± 5.4 µmol/L, respectively, across all three
experiments. Uptake by blood leukocytes.
As with plasma, ß-carotene was not detectable in whole lymphocytes
and neutrophils prior to ß-carotene supplementation or in
unsupplemented dogs (Table 1)
. In contrast, dogs fed ß-carotene
showed significant (P < 0.05) uptake of ß-carotene
by both lymphocytes and neutrophils. ß-Carotene concentrations in
lymphocytes increased through d 30 in ß-carotene–supplemented dogs
where concentrations were ~2.5 times higher than on d 10. There was
no significant difference in lymphocyte ß-carotene concentrations,
during any of the sampling periods, between dogs fed 50 or 100 mg
ß-carotene. In contrast to lymphocytes, ß-carotene concentrations
in neutrophils remained constant between d 10 and d 30 and were not
different in dogs fed 50 or 100 mg ß-carotene.
ß-Carotene was not detectable in all lymphocyte subcellular fractions
from unsupplemented dogs and in supplemented dogs prior to ß-carotene
feeding (Table 1)
. In contrast, ß-carotene was taken up by all
subcellular fractions of blood lymphocytes isolated from
ß-carotene–supplemented dogs. There was no significant difference in
ß-carotene distribution among the lymphocyte subcellular fractions
between dogs fed 50 or 100 mg ß-carotene. The ß-carotene in the
cytosol fraction accounted for 52–62% of the total ß-carotene in
the lymphocytes whereas the mitochondria and microsomes contained
~14–23% of total ß-carotene. ß-Carotene was lowest in the
nuclear fraction.
As in lymphocytes, the cytosolic fraction of blood neutrophils
contained the highest proportion of total ß-carotene (Table 1)
.
Again, the mitochondria and microsomal fractions accounted for 12–23%
of total ß-carotene in the neutrophils whereas no ß-carotene was
detected in the nuclear fraction. There was no significant difference
in neutrophil subcellular ß-carotene content between dogs fed 50 or
100 mg ß-carotene.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, Frohring 1935
,
Steenbock et al. 1921
, Turner 1934
) or
undetectable concentrations in wild canids (Slifka et al. 1999
). In this study, peak plasma ß-carotene concentration in
dogs after a single dose of ß-carotene was observed ~6 h
postfeeding. The timing of this peak is earlier than observed in
preruminant calves (12–30 h) (Poor et al. 1992
), humans
(24–48 h) (Brown et al. 1989
, Cornwell et al. 1961
), ferrets (Gugger et al. 1992
,
Ribaya-Mercado et al. 1989
) and cats (12 h) (Chew et al. 1997
). Also, the peak concentration of plasma
ß-carotene in dogs after a single 200-mg dose (~14 mg
ß-carotene/kg body weight) was 0.065 µmol/L; this is ~10-fold
lower than in preruminant calves given 0.44 mg ß-carotene/kg body
weight (Poor et al. 1992
), ferrets given 10–20 mg
ß-carotene/kg body weight (Gugger et al. 1992
,
Ribaya-Mercado et al. 1989
) and the domestic cat given
1.5–3 mg ß-carotene/kg body weight (Chew et al. 1997
). However, plasma ß-carotene concentrations in dogs
reached 0.2–0.3 µmol/L after 30 d of oral supplementation;
plasma saturation could not be determined from this study. However,
Chew et al. (1998)
reported blood ß-carotene
concentrations of 0.4–0.6 µmol/L in beagle dogs fed 50 mg
ß-carotene daily for 8 wk.
Plasma ß-carotene in dogs decreased rapidly to reach prefeeding
concentrations by 24-h postfeeding. In contrast, plasma ß-carotene in
humans (Kubler 1969
), preruminant calves (Poor et al. 1992
) and ferrets (Gugger et al. 1992
)
decreased much more gradually after a single dose.
Circulating ß-carotene was taken up in significant concentrations by
blood lymphocytes and neutrophils. Maximal ß-carotene uptake by
lymphocytes occurred around d 30 whereas in neutrophils uptake occurred
by d 10. Maximal uptake of ß-carotene was already achieved with the
lower (50 mg ß-carotene) dietary dose. ß-Carotene uptake by
lymphocyte subcellular fractions in dogs is similar to that reported
with cats (Chew et al. 1997
), calves (Chew et al. 1993
) and pigs (Chew et al. 1991a
and 1991b
).
Uptake of ß-carotene by blood neutrophils was similar in humans
(Mathews-Roth 1978
) but not in calves (Chew et al. 1993
) and pigs (Chew et al. 1991a
).
ß-Carotene was highest in the cytosol fraction of dog lymphocytes
(this study) but was highest in the mitochondria of calves (Chew et al. 1993
), cats (Chew et al. 1997
) and in the
nuclei of pigs (Chew et al. 1991b
). Mitochondrial and
microsomal fractions individually still account for 15–20% of total
cellular ß-carotene in lymphocytes and neutrophils. Whether the
relative uptake of ß-carotene by different subcellular fractions
reflects cellular requirements by various compartments is not known. It
is known that mitochondria are required for ATP production and
constitute the most important source of reactive oxygen species
(Shigenaga et al. 1994
). In fact, the mitochondria
electron transport system utilizes ~85% of the oxygen consumed by
cells. Decreased mitochondrial and plasma membrane potential appears to
suppress mitogen responsiveness whereas increasing mitochondrial ATP
production enhanced cell-mediated immunity (Shigenaga et al. 1994
).
In summary, beagle dogs can absorb dietary ß-carotene which is
subsequently taken up by peripheral blood lymphocytes and neutrophils.
In the blood leukocytes, ß-carotene is distributed in all subcellular
organelles. The physiological importance of these findings is not
known. However, it is likely that ß-carotene may serve to protect the
immune cells against mitochondrial dysfunction and membrane rigidity.
Obvious differences in blood uptake kinetics of ß-carotene in dogs
compared to humans makes the beagle dog an unsuitable model for
studying the absorption and metabolism of ß-carotene in the human.
However, there are >53 million dogs in the United States alone
(Wise 1991
) and dogs have become an important companion
animal. In addition, information contained within can be useful in
understanding the nutrition of wild endangered canids.
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FOOTNOTES |
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2 To whom reprint requests should be addressed.
Manuscript received January 6, 2000. Revision accepted March 6, 2000.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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