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Department of Animal Sciences, Washington State University, Pullman, WA and * The Iams Company, Lewisburg, OH
2To whom correspondence and reprint requests should be addressed.
 |
ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: • ß-carotene • uptake • cats • blood • leukocytes
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), reproduction (Chew et al. 1995b
), cancer
(Weisburger 1991
) and atherosclerosis (Esterbauer et al. 1989
). For instance, ß-carotene increased the number
of T-helper cells (Alexander et al. 1985
), increased
the expression of interleukin-2 receptors on natural killer cells
(Prabhala et al. 1989
) in humans, and enhanced cytotoxic
T-cell proliferation and induction in mice (Seifter et al. 1982
). Dietary ß-carotene similarly enhanced both humoral and
cell-mediated immune responses (Chew et al. 2000b
).
Because of the importance of ß-carotene in health, efforts to
identify animal models appropriate for studying carotenoid absorption
and metabolism in humans have continued. Species suggested as suitable
models include 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
). Little is known
concerning the use of domestic cats for such studies. The cat is a
useful research model for studying various human genetic and metabolic
anomalies (Migaki 1982
, O’Brien 1993
),
and in applying assisted reproduction to endangered felid species
(Wildt et al. 1986
). There are >57 million cats in the
U.S. alone (Wise 1991
) and cats have become an important
part of the human life experience. Unfortunately, no systematic study
is available at present on the absorption and transport of ß-carotene
in domestic cats. Cats are unique in that they cannot utilize
ß-carotene for vitamin A synthesis (Lakshman et al. 1972
). ß-Carotene concentrations in the blood of exotic
felids are generally high even though their diets contain low amounts
of carotenoids (Slifka et al. 1999
). Our objective was
to evaluate the kinetic uptake of ß-carotene into blood plasma and
leukocytes in domestic cats supplemented with oral ß-carotene. |
MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Mature female short-haired Tabby cats (12 mo of age; 3–3.5 kg body
weight; Liberty Research, Waverly, NY) had free access to a basal diet
(Table 1
; The Iams, Lewisburg, OH) that was balanced for all essential
nutrients. The diet composition was as follows (g/kg): 72 moisture, 310
protein, 52 ash, 217 fat, 14 crude fiber, 11.5 Ca and 8.3 P. Cats were
group-housed indoors in light- (14 h light, 10 h dark) and
temperature-controlled (20–22°C) rooms. The research protocol
was approved by the Washington State University Institutional Animal
Care and Use Committee. Three experiments were conducted to study the
uptake profile of ß-carotene after a single oral dose or after
multiple oral doses of ß-carotene.
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To study plasma uptake of ß-carotene after a single oral dose, cats
(n = 6/treatment) were administered perorally 0,
10, 20 or 50 mg ß-carotene (cold water dissolvable; BASF,
Ludwigshafen, Germany). The appropriate dose of ß-carotene was
dissolved in 0.6 mL of water and fed with a syringe. Blood was sampled
from the jugular vein at 0, 12, 24, 30, 36, 42, 48 and 72 h after
dosing. These sampling times were chosen on the basis of results from a
preliminary study using three cats administered 50 mg ß-carotene
perorally. Plasma was analyzed for ß-carotene, retinol and
-tocopherol content by HPLC.
Experiment 2.
Cats (n = 6/treatment) were dosed perorally with 0, 1, 2, 5 or 10 mg ß-carotene at 0800 h daily for six consecutive days to study ß-carotene uptake from repeated doses. Blood was sampled once daily 12 h after each feeding (d 0 = immediately before ß-carotene feeding) because Experiment 1 showed peak blood ß-carotene concentrations 12 h postdosing. Also, lower oral doses were selected in this study because Experiment 1 showed high uptake of oral ß-carotene.
Experiment 3.
This experiment was designed to study the uptake of ß-carotene by
blood leukocytes. Cats (n = 8/treatment) were fed
0, 5 or 10 mg of ß-carotene daily for 14 d. Blood was sampled
from the jugular vein on d 7 and 14; lymphocytes and neutrophils were
separated by using percol (Histopaque 1077 and 1072, Sigma Chemical,
St. Louis, MO; Chew et al. 1993
). However, these
separation procedures failed to yield consistently pure populations of
lymphocytes and neutrophils. Therefore, the blood leukocytes were
pooled and analyzed as total leukocytes. Whole-leukocyte content
and leukocyte subcellular fractions were prepared for HPLC analyses as
previously described (Chew et al. 2000a
). Plasma and
leukocytes were extracted and analyzed using the Alliance 2690 Waters
HPLC system fitted with a photodiode array detector (Waters, Milford,
MA). trans-ß-Apo-8'carotenal (Sigma Chemical) was used
as the internal standard.
Statistical analysis.
Treatment differences across sampling periods were analyzed by
repeated-measures ANOVA using the General Linear Models procedure
of SAS (1991)
. The statistical model was
Yijk = µ +
treatmenti +
catj(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
and considered significant at P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Concentrations of plasma ß-carotene were undetectable in
unsupplemented cats in all experiments. In contrast, plasma
ß-carotene concentrations in cats given an oral dose of ß-carotene
increased (P < 0.01) in a dose-dependent manner
(Fig. 1
). Peak plasma concentrations were observed at 12 h (10 and 20 mg
ß-carotene) to 24 h (50 mg ß-carotene) after the dose.
However, analysis of ß-carotene uptake by individual cats revealed
peak concentrations at 30 h in some cats. Cats fed 50 mg
ß-carotene had peak plasma ß-carotene concentrations 
100%
higher than those given 20 mg ß-carotene. Subsequently, plasma
ß-carotene concentrations declined (P < 0.01)
gradually and reached baseline concentrations by 72 h in cats fed
10 or 20 mg ß-carotene. However, plasma ß-carotene was still
significantly higher (P < 0.05, 0.23
µmol/L) at 72 h in cats fed 50 mg ß-carotene
compared with unsupplemented cats. The half-life of ß-carotene in
the plasma ranged from 12 h (10 mg ß-carotene) to 20 h (20
and 50 mg ß-carotene).
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). However, two cats fed 5 mg ß-carotene did not show significant
changes in plasma ß-carotene concentrations; these cats were,
therefore, deleted from the final data analysis. It was interesting to
note that both cats showed significant increases in plasma ß-carotene
when a higher oral dose (20 mg) was administered subsequently. Cats fed
10 mg ß-carotene had the greatest relative increase in plasma
ß-carotene concentration. Plasma ß-carotene had not reached
saturation by d 6, and concentrations across d 1–6 averaged 9, 9 and
1.3 times higher in cats fed 10 mg ß-carotene compared with those fed
1, 2 or 5 mg ß-carotene, respectively. These relative values
generally were similar when concentrations were compared on d 6 alone
(10.4, 6.6 and 1.3 times, respectively). In this study, the plasma
concentration of ß-carotene (0.36 ± 0.05 µmol/L)
after the first dose in cats fed 10 mg ß-carotene was similar to that
observed in Experiment 1 when blood was taken at 12 h postdosing
(0.42 ± 0.11 µmol/L, Fig. 1
).
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In Experiment 3, the plasma concentration of ß-carotene on d 14 in
cats fed 5 or 10 mg ß-carotene (Table 1)
was higher than that
observed on d 6 (Experiment 2, Fig. 2
). This indicates a continued
increase in plasma ß-carotene after 6 d of feeding. On d 14,
plasma ß-carotene concentration in cats fed 10 mg ß-carotene was
60% higher than that in cats fed 5 mg ß-carotene; this
relationship is similar to concentrations observed during the first
6 d of feeding (Fig. 2)
. ß-Carotene feeding did not influence
plasma -tocopherol and retinol concentrations, which were 20.9
± 0.6 and 0.69 ± 0.02 µmol/L, respectively.
ß-Carotene was undetectable in peripheral blood leukocytes in all
unsupplemented cats (Table 1)
. On d 7, there was a significant
(P < 0.01) uptake of ß-carotene by blood leukocytes.
However, no additional accumulation of ß-carotene was apparent with
higher oral doses (10 mg ß-carotene) or with longer ß-carotene
supplementation (Table 1)
.
Upon fractionation of the peripheral blood leukocytes, ß-carotene was
found to accumulate in all subcellular fractions (Table 1)
. On d 7,
concentrations of ß-carotene in the leukocytes were highest in the
mitochondria (45–49% of total leukocyte ß-carotene) and somewhat
lower in the microsomes (20–35%) (Table 1)
. The nuclear fraction
contained 5% of total leukocyte ß-carotene. There was no
difference in ß-carotene concentration in cats fed 5 or 10 mg
ß-carotene. Also, there was no further increase in ß-carotene
concentration in any subcellular fraction on d 14 compared with d 7.
The maximal uptake that was observed on d 7 in cats fed 5 mg
ß-carotene was in agreement with changes observed in whole leukocytes
and in plasma.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), and in preruminant calves
(Hoppe et al. 1996
, Poor et al. 1992
). In
this study, peak concentrations of plasma ß-carotene in cats were
observed 12–24 h after a single oral dose of ß-carotene. This is
similar to peak concentrations reported in humans (24–48 h;
Brown et al. 1989
, Cornwell et al. 1961
)
and preruminant calves (12–30 h, Poor et al. 1992
).
Also, peak concentration of ß-carotene in cats (0.4–0.7
µmol/L) given a single dose of 1.5 to 3 mg ß-carotene/kg
body weight was similar to that reported in preruminant calves (0.5
µmol/L) given 0.44 mg ß-carotene/kg body (Poor et al. 1992
), and ferrets (0.7 µmol/L) fed a single
dose of 10 mg ß-carotene/kg body (Gugger et al. 1992
).
In cats, the concentration of plasma ß-carotene decreased gradually
after a single dose, to reach baseline concentrations by 72 h
after the dose. The rate of decrease in plasma ß-carotene
concentration after the initial peak in cats was similar to that
observed in ferrets (75 h, Gugger et al. 1992
) but
shorter than that in preruminant calves (240 h; Poor et al. 1992
) and humans (Kubler 1969
).
Plasma ß-carotene concentrations continued to increase in a
dose-dependent manner through d 14. However, maximal uptake of
ß-carotene by whole leukocytes and their subcellular fractions was
observed by d 7 in cats fed 5 mg ß-carotene. A higher oral dose (10
mg ß-carotene) or a longer feeding period (14 d) did not result in
further increase in ß-carotene uptake. In the leukocyte subcellular
fractions, ß-carotene concentrations were highest in the
mitochondria, accounting for 40–52% of the total ß-carotene. The
microsomes took up 16–22%, whereas the nuclei accounted for 2–6%
of the total ß-carotene. These results are similar to those reported
in calves (Chew et al. 1993
). In that study, calves
administered ß-carotene orally showed maximal uptake of ß-carotene
in each of the subcellular fractions on d 7; the highest proportion of
total ß-carotene in the leukocytes were found in the mitochondria.
Uptake of ß-carotene by blood leukocytes and leukocyte subcellular
fractions also has been reported in humans (Mathews-Roth 1978
), dogs (Chew et al. 2000a
), calves
(Chew et al. 1993
) and pigs (Chew et al. 1991
). High concentrations of ß-carotene in the mitochondria
are of particular importance in that the mitochondrial electron
transport system utilizes 85% of the oxygen consumed by the cell to
produce ATP. Thus, the mitochondria constitute the most important
source of reactive oxygen species (Shigenaga et al. 1994
) whose overproduction can damage cell membranes, DNA and
other subcellular structures. Decreased mitochondrial and plasma
membrane potential appears to suppress mitogen responsiveness, whereas
increasing mitochondrial ATP production enhances cell-mediated
immunity (Shigenaga et al. 1994
). Machlin and Bendich (1987)
reported that ß-carotene reduced free radical
formation in lipid membranes, lysosomes and endoplasmic reticulum in
normal tissues. Therefore, the intracellular accumulation of
antioxidants, in this case ß-carotene, can maintain normal
mitochondrial function, membrane fluidity and nuclear DNA integrity.
Results in this study contradict an earlier report that indicated that
domestic cats are unable to absorb dietary ß-carotene (Ahmad 1931
). However, exotic felids (jaguars and bobcats) have
similarly shown high concentrations of blood ß-carotene (0.1–0.7
µmol/L) in spite of being fed diets seemingly low in
carotenoids (Slifka et al. 1999
). Cats do not possess
the necessary intestinal enzyme to convert ß-carotene to vitamin A
(Lakshman et al. 1972
). Lack of ß-carotene cleavage
enzymes has been suggested as an explanation for the presence of high
concentrations of ß-carotene in the general circulation. However, it
is very unlikely that this physiologic difference can directly account
for the cat’s ability to absorb ß-carotene. For instance, humans,
cattle, pigs and rodents possess active intestinal ß-carotene
cleavage enzyme systems. Yet, only humans and cattle have high
concentrations of blood ß-carotene. Thus, the absorption of
ß-carotene from the intestinal mucosa is more likely due to the
presence of a specific intestinal ß-carotene transport mechanism.
Two cats fed 5 mg ß-carotene for six consecutive days did not show a
significant increase in concentrations of plasma ß-carotene. However,
these cats responded to a subsequent higher single dose of 20 mg
ß-carotene. It is possible that humans (Dimitrov et al. 1988
) or animals (Poor et al. 1992
) initially
classified as "low responders" may respond to higher doses of
ß-carotene. This remains to be demonstrated. On the other hand,
others have failed to report "low responders" in preruminant calves
(Hoppe et al. 1996
) and Beagle dogs (Chew et al. 2000a
).
In summary, these studies provide the first available evidence that domestic cats can readily absorb ß-carotene across the intestinal mucosa. ß-Carotene also is taken up by peripheral blood leukocytes and is distributed into subcellular organelles, notably the mitochondria. In the leukocytes, ß-carotene may play an important role in maintaining their structural and functional integrity. Therefore, some aspects of the biokinetic uptake profile of ß-carotene in domestics cat are similar to those of humans. This similarity suggests that domestic cats may be an appropriate animal model for studying ß-carotene absorption and metabolism in humans. Furthermore, the present data suggest a similarity between domestic cats and endangered wild felids in carotenoid uptake.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Manuscript received February 29, 2000. Revision accepted April 11, 2000.
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