Waltham International Symposium:

Pet Nutrition Coming of Age

Mineral and Trace Element Absorption from Dry Dog Food by Dogs,
Determined Using Stable Isotopes1,2
Peter Kastenmayer,3 Gail L. Czarnecki-Maulden* and Wayne King*
Nestle Research Center Lausanne, Lausanne, Switzerland and *Friskies Product Technology Center,
St. Joseph, MO
EXPANDED ABSTRACT

KEY WORDS:

minerals

absorption

stable isotopes

dogs

dog food

Experimental design
At the end of the equilibration period apparent Ca, Fe, Cu and Zn
absorption was determined from a single test meal. For each dog,
100 g of dog food was extrinsically labeled by pipetting tracer solutions containing 101 mg 44Ca (as CaCl2) and 0.72 mg 58Fe, 2.1 mg
65
Cu and 0.60 mg 70Zn (as sulfate) onto the slightly crushed kibbles.
Dysprosium (1.5 mg as DyCl3) was added as a nonabsorbable fecal
marker. Care was taken to make sure that all isotope solution was
taken up by the food. After all of the labeled food had been consumed, the remainder of the daily food portion was added to the bowls
and fed to the dogs; this ensured complete consumption of the
isotopes. After the meal, bowls were wiped with filter paper and
rinsed with distilled water to recover trace amounts of residual isotope. From the time of isotope administration complete feces were
collected for 5 d. A post sample was taken on d 6 to verify that all
nonabsorbed isotope had been excreted (i.e., isotope enrichment
returned to baseline). Fecal samples were collected in the kennels
several times a day and frozen until analysis. The protocol was
approved by the Friskies Pet Care Committee.

MATERIALS AND METHODS

Animals and diets
Fifteen beagles aged 9 15 y (7 males, 8 females) were fed a
standard dry dog food for 4 wk. Maintenance feeding amounts were
calculated using the equation: ME requirement 132 (body
weight in kg)0.75 as a guide. Because activity level and metabolic rate
vary between dogs, feeding amounts were adjusted to meet each
individual dogs needs. Food intake was measured daily throughout
the trial. The food supplied (dry matter basis) the following: 20.1%
protein, 11.1% fat, 1.5% fiber, 3.49 kcal/g metabolizable energy,
1.72% Ca, 218 mg/kg Fe, 14.5 mg/kg Cu and 148 mg/kg Zn. Water
was provided ad libitum throughout the trial.

Materials and analysis

Isotopically enriched elemental Fe (93.3%), Cu (99.6%), Zn
(95.8%) and 44Ca-enriched CaCO3 (96.9%) were obtained from
Chemgas (Boulogne, France). Metals were dissolved in 0.5 mol/L
H2SO4 to obtain the corresponding sulfates, and CaCO3 was dissolved in a stoichiometric amount of 3 mol/L HCl to give CaCl2.
Total Ca, Fe, Cu and Zn concentrations in tracer solutions were
determined by flame atomic absorption spectroscopy (FAAS) (SpectrAA 400; Varian, Mulgrave, Australia). Isotopic composition of
enriched isotopes was verified by inductively coupled plasma mass
spectrometry (ICP-MS) (Elan 6000; Perkin Elmer, Rotkreuz, Switzerland).
Fecal post samples and pooled feces for d 13 and d 4/5 were
freeze-dried and homogenized in a standard grinder (Compact robot;
Tefal, Selongey, France). Duplicate freeze-dried fecal samples (400
mg) and unconsumed food were ashed in silica Erlenmeyer flasks in a
muffle furnace (Gallenkamp Size 3; Kleiner, Wohlen, Switzerland) at
520C for 48 h. A 2-mL aliquot of 65% HNO3 was added and samples
were ashed overnight a second time. Ash was dissolved in 2.5 mL
65% HNO3 and diluted to 25 mL with ultrapure water. Total Ca, Cu
and Zn in samples was determined by inductively coupled atomic

1
Presented as part of the Waltham International Symposium: Pet Nutrition
Coming of Age held in Vancouver, Canada, August 6 7, 2001. This symposium
and the publication of symposium proceedings were sponsored by the Waltham
Centre for Pet Nutrition. Guest editors for this supplement were James G. Morris,
University of California, Davis, Ivan H. Burger, consultant to Mars UK Limited, Carl
L. Keen, University of California, Davis, and DAnn Finley, University of California,
Davis.
2
Supported by Nestle Research Center and Friskies Product Technology
Center, Nestec Ltd.
3
To whom correspondence should be addressed.
E-mail: peter.kastenmayer@rdls.nestle.com.

0022-3166/02 $3.00 2002 American Society for Nutritional Sciences. J. Nutr. 132: 1670S1672S, 2002.

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To date only very limited data on bioavailability of minerals and trace elements in dogs fed dog food are available,
although such information is essential for establishing correct
mineral requirements (1). This is partially attributable to
difficulties (precision of analysis, contamination problems)
encountered using the standard balance techniques. We have
determined apparent fractional absorption of Ca, Fe, Cu and
Zn from a standard dry dog food in Beagles using the fecalexcretion stable-isotope technique. The stable-isotope technique provides precise results and minimizes errors incurred as
a result of endogenous excretion (2). To compare standard
methodology to the stable isotope technique, apparent Ca
digestibility was also determined using a standard digestibility
trial.

MINERAL AND TRACE ELEMENT ABSORPTION IN DOGS

Calculations
Fractional apparent absorption of 44Ca, 58Fe, 65Cu and 70Zn was
calculated based on 5-d excretion of the isotopes using total fecal
element content and isotope ratios, as described by Turnlund and
coworkers (4). Tracer amounts recovered in feces were corrected for
a Dy recovery 100%, if required (5). Apparent digestibility of Ca
was calculated based on total amount of Ca in diet and feces collected
during the 5-d digestibility period.

RESULTS AND DISCUSSION

Mean Dy recovery ( SEM) in fecal pools was 100.8
3.1%. Dy found in post samples and enrichment of 44Ca,
58
Fe, 65Cu and 70Zn were negligible, indicating an adequate
fecal collection time. Mean fractional absorption for Ca, Fe,
Cu and Zn and amount of mineral absorbed are given in Table
1. With the exception of Cu, absorption values found were
comparatively low. This might be ascribed to high total element intake. For example, the calcium level in the diet used in
this study was more than double the recommended minimum
level of calcium for dogs (6). Ca absorption in dogs was
previously found to be quite variable (0 90%), depending on
Ca content and composition of food (7). In general, calcium

TABLE 1
Fractional absorption and total amount of Ca, Fe, Cu and Zn
absorbed from test meal1
Element
Ca
Fe
Cu
Zn

Intake
(mg)
4300
54.5
3.63
37.0

Absorption2
(%)

Absorbed amount
(mg)

10.1 1.1
8.8 2.1
23.1 2.0
11.5 1.4

434
4.8
0.8
4.3

1 Based on 250 g food intake.

2 Mean SEM.

TABLE 2
Comparison of calcium digestibility based on standard
and stable isotope procedures
Procedure
Stable isotope
Standard digestibility

Digestibility
(%)

Standard
deviation

Coefficient of
variation

10.1
4.8

4.1
7.5

41.1
155.8

digestibility decreases as calcium intake increases. Nap and

coworkers (8) reported that Ca absorption in growing Miniature Poodles fed a low Ca diet (0.05 or 0.33%) was 70 96%.
At dietary Ca levels of 1.1 or 3.3% digestibility was reduced to
28 53%. Hazewinkel and coworkers (9) studied Ca metabolism in Great Dane dogs fed dry dog food with various Ca and
P levels. Ca absorption in a group of control dogs fed normal
Ca and P levels (1.1% Ca and 0.9% P) was 45 66%. At high
(3.3%) and low (0.55%) Ca levels in the diet, Ca absorption
changed to 23 43% and 70 97%, irrespectively, of the P
content.
In contrast to Ca, only scanty information on Fe, Cu and
Zn absorption in dogs is currently available in the literature.
Lipschitz and coworkers (10) measured absorption of nonheme
Fe from a human mixed meal and a semisynthetic meal in
adult mongrel dogs using radioisotopes. Fe absorption was 19.3
and 7.1%, respectively, and varied widely among animals, an
observation also made in human studies. Fe absorption for the
purified diet was thus similar to the value we obtained for Fe
absorption from dry dog food. Lowe and coworkers (11) compared absorption of ZnO and an amino-acid chelate from dry
dog food containing 56 mg/kg Zn by measuring plasma-appearance curves of 65Zn. Absorption for the Zn amino-acid chelate
was 88% and twice as high as that for ZnO. The fact that we
observed a much lower Zn absorption for inorganic Zn in this
study (11.5%) can probably be explained by the threefold
higher Zn intake and possible differences in the methodology
used to assess Zn absorption. In other species, such as pig and
rat, mineral bioavailability from different diets has been established. Zinn and coworkers (12) measured the effect of iron
supplementation on apparent Ca, Cu and Zn absorption in
young piglets using radioisotopes. A cereal-based diet containing 9% Ca, 100 mg/kg Zn and 8.3 mg for Cu was fortified with
100 mg/kg Fe. Values obtained for apparent absorption of Ca,
Fe, Cu and Zn were 53, 26, 42 and 26%, respectively, and
considerably higher than the values we found for adult dogs.
Coudray and coworkers (13) measured Cu and Zn absorption
in adult Wistar rats by applying a stable-isotope technique. In
this study Cu and Zn absorption from the purified diet fed was
13.4 and 19.3%, respectively.
Ca digestibility was approximately 5% lower than apparent
Ca stable isotope absorption (Table 2). This is most likely the
result of endogenous losses of Ca that are not corrected for in
standard digestibility trials. Variability was much higher when
Ca absorption was estimated using the standard digestibility
technique rather than the stable-isotope technique. The stable-isotope technique thus provided a more precise method of
measuring Ca absorption in dogs.
In conclusion, we have shown in the present study that
apparent absorption of Ca, Fe and Zn, but not Cu, from
commercial dry dog food by adult dogs is low. The low absorption observed for Ca, Fe and Zn should be taken in account
when recommendations for requirements of these elements are
established.

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emission spectrometry (ICP-AES). Total Fe was analyzed by FAAS

and Dy by ICP-MS. Accuracy of Ca, Fe, Cu and Zn determinations
was verified by analyzing the NIST standard reference materials
Typical Diet (SRM1548a), Bovine Liver (SRM1577b) and a pooled
fecal sample as laboratory standard.
Isotope enrichment of the tracer isotopes in feces was determined
by ICP-MS. The 65Cu/63Cu isotope ratio was measured directly in
mineralized samples diluted to give a Cu concentration of 100 ppb in
0.1 mol/L HNO3. Ca, Fe and Zn ratios were determined after separation of elements from matrix elements. Ca was precipitated using
ammonium oxalate; Fe and Zn were purified using anion-exchange
chromatography as described previously (3). The 44Ca/43Ca and
58
Fe/56Fe ratios were measured using cool plasma conditions (600 W)
at a concentration of 7.5 mg/L for Ca and 1 mg/L for Fe. The
70
Zn/68Zn isotope ratio was determined using normal plasma conditions and a concentration of 0.5 mg/L. Instrumental mass bias was
corrected for by analyzing isotope ratios of Ca, Fe, Cu and Zn
standards solutions (AAS standard; Merck, Darmstadt, Germany)
with natural isotopic composition. After correction for instrumental
mass bias, isotope ratios of samples with natural isotopic composition
were within 1% of accepted IUPAC values. To verify the accuracy of
enrichments measured in feces, standards with known enrichment in
44
Ca, 58Fe and 70Zn were analyzed together with the samples. Good
agreement was found between calculated and measured enrichments.
All acids used were purified by subboiling distillation. Other
chemicals were analytical-grade purity. All materials used for sample
collection and storage were acid washed in 1 mol/L HNO3 for 24 h
followed by rinsing in ultrapure water. Ultrapure water (18 M)
came from a Millipore system (Millipore AG, Zurich, Switzerland).

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SUPPLEMENT

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ACKNOWLEDGMENT
We thank Peter Zeltner and Susanne Berger (Nestle Product
Technology Center Konolfingen, Switzerland) for the ICP-AES measurements and Mario Vigo for expert technical assistance.

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