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Nestlé Research Center Lausanne, Lausanne, Switzerland and * Friskies Product Technology Center, St. Joseph, MO
3To whom correspondence should be addressed. E-mail: peter.kastenmayer{at}rdls.nestle.com.
KEY WORDS: • minerals • absorption • stable isotopes • dogs • dog food
EXPANDED ABSTRACT
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 fecal-excretion 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.
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MATERIALS AND METHODS |
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 TOP MATERIALS AND METHODS RESULTS AND DISCUSSIONLITERATURE CITED |
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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 x (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 dog’s 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.
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 65Cu 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 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 1–3 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 520°C 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 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 58Fe/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 70Zn/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 44Ca, 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).
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.
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RESULTS AND DISCUSSION |
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TOPMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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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.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Supported by Nestlé Research Center and Friskies Product Technology Center, Nestec Ltd.
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LITERATURE CITED |
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TOPMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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1. Morris, J. G. & Rogers, Q. R. (1994) Assessment of the nutritional adequacy of pet foods through the life cycle. J. Nutr. 124:2520S-2534S.
2. Sandström, B. (1996) Methods for studying the absorption and metabolism of inorganic nutrients. Mellon, F. A. Sandström, B. eds. Stable Isotopes in Nutrition 1996:11-14 Academic Press London, UK .
3. Davidsson, L., Kastenmayer, P., Szajewska, H., Hurrell, R. F. & Barclay, D. (2000) Iron bioavailability in infants from an infant cereal fortified with ferric pyrophophate or ferrous fumarate. Am. J. Clin. Nutr. 71:1597-1602.
4. Sturnlund, J. R., Michel, M. C., Keyes, W. R., King, J. C. & Margen, S. (1982) Use of enriched stable isotopes to determine zinc and iron absorption in elderly men. Am. J. Clin. Nutr. 35:1033-1040.
5. Schuette, S. A., Janghorbani, M., Young, V. R. & Weaver, C. (1993) Dysprosium as nonabsorbable marker for studies of mineral absorption with stable isotope tracers in human subjects. J. Am. Coll. Nutr. 12:307-315.[Abstract]
6. AAFCO (2001) Official Publication 2001 Association of American Feed Control Officials West Lafayette, IN .
7. Hazewinkel, H.A.W. (1989) Ca metabolism and skeletal development in dogs. Burger, I. H. Rivers, J.P.W. eds. Nutrition of the Dog and Cat 1989:293-302 .
8. Nap, R. C., Hazewinkel, H. A. & van den Brom, W. E. (1993) 45Ca kinetics in growing miniature poodles challenged by four different dietary levels of calcium. J. Nutr. 123:1826-1833.
9. Hazewinkel, H. A., van den Brom, W. E., van T’Klooster, A. T., Voorhout, G. & Van Wees, A. (1991) Calcium metabolism in Great Dane dogs fed diets with various calcium and phosphorus levels. J. Nutr. 121:99S-106S.
10. Lipschitz, D. A., Simpson, K. M., Cook, J. D. & Morris, E. R. (1978) Absorption of monoferric phytate by dogs. J. Nutr. 109:1154-1160.
11. Lowe, J. A., Wiseman, J. & Cole, D.J.A. (1994) Absorption and retention of Zn when administered as an amino-acid chelate in the dog. J. Nutr. 124:2572S-2574S.
12. Zinn, K. R., Chaudhuri, T. R., Mountz, J. M., van den Berg, G. J., Gordon, D. T. & Johanning, G. L. (1999) 59Fe is retained from an elemental 59Fe powder supplement without effects on 65Zinc, 47Calcium and 67Copper in young pigs. J. Nutr. 129:181-187.
13. Coudray, Ch, Bousset, C., Tressol, J. C., Pépin, D. & Rayssiguier, Y. (1998) Short-term ingestion of chlorogenic acid or caffeic acid decreases zinc but not copper absorption in rats, utilization of stable isotopes and inductively-coupled plasma mass spectrometry technique. Br. J. Nutr. 80:575-584.[Medline]
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