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Supplement: Waltham International Symposium

Predictive Equations for the Quantitation of Polyunsaturated Fats in Dog Plasma and Neutrophils from Dietary Fatty Acid Profiles

Comparative Nutrition Laboratory, Texas A&M University, College of Veterinary Medicine, College Station, TX and ^* Nestle-Purina Pet Care, St. Louis, MO

³To whom correspondence should be addressed. E-mail: jbauer{at}cvm.tamu.edu.

KEY WORDS: • dog • polyunsaturated fatty acids • equations • plasma • neutrophil

EXPANDED ABSTRACT

Polyunsaturated fatty acids (PUFA) of the (n-3) or (n-6) type are not synthesized de novo in animal tissues. When they are fed, displacement of endogenous fatty acids [20:3(n-9) and 20:4(n-7) types] occurs, resulting in enrichment of (n-3) and (n-6) long-chain PUFA (LCPUFA). Competition between the (n-3) and (n-6) fatty acid (FA) precursors for conversions exists and is significant because LCPUFA are important as precursors and antagonists of eicosanoid biosynthesis. Thus, dietary modification of LCPUFA products may alter certain eicosanoid-related disorders (1,2) and this provides a basis for making diet recommendations. Studies with rat plasma and tissues and with human plasma have revealed a quantitative relationship between diet and tissue FA (3,4). The present study examined whether similar relationships exist in dog species and equations to predict such relationships were derived. These relationships can be used to estimate the plasma and tissue accumulation of LCPUFA. They also enable diet formulation to be conducted in such a way as to reasonably predict FA tissue enrichment prior to actual feeding and thus preclude the need for expensive and tedious feeding trials.

	MATERIALS AND METHODS

TOP MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Sixty clinically normal, adult dogs of both genders (24 females, 16 males) were fed known basal diets supplemented with beef tallow (40-BTO), safflower oil (40-SAF), linseed oil (40-LSO) or menhaden fish oil (40-MHO) [all 40 energy % (en%) fat] or plus beef tallow (20-BTO) or safflower oil (20-SAF) (20 en% fat, n = 10 each group) (5). Two basal diets were used ("40" series and "20" series). They delivered equivalent amounts of amino acids, vitamins and minerals on an energy basis, after fat supplementation at either 20 or 40 en%. Because the basal diets contained a small amount of fat from other ingredients, differences in fatty acid profiles of the 40-BTO vs. 20-BTO and 40-SFO vs. 20-SFO were not simply twofold but reflected the total fat contents (Table 1).

Concentrations of LA [18:2(n-6)] and ALA [18:3(n-3)] in plasma triglyceride (TG) fractions were expressed as weight % and plasma PL data were expressed as (n-6) and (n-3) LCPUFA as a percentage of total LCPUFA.

	RESULTS

TOP MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
All animals maintained their adult body weights during the 28-d feeding period. The diets used varied in total calories from fat (i.e., 40.8 and 20.6% in the formulated diets and 23.8% in the supplemented diets). They also contained variable amounts of saturated and unsaturated FA. The average FA compositions of the plasma triglyceride fractions were directly influenced by the dietary FA content. Specifically, a relationship between the en% contributions of diet LA and ALA and plasma LA and ALA expressed as relative weight % was found. As with rats (3), a dose response was found between plasma TG-LA and TG-ALA and the dietary amounts of these FA within the ranges of 2.5 to 28 en% LA and 0.26 to 19.5 en% ALA (Fig. 1). These relationships were not linear and appeared to plateau at the highest levels of FA inclusion. Regression analysis of the TG data revealed a best-curve fit using a second-order polynomial equation with r² values of 0.999 and 0.997 (P < 0.05) for the ALA and LA acids, respectively. These equations appeared to be independent of the other dietary FA and different concentrations of total dietary calories from fat.

View larger version (28K): [in this window] [in a new window]   FIGURE 1 Plasma triacylglycerol linoleic [18:2(n-6)] and -linoleic [18:3(n-3)] acid content in weight % as a function of dietary 18:2(n-6) and 18:3(n-3) of dogs. Symbols are as follows: Closed circles, predicted values according to the linear model of Lands et al. (3); closed diamonds with white dot, actual values of Lands et al. for rats (3); open squares, actual values for dogs in this study. Inset graph is expanded scale for 18:3(n-3) for <1 en%.

(1)

In this equation, V_max describes the maximal velocity of a saturable process (e.g., tissue saturation); K_m is the substrate concentration at half V_max; en%S is the substrate concentration (e.g., LA concentration); en%I is the concentration of inhibitor (e.g., ALA concentration); and K_i is the concentration of inhibitor needed to reduce V_max by half of the uninhibited value.

Lands et al. (3) employed a version of ^{eq. (1)} and fit FA data from several dietary studies in rats. They found that only very small amounts of the dietary LA or ALA (e.g., 0.1 en%) were needed to fit this equation. These concentrations, referred to as C₃ and C₆ [see ^{eqs. (2)} and ⁽³⁾ below], were used analogously in place of either K_m and K_i in ^{eq. (1)}. The terms en%3, en%6 and en%O in ^{eqs. (2)} and ⁽³⁾ refer to energy % of ALA, LA and other FA (i.e., saturated and monounsaturated), respectively. Terms HC₃ and HC₆ are constants for the efficiency of direct esterification of diet (n-3) and (n-6) LCPUFA, respectively. Later, these workers (4) added additional terms: C₀, to account for the influence of other FA on the calculated plasma and tissue LCPUFA; K_s, to modify the hyperbolic curve to better fit the observed shape; en%H₃ and en%H₆, to account for the competitive interactions of the LCPUFA in direct esterifications; and HI₃ and HI₆, to explain competitive inhibition by dietary LCPUFA for elongation and desaturation of the respective (n-3) and (n-6) acids. Although the derived equations fit the rat data, modification of the constants was needed to fit data generated from human investigations (4).

In the present study it was also necessary to change the constants to provide a best fit of the dog data. These modifications were performed by trial-and-error fitting of plasma PL data generated by feeding the six formulated diets. These exercises resulted in a unique set of constants for this species (data not shown). An additional modification used for dog was to assign unique values for the analogous K_i values (i.e., KI₃ and KI₆) rather than simply using the same values of C₃ and C₆ for both the analogous K_m and K_i, in contrast to earlier studies (3,4). The final equations for dog took the form

(2)

(3)

The (n-3) as % total LCPUFA in ^{eq. (2)} is equivalent to the sum of 20:5(n-3) plus 22:5(n-3) divided by total LCPUFA, and the (n-6) as % total LCPUFA value in ^{eq. (3)} is equivalent to 20:3(n-6) plus 20:4(n-6) divided by total LCPUFA amounts. One similarity of the dog equations with the other species is that the magnitude of constants representing the effective concentrations of dietary ALA and LA (C₃ and C₆) were low (0.29 and 0.036, respectively) (3,4).

Constants for the equations were derived using plasma data from the six oil-supplemented diets. The resultant calculated values of the (n-6) and (n-3) LCPUFA using these constants and the dietary fat profiles compared favorably with the actual values obtained for each diet group (Fig. 2). The usefulness of these equations with constants derived from the plasma data of the six oil-supplemented diets was then evaluated using plasma data from the two oilseed-supplemented diets and the neutrophil membrane data from the six oil-supplemented diets. In the case of the FLX and SUN diets excellent agreement between the predicted and observed (n-3) and (n-6) LCPUFA as a percentage of total LCPUFA was found (Fig. 2). Similarly, neutrophils isolated from dogs fed the varying PUFA diets were also found to closely mimic their predicted values (Fig. 3). It should be noted that in the latter case different values of KI₃ and KI₆ were needed to provide a best fit.

View larger version (30K): [in this window] [in a new window]   FIGURE 2 Comparison of actual (gray bars) vs. calculated (black bars) values after curve fitting to derive the equation constants described in the text. Plasma (n-6) LCPUFA and (n-3) LCPUFA are expressed as percentage of total plasma LCPUFA of dogs fed the six oil-supplemented diets (40-BTO, 40-SAF, 40-LSO, 40-MHO, 20-SAF and 20-BTO). Comparison of actual and predicted LCPUFA values is also shown using data from dogs fed FLX and SUN diets from a separate study (7). Error bars indicate standard deviations of actual mean values, n = 9 for flaxseed and sunflower seed diets; n = 10 for each of the remaining diets.

View larger version (31K): [in this window] [in a new window]   FIGURE 3 Comparison of actual (gray bars) vs. predicted (black bars) values of neutrophil membrane (n-6) LCPUFA and (n-3) LCPUFA as a percentage of total neutrophil LCPUFA of dogs fed the six oil-supplemented diets. Error bars indicate standard deviations of actual mean values, n = 10 for each diet.

	DISCUSSION

TOP MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Analysis of the PL-FA in this study extends findings in humans and rats to dogs and supports the concept of a competitive and "saturable" hyperbolic relationship between dietary PUFA and plasma and tissue LCPUFA accumulation in dogs. Curve fitting of the data to the equations consistently found that the magnitude of the constants representing effective concentrations of dietary LA and ALA was low. These results support the long-held belief that adequate amounts of dietary essential FA may not be >0.5 en%. Furthermore, an eightfold relative difference between these constants was seen. This difference is consistent with the observed low metabolic conversion rates of ALA compared to those of LA from numerous other studies (8,9).

The set of constants determined for dogs in this study was similar to those of Lands et al., yet each was different from those values. A further refinement adapted here for dog plasma and neutrophil PL fatty acid enrichment was that unique values of the competitive inhibitor constant K_i were used to account for effects of (n-3) acids on (n-6) acids and vice versa. In other "saturable"-type systems values of K_m and K_i are also unique from one another and generally different from the concentration of substrate available for metabolic reaction. Thus ^{eqs. (2)} and ⁽³⁾ incorporate an appropriate inhibitor constant (i.e., KI₃ or KI₆) rather than using C₃ or C₆, as was done for rats.

In spite of many similarities in plasma and cell membrane fatty acid accumulation with diet, some cell types may accumulate FA differently from plasma. For example, when ALA is fed, neutrophils contain no docosahexaenoic acid (DHA), whereas this FA is seen in small amounts in plasma and found in high amounts in neurologic tissues (10,11). Thus, it is necessary to account for these differences when developing quantitative equations to estimate FA enrichment. The use of unique KI₃ and KI₆ values for individual cell types, while maintaining all other terms constant, provides this correction in the current approach. It also recognizes that differences in FA concentrations exist among cell types, which may reflect specific cell functions. For neutrophils, values of KI₃ and KI₆ different from those used for plasma PL were necessary to fit the cell membrane data. It is expected that estimation of FA enrichment of other cell types may also require some modification of the KI₃ and KI₆ values used in the predictive equations.

In studies by Lands et al. (4), several unique sets of constants overall provided similar good fits to plasma PL-FA data in both rats and humans. This phenomenon is most likely the result of inherent complexities of mammalian FA metabolism in multisystemic animal species. One difference between human and dog diets is that dogs are generally fed complete and balanced formulated diets of fairly predictable variability. Also, the diets used in the present analysis spanned a wide range of FA concentrations and likely encompass most of those that will be encountered in any practical feeding setting. Thus the set of constants derived in this study would be expected to reasonably predict dog plasma and possibly tissue enrichments achieved when diets of varying (n-3) and (n-6) PUFA composition are fed. This knowledge will help evaluate the extent to which dietary essential FA and their competitively formed metabolites accumulate in plasma and tissues as substrates for subsequent eicosanoid production or other roles. Analysis of a single blood sample or dietary FA profile and these equations allow important new interpretations to be made regarding dietary recommendations.

	FOOTNOTES

 
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 D’Ann Finley, University of California, Davis.

² Supported, in part, by Ralston-Purina Company and the Mark L. Morris Professorship in Clinical Nutrition at Texas A&M University.

⁴ Present address: Nestle-Purina Pet Care, St. Louis, MO.

⁵ Abbreviations used: ALA, -linolenic acid; FA, fatty acid; FLX, flaxseed-supplemented diet; LA, linoleic acid; LCPUFA, long-chain polyunsaturated fatty acid; PL, phospholipid; PUFA, polyunsaturated fatty acid; SUN, sunflower seed-supplemented diet; TG, triacylglycerol; 40-BTO, beef tallow-supplemented 40 energy percent diet; 40-LSO, linseed oil-supplemented 40 energy percent diet; 40-MHO, menhaden oil-supplemented 40 energy percent diet; 40-SAF, safflower oil-supplemented 40 energy percent diet; 20-BTO, beef tallow-supplemented 20 energy percent diet; 20-SAF, safflower oil-supplemented 20 energy percent diet.

	LITERATURE CITED

TOP MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 

1. Bauer, J. E. (1994) The potential for dietary polyunsaturated fatty acids in domestic animals. Aust. Vet. J. 71:342-345.[Medline]

2. Watson, T.D.G. (1998) Diet and skin disease in dogs and cats. J. Nutr. 128:2783S-2789S.[Abstract/Free Full Text]

3. Lands, W.E.M., Morris, A. & Libelt, B. (1990) Quantitative effects of dietary polyunsaturated fats on the composition of fatty acids in rat tissues. Lipids 25:505-516.[Medline]

4. Lands, W.E.M., Libelt, B., Morris, A., Kramer, N. C., Prewitt, T. E., Bowen, P., Schmeisser, D., Davidson, M. H. & Burns, J. H. (1992) Maintenance of lower proportions of (n-6) eicosanoid prescursors in phospholipids of human plasma in response to added dietary (n-3) fatty acids. Biochim. Biophys. Acta 1180:147-162.[Medline]

5. Waldron, M. K. (1999) Dietary fat effects on neutrophil membrane fatty acid composition and cell functions 1999 Ph.D. Dissertation Texas A&M University.

6. Markert, M., Andrews, P. C. & Babior, B. M. (1984) Measurement of O₂^- production by human neutrophils. The preparation and assay of NADPH oxidase-containing particles from human neutrophils. Methods Enzymol. 105:358-365.[Medline]

7. Bauer, J. E., Dunbar, B. L. & Bigley, K. E. (1998) Dietary flaxseed in dogs results in differential transport and metabolism of (n-3) polyunsaturated fatty acids. J. Nutr. 128:2641S-2644S.[Free Full Text]

8. Adam, O., Wolfram, G. & Zollner, N. (1986) Effect of alpha-linolenic acid in the human diet on linoleic acid metabolism and prostaglandin synthesis. J. Lipid Res. 27:421-426.[Abstract]

9. Nettleson, J. A. (1991) w-3 Fatty acids: comparison of plant and seafood sources in human nutrition. J. Am. Diet. Assoc. 91:331-337.[Medline]

10. Waldron, M. K, Bauer, J. E., Dunbar, B. L. & Bigley, K. E. (1999) 18 and 20 Carbon (n-3) fatty acids differentially affect dog neutrophil function at the same (n-6)/(n-3) ratio. J. Vet. Int. Med. 13:262.

11. Abedin, L., Lein, I. L., Vingrys, A. J. & Sinclair, A. J. (1999) The effects of dietary -linolenic acid compared with docosahexaenoic acid on brain, retina, and heart in the Guinea pig. Lipids 34:475-482.[Medline]

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