The Journal of Nutrition Vol. 128 No. 12 December 1998, pp. 2645S-2647S

Manipulation of Dietary (n-6) and (n-3) Fatty Acids Alters Platelet Function in Cats^1,2

Korinn E. Saker³, Alison L. Eddy, Craig D. Thatcher, and Joan Kalnitsky^*

Department of Large Animal Clinical Sciences and ^* Department of Biosciences, Virginia-Maryland Regional College of Veterinary Medicine, Blacksburg, VA 24061

KEY WORDS: (n-6) fatty acids · (n-3) fatty acids · platelets · feline · diet

	INTRODUCTION

Introduction References

Consuming diets enriched with either (n-6) or (n-3) fatty acids (FA)⁴ will subsequently influence the proportion of cell membrane-associated lipids, arachidonic acid (AA) and eicosapentaenoic acid (EPA). Recent investigations suggest that the mechanism underlying the effectiveness of (n-3) FA supplementation involves the reduction of inflammatory mediators (Vaughn et al. 1994). In addition to reducing levels of inflammatory mediators, (n-3) FA have been associated with altered platelet function. Activation of platelets with high concentrations of (n-6) or (n-3) FA in the cell membrane results in the release of increased amounts of proaggregatory thromboxane A₂ (TXA₂) or antiaggregatory thromboxane A₃ (TXA₃) metabolites (Hall 1996), respectively. Studies regarding the possible detrimental effects that may occur from intake of dietary (n-3) FA have yielded conflicting results regarding platelet function (Casali et al. 1986, Kristensen et al. 1989, Landhmore et al. 1986).

Currently, pet foods are being formulated to maximize the anti-inflammatory effect of (n-3) FA. Although the benefits of increasing dietary (n-3) FA have been studied, potential adverse effects of feeding diets enriched in (n-3) FA on a long-term basis have not been evaluated. Therefore, the objective of this study was to determine the effects of altering dietary (n-6) to (n-3) FA ratios on platelet aggregation, platelet activation, blood coagulation indices and bleeding time in cats.

Materials and methods. 

Animals and facilities.  Twelve domestic short-haired cats, six neutered males and six spayed females, (average age 4 y) were used in this experiment. Cats were housed and fed in individual metal cages in a climate-controlled environment at the Virginia-Maryland Regional College of Veterinary Medicine for the duration of the study. All cats were fed a nutritionally complete dry diet⁵ to maintain an optimal body weight and condition score before the start of the study. The prestudy diet contained a (n-6):(n-3) fatty acid ratio of 12:1. The experimental protocol was reviewed and approved by the Virginia Tech Animal Care and Use Committee according to NIH guidelines (NRC 1985).

Experimental protocol.  Cats were allotted by gender and weight to dietary treatments as follows: enriched (n-6), [(n-6):(n-3) FA ratio 25:1]; control, (12:1); or enriched (n-3), (1.3:1). Experimental diets were formulated by Hill's Science and Technology Center, Topeka, KS. Fatty acid ratios were manipulated by incorporating specific FA sources into each of the three treatment diets (corn oil, choice white grease or menhaden oil, respectively). During the 16-wk study period, whole blood was collected via jugular venipuncture on d 0, 56 and 112 for evaluation of platelet function, coagulation parameters and fibrinogen concentration. Toenail bleeding time was recorded on d 0 and 112.

Platelet isolation.  Whole blood was collected via jugular venipuncture into syringes containing 3.8% trisodium citrate (9 parts blood:1 part anticoagulant), and platelet-rich plasma (PRP) was harvested according to the procedure described by Welles et al. (1994). Platelet counts were determined by using a Baker System 9000 Hematology Series Cell Counter (Allentown, PA). Samples of PRP were diluted to 300,000 platelets/µL with autologous platelet-poor plasma (PPP).

View larger version (53K): [in this window] [in a new window]   Fig 1. Peak aggregation of platelet-rich plasma stimulated with 50 µmol/L ADP. Platelets were harvested from peripheral blood collected from cats fed an enriched (n-6), control or enriched (n-3) FA diet for 112 d. Column values represent the mean ± SEM of four cats per dietary treatment group. Error bars with different letters indicate dietary treatment differences (P < 0.05) within day.

View larger version (48K): [in this window] [in a new window]   Fig 2. Platelet activation measured as the percentage of platelets bound with fluorescein-labeled antifibrinogen monoclonal antibody. Platelet-rich plasma was harvested from cats fed an enriched (n-6), control or enriched (n-3) FA diet for 112 d and incubated with prostaglandin E1 (0.03 µmol/L) and 20 µmol/L fluorescein-labeled antihuman fibrinogen for 30 min at 25°C. Monoclonal antibody bound to fibrinogen receptors on the platelet surface to initiate activation. Column values represent the mean ± SEM of four cats per dietary treatment group. The value for the enriched (n-3) dietary treatment group tended to be less than that of the other two groups on d 56 and 112 (P = 0.15).

Platelet aggregation.  Platelet aggregation was performed on diluted PRP stimulated with 50 µmol/L ADP (grade I: Sigma Chemical, St. Louis, MO) using a dual-channel aggregometer (Payton Series 300BD-5, Buffalo, NY). The procedure has been previously described (Welles et al. 1994).

Platelet activation.  Platelet activation was measured using a modified technique previously reported by Welles et al. (1994). Briefly, PRP stimulated with 50 µmol/L ADP was labeled with fluorescein- conjugated goat F[Ab] monoclonal antibody (Cappel-Organon Teknika, Durham, NC). Fluorescence was measured on the platelets bound with fluorescein-labeled antifibrinogen using a Coulter Epics XL-MCL Flow Cytometer.

Determination of bleeding time, coagulation indices, and fibrinogen.  Cats were sedated with a combination of acepromazine (0.11 mg/kg, intramuscular) and ketamine HCl (22 mg/kg, intramuscular); one nail was trimmed 2 mm into the capillary bed and gently blotted every 10 s until a clot formed. Time to clot formation was recorded. Whole blood was collected via jugular venipuncture for determination of activated partial thromboplastin time (APTT), one-stage prothrombin time (OSPT) and fibrinogen concentration with the use of a Sysmex Coagulation Analyzer 1000.

Statistical analysis.  Data were tested for normality and homogeneity of variance. An ANOVA (SAS/STAT Version 5.0, SAS Institute, Cary, NC) was used to determine dietary treatment effects on platelet count, platelet aggregation, platelet activation, bleeding time, fibrinogen concentration, APTT and OSPT. Tukey's studentized range test (HSD) was used for means comparisons. The General Linear Models procedure for repeated-measures ANOVA was used to determine within-subject effects and compare response changes over time. Data were considered significant at P < 0.05.

Results.  Diet analyses are presented in Table 1. Cats fed the enriched (n-3) FA diet for 112 d exhibited an increased (P < 0.05) toenail bleeding time compared with cats fed either the enriched (n-6) FA or control diet (6.1 vs. 3.2 and 3.2 min, respectively). Peak aggregation of platelets from cats fed the enriched (n-3) FA diet was lower (P < 0.05) compared with both the enriched (n-6) FA and control diet on d 112 (Fig. 1). Platelet activation (Fig. 2) tended (P = 0.15) to be higher in the enriched (n-6) FA and control diet groups on d 56 and 112 compared with the enriched (n-3) FA diet group. There was no difference between treatment groups for OSPT, APTT and fibrinogen concentration throughout the study period (data not shown).

Discussion.  Manipulation of dietary (n-6):(n-3) ratios does not appear to affect coagulation indices such as APTT and OSPT or platelet count in human studies, but platelet function does appear to be altered (Kristensen et al. 1989). The effects of FA supplementation for 4 or 6 wk on bleeding time in dogs have been contradictory (Casali et al. 1986, Landhmore et al. 1986). In this study, bleeding time was significantly increased in cats fed an enriched (n-3) FA diet for 16 wk. Indices of the intrinsic, extrinsic and common coagulation pathways (APTT and OSPT) were not different among cats in the dietary treatment groups throughout the study. Therefore, it is unlikely that alterations in bleeding time were a function of abnormalities in the coagulation cascade. Alterations in bleeding time due to increased dietary (n-3) FA levels may occur in less time than 16 wk; however, that information could not be determined from our study design.

The ability of activated platelets to aggregate appeared to be influenced by the ratio of (n-6) to (n-3) FA in the diet as shown in Figure 1. As the level of dietary (n-6) FA decreased, there would be proportionately less AA incorporated into platelet membranes and, therefore, less AA available for conversion to the proaggregatory TXA₂. As the concentrations of TXA₂ decreased, the ability of platelets to aggregate would diminish. Kristensen et al. (1989) showed a similar effect on human platelet aggregation when fish-based diets were fed. Aggregation of platelets is dependent on or enhanced by fibrinogen binding to platelets. Alteration of platelet membrane glycoprotein complexes, which form a functional receptor for fibrinogen, is an activation-dependent platelet surface change (Welles et al. 1994). The method of measuring platelet activation in this study used an antifibrinogen antibody, which requires the presence of adequate fibrinogen to first bind to the exposed fibrinogen receptors of activated platelets. Our results indicated that platelets derived from cats fed the enriched (n-3) FA diet tended to exhibit decreased activation compared with platelets from cats fed the enriched (n-6) or control diet; fibrinogen concentration did not differ among treatment groups during the study, and values were within the normal range for cats (Duncan and Prasse 1986). This suggested that a decrease in platelet activation resulted in a decrease in aggregatory ability and that this sequence of events was not limited by fibrinogen concentration. Saynor and Gillott (1992) reported that intake by human subjects of diets containing high concentrations of fish oil for over a 7-y period significantly reduced fibrinogen. Possible longer-term effects on fibrinogen concentration of felines fed (n-3) FA-enriched diets may warrant further study with a larger sample size.

The results from this study suggest that prolonged (112 d) intake of enriched (n-3) FA diets in cats alters platelet aggregation and bleeding time, such that there were detrimental effects on platelet function. Specific medical conditions in which an enriched (n-3) FA diet or FA supplementation may be indicated would require long-term feeding to confirm the viability of a desired effect.

	FOOTNOTES

	LITERATURE CITED

Introduction References

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