ω3 polyunsaturated fatty acids and maize flour diets

BASIC NUTRITIONAL INVESTIGATION

␻3 Polyunsaturated Fatty Acids and Maize Flour Diets I. Fernandez, MD, A. N. Pallaro, PhD, and N. H. Slobodianik, PhD From the Laboratory of Experimental Nutrition, Department of Nutrition, School of Pharmacy and Biochemistry, University of Buenos Aires, Buenos Aires, Argentina OBJECTIVES: Nutrition disorders caused by a 6.5% maize protein diet (M), unbalanced in its indispensable amino acid pattern, provokes an arrest on cellular proliferation and maturation in the thymus of growing rats. We investigated the effect of diet supplementation with different amounts of ␻3 polyunsaturated fatty acid (M ⫽ 0 mg/d, M1 ⫽ 12 mg/d, M2 ⫽ 24 mg/d, respectively) on thymus, plasma lipid concentrations, and hepatic tissue. METHODS: A well-nourished age-matched control group received stock diet from weaning. The animals (8 to 10 per group) received the diets for 9 d. At the end of the experimental period, they were killed, thymuses were removed, and cell number, absolute number of T cells labeled by the monoclonal antibody W3/13, plasma lipid profile (total cholesterol, triacylglycerols, high- and low-density lipoprotein cholesterols), and oxidative stress in hepatic tissue were determined. RESULTS: Only M2 reached the values of the control group when cell number and absolute number of T cells were compared. No statistical differences were observed among the M, M1, M2, and control group when high- and low-density lipoprotein cholesterols and hepatic lipid peroxidation were considered. CONCLUSIONS: The supplementation of a 6.5% maize protein diet with 24 mg/d of ␻3 polyunsaturated fatty acid can recover the proliferation and absolute number of T cells labeled with W3/13 without affecting lipid profile and hepatic lipid peroxidation. Nutrition 2001;17:944 –947. ©Elsevier Science Inc. 2001 KEY WORDS: ␻3 polyunsaturated fatty acid, maize supplementation, thymus, plasma lipids, hepatic peroxidation, oxidative stress

INTRODUCTION The relation between nutrition and immunity has been studied extensively. Many cells of the immune system depend for their function on metabolic pathways that use various nutrients as critical cofactors. Several studies have documented the adverse effects of protein malnutrition in human and experimental models. Specific amino acids modulate immune responses in many different ways; for example, dietary deficiencies of selected amino acids decrease antibody responses. The thymus, lymphoid tissue that has rapid cell turnover, is severely affected during malnutrition; the size and weight are reduced; there is a loss of corticomedullary differentiation, with fewer lymphoid cells; and the hassal bodies are enlarged, degenerated, and, occasionally, calcified.1,2 In previous papers we reported that nutrition disorders, i.e., protein restriction or unbalanced diet due to the deficiency of lysine, arrests cellular proliferation and maturation on thymus of growing rats.3,4 Moreover, evidence obtained from human studies, in vitro and in vivo, suggests that certain unsaturated fatty acids may be safe and effective antiinflammatory and immunomodulatory agents.5 The nature of the behavior depends on the type of fatty acid, age, antioxidant status, and health status of the subject.6 Moreover, it is well known that the consumption of fish or fish oil modifies blood lipid levels in human subjects and experimental

Supported by the University of Buenos Aires. (TB-077). Correspondence to: I. Fernandez, MD, Laboratory of Experimental Nutrition, Department of Nutrition, Junin 956-2do piso- (1113), Capital Federal, Buenos Aires, Argentina. E-mail:[email protected] Nutrition 17:944 –947, 2001 ©Elsevier Science Inc., 2001. Printed in the United States. All rights reserved.

animals.7–12 We also reported that the addition of ␻3 polyunsaturated fatty acid (PUFA) in the recovery diet could reverse the effect of a severe protein deprivation on the thymuses of weanling rats.13,14 We investigated the effect of different amounts of ␻3 PUFA on

TABLE I. DIET COMPOSITION

Components

M (g/kg)

M1 (g/kg)

M2 (g/kg)

C* (g/kg)

Protein Minerals Fat Total vitamins Choline Fiber Glucides ␻3 PUFA Ratio ␻6:␻3

65 50 45 7.5† 1.5 — 831‡ — —

65 50 45 7.5† 1.5 — 831‡ 12 mg/d 11.7

65 50 45 7.5† 1.5 — 831‡ 24 mg/d 5.8

180–200 40–50 60 5–10 1.9 55 370–400 — —

* The composition of the stock diet was provided by the manufacturer (Cargill S.A.C.I.-Animal Nutrition Division). † Fat-soluble vitamin composition of the experimental maize diets: vitamin A, 400 mg/100 g of diet; vitamin E, 10 mg/100 g of diet; vitamin D, 200 mg/100 g of diet. ‡ As dextrins. PUFA, polyunsaturated fatty acid. 0899-9007/01/$20.00 PII S0899-9007(01)00669-4

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TABLE II. FOOD, PROTEIN, AND ENERGY INTAKES OF THE EXPERIMENTAL GROUPS* Protein intake Group M (n ⫽ 10) M1 (n ⫽ 8) M2 (n ⫽ 9)

Energy intake

Food intake (g/d)

g/d

mg/d/W0.75

kcal/d

kcal/d/W0.75

5.8 ⫾ 0.7 6.3 ⫾ 1.0 6.1 ⫾ 0.9

0.38 ⫾ 0.04 0.41 ⫾ 0.06 0.39 ⫾ 0.06

23.7 ⫾ 1.9 24.4 ⫾ 3.2 24.2 ⫾ 2.8

23.2 ⫾ 2.8 25.6 ⫾ 3.9 25.0 ⫾ 3.8

1.5 ⫾ 0.1 1.5 ⫾ 0.2 1.5 ⫾ 0.2

* Data are presented as mean ⫾ standard deviation.

the thymuses of rats fed a maize protein diet from weaning and analyzed their influence on plasma lipid profile and the potential oxidative stress as a consequence of this supplementation.

MATERIALS AND METHODS Wistar rats from a closed colony from the breeding unit kept at the Department of Nutrition were assigned to three groups that received from weaning and over 9 d a 6.5% maize flour protein diet with (M1 ⫽ 12 mg/d, M2 ⫽ 24 mg/d) and without (M ⫽ 0 mg/d) supplementation with different amounts of ␻3 PUFA. M1 rats received 39.3 mg/d and M2 rats received 77.8 mg/d of a commercial fish oil (Regulip, Raffo) that contains 12 and 24 mg of ␻3 PUFA, respectively. Experimental diets were isocaloric (4 kcal/g of diet) and complete in all indispensable nutrients according to the recommendations of the American Institute of Nutrition and prepared as previously reported3– 4 (Table I). Animals were housed individually in screen-bottom cages and throughout the experiment exposed to a 12-h cycle of light and dark (7:00 AM to 7:00 PM); room temperature was kept at 21 ⫾ 1.0°C. Water and diet were offered ad libitum. Food intake was recorded daily, and total and complete protein intakes and energy intake were calculated and expressed as milligrams and kilocalories, respectively, per gram of body weight ⫻ 0.75 per day, where body weight is defined as body weight at weaning plus body weight at the end of the experiment (Table II). A well-nourished, age-matched control group (C) received a stock diet from weaning (Cargill S.A.C.I., Animal Nutrition Division). At the end of the experimental feeding period and after 4 h of fasting, body weight was determined; animals were exsanguinated

by venous puncture under anesthesia, and livers and thymuses were removed and weighed. Cell suspensions of the thymuses were prepared in RPMI-1640 medium plus 10% fetal calf serum. Cell recovery and viability were determined by trypan blue exclusion (0.05% saline) using an hemocytometer counting chamber. The absolute number of T cells labeled with the monoclonal antibody W3/13 (Accurate Chemical) was counted with an indirect immunofluorescence technique.15 Total cholesterol, triacylglycerols, and high-density lipoprotein (HDL) cholesterol concentrations were measured in serum by enzymatic methods using Wiener kits (Enzymatic Cholestat AA, Color Triacylglycerols GPO/PAP AA, and Monofase Cholesterol HDL AA) in a Ciba-Corning 550 Express Autoanalyzer, according to the manufacturer’s instructions. The low-density lipoprotein (LDL) cholesterol fraction was determined with the Friedewald equation: LDL-cholesterol ⫽ total cholesterol ⫺ (triacylglycerols/5 ⫹ HDL-cholesterol) Oxidative stress was evaluated in the liver with two methods: 1) fluorometric determination of malondialdehyde using 2thiobarbituric acid16,17 and 2) tert-butyl hydroperoxide–initiated chemiluminescence as a sensitive microassay.18 The statistical analysis of the data was done with analysis of variance with Instat software.19,20

RESULTS No statistical differences were observed among M, M1, and M2 groups when food, protein, and energy intakes were considered (Table II). The results of body weight, thymus weight, cell number, and

TABLE III. BODY WEIGHT, THYMUS WEIGHT, CELL NUMBER, AND ABSOLUTE NUMBER OF T CELLS LABELED WITH W3/13 IN THE EXPERIMENTAL GROUPS (M, M1, M2) AND THE CONTROL GROUP C*

Body weight (g) Thymus weight (mg) Thymus weight (mg/WF0.75) Cell number, 10⫺5/thymus weight (g) Absolute number of W3/13⫹ T cells,† 10⫺5/thymus weight (g) * Data are presented as mean ⫾ standard deviation. † 400 – 800 cells were read. ‡ P ⱕ 0.01 versus C. § P ⬍ 0.05 versus C.

M (n ⫽ 10)

M1 (n ⫽ 8)

M2 (n ⫽ 9)

C (n ⫽ 10)

41.1 ⫾ 6.6‡ 90.3 ⫾ 25.0‡ 7.0 ⫾ 0.6‡ 1.7 ⫾ 0.6§ 1.1 ⫾ 0.3‡

43.5 ⫾ 3.9‡ 118.4 ⫾ 17.4‡ 7.0 ⫾ 0.9‡ 1.3 ⫾ 0.6§ 0.8 ⫾ 0.3‡

41.8 ⫾ 5.8‡ 108.6 ⫾ 26.7‡ 6.6 ⫾ 1.3‡ 2.1 ⫾ 0.7 1.5 ⫾ 0.5

64.2 ⫾ 6.4 246.1 ⫾ 38.5 11.2 ⫾ 1.3 2.6 ⫾ 0.7 1.8 ⫾ 0.5

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Fernandez et al.

Nutrition Volume 17, Number 11/12, 2001 TABLE IV. SERUM LIPID CONCENTRATIONS OF THE EXPERIMENTAL AND CONTROL GROUPS*

Cholesterol (mg%) Triacylglycerol (mg%) High-density lipoprotein cholesterol (mg%) Low-density lipoprotein cholesterol (mg%)

M (n ⫽ 10)

M1 (n ⫽ 8)

M2 (n ⫽ 9)

C (n ⫽ 10)

64.2 ⫾ 6.9† 45.9 ⫾ 22.6† 27.4 ⫾ 4.1 27.6 ⫾ 6.4

65.9 ⫾ 4.9† 42.5 ⫾ 17.6† 30.1 ⫾ 3.4 27.3 ⫾ 4.7

61.3 ⫾ 5.0† 40.0 ⫾ 9.4† 27.5 ⫾ 3.2 25.8 ⫾ 5.4

78.9 ⫾ 6.7 109.8 ⫾ 35.8 30.4 ⫾ 2.6 26.5 ⫾ 8.1

* Data are presented as mean ⫾ standard deviation. † P ⬍ 0.001 versus C.

absolute number of T cells labeled with W3/13 in the experimental and control groups are presented in Table III. No statistical differences among the experimental groups (M, M1, M2) were observed in body and thymus weights; these values were far below those of the age-matched C group. Only the M2 group showed no statistical differences from the C group when cell number and absolute number of T cells labeled with W3/13 were studied. Of note, groups M and M1 were statistically different from group M2 when cell number and absolute number of T cells labeled with W3/13 were examined. Serum lipid profiles of the experimental and control groups are presented in Table IV. The cholesterol and triacylglycerol values of the three experimental groups were lower than those in group C; we found no differences in HDL and LDL cholesterol. Hepatic levels measured with the 2-thiobarbituric acid and tert-butyl hydroperoxide techniques are presented in Table V. The test used to evaluate oxidative stress showed no differences among groups.

DISCUSSION Growth can be defined as a complex process determined by genetic and environmental influences, of which diet is a major determinant. It depends on adequate nutritional substrates, the action of a variety of hormones (insulin, cortisol, thyroid hormones, growth hormone) and growth factors such as insulin growth factors I and II.21 Previous reports have shown that Wistar rats that received maize flour protein diets from weaning present a lower body and thymus weight when compared with a well-nourished, agematched control group. This diet, deficient in an indispensable amino acid, lysine, arrests the cellular proliferation and maturation of thymuses in growing rats.3,4

TABLE V. HEPATIC LEVELS OF TBARS AND TBOOH*

TBARS (nmol/mg protein) TBOOH (103cps/ mg protein)

M (n ⫽ 10)

M1 (n ⫽ 8)

M2 (n ⫽ 9)

1.0 ⫾ 0.6

1.1 ⫾ 0.4

1.1 ⫾ 0.5

68.7 ⫾ 8.4

61.9 ⫾ 19.1

69.2 ⫾ 15.2

* Data are presented as mean ⫾ standard deviation. TBARS, fluorometric determination of malondialdehyde using 2-thiobarbituric acid; TBOOH, tert-butyl hydroperoxide–initiated chemiluminescence as a sensitive microassay.

PUFAs modulate several steps of the immune response. The mechanism behind the potentially beneficial effect of very– long-chain ␻3 fatty acids may be related to altered eicosanoid formation. The effect of pure fatty acids depends on its concentration, chain length, and number of double bonds within each fatty acid family.22,23 In the present paper, even though the experimental groups showed no statistical differences in food, energy, and protein intakes, the addition of 24 mg/d of ␻3 PUFA to a 6.5% maize protein diet recovered the cell number and absolute number of T cells labeled with W3/13 in the thymuses of growing rats. These data point out the beneficial effect of ␻3 PUFA on the thymus, with a daily ratio of ␻6:␻3 ⫽ 5.8 (Table II). International data have reported that intakes of moderate amounts of ␻3 PUFA modify plasma lipid pattern. In most human studies of lipoprotein metabolism in which very–long-chain ␻3 fatty acids are fed, there is a small but significant increase in HDL levels accompanied with a simultaneous fall in VLDL and triacylglycerol values. Conversely, LDL cholesterol levels increase. Although there is no unique effect of ␻3 fatty acids on lipid levels, supplementation with fish oil would be expected to produce changes when compared with no supplementation.11,23 In this report, due to the small amount of ␻3 PUFA added, no differences among the experimental groups were observed in LDL and HDL cholesterols. However, cholesterol and triacylglycerol values were lower in all the experimental groups compared with the control group. These findings may be due to the amount of animal fat present in the stock diet (58% and 42% of animal and vegetable sources, respectively, according to the manufacturer’s information). The risks associated with high intakes of ␻3 PUFA, such as increased free radical activity, can be minimized by the intake of appropriate levels of an antioxidant nutrient such as vitamin E without compromising its beneficial effects.6,24,25 Data from the literature indicate that 2.24 and 2.68 mg of supplementary dL-␣ tocopherol are needed for each gram of dietary EPA and DHA, respectively.26 The evaluation of oxidative stress in vivo and its influence on hepatic tissue were assessed. The amounts of ␻3 PUFA used were not enough to improve lipid peroxidation. The total vitamin E intake (M2 ⫽ 0.61 mg of vitamin E per day, amount provided by 6.1 g of diet) was well above the level needed to protect against the effect of ␻3 PUFA supplementation (24 mg of EPA and DHA would need only 59 ␮g of vitamin E/d). We can conclude that the supplementation with 24 mg of ␻3 PUFA of a maize flour protein diet reversed the growth arrest of thymuses in growing rats and neither affected plasma lipid profile nor caused hepatic oxidative damage. These observations suggest the importance of including ␻3 PUFA (fish, fish oils) in children’s basic diets which contains maize flour as only source of protein.

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ACKNOWLEDGMENTS The authors thank Mrs. Lia Culotta de Calafat for the preparation of the experimental diets and the care of the animals.

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