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Essentiality of arachidonic acid intake in murine early development
Prostaglandins, Leukotrienes and Essential Fatty Acids 108 (2016) 51–57
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Prostaglandins, Leukotrienes and Essential Fatty Acids journal homepage: www.elsevier.com/locate/plefa
Essentiality of arachidonic acid intake in murine early development Erisa Hatanaka a, Akiko Harauma a, Hidemi Yasuda a, Junnosuke Watanabe a, Manabu T. Nakamura b, Norman Salem Jr.c, Toru Moriguchi a,n a
School of Life and Environmental Science, Azabu University, 1-17-71 Fuchinobe, Sagamihara, Kanagawa 252-5201, Japan Division of Nutritional Science, University of Illinois at Urbana-Champaign, 905 South Goodwin Avenue, Urbana, IL 61801, USA c Nutritional Lipids, DSM Nutritional Products, Columbia, MD 21045, USA b
art ic l e i nf o
a b s t r a c t
Article history: Received 8 February 2016 Received in revised form 17 March 2016 Accepted 17 March 2016
We previously reported the importance of long-chain polyunsaturated fatty acid (LC-PUFA ( 4C20)) intake, including arachidonic acid (ARA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA), for growth. This follow-up study focuses on ARA using a novel artificial rearing model during the lactation period in delta-6-desaturase knockout (D6D-KO) mice. Newborn D6D-KO male mouse pups were separated from dams within 48 hours and fed artificial milks containing 18-C essential fatty acids (EFAs) (16–17% LA, 3.8–4.1% ALA) with or without 1.2% ARA. After weaning, mice were maintained on similar diets: 15% LA, 2.3–2.4% ALA with or without 1.9% ARA. As a reference group, new born wild type (WT) male mouse pups were maintained by artificial milk and diet containing LA and ALA without ARA. Aspects of brain function were measured behaviorally (motor activity and rota-rod test) when mice were age 9 weeks. Body weight in the KO-Cont group was significantly lower (approximately 30%) than in the WT-Cont group, but this decrease was ameliorated by providing ARA in the KO-ARA group. The motor activity and coordination in the KO-Cont group decreased markedly compared to the WT-Cont group. The KO-ARA group had a tendency toward deteriorated motor coordination, although the motor activity was significantly enhanced compared to the KO-Cont group. In KO-ARA group brains, the level of ARA was increased and DHA decreased compared to WT-Cont. These results suggest that intake of LA and ALA only is insufficient to support healthy growth, and that ARA is also required, at least during the lactation period. These findings also suggested that continued intake of relatively high levels of ARA and without supplemental DHA during development led to an increased motor activity above that of WT animals. These studies indicate that both ARA dose and proper combination with DHA must be delineated to define optimal growth and behavioral function. & 2016 Elsevier Ltd. All rights reserved.
Keywords: Delta-6-desaturase Knock-out mouse Polyunsaturated fatty acid Arachidonic acid Docosahexaenoic acid
1. Introduction Two families of polyunsaturated fatty acids (PUFAs), n-6 and n-3 fatty acids, are essential fatty acids, as they cannot be biosynthesized and therefore must be ingested in the diet. Although a lack of n-6 fatty acids causes growth disorders and dermopathy [1,2], linoleic acid (LA, 18:2n-6) and arachidonic acid (ARA, 20:4n6) are contained in many types of foods, especially animal fats and vegetable oils, and therefore such deficiencies rarely occur. α-Linolenic acid (ALA, 18:3n-3), an n-3 fatty acid, is contained in perilla Abbreviations: D6D, delta-6-desaturase; KO, knock-out; PUFA, polyunsaturated fatty acid; LA, linoleic acid; ARA, arachidonic acid; ALA, α-linolenic acid; DHA, docosahexaenoic acid n Correspondence to: Laboratory of Food and Nutritional Science, Department of Food and Life Science, School of Life and Environmental Science, Azabu University, 1-17-71 Fuchinobe, Chuo, Sagamihara, Kanagawa 252-5201, Japan. E-mail address: [email protected] (T. Moriguchi). http://dx.doi.org/10.1016/j.plefa.2016.03.007 0952-3278/& 2016 Elsevier Ltd. All rights reserved.
oil and linseed oil, and its metabolites eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3) are abundant in fish and seafood [3]. DHA can pass through the blood-brain barrier [4] and the blood-retinal barrier to accumulate in these organs, thereby improving brain function (e.g. learning ability), mental condition, and visual function [5–7]. It has also been reported to have preventive and therapeutic effects on cardiovascular diseases [8]. However, n-3 fatty acid–rich foods are relatively uncommon, and thus, resulting impairment of brain function by deficiency of n-3 fatty acids is a concern. Dietary LA is converted to ARA through a reaction catalyzed by delta-6-desaturase (D6D) and chain elongating enzymes, and then to the n-6 end product docosapentaenoic acid (DPAn-6, 22:5n-6) via the reactions catalyzed by chain elongating enzymes, D6D and β-oxidation [9]. Similar fatty acid–metabolizing enzymes convert dietary ALA to EPA and DHA. On the other hand, it has been reported that DPAn-6 and DHA may be synthesized by direct delta-4-desatuation in human
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cells [10]. However in human infants, the activities of these desaturases are still too low to sufficiently convert LA and ALA to ARA and DHA, respectively, requiring direct intake of ARA and DHA [11]. Infants undergo marked growth and development of the body and the brain, and several studies have demonstrated the efficacy of ARA and DHA intake in infancy [12–14]; in fact, breast milk is rich in these fatty acids [15–17]. PUFAs are necessary to maintain biological functions throughout all life stages, but its intake is likely to be particularly essential in infancy. Yet, the European Food Safety Authority (EFSA) recently expressed the opinion that there is no need to add ARA and EPA to infant and follow-on formulae [18]. The effect of PUFA in infants has been examined only through administration of ARA plus DHA [13,14], and through administration of DHA only, and the effect of PUFA intervention in infancy after the initial growth period have been studied only rarely. Thus, a careful evaluation of the EFSA claim using carefully controlled diets in infants is necessary. D6D knockout mice (D6D-KO mice), cannot convert either n-6 or n-3 fatty acids to their more unsaturated metabolites [9,19,20] and thus serve as an ideal model for investigating requirements for various PUFAs [21]. In this study, we examined the importance of ARA in infancy using a D6D-KO mouse model and an artificial feeding technique from the second day of life.
2. Materials and methods The experimental protocol was approved by the Institutional Animal Care and Use Committee of Azabu University (No. 1410072). 2.1. D6D-KO mice D6D-KO mice were back-crossed to the C57BL/J background were transferred from the University of Illinois. Heterozygous male and female fed a custom pelleted diet (termed “MF”, Oriental Yeast Co., Ltd., Tokyo, Japan, Table 1), were mated, and resultant male WT and homozygous KO were used for the study. Tail DNA genotyping by PCR was performed to confirm D6D KO status in mice [9]. DNA was extracted from tails of 2-day-old mice using a tissue PCR kit (Extract-N-Amp™, Sigma-Aldrich, Tokyo, Japan), and PCR was performed using a thermal cycler (T100™, Bio-Rad Laboratories K.K., Tokyo, Japan). After DNA genotyping, both homozygous Table 1 Composition of custom pelleted diet.1 Amount (g/100g diet) Pelleted diet Casein, vitamin free Carbohydrate: Cellulose Mineral-Salt mix Fat
23.1 55.3 2.8 5.8 5.1
Fatty acid composition (% of total fatty acids) Saturates Monounsaturates 18:2n-6 20:4n-6 Total n-6 FAs 18:3n-3 20:5n-3 22:6n-3 Total n-3 FAs Total FA (μg/mg)
19.6 28.2 42.0 0.13 42.3 3.9 1.5 1.3 6.3 47.0
1
Pelleted chow is made by Oriental Yeast Co., Ltd., Tokyo, Japan.
(D6D-KO, / ) and wild type (WT, þ/þ) mice were used in this study. The study design of this experiment is shown as Fig. 1. Newborn male pups of D6D-KO mice were separated from their dams within 48 h, and were fed artificial milks containing 18-C EFAs (LA and LA) with or without ARA. The fat content of two experimental milks (Control and ARA) is approximately 16% and their fatty acid compositions are shown in Table 2. After weaning, mice were housed in an artificially regulated environment at 2373 °C, 55 710% humidity, under a 12-h light/dark cycle (lights on between 07:00 and 19:00), and fed a similar diet with or without ARA as during lactation (Table 3). The fatty acid compositions of these artificial milks and diets are shown in Table 4. As reference group, new born WT male mouse pups were maintained by artificial milk and diet containing LA and ALA without ARA (WT-Cont). When mice were age 9 weeks, motor coordination and spontaneous motor activity were assessed. At age 10 weeks, blood and brain tissue was collected and fatty acid composition was analyzed using gas chromatography (GC) (Fig. 1). 2.1.1. Artificial mouse milk Artificial milk formula was developed based on the methods of Yajima et al. and Hussein et al. [22,23], with slight modifications of fat content (Table 2). Casein and whey protein were used as protein sources and lactose was used as the carbohydrate. Fat was adjusted to 16% by mixing several oils. For complete dissolution, ingredients were mixed in the order described by Yajima et al. [22] using a sonicator (Ultrasonic Processor S-4000, Misonix, Inc., Farmingdale, NY, USA). Milk was homogenized twice under high pressure (800–1000 bar) using a high-pressure homogenizer (Panda PLUS 2000, Niro Soavi S.p.A., Parma, Italy) resulting in emulsified, sterilized, and smoothed milk. The homogenized milk was stored at 80 °C. 2.1.2. Artificial rearing system The artificial rearing procedure used a hand-feeding technique with specially constructed nursing bottles [24], (Fig. 2). Pups were separated from their dams on postnatal day 2 and fed artificial milk by hand using a nursing bottle every 3 h (5 times/day). Pups were capable of suckling from silicon nipples connected to the nursing bottles. Pups were placed in a cage with an ovariectomized foster mother for maternal care and warmth except at feeding times. From day 14, pups were fed artificial milk from a nursing bottle in combination with a pelleted diet. Infant diets were made by mixing a crushed control diet and their respective artificial milks. Pups in all groups were weaned to the pelleted diet at day 21. Pups were housed in an artificially regulated environment at 23 73 °C, 557 10% humidity after a 12-h light/dark cycle was used (lights on between 07:00 and 19:00). 2.2. Behavioral experiments 2.2.1. Motor activity test Motor activity was measured using cages (19 30 13 cm) equipped with running wheels (Wireless Low Profit Running Wheel, ENV-044 wheel, and SOF-860 software, Neuro-science Co., Ltd., Tokyo, Japan). Mice were assessed individually at the same time of day by recording the number of wheel rotations over a 30 min period (08:00–11:00) [25]. 2.2.2. Motor coordination test The motor coordination test was carried out using a rota-rod setup (Rota-Rod Treadmill, ENV-575M, Neuro-science Co., Ltd., Tokyo, Japan). Mice were allowed a trial run of 5 min on the rotor, set at 4 rpm, on the day preceding the measurement. On the following day, motor coordination ability was assessed with rotational speed set to increase from 4 rpm to 40 rpm over 5 min. The
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Fig. 1. Schematic diagram showing study design of D6D-KO mouse experiment. Newborn D6D-KO male pups were separated from their dams within 48 hours and fed artificial milks. After weaning, mice were fed diets similar to these during lactation. Spontaneous motor activity and the rota-rod test were conducted at 9 weeks of age. At 10 weeks of age mice were killed by decapitation, and plasma and brain tissue were collected and analyzed to ascertain fatty acid composition.
maximum duration that the mice remain on the rotor, up to a limit of 300 s, was recorded [25]. 2.3. Lipid extraction and transmethylation
22:5n-6, and 23:0. The standard fatty acid was used to verify the identity and assignment of retention times. Fatty acid quantification was checked using quantitative standard mixtures. Internal standards were used to calculate tissue fatty acid concentrations.
The transmethylation method developed by Lepage and Roy was used [26]. A half-brain (approximately 200 mg) was homogenized with 6 ml methanol:hexane (4:1) solvent containing 50 mg/ml butylhydroxytoluene to prevent lipid oxidation, and an additional 60 mg docosatrienoic acid methyl ester (22:3n-3 methyl ester) was added as an internal standard. After homogenization, 200 ml acetylchloride was added to each 2 ml homogenate in tubes (13 mm 100 mm). Tubes were capped under nitrogen gas and transferred to a heating block at 100 °C for 60 min to transmethylate fatty acids. Samples were then placed on ice to remove unmethylated acids by addition of 5 ml of 6% solution of K2CO3. The tubes were vortexed and centrifuged for 15 min at 2200 g to remove emulsion and separate the mixture into two phases. The upper phase of hexane was collected into micro-vials for GC injection [25]. For plasma and erythrocytes (100 ml of each sample), 2 ml methanol:hexane solvent and 20 mg of the internal standard were added to tubes and processed further as for brain tissue.
2.5. Statistical analysis
2.4. GC analysis
There was no difference in body weight change among the three groups during the lactation period. Body weight in the KOARA group was almost identical to that in the WT-Cont group during the experimental period for 10 weeks (P ¼0.85). However, the KO-Cont group had clearly lower body weight than the WTCont and KO-ARA groups after 3 weeks of age (P o0.01, Fig. 3). At 10 weeks of age, the mean body weights were 25.5 70.3 g for the WT-Cont, 17.37 0.5 g for the KO-Cont (Po 0.01 vs WT-Cont) and 25.2 70.6 g for the KO-ARA (Po 0.01 vs KO-Cont). The KO-Cont group (341.27 4.7 mg) had also significantly lower brain weights at 10 weeks of age compared with the WT-Cont and KO-ARA groups (389.0 73.6 and 377.7 7.3 mg, respectively, P o0.01), while those were not significantly different between WT-Cont and KOARA groups (body weight; P ¼0.84, brain weight; P ¼0.28). The spontaneous motor activity for 30 min in the KO-Cont group decreased markedly compared to the WT-Cont group (P o0.01, Fig. 4). For motor coordination, the KO-Cont group had a shorter duration before falling compared with the WT-Cont group
Fatty acid methyl esters were analyzed using an Agilent 7890A Network Gas Chromatograph (Agilent Technologies; Palo Alto, CA) equipped with a split injector, a 7693A automatic liquid sampler, an FID, and a 208 V power supply to enable rapid temperature ramping. The instrument was controlled, and data were collected using GC Chemstation Rev. B0.040.01. SP1 (Agilent Technologies). The column used was a DB-FFAP (15 m 0.1 mm i.d. 0.1 mm film thickness, J&W Scientific, Agilent Technologies). The detector and injector temperatures were set to 250 °C. The oven temperature program began at 150 °C with a 0.25 min hold, was increased at a rate of at 35 °C/min to 200 °C followed by 8 °C/min to 225 °C with a 3.2 min hold and 80 °C/min to 245 °C with a 2.75 min hold. Hydrogen was used as the carrier gas at a linear velocity of 56 cm/s [25,27]. A standard mixture of 30 components was supplemented with a custom standard (NuChek Prep 462, Elysian, MN) containing 10–24 carbons and 0–6 double bonds and 16:1n-9, 22:3n-3,
Data are expressed as means 7standard errors. Body weight gain for the 10-week period was analyzed using a repeated measures two-way ANOVA followed by Tukey's test. Body and brain weight at 10 weeks of age and content of each fatty acid in plasma, brain and erythrocyte were analyzed using one-way ANOVA followed by Tukey's test. The results of motor activity and the motor coordination test were analyzed using one-way ANOVA followed by Duncan's test (Statistica, Statsoft Japan, Tokyo, Japan). Significance was set at p o0.05. Total fatty acids values o0.01% were considered to indicate “not detected; ND.”
3. Results 3.1. D6D-KO mice
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Table 2 Composition of artificial milk.2 Ingredient
Amount (weight/100 mL milk)
Whey protein isolate (BIPRO)a Whey protein hydrolyzed (PEPTIGENs IF-3090)b Casein (Acid Casein LACTIC CASEIN720)c Serined Cystined Tryptophand Methionined Carbohydrate (g) Lactosee Minerals (mg) NaOHd KOHe GlyCaPO4d MgCl2 6H2Od CaCl2 2H2Od CaCo3d Ca-Citratee Na2HPO4d KH2PO4d FeSO4d Citrate H2Oe ZnSO4e CuSO4e MnSO4e NaFd KId K2SO4e Na2SiO3 9H2Od Na2O4Sed H8MoN2O4 4H2Oe KCr(SO4)2 12H2Od LiCle H3BO3d NiCO3d NH4VO3e Vitamins (mg) Vitamin mixf Vitamin Ce Vitamin K3d Vitamin Ae Vitamin Dd Vitamin Ed Others (mg) Carnitined Picolinated Ethanolamined Taurined Tricholine Citrated
Protein (g)
Fat (g)
Table 3 Composition of experimental diets.3
4.0 5.0
Amount (g/100g diet)
Casein, vitamin free Carbohydrate: Cornstarch Sucrose Glucose Dextrose Maltose Cellulose Mineral-Salt mix Vitamin mix L-Cystine Choline bitartrate TBHQ Fat Hydrogenated coconut oil Safflower oil Flaxseed oil s ARASCO
4.0 0.02875 0.3 0.027 0.0045 1.89 25 150 800 190 170 184 120 80 8 24 0.5 6 1.5 0.25 0.155 0.25 163.5 5.075 0.035 0.0275 0.975 0.05 0.285 0.1125 0.0225 400 200 1.9825 0.1284 23.46 0.0025 4 2 3.5 15 147
Control
ARA
20 63 15 13 20 7.5 7.5 5 3.5 1 0.3 0.25 0.002 7 5.4 1.2 0.3 none
20 63 15 13 20 7.5 7.5 5 3.5 1 0.3 0.25 0.002 7.2 5.4 1.2 0.3 0.2
3 The experimental diet was based on the AIN-93 formulation with modificas tion to the fat composition (Oriental Yeast Co., Ltd., Tokyo, Japan). ARASCO was used as ARA source (DSM Nutritional Products, Columbia, MD, USA).
Table 4 Fatty acid composition of artificial milk and experimental diet (% of total fatty acid). Artificial milk
(16.04 g)
Control
ARA
MCTg Palm oilh Coconut oile Corn oile Soybean oile Linseed oile s ARASCO i Cholesterole
1.25 8.25 2.5 0.5 2.75 0.75 none 0.04
1.25 7.75 2.5 0.5 2.75 0.75 0.5 0.04
2 Artificial milk formula was made following the method of Yajima et al. and Hussein et al. Component sources were as follows: a Davisco Foods International, Inc. MN, USA. b Arla Foods Ingredients,Viby, Denmark. c Fonterra Co-operative Group Limited, Auckland, New Zealand. d Sigma-Aldrich Corp. MO, USA. e Wako Pure Chemical Industries, Ltd., Osaka, Japan. f Oriental Yeast Co., Ltd., Tokyo, Japan. g The Nisshin Oillio Group,Ltd., Tokyo, Japan. h Spectrum Laboratory Products, Inc., New York, USA. i DSM Nutritional Products, Columbia, MD, USA.
8:0 10:0 12:0 14:0 16:0 18:0 20:0 22:0 24:0 Total Sat. 14:1 16:1n-7 18:1n-7 18:1n-9 20:1n-9 22:1n-9 Total Mono. 18:2n-6 18:3n-6 20:3n-6 20:4n-6 22:5n-6 Total n-6 FAs 18:3n-3 20:5n-3 22:5n-3 22:6n-3 Total n-3 FAs Total Fas (lg/mg)
Diet
Control
ARA
Control
ARA
5.40 7 0.02 2.31 7 0.01 6.677 0.02 3.17 7 0.02 25.94 7 0.20 3.68 7 0.02 0.29 7 0.005 0.13 7 0.005 0.09 7 0.002 47.72 7 0.25 0.077 0.001 0.11 7 0.002 0.68 7 0.003 25.97 7 0.18 0.15 7 0.01 0.12 7 0.04 27.12 7 0.12 17.41 7 0.13 ND ND ND ND 17.41 7 0.13 4.09 7 0.03 ND ND ND 4.09 7 0.03 133.467 6.97
5.497 0.14 2.36 7 0.01 6.84 7 0.04 3.25 7 0.03 26.377 0.19 3.94 7 0.02 0.317 0.01 0.177 0.01 0.127 0.005 48.887 0.15 0.077 0.003 0.127 0.003 0.687 0.01 25.94 7 0.19 0.147 0.01 0.077 0.04 27.027 0.17 16.197 0.13 0.08 7 0.001 0.107 0.001 1.217 0.02 ND 17.597 0.12 3.767 0.03 ND ND ND 3.767 0.03 130.167 3.79
5.007 0.07 4.43 7 0.07 33.107 0.19 13.96 7 0.02 10.39 7 0.06 9.767 0.08 0.187 0.004 0.09 7 0.004 0.03 7 0.03 76.95 7 0.14 ND ND 0.34 7 0.01 4.56 7 0.04 0.067 0.01 ND 4.96 7 0.04 14.977 0.04 ND ND ND ND 14.977 0.04 2.417 0.01 ND ND ND 2.417 0.01 63.78 7 1.95
4.747 0.01 4.25 7 0.01 31.46 7 0.02 13.247 0.02 10.337 0.01 9.727 0.02 0.20 7 0.001 0.147 0003 0.127 0.003 74.287 0.01 ND ND 0.34 7 0.01 5.45 7 0.01 0.067 0.003 ND 5.877 0.04 14.617 0.01 0.137 0.001 0.157 0.001 1.87 7 0.01 ND 16.807 0.01 2.32 7 0.003 ND ND ND 2.327 0.003 68.767 3.14
Fatty acid methyl esters from 8:0 to 24:0 were analyzed. Each parameter is presented as the mean 7SEM. Total fatty acids values o 0.01% are indicate as “not detected; ND”.
(P o0.05, Fig. 5). The KO-ARA group had a tendency toward deteriorated motor function in the motor coordination test (P o0.10), although the administration of arachidonic acid for 9 weeks
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resulted in elevated spontaneous motor activity compared to the KO-Cont group (P o0.01, Figs. 4 and 5). Plasma, erythrocytes, and brain were collected at 10 weeks of age and fatty acid composition was measured. In the brain, the concentration of ARA in the KO-ARA group was much higher than
Fig. 2. Artificial feeding apparatus and technique.
Fig. 5. Motor coordination test on rota-rod. The KO-Cont group was significantly lower than the WT-Cont group. The KO-ARA group was lower than the WT-Cont group ((n) Po 0.10, nP o0.05, Duncan's test after one-way ANOVA).
Table 5 The fatty acid composition of brain in D6D mice (% of total fatty acid).
Fig. 3. Restoration of body weight loss by ARA in D6D-KO mice fed LA and ALA. Body weight is presented as mean 7 SEM. The KO-Cont group was significantly lower than the WT-Cont and KO-ARA groups (nnPo 0.01 vs, WT-Cont, ##Po 0.01 vs, KO-ARA, Tukey's test after two-way ANOVA).
Fig. 4. Spontaneous motor activity. The KO-Cont group was significantly lower than the WT-Cont and KO-ARA groups (**Po 0.01, Duncan’s test after one-way ANOVA).
14:0 16:0 16:0DMA 18:0 18:0DMA 20:0 22:0 23:0 24:0 Total sat. 16:1n-7 16:1n-9 18:1DMA 18:1n-7 18:1n-9 20:1n-7 20:1n-9 22:1n-9 24:1n-9 Total mono. 18:2n-6 18:3n-6 20:2n-6 20:3n-6 20:4n-6 22:4n-6 22:5n-6 Total n-6 FAs 18:3n-3 22:5n-3 22:6n-3 Total n-3 FAs 7,11,14-20:3 7,11,14,17-20:4 9,13,16,19-22:4 Total D5D products Total PUFA n-6/n-3 Total FAs (lg/mg)
WT
D6D-KO
Control (n¼ 24)
Control (n¼ 6)
ARA (n ¼7)
0.20 70.004 18.18 70.05 2.15 70.01 19.76 70.05 3.62 70.02 0.45 70.01 0.63 70.02 0.19 70.004 0.81 70.02 45.99 70.09 0.56 70.01 0.18 70.01 1.20 70.01 3.68 70.03 14.30 70.05 ND 1.57 70.02 0.19 70.004 2.02 70.04 23.7070.11 0.40 70.01 ND 0.11 70.002 0.42 70.01 8.84 70.05 2.21 70.01 0.42 70.02 12.39 70.06 ND 0.17 70.003 14.84 70.05 15.06 70.06 ND ND ND ND 27.45 70.07 0.82 70.01 38.40 70.27
0.32 7 0.01 19.45 7 0.06 2.93 7 0.01 17.317 0.08 2.94 7 0.01 0.42 7 0.01 0.42 7 0.01 0.137 0.004 0.52 7 0.03 44.45 7 0.15nn 0.99 7 0.05 0.177 0.004 1.09 7 0.01 4.84 7 0.06 16.54 7 0.07 0.46 7 0.02 2.03 7 0.04 0.177 0.01 1.85 7 0.06 28.157 0.20nn 3.447 0.10 nn ND 1.40 7 0.05nn ND 1.94 7 0.08nn 0.40 7 0.01nn ND 7.20 7 0.12nn ND 0.517 0.01nn 5.90 7 0.10nn 6.42 7 0.10 nn 8.82 7 0.13 0.217 0.01 0.56 7 0.02 10.42 7 0.12 23.217 0.16nn 1.127 0.03nn 36.03 7 0.41nn
0.25 70.01 18.82 70.09 2.43 70.02 19.32 70.06 3.36 70.03 0.44 70.01 0.50 70.02 0.16 70.005 0.7070.04 45.99 70.10## 0.58 70.02 0.09 70.002 1.08 70.03 4.30 70.05 14.29 70.07 ND 1.62 70.04 0.18 70.01 1.98 70.07 24.11 70.18## 0.66 70.02nn,## ND 0.2470.01nn,## 0.10 70.002nn 13.87 70.09nn,## 6.20 70.08nn,## ND 21.09 70.16nn,## ND 0.48 70.01nn,## 4.91 70.16nn,## 5.39 70.16nn,## 0.13 70.01## ND 0.04 70.02## 0.17 70.02## 26.65 70.16nn,## 3.94 70.14nn,## 37.94 70.47#
Fatty acid methyl esters from 8:0 to 24:1 were analyzed. Each parameter is presented as the mean 7SEM. Total fatty acids values o 0.01% are indicate as “not detected; ND”. Some minor and unidentified peaks are not listed. nPo 0.05, nn Po 0.01 vs WT-Cont, #P o0.05, ##Po 0.01 vs KO-Cont (one-way ANOVA followed by Tukey's test).
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Table 6 Fatty acid composition of erythrocytes in D6D mice (% of total fatty acid).
12:0 14:0 16:0 16:DMA 18:0 18:DMA 20:0 22:0 23:0 24:0 Total sat. 16:1n-7 16:1n-9 18:1DMA 18:1n-7 18:1n-9 20:1n-7 20:1n-9 22:1n-9 24:1n-9 Total mono. 18:2n-6 18:3n-6 20:2n-6 20:3n-6 20:4n-6 22:2n-6 22:4n-6 22:5n-6 Total n-6 FAs 18:3n-3 20:3n-3 20:5n-3 22:5n-3 22:6n-3 Total n-3 FAs 7,11,14-20:3 7,11,14,17-20:4 9,13,16,19-22:4 Total D5D Products Total PUFA n-6/n-3 Total FAs (lg/ll)
WT
D6D-KO
Control (n¼ 24)
Control (n¼ 6)
ARA (n¼ 7)
0.317 0.04 0.81 7 0.06 24.187 0.32 1.39 7 0.03 11.727 0.19 0.737 0.02 0.28 7 0.01 0.95 7 0.02 0.157 0.02 1.777 0.05 42.287 0.49 1.217 0.07 0.56 7 0.12 0.317 0.01 2.137 0.06 11.007 0.33 0.417 0.01 0.127 0.02 0.08 7 0.02 1.45 7 0.03 17.287 0.37 8.36 7 0.14 0.02 7 0.01 0.157 0.01 1.38 7 0.05 16.28 7 0.34 ND 1.50 7 0.04 1.007 0.07 28.697 0.46 0.177 0.01 ND 0.43 7 0.02 0.677 0.02 5.53 7 0.13 6.807 0.15 ND ND ND ND 35.497 0.51 4.277 0.12 3.307 0.06
0.217 0.04 0.89 7 0.09 23.337 0.28 1.60 7 0.06n 10.117 0.34nn 0.63 7 0.03 0.28 7 0.01 0.87 7 0.03 0.08 7 0.02 1.577 0.6 39.577 0.30n 1.56 7 0.19 0.277 0.08 0.447 0.03nn 5.147 0.27nn 14.52 7 0.42nn 0.56 7 0.05nn 1.667 0.16nn 0.25 7 0.01nn 2.58 7 0.12nn 26.977 0.68nn 15.95 7 0.45nn ND 1.34 7 0.10nn ND 1.60 7 0.50nn 0.137 0.005 0.05 7 0.03nn ND 19.097 0.83nn 0.377 0.02nn 0.05 7 0.02 0.28 7 0.02nn 0.23 7 0.02nn ND 0.937 0.05nn 7.93 7 0.34 0.767 0.07 2.02 7 0.13 10.717 0.39 30.737 0.72nn 20.917 1.69 3.587 0.38
0.32 7 0.07 0.85 7 0.08 24.197 0.31 1.317 0.09 # 13.047 0.18nn,## 0.717 0.05 0.32 7 0.03 0.95 7 0.03 0.08 7 0.03 1.58 7 0.04 43.34 7 0.25## 1.05 7 0.07 # 0.047 0.02 n 0.32 7 0.03## 2.52 7 0.07n,## 11.337 0.33## ND 0.54 7 0.02## 0.147 0.01 # 1.717 0.03nn,## 17.65 7 0.30## 8.23 7 0.22 ## ND 0.29 7 0.01nn,## 0.50 7 0.01nn,## 24.357 0.18nn,## ND 3.667 0.14nn,## ND 37.03 7 0.19nn,## 0.23 7 0.01## ND ND ND ND 0.277 0.03nn 0.36 7 0.02## 0.117 0.02## 0.36 7 0.03## 0.837 0.05## 38.137 0.16n,## 146.917 15.06nn,## 3.38 7 0.21
Fatty acid methyl esters from 8:0 to 24:1 were analyzed. Each parameter is presented as the mean 7 SEM. Total fatty acids values o0.01% are indicate as “not detected; ND”. Some minor and unidentified peaks are not listed. nP o 0.05, nn P o0.01 vs WT-Cont, ##Po 0.01 vs KO-Cont (one-way ANOVA followed by Tukey's test).
in the WT-Cont group (Po0.01), and moreover, docosatetraenoic acid (22:4n-6) derived from ARA elongation was significantly increased (P o0.01). However, in the KO-Cont group the concentration of LA and eicosadienoic avid (20:2n-6) derived from elongase was greater than in the WT-Cont and KO-ARA groups (P o0.01), although the concentration of ARA and 22:4n-6 in the KO-Cont group was lower compared to the WT-Cont and KO-ARA groups (P o0.01). The concentrations of DHA in both the KO-Cont and KO-ARA groups were significantly lower than that in the WTCont group (P o0.01). It should be noted that DHA in the KO-ARA group was significantly lower than that in the KO-Cont group (P o0.01). Total PUFA in the KO-Cont and KO-ARA groups was lower than in the WT-Cont group (Po 0.01), the KO-Cont group was especially lower than the KO-ARA (Po0.01). To compensate for them, the minor fatty acids derived from D5D had accumulated in both KO groups. In addition, the total fatty acid content of the KO-Cont was clearly reduced with respect to the WT-Cont and KO-
ARA groups (P o0.01, Table 5). The fatty acid compositions of erythrocyte and plasma followed a very similar pattern to the brain, except that these changes were more dramatic, as the blood was directly exposed to the influence of diet rather than being filtered through the blood-brain barrier (erythrocyte, Table 6; plasma, data not shown). Most notable were the very large increases in ARA in the KO-ARA group as well as the absence of DHA in both KO groups.
4. Discussion This study evaluated body and brain weight growth as well as behavioral changes with ARA intake as the independent variable. Intracranial and bloodstream fatty acid composition were also examined in wild type and D6D-KO mice at the end of the 10 week experiment. We tested newborn pups, separated from their dams within 48 hours and fed either artificial milk containing LA and ALA, or that supplemented with ARA [24]. The KO-Cont group did not require ARA and DHA intake for survival but consistently showed lower body weight than the other groups; body weight at 10 weeks of age was approximately 30% lower in the KO-Cont group than in the WT-Cont group, indicating suppressed physical growth. However, increases in body weight between the KO-ARA group and the WT-Cont group were comparable. These results suggest that supplementation with the 18-C essential fatty acids LA and ALA only is insufficient, and that added contribution of ARA is essential for mammalian physical growth. A previous study noted that a diet containing only two types of PUFA (LA and ALA), given after weaning, caused dermatitis, formation of intestinal tumors, and fatty liver after 4 months of age in D6D-KO mice, but supplementation with ARA prevented these symptoms [9]. Prostaglandin E2, derived from ARA, exhibits growth hormone–like actions and is involved in skeletal muscle increases and bone metabolism [28]. It is also known to protect the gastrointestinal mucosa in cases of ulceration [29]. At the very least, the action of ARA on the gastrointestinal mucosa and resulting favorable nutrient absorption may explain comparable weight gain between the KO-ARA and WT-Cont groups in this study. Even more concerning was the decrease in brain weights in the KO-Cont group relative to either of the other groups. This deficit was corrected in the artificially reared group to which ARA was added (KO-ARA group). These data clearly show that ARA is capable of restoring normal brain and body growth. The artificially reared groups did not receive preformed DHA and what is not clear is whether in the DHA sufficient animal whether these ARA effects would still be obtained. Motor function assessment indicated that spontaneous motor activity and motor coordination were impaired in the KO-Cont group, probably due to the strong influence of poor physical growth. However, body weight increases and spontaneous motor activity were similar between the KO-ARA and the WT-Cont group, but motor coordination was not increased above the WT-Cont group in the KO-ARA group. Long-term oral ARA has been reported to increase brain ARA levels in n-3 deficient mice, thereby enhancing spontaneous motor activity significantly while impairing motor coordination [25]. These findings suggest that ARA is essential for infants’ growth in the lactation period, but continuing a high level of ARA intake in the absence of dietary DHA results in increased brain ARA levels, and possibly decreased motor function control. DHA was detected in the brain in both the KO-Cont and the KOARA groups, likely originating from maternal DHA passed to pups via the placenta or breast milk before formula feeding began at 48 hours after birth. However, the KO-ARA group showed higher brain ARA levels than the WT-Cont group, but lower brain DHA
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level than the KO-Cont group. Excess accumulation of ARA in the brain was associated with decreases in brain DHA levels [25] consistent with the known antagonism between these two long chain PUFAs. In the KO-Cont group, fatty acids were not metabolized after the LA and ALA stages due to the lack of D6D, resulting in marked decreases in the amounts of both n-6 and n-3 fatty acids having 4 20-carbon chain. Minor fatty acids such as 20:3 (Δ7, 11, 14), 20:4 (Δ7, 11, 14, 17) and 22:4 (Δ9, 13, 16 and 19), which have been found in the cat, a natural limited D6D animal requiring D5D activity in the body were accumulated in apparent compensation for the reduction in the above PUFA [9,30]. In the lactation period, the body grows rapidly but enzymatic activities are still low. ARA intake appears essential in such a period at least in mice, to supplement immature lipid metabolizing enzymes and thereby support rapid physical growth. However, selective intake of high levels of ARA and in the absence of dietary DHA after the lactation period may cause an imbalance between n-6 and n-3 fatty acids in the brain and bloodstream. Further studies on the optimum timing and duration of ARA intake, in relation to the balance between n-6 and n-3 fatty acids, are necessary to support healthier growth and development in infants.
[10]
[11]
[12]
[13]
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[15]
[16]
[17]
Acknowledgments This research was supported in part by The Ministry of Education, Culture, Sports, Science and Technology (Grant-in-Aid for Scientific Research C-22500777) and by funding from DSM Nutritional Products Inc., Columbia, Maryland, USA.
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Contents lists available at ScienceDirect
Prostaglandins, Leukotrienes and Essential Fatty Acids journal homepage: www.elsevier.com/locate/plefa
Essentiality of arachidonic acid intake in murine early development Erisa Hatanaka a, Akiko Harauma a, Hidemi Yasuda a, Junnosuke Watanabe a, Manabu T. Nakamura b, Norman Salem Jr.c, Toru Moriguchi a,n a
School of Life and Environmental Science, Azabu University, 1-17-71 Fuchinobe, Sagamihara, Kanagawa 252-5201, Japan Division of Nutritional Science, University of Illinois at Urbana-Champaign, 905 South Goodwin Avenue, Urbana, IL 61801, USA c Nutritional Lipids, DSM Nutritional Products, Columbia, MD 21045, USA b
art ic l e i nf o
a b s t r a c t
Article history: Received 8 February 2016 Received in revised form 17 March 2016 Accepted 17 March 2016
We previously reported the importance of long-chain polyunsaturated fatty acid (LC-PUFA ( 4C20)) intake, including arachidonic acid (ARA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA), for growth. This follow-up study focuses on ARA using a novel artificial rearing model during the lactation period in delta-6-desaturase knockout (D6D-KO) mice. Newborn D6D-KO male mouse pups were separated from dams within 48 hours and fed artificial milks containing 18-C essential fatty acids (EFAs) (16–17% LA, 3.8–4.1% ALA) with or without 1.2% ARA. After weaning, mice were maintained on similar diets: 15% LA, 2.3–2.4% ALA with or without 1.9% ARA. As a reference group, new born wild type (WT) male mouse pups were maintained by artificial milk and diet containing LA and ALA without ARA. Aspects of brain function were measured behaviorally (motor activity and rota-rod test) when mice were age 9 weeks. Body weight in the KO-Cont group was significantly lower (approximately 30%) than in the WT-Cont group, but this decrease was ameliorated by providing ARA in the KO-ARA group. The motor activity and coordination in the KO-Cont group decreased markedly compared to the WT-Cont group. The KO-ARA group had a tendency toward deteriorated motor coordination, although the motor activity was significantly enhanced compared to the KO-Cont group. In KO-ARA group brains, the level of ARA was increased and DHA decreased compared to WT-Cont. These results suggest that intake of LA and ALA only is insufficient to support healthy growth, and that ARA is also required, at least during the lactation period. These findings also suggested that continued intake of relatively high levels of ARA and without supplemental DHA during development led to an increased motor activity above that of WT animals. These studies indicate that both ARA dose and proper combination with DHA must be delineated to define optimal growth and behavioral function. & 2016 Elsevier Ltd. All rights reserved.
Keywords: Delta-6-desaturase Knock-out mouse Polyunsaturated fatty acid Arachidonic acid Docosahexaenoic acid
1. Introduction Two families of polyunsaturated fatty acids (PUFAs), n-6 and n-3 fatty acids, are essential fatty acids, as they cannot be biosynthesized and therefore must be ingested in the diet. Although a lack of n-6 fatty acids causes growth disorders and dermopathy [1,2], linoleic acid (LA, 18:2n-6) and arachidonic acid (ARA, 20:4n6) are contained in many types of foods, especially animal fats and vegetable oils, and therefore such deficiencies rarely occur. α-Linolenic acid (ALA, 18:3n-3), an n-3 fatty acid, is contained in perilla Abbreviations: D6D, delta-6-desaturase; KO, knock-out; PUFA, polyunsaturated fatty acid; LA, linoleic acid; ARA, arachidonic acid; ALA, α-linolenic acid; DHA, docosahexaenoic acid n Correspondence to: Laboratory of Food and Nutritional Science, Department of Food and Life Science, School of Life and Environmental Science, Azabu University, 1-17-71 Fuchinobe, Chuo, Sagamihara, Kanagawa 252-5201, Japan. E-mail address: [email protected] (T. Moriguchi). http://dx.doi.org/10.1016/j.plefa.2016.03.007 0952-3278/& 2016 Elsevier Ltd. All rights reserved.
oil and linseed oil, and its metabolites eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3) are abundant in fish and seafood [3]. DHA can pass through the blood-brain barrier [4] and the blood-retinal barrier to accumulate in these organs, thereby improving brain function (e.g. learning ability), mental condition, and visual function [5–7]. It has also been reported to have preventive and therapeutic effects on cardiovascular diseases [8]. However, n-3 fatty acid–rich foods are relatively uncommon, and thus, resulting impairment of brain function by deficiency of n-3 fatty acids is a concern. Dietary LA is converted to ARA through a reaction catalyzed by delta-6-desaturase (D6D) and chain elongating enzymes, and then to the n-6 end product docosapentaenoic acid (DPAn-6, 22:5n-6) via the reactions catalyzed by chain elongating enzymes, D6D and β-oxidation [9]. Similar fatty acid–metabolizing enzymes convert dietary ALA to EPA and DHA. On the other hand, it has been reported that DPAn-6 and DHA may be synthesized by direct delta-4-desatuation in human
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cells [10]. However in human infants, the activities of these desaturases are still too low to sufficiently convert LA and ALA to ARA and DHA, respectively, requiring direct intake of ARA and DHA [11]. Infants undergo marked growth and development of the body and the brain, and several studies have demonstrated the efficacy of ARA and DHA intake in infancy [12–14]; in fact, breast milk is rich in these fatty acids [15–17]. PUFAs are necessary to maintain biological functions throughout all life stages, but its intake is likely to be particularly essential in infancy. Yet, the European Food Safety Authority (EFSA) recently expressed the opinion that there is no need to add ARA and EPA to infant and follow-on formulae [18]. The effect of PUFA in infants has been examined only through administration of ARA plus DHA [13,14], and through administration of DHA only, and the effect of PUFA intervention in infancy after the initial growth period have been studied only rarely. Thus, a careful evaluation of the EFSA claim using carefully controlled diets in infants is necessary. D6D knockout mice (D6D-KO mice), cannot convert either n-6 or n-3 fatty acids to their more unsaturated metabolites [9,19,20] and thus serve as an ideal model for investigating requirements for various PUFAs [21]. In this study, we examined the importance of ARA in infancy using a D6D-KO mouse model and an artificial feeding technique from the second day of life.
2. Materials and methods The experimental protocol was approved by the Institutional Animal Care and Use Committee of Azabu University (No. 1410072). 2.1. D6D-KO mice D6D-KO mice were back-crossed to the C57BL/J background were transferred from the University of Illinois. Heterozygous male and female fed a custom pelleted diet (termed “MF”, Oriental Yeast Co., Ltd., Tokyo, Japan, Table 1), were mated, and resultant male WT and homozygous KO were used for the study. Tail DNA genotyping by PCR was performed to confirm D6D KO status in mice [9]. DNA was extracted from tails of 2-day-old mice using a tissue PCR kit (Extract-N-Amp™, Sigma-Aldrich, Tokyo, Japan), and PCR was performed using a thermal cycler (T100™, Bio-Rad Laboratories K.K., Tokyo, Japan). After DNA genotyping, both homozygous Table 1 Composition of custom pelleted diet.1 Amount (g/100g diet) Pelleted diet Casein, vitamin free Carbohydrate: Cellulose Mineral-Salt mix Fat
23.1 55.3 2.8 5.8 5.1
Fatty acid composition (% of total fatty acids) Saturates Monounsaturates 18:2n-6 20:4n-6 Total n-6 FAs 18:3n-3 20:5n-3 22:6n-3 Total n-3 FAs Total FA (μg/mg)
19.6 28.2 42.0 0.13 42.3 3.9 1.5 1.3 6.3 47.0
1
Pelleted chow is made by Oriental Yeast Co., Ltd., Tokyo, Japan.
(D6D-KO, / ) and wild type (WT, þ/þ) mice were used in this study. The study design of this experiment is shown as Fig. 1. Newborn male pups of D6D-KO mice were separated from their dams within 48 h, and were fed artificial milks containing 18-C EFAs (LA and LA) with or without ARA. The fat content of two experimental milks (Control and ARA) is approximately 16% and their fatty acid compositions are shown in Table 2. After weaning, mice were housed in an artificially regulated environment at 2373 °C, 55 710% humidity, under a 12-h light/dark cycle (lights on between 07:00 and 19:00), and fed a similar diet with or without ARA as during lactation (Table 3). The fatty acid compositions of these artificial milks and diets are shown in Table 4. As reference group, new born WT male mouse pups were maintained by artificial milk and diet containing LA and ALA without ARA (WT-Cont). When mice were age 9 weeks, motor coordination and spontaneous motor activity were assessed. At age 10 weeks, blood and brain tissue was collected and fatty acid composition was analyzed using gas chromatography (GC) (Fig. 1). 2.1.1. Artificial mouse milk Artificial milk formula was developed based on the methods of Yajima et al. and Hussein et al. [22,23], with slight modifications of fat content (Table 2). Casein and whey protein were used as protein sources and lactose was used as the carbohydrate. Fat was adjusted to 16% by mixing several oils. For complete dissolution, ingredients were mixed in the order described by Yajima et al. [22] using a sonicator (Ultrasonic Processor S-4000, Misonix, Inc., Farmingdale, NY, USA). Milk was homogenized twice under high pressure (800–1000 bar) using a high-pressure homogenizer (Panda PLUS 2000, Niro Soavi S.p.A., Parma, Italy) resulting in emulsified, sterilized, and smoothed milk. The homogenized milk was stored at 80 °C. 2.1.2. Artificial rearing system The artificial rearing procedure used a hand-feeding technique with specially constructed nursing bottles [24], (Fig. 2). Pups were separated from their dams on postnatal day 2 and fed artificial milk by hand using a nursing bottle every 3 h (5 times/day). Pups were capable of suckling from silicon nipples connected to the nursing bottles. Pups were placed in a cage with an ovariectomized foster mother for maternal care and warmth except at feeding times. From day 14, pups were fed artificial milk from a nursing bottle in combination with a pelleted diet. Infant diets were made by mixing a crushed control diet and their respective artificial milks. Pups in all groups were weaned to the pelleted diet at day 21. Pups were housed in an artificially regulated environment at 23 73 °C, 557 10% humidity after a 12-h light/dark cycle was used (lights on between 07:00 and 19:00). 2.2. Behavioral experiments 2.2.1. Motor activity test Motor activity was measured using cages (19 30 13 cm) equipped with running wheels (Wireless Low Profit Running Wheel, ENV-044 wheel, and SOF-860 software, Neuro-science Co., Ltd., Tokyo, Japan). Mice were assessed individually at the same time of day by recording the number of wheel rotations over a 30 min period (08:00–11:00) [25]. 2.2.2. Motor coordination test The motor coordination test was carried out using a rota-rod setup (Rota-Rod Treadmill, ENV-575M, Neuro-science Co., Ltd., Tokyo, Japan). Mice were allowed a trial run of 5 min on the rotor, set at 4 rpm, on the day preceding the measurement. On the following day, motor coordination ability was assessed with rotational speed set to increase from 4 rpm to 40 rpm over 5 min. The
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Fig. 1. Schematic diagram showing study design of D6D-KO mouse experiment. Newborn D6D-KO male pups were separated from their dams within 48 hours and fed artificial milks. After weaning, mice were fed diets similar to these during lactation. Spontaneous motor activity and the rota-rod test were conducted at 9 weeks of age. At 10 weeks of age mice were killed by decapitation, and plasma and brain tissue were collected and analyzed to ascertain fatty acid composition.
maximum duration that the mice remain on the rotor, up to a limit of 300 s, was recorded [25]. 2.3. Lipid extraction and transmethylation
22:5n-6, and 23:0. The standard fatty acid was used to verify the identity and assignment of retention times. Fatty acid quantification was checked using quantitative standard mixtures. Internal standards were used to calculate tissue fatty acid concentrations.
The transmethylation method developed by Lepage and Roy was used [26]. A half-brain (approximately 200 mg) was homogenized with 6 ml methanol:hexane (4:1) solvent containing 50 mg/ml butylhydroxytoluene to prevent lipid oxidation, and an additional 60 mg docosatrienoic acid methyl ester (22:3n-3 methyl ester) was added as an internal standard. After homogenization, 200 ml acetylchloride was added to each 2 ml homogenate in tubes (13 mm 100 mm). Tubes were capped under nitrogen gas and transferred to a heating block at 100 °C for 60 min to transmethylate fatty acids. Samples were then placed on ice to remove unmethylated acids by addition of 5 ml of 6% solution of K2CO3. The tubes were vortexed and centrifuged for 15 min at 2200 g to remove emulsion and separate the mixture into two phases. The upper phase of hexane was collected into micro-vials for GC injection [25]. For plasma and erythrocytes (100 ml of each sample), 2 ml methanol:hexane solvent and 20 mg of the internal standard were added to tubes and processed further as for brain tissue.
2.5. Statistical analysis
2.4. GC analysis
There was no difference in body weight change among the three groups during the lactation period. Body weight in the KOARA group was almost identical to that in the WT-Cont group during the experimental period for 10 weeks (P ¼0.85). However, the KO-Cont group had clearly lower body weight than the WTCont and KO-ARA groups after 3 weeks of age (P o0.01, Fig. 3). At 10 weeks of age, the mean body weights were 25.5 70.3 g for the WT-Cont, 17.37 0.5 g for the KO-Cont (Po 0.01 vs WT-Cont) and 25.2 70.6 g for the KO-ARA (Po 0.01 vs KO-Cont). The KO-Cont group (341.27 4.7 mg) had also significantly lower brain weights at 10 weeks of age compared with the WT-Cont and KO-ARA groups (389.0 73.6 and 377.7 7.3 mg, respectively, P o0.01), while those were not significantly different between WT-Cont and KOARA groups (body weight; P ¼0.84, brain weight; P ¼0.28). The spontaneous motor activity for 30 min in the KO-Cont group decreased markedly compared to the WT-Cont group (P o0.01, Fig. 4). For motor coordination, the KO-Cont group had a shorter duration before falling compared with the WT-Cont group
Fatty acid methyl esters were analyzed using an Agilent 7890A Network Gas Chromatograph (Agilent Technologies; Palo Alto, CA) equipped with a split injector, a 7693A automatic liquid sampler, an FID, and a 208 V power supply to enable rapid temperature ramping. The instrument was controlled, and data were collected using GC Chemstation Rev. B0.040.01. SP1 (Agilent Technologies). The column used was a DB-FFAP (15 m 0.1 mm i.d. 0.1 mm film thickness, J&W Scientific, Agilent Technologies). The detector and injector temperatures were set to 250 °C. The oven temperature program began at 150 °C with a 0.25 min hold, was increased at a rate of at 35 °C/min to 200 °C followed by 8 °C/min to 225 °C with a 3.2 min hold and 80 °C/min to 245 °C with a 2.75 min hold. Hydrogen was used as the carrier gas at a linear velocity of 56 cm/s [25,27]. A standard mixture of 30 components was supplemented with a custom standard (NuChek Prep 462, Elysian, MN) containing 10–24 carbons and 0–6 double bonds and 16:1n-9, 22:3n-3,
Data are expressed as means 7standard errors. Body weight gain for the 10-week period was analyzed using a repeated measures two-way ANOVA followed by Tukey's test. Body and brain weight at 10 weeks of age and content of each fatty acid in plasma, brain and erythrocyte were analyzed using one-way ANOVA followed by Tukey's test. The results of motor activity and the motor coordination test were analyzed using one-way ANOVA followed by Duncan's test (Statistica, Statsoft Japan, Tokyo, Japan). Significance was set at p o0.05. Total fatty acids values o0.01% were considered to indicate “not detected; ND.”
3. Results 3.1. D6D-KO mice
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E. Hatanaka et al. / Prostaglandins, Leukotrienes and Essential Fatty Acids 108 (2016) 51–57
Table 2 Composition of artificial milk.2 Ingredient
Amount (weight/100 mL milk)
Whey protein isolate (BIPRO)a Whey protein hydrolyzed (PEPTIGENs IF-3090)b Casein (Acid Casein LACTIC CASEIN720)c Serined Cystined Tryptophand Methionined Carbohydrate (g) Lactosee Minerals (mg) NaOHd KOHe GlyCaPO4d MgCl2 6H2Od CaCl2 2H2Od CaCo3d Ca-Citratee Na2HPO4d KH2PO4d FeSO4d Citrate H2Oe ZnSO4e CuSO4e MnSO4e NaFd KId K2SO4e Na2SiO3 9H2Od Na2O4Sed H8MoN2O4 4H2Oe KCr(SO4)2 12H2Od LiCle H3BO3d NiCO3d NH4VO3e Vitamins (mg) Vitamin mixf Vitamin Ce Vitamin K3d Vitamin Ae Vitamin Dd Vitamin Ed Others (mg) Carnitined Picolinated Ethanolamined Taurined Tricholine Citrated
Protein (g)
Fat (g)
Table 3 Composition of experimental diets.3
4.0 5.0
Amount (g/100g diet)
Casein, vitamin free Carbohydrate: Cornstarch Sucrose Glucose Dextrose Maltose Cellulose Mineral-Salt mix Vitamin mix L-Cystine Choline bitartrate TBHQ Fat Hydrogenated coconut oil Safflower oil Flaxseed oil s ARASCO
4.0 0.02875 0.3 0.027 0.0045 1.89 25 150 800 190 170 184 120 80 8 24 0.5 6 1.5 0.25 0.155 0.25 163.5 5.075 0.035 0.0275 0.975 0.05 0.285 0.1125 0.0225 400 200 1.9825 0.1284 23.46 0.0025 4 2 3.5 15 147
Control
ARA
20 63 15 13 20 7.5 7.5 5 3.5 1 0.3 0.25 0.002 7 5.4 1.2 0.3 none
20 63 15 13 20 7.5 7.5 5 3.5 1 0.3 0.25 0.002 7.2 5.4 1.2 0.3 0.2
3 The experimental diet was based on the AIN-93 formulation with modificas tion to the fat composition (Oriental Yeast Co., Ltd., Tokyo, Japan). ARASCO was used as ARA source (DSM Nutritional Products, Columbia, MD, USA).
Table 4 Fatty acid composition of artificial milk and experimental diet (% of total fatty acid). Artificial milk
(16.04 g)
Control
ARA
MCTg Palm oilh Coconut oile Corn oile Soybean oile Linseed oile s ARASCO i Cholesterole
1.25 8.25 2.5 0.5 2.75 0.75 none 0.04
1.25 7.75 2.5 0.5 2.75 0.75 0.5 0.04
2 Artificial milk formula was made following the method of Yajima et al. and Hussein et al. Component sources were as follows: a Davisco Foods International, Inc. MN, USA. b Arla Foods Ingredients,Viby, Denmark. c Fonterra Co-operative Group Limited, Auckland, New Zealand. d Sigma-Aldrich Corp. MO, USA. e Wako Pure Chemical Industries, Ltd., Osaka, Japan. f Oriental Yeast Co., Ltd., Tokyo, Japan. g The Nisshin Oillio Group,Ltd., Tokyo, Japan. h Spectrum Laboratory Products, Inc., New York, USA. i DSM Nutritional Products, Columbia, MD, USA.
8:0 10:0 12:0 14:0 16:0 18:0 20:0 22:0 24:0 Total Sat. 14:1 16:1n-7 18:1n-7 18:1n-9 20:1n-9 22:1n-9 Total Mono. 18:2n-6 18:3n-6 20:3n-6 20:4n-6 22:5n-6 Total n-6 FAs 18:3n-3 20:5n-3 22:5n-3 22:6n-3 Total n-3 FAs Total Fas (lg/mg)
Diet
Control
ARA
Control
ARA
5.40 7 0.02 2.31 7 0.01 6.677 0.02 3.17 7 0.02 25.94 7 0.20 3.68 7 0.02 0.29 7 0.005 0.13 7 0.005 0.09 7 0.002 47.72 7 0.25 0.077 0.001 0.11 7 0.002 0.68 7 0.003 25.97 7 0.18 0.15 7 0.01 0.12 7 0.04 27.12 7 0.12 17.41 7 0.13 ND ND ND ND 17.41 7 0.13 4.09 7 0.03 ND ND ND 4.09 7 0.03 133.467 6.97
5.497 0.14 2.36 7 0.01 6.84 7 0.04 3.25 7 0.03 26.377 0.19 3.94 7 0.02 0.317 0.01 0.177 0.01 0.127 0.005 48.887 0.15 0.077 0.003 0.127 0.003 0.687 0.01 25.94 7 0.19 0.147 0.01 0.077 0.04 27.027 0.17 16.197 0.13 0.08 7 0.001 0.107 0.001 1.217 0.02 ND 17.597 0.12 3.767 0.03 ND ND ND 3.767 0.03 130.167 3.79
5.007 0.07 4.43 7 0.07 33.107 0.19 13.96 7 0.02 10.39 7 0.06 9.767 0.08 0.187 0.004 0.09 7 0.004 0.03 7 0.03 76.95 7 0.14 ND ND 0.34 7 0.01 4.56 7 0.04 0.067 0.01 ND 4.96 7 0.04 14.977 0.04 ND ND ND ND 14.977 0.04 2.417 0.01 ND ND ND 2.417 0.01 63.78 7 1.95
4.747 0.01 4.25 7 0.01 31.46 7 0.02 13.247 0.02 10.337 0.01 9.727 0.02 0.20 7 0.001 0.147 0003 0.127 0.003 74.287 0.01 ND ND 0.34 7 0.01 5.45 7 0.01 0.067 0.003 ND 5.877 0.04 14.617 0.01 0.137 0.001 0.157 0.001 1.87 7 0.01 ND 16.807 0.01 2.32 7 0.003 ND ND ND 2.327 0.003 68.767 3.14
Fatty acid methyl esters from 8:0 to 24:0 were analyzed. Each parameter is presented as the mean 7SEM. Total fatty acids values o 0.01% are indicate as “not detected; ND”.
(P o0.05, Fig. 5). The KO-ARA group had a tendency toward deteriorated motor function in the motor coordination test (P o0.10), although the administration of arachidonic acid for 9 weeks
E. Hatanaka et al. / Prostaglandins, Leukotrienes and Essential Fatty Acids 108 (2016) 51–57
55
resulted in elevated spontaneous motor activity compared to the KO-Cont group (P o0.01, Figs. 4 and 5). Plasma, erythrocytes, and brain were collected at 10 weeks of age and fatty acid composition was measured. In the brain, the concentration of ARA in the KO-ARA group was much higher than
Fig. 2. Artificial feeding apparatus and technique.
Fig. 5. Motor coordination test on rota-rod. The KO-Cont group was significantly lower than the WT-Cont group. The KO-ARA group was lower than the WT-Cont group ((n) Po 0.10, nP o0.05, Duncan's test after one-way ANOVA).
Table 5 The fatty acid composition of brain in D6D mice (% of total fatty acid).
Fig. 3. Restoration of body weight loss by ARA in D6D-KO mice fed LA and ALA. Body weight is presented as mean 7 SEM. The KO-Cont group was significantly lower than the WT-Cont and KO-ARA groups (nnPo 0.01 vs, WT-Cont, ##Po 0.01 vs, KO-ARA, Tukey's test after two-way ANOVA).
Fig. 4. Spontaneous motor activity. The KO-Cont group was significantly lower than the WT-Cont and KO-ARA groups (**Po 0.01, Duncan’s test after one-way ANOVA).
14:0 16:0 16:0DMA 18:0 18:0DMA 20:0 22:0 23:0 24:0 Total sat. 16:1n-7 16:1n-9 18:1DMA 18:1n-7 18:1n-9 20:1n-7 20:1n-9 22:1n-9 24:1n-9 Total mono. 18:2n-6 18:3n-6 20:2n-6 20:3n-6 20:4n-6 22:4n-6 22:5n-6 Total n-6 FAs 18:3n-3 22:5n-3 22:6n-3 Total n-3 FAs 7,11,14-20:3 7,11,14,17-20:4 9,13,16,19-22:4 Total D5D products Total PUFA n-6/n-3 Total FAs (lg/mg)
WT
D6D-KO
Control (n¼ 24)
Control (n¼ 6)
ARA (n ¼7)
0.20 70.004 18.18 70.05 2.15 70.01 19.76 70.05 3.62 70.02 0.45 70.01 0.63 70.02 0.19 70.004 0.81 70.02 45.99 70.09 0.56 70.01 0.18 70.01 1.20 70.01 3.68 70.03 14.30 70.05 ND 1.57 70.02 0.19 70.004 2.02 70.04 23.7070.11 0.40 70.01 ND 0.11 70.002 0.42 70.01 8.84 70.05 2.21 70.01 0.42 70.02 12.39 70.06 ND 0.17 70.003 14.84 70.05 15.06 70.06 ND ND ND ND 27.45 70.07 0.82 70.01 38.40 70.27
0.32 7 0.01 19.45 7 0.06 2.93 7 0.01 17.317 0.08 2.94 7 0.01 0.42 7 0.01 0.42 7 0.01 0.137 0.004 0.52 7 0.03 44.45 7 0.15nn 0.99 7 0.05 0.177 0.004 1.09 7 0.01 4.84 7 0.06 16.54 7 0.07 0.46 7 0.02 2.03 7 0.04 0.177 0.01 1.85 7 0.06 28.157 0.20nn 3.447 0.10 nn ND 1.40 7 0.05nn ND 1.94 7 0.08nn 0.40 7 0.01nn ND 7.20 7 0.12nn ND 0.517 0.01nn 5.90 7 0.10nn 6.42 7 0.10 nn 8.82 7 0.13 0.217 0.01 0.56 7 0.02 10.42 7 0.12 23.217 0.16nn 1.127 0.03nn 36.03 7 0.41nn
0.25 70.01 18.82 70.09 2.43 70.02 19.32 70.06 3.36 70.03 0.44 70.01 0.50 70.02 0.16 70.005 0.7070.04 45.99 70.10## 0.58 70.02 0.09 70.002 1.08 70.03 4.30 70.05 14.29 70.07 ND 1.62 70.04 0.18 70.01 1.98 70.07 24.11 70.18## 0.66 70.02nn,## ND 0.2470.01nn,## 0.10 70.002nn 13.87 70.09nn,## 6.20 70.08nn,## ND 21.09 70.16nn,## ND 0.48 70.01nn,## 4.91 70.16nn,## 5.39 70.16nn,## 0.13 70.01## ND 0.04 70.02## 0.17 70.02## 26.65 70.16nn,## 3.94 70.14nn,## 37.94 70.47#
Fatty acid methyl esters from 8:0 to 24:1 were analyzed. Each parameter is presented as the mean 7SEM. Total fatty acids values o 0.01% are indicate as “not detected; ND”. Some minor and unidentified peaks are not listed. nPo 0.05, nn Po 0.01 vs WT-Cont, #P o0.05, ##Po 0.01 vs KO-Cont (one-way ANOVA followed by Tukey's test).
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E. Hatanaka et al. / Prostaglandins, Leukotrienes and Essential Fatty Acids 108 (2016) 51–57
Table 6 Fatty acid composition of erythrocytes in D6D mice (% of total fatty acid).
12:0 14:0 16:0 16:DMA 18:0 18:DMA 20:0 22:0 23:0 24:0 Total sat. 16:1n-7 16:1n-9 18:1DMA 18:1n-7 18:1n-9 20:1n-7 20:1n-9 22:1n-9 24:1n-9 Total mono. 18:2n-6 18:3n-6 20:2n-6 20:3n-6 20:4n-6 22:2n-6 22:4n-6 22:5n-6 Total n-6 FAs 18:3n-3 20:3n-3 20:5n-3 22:5n-3 22:6n-3 Total n-3 FAs 7,11,14-20:3 7,11,14,17-20:4 9,13,16,19-22:4 Total D5D Products Total PUFA n-6/n-3 Total FAs (lg/ll)
WT
D6D-KO
Control (n¼ 24)
Control (n¼ 6)
ARA (n¼ 7)
0.317 0.04 0.81 7 0.06 24.187 0.32 1.39 7 0.03 11.727 0.19 0.737 0.02 0.28 7 0.01 0.95 7 0.02 0.157 0.02 1.777 0.05 42.287 0.49 1.217 0.07 0.56 7 0.12 0.317 0.01 2.137 0.06 11.007 0.33 0.417 0.01 0.127 0.02 0.08 7 0.02 1.45 7 0.03 17.287 0.37 8.36 7 0.14 0.02 7 0.01 0.157 0.01 1.38 7 0.05 16.28 7 0.34 ND 1.50 7 0.04 1.007 0.07 28.697 0.46 0.177 0.01 ND 0.43 7 0.02 0.677 0.02 5.53 7 0.13 6.807 0.15 ND ND ND ND 35.497 0.51 4.277 0.12 3.307 0.06
0.217 0.04 0.89 7 0.09 23.337 0.28 1.60 7 0.06n 10.117 0.34nn 0.63 7 0.03 0.28 7 0.01 0.87 7 0.03 0.08 7 0.02 1.577 0.6 39.577 0.30n 1.56 7 0.19 0.277 0.08 0.447 0.03nn 5.147 0.27nn 14.52 7 0.42nn 0.56 7 0.05nn 1.667 0.16nn 0.25 7 0.01nn 2.58 7 0.12nn 26.977 0.68nn 15.95 7 0.45nn ND 1.34 7 0.10nn ND 1.60 7 0.50nn 0.137 0.005 0.05 7 0.03nn ND 19.097 0.83nn 0.377 0.02nn 0.05 7 0.02 0.28 7 0.02nn 0.23 7 0.02nn ND 0.937 0.05nn 7.93 7 0.34 0.767 0.07 2.02 7 0.13 10.717 0.39 30.737 0.72nn 20.917 1.69 3.587 0.38
0.32 7 0.07 0.85 7 0.08 24.197 0.31 1.317 0.09 # 13.047 0.18nn,## 0.717 0.05 0.32 7 0.03 0.95 7 0.03 0.08 7 0.03 1.58 7 0.04 43.34 7 0.25## 1.05 7 0.07 # 0.047 0.02 n 0.32 7 0.03## 2.52 7 0.07n,## 11.337 0.33## ND 0.54 7 0.02## 0.147 0.01 # 1.717 0.03nn,## 17.65 7 0.30## 8.23 7 0.22 ## ND 0.29 7 0.01nn,## 0.50 7 0.01nn,## 24.357 0.18nn,## ND 3.667 0.14nn,## ND 37.03 7 0.19nn,## 0.23 7 0.01## ND ND ND ND 0.277 0.03nn 0.36 7 0.02## 0.117 0.02## 0.36 7 0.03## 0.837 0.05## 38.137 0.16n,## 146.917 15.06nn,## 3.38 7 0.21
Fatty acid methyl esters from 8:0 to 24:1 were analyzed. Each parameter is presented as the mean 7 SEM. Total fatty acids values o0.01% are indicate as “not detected; ND”. Some minor and unidentified peaks are not listed. nP o 0.05, nn P o0.01 vs WT-Cont, ##Po 0.01 vs KO-Cont (one-way ANOVA followed by Tukey's test).
in the WT-Cont group (Po0.01), and moreover, docosatetraenoic acid (22:4n-6) derived from ARA elongation was significantly increased (P o0.01). However, in the KO-Cont group the concentration of LA and eicosadienoic avid (20:2n-6) derived from elongase was greater than in the WT-Cont and KO-ARA groups (P o0.01), although the concentration of ARA and 22:4n-6 in the KO-Cont group was lower compared to the WT-Cont and KO-ARA groups (P o0.01). The concentrations of DHA in both the KO-Cont and KO-ARA groups were significantly lower than that in the WTCont group (P o0.01). It should be noted that DHA in the KO-ARA group was significantly lower than that in the KO-Cont group (P o0.01). Total PUFA in the KO-Cont and KO-ARA groups was lower than in the WT-Cont group (Po 0.01), the KO-Cont group was especially lower than the KO-ARA (Po0.01). To compensate for them, the minor fatty acids derived from D5D had accumulated in both KO groups. In addition, the total fatty acid content of the KO-Cont was clearly reduced with respect to the WT-Cont and KO-
ARA groups (P o0.01, Table 5). The fatty acid compositions of erythrocyte and plasma followed a very similar pattern to the brain, except that these changes were more dramatic, as the blood was directly exposed to the influence of diet rather than being filtered through the blood-brain barrier (erythrocyte, Table 6; plasma, data not shown). Most notable were the very large increases in ARA in the KO-ARA group as well as the absence of DHA in both KO groups.
4. Discussion This study evaluated body and brain weight growth as well as behavioral changes with ARA intake as the independent variable. Intracranial and bloodstream fatty acid composition were also examined in wild type and D6D-KO mice at the end of the 10 week experiment. We tested newborn pups, separated from their dams within 48 hours and fed either artificial milk containing LA and ALA, or that supplemented with ARA [24]. The KO-Cont group did not require ARA and DHA intake for survival but consistently showed lower body weight than the other groups; body weight at 10 weeks of age was approximately 30% lower in the KO-Cont group than in the WT-Cont group, indicating suppressed physical growth. However, increases in body weight between the KO-ARA group and the WT-Cont group were comparable. These results suggest that supplementation with the 18-C essential fatty acids LA and ALA only is insufficient, and that added contribution of ARA is essential for mammalian physical growth. A previous study noted that a diet containing only two types of PUFA (LA and ALA), given after weaning, caused dermatitis, formation of intestinal tumors, and fatty liver after 4 months of age in D6D-KO mice, but supplementation with ARA prevented these symptoms [9]. Prostaglandin E2, derived from ARA, exhibits growth hormone–like actions and is involved in skeletal muscle increases and bone metabolism [28]. It is also known to protect the gastrointestinal mucosa in cases of ulceration [29]. At the very least, the action of ARA on the gastrointestinal mucosa and resulting favorable nutrient absorption may explain comparable weight gain between the KO-ARA and WT-Cont groups in this study. Even more concerning was the decrease in brain weights in the KO-Cont group relative to either of the other groups. This deficit was corrected in the artificially reared group to which ARA was added (KO-ARA group). These data clearly show that ARA is capable of restoring normal brain and body growth. The artificially reared groups did not receive preformed DHA and what is not clear is whether in the DHA sufficient animal whether these ARA effects would still be obtained. Motor function assessment indicated that spontaneous motor activity and motor coordination were impaired in the KO-Cont group, probably due to the strong influence of poor physical growth. However, body weight increases and spontaneous motor activity were similar between the KO-ARA and the WT-Cont group, but motor coordination was not increased above the WT-Cont group in the KO-ARA group. Long-term oral ARA has been reported to increase brain ARA levels in n-3 deficient mice, thereby enhancing spontaneous motor activity significantly while impairing motor coordination [25]. These findings suggest that ARA is essential for infants’ growth in the lactation period, but continuing a high level of ARA intake in the absence of dietary DHA results in increased brain ARA levels, and possibly decreased motor function control. DHA was detected in the brain in both the KO-Cont and the KOARA groups, likely originating from maternal DHA passed to pups via the placenta or breast milk before formula feeding began at 48 hours after birth. However, the KO-ARA group showed higher brain ARA levels than the WT-Cont group, but lower brain DHA
E. Hatanaka et al. / Prostaglandins, Leukotrienes and Essential Fatty Acids 108 (2016) 51–57
level than the KO-Cont group. Excess accumulation of ARA in the brain was associated with decreases in brain DHA levels [25] consistent with the known antagonism between these two long chain PUFAs. In the KO-Cont group, fatty acids were not metabolized after the LA and ALA stages due to the lack of D6D, resulting in marked decreases in the amounts of both n-6 and n-3 fatty acids having 4 20-carbon chain. Minor fatty acids such as 20:3 (Δ7, 11, 14), 20:4 (Δ7, 11, 14, 17) and 22:4 (Δ9, 13, 16 and 19), which have been found in the cat, a natural limited D6D animal requiring D5D activity in the body were accumulated in apparent compensation for the reduction in the above PUFA [9,30]. In the lactation period, the body grows rapidly but enzymatic activities are still low. ARA intake appears essential in such a period at least in mice, to supplement immature lipid metabolizing enzymes and thereby support rapid physical growth. However, selective intake of high levels of ARA and in the absence of dietary DHA after the lactation period may cause an imbalance between n-6 and n-3 fatty acids in the brain and bloodstream. Further studies on the optimum timing and duration of ARA intake, in relation to the balance between n-6 and n-3 fatty acids, are necessary to support healthier growth and development in infants.
[10]
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[12]
[13]
[14]
[15]
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[17]
Acknowledgments This research was supported in part by The Ministry of Education, Culture, Sports, Science and Technology (Grant-in-Aid for Scientific Research C-22500777) and by funding from DSM Nutritional Products Inc., Columbia, Maryland, USA.
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