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* Laboratory of Membrane Biochemistry and Biophysics, National Institutes on Alcohol Abuse and Alcoholism, National Institutes of Health
Division of Ocean Science, Korea Maritime University, Busan, Korea
Uniformed Services University of the Health Sciences, Bethesda, MD
** Department of Animal Resources, Advanced Science Research Center, Okayama University, Okayama, Japan
Published, JLR Papers in Press, June 1, 2004. DOI 10.1194/jlr.M400087-JLR200
1 To whom correspondence should be addressed. e-mail: nsalem{at}niaaa.nih.gov
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-linolenic acid (LNA) were fed, using an artificial rearing system, either an n-3-deficient (n-3-Def) or an n-3-adequate (n-3-Adq) diet. Both diets contained 17.1% linoleic acid, but the n-3-Adq diet also contained 3.1% LNA. The percentage of brain docosahexaenoic acid (DHA) continuously decreased (71%) with time over the 29 days of the experiment, with concomitant increases in docosapentaenoic acid (DPAn-6). In the retina, the percentage of DHA rose in the n-3-Adq group, with an apparent increased rate around the time of eye opening. However, there was a flat curve for the percentage of DHA in the n-3-Def group and a rising DPAn-6 with time. Liver DHA was highest at the time of birth in the n-3-Adq group but fell off somewhat over the course of 29 days. This decrease was more pronounced in the n-3-Def group, and the DPAn-6 rose considerably during the second half of the experiment. This method presents a first-generation model for n-3 deficiency that is more similar to the case of human nutrition than is the commonly employed two-generation model.
Abbreviations: AA, arachidonic acid (20:4n6); DHA, docosahexaenoic acid (22:6n3); DPAn-6, docosapentaenoic acid (22:5n6); EPA, eicosapentaenoic acid (20:5n3); LA, linoleic acid, (18:2n6); LNA, linolenic acid (18:3n3); n-3 Adq, n-3 adequate; n-3 Def, n-3 deficient
Supplementary key words artificial rearing • docosahexaenoic acid • docosapentaenoic acid • fatty acid composition
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INTRODUCTION |
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50% loss in the first generation (1). However, this method has some disadvantages, including the stress of surgery and forced feeding, and the absence of social interaction and suckling and swallowing behavior. But perhaps its greatest limitation is that this method cannot begin until day 4–5 (1, 3, 4).
Hoshiba (5, 6) developed a completely new system for the artificial rearing of rat pups based on the use of natural suckling behavior together with a bottle-nipple system devised to control the resistance required for milk flow. He has shown that this system can be used to feed rats an artificial milk beginning within 12 h of birth (6). This system has recently been adapted to the study of essential fatty acids by Lim et al. (7). In this work, Lim et al. demonstrated that rat pups fed on an n-3-deficient (n-3-Def) diet from postnatal day 2 to adulthood had spatial learning deficits. These pups were artificially reared using artificial milk modeled on that used by Kanno et al. (4) but modified so as to control the n-3 fatty acid content. The objective of the present study was to demonstrate that a marked level of DHA deficiency could be achieved in the nervous system by the use of this system without resorting to the multiple generation approach that has heretofore been standard. This paper describes the time course of tissue fatty acid responses during n-3-Def or n-3-adequate (n-3-Adq) diets. In this model, a diet that contains -linolenic acid (LNA) is fed to the dam so that n-3 deficiency is induced only after birth. An experiment was performed to determine whether the controlled feeding of the maternal diet could be shortened so that it began only after conception. In this way, first generation n-3-Def rat pups could be generated in a relatively short period of time.
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MATERIALS AND METHODS |
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Lipid extraction, transmethylation, and gas chromatography
Tissue samples were thawed, weighed, and homogenized in methanol-hexane, and methylated in acetyl chloride according to the method of Lepage and Roy (9). Varying amounts of methyl docosatrienoate (22:3n-3) for brain, retina, and liver, and methyl heneicosanoate (21:0) for the stomach contents of dam-reared pups were added as internal standards to each sample to compensate for differences in tissue weight and lipid concentration. As an aid to avoiding lipid oxidation during the procedures, 50 µg/ml butylated hydroxytoluene was added to the methanol-hexane homogenizing solvent. The hexane extracts were concentrated to a small volume with a stream of nitrogen and transferred to microvials for GC injection.
Fatty acid methyl esters were analyzed with an HP-5890B gas chromatograph equipped with a flame ionization detector (Agilent, Palo Alto, CA) and a fused silica capillary column (DB-FFAP, 30 m x 0.25 mm inner diameter x 0.25 µm film thickness; J and W Scientific, Folsom, CA). The detector and injector temperatures were set to 250°C. The oven temperature program began at 130°C and increased to 175°C at 4°C/min, then increased at 1°C/min to 210°C, and finally increased at 30°C/min to 245°C, with a final hold for 15 min. Hydrogen was used as carrier gas at a linear velocity of 50 cm/sec. A custom-mixed, 30 component, quantitative methyl ester standard containing 10–24 carbons and 0–6 double bonds was used for assignment of retention times and to ensure accurate quantification (Nu-Chek Prep 462, Elysian, MN). Fatty acid data were expressed as percent of total peak area, which corresponded to weight % to within 5%, as demonstrated by quantitative standard mixtures. Internal standards were used to calculate tissue fatty acid concentrations.
Analysis of data
The time course curves for DHA and docosapentaenoic acid (DPAn-6) data from each tissue were fitted by regression analysis using the Sigma Plot program (SPSS Science Inc., Chicago, IL). Data on the stomach contents of dam-reared pups are expressed as mean ± SEM. These fatty acid data were analyzed by one-way ANOVA using Statistica (Statsoft, Tulsa, OK). Body weight differences between the artificially reared and the dam-reared groups were compared at discrete time points using Student's t-test.
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RESULTS |
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Artificial rearing
The body weights of the artificially reared rats were generally less than those of the dam-reared animals during the initial milk-feeding phase. For example, at day 20, the body weights of the artificially reared animals were 43.4 ± 1.8 g and those of the dam-reared were 60.6 ± 3.5 g (t(5) = 2.94, P < 0.05). However, after 29 days, there was no difference in body weight between the dam-reared and the artificially reared pups (dam-reared, 109.7 ± 3.9 g; artificially reared, 106.1 ± 0.3 g; t(5) = 0.56, P = 0.60). There were no differences in body weight between the two artificially reared groups related to the n-3 content of the diet. Artificial rearing led to some difficulties with intestinal bloating that led to death in some cases. This difficulty was believed to be related to excessive air intake; the bacterial milieu of the milk and the rat tissues was tested but revealed no unusual flora. The artificial rat milk diets employed ethyl esters of unsaturated fatty acids so as to maintain a very low level of n-3 fatty acids (Table 2). Also, sources of proteins were tested, and those with the lowest levels of n-3 polyunsaturates were used. Although the pelleted diet did not employ ethyl ester sources of unsaturated fatty acids, the n-3 level was also very low, and this diet was used only for the last 9 days.
Brain
The pup brain DHA and DPAn-6 levels at the time of birth were 10.45 ± 0.24% and 2.98 ± 0.15%, respectively (Table 3). Thereafter, the DHA level in the dam-reared group increased gradually and reached 11.88 ± 0.32% (n = 5) at 29 days of age. The time course curve for the brain DHA in the n-3-Adq group was very similar to that for the dam-reared group, with its level rising to 12% (Fig. 3). On the other hand, the brain DHA level of the n-3-Def group decreased rapidly from about day 10 to day 17 and then continued to gradually decrease percentage-wise to a level of 3.4% at the end of the experiment. The brain DPAn-6 of the dam-reared and n-3-Adq groups decreased gradually to less than 1% by 29 days of age. For example, the brain DPAn-6 in the dam-reared group at 29 days was 0.86 ± 0.02% (n = 5). The level of brain DPAn-6 in the n-3-Def group showed a rapid increase after 5 days of age and was 9.0% at 29 days (Fig. 3).
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10 days of age (Fig. 4). Eye opening at 12–14 days of age was accompanied by a jump in the DHA level in the 10–14 day period, with the level increasing by 15 percentage points within 5 days. The increase in the percentage of retinal DHA appeared to be sigmoid, reaching its adult level by the end of the experiment at 29 days. There was a slight lag in the DHA increase in the n-3-Adq group, but the same level was reached by 29 days, and the curves were similar. The DPAn-6 in these two groups began low at birth (1.63 ± 0.25%) and remained constant. In the n-3-Def group, there was also a slight increase in DHA up to 10 days of age; however, in the period of eye opening and afterwards, there was no further increase in the retinal DHA. After day 13, the DPAn-6 in the n-3-Def group rose quickly to 28.4% at day 29.
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4% of fatty acids by day 20 (Fig. 5). There was more biological variability in the liver; for example, between days 5 and 15, values in the n-3-Adq group ranged from 4% to 11%. Thereafter, there appeared to be a slight decline in the DHA level, ending at 4.4% at 29 days. A smooth curve was obtained for the liver DHA in the n-3-Def group, which exhibited a decline by day 10 to 1%. The curve asymptotically declined thereafter, falling to 0.3% at day 29. The DPAn-6 in the dam-reared and n-3-Adq groups gradually and linearly declined from a low initial level (1.42 ± 0.11%). However, the liver DPAn-6 in the n-3-Def group followed a time course curve much like that of the retina, inasmuch as it increased linearly at first and then with increasing slope.
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20% or less of their values on day 1. LA was constant, but LNA increased significantly. This pattern continued in the day 20 stomach content samples, with long-chain polyunsaturates decreasing further and LNA increasing further. The percentage of monounsaturated fatty acid fell, whereas the saturates increased significantly between days 0 and 10. It should be noted that the absolute concentration of total fatty acids was falling precipitously, inasmuch as it fell to about half of the birth level by 20 days of age.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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69% lower in the pups fed the n-3-Def milk formula relative to those fed the same formula to which 3.1% LNA had been added. If this diet were to be continued, these low levels of nervous system DHA could be maintained into adulthood. It was of interest that the dam-reared animals were receiving preformed DHA from their dams (as evidenced by their stomach contents) even though the dams had been fed a diet with LNA as the sole source of n-3 fatty acids for an extended period. Thus, the dam-reared pups received DHA, whereas the artificially reared pups maintained on the n-3-Adq diet did not. However, the level of LNA in the stomach contents of the dam-reared pups (0.5–1.3%) was lower than that fed to the n-3-Adq pups (3.1%). These factors may in some manner counterbalance each other. Still, the initially slightly higher levels of DHA observed in the nervous system in the dam-reared pups relative to the n-3-Adq pups were likely due to the presence of preformed DHA in their diets.
An important issue is whether this "first generation" model can generate large enough losses in nervous system DHA so that functional changes can be observed. Few such studies are available, but Lim et al. (7) have recently shown, in an experiment similar in design to the present one, that adult rats with a 70% loss in brain DHA performed more poorly in spatial tasks. The degree of n-3 deficiency here can be compared with other two- or three-generational studies in which functional effects were observed. For example, Weisinger et al. (13) were able to show that there was a loss in retinal sensitivity and b-wave implicit times in rats after three generations of n-3 deficiency. However, these rats had only a 55% loss of retinal DHA, probably owing to their age and the presumed slow accretion of n-3 fatty acids from the periphery, and showed no differences in a-wave amplitudes or in most of the phototransduction parameters measured. Wainwright et al. (14, 15) reported that rats with 40–50% loss of brain DHA had no alteration in spatial task performance. Also, Moriguchi and Salem (10) have recently demonstrated, in a DHA repletion paradigm, that spatial task performance is altered when the brain level of DHA declines by 40% or more. It thus appears that many of the available measures of retinal physiology and brain behavior generally require a 50% decrease or more in brain or retinal DHA to observe a loss in function. The artificial rearing method presented here achieves a somewhat greater loss in DHA than this and so may be expected to produce animals that exhibit functional alterations of the nervous system.
It was of great interest that the percentage of brain DHA at birth was high whereas the retinal value was relatively low (cf. Figs. 3, 4). Thus, the time courses for these two nervous system organs with respect to DHA content are quite distinct. The "normal" retinal development entails a steady accretion of DHA, as reflected by the steady rise in the percentage of DHA. In the case of n-3 deficiency, however, retinal DHA remains near the low level characteristic at birth. The "normal" pattern of brain accretion of DHA, however, is one in which there is only a slight increase in the percentage of DHA during the first 4 weeks of development. When n-3 fatty acids are not fed, there is a rapid diminution in the percentage of brain DHA in the first 3 weeks of life.
It must be noted here that the percentages of DHA in various organs at birth has little meaning without reference to the maternal diet during gestation. One example of this was shown in the higher levels of milk AA and DHA on the first day of birth in pups of time-pregnant dams who had been fed EPA and DHA prior to pregnancy. This is evidence that the maternal body complement of these long-chain polyunsaturates influences the dam's ability to output AA and DHA in milk. The controlled maternal diet employed in this study was the n-3-Adq diet that contained 3.1% of fatty acids as LNA (and no EPA or DHA). This diet was chosen to provide an ample source of n-3 fatty acids during fetal development so that pups would be born with a supply of tissue DHA and other n-3 fatty acids. This situation models fairly well that of humans in the Western world, where most of the n-3 fatty acids are supplied by LNA. It would thus be expected that human infants who receive vegetable oil-based formulas in which the essential fatty acids are supplied by corn oil or safflower oil would exhibit a marked decline in brain (16–18) and retinal DHA within the first months of life, similar to that observed for the n-3-Def rats in the present study.
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ACKNOWLEDGMENTS |
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Manuscript received March 1, 2004 and in revised form May 20, 2004.
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