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- Materials and methods
Adult male unanesthetized rats, reared on a diet enriched in both α-linolenic acid (α-LNA) and docosahexaenoic acid (DHA), were infused intravenously for 5 min with [1-14C]α-LNA. Timed arterial samples were collected until the animals were killed at 5 min and the brain was removed after microwaving. Plasma and brain lipid concentrations and radioactivities were measured. Within plasma lipids, > 99% of radioactivity was in the form of unchanged [1-14C]α-LNA. Eighty-six per cent of brain radioactivity at 5 min was present as β-oxidation products, whereas the remainder was mainly in ‘stable’ phospholipid or triglyceride as α-LNA or DHA. Equations derived from kinetic modeling demonstrated that unesterified unlabeled α-LNA rapidly enters brain from plasma, but that its incorporation into brain phospholipid and triglyceride, as in the form of synthesized DHA, is ≤ 0.2% of the amount that enters the brain. Thus, in rats fed a diet containing large amounts of both α-LNA and DHA, the α-LNA that enters brain from plasma largely undergoes β-oxidation, and is not an appreciable source of DHA within brain phospholipids.
α-Linolenic acid (α-LNA; 18 : 3n-3) is a dietary essential n-3 polyunsaturated fatty acid (PUFA) that is a precursor for docosahexaenoic acid (DHA; 22 : 6n-3). Conversion of α-LNA to DHA occurs by a series of elongation, desaturation and β-oxidation steps, passing through eicosapentaenoic acid (EPA; 20 : 5n-3) and docosapentaenoic acid (DPA; 22 : 5n-3) intermediates (Sprecher 2000). DHA also can be directly obtained from dietary sources. Within brain, DHA is largely esterified at the stereospecifically numbered (sn)-2 position of phospholipids (Sastry 1985). It is thought to modulate membrane elasticity (Salem et al. 2001), activation of G-protein coupled receptors, ion channel flow, and neurotransmitter release (Innis 2003). Depletion of brain DHA by prolonged dietary deficiency of n-3 PUFAs can impair brain function in mammals (Innis 2000; Youdim et al. 2000).
Like other long-chain fatty acids (C16–C22) (Washizaki et al. 1994; Grange et al. 1995; Contreras et al. 2000), unesterified plasma α-LNA can rapidly diffuse from plasma into brain (Spector 2001). The extent, however, to which it is converted to DHA within brain is uncertain. Cultured neurons from fetal rat brain were reported to elongate α-LNA only to 20 : 3n-3 (homo-di-α-linolenic acid), suggesting an absence of desaturase activity, whereas cultured fetal rat astrocytes could synthesize the final DHA product as well as the EPA and DPA intermediates (Moore et al. 1991; Moore 2001; Williard et al. 2001). In the astrocytic cultures, about 50% of radiolabeled α-LNA was converted to DHA after 48 h of exposure. Likewise, it has also been noted that cultured rat C6 glioma cells, which are adult-derived but undifferentiated, can convert stable-isotope labeled α-LNA to DHA, with 3% of label in phospholipids as DHA after 16 h of incubation (Cook et al. 1991).
As most studies have focused on α-LNA metabolism in the immature brain, we thought it of importance to quantify the ability of the adult mammalian brain to synthesize DHA from plasma-derived α-LNA, when substantial DHA is included in the diet. We determined rates of uptake of α-LNA from plasma into the brain of unanesthetized adult male rats fed a diet enriched in DHA, as well as the extent to which α-LNA is incorporated into brain phospholipids per se or incorporated after being elongated to DHA inside the brain, as opposed to being lost through diversion to β-oxidation. To do this, we extended to α-LNA our in vivo fatty acid method for examining incorporation and turnover of circulating long chain fatty acids in brain phospholipids (Robinson et al. 1992; Rapoport et al. 2001). An abstract of part of this work has been presented (DeMar et al. 2004b).
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- Materials and methods
This study demonstrates that unesterified radiolabeled α-LNA, circulating in the blood, rapidly enters the brain of unanesthetized rats fed high amounts of DHA, as has been reported for plasma unesterified radiolabeled DHA, ARA (arachidonic acid; 20 : 4n-6) and palmitic (16 : 0) acids (DeGeorge et al. 1991; Washizaki et al. 1994; Grange et al. 1995). Most (∼86%) of the radiolabeled α-LNA that enters the brain rapidly undergoes β-oxidation, whereas only a small fraction is incorporated unchanged into phospholipid (10%) and triglycerides (2%) or as newly synthesized DHA (≤ 0.2%) within these lipids (Table 4, Figs 3 and 5).
Our intravenous infusion conditions followed standard tracer techniques, and rapidly produced a steady-state level of [1-14C]α-LNA in the plasma. The endogenous plasma unesterified α-LNA (41 ± 13 nmol/mL) was not significantly perturbed by infusion of [1-14C]α-LNA, which provided 2.8 ± 0.4 nmol total unlabeled α-LNA. The quantity, if distributed only in plasma, would have raised the plasma unesterified α-LNA concentration by 0.9 ± 0.4%; and thus did not produce a non-tracer effect. The values above were calculated using the amount of [1-14C]α-LNA infused per rat (0.5 mCi/kg), specific radioactivity of the [1-14C]α-LNA (54 mCi/mmol), and estimated whole body plasma volume of rats (29 ± 3 mL/kg) (NIH Guide for the Care and Use of Laboratory Animals; Pub. no. 80–23).
Many studies have examined α-LNA metabolism in mammals by giving the tracer orally, which is the normal route for its entry into the body (Su et al. 1999; Cunnane 2001; Pawlosky et al. 2001; Lefkowitz et al. 2005). We chose a rapid 5-min intravenous infusion to deliver the [1-14C]α-LNA, because an oral route is not able to bypass liver metabolism and leads to a marked delay in brain uptake (Purdon et al. 1997). With an oral dose, it would be very difficult to distinguish synthesized DHA contributed by the liver, via plasma, to the brain from any DHA synthesized within the brain itself. In our study, [14C-DHA] was essentially absent from the plasma at the end of the 5-min infusion (Fig. 2). The albumin-bound unesterified [1-14C]α-LNA that we infused is the normal form that the brain is exposed to for fastest uptake under physiological conditions where brain lipid metabolism is at a constant flow (Rapoport 2001). Additionally, determination of the tracer kinetics of [1-14C]α-LNA uptake and metabolism within a specific organ alone (brain) requires fast delivery of the tracer and tracee at steady-state levels to that compartment (Fig. 1).
Equations derived from our published kinetic incorporation model (Robinson et al. 1992; Rapoport et al. 1997, 2001), when applied to our current experimental data, provided values for incorporation coefficients ki* and incorporation rates Jin,i of plasma-derived unesterified α-LNA into brain phospholipids and triglycerides, and of DHA synthesized within brain from plasma-derived α-LNA into brain phospholipid and triglycerides. The latter equations were valid because [14C]DHA was essentially absent in plasma during the 5-min infusion period. Our equations also provided values for incorporation rates of unlabeled α-LNA from the brain precursor α-LNA-CoA pool, JFA,i, and for the turnover rate and half-life of α-LNA in brain phospholipid, at steady-state unlabeled concentrations.
As illustrated in Table 4, the sum (over i) of Jin,i(α-LNA) for α-LNA in brain phospholipid and trigylceride equaled 12.4 nmol/s/g brain × 10−4, whereas Jin,i(α–LNADHA) for DHA synthesized from plasma-derived α-LNA equaled 0.24 nmol/s/g brain × 10−4. The 50 : 1 ratio of these incorporation rates indicates that plasma-derived α-LNA is incorporated into ‘stable’ brain lipids at a much greater rate than is the DHA synthesized from it, in DHA-fed rats. The rate of incorporation of α-LNA and synthesized DHA, however, represents ∼12% of the rate of entry of α-LNA into brain, as the remainder mostly undergoes β-oxidation. A high fractional β-oxidation of α-LNA in brain is consistent with a reported high percentage of α-LNA β-oxidation in the body as a whole (Menard et al. 1998; Cunnane et al. 1999; Cunnane 2001).
The incorporation rate of unesterified plasmaα-LNA in the form of DHA, Jin,i(α–LNADHA), into brain phospholipids, 0.22 nmol/s/g brain × 10−4, is 1% of the published incorporation rate of unesterified plasma DHA into phospholipids, which is 17.4 nmol/s/g brain × 10−4 (Chang et al. 1999). This difference arises, not because α-LNA enters brain more slowly than does DHA, but because most (∼86%) of the α-LNA that enters undergoes β-oxidation, compared with only 10% of the DHA that enters (Rapoport 2001). It remains to be seen, however, whether shorter-chain n-3 PUFA precursors of DHA, including both α-LNA and EPA, can be efficiently converted to DHA when using them to treat brain disorders in which DHA may be deficient (Pawlosky and Salem 1999; Conquer et al. 2000; Tanskanen et al. 2001; Morris et al. 2003; Noaghiul and Hibbeln 2003; Kack et al. 2004).
Our rats were fed a diet with a high DHA content, and DHA is reported to directly bind to and activate transcription factors that increase expression of β-oxidation enzyme, and decrease expression of the desaturases (Δ-5 and Δ-6) that elongate it from α-LNA (Cho et al. 1999; Price et al. 2000; Clarke 2001; Nakamura and Nara 2003). In neuronal tumor cell cultures (retinoblastoma), when the cellular content of DHA in phospholipids was increased, the transcription factor PPAR-δ (peroxisome proliferator-activated receptor, subtype-δ) up-regulates the expression of oxidative enzymes (Langelier et al. 2003). Mouse brain expression of PPAR-γ (γ-subtype), but not of other PPARs, is elevated in vivo by a diet containing fish oil rich in DHA (Puskas et al. 2004). In contrast, expression of Δ6-desaturase has been shown in the liver to be increased by PPAR-α (α-subtype) activation (Tang et al. 2003). Instead, Δ-5 and Δ-6 desaturase expression in liver are positively controlled by the transcription factors SREBP-1 and NF-Y (sterol regulatory element binding protein-1 and nuclear factor-Y), which are both turned off when DHA is abundant (Matsuzaka et al. 2002; Nara et al. 2002). Thus, if the diet instead contained α-LNA alone without DHA, it is likely that the rate of conversion of α-LNA to DHA in both brain and liver would have been elevated and its rate of β-oxidation reduced. The likelihood for this is predicted from a number of studies where active DHA synthesis is detected in the absence of DHA (Dwyer and Bernsohn 1979; Emken et al. 1999; Moore 2001; Williard et al. 2001). We could test whether conversion of α-LNA to DHA within brain or liver is up-regulated in animals fed α-LNA but not other n-3 PUFAs with the method of this paper. DHA synthesis might also be induced in the brain and liver of rats by subjecting them to prolonged n-3 PUFA deprivation over a single generation (DeMar et al. 2004a) and then abruptly feeding them α-LNA.
We have hypothesized (Rapoport et al. 2001; Rapoport 2003) that the rate of metabolic loss of DHA in brain should equal the rate of its replacement by plasma-derived unesterified DHA plus α-LNA, as n-3 PUFAs cannot be completely synthesized de novo from acetyl-CoA in vertebrate tissues. Now that we have shown, under DHA enriched dietary conditions, that the sum (over i) Jin,i(DHA) >> Jin,i(α–LNADHA), we have proven that the sum of Jin,i(DHA) (over i) is identical to the rate of DHA loss from brain. This conclusion is supported by evidence that DHA loss from rat brain, as calculated from its measured half-life of disappearance, approximates Jin,i(DHA) (Contreras et al. 2000; DeMar et al. 2004b).
The major determinant of the diffusion rate into brain of a circulating unesterified long chain fatty acid is its rate of disassociation from plasma albumin, whereas a major factor that governs its retention in brain is its esterification to Coenzyme A (acyl-CoA) by long chain acyl-CoA synthetase (Marcel and Suzue 1972; Robinson and Rapoport 1986; Robinson et al. 1992; Waku 1992; Rapoport 2003). From the acyl-CoA pool, the fatty acid can be trans-esterified into ‘stable’ triglycerides and phospholipids (Yamashita et al. 1997), or be shuttled into mitochondria after transfer to carnitine by carnitine O-palmitoyl transferase (CPT) and undergo β-oxidation (Fig. 5). The catalytic efficiency of CPT for α-LNA-CoA (Vmax/Km), as bound to albumin, was found to be threefold higher than that for DHA-CoA (Gavino and Gavino 1991). This selectively may provide ‘an enzymatic rationale’ for the relatively low content of α-LNA in esterified lipid, and for the high fractional rate of β-oxidation of α-LNA in brain compared with that of DHA (∼9-fold higher than that of DHA). As in brain, the body as a whole diverts most ingested α-LNA (> 80%) towards β-oxidation (Yang and Cunnane 1994; Cunnane 2001).
Reported turnover rates of DHA and ARA in net brain phospholipid are 0.9 and 3.6% per h, respectively (Rapoport 2001). They correspond to half-lives of 77 and 19 h, although some turnover rates in individual phospholipids, such as transitory molecular species of PI, are higher with corresponding half-lives of a few hours (Washizaki et al. 1994; Shetty et al. 1996; Chang et al. 1999; Contreras et al. 2000, 2001). Reported values for λacyl–CoA (Equation 5) of DHA and ARA are 0.02–0.04, suggesting, according to our model (Robinson et al. 1992; Rapoport 2001), that 96–98% of brain ARA-CoA or DHA-CoA comes from fatty acid released from phospholipid and triglycerides, compared with 2–4% from plasma. In contrast, the half-life for α-LNA in net brain phospholipid equaled 1.3 h, and λα–LNA–CoA equaled 0.77 (Table 5), implying that plasma contributes to about 77% of brain α-LNA-CoA, compared with 23% from hydrolysis of phospholipids.
Rat brain phospholipids contain only a trace of esterified α-LNA (Table 3), in agreement with the literature (Sastry 1985). Nevertheless, 10% of radioactivity was in brain phospholipid after 5 min of constant [1-14C]α-LNA infusion (Fig. 5). These observations, and our finding that the half-life of unlabeled α-LNA in phospholipid equals 1.3 h (Table 5), show that α-LNA in brain phospholipids is rapidly recycled and, when released, undergoes extensive β-oxidation similar to what we found for plasma-derived α-LNA. Recycling (deacylation and reacylation) of polyunsaturated fatty acids ARA and DHA in brain phospholipids is an ongoing and active process (Lands and Crawford 1976; Rapoport 2001), but unlike α-LNA they are much less susceptible to β-oxidation and thus are conserved in the phospholipids by the recycling process. In pregnant rhesus monkeys, DHA undergoes ∼80% less β-oxidation than α-LNA, as determined by recycling of β-oxidation products into maternal plasma and fetal brain fatty acids (Sheaff Greiner et al. 1996). Physiological differences in the potential for β-oxidation thus may largely account for the maintained abundance of ARA and DHA, and the near absence of α-LNA, in neuronal membranes. Enrichment of specific polyunsaturated fatty acids in brain does not appear to be as a result of any discriminatory uptake by PUFA transporters at the blood–brain barrier (Edmond 2001).
Below normal blood levels of DHA or reduced dietary n-3 PUFA intake has been reported to be associated with bipolar disorder, Alzheimer disease, depression, chronic alcoholism, and age-related cognitive dysfunction (Pawlosky and Salem 1999; Conquer et al. 2000; Tanskanen et al. 2001; Morris et al. 2003; Noaghiul and Hibbeln 2003; Whalley et al. 2004). Furthermore, although some clinical trials have suggested that feeding long chain n-3 PUFAs, such as EPA and DHA, is therapeutically effective in bipolar disorder (Stoll et al. 1999), a randomized control trial with dietary EPA did not show a beneficial outcome (Kack et al. 2004). Rigorously designed trials are lacking and it is not certain whether DHA itself, one of its more immediate n-3 precursors such as EPA, or both, should be used in these trials (Kidd 2004; Marangell et al. 2004). In this regard, the results of our study suggest α-LNA does not have great potential to effectively serve as a precursor for synthesizing DHA within the adult brain, when substantial DHA is found in the diet.
In summary, we have demonstrated quantitatively that α-LNA circulating in the plasma can enter the brain of unanesthetized adult rats fed a DHA-enriched diet. However, the amount that enters is largely lost to β-oxidation products and little if any is used for synthesizing DHA found esterified into phospholipids. Because of this, the rate of influx of plasma unesterified DHA across the blood–brain barrier and into brain phospholipids, Jin,i(DHA), alone represents the rate of loss by metabolism of brain DHA.
Additionally, these observations suggest that dietary supplementation with n-3 PUFAs to increase brain DHA content might consist of only DHA and not have to include its precursors, when DHA is already present in the diet. It should be recognized, however, that consumption of large amounts of α-LNA is capable of supporting nearly normal levels of brain DHA, when DHA is completely absent from the diet (Abedin et al. 1999; DeMar et al. 2004a; Lefkowitz et al. 2005). In this case, it is possible that the β-oxidation of α-LNA is greatly suppressed and DHA synthesis strongly up-regulated. Our study also did not directly address the degree to which the brain oxidizes, stores, and elongates EPA or DPA, two more immediate dietary precursors to DHA.