α-Linolenate reduces the dietary requirement for linoleate in the growing rat.

BACKGROUND: We hypothesized that due to the absence of a dietary source of omega-3 fatty acids, the essential fatty acid (EFA) deficiency model leads to an overestimate of linoleic acid (LA) requirements. METHODS: over 7wk, young rats consumed an EFA diet containing either 0en% linoleate (0LA) and 0en% α-linolenate (0LNA) or a diet containing 0.5en% LNA plus one of seven levels of added LA (0.12-4.0en%; n=6/group). RESULTS: Rats consuming the 0LA-0LNA diet had the lowest final body weight, 34-68% lower LA and arachidonate in plasma and liver, 87% lower LA in epididymal fat, and an 8-20 fold higher eicosatrienoate in plasma, liver and muscle lipids. 0.5LNA completely prevented the lower growth and partly prevented the rise in eicosatrienoate seen in the 0LA-0LNA group. CONCLUSION: Providing dietary LNA at 0.5 en% reduces the rat's physiological requirement for LA by an estimated factor of at least four (0.5en% instead of 2en%). Since LA requirements in humans are also based on the same flawed model of EFA deficiency, it is plausible that they too have been overestimated and should therefore be reinvestigated.

Brain and systemic glucose metabolism in the healthy elderly following fish oil supplementation.

Cerebral metabolic rate of glucose (CMRg) is lower in individuals affected by cognitive decline and dementia, especially in Alzheimer's disease. However, as yet there is no consensus as to whether CMRg decreases during healthy aging. Epidemiological studies show that weekly consumption of fish abundant in ω3 fatty acids has a protective effect on cognition during aging. Thus, the primary objective of this human study was to use positron emission tomography analysis with (18)F-fluorodeoxyglucose to evaluate whether supplementation with a fish oil rich in ω3 fatty acids increases cerebral glucose metabolism in young or elderly adults. Healthy young (23±5y old; n=5) and elderly (76±3y old; n=6) women and men were included in the study. Semi-quantitative expression of the data as 'standardized uptake values' showed that elderly participants had significantly lower cerebral glucose metabolism compared with the young group. However, when expressed quantitatively a CMRg, there was no effect of age or ω3 supplementation on glucose metabolism in any of the brains regions studied. Higher plasma triglyceride levels and higher plasma insulin levels were associated with lower CMRg in several regions, suggesting that a trend towards the metabolic syndrome may be associated with cerebral hypometabolism. We conclude that under these experimental conditions, ω3 supplementation did not affect brain glucose metabolism in the healthy elderly. Future studies in this area should address whether glucose intolerance or other conditions linked to the metabolic syndrome impact negatively on brain glucose metabolism and cognition.

Higher plasma n-3 fatty acid status in the moderately healthy elderly in southern Québec: higher fish intake or aging-related change in n-3 fatty acid metabolism?

The elderly reportedly have a significantly higher % of eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids in plasma and red cell lipids. However, these observations are from a few small studies and the health status of the elderly in these studies is for the most part unclear. Since the elderly are susceptible to cardiovascular and neurological illnesses that seem to be related in part to lower intake of n-3 fatty acids it seems paradoxical that their blood levels of EPA and DHA would be higher than in young adults. We report here plasma fatty acid profiles and their response to supplementation with two types of fish oils from several of our recent studies in the moderately healthy elderly. We define the moderately healthy elderly as those who were in good physical condition, had no cognitive decline and, if present, in whom hypothyroidism, hyperlipidemia and/or hypertension were well-controlled. As shown previously, we confirm the higher % EPA and % total n-3 fatty acids (but not DHA) in fasting plasma and extend these findings to include higher plasma concentrations (mg/L) of n-3 fatty acids as well. The EPA-predominant supplement raised DHA only in the young, whereas the DHA-predominant supplement raised EPA more in the young than in the elderly. The moderately healthy elderly clearly have higher plasma n-3 fatty acids but whether this reflects differences in intake versus aging-related changes in n-3 fatty acid metabolism remains to be elucidated.

Potential role of polyunsaturates in seizure protection achieved with the ketogenic diet.

Epilepsy is a serious neurological disease that responds to two very different treatments involving lipids. Clinically, it responds to a state of ketosis induced by a very high-fat 'ketogenic' diet. Experimentally, in vitro and in vivo models demonstrate that injection or infusion of free (non-esterified) polyunsaturates such as arachidonate and docosahexaenoate also reduces seizure susceptibility. In our experience, rats on a very high-fat ketogenic diet not only have mild-to-moderate ketosis, but also have raised serum free fatty acids. Some polyunsaturates, particularly linoleate and alpha-linolenate, are relatively easily beta-oxidized and are therefore ketogenic. We conclude that raised levels of free plasma polyunsaturates could contribute to the beneficial effect of the ketogenic diet in refractory epilepsy not only by helping sustain ketosis, but also by their own direct (though poorly defined) antiseizure effects.

Lipid and fatty acid profiles in rats consuming different high-fat ketogenic diets.

High-fat ketogenic diets are used to treat intractable seizures in children, but little is known of the mechanism by which these diets work or whether fats rich in n-3 polyunsaturates might be beneficial. Tissue lipid and fatty acid profiles were determined in rats consuming very high fat (80 weight%), low-carbohydrate ketogenic diets containing either medium-chain triglyceride, flaxseed oil, butter, or an equal combination of these three fat sources. Ketogenic diets containing butter markedly raised liver triglyceride but had no effect on plasma cholesterol. Unlike the other fats, flaxseed oil in the ketogenic diet did not raise brain cholesterol. Brain total and free fatty acid profiles remained similar in all groups, but there was an increase in the proportion of arachidonate in brain total lipids in the medium-chain triglyceride group, while the two groups consuming flaxseed oil had significantly lower arachidonate in brain, liver, and plasma. The very high dietary intake of alpha-linolenate in the flaxseed group did not change docosahexaenoate levels in the brain. Our previous report based on these diets showed that although ketosis is higher in rats consuming a ketogenic diet based on medium-chain triglyceride oil, seizure resistance in the pentylenetetrazol model is not clearly related to the degree of ketosis achieved. In combination with our present data from the same seizure study, it appears that ketogenic diets with widely differing effects on tissue lipids and fatty acid profiles can confer a similar amount of seizure protection.

The effect on serum lipids and oxidized low-density lipoprotein of supplementing self-selected low-fat diets with soluble-fiber, soy, and vegetable protein foods.

An increased intake of soluble fiber and soy protein may improve the blood lipid profile. To assess any additional benefit on serum lipids of providing soy protein and soluble-fiber foods to hyperlipidemic subjects already consuming low-fat, low-cholesterol therapeutic diets, 20 hyperlipidemic men and postmenopausal women completed 8-week test and control dietary treatments in a randomized crossover design as part of an ad libitum National Cholesterol Education Program (NCEP) step 2 therapeutic diet (<7% saturated fat and <200 mg/d cholesterol). During the test phase, foods high in soy, other vegetable proteins, and soluble fiber were provided. During the control phase, low-fat dairy and low-soluble-fiber foods were provided. Fasting blood lipid and apolipoprotein levels were measured at 4 and 8 weeks of each phase. On the test diet, 12 +/- 2 g/d soy protein was selected from the foods chosen. Direct comparison of test and control treatments indicated an elevated high-density lipoprotein (HDL) cholesterol concentration on the test diet (6.4% +/- 2.4%, P = .013) and a significantly reduced total to HDL cholesterol ratio (-5.9% +/- 2.3%, P = .020). The proportion of conjugated dienes in the low-density lipoprotein (LDL) cholesterol fraction was significantly reduced (8.5% +/- 3.3%, P = .020) as a marker of oxidized LDL. A combination of acceptable amounts of soy, vegetable protein, and soluble-fiber foods as part of a conventional low-fat, low-cholesterol therapeutic diet is effective in further reducing serum lipid risk factors for cardiovascular disease.

Substantial carbon recycling from linoleate into products of de novo lipogenesis occurs in rat liver even under conditions of extreme dietary linoleate deficiency.

A significant portion of the beta-oxidized carbon skeleton of some polyunsaturated fatty acids can be recycled into de novo lipogenesis, i.e., cholesterol, saturates and monounsaturates. The recycling of carbon from linoleate was quantified in liver lipids of severely linoleate-deficient rats to determine whether it is more likely to be a function of redundancy or could be obligatory. After 13 wk on a control (2 energy % linoleate) or severely linoleate-deficient (<0. 05 energy % linoleate) diet, 7 muCi [1-14C]linoleate was given by gavage and the rats were killed 48 h later. A second linoleate-deficient group received an oral bolus of 256 mg linoleate as a supplement with the radiotracer. In comparison to the controls, 14C recovery in liver total lipids of the linoleate deficient group was increased about 5-fold with increased dpm/g in linoleate (13.7-fold higher), arachidonate (2.7-fold higher) and products of de novo lipogenesis (3.5-fold higher). In livers of control rats, 14C distribution was: 41% arachidonate, 29% linoleate, 22% sterols, 3% oleate, 3% palmitate, and 2% stearate. In livers of linoleate-deficient rats, 14C distribution was: 63% linoleate, 19% arachidonate, 11% sterols, 4% oleate, 3% palmitate, and <1% stearate. Thus, in controls, equivalent amounts of 14C were in products of de novo lipogenesis as in linoleate (29-30%), and in livers of linoleate-deficient rats, a similar proportion of 14C was in products of de novo lipogenesis as was converted to arachidonate (18-19%). We conclude that carbon recycling into de novo lipogenesis accounts for a significant, obligatory component of linoleate metabolism even during extreme linoleate deficiency.

Effect of psyllium in hypercholesterolemia at two monounsaturated fatty acid intakes.

We performed two studies to determine whether the lipid-lowering effect of viscous soluble fiber was modified by monounsaturated fatty acid (MUFA). First, psyllium (1.4 g/MJ) was compared with wheat bran (control) in 1-mo metabolic diets by using a randomized crossover design (n = 32 hyperlipidemic subjects). The background diet contained approximately 6% of energy as MUFA (20% of total fat). The second study (n = 27 hyperlipidemic subjects) was similar to the first but the background diet contained approximately 12% MUFA (29% of total fat) because of the addition of canola oil. At both fat intakes, psyllium resulted in significant reductions in total, low-density-lipoprotein (LDL), and high-density-lipoprotein (HDL) cholesterol compared with the wheat bran control. For the psyllium diet at 6% compared with 12% MUFA, the decreases in LDL cholesterol were 12.3 +/- 1.5% (P < 0.001) and 15.3 +/- 2.4% (P < 0.001), respectively. With the higher-MUFA diet triacylglycerol fell significantly over the control phase (16.6 +/- 5.5%, P = 0.006) and the ratio of LDL to HDL cholesterol fell significantly over the psyllium phase (7.3 +/- 2.8%, P = 0.015). Psyllium and MUFA intakes were negatively related to the percentage change in the ratio of LDL to HDL cholesterol (r = -0.34, P = 0.019 and r = -0.44, P = 0.002, respectively). Chenodeoxycholate synthesis rate increased (30 +/- 13%, P = 0.038) with the psyllium diet in the 12 subjects in whom this was assessed. We conclude that psyllium lowered LDL- and HDL-cholesterol concentrations similarly at both MUFA intakes. However, there may be some advantage in combining soluble fiber and MUFA to reduce the ratio of LDL to HDL cholesterol.

Pure linoleate deficiency in the rat: influence on growth, accumulation of n-6 polyunsaturates, and [1-14C]linoleate oxidation.

Essential fatty acid deficiency has been widely studied but the extent to which its effects are attributable specifically to deficiency of linoleate as opposed to deficiency of all unsaturated fatty acids is unknown. Our objective was to evaluate the effect of pure linoleate deficiency on growth as well as changes in the metabolism and oxidation of n-6 polyunsaturates. The diets contained 20 energy % fat blended from 3 energy % pure oleate, 2 energy % linoleate (0.01 energy % in the linoleate-deficient group), 0.3 energy % pure alpha-linolenate, and the balance as palmitate and stearate from fully hydrogenated soybean oil. Thirty-five-day-old rats consumed the two diets for 84 days, after which the linoleate-deficient rats weighed 15% less than the controls (P < 0.05), had mild scaling on the paws, and visible hair loss (in a few rats). Compared with the controls, the ratio of eicosatrienoate to arachidonate after 84 days was elevated in liver (170-fold) and serum (520-fold) phospholipids of the linoleate-deficient group. In total, linoleate-deficient rats consumed 122 mg of linoleate and had a net whole body loss of 479 mg n-6 polyunsaturates compared with an intake of 24,130 mg and a net whole body gain of 7206 mg n-6 polyunsaturates in the control group. Linoleate-deficient rats oxidized 1% of an oral bolus of [1-14C]linoleate over 8 h compared with 34% in the control rats (P < 0.05). We conclude that pure linoleate deficiency has marked effects on accumulation of n-6 polyunsaturates but induces milder gross symptoms, particularly growth retardation, than classical essential fatty acid deficiency. alpha-Linolenate and possibly oleate may have a sparing effect on linoleate oxidation from body stores during linoleate deficiency.

Effects of n-3 fatty acids on the NMR profile of plasma lipoproteins.

The effects of fish oil supplementation (14.5 g n-3 fatty acids/day) on plasma lipoprotein particles in healthy volunteers were assessed by high resolution 13C and 1H nuclear magnetic resonance (NMR) spectroscopy. Resonances not previously observed in the 13C and 1H spectra of plasma and isolated lipoproteins were detected after fish oil ingestion. The 13C resonances, centered at 14.3, 127.1, and 131.6 ppm, have been assigned to specific carbon groups (CH3-CH2-CH = CH-, CH3-CH2-CH = CH-CH2-, CH3-CH2-CH = CH-CH2-, respectively) in eicosapentaenoic acid (C20:5n-3) and docosahexaenoic (C22:6n-3) DHA. The new lipid resonance observed in the 1H spectra of plasma (0.941 ppm) is consistent with the incorporation of these n-3 fatty acids into lipoprotein particles. The presence of increased EPA and DHA in plasma lipids was confirmed by gas-liquid chromatography. A marked reduction in the intensity of the methylene signal from very low density lipoproteins (VLDL) was also observed with fish oil. This reduction arises from a decrease in plasma triglyceride concentration (ca. 18%) and a reduction in the number of VLDL particles. Transverse relaxation studies of isolated VLDL and low density lipoprotein (LDL) showed significant elevation in the T2 of the -(CH2)n- and CH3- signals from non-n-3 fatty acids. The relaxation characteristics and signal intensity of the novel 1H peak (0.941 ppm) point to the existence of n-3 enriched microenvironments within lipoprotein particles. These findings suggest that incorporation of EPA and DHA into VLDL and LDL, after fish oil ingestion, leads to significant alteration in the molecular architecture of lipoprotein particles.

13C NMR spectroscopy and gas chromatograph--combustion--isotope ratio mass spectrometry: complementary applications in monitoring the metabolism of 13C-labelled polyunsaturated fatty acids.

This review aims to describe some recent and novel applications of stable isotope tracer technology to study the metabolism of 13C-polyunsaturated fatty acids. Stable isotope methodology has existed for several decades, and in that sense, it is not novel per se. However, in the past 10 years, developments in the fields of isotope ratio mass spectrometry, nuclear magnetic resonance (NMR) spectroscopy, and biological approaches to labelling tracer compounds with stable isotopes have provided new opportunities in fatty acid research. Sample preparation for isotope ratio mass spectrometry has been converted from a manual combustion method to an on-line or continuous flow method, making it much more versatile and easier to use. Similarly, 13C-NMR spectroscopy has recently developed as a remarkably useful method for monitoring metabolic steps and pathways both in vivo and at the molecular level. Coincident with these improvements in instrumentation, the commercial availability of numerous uniformly 13C-labelled compounds has made these studies more affordable. The application of some of these developments to questions in the field of polyunsaturated fatty acid metabolism is described.

Utilization of carbon from dietary polyunsaturates for brain cholesterol synthesis during early postnatal development in the rat: a 13C NMR study.

Incorporation of 13C from a dietary precursor into cholesterol was studied in neonatal rats. Rats were given uniformly 13C-enriched polyunsaturated fatty acids intragastrically and total lipid extracts of liver and brain were analyzed by 13C-NMR 1, 4, 8, and 15 days later. 13C-enrichment was detected in brain but not in liver cholesterol. Maximal 13C-labeling was observed 4 days after injection of the label. Spectra revealed that 70% of newly incorporated 13C had 13C as an adjacent neighbor, the other 30% had 12C as the neighbor. Double quantum NMR revealed the arrangement in the cholesterol skeleton of the 13C-13C pairs transferred from precursors to cholesterol. Desmosterol, an intermediate of cholesterol synthesis, was identified in the spectra of brain lipids. Comparison of 13C-13C unit arrangements in both cholesterol and desmosterol allowed carbons 26 and 27 of desmosterol to be unambiguously assigned.

Similar distribution of trans fatty acid isomers in partially hydrogenated vegetable oils and adipose tissue of Canadians.

The objective of this study was to evaluate the composition of trans fatty acids in the subcutaneous fat of Canadians relative to the composition of dietary sources of trans fatty acids. The fatty acid composition, total trans acid content, and the geometric and positional isomer distribution of unsaturated fatty acids of subcutaneous adipose tissue of Canadians were determined using a combination of capillary gas-liquid chromatography and silver nitrate thin-layer chromatography. The mean total trans fatty acid content was 6.80% at the abdominal site and 5.80% at the lateral thigh site. Total trans isomers of linoleic acid (18:2n-6) were present at 1.17% in abdominal and 1.59% in thigh adipose tissue, with 9c.12t-18:2 being the most prevalent isomer followed by 9c-13t-18:2 and 9t,12c,-18:2. The oleic acid (18:1) trans isomer distribution in adipose tissue differed from that in butter fat, but it was similar to that in partially hydrogenated vegetable oils. The reverse was true for the 18:1 cis isomers. Total 18:1 trans isomers were inversely related to 18:2n-6 content in adipose tissue, suggesting the trans fatty acid intake is inversely related to the intake of linoleic acid. Partially hydrogenated vegetable oils appear to be the major source of trans fatty acids in adipose tissue of Canadians.

Refeeding after fasting increases apparent oxidation of N-3 and N-6 fatty acids in pregnant rats.

The intake, excretion, and accumulation of long-chain fatty acids was analyzed to test the hypothesis that during pregnancy in the rat whole-body partitioning of n-6 and n-3 fatty acids between net accumulation or disappearance (apparent oxidation) is determined by both maternal energy status and fetal development. From midpregnancy to term in ad libitum-fed rats consuming-rodent chow, 30% more saturates and monounsaturates accumulated in the whole body than were consumed, whereas 28% of dietary n-6 fatty acids and 55% of dietary n-3 fatty acids were apparently oxidized. After 48 hours of fasting during midpregnancy (days 13 to 15) followed by refeeding to term, net accumulation of saturates and monounsaturates was equivalent to intake, but whole-body disappearance of n-6 fatty acids exceeded intake by 6% (NS), whereas n-3 fatty acid disappearance exceeded intake by 43%. Thus during refeeding after fasting, dietary n-6 and n-3 fatty acids were apparently completely oxidized and there was actually a net loss of n-3 fatty acids from whole-body stores. Fasting during midpregnancy did not significantly affect fetal weight gain or maternal gestational hyperlipidemia toward term. We conclude that during pregnancy n-6 and n-3 fatty acids are not only required for maternal and fetal structural and storage lipids, but are also used to meet energy requirements, especially during refeeding after fasting.

High alpha-linolenic acid flaxseed (Linum usitatissimum): some nutritional properties in humans.

Although high alpha-linolenic acid flaxseed (Linum usitatissimum) is one of the richest dietary sources of alpha-linolenic acid and is also a good source of soluble fibre mucilage, it is relatively unstudied in human nutrition. Healthy female volunteers consumed 50 g ground, raw flaxseed/d for 4 weeks which provided 12-13% of energy intake (24-25 g/100 g total fat). Flaxseed raised alpha-linolenic acid and long-chain n-3 fatty acids in both plasma and erythrocyte lipids, as well as raising urinary thiocyanate excretion 2.2-fold. Flaxseed also lowered serum total cholesterol by 9% and low-density-lipoprotein-cholesterol by 18%. Changes in plasma alpha-linolenic acid were equivalent when 12 g alpha-linolenic acid/d was provided as raw flaxseed flour (50 g/d) or flaxseed oil (20 g/d) suggesting high bioavailability of alpha-linolenic acid from ground flaxseed. Test meals containing 50 g carbohydrate from flaxseed or 25 g flaxseed mucilage each significantly decreased postprandial blood glucose responses by 27%. Malondialdehyde levels in muffins containing 15 g flaxseed oil or flour/kg were similar to those in wheat-flour muffins. Cyanogenic glycosides (linamarin, linustatin, neolinustatin) were highest in extracted flaxseed mucilage but were not detected in baked muffins containing 150 g flaxseed/kg. We conclude that up to 50 g high-alpha-linolenic acid flaxseed/d is palatable, safe and may be nutritionally beneficial in humans by raising n-3 fatty acids in plasma and erythrocytes and by decreasing postprandial glucose responses.

Specific types of colonic fermentation may raise low-density-lipoprotein-cholesterol concentrations.

To assess the effects of increased colonic fermentation on serum lipids, eight healthy volunteers were placed on two identical 2-wk metabolic diets, one of which was supplemented with lactulose (18-25 g/d). Lactulose raised day-long concentrations of breath hydrogen and serum glutamine as indicators of increased colonic fermentation by 78 +/- 13% (P less than 0.001) and 24.7 +/- 9.5% (P less than 0.05), respectively). Unexpectedly, however, fasting serum total and low-density-lipoprotein cholesterol and apolipoprotein B concentrations were higher at 2 wk by 8.9 +/- 1.5% (P less than 0.001), 10.9 +/- 2.2% (P less than 0.005), and 18.9 +/- 5.9% (P less than 0.02), respectively, compared with the control diet. With lactulose, mean free fatty acid concentrations were reduced over the day by 19.5 +/- 5.9% (P less than 0.02), with no change in mean day-long blood glucose, serum insulin, or C-peptide concentrations. We conclude that certain rapidly fermented substrates may raise rather than lower serum lipids, possibly through increasing the amount of acetate absorbed from the colon.

Differential utilization of long chain fatty acids during fasting-induced triacylglycerol depletion. III. Comparison of n-3 and n-6 fatty acids in rat plasma and liver.

Previous research has shown that arachidonic acid (20:4(n-6)) is preferentially retained in liver triacylglycerol in fasted compared to fed rats consuming a diet containing n-6 fatty acids. It was hypothesized that eicosapentaenoic (20:5(n-3)) and docosahexaneoic acids (22:6(n-3)) would be similarly retained in liver and plasma triacylglycerol of fasted rats consuming a diet containing n-3 fatty acids. In comparison with fed rats, it was observed that in partially fasted rats consuming diets which contained 5% sunflower oil (78% 18:2(n-6)) or 5% marine fish oil (30% 20:5(n-3) and 22:6(n-3)), both liver and plasma had significantly depleted triacylglycerol levels containing higher proportions of both arachidonic and docosahexaneoic acids but a lower proportion of eicosapentaenoic acid (marine fish oil group only). Separation of liver and plasma triacylglycerol by silicic acid column chromatography and silver nitrate TLC showed that the majority of long chain fatty acids utilized during fasting were derived from the triacylglycerol subclasses containing palmitic acid (16:0), palmitoleic acid (16:1(n-7)) and oleic acid (18:1(n-9)) with retention o both highly saturated and polyunsaturated subclasses. Greater utilization of eicosapentaenoic acid than either arachidonic acid or docosahexaenoic acid during fasting may be due to triacylglycerol speciation of the former with readily oxidized monounsaturated fatty acids.

Long-chain fatty acid composition of maternal liver lipids during pregnancy and lactation in the rat: comparison of triglyceride to phospholipid.

The change in long-chain fatty acid composition in maternal liver was studied during pregnancy and lactation in the rat. Maternal liver triglycerides and phospholipids transiently accumulated and were depleted of long-chain fatty acids during pregnancy and lactation. During pregnancy, maternal liver accumulated triglyceride, but triglyceride fatty acid composition changed little. However, maternal liver total phospholipid fatty acid composition changed significantly without a change in the total pool size throughout pregnancy or lactation. The change in composition of (n-3) and (n-6) essential fatty acids in maternal liver triglyceride and total phospholipid occurred in an apparently dyssynchronous manner throughout pregnancy and lactation.