Sharon F. Majchrzak-Hong

Use of dietary linoleic acid for secondary prevention of coronary heart disease and death: Evaluation of recovered data from the Sydney Diet Heart Study and updated meta-analysis

Objective To evaluate the effectiveness of replacing dietary saturated fat with omega 6 linoleic acid, for the secondary prevention of coronary heart disease and death. Design Evaluation of recovered data from the Sydney Diet Heart Study, a single blinded, parallel group, randomized controlled trial conducted in 1966-73; and an updated meta-analysis including these previously missing data. Setting Ambulatory, coronary care clinic in Sydney, Australia. Participants 458 men aged 30-59 years with a recent coronary event. Interventions Replacement of dietary saturated fats (from animal fats, common margarines, and shortenings) with omega 6 linoleic acid (from safflower oil and safflower oil polyunsaturated margarine). Controls received no specific dietary instruction or study foods. All non-dietary aspects were designed to be equivalent in both groups. Outcome measures All cause mortality (primary outcome), cardiovascular mortality, and mortality from coronary heart disease (secondary outcomes). We used an intention to treat, survival analysis approach to compare mortality outcomes by group. Results The intervention group (n=221) had higher rates of death than controls (n=237) (all cause 17.6% v 11.8%, hazard ratio 1.62 (95% confidence interval 1.00 to 2.64), P=0.05; cardiovascular disease 17.2% v 11.0%, 1.70 (1.03 to 2.80), P=0.04; coronary heart disease 16.3% v 10.1%, 1.74 (1.04 to 2.92), P=0.04). Inclusion of these recovered data in an updated meta-analysis of linoleic acid intervention trials showed non-significant trends toward increased risks of death from coronary heart disease (hazard ratio 1.33 (0.99 to 1.79); P=0.06) and cardiovascular disease (1.27 (0.98 to 1.65); P=0.07). Conclusions Advice to substitute polyunsaturated fats for saturated fats is a key component of worldwide dietary guidelines for coronary heart disease risk reduction. However, clinical benefits of the most abundant polyunsaturated fatty acid, omega 6 linoleic acid, have not been established. In this cohort, substituting dietary linoleic acid in place of saturated fats increased the rates of death from all causes, coronary heart disease, and cardiovascular disease. An updated meta-analysis of linoleic acid intervention trials showed no evidence of cardiovascular benefit. These findings could have important implications for worldwide dietary advice to substitute omega 6 linoleic acid, or polyunsaturated fats in general, for saturated fats. Trial registration Clinical trials NCT01621087.

Re-evaluation of the traditional diet-heart hypothesis: analysis of recovered data from Minnesota Coronary Experiment (1968-73)

Objective To examine the traditional diet-heart hypothesis through recovery and analysis of previously unpublished data from the Minnesota Coronary Experiment (MCE) and to put findings in the context of existing diet-heart randomized controlled trials through a systematic review and meta-analysis. Design The MCE (1968-73) is a double blind randomized controlled trial designed to test whether replacement of saturated fat with vegetable oil rich in linoleic acid reduces coronary heart disease and death by lowering serum cholesterol. Recovered MCE unpublished documents and raw data were analyzed according to hypotheses prespecified by original investigators. Further, a systematic review and meta-analyses of randomized controlled trials that lowered serum cholesterol by providing vegetable oil rich in linoleic acid in place of saturated fat without confounding by concomitant interventions was conducted. Setting One nursing home and six state mental hospitals in Minnesota, United States. Participants Unpublished documents with completed analyses for the randomized cohort of 9423 women and men aged 20-97; longitudinal data on serum cholesterol for the 2355 participants exposed to the study diets for a year or more; 149 completed autopsy files. Interventions Serum cholesterol lowering diet that replaced saturated fat with linoleic acid (from corn oil and corn oil polyunsaturated margarine). Control diet was high in saturated fat from animal fats, common margarines, and shortenings. Main outcome measures Death from all causes; association between changes in serum cholesterol and death; and coronary atherosclerosis and myocardial infarcts detected at autopsy. Results The intervention group had significant reduction in serum cholesterol compared with controls (mean change from baseline −13.8% v −1.0%; P<0.001). Kaplan Meier graphs showed no mortality benefit for the intervention group in the full randomized cohort or for any prespecified subgroup. There was a 22% higher risk of death for each 30 mg/dL (0.78 mmol/L) reduction in serum cholesterol in covariate adjusted Cox regression models (hazard ratio 1.22, 95% confidence interval 1.14 to 1.32; P<0.001). There was no evidence of benefit in the intervention group for coronary atherosclerosis or myocardial infarcts. Systematic review identified five randomized controlled trials for inclusion (n=10 808). In meta-analyses, these cholesterol lowering interventions showed no evidence of benefit on mortality from coronary heart disease (1.13, 0.83 to 1.54) or all cause mortality (1.07, 0.90 to 1.27). Conclusions Available evidence from randomized controlled trials shows that replacement of saturated fat in the diet with linoleic acid effectively lowers serum cholesterol but does not support the hypothesis that this translates to a lower risk of death from coronary heart disease or all causes. Findings from the Minnesota Coronary Experiment add to growing evidence that incomplete publication has contributed to overestimation of the benefits of replacing saturated fat with vegetable oils rich in linoleic acid.

Lowering dietary linoleic acid reduces bioactive oxidized linoleic acid metabolites in humans

Linoleic acid (LA) is the most abundant polyunsaturated fatty acid in human diets, a major component of human tissues, and the direct precursor to the bioactive oxidized LA metabolites (OXLAMs), 9- and 13 hydroxy-octadecadienoic acid (9- and 13-HODE) and 9- and 13-oxo-octadecadienoic acid (9- and 13-oxoODE). These four OXLAMs have been mechanistically linked to pathological conditions ranging from cardiovascular disease to chronic pain. Plasma OXLAMs, which are elevated in Alzheimers dementia and non-alcoholic steatohepatitis, have been proposed as biomarkers useful for indicating the presence and severity of both conditions. Because mammals lack the enzymatic machinery needed for de novo LA synthesis, the abundance of LA and OXLAMs in mammalian tissues may be modifiable via diet. To examine this issue in humans, we measured circulating LA and OXLAMs before and after a 12-week LA lowering dietary intervention in chronic headache patients. Lowering dietary LA significantly reduced the abundance of plasma OXLAMs, and reduced the LA content of multiple circulating lipid fractions that may serve as precursor pools for endogenous OXLAM synthesis. These results show that lowering dietary LA can reduce the synthesis and/or accumulation of oxidized LA derivatives that have been implicated in a variety of pathological conditions. Future studies evaluating the clinical implications of diet-induced OXLAM reductions are warranted.

Dietary omega-6 fatty acid lowering increases bioavailability of omega-3 polyunsaturated fatty acids in human plasma lipid pools.

BACKGROUND Dietary linoleic acid (LA, 18:2n-6) lowering in rats reduces n-6 polyunsaturated fatty acid (PUFA) plasma concentrations and increases n-3 PUFA (eicosapentaenoic (EPA) and docosahexaenoic acid (DHA)) concentrations. OBJECTIVE To evaluate the extent to which 12 weeks of dietary n-6 PUFA lowering, with or without increased dietary n-3 PUFAs, alters unesterified and esterified plasma n-6 and n-3 PUFA concentrations in subjects with chronic headache. DESIGN Secondary analysis of a randomized trial. Subjects with chronic headache were randomized for 12 weeks to (1) average n-3, low n-6 (L6) diet; or (2) high n-3, low n-6 LA (H3-L6) diet. Esterified and unesterified plasma fatty acids were quantified at baseline (0 weeks) and after 12 weeks on a diet. RESULTS Compared to baseline, the L6 diet reduced esterified plasma LA and increased esterified n-3 PUFA concentrations (nmol/ml), but did not significantly change plasma arachidonic acid (AA, 20:4n-6) concentration. In addition, unesterified EPA concentration was increased significantly among unesterified fatty acids. The H3-L6 diet decreased esterified LA and AA concentrations, and produced more marked increases in esterified and unesterified n-3 PUFA concentrations. CONCLUSION Dietary n-6 PUFA lowering for 12 weeks significantly reduces LA and increases n-3 PUFA concentrations in plasma, without altering plasma AA concentration. A concurrent increase in dietary n-3 PUFAs for 12 weeks further increases n-3 PUFA plasma concentrations and reduces AA.

All PUFAs Are Not Created Equal: Absence of CHD Benefit Specific to Linoleic Acid in Randomized Controlled Trials and Prospective Observational Cohorts

Advice to maintain or increase consumption of the omega-6 polyunsaturated fatty acid (n–6 PUFA) linoleic acid (LA) should be derived from interventional and observational trials evaluating the specific effects of dietary LA, rather than effects of n–3 PUFAs or total PUFAs. Failure to make a clear distinction among PUFA species may result in inadvertently attributing health effects of n–3 PUFAs to linoleic acid. Pooled analyses of randomized controlled trials (RCTs) of clinical CHD events [1] and intermediate risk factors [2] and pooled analyses of nonrandomized prospective observational trials of clinical CHD events [3] are often cited as providing strong concordant evidence [1, 4] that LA is cardioprotective. These pooled analyses [1–3] form the primary basis for recent population-wide advice to maintain or increase n–6 PUFA [5–7]. However, total PUFA rather than n–6 LA, was defined as the independent variable for statistical calculations in all three pooled analyses [1–3] (table 1), then interpreted as attributable to LA [4–8]. In this paper we: (1) establish that a clear distinction was not made between n–3 and n–6 PUFAs in pooled analyses of randomized and nonrandomized trials (tables 2, ​,3),3), (2) report whether a clear distinction was made between n–3 and n–6 PUFAs in each individual trial before pooling, (3) assess strengths and limitations of randomized and nonrandomized study designs for disentangling respective intakes of n–6 and n–3 PUFA species, and (4) highlight the necessity of making a clear distinction between PUFA species for interpreting the results of clinical trials and formulating dietary guidelines. Table 1 Defining the independent variable in pooled analyses cited as concordant evidence for CHD benefits of n–6 PUFAs Table 2 General characteristics of 8 randomized controlled dietary intervention trials that substituted PUFAs for saturated and trans fatty acids Table 3 Characteristics of 11 nonrandomized observational trials that assessed PUFA intake

Brain Docosahexaenoic Acid [DHA] Incorporation and Blood Flow Are Increased in Chronic Alcoholics: A Positron Emission Tomography Study Corrected for Cerebral Atrophy

Objective Chronic alcohol dependence has been associated with disturbed behavior, cerebral atrophy and a low plasma concentration of docosahexaenoic acid (DHA, 22∶6n-3), particularly if liver disease is present. In animal models, excessive alcohol consumption is reported to reduce brain DHA concentration, suggesting disturbed brain DHA metabolism. We hypothesized that brain DHA metabolism also is abnormal in chronic alcoholics. Methods We compared 15 non-smoking chronic alcoholics, studied within 7 days of their last drink, with 22 non-smoking healthy controls. Using published neuroimaging methods with positron emission tomography (PET), we measured regional coefficients (K*) and rates (Jin) of DHA incorporation from plasma into the brain of each group using [1-11C]DHA, and regional cerebral blood flow (rCBF) using [15O]water. Data were partial volume error corrected for brain atrophy. Plasma unesterified DHA concentration also was quantified. Results Mean K* for DHA was significantly and widely elevated by 10–20%, and rCBF was elevated by 7%–34%, in alcoholics compared with controls. Unesterified plasma DHA did not differ significantly between groups nor did whole brain Jin, the product of K* and unesterified plasma DHA concentration. Discussion Significantly higher values of K* for DHA in alcoholics indicate increased brain avidity for DHA, thus a brain DHA metabolic deficit vis-à-vis plasma DHA availability. Higher rCBF in alcoholics suggests increased energy consumption. These changes may reflect a hypermetabolic state related to early alcohol withdrawal, or a general brain metabolic change in chronic alcoholics.

Don’t disregard the essential distinction between PUFA species

Harris et al.(1) apparently overlooked the central finding of our analysis(2), that n-6 specific PUFA interventions and mixed n-3/n-6 PUFA interventions have significantly different effects on risk of non-fatal myocardial infarction (MI) and CHD death in heterogeneity analysis (P=0·02). Given this clear statistical distinction, it is not valid to assess these two heterogeneous interventions together as a single unit(3). This key finding of heterogeneity calls into question the validity of interpreting results of any pooled analysis of the effects of mixtures of n-3 and n-6 PUFA to support advice specific to n-6 PUFA. Yet Harris et al. have repeatedly cited(4 – 8) three such pooled analyses, that did not make this essential distinction, as the primary evidence base for American Heart Association (AHA) advice to maintain or increase intake of n-6 PUFA: (1) the meta-analysis of randomised controlled trials (RCT) and CHD outcomes by Mozaffarian et al.(8); (2) the analysis of prospective observational cohorts by Jakobsen et al.(9); and (3) the analysis of intermediate risk factors by Mensink et al.(10). Each of these analyses evaluated the effects of increasing n-3 and n-6 PUFA together (Tables 1 and ​and2),2), without specificity for n-6 linoleic acid (LA), as we recently reviewed in detail(11). In relying on these non-specific PUFA data, rather than data selective to n-6 LA, Harris et al.(1) and the AHA Advisory Group were willing to assume that the modest but ‘remarkably consistent’ CHD benefits were attributable to n-6 LA, rather than to n-3 EPA + DHA and/or α-linolenic acid (ALA). We were unwilling to make this assumption without valid data evaluating the effects of selectively increasing LA. Table 1 Pooled analyses cited as the primary evidence base for CHD benefits of n-6 linoleic acid (LA): no clear distinction made between n-3 and n-6 PUFA species (adapted with permission from Ramsden et al.(11))* Table 2 Limited capacity of food-frequency questionnaires in the Nurses’ Health Study (NHS) to disentangle respective intakes of n-3 α-linolenic acid (ALA) and n-6 linoleic acid (LA) (adapted with permission from Ramsden et al.(11))* Our meta-analysis of four n-6 specific PUFA RCT data-sets with 9569 participants(2) is therefore unique because it is the first analysis to compile the detailed dietary fatty acid data necessary to evaluate the CHD effects of selectively increasing n-6 LA (in place of trans-fatty acids and SFA). These n-6 specific PUFA data, which were not available to Mozaffarian et al.(8), were obtained via an extensive search of published and unpublished data sources enabling identification of the specific study oils and n-3 and n-6 PUFA content of RCT diets. Of the total RCT participants (n 11 275), 85 % were in the n-6 specific PUFA RCT (n 9569); therefore only 15 % of all RCT participants (n 1706) were in the mixed n-3/n-6 PUFA RCT. Harris et al.(1) seem preoccupied with the results of the latter 15 % of RCT participants, which they interpret as ‘confirming’ that ‘vegetable oils rich in n-6 PUFA’ lower CHD(1). However, they apparently dismiss our pivotal finding that there was no indication of benefit in any of the four n-6 specific PUFA datasets representing 85 % of the total RCT participants. There was a non-significant, but relatively consistent, signal toward harm in each of these four n-6 specific PUFA datasets (Figs. 1 and ​and2),2), as well as a pooled analysis for both CHD outcomes and death from all causes, despite substitution of n-6 LA for trans-fatty acids and SFA, which are generally considered atherogenic. By highlighting only the two n-6 specific PUFA RCT (three datasets) that provided corn oil(12,13), and ignoring the unfavourable results of the fourth n-6 specific PUFA RCT dataset that provided safflower-seed oil(14,15), Harris et al. have inaccurately portrayed our n-6 specific PUFA evaluation of four datasets and 9569 participants as a ‘two-trial analysis’ with ‘limited statistical power’ useful for ‘hypothesis generation but insufficient for deriving meaningful conclusions’. This fourth dataset, the Sydney Diet Heart Study (SDHS)(14), reported a 49 % increase in risk of death in the n-6 specific intervention group that approached significance (risk ratio (RR) 1·49 (95 % CI 0·95, 2·34); P=0·08). Nearly all SDHS deaths were attributed to CHD (91 %); however, non-fatal MI and CHD death were not reported by group. Using reported SDHS data, the lowest possible increase in CHD death in the SDHS was + 26 % (RR 1·26 (95 % CI 0·79, 2·02); P=0·33); the highest possible increase was + 90 % (RR 1·90 (95 % CI 1·17, 3·10); P=0·01). Therefore, the 13 % increase in CHD risk from only three of the four n-6 specific PUFA interventions underestimates potential harm. In modelling the equitable assumption that the same percentage of SDHS deaths (91 %) were CHD deaths in each group, the risk of CHD death was increased by 49 % (RR 1·49 (95 % CI 0·93, 2·40); P=0·10), and the pooled 28 % increase in risk of CHD death from n-6 specific PUFA interventions was nearly significant (RR 1·28 (95 % CI 0·96, 1·71); P=0·09). Pooled data from all four n-6 specific PUFA datasets (n 9569; 85 % of all RCT participants) provide a relatively consistent, albeit non-significant, trend toward increased risks for CHD death (+28 %) (RR 1·28 (95 % CI 0·96, 1·71); P=0·09), total CHD events (+23 %) (RR 1·23 (95 % CI 0·94, 1·61); P=0·13), and death from all causes (+16 %) (RR 1·16 (95 % CI 0·95, 1·42); P=0·15). With no indication of benefit in any n-6 specific PUFA RCT or the pooled analysis of all four datasets, and a relatively consistent signal in the opposite direction for each dataset and for the pooled analysis, these data provide no justification for population-wide advice to maintain or increase n-6 PUFA consumption. Fig. 1 No indication of benefit, and a signal toward harm, in non-fatal myocardial infarction (MI) + CHD death (a) and in CHD death (b) shown by n-6 specific PUFA interventions. PUFA replaced a combination of trans-fatty acids and SFA in each randomised controlled ... Fig. 2 No indication of benefit, and a signal toward harm, in deaths from all causes shown by n-6 specific PUFA interventions. PUFA replaced a combination of trans-fatty acids and SFA in each randomised controlled trial (RCT). ‘A’ indicates mixed ... Harris et al.(1) have not fully appreciated the essential distinction between n-3 and n-6 PUFA species in attributing benefits from our pooled analysis of mixed n-3/n-6 PUFA RCT datasets to ‘vegetable oils rich in n-6 PUFA’ or simply ‘soybean oil’, despite substantial increases in n-3 EPA + DHA in two(16 – 18) of the four datasets. The experimental dieters in the Oslo Diet Heart Study (ODHS) were actually provided with ‘substantial quantities of Norwegian sardines canned in cod liver oil’(16) and not simply ‘encouraged’ as Harris et al. state here(1) and elsewhere(4,19,20). Oslo dieters consumed about 5 g EPA + DHA per d (thirty times the average US intake)(21); therefore it is not valid to attribute CHD benefits to LA or soybean oil. After exclusion of the ODHS and the St Thomas Atherosclerosis Regression Study (STARS), the remaining two datasets(22 – 24) that substantially increased both n-3 ALA and n-6 LA from soybean oil without increasing n-3 EPA + DHA have a non-significant signal toward benefit, and lack the signal toward harm of the n-6 specific PUFA interventions. The structure of all RCT data is shown in Fig. 1 and Fig. 2, as follows: (A) mixed n-3/n-6 interventions including EPA + DHA are beneficial; (B) mixed n-3/n-6 interventions including ALA provide an intermediate signal toward benefit; and (C) interventions specific to n-6 LA provide no indication of benefit and a relatively consistent signal toward harm. Given this consistent pattern for CHD deaths and total CHD events (Fig. 1), and for deaths from all causes (Fig. 2), it is tempting to speculate that CHD benefits previously attributed to n-6 PUFA or total PUFA in general(4,6 – 8,25) were actually due to substantial increases in n-3 PUFA in general, and n-3 EPA + DHA in particular, and that the accompanying increases in n-6 LA attenuated these benefits. If this testable hypothesis is correct, lowering dietary n-6 LA is likely to potentiate the benefits of n-3 PUFA and/or have independent CHD benefits. However, we have no RCT data evaluating the effects of lowering n-6 LA as a controlled variable on clinical CHD outcomes. This is clearly a critical evidence gap with major public health implications. The next logical step is to evaluate whether lowering dietary n-6 LA as a controlled variable to about 2 % of energy (consistent with evolutionary(26) and historical US diets(27)) reduces the risk of CHD in a large RCT. This can be accomplished via substitution of a high-oleic version of sunflower-seed oil (3·6 g LA per 100 g)(28) for a typical high-LA version of the same oil (65·7 g LA per 100 g)(28) with otherwise identical background diets. This project was supported by the intramural research program of the National Institute on Alcohol Abuse and Alcoholism. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the National Institute on Alcohol Abuse and Alcoholism or the National Institutes of Health.

Lipidomic profiling of targeted oxylipins with ultra-performance liquid chromatography-tandem mass spectrometry

Oxylipins are bioactive mediators that play diverse roles in (patho)physiology. We developed a sensitive and selective ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for the simultaneous profiling of 57 targeted oxylipins derived from five major n-6 and n-3 polyunsaturated fatty acids (PUFAs) that serve as oxylipin precursors, including linoleic (LA), arachidonic (AA), alpha-linolenic (ALA), eicosapentaenoic (EPA), and docosahexaenoic (DHA) acids. The targeted oxylipin panel provides broad coverage of lipid mediators and pathway markers generated from cyclooxygenases, lipoxygenases, cytochrome P450 epoxygenases/hydroxylases, and non-enzymatic oxidation pathways. The method is based on combination of protein precipitation and solid-phase extraction (SPE) for sample preparation, followed by UPLC-MS/MS. This is the first methodology to incorporate four hydroxy-epoxy-octadecenoic acids and four keto-epoxy-octadecenoic acids into an oxylipin profiling network. The novel method achieves excellent resolution and allows in-depth analysis of isomeric and isobaric species of oxylipin extracts in biological samples. The method was quantitatively characterized in human plasma with good linearity (R = 0.990–0.999), acceptable reproducibility (relative standard deviation (RSD) < 20% for the majority of analytes), accuracy (67.8 to 129.3%) for all analytes, and recovery (66.8–121.2%) for all analytes except 5,6-EET. Ion enhancement effects for 28% of the analytes in tested concentrations were observed in plasma, but were reproducible with RSD < 17.2%. Basal levels of targeted oxylipins determined in plasma and serum are in agreement with those previously reported in literature. The method has been successfully applied in clinical and preclinical studies.

Quantitation of Human Whole-Body Synthesis-Secretion Rates of Docosahexaenoic Acid and Eicosapentaenoate Acid from Circulating Unesterified α-Linolenic Acid at Steady State

The rate at which dietary α-linolenic acid (ALA) is desaturated and elongated to its longer-chain n-3 polyunsaturated fatty acid (PUFA) in humans is not agreed upon. In this study, we applied a methodology developed using rodents to investigate the whole-body, presumably hepatic, synthesis-secretion rates of esterified n-3 PUFA from circulating unesterified ALA in 2 healthy overweight women after 10 weeks of low-linoleate diet exposure. During continuous iv infusion of d5-ALA, 17 arterial blood samples were collected from each subject at -10, 0, 10, 20, 40, 60, 80, 100, 120, 150, 180, and 210 min, and at 4, 5, 6, 7, and 8 h after beginning infusion. Plasma esterified d5-n-3 PUFA concentrations were plotted against the infusion time and fit to a sigmoidal curve using nonlinear regression. These curves were used to estimate kinetic parameters using a kinetic analysis developed using rodents. Calculated synthesis-secretion rates of esterified eicosapentaenoate, n-3 docosapentaenoate, docosahexaenoic acid, tetracosapentaenate, and tetracosahexaenoate from circulating unesterified ALA were 2.1 and 2.7; 1.7 and 5.3; 0.47 and 0.27; 0.30 and 0.30; and 0.32 and 0.27 mg/day for subjects S01 and S02, respectively. This study provides new estimates of whole-body synthesis-secretion rates of esterified longer-chain n-3 PUFA from circulating unesterified ALA in human subjects. This method now can be extended to study factors that regulate human whole-body PUFA synthesis-secretion in health and disease.