Abstract & Background

Omega-3 and -6 polyunsaturated fatty acids (LCPUFA), are important for good health conditions. hey are present in membrane phospholipids.

The ratio of total n-6:n-3 LCPUFA and arachidonic acid:eicosapentaenoic acid (AA and EPA), should not exceed 5:1.

Increased intake of n-6 and decreased consumption of n-3 has resulted in much higher, ca 10/15:1 ratio in RBC fatty acids with the possible appearance of a pathological "scenario".

The determination of RBC phospholipid LCPUFA contents and ratios is the method of choice for assessing fatty acid status but it is labour intensive and time consuming.

Aims of the study

[i] To describe and validate a rapid method, suitable for large scale population studies, for total blood fatty acid assay;

[ii] to verify a possible correlation between total n-6:n-3 ratio and AA:EPA ratios in RBC phospholipids and in whole-blood total lipids, [iii] to assess usefulness of these ratio as biomarkers of LCPUFA status.

Methods [1] Healthy volunteers and patients with various pathologies were recruited. [2]

Fatty acid analyses by GC of methyl esters from directly derivatized whole blood total lipids and from RBC phospholipids were performed on fasting blood samples from 1432 subjects categorised according to their age, sex and any existing pathologies.

AA:EPA ratio and the total n-6:n-3 ratio were determined.

Results

• AA:EPA ratio
• is a more sensitive and reliable index for determining changes in total blood fatty acid and it
• is correlated with the ratio derived from extracted RBC phospholipids.

Conclusions

The described AA:EPA ratio is a simple, rapid and reliable method for determining n-3 fatty acid status.

Background

The evidence that diet is a key component of general health is well accepted.

Governments are developing health strategies that propose to modify the diet and lifestyle of their citizens in order

To reduce the incidence of diet-related conditions such as obesity, cardiovascular disease, cancer, type 2 diabetes and mental health problems and reduce the health-associated social and healthcare costs.

Food manufacturers have reacted to the new market demands for healthier products in two ways.

Firstly, they have attempted to eliminate or reduce "negative" nutrients, such as trans-fats, saturated fat, sugars with high glycaemic index and salt, and secondly, they are adding ingredients to their products with known and substantiated health benefits.

An example of the latter approach is the fortification of many foods with the long-chain, omega-3 polyunsaturated fatty acids (omega-3 LCPUFAs), [eicosapentaenoic acid (EPA) and docosohexaenoic acid (DHA)] found naturally in human breast milk, in marine algae and in the oil of fish such as tuna, mackerel, herring, salmon and sardines.

Numerous studies have reported a variety of beneficial effects of omega-3 fatty acids on human health [1].

In humans, EPA (20:5n-3) and DHA (22:6n-3) can be biosynthesized from the parent essential fatty acid alpha-linolenic acid (ALA; 18:3n-3).

However, due to low conversion rates of ALA into EPA, and particularly DHA, the dietary intake of ALA must be relatively high and the intake of LA, a metabolic competitor for the biosynthetic pathway, low if this pathway was to meet the body's needs for the longer chain derivatives.

Fish do not synthesize the greater proportion of these compounds found in their tissues. They are obtained from single-cell marine organisms that fish and shellfish regularly consume. EPA and DHA are found mainly in fish that live in cold, deep seas [2].

The omega-6 PUFAs can be found in vegetable oils, and they are present in smaller amounts in breast milk.

The parent essential fatty acid of the omega-6 family is linoleic acid (C18:2n-6, LA). LA, ALA and their metabolic products, arachidonic acid (C20:4n-6, AA) together with EPA and DHA play a key role as structural and functional components of cellular and intracellular membranes throughout the human body, but especially in brain, heart, retina, and testes.

Many international agencies suggest that LCPUFAs should provide about 7% of total calories and that the omega-6/omega-3 ratio should be no more than 5:1 [3].

Recent reports suggest that the total dietary amounts of these fatty acids are also important and that diets with similar ratios of n-6:n-3 elicit different metabolic effects depending on their actual amounts.

Increasing absolute amounts of α-linolenic acid whilst keeping the ratios of n-6:n-3 the same resulted in a greater conversion of the precursor fatty acids to EPA and DHA which highlighted the competitive interactions of these substrates for the elongation/desaturation/membrane incorporation pathways in man.

The amounts of other fatty acids in membrane lipids would reduce the availability of the AA and EPA after phospholipolysis and would be expected to alter EFA (Essential Fatty Acids) metabolism.

This does not preclude the usefulness of the AA:EPA ratio in membrane and whole blood phospholipids as a biomarker for omega-6:omega-3 status in man.

AA is released from phospholipids by phospholipase A2 and is the precursor of the eicosanoids, which include prostaglandins of the 2 series (PGE₂, PGD₂), leukotrienes of the 4 series (LTA₄, LTB₄, LTC₄, LTD₄, LTE₄), and lipoxines [4].

Their production is catalyzed by cyclooxygenase, lipooxygenase and epoxygenase enzymes respectively.

These omega-6 derived eicosanoids have numerous, physiologically important roles including augmenting inflammation, modulating immunity and promoting platelet aggregation and vasoconstriction.

The omega-3 EPA can compete with AA for the same enzymes to form different classes of eicosanoids, namely 3 series PGs and 5 series LTs which can counteract the deleterious effects of 2-series prostanoids.

Omega-6 and omega-3 fatty acids are both incorporated into membrane phospholipids when the AA/EPA ratio is between 1:1 to 5-10:1. When the ratio is higher, the incorporation of AA is preferred, suggesting a greater affinity of the enzymes for EPA [5].

The predicted production of the 2-series eicosanoids therefore increases, giving rise to pro-inflammatory and pro-aggregatory conditions. The omega-3 LCPUFAs present a wide range of beneficial properties.

Studies in vitro and with animal models have indicated that they affect membrane lipid composition, blood lipid profiles, eicosanoids biosynthesis, cell signalling cascades, gene expression and the functioning of the cardiovascular system; they are involved in ameliorating or preventing the aetiology of a number of different pathologies [6-9].

It is now recognized that omega-3 LCPUFAs have an important role in growth and development of the nervous system and retina and are involved in the regulation of cognitive and visual functions and in general mental health [10-12].

The highest concentrations of DHA are found in the cerebral cortex, synaptic vesicles and synaptosomes [13-16].

DHA represents the prevalent fatty acid in cerebral gray matter phospholipids, comprising 45% to 65% of the total fatty acid content in phosphatidylserine of the CNS [7].

An increasing number of reports demonstrate that DHA-containing phospholipids can influence membrane properties such as permeability, fusion, and plane elasticity [6].

The effect of omega-3 LCPUFAs on gene expression is shown by their influence on the plasma triglyceride (TG) profile.

The only fully recognized therapeutic action of omega-3 is the clinically certified decrease of plasma TG concentration following their supplementation [17-19] which is due to the up-regulation of enzymes involved in fatty acid β-oxidation and down-regulation of enzymes of fatty acid synthesis [20].

Another positive effect of omega-3 LCPUFAs is the control of cardiac arrhythmia that is linked to the modulation of specific calcium ion channels in cardiomocytes [21,22].

Many observational studies (e.g. in cancer and neurodegenerative diseases) have postulated the possible therapeutic potential of omega-3 LCPUFAs supplementation, due to the observation that plasma and tissues of the affected subjects showed a low content of omega-3 LCPUFAs, in particular of EPA [23-28].

However, the usefulness of the clinico-chemical methods and indices used to assess the LCPUFA content in the cells and blood of subjects are labour-intensive and difficult to compare.

Total omega-6 versus total omega-3 fatty acid ratios in different circulating cells and in blood plasma has been used, but comparisons with the more specific AA/EPA ratio are few and show some discrepancies.

The primary aim of this study was to evaluate the AA/EPA ratio in directly derivatised whole blood as a simple metabolic index of the LCPUFA status of subjects of different ages, omega-3 intake and pathologies by comparing it with the total omega-6/omega-3 fatty acid ratios in whole blood and in RBC membrane phospholipids, which has historically been the standard for assessing longer-term intake of the LCPUFA in man.

We analysed whole blood samples from subjects classified by age, sex and health status, with or without self-motivated omega-3 supplement consumption, using a simple, direct derivatisation method.

RBC cell membranes were isolated, their phospholipids were extracted by standard chromatographic procedures, their fatty acids derivatised and analysed by gas chromatography to assess specific n-6:n-3 ratios in cell membrane phospholipids by the standard method.

Lipids Health Dis. 2010; 9: 7. doi: 10.1186/1476-511X-9-7.  

Angela M Rizzo,¹ Gigliola Montorfano,¹ Manuela Negroni,¹ Laura Adorni,¹ Patrizia Berselli,¹ Paola Corsetto,¹ Klaus Wahle,² and Bruno Berra¹

¹Dipartimento di Scienze Molecolari Applicate ai Biosistemi, Università degli Studi di Milano, Italy ²School of Medicine and Dentistry, University of Aberdeen, UK Corresponding author. Angela M Rizzo: angelamaria.rizzo@unimi.it ;

Gigliola Montorfano: gilgiola.montorfano@unimi.it ;

Manuela Negroni: manuela.negroni@unimi.it ;

Laura Adorni: laura.adorni@unimi.it ;

Patrizia Berselli: patrizia.berselli@unimi.it ;

Paola Corsetto: paola.corsetto@unimi.it ;

Klaus Wahle: k.wahle@abdn.ac.uk ;

Bruno Berra: bruno.berra@unimi.it

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Abstract