Chapter  92:  Effects of Omega-3 Fatty Acids on Arrhythmogenic Mechanisms in Animal and Isolated Organ/Cell Culture Studies

Metabolism and Biological Effects of Essential Fatty Acids

Dietary fat is an important source of energy for biological activities in human beings. Dietary fat encompasses saturated fatty acids, which are usually solid at room temperature, and unsaturated fatty acids, which are liquid at room temperature. Unsaturated fatty acids can be further divided into monounsaturated and polyunsaturated fatty acids. Polyunsaturated fatty acids (PUFAs) can be classified on the basis of their chemical structure into two groups: omega-3 (n-3) fatty acids and omega-6 (n-6) fatty acids. The omega-3 or n-3 notation means that the first double bond from the methyl end of the molecule is in the third. The same principle applies to the omega-6 or n-6 notation. Despite their differences in structure, all fats contain the same amount of energy (9 kcal/g or 37 kJ/g).

Of all fats found in food, 2 — alpha-linolenic acid (chemical abbreviation: ALA, 18:3 n-3) and linoleic acid (LA, 18:2 n-6) — cannot be synthesized in the human body, yet are necessary for proper physiological functioning. These 2 fats are called essential fatty acids. The essential fatty acids can be converted in the liver to long-chain polyunsaturated fatty acids (LC PUFAs), which have a higher number of carbon atoms and double bonds. These LC PUFAs retain the omega type (n-3 or n-6) of the parent essential fatty acids.

ALA and LA comprise the bulk of the total PUFAs consumed in a typical North American diet. Typically, LA comprises 89% of the total PUFAs consumed, while ALA comprises 9%. Smaller amounts of other PUFAs make up the remainder ¹. Both ALA and LA are present in a variety of foods. For example, LA is present in high concentrations in many commonly used oils, including safflower, sunflower, soy, and corn oil. ALA, which is consumed in smaller quantities, is present in leafy green vegetables and in some commonly used oils, including canola and soybean oil. Some novelty oils, such as flaxseed oil, contain relatively high concentrations of ALA, but these oils are not commonly found in the food supply.

The Institute of Medicine suggests that, for adults 19 and older, an adequate intake (AI) of ALA is 1.1–1.6 g/day, while an adequate daily intake of LA is 11–17 g/day ². Recommendations regarding AI differ by age and gender groups, and for special conditions such as pregnancy and lactation.

Figure 1. Classical omega-3 and omega-6 fatty acid (more...)

   Figure 1. Classical omega-3 and omega-6 fatty acid synthesis pathways and the role of omega-3 fatty acid in regulating health/disease markers

Figure 1.1

Metabolic Pathways of Omega-3 and Omega-6 Fatty Acids

Figure 1.1

The specific biological functions of fatty acids depend on the number and position of double bonds and the length of the acyl chain. Both EPA and AA are 20-carbon fatty acids and are precursors for the formation of prostaglandins, thromboxane, and leukotrienes — hormone-like agents that are members of a larger family of substances called eicosanoids. Eicosanoids are localized tissue hormones that seem to be one of the fundamental regulatory classes of molecules in most higher forms of life. They do not travel in the blood, but are created in the cells to regulate a large number of processes, including the movement of calcium and other substances into and out of cells, dilation and contraction of muscles, inhibition and promotion of clotting, regulation of secretions including digestive juices and hormones, and control of fertility, cell division, and growth ⁴.

Figure 1.1

In addition to affecting eicosanoid production as described above, EPA also affects lipoprotein metabolism and decreases the production of other compounds — including cytokines, interleukin 1ß (IL-1ß), and tumor necrosis factor a (TNF-a) — that have pro-inflammatory effects. These compounds exert pro-inflammatory cellular actions that include stimulating the production of collagenases and increasing the expression of adhesion molecules necessary for leukocyte extravasation ⁶. The mechanism responsible for the suppression of cytokine production by omega-3 LC PUFAs remains unknown, although suppression of eicosanoid production by omega-3 fatty acids may be involved. EPA can also be converted into the longer chain omega-3 form of docosapentaenoic acid (DPA, 22:5 n-3), and then further elongated and oxygenated into DHA. EPA and DHA are frequently referred to as very long chain omega-3 fatty acids. DHA, which is thought to be important for brain development and functioning, is present in significant amounts in a variety of food products, including fish, fish liver oils, fish eggs, and organ meats. Similarly, AA can convert into an omega-6 form of DPA. Studies have reported that omega-3 fatty acids decrease triglycerides (Tg) and very low density lipoprotein (VLDL) in hypertriglyceridemic subjects, with a concomitant increase in high density lipoprotein (HDL). However, they appear to increase or have no effect on low density lipoprotein (LDL). Omega-3 fatty acids apparently lower Tg by inhibiting VLDL and apolipoprotein B-100 synthesis and decreasing post-prandial lipemia ⁷. Omega-3 fatty acids, in conjunction with transcription factors (small proteins that bind to the regulatory domains of genes), target the genes governing cellular Tg production and those activating oxidation of excess fatty acids in the liver. Inhibition of fatty acid synthesis and increased fatty acid catabolism reduce the amount of substrate available for Tg production ⁸

As noted earlier, omega-6 fatty acids are consumed in larger quantities (>10 times) than omega-3 fatty acids. Maintaining a sufficient intake of omega-3 fatty acids is particularly important since many of the body's physiologic properties depend upon their availability and metabolism.

Overview of the Electrophysiology of the Heart

In this report, we examine evidence that omega-3 fatty acids affect cell organelles — such as cardiac ion channels, pumps, or exchange mechanisms — that are involved in cardiac electrophysiology or electrogenesis. This section of the report reviews some basic aspects of electrogenesis and omega-3 fatty acids., and discusses the analytic framework that guided our systematic review of the literature. Two accompanying reports --- Effects of Omega-3 Fatty Acids on Cardiovascular Disease Risk Factors and Effects of Omega-3 Fatty Acids on Cardiovascular Disease review evidence from clinical studies focused on the relationship between omega-3 fatty acids and outcomes in humans including sudden death.

Potential Impact of Omega-3 Fatty Acids on Arrhythmogenesis

As described above, cell organelles such as cardiac ion channels, pumps, currents, and exchange mechanisms are essential electrophysiological processes that ensure normal heart rate and coronary blood flow. These processes depend upon the concentration gradient of sodium, potassium, and calcium, and associated enzymes. Disruptions in these concentrations can lead to asynchronous contractility of the myocardium and result in arrhythmias. Clinically, the main causes of arrhythmia are ischemia, electrolyte disturbances, drugs, and underlying structural problems (e.g. bypass tracts). The physiologic mechanisms underlying these effects involve such mechanisms as ion channels and pumps and membrane currents.

Omega-3 LC PUFAs may exert an anti-arrhythmic effect on cardiac cells in several ways. For example, they can affect the adrenoceptors that transmit neuroendocrine messages sent by catecholamines. The omega-3 fatty acid, DHA, for instance, causes both a decrease in the production of cyclic adenosine monophosphate (cAMP), the main ß-adrenergic messenger, and an increase in chronotropic response or heart rate¹¹. Omega-3 LC PUFAs also appear to act like another group of cardiovascular drugs, calcium channel blockers, by increasing intracellular calcium sequestration and interfering with receptor-operated calcium channels, influx ¹².

Overview

This evidence report on the effect of omega-3 fatty acids on cardiac electrogenesis and arrhythmias is based on a systematic review of the literature. To identify the specific issues central to this report, the Tufts-New England Medical Center (Tufts-NEMC) evidence-based practice center (EPC) held meetings and teleconferences with a Technical Expert Panel (TEP) formed for this project and with participants from the Agency for Healthcare Research and Quality (AHRQ) and the Office of Dietary Supplements (ODS). In addition, teleconferences with the Southern-California RAND (SC-RAND) and University of Ottawa (UO) EPCs were held to discuss common methodological issues associated with the production of the evidence report. A comprehensive search of the scientific literature was conducted to identify studies addressing the key questions. Evidence tables of study characteristics and results were compiled, and the methodological quality and applicability of the studies were appraised. Results were summarized with both qualitative reviews of the evidence and quantitative meta-analyses, as appropriate.

The TEP served in an advisory capacity for this project. It helped to refine key questions, identify important issues, and define parameters of the report. Additional domain expertise was obtained through consultation with lipid/nutrition experts.

Analytic Framework of This Evidence Report

Figure 2.1 Analytic framework for animal studies

   Figure 2.1 Analytic framework for animal studies

Question: What is the evidence from whole animal studies that omega-3 fatty affect arrhythmogenic outcomes (and intermediate outcomes)?

Figure 2.2 Analytic framework for intact animal isolated (more...)

   Figure 2.2 Analytic framework for intact animal isolated organ and cell studies

Question: What is the evidence from intact {intact, whole animal} cell culture and tissue studies (including animal and human cardiac tissue). that omega-3 fatty acids directly affect cell organelles such as cardiac ion channels, pumps, or exchange mechanisms involved in electrogenesis?

Figure 2.3 Analytic framework for cell culture studies (more...)

   Figure 2.3 Analytic framework for cell culture studies

Question: What is the evidence from cell culture and tissue studies (including animal and human cardiac tissue). that omega-3 fatty acids directly affect cell organelles such as cardiac ion channels, pumps, or exchange mechanisms involved in electrogenesis?

Figure 2.1

Figure 2.2

Figure 2.3

• What is the evidence from whole animal studies that omega-3 fatty acids affect arrhythmogenic outcomes (and intermediate outcomes)?

• What is the evidence from cell culture and tissue studies (including animal and human cardiac tissue) that omega-3 fatty acids directly affect cell organelles such as cardiac ion channels, pumps, or exchange mechanisms involved in electrogenesis?

Figure 2.1

Figure 2.2

Figure 2.3

Potential mechanisms suggested by different investigators to explain the antiarrhythmic action of omega-3 fatty acids can be broadly classified into several categories (See list of Acronyms, Abbreviations, and Parameters). These include the effects of omega-3 fatty acids on:

• Contractile parameters (e.g. contractility)

• Basoelectromechanical parameters (e.g. action potential)

• Ion channels and pumps (e.g. calcium channels)

• Membrane currents (e.g. depolarizing current)

• Receptors (e.g. beta adrenergic)

• Membrane characteristics (e.g. fluidity and composition)

• Enzymes (e.g. sodium, potassium ATPases, adenosine triphosphatase)

• Eicosanoid system (e.g. prostaglandins)

Our focus in this report is limited to contractile parameters, basoelectromechanical parameters, ion pumps, channels, and membrane currents.

Literature Search Strategy

A comprehensive literature search was conducted to address the key questions. Relevant studies were identified primarily through search strategies conducted in collaboration with the UO EPC. Preliminary searches were conducted at the Tufts-NEMC EPC using the OVID search engine on the Medline database. The final searches used five databases including:

- Medline from 1966 to week 2 of February 2003

- PRE-MEDLINE from February 7, 2003

- Embase from 1980 to week 6 of 2003

- Biological Abstracts 1990 - December 2002

- Commonwealth Agricultural Bureau (CAB) Health from 1973 to December 2002

Subject headings and text words were selected so that the same set could be applied to each of the different databases with their varying attributes. Supplemental search strategies were conducted as needed. Additional publications were referred to us by the TEP and the other two EPCs.

A targeted search was conducted to retrieve articles that examined the effects of omega-3 fatty acids on cell organelles involved in electrophysiology. This search included in-vivo as well as in vitro animal studies. MeSH subject headings and text words were defined by reviewing key articles supplied by researchers and members of the TEP. In addition, citation analyses of key articles were conducted using the Science Citation Index database from the Institute for Scientific Information's Web of Science. Publications that cited the key articles were scanned for appropriateness and for additional subject headings or text words. These additional headings and text words were then added to those used in the search strategy.

Numbers for the final results of the database search strategies are approximate. Because the 5 main databases used in the search employ different citation formats, a number of duplicate publications were encountered. Although the UO EPC eliminated some of the duplicates, it was impossible to identify all of them. We eliminated additional duplicate publications as they were discovered. The database searches were updated regularly. The last update was conducted on April 18, 2003.

Study Selection

Abstracts identified through the literature search were screened using eligibility criteria defined to include all English language primary experimental studies that evaluated the impact of omega-3 fatty acids on arrhythmia, intermediate mechanisms of arrhythmia, and electrogenesis. Reports published only as letters or abstracts were excluded.

Articles associated with abstracts that passed these screens were retrieved and screened once more for eligibility. Inclusion and exclusion criteria used in this round of review are summarized below.

Inclusion Criteria

Studies were included if they examined the effect of omega-3 fatty acids on one of the following:

• Arrhythmia

• Adenosine triphophatase (ATPase, either Calcium, Sodium, Potasssium, or Magnesium)

• Beating rate

• Cardiac dynamics

• Cardiac or myocyte contraction

• Cardiomyocytes

• Cell organelles in cardiac tissue (sarcoplasmic reticulum or endoplasmic reticulum; mitochondria)

• Cell signaling

• Coronary perfusion pressure

• Cultured myocytes

• Electrogenesis in cardiac myocyte

• Electrophysiology

• Heart rate or rhythm

• Ion channels, pumps, currents, voltage dependant/sensitive channels (Calcium (Ca²⁺), Sodium (Na⁺), Potassium (K⁺), K⁺ transient outward current, delayed rectifier current, inward rectifier current, L-type Ca²⁺ current or channel)

• Ischemia/ischemic reperfusion in heart

• Sudden cardiac death

• Ventricular fibrillation (VF)

• Ventricular fibrillation threshold (VFT)

• Ventricular ectopic beats (VEB)

• Ventricular premature beats (VPB); sometimes referred to as premature ventricular complex (PVC)

The TEP agreed that given the wide range and number of studies of potential relevance, prioritization of which to include was important. We therefore identified studies of the following mechanisms related to the antiarrhythmic action of omega-3 fatty acids but judged them not immediately relevant to the scope of the key questions to include in this report. Mechanisms excluded were:

• Eicosanoid production (prostaglandins, leucotrienes, thromboxanes)

• Enzymes (5′nuclotidase, phospholipase, cyclo-oxygenase)

• Receptors (ß-adrenergic, thromboxane)

• Membrane composition, fluidity, or phospholipids

For articles identified through the review, grounds for rejection included: non-mammalian animals or cell lines, no outcome of interest reported (see below), no omega-3 fatty acid intervention, review article, non-English article, and toxicology study/safety assessment. For each study that was rejected, the reason(s) for rejection was noted. Basic information about all studies that addressed relevant outcomes was recorded.

Data Extraction

A standardized data extraction process was followed to ensure consistency across reviewers. Definitions for terms used in the extraction process were specified by consensus. As part of the training process, data extractors extracted data from 2 of the same studies to compare interpretations. After this process, each study was partially screened to determine whether it met eligibility criteria and addressed relevant outcomes. Studies deemed eligible were then fully extracted by a single reviewer. Issues and discrepancies encountered during the extraction process were addressed at weekly meetings.

For both animal and in vitro studies, general items extracted included country in which the experiment occurred, funding source, and sample size. Extraction of additional data relating to the intervention, intermediate outcomes, potential mechanisms, and arrhythmogenic outcomes was guided by the analytic framework described in Chapter 1.

For animal and animal in vitro studies, data extracted regarding the intervention included species of animal, animal characteristics, control and experimental diets (including detailed description of any omega-3 fatty acids), and dosage and duration of feeding or infusion. For animal studies, data extracted about intermediate outcomes included heart rate, coronary flow, and electrocardiogram (ECG) changes. Data extracted about arrhythmogenic outcomes included induced arrhythmia, ventricular ectopic beats, ventricular fibrillation, and atrial fibrillation.

For in vitro studies, data extracted regarding the intervention included species of animal, animal characteristics, cell line, sample sizes, number of experiments, detailed description of any omega-3 fatty acids, and whether the fatty acids were free (directly added to the cell culture medium) or bound (incubated with the fatty acid and incorporated into membrane phospholipid). Data extracted about potential mechanisms of arrhythmia included ion channels, ion pumps, and ion movement, as well as ion currents, contractility, and basoelectromechanical parameters.

Format for Reporting Evidence

We report the evidence in three forms: (1) Evidence tables offer a detailed description of the studies we identified that address each of the key questions. These tables provide detailed information about the study design, characteristics of the animal and in vitro model used in the study, inclusion and exclusion criteria, intervention or test evaluated, and outcomes. Where appropriate, we graded the studies according to the methodological quality, applicability, size, and the effect or test performance. (2) Summary tables report on each study in an abbreviated form using summary measures of the main outcomes. These tables were developed by condensing information from the evidence tables to provide a concise overview of study quality and results, and are designed to facilitate comparisons across studies. Summary tables include important variables including study size, omega-3 fatty acids evaluated in the study, study dosages and duration, the animal model, outcomes, and methodological quality. (3) Additional tables were developed to provide an overall synthesis of information related to several key questions.

Methods of Analysis

For the whole animal studies, wherever feasible, we performed meta-analyses combining the results from individual experiments. It is important, however, to interpret results cautiously when combining data that are highly variable. We identified key measures and subgroups to construct random effects meta-analysis models ¹³.

For the isolated organ and cell studies, we frequently developed a qualitative summary of data presented in the articles. When possible, we report percentage changes in evidence tables. When a treatment group was compared to a control group, the difference in percentage change between the treatment group and control group was calculated. When one omega-3 fatty acid was compared to another fatty acid, we first report results of the comparisons to omega-6 fatty acids, followed by comparisons to monounsaturated fatty acids (MUFAs), then to saturated fatty acids (SFAs), and finally to other omega-3 fatty acids. In the summary tables, percentage changes are characterized as a statistically significant (P<.05) increase, decrease, improvement, or no change (i.e. change not statistically significant (P>.05).

Diet Classification

The criteria used to assess the methodological quality of animal studies are different from those used for human studies. Compared to human clinical trials, methods used to evaluate animal studies are not as advanced and there are no quality assessment rating schemes in widespread use. It is, however, important to stratify analyses, where possible, by the rigor of the study design and by the conduct, analysis, and reporting of the study. Since diet composition and the structure of the comparisons is a key aspect of study design in studies using intact animals fed different diets, we devised a four level categorization schema that is based on the fatty acid and/or level of fat contained in the comparison diet. The levels range from A to D, where the comparison diet in level A is most similar to eicosapentaenoic acid (EPA, 20:5 n-3) and decosahexaenoic acid (DHA, 22:6 n-3), and the comparison diet in level D is least similar. Specifically:

1. Omega-3 (fish, soybean, canola, linseed oils) vs. omega-6 (e.g. corn, safflower, sunflower oils) fatty acids. The omega-6 comparison oils have the longest fatty acid chains normally consumed by humans, and are most similar to EPA and DHA. They provide a similar level of dietary fat and have a similar number of double bonds.

2. Omega-3 fatty acids vs. MUFAs (e.g. olive oil). As with omega-6 comparison oils, MUFA oils have the longest fatty acid chains normally consumed by humans. They contain at least one double bond and provide a similar level of dietary fat.

3. Omega-3 fatty acids vs. SFA (e.g. butter, lard, palm oil, coconut oil, sheep fat). These saturated fatty acids provide a level of dietary fat in the comparison diet that is similar to the level obtained with omega-3 fatty acids.

4. Omega-3 fatty acids vs. control (e.g. standard chow). Standard chow is most different from the omega-3 enriched diet because no “counter-balancing” fatty acids are contained in this comparison diet.

In some studies, certain dietary comparisons conducted by the article authors were not relevant to this report. In such instances, only those components of the analysis that addressed the objectives of this report were extracted, using the scheme described above (order of comparison: omega-3 fatty acids to omega-6 fatty acids, MUFA, SFA, other omega-3 fatty acid).

Data Presentation

Data from the whole animal isolated organ and cell studies and the pure isolated organ and cell studies are presented in the evidence and summary tables in a specific order. Studies and/or comparisons are presented in the rows, and results or outcomes (e.g., contractile parameters [CP], basoelectromechanical parameters [BEP], ion pumps and ion movements, [IPIM], ion currents [ICU], and ion channels [ICH]) are presented in the table columns. For each outcome, the omega-3 fatty acid used, the dose, and the experimental condition under which the study was performed, is noted. Outcomes or results obtained under ‘ambient’ (no perturbation) conditions are presented first, followed by outcomes or results under other conditions. Presenting results in this order is similar to the order followed in the studies themselves: after observations were made in the ambient condition, specific blocking or facilitating agents (e.g., antagonists such as iosproteronol and agonists such as BAY8644 (BAY), respectively) were often introduced to investigate specific mechanisms (e.g., receptors) that are affected by the fatty acids. For example, isoproteronol was used in some studies to produce arrhythmia. This approach provides an understanding about which specific receptors are affected by omega-3 fatty acids and which omega-3 fatty acids might yield anti-arrhythmogenic effects. The parameter of interest in some studies is electrical current, which must be elicited by electrical stimulation. For the purposes of this report electrical stimulation is not considered an ‘agent’.

Literature Search Overview

Through the literature search, we identified 1,807 abstracts that met our search criteria. After screening the abstracts, we retrieved 274 articles. Of these, 184 were rejected after reviewing the full text. The reasons for rejection are as follows: no omega-3 fatty acids (30), not specific to arrhythmia (31), no cardiac cells (4), fatty acid composition or products only (34), other reasons (90). Details associated with the reasons for rejection are summarized in the reasons for rejection section. At the end of this process, 89 articles were accepted and reviewed.

Table 3-1

Summary Table 3-1. Summary of Study Design and Outcomes (more...)

Table 3-1

Summary Table 3-1. Summary of Study Design and Outcomes Evaluated in Whole Animal Studies (23 feeding and 3 infusion studies) *

Author, Year	Omega-3 Arm(s)	Control Arm*	Animals	Outcomes Evaluated
				VF	VT	VPB	AS	Deaths	IS	TSR	VFT
Feeding studies:
Omega-3 PUFAs vs Omega-6 PUFAs
Abeywardena, 1995	Soybean, MaxEPA™	SSO	Rats	v	v	v	v	v
Anderson, 1996	MaxEPA™	Safflower	Rats	v	v	v	v
Charnock, 1992	Fish oil	SSO	Monkeys	v							v
Charnock, 1991	Fish oil	SSO	Rats	v	v	v	v
Hock, 1990	Menhaden	Corn	Rats	v			v	v
Hock, 1987	Menhaden	Corn	Rats			v		v
Isensee, 1994	Linseed, Fish oil	Corn	Rats	v	v				v	v
McLennan, 1995	Canola, Soybean	SSO	Rats	v	v	v	v	v
McLennan, 1992	Tuna	SSO	Monkeys	v							v
McLennan, 1993	Fish oil	SSO	Rats	v	v	v	v	v		v
McLennan, 1990	Tuna	SSO	Rats	v	v	v	v	v		v
McLennan, 1988	Tuna	SSO	Rats	v	v		v	v	v
McLennan, Bridle, 1993	Fish oil	SSO	Monkeys	v							v
Omega-3 PUFAs vs MUFAs
McLennan, 1996	EPA-e, DHA-e, EPA-e+DHA-e	Olive	Rats	v			v
Omega-3 PUFAs vs SFAs
al Makdessi, 1995	Sardine	Coconut	Rats						v	v
Chen, 1994	Fish oil	Coconut	Rabbits			v		v
Hartog, 1987	Mackerel	Lard	Piglets	v	v	v		v
Pepe, 1996	Fish oil	Sheep fat	Rats	v	v	v					v
Yang, 1993	Fish oil	Butter	Rats	v	v
Omega-3 PUFAs vs Chows
Culp, 1980	Menhaden	Friskies Dinner	Dogs			v		v	v
Kinoshita, 1994	EPA-e	Oriental Yeast Co.	Dogs	v	v	v	v
Oskarsson, 1993	MaxEPA™	Chows	Dogs						v
Otsuji, 1993	EPA-e	Oriental Yeast Co.	Dogs					v	v	v
	Total =			17	12	12	10	11	6	5	4
Infusion studies:
Omega-3 PUFAs vs Omega-6 PUFAs
Billman, 1999	Albumin-bound ALA, EPA, DHA	Soybean or saline	Dogs	v
Billman, 1994	Fish oil emulsion	Soybean	Dogs	v
Omega-3 PUFAs vs Chows
Lo, 1991	ALA	Buffer	Dogs		v	v
	Total =			2	1	1

SSO = sunflower seed oil; VF=ventricular fibrillation; VT=ventricular tachycardia; VPB=ventricular premature beats; AS=arrhythmia score; IS=infarct size; VFT=ventricular fibrillation threshold, measured only in VF inducible animals; TSR =length of time in normal sinus rhythm; EPA-e = EPA esters; DHA-e = DHA esters

*

For the purposes of our evidence review, only optimal comparison group was chosen. See Chapter 2: Methods.

Summary Table 3-3. Summary of Study Design and Outcomes (more...)

Summary Table 3-3. Summary of Study Design and Outcomes Evaluated in Isolated Organ and cell Studies

Author	Species	Stage*	ICU	ICH	IPIM	BEP	CP
Bogdanov, 1998	Rat	Adult	v			v
Courtois, 1992	Rat	W					v
De Jonge, 1996	Rat	W					v
Hallaq, 1990	Rat	W			v		v
Hallaq, 1992	Rat	W		v	v		v
Honore, 1994	Mouse	W	v	v
Jahangiri, 2000	Rat	Adult					v
Kang, 1994	Rat	W					v
Juan, 1987	Guinea pig	Adult					v
Xiao, 2002	Ferret	Adult	v
Kang, 1996	Rat	W			v		v
Leifert, 1999	Rat	Adult	v
Leifert, 2000	Rat	Adult					v
Rodrigo, 1999	Rat, guinea pig	ND	v				v
MacLeod, 1998	Rat, guinea pig	Adult	v				v
O'Neill, 2002	Rat	Not sure	v		v
Durot, 1997	Rat	W				v	v
Grynberg, 1988	Rat	W				v	v
Kang, 1995a	Rat	W				v
Kang, 1997	Rat	W		v
Li, 1997	Rat	W					v
Negretti, 2000	Rat	ND	v		v		v
Pepe, 1994	Rat	2–3 mo	v		v		v
Phillipson, 1985	Dog	ND			v
Phillipson, 1987	Dog	ND			v
Grynberg, 1996	Rat	W				v	v
Kang, 1995b	Rat	W					v
Fournier, 1995	Rat	W				v	v
Grynberg, 1995	Rat	W					v
Ferrier, 2002	Guinea pig	Adult	v				v
Reithman, 1996	Rats	W				v	v
Ponsard, 1999	Rats	W					v
Xiao, 1997	Rats	Adult	v		v
Xiao, 1995	Rats	W	v
Goel, 2002	Pig	Adult			v
Vitelli, 2002	Rats	Adult			v
Weylandt,1996	Rats	W				v
Rinaldi, 2002	Rats	Adult			v	v
Bayer, 1979	Cat	Adult				v
Total			12	3	12	10	23

ICU=ion currents; ICH=ion channels; IPIM=ion pumps and ion channel movement; BEP=basal electromechanical parameters; CP=contractile parameters.

*

Stage: ND=no data; W = weanling

Whole Animal Studies

Summary Table 3-4. Total Deaths in Ischemia-Reperfusion-Induced (more...)

Summary Table 3-4. Total Deaths in Ischemia-Reperfusion-Induced Arrhythmia: Comparison of Rates Fed Omega-3 Fatty Acids With Controls Fed Omega-6 PUFA Oils

Author, Year	Omega-3 Arms	Dosage, g/100 g	Duration	Omega-3 Fatty Acids		Control		RR (95% CI)	Experiment Protocols
				Event	Total	Event	Total
ALA Oils
Abeywardena, 1995	Soybean	0.4	9 months	2	18	1	18	2.0 (1.5–20)	5-min ischemia; 10-min reperfusion
McLennan, 1995	Soybean	1.1	5 weeks	3	10	2	10	1.5 (0.32–7.1)	5-min ischemia; reperfusion
								1.3 (0.25–6.8)
McLennan, 1995	Soybean	1.1	5 weeks	2*	13	2†	14	0.20 (0.01–3.7)	15-min ischemia; reperfusion
								1.3 (0.25–6.8)
McLennan, 1995	Canola	1.2	5 weeks	0	10	2	10	0.20 (0.01–3.7)	5-min ischemia; reperfusion
McLennan, 1995	Canola	1.2	5 weeks	3‡	16	2†	14	1.1 (0.18–6.6)	15-min ischemia; reperfusion
Meta-analysis: Total subjects = 133				10	67	9	66	1.2 (0.51–2.6)	Random-effect model
Fish Oils (EPA+DHA)
Hock, 1987	Menhaden	1.0	4 weeks	2‡	13	2‡	14	1.1 (0.18–6.6)	15-min after ischemia without reperfusion
Hock, 1990	Menhaden	1.0	4 weeks	5	21	13	22	0.40 (0.17–0.93)	15-min ischemia; 24 h reperfusion
McLennan, 1993	Fish oil	2.6	12 weeks	0	10	1*	12	0.39 (0.02–8.7)	5-min ischemia; 5-min reperfusion
McLennan, 1993	Fish oil	2.6	12 weeks	0	14	1*	13	0.31 (0.01–7.0)	15-min ischemia; 5-min reperfusion
Abeywardena, 1995	MaxEPA™	3.3	9 months	0	18	1	18	0.33 (0.01–7.7)	5-min ischemia; 10-min reperfusion
McLennan, 1988	Tuna	3.7	12 months	0	10	0	10	1.0 (0.02–46)	15-min ischemia; reperfusion
McLennan, 1990	Tuna	3.7	18 months	0	7	0	7	1.0 (0.02–45)	15-min ischemia; reperfusion
Meta-analysis: Total subjects = 169				7	83	18	86	0.47 (0.23–0.93)	Random-effect model

RR=risk ratio=(omega-3 FA event rate)/(control's event rate)

*

All deaths occured during ischemia procedure

†

One death occured during ischemia procedure

‡

Deaths were observed 15-min after ischemia procedure without reperfusion

Summary Table 3-20. Effects of Intravenously Infused (more...)

Summary Table 3-20. Effects of Intravenously Infused Omega-3 Fatty Acids on Ischemia-Induced or Spontaneous Arrhythmias in Mongrel Dogs

Author, Year	Omega-3 Arms (N)	Dosage	Controls (N)	Results				Experiment Protocols
Billman, 1994	10 ml fish oil conc (n=4), or 5 ml fish oil + 5 ml Tg conc (n=4)	Fish oil conc.: 70% EPA+DHA	Saline (n=3) or lipid emulsion (n=5)		N	VF incidence		Exercise-ischemia (2-min) test
		T conc.: 65% EPA+DHA		Fish oil infusion	8	13%*
				Controls	8	100%
				*P<0.005 compared to controls
Billman, 1999	Albumin-bound	98% EPA	SBO lipid emulsion, containing 7%~8% ALA (n=7)		N	VF incidence		Exercise-ischemia (2-min) test
	ALA (n=8)	91% DHA		ALA	8	25%*
	EPA (n=7)	>99% ALA		EPA	7	29%*
	DHA (n=8)	No data on the amount (ml) infused		DHA	8	25%*
				Controls	7	100%
				*P<0.05 compared to controls
Lo, 1991	ALA (n=8)1	1, 5, 10, 20, 30, or 60 mg/kg	Control buffer, pH 8.1 (no lipids)	Eight dogs were infused control buffer or different doses of ALA. No events of VT or VPB were observed when infusing control buffer, or ALA up to 10 mg/kg.				Normal condition
				ALA (mg/kg)	20	30	60
				VPC	25%	75%	88%
				VT	13%	38%	63%
				*P<0.05 compared to control buffer

Tg = triglyceride; VF = ventricular fibrillation; VPC = ventricular premature complex; VT = ventricular tachycardia; conc = concentrate; SBO = soybean oil

1

A left atrial injection instead of intravenous injection was used as the route of administration in this study

Table 1

Evidence Table: Whole Animal Studies Part 1

Table 1

Evidence Table: Whole Animal Studies Part 1

Author, yr	Study Characteristics	Animal Model	Exposure Duration	Ref. Diet	Dietary Characteristics
					Groups	Total Fat	% of Total Fatty Acids					Other
							ALA	E+D	n-6	SFA	MUFA
Abeywardena, 1995	Country: Australia	Mean age: ND	9 months	Standard rat chow (Milling Industries, Adelaide, Australia)	SSO	15* %w/w or 32* %kcal	0.9	0	59*	13*	22*
	Animal: Wistar rats	Age grp: ND			SBO		2.8	0	44*	21*	25*
	Funding: Industry	Sex: Males			FO (MaxEPA)		1.4	22	7*	28*	15*
al Makdessi, 1995	Country: Germany	Mean age: ND	10 weeks	Low-fat (<1% w/w) standard chow from (Altromin GmbH & Co.)	FO (sardine oil)	10% w/w	1.3	29	ND	31*	ND
	Animal: Wistar rats	Age grp: Young			Coconut oil		0.9	0	ND	>60*	ND
	Funding: ND	Sex: Males
Anderson, 1996	Country: Australia	Mean age: ND	8 weeks	Total fat: 3.5%	FO (MaxEPA)	Initially given at 0.6 ml and + 0.1 ml/wk up to 1.0 ml w/ increasing body weight	ND	ND	0	10	28	Total n3 = 41%
	Animal: Sprague-Dawley rats	Age grp: Adult			Safflower oil		0	0	75	25	15
	Funding: Government	Sex: Males
Billman, 1994	Country: US	Mean age: ND	Infusion study	ND	Saline (n=3) or I.V. infusion (n=5)	100 ml of Intralipid, a 10% lipid emulsion	7	0	ND	ND	ND
	Animal: Mongrel dogs	Age grp: ND			Emulsion of fish oil	10 ml FO concentrate (n=4)5 ml same FO concentrate + 5 ml TG concentrate (n=4)	ND	70	ND	ND	ND
	Funding: Government	Sex: ND					ND	65	ND	ND	ND
Billman, 1999	Country: US	Mean age: ND	Infusion study	ND	SBO lipid emulsion (n=7) or saline (n=7)	ND	7~8 in SBO	0	ND	ND	ND
	Animal: Mongrel dogs	Age grp: ND			EPA		0	E=98	ND	ND	ND
	Funding: Government	Sex: ND						D=1
					DHA		0	E=1	ND	ND	ND
								D=91
					ALA		>99	0
Charnock, 1992	Country: Australia	Mean age: > 3 years old	30 months	ND	SSO	12* %w/w or 28 %kcal	1.1	0	ND	23*	23*	N3/n6 = 0.02= 2.0
	Animal: Wistar rats	Age grp: Adult			FO		1.4	20	ND	29*	26*
	Funding: ND	Sex: ND
Charnock, 1991	Country: Australia	Mean age: near 1 yr old	12 months	NDMilling Industries Australia. Total fat: 3 %w/w	SF/SSO	16* %w/w or 35 %kcal	ND	ND	19	45	29	TT n-3 = 1.1
	Animal: Wistar rats	Age grp: Adult										TT n6 = 19
	Funding: ND	Sex: males			SF/FO		ND	ND	12	41	29	TT n-3 = 13
												TT n6 = 12
Chen, 1994	Country: Taiwan	Mean age: ND	2 weeks.	Standard rabbit chow (Purina 5321, St. Louis, MO, USA)	HC (1% CHOL-enriched diet)	40 %kcal (1% chol)	ND	ND	ND	ND	ND
	Animal: rabbits	Age grp: ND			HCF (1% CHOL and 10% FO)	40% energy (1% chol +10% fish oil)	ND	52	ND	ND	ND
	Funding: Government	Sex Males
Culp, 1980	Country: US	Mean age: ND	36 to 45 days	Standard dog chow (Friskies Dinner)	Standard dog chow	ND	ND	0.1	ND	32	34
	Animal: Mongrel dogs	Age grp: ND			FO (Menhaden)	+25 %kcal	ND	13	ND	32	20
	Funding: Government	Sex: ND
Germain, 2003	Country: France	Mean age: ND	>= 3 weeks	APAE, Jouy en Josas, France.	Palm oil	+15% of total fat	ND	ND	ND	high MUFA level	ND
	Animal: Sprague-Dawley rats	Age grp: ND			DHASCO (DHA fom purified TGs)		ND	ND	ND	ND	ND
	Funding: Government	Sex: Females
Hartog JM 1987	Country: Netherlands	Mean age: 5 weeks	16 weeks	ND	Lard fat (9% w/w)	ND	1	0	ND	36	ND
	Animal: Yorkshire piglets	Age grp: Unclear			ML (4.5% mackerel oil + 4.5% lard fat)		1	13	ND	32	ND
	Funding: Dutch Heart Foundation	Sex: ND
Hock, 1990	Country: US	Mean age: ND	4 weeks	Fat-free purified diet	CO (corn oil)	12 %kcal or 5 %w/w	1	0	59	14*	25*	n3/n6= 0.02
	Animal: Sprague-Dawley rats	Age grp: Weanling			MO (Menhaden oil)		2	20	5	33*	27*	=6.06
	Funding: Government	Sex: ND
Hock, 1987	Country: US	Mean age: ND	4 weeks	Fat-free purified diet	CO (corn oil)	12 %kcal or 5 %w/w	1	0	59	14*	25*	n3/n6= 0.02
	Animal: Sprague-Dawley rats	Age grp: Adult			MO (Menhaden oil)		2	21	4	31*	27*	=7.99
	Funding: Government	Sex: Males
Isensee H 1994	Country: Germany	Mean age: 2 months	10 weeks	Low-fat (<1 %w/w ) basic diet (Altromin GmbH, Lage, Germany)	CO	10 %w/w	1	0	50	16	31
	Animal: Wistar rats	Age grp: Young			LO (Linseed oil)		52	0	20	9	16
	Design: A	Sex: Males			FO		0.3	29	12	31	26
	Funding: Alfred Teufel-Stiftung research foundation
Kinoshita, 1994	Country: Japan	Mean age: ND	8 weeks	Standard diet (Oriental Yeast Co.)	Standard diet	ND	ND	ND	ND	ND	ND
	Animal: Mongrel dogs	Age grp: Adult			EPA ester	Mochida Pharmaceutical Co	ND	100 mg/kg BW/d	ND	ND	ND
	Diseased: Funding: ND	Sex: ND
Lo, 1991	Country: Taiwan	Mean age: ND	Infusion study	Same dogs were infused control buffer or different dosages of ALA.	Control buffer	ND	ND	ND	ND	ND	ND
	Animal: Mongrel dogs	Age grp: ND			ALA infusion		1, 5, 10, 20, 30, or 60 mg/kg	ND	ND	ND	ND
	Funding: ND	Sex: MixSex : “either sex”
McLennan, 1996	Country: Australia, Switzerland	Mean age: ND	5 weeks	ND	Olive oil	5% w/w from olive oil	ND	ND	ND	ND	ND
	Animal: spontaneously hypertensive Wistar rats	Age grp: ND			EPA	0.5% from n-3; 4.5% from olive oil	ND	E:0.5 w/w	ND	ND	ND
	Funding: ND	Sex: Males			DHA		ND	D:0.5w/w	ND	ND	ND
					EPA+DHA		ND	ND	ND	ND	ND
McLennan, 1995	Country: Australia	Mean age: 12 weeks	12 weeks	Nonpurified lab rat diet. Total fat = 4% w/w	CAN	15 %w/w or 32 %kcal	8	0	21	12	60	N3/n6 = 0.37
	Animal: Sprague-Dawley rats	Age grp: Adult			SBO		7	0	52	19	22	= 0.14
	Funding: ND	Sex: Males			SSO		5	0	64	12	23	=0.008
McLennan, Bridle, 1993	Country: Australia	Mean age: ND	16 weeks	Low-fat marmoset diet (Milling Industries, Adelaide, Australia) Total fat = 6% w/w	SF/SSO (8% sheep perirenal fat + 2% SSO)	16 %w/w	0.8	1	20	48	ND	N3/n6 = 0.12
	Animal: Marmoset monkeys	Age grp: Old			SF/FO (7% SF + 3% FO)		0.8	11	10	47	ND	=1.25
	Funding: Government	Sex: 50% Males
McLennan, 1993	Country: Australia	Mean age: 30 weeks	12 weeks	Basic laboratory diet (Milling Industries, Adelaide, Australia) Total fat = 4% w/w	SSO	15 %w/w or 32 %kcal	ND	ND	56	15	25	Total n3 = 4%
	Animal: Sprague-Dawley rats	Age grp: Old			FO		ND	ND	8	40	25	=17%
	Funding: International Olive Oil Council	Sex: ND
McLennan, 1992	Country: Australia	Mean age: 2 years	30 months	Total fat: 4 %w/w SFA: 37.3%MUFA: NDPUFA: 18.3%	SSO	12 %w/w or 29 %kcal	ND	ND	54	23	ND	ND
	Animal: Marmoset monkeys	Age grp: Unclear			TFO (tuna fish oil)		ND	ND	11	29	ND	Total n3 = 23%
	Funding: ND	Sex: breeding pairs
McLennan, 1990	Country: Australia	Mean age: 2 months	18 months	Standard lab rat diet. Total fat = 4% w/w	SF+SSO	16 %w/w or 35 %kcal	0	0	58	16	ND
	Animal: Sprague-Dawley rats	Age grp: Adult			SF+TFO		ND	23	9	31	ND
	Funding: Government	Sex: Males
McLennan, 1988	Country: Australia	Mean age: “age-matched”	12 months	Standard lab rat diet. Total fat = 4% w/w	SSO	16 %w/wor 35 %kcal	ND	0	58	ND	ND
	Animal: Wistar rats	Age grp: Unclear			TFO		ND	23	9	ND	ND
	Funding: Government	Sex: Males
Oskarsson, 1993	Country: US	Mean age: ND	6 weeks	ND	No fish oil Rx	ND	ND	ND	ND	ND	ND
	Animal: Mongrel dogs	Age grp: ND			MaxEPA		ND	0.1 g/kg/d	ND	ND	ND
	Funding: ND	Sex: “Mixed Sex”
Otsuji, 1993	Country: Japan	Mean age: ND	8 weeks	Standard diet prepared by Oriental Yeast Co.	Standard dog chow	30 g/kg BW /day	ND	ND	ND	ND	ND
	Animal: Mongrel dogs	Age grp: Adult			EPA ester	100 mg/kg BW/day	ND	ND	ND	ND	ND
	Funding: ND	Sex: MixSex : No data on the distribution
Pepe, 1996	Country: Australia	Mean age: 16 weeks	16 weeks	Nonpurified diet fed to all rats (Milling Industries, Adelaide, Australia).Total fat = 7.6%	SAT (sheep perirenal fat)	15.3% w/w	1.5	1	8	55	ND
	Animal: Wistar rats	Age grp: Young			FO		1.2	36	8	25	ND
	Funding: ND	Sex: Males
Yang, 1993	Country: US	Mean age: ND	5 days	Standard rat nonpurified diet (Purina Mills, St. Louis, MO)Total fat = 5 %kcal	Butter	17 %kcal	ND	ND	ND	ND	ND
	Animal: Sprague-Dawley rats	Age grp: ND			FO (fish oil rich pellets		ND	32	23	25	15
	Funding: Government	Sex: Males

*

estimated values, not reported in original paper

Types of study design:

A = N-3 PUFAs vs. n-6 PUFAs

B = N-3 PUFAs vs. MUFAs

C = N-3 PUFAs vs. SFAs

D = N-3 PUFAs vs. Standard chows

Individual summary tables were created to show the effects of omega-3 fatty acids on various arrhythmic outcomes. Studies were grouped first by outcomes, then by species, and finally by experimental protocols (or mechanisms) for induced arrhythmias. Within each table, comparisons were first clustered into alpha linolenic acid (ALA, 18:3 n 3) oils or fish oils, and then sorted by the dose of omega-3 fatty acids. Frequently, studies had more than one comparison and used more than one experimental protocol. As a result, some studies appear multiple times in one table (once for each comparison group) or appear in several different tables.

In general, the arrhythmic outcomes assessed were defined consistently across the 26 whole-animal studies with the exception of the definition for arrhythmia score, which varied somewhat across studies. The following arrhythmic outcome measures and general definitions were used in the original studies:

• Ventricular Tachycardia (VT): A run of four or more consecutive ventricular premature beats ¹⁴.

• Ventricular Fibrillation (VF): A signal for which individual QRS deflections can no longer be distinguished from one another (implying morphological instability) and for which a rate can no longer be measured ¹⁴.

• Ventricular Premature Beats (VPB): Isolated ventricular premature beats are generally defined as discrete and identifiable premature QRS complexes (premature in relation to the P wave) ¹⁴.

• Arrhythmia Score (AS): A hierarchical scale of 0 to 9 during occlusion as most described by Curtis et al., 1987 ¹⁵, and during reperfusion using a slightly modified version of the scale as described by McLennan et al., 1988 ¹⁶.

• Infarct Size (IS): The under-perfused ischemic regions determined by dye exclusion and expressed as a percentage of wet weight in both ventricles ¹⁶. In the studies examined for this report, infarct size reflects myocardial tissue that has sustained damage due to the ischemia procedures that were used to induce arrhythmias.

We performed meta-analyses for each of the outcomes. In these analyses, fish oils and ALA oils were analyzed separately and in combination.

In the following sections, the 23 pre-fed studies are discussed first and are grouped according to the comparison substance. Studies comparing omega-3 polyunsaturated fatty acids (PUFAs) to omega-6 PUFAs are presented first, followed by studies comparing omega-3 PUFAs to a-linolenic acid, monounsaturated fatty acids (MUFAs), saturated fatty acids (SFAs), and no treatment. The 3 studies that infused free form omega-3 fatty acids are reviewed at the end of the Whole Animal Studies section.

Studies Comparing Pre-Fed Omega-3 PUFAs to Omega-6 PUFAs

This section summarizes 13 studies that compared pre-fed omega-3 PUFAs to pre-fed omega-6 PUFAs (see Table 3 and Evidence Table 1). In each study, the same amount of experimental and control oil was added to each animal's basic diet. Therefore, all comparisons have iso-caloric intake from fat. The dose of omega-3 fatty acids ranged from 0.4 to 3.7g/100g. Fish oils (menhaden, tuna fish oils, or MaxEPA---a commercial preparation of EPA), soybean oil, or canola oil were used as the source of omega-3 PUFAs in the experimental groups, while controls were fed sunflower seed oil, corn oil or safflower oil. The effects of omega-3 PUFAs on arrhythmia deaths, ventricular fibrillation, ventricular premature beats, arrhythmia scores, infarct size, and length of time in sinus rhythm are reviewed below.

Summary Table 3-6. Total VF Deaths: Comparison of Monkeys (more...)

Summary Table 3-6. Total VF Deaths: Comparison of Monkeys Fed Tuna Fish Oil With Controls Fed Sunflower Seed Oil (Omega-6 PUFA)*

Author, Year	Omega-3 Arms	Dosage, g/100 g	Duration	Omega-3 Fatty Acids		Control		Experiment Protocols
				Event	Total	Event	Total
McLennan, 1992	Tuna	2.8	30 months	0	16	3	13	Control condition, ischemia, and isoproterenol (0.5 ug/kg body weight/minute) models

*

Total ventricular fibrillation (VF) deaths were combined in control condition, ischemia, and isoproterenol models.

Effect on incidence of arrhythmia deaths. Seven studies in rats (Table 3-4) and one study in monkeys (Table 3-6) reported arrhythmia deaths. In the rat studies, investigators looked for deaths in ischemia-reperfusion-induced arrhythmias in 12 comparisons. In the monkey study, investigators looked for deaths after induced arrhythmias in 1 comparison (Table 3-6).

Meta-analyses of risk ratio of total deaths in ischemia-reperfusion-induced arrhythmias. As shown in Table 3-4, 12 comparisons in 7 studies were included in the meta-analyses. The 7 studies involved 150 rats that were fed omega-3 PUFAs and 152 rats fed omega-6 PUFAs. In all but one ¹⁷ of the studies, deaths during reperfusion after an ischemia procedure were monitored. Two studies ^{18, 19} also looked for deaths that occurred during the ischemia procedure. They all found that deaths occurred only during the ischemia procedure; no deaths occurred during reperfusion in either the omega-3 PUFA or control groups The ischemia deaths in these two studies were combined into total deaths for ischemia-reperfusion-induced arrhythmia.

Of the 12 comparisons, 5 compared ALA oils to omega-6 PUFA oils (Figure 1). The combined risk ratio of deaths in ischemia-reperfusion-induced arrhythmias in these 5 comparisons was 1.2 (95% CI: 0.51–2.6). There was no statistically significant heterogeneity between studies.

Summary Table 3-5. Sensitivity Analysis on Total Deaths (more...)

Summary Table 3-5. Sensitivity Analysis on Total Deaths In Ischemia-ReperfusionIinduced Arrhythmia: Comparison of Rats Fed Fish Oil With Controls Fed Omega-6 PUFA Oils

Sensitivity Analysis - Sequential Dropping of One Study Random Effects Model - Risk Ratio (D&L method)
Study Dropped	Study Year	Size	Total N	Risk Ratio	95% CI		2P
					Low	High
Hock	1987	27	142	0.41	0.19	0.86	0.018
Hock	1990	43	126	0.64	0.19	2.14	0.47
McLennan	1993	22	147	0.47	0.23	0.96	0.038
McLennan	1993	27	142	0.48	0.24	0.97	0.041
Abeywardena	1995	36	133	0.48	0.23	0.97	0.040
McLennan	1990	14	155	0.46	0.23	0.92	0.028

The other 7 comparisons were combined to assess the effects of fish oils on deaths in ischemia-reperfusion-induced arrhythmias (Figure 2). The combined risk ratio of deaths in these 7 comparisons was 0.47 (95% CI: 0.23–0.93). There was no statistically significant heterogeneity between studies. However, the significantly reduced risk ratio of deaths was due to a single study ²⁰ as shown by a sensitivity analysis (Table 3-5). When this study was removed, the combined risk ratio of deaths became 0.64 (95% CI: 0.19–2.1).

A separate meta-analysis combined comparisons involving ALA with comparisons involving eicosapentaenoic acid (EPA, 20:5 n-3) plus decosahexaenoic acid (DHA, 22:6 n-3). The overall risk ratio of deaths in this analysis was 0.68 (95% CI: 0.40–1.2).

Deaths from ventricular fibrillation in monkeys. One study examined total VF deaths — which combined deaths in the control condition, ischemia model, and isoproterenol model — among marmoset monkeys (Table 3-6). For the purpose of our evidence review, we evaluated only the results from a comparison between 16 monkeys fed fish oil and 13 monkeys fed sunflower seed oil. The fish oil and sunflower seed oil diets both had 12% weight-by-weight (w/w) of total fat or 29% kcal of fat. The fish-oil diet contained 2.8g/100g EPA plus DHA. The animals were fed for 30 months in both studies. No VF deaths occurred in the monkeys that were fed fish oil, while 3 deaths (23%) occurred in those fed sunflower seed oil.

Summary Table 3-7. Ventricular Tachycardia in Ischemia-Induced (more...)

Summary Table 3-7. Ventricular Tachycardia in Ischemia-Induced Arrhythmias: Comparison of Rats Fed Omega-3 Fatty Acids With Controls Fed Omega-6 PUFA Oils

Author, Year	Omega-3 Arms	Dosage, g/100 g	Duration	Omega-3 Fatty Acids		Control		RR (95% CI)	Experiment Protocols
				Event	Total	Event	Total
ALA Oils
Abeywardena, 1995	Soybean	0.4	9 months	8	18	7	18	1.1 (0.53–2.5)	5-min ischemia
McLennan, 1995	Soybean	1.1	12 weeks	8	13	13	14	0.66 (0.42–1.0)	15-min ischemia
McLennan, 1995	Canola	1.2	12 weeks	12	16	13	14	0.81 (0.59–1.1)	15-min ischemia
Isensee, 1994	Linseed	5.2	10 weeks	6	10	4	9	1.4 (0.56–3.3)	20-min ischemia
Meta-analysis: Total subjects = 112				34	57	37	55	0.82 (0.65–1.0)	Random-effect model
Fish Oils (EPA+DHA)
Charnock, 1991	Fish oil	2.1	12 months	7	10	10	10	0.71 (0.41–1.1)	15-min ischemia
McLennan, 1993	Fish oil	2.6	12 weeks	5	14	12	13	0.39 (0.19–0.79)	15-min ischemia
Isensee, 1994	Fish oil	3.0	10 weeks	0	10	4	9	0.10 (0.01–1.7)	20-min ischemia
Abeywardena, 1995	MaxEPA	3.3	9 months	1	18	7	18	0.14 (0.02–1.1)	5-min ischemia
McLennan, 1988	Tuna	3.7	12 months	2	10	8	10	0.25 (0.07–0.90)	15-min ischemia
McLennan, 1990	Tuna	3.7	18 months	4	7	4	7	1.0 (0.40–2.5)	15-min ischemia
Meta-analysis: Total subjects = 136				19	69	45	67	0.49 (0.29–0.83)	Random-effect model

Summary Table 3-8. Ventricular Tachycardia in Reperfusion-Induced (more...)

Summary Table 3-8. Ventricular Tachycardia in Reperfusion-Induced Arrhythmias: Comparison of Rats Fed Omega-3 Fatty Acids With Controls Fed Omega-6 PUFA Oils

Author, year	Omega-3 Arms	Dosage, g/100 g	Duration	Omega-3 Fatty Acids		Control		RR (95% CI)	Experiment Protocols
				Event	Total	Event	Total
ALA oils
Abeywardena, 1995	Soybean	0.4	9 months	13	17	7	18	2.0 (1.0–3.7)	5-min Ischemia; 10-min Reperfusion
McLennan, 1995	Soybean	1.1	12 weeks	9	10	7	10	1.3 (0.82–2.0)	5-min Ischemia; 10-min Reperfusion
McLennan, 1995	Soybean	1.1	12 weeks	7	11	9	13	0.92 (0.52–1.6)	15-min Ischemia; 10-min Reperfusion
McLennan, 1995	Canola	1.2	12 weeks	7	10	7	10	1.0 (0.56–1.8)	5-min Ischemia; 10-min Reperfusion
McLennan, 1995	Canola	1.2	12 weeks	4	13	9	13	0.44 (0.18–1.1)	15-min Ischemia; 10-min Reperfusion
Meta-analysis: Total subjects = 125				40	61	39	64	1.1 (0.73–1.6)	Random-effect model
Fish Oils (EPA+DHA)
Anderson, 1996	MaxEPA	41% of TT FAs	8 weeks	3*	8	3*	6	0.75 (0.23–2.5)	20-min ischemia; reperfusion
McLennan, 1993	Fish oil	2.6	12 weeks	6	10	10	12	0.72 (0.41–1.3)	5-min Ischemia; 5-min Reperfusion
McLennan, 1993	Fish oil	2.6	12 weeks	3	14	8	12	0.32 (0.11–1.0)	15-min ischemia; 5-min reperfusion
Abeywardena, 1995	MaxEPA	3.3	9 months	4	18	7	18	0.57 (0.20–1.6)	5-min ischemia; 10-min reperfusion
McLennan, 1988	Tuna	3.7	12 months	5	10	8	10	0.63 (0.31–1.3)	15-min ischemia; reperfusion
McLennan, 1990	Tuna	3.7	18 months	5	7	6	7	0.83 (0.48–1.5)	15-min ischemia; 10-min reperfusion
Meta-analysis: Total subjects = 132				26	67	42	65	0.68 (0.50–0.91)	Random-effect model

TT FAs = total fatty acids; RR = risk ratio; VF = ventricular fibrillation; VT = ventricular tachycardia

*

Sustained VT and/or VF were excluded from the analyses

Effects on incidence of ventricular tachycardia. Eight studies, representing 21 comparisons, reported the incidence of VT among rats fed omega-3 PUFA oils vs. those fed omega-6 PUFA oils (Table 3-7 and Table 3-8). In 10 of the comparisons, the incidence of VT in ischemia-induced arrhythmias was monitored (Table 3-7). In the other 11 comparisons, the incidence of VT during reperfusion-induced arrhythmias was monitored (Table 3-8). Only ischemia-induced VT was assessed in 2 ^{21, 22} of the 8 studies. The remaining 6 studies assessed both ischemia-induced and reperfusion-induced VT.)

Meta-analyses of risk ratio of ventricular tachycardia in ischemia-induced arrhythmias. As shown in Table 3-7, 10 comparisons in 6 studies were included in the meta-analyses. Of the 248 rats used in the studies, 126 were in the omega-3 PUFA groups and 122 were in the omega-6 PUFA control groups.

Figure 3-1. Total deaths in ischemia-reperfusion-induced (more...)

   Figure 3-1. Total deaths in ischemia-reperfusion-induced arrhythmia: comparison of rats fed, alpha linolenic acid (ALA) with controls fed omega-6 PUFA oils

Figure 3-2. Total deaths in ischemia-reperfusion-indued (more...)

   Figure 3-2. Total deaths in ischemia-reperfusion-indued arrhythmia: comparison of rats fed fish oils with controls fed omega-6 PUFA oils

Figure 3-3. Ventricular tachycardia in ischemia-induced (more...)

   Figure 3-3. Ventricular tachycardia in ischemia-induced arrhythmias: comparison of rats fed alpha linolenic acid (ALA) with controls fed omega-6 PUFA oils

Figure 3-4. Ventricular tachycardia in ischemia-induced (more...)

   Figure 3-4. Ventricular tachycardia in ischemia-induced arrhythmias: comparison of rats fed fish oils with controls fed omega-6 PUFA oils

Figure 3-5. Ventricular tachycardia in reperfusion-induced (more...)

   Figure 3-5. Ventricular tachycardia in reperfusion-induced arrhythmias: comparison of rats fed alpha linolenic acid (ALA) with controls fed omega-6 PUFA oils

Figure 3-6. Ventricular tachycardia in reperfusion-induced (more...)

   Figure 3-6. Ventricular tachycardia in reperfusion-induced arrhythmias: comparison of rats fed fish oils with controls fed omega-6 PUFA oils

Figure 3-7. Ventricular fibrillation in ischemia-induced (more...)

   Figure 3-7. Ventricular fibrillation in ischemia-induced arrhythmias: comparison of rats fed alpha linolenic acid (ALA) with controls fed omega-6 PUFA oils

Figure 3-8/ Ventricular fibrillation in ischemia-induced (more...)

   Figure 3-8/ Ventricular fibrillation in ischemia-induced arrhythmias: comparison of rats fed fish oils with controls fed omega-6 PUFA oils

Figure 3-9. Ventricular fibrillation in reperfusion-induced (more...)

   Figure 3-9. Ventricular fibrillation in reperfusion-induced arrhythmias: comparison of rats fed alpha linolenic acid (ALA) with controls fed omega-6 PUFA oils

Figure 3-10. Ventricular fibrillation in reperfusion-induced (more...)

   Figure 3-10. Ventricular fibrillation in reperfusion-induced arrhythmias: comparison of rats fed fish oils with controls fed omega-6 PUFA oils

Among the 10 comparisons, 4 examined the effects of ALA vs. omega-6 PUFA oils on the incidence of VT in ischemia-induced arrhythmias (Figure 3). The dose of ALA ranged from 0.4 to 5.2g/100g. The combined risk ratio of deaths was 0.82 (95% CI: 0.65–1.0). There was no statistically significant heterogeneity between studies.

The other 6 comparisons were combined to evaluate the effects of fish oils (EPA plus DHA) on the incidence of VT in ischemia-induced arrhythmias (Figure 4). The combined risk ratio of deaths in this analysis was 0.49 (95% CI: 0.29–0.83). The studies were heterogeneous. Sensitivity analysis did not show that any single study had a dominating effect.

A separate meta-analysis combined comparisons involving ALA with comparisons involving EPA plus DHA. In this meta-analysis, the overall risk ratio of VT in ischemia-induced arrhythmias was 0.70 (95% CI: 0.53–0.92).

Meta-analyses of risk ratio of ventricular tachycardia in reperfusion-induced arrhythmias. As shown in Table 3-8, 11 comparisons in 7 studies were included in these meta-analyses. Of the 257 rats used in the studies, 128 were in the omega-3 PUFA groups and 129 were in the omega-6 PUFA control groups.

Among the 11 comparisons, 5 examined the effects of ALA vs.omega-6 PUFA oils on the incidence of VT in reperfusion-induced arrhythmias (Figure 5). The combined risk ratio of deaths was 1.1 (95% CI: 0.73–1.6). The studies were heterogeneous.

The other 6 comparisons were combined to evaluate the effects of fish oils (EPA plus DHA) on the incidence of VT in reperfusion-induced arrhythmias (Figure 6). The combined risk ratio of deaths was 0.68 (95% CI: 0.50–0.91). There was no statistically significant heterogeneity between studies.

In the meta-analysis that combined comparisons involving ALA with comparisons involving EPA plus DHA, the overall risk ratio of VT in reperfusion-induced arrhythmias was 0.85 (95% CI: 0.65–1.1).

Summary Table 3-9. Ventricular Fibrillation in Ischemia-Induced (more...)

Summary Table 3-9. Ventricular Fibrillation in Ischemia-Induced Arrhythmias: Comparison of Rats Fed Omega-3 Fatty Acids With Controls Fed Omega-6 PUFA Oils

Author, Year	Omega-3 Arms	Dosage, g/100 g	Duration	Omega-3 Fatty Acids		Control		RR (95% CI)	Experiment Protocols
				Event	Total	Event	Total
ALA oils
McLennan, 1995	Soybean	1.1	12 weeks	5	13	6	14	0.90 (0.36–2.2)	15-min ischemia
McLennan, 1995	Canola	1.2	12 weeks	7	16	6	14	1.0 (0.45–2.3)	15-min ischemia
Isensee, 1994	Linseed	5.2	10 weeks	4	10	4	9	0.90 (0.31–2.6)	20-min ischemia
Meta-analysis: Total subjects = 76				16	39	16	37	0.95 (0.56–1.6)	Random-effect model
Fish Oils (EPA+DHA)
Charnock, 1991	Fish oil	2.1	12 months	0	10	6	10	0.08 (0.00–1.2)	15-min ischemia
McLennan, 1993	Fish oil	2.6	12 weeks	0	14	5	13	0.08 (0.01–1.4)	15-min ischemia
Isensee, 1994	Fish oil	3.0	10 weeks	1	10	4	9	0.22 (0.03–1.7)	20-min ischemia
McLennan, 1988	Tuna	3.7	12 months	0	10	1*	10	0.33 (0.02–7.3)	15-min ischemia
McLennan, 1990	Tuna	3.7	18 months	1	7	2	7	0.50 (0.06–4.3)	15-min ischemia
Meta-analysis: Total subjects = 100				2	51	18	49	0.21 (0.07–0.63)	Random-effect model

*

Estimated from graph

Summary Table 3-10. Ventricular Fibrillation in Reperfusion-Induced (more...)

Summary Table 3-10. Ventricular Fibrillation in Reperfusion-Induced Arrhythmias: Comparison of Rats Fed Omega-3 Fatty Acids With Controls Fed Omega-6 PUFA Oils

Author, Year	Omega-3 Arms	Dosage, g/100 g	Duration	Omega-3 Fatty Acids		Control		RR (95% CI)	Experiment Protocols
				Event	Total	Event	Total
ALA Oils
Abeywardena, 1995	Soybean	0.4	9 months	4	17	2	18	2.1 (0.44–10)	5-min ischemia; 10-min reperfusion
McLennan, 1995	Soybean	1.1	12 weeks	5	10	5	10	1.0 (0.42–2.4)	5-min Ischemia; Reperfusion
McLennan, 1995	Soybean	1.1	12 weeks	3	11	3	13	1.2 (0.30–4.7)	15-min ischemia; reperfusion
McLennan, 1995	Canola	1.2	12 weeks	1	10	5	10	0.20 (0.03–1.4)	5-min ischemia; reperfusion
McLennan, 1995	Canola	1.2	12 weeks	0	13	3	13	0.14 (0.01–2.5)	15-min ischemia; reperfusion
Isensee, 1994	Linseed	5.2	10 weeks	6	10	7	9	0.77 (0.42–1.4)	20-min ischemia; 20-min reperfusion
Meta-analysis: Total subjects = 144				19	71	25	73	0.84 (0.52–1.3)	Random-effect model
Fish Oils (EPA+DHA)
Anderson, 1996	MaxEPA™	41% of TT FAs	8 weeks	1*	8	2*	6	0.38 (0.04–3.2)	20-min ischemia; reperfusion
Hock, 1990	Menhaden	1.2	4 weeks	1†	7	9†	10	0.16 (0.03–0.99)	15-min ischemia; 6-hr reperfusion
McLennan, 1993	Fish oil	2.6	12 weeks	1	10	3	12	0.40 (0.05–3.3)	5-min ischemia; 5-min reperfusion
McLennan, 1993	Fish oil	2.6	12 weeks	0	14	1	12	0.29 (0.01–6.5)	15-min ischemia; 5-min reperfusion
Isensee, 1994	Fish oil	3.0	10 weeks	4	10	7	9	0.51 (0.22–1.2)	20-min ischemia; 20-min reperfusion
Abeywardena, 1995	MaxEPA™	3.3	9 months	1	18	2	18	0.50 (0.05–5.0)	5-min ischemia; 10-min reperfusion
McLennan, 1988	Tuna	3.7	12 months	0	10	3	10	0.14 (0.01–2.5)	15-min ischemia; reperfusion
McLennan, 1990	Tuna	3.7	18 months	2	7	2	7	1.0 (0.19–5.2)	15-min ischemia; reperfusion
Meta-analysis: Total subjects = 168				10	84	29	84	0.4 (0.25–0.79)	Random-effect model

TT FA = total fatty acids; VT = ventricular tachycardia; VF = ventricular fibrillation

*

Sustained VT and/or VF were excluded from the analyses

†

VT or VF (%)

Summary Table 3-11. Ventricular Fibrillation in Induced (more...)

Summary Table 3-11. Ventricular Fibrillation in Induced Arrhythmia: Comparison of Monkeys Fed Fish Oils With Controls Fed Sunflower Seed Oil (Omega-6 PUFA)

Author, Year	Omega-3 Arms	Dosage, (g/100 g)	Duration	Omega-3 Fatty Acids		Control		VFT¶	Experiment Protocols
				Event	Total	Event	Total
Electrical-Stimulation Arrhythmias†
McLennan, Bridle, 1993	Fish oil	1.8	16 weeks	6	10	5	9	+133% *	Electrical stimulation in control condition
Charnock, 1992	Fish oil	2.4	16 weeks	8%	ND	13%	ND	NS	Electrical stimulation in control condition
McLennan, 1992	Tuna	2.8	30 months	10	16	8	13	NS	Electrical stimulation in control condition
Electrical-Stimulation Arrhythmias in Ischemic Hearts†
McLennan, Bridle, 1993	Fish oil	1.8	16 weeks	10	10	9	9	+79% *	Electrical stimulation + 5-min ischemia
Charnock, 1992	Fish oil	2.4	16 weeks	Nil	ND	13%	ND	NS	Electrical stimulation + ischemia
McLennan, 1992	Tuna	2.8	30 months	12	16	8	13	NS	Electrical stimulation + 5-min ischemia
Electrical-Stimulation Arrhythmias With Isoproterenol†
McLennan, Bridle, 1993	Fish oil	1.8	16 weeks	3	10‡	7	9‡	+55% *	Electrical stimulation + 30-min isoproterenol (0.5 ug/kg BW/min)
McLennan, Bridle, 1993	Fish oil	1.8	16 weeks	5	10‡	9	9‡	+75%	Electrical stimulation + 30-min isoproterenol (2.0 ug/kg BW/min)
McLennan, 1992	Tuna	2.8	30 months	7	16	10	13	NS	Electrical stimulation + 30-min Iisoproterenol (0.5 ug/kg BW/min)

ND = no data; BW = body weight; min = minute; VFT = ventricular fibrillation threshold, measured only in VF inducible animals; NS = no significant difference compared to controls

*

P<0.05 compared to control animals

†

Same monkeys underwent electrical stimulation in control condition, 5 minutes after ischemia. procedure, and 30 minutes after restoration of coronary blood flow during the infusion of isoproterenol.

‡

Same monkeys injected 0.5 ug/kg BW/min isoproterenol, then the dosage of isoproterenol was increased to 2.0 ug/kg BW/min.

¶

An increase in VFT is a desirable outcome for antiarrhythimic effects. See Chapter 2: Methods for the effects expressed as percent change.

Effects on incidence of ventricular fibrillation. Nine studies in rats with 22 comparisons (Table 3-9 & Table 3-10), and 3 studies in monkeys with 9 comparisons (Table 3-11), reported the incidence of VF in induced arrhythmias. All the rat studies used ischemia-reperfusion models. In the monkey studies, arrhythmias were induced by electrical stimulation in normal or ischemic conditions and/or with the injection of isoproterenol.

In the rat studies, the incidence of VF in ischemia-induced arrhythmias was monitored in 8 comparisons (Table 3-9), while in the other 14 comparisons, the incidence of VF during reperfusion after an induced-ischemia procedure was monitored (Table 3-10). (Four ^20–23 of the nine rat studies monitored only the incidence of reperfusion-induced VF. The remaining 5 studies monitored the incidence of both ischemia-induced and reperfusion-induced VF.)

Meta-analyses of risk ratio of ventricular fibrillation in ischemia-induced arrhythmias. As shown in Table 3-9, a total of 8 comparisons from 6 studies were included in the meta-analyses. Of the 176 rats used in the studies, 90 were in the omega-3 PUFA groups and 86 were in the omega-6 PUFA control groups.

Among the 8 comparisons, 3 examined the effects of ALA vs. omega-6 PUFA oils on the incidence of VF in ischemia-induced arrhythmias (Figure 7). The combined risk ratio of deaths was 0.95 (95% CI: 0.56–1.6). There was no statistically significant heterogeneity between studies.

The other 5 comparisons were combined to evaluate the effect of fish oils on the incidence of VF in ischemia-induced arrhythmias (Figure 8). The combined risk ratio of deaths was 0.21 (95% CI: 0.07–0.63). There was no statistically significant heterogeneity between studies.

In the meta-analysis that combined ALA comparisons and EPA plus DHA comparisons, the overall random-effect risk ratio of VF in ischemia-induced arrhythmias was 0.69 (95% CI: 0.41–1.24).

Meta-analyses of risk ratio of ventricular fibrillation in reperfusion-induced arrhythmias. As shown in Table 3-10, a total of 14 comparisons in eight studies were included in these meta-analyses. Of the 312 rats used in the studies, 155 were in the omega-3 PUFA groups and 157 were in the omega-6 PUFA control groups.

Among the 14 comparisons, 6 examined the effects of ALA vs. omega-6 PUFA oils on the incidence of VF in reperfusion-induced arrhythmias (Figure 9). The combined risk ratio of deaths was 0.84 (95% CI: 0.52–1.3). The studies were heterogeneous.

The other 8 comparisons were combined to evaluate the effects of fish oils on the incidence of VF in reperfusion-induced arrhythmias (Figure 10). The combined risk ratio of deaths was 0.44 (95% CI: 0.25–0.79). There was no statistically significant heterogeneity between studies.

In the meta-analysis combining ALA comparisons and EPA plus DHA comparisons, the overall random-effect risk ratio of VT in reperfusion-induced arrhythmias was 0.85 (95% CI: 0.65–1.1).

Ventricular fibrillation and ventricular fibrillation threshold in induced arrhythmia among monkeys. Table 3-11 shows results from 3 studies that compared monkeys fed fish oils to controls fed sunflower seed oil (omega-6 PUFA), and that examined the incidence of VF and ventricular fibrillation threshold (VFT) in induced arrhythmia. The dose of EPA plus DHA ranged from 1.8 to 2.8g/100g. The feeding duration ranged from 16 weeks to 30 months. Three different arrhythmia-induction protocols were used. In the first protocol, arrhythmias were induced by electrical stimulation in the control condition. In the second, arrhythmias were induced 5 minutes after an ischemia procedure, and in the third, arrhythmias were induced 30 minutes after restoration of coronary blood flow and during the infusion of isoproterenol. The three arrhythmia-induction protocols were not independent of each other, that is, the same monkeys underwent the series of experimental procedures in sequence. Thus, the cumulative effects of induced arrhythmias must be considered. (Note also be noted that the same group of investigators from one laboratory authored all three studies).

For each of the arrhythmia induction protocols, the investigators compared the proportion of monkeys from the fish oil group that experienced inducible VF to the proportion in the sunflower seed oil group that experienced VF. In the first protocol (electrical stimulation in control condition), the investigators found no difference between groups. In the second protocol (electrical stimulation five minutes after an ischemia procedure), 2 of the 3 studies found no difference in the proportion of monkeys that had inducible VF ^{24, 25}. One study ²⁶, however, reported that while no VF was inducible in the monkeys fed fish oil, VF was induced in 13% of the monkeys fed sunflower seed oil . In the third protocol (electrical stimulation during the infusion of isoproterenol), VF was induced in 30% to 50% of the monkeys fed fish oil compared to 77% to 100% of the monkeys fed sunflower seed oil.

Ventricular fibrillation thresholds (VFTs) were measured only among the VF inducible monkeys. In 2 studies^{24, 26}, VFTs remained unchanged in both groups in all conditions. However, one study ²⁵ found that VFTs were significantly increased among monkeys that were fed fish oil relative to those fed sunflower seed oil in all conditions (note that an increased threshold indicates a desirable outcome).

Summary Table 3-12. Ventricular Premature Beats/Complex, (more...)

Summary Table 3-12. Ventricular Premature Beats/Complex, Infarct Size, Arrhythmia Score and Length of Time in Normal Sinus Rhythm: Comparison of Rats Fed Omega-3 Fatty Acids With Controls Fed Omega-6 PUFA Oils

Author, Year	Omega-3 Arms	Dosage, g/100 g	Duration	Total N	Arrhythmia Outcomes1				Experimental Protocols
					VPB	AS2	IS	TSR3
ALA Oils
Abeywardena, 1995	Soybean	0.4	9 months	36	+176%	+107%*	-	-	5-min ischemia; 10-min reperfusion
McLennan, 1995	Canola	1.1	5 weeks	30	-13%	-11%	-	-	15-min ischemia
					-43%	-64%	-	-	10-min reperfusion
McLennan, 1995	Canola	1.1	5 weeks	20	-19%	-41%*	-	-	5-min ischemia; 10-min reperfusion
Isensee, 1994	Linseed	5.2	10 weeks	20	-	-	NS	NS	20-min ischemia
McLennan, 1995	Soybean	1.2	5 weeks	27	-14%	-18%	-	-	15-min ischemia
					-2%	-12%	-	-	10-min reperfusion
McLennan, 1995	Soybean	1.2	5 weeks	20	+34%	+30%	-	-	5-min ischemia; 10-min reperfusion
Fish Oils (EPA+DHA)
Anderson, 1996	MaxEPA	41% of TT FAs	8 weeks	14	-31%	-54%	-	-	20-min ischemia; reperfusion
Hock, 1990	Menhaden	1.0	4 weeks	17	-	-77%†	-	-	15-min ischemia; 6-hr reperfusion
Hock, 1987	Menhaden	1.0	4 weeks	23	NC	-	-	-	15-min after ischemia w/o reperfusion
Charnock, 1991	Fish oil	2.1	12 months	20	-72%*	-59%*	-	-	15-min ischemia
McLennan, 1993	Fish oil	2.6	12 weeks	27	-10%	-41%*	-	+12%	15-min ischemia
					-31%	-63%*	-	+2%	5-min reperfusion
McLennan, 1993	Fish oil	2.6	12 weeks	22	-27%	-48%	-	+16%	5-min ischemia; 5-min reperfusion
Isensee, 1994	Fish oil	3.0	10 weeks	20	-	-	NS	Increased*	20-min ischemia
Abeywardena, 1995	MaxEPA	3.3	9 months	36	-13%	-40%	-	-	5-min ischemia; 10-min reperfusion
McLennan, 1990	Tuna	3.7	18 months	14	+6%	NS	-	-5%	15-min ischemia
					-24%*	NS		+21%	10-min reperfusion
McLennan, 1988	Tuna	3.7	12 months	20	-	NS -44%*	+7%, NS	-	15-min ischemia reperfusion

TT FAs = total fatty acids; VPB = ventricular premature beats/complex; IS = infarct size/size of ischemia zone; AS = arrhythmia score (according to Curtis et. al.); TSR =length of time in normal sinus rhythm; ISO = Isoproterenol

-

Not reported NS = no significant difference compared to controls

*

P<0.05 compared to controls

†

P<0.01 compared to controls

1

See Methods for the effects expressed as percent change.

2

AS in all studies were calculated according to Curtis et al [Cardiovascular Research 22, 656–665], except Hock, 1990 used a modified method

3

An increase in TSR is a desirable outcomes for antiarrhythmic effects.

Effects on ventricular premature beats. As shown in Table 3-12, 7 studies with 16 comparisons evaluated the number of VPBs in ischemia-induced and/or reperfusion-induced arrhythmias. Rats were used in all 7 studies. No consistent results were found in the studies comparing rats fed ALA oils (soybean, linseed or canola oils) to rats fed omega-6 PUFA oils. However, studies comparing rats fed fish oils to rats fed omega-6 PUFA oils suggest that rats fed fish oils might have reduced numbers of VPBs in ischemia-induced and/or reperfusion-induced arrhythmias relative to rats fed omega-6 PUFA oils.

Effects on arrhythmia scores or severity of arrhythmias. As shown in Table 3-12, 8 studies with 18 comparisons evaluated the arrhythmia scores associated with the ischemia-induced and/or reperfusion-induced arrhythmias. Rats were used in all studies. More severe arrhythmias are associated with higher scores.

No consistent results were found in studies that compared rats fed ALA oils (soybean, linseed or canola oils) to rats fed omega-6 PUFA oils. However, when rats fed fish oils were compared to rats fed omega-6 PUFA oils, the studies found that most of the fish oil fed rats had less severe ischemia-induced and/or reperfusion-induced arrhythmias than the omega-6 PUFA fed rats.

Effects on infarct size. Infarct size, or size of the ischemic region, was evaluated in only 2 studies ^{16, 27}. The results showed no significant difference in the infarct size between rats fed omega-3 PUFA oils and rats fed omega-6 PUFA oils (Table 3-12).

Effects on length of time in sinus rhythm. As shown in Table 3-12, 3 studies with 7 comparisons evaluated length of time in sinus rhythm (TSR) in ischemia-induced and/or reperfusion-induced arrhythmias. Rats were used in all studies. One study ²⁷ that compared rats fed linseed oil (rich in ALA) to rats fed corn oil found no significant difference in TSR. In the same study, however, TSR was significantly increased in rats fed fish oil compared to rats fed corn oil. Two other studies compared rats fed fish oils to rats fed omega-6 PUFA oils. These studies found no significant difference in TSR in ischemia-induced and/or reperfusion-induced arrhythmias ^{19, 28}.

Studies Comparing Pre-fed Omega-3 Long-Chain PUFAS to a-Linolenic Acid

Summary Table 3-13. Arrhythmic Effects in Studies Comparing (more...)

Summary Table 3-13. Arrhythmic Effects in Studies Comparing Omega-3 Long-Chain PUFAs with a Linolenic Acid

Author, Year	Omega-3 Arms	Dosage, g/100 g	Duration	Animal	Sample Size	Arrhythmia Outcomes							Experimental Protocols
						Deaths	VT	VF	VPB/10 min	IS	AS	TSR¶, min
Abeywardena, 1995	Soybean	0.4	9 months	Rats	18	11%	76%	23%	298	-	3.1	-	5-min ischemia
	MaxEPA	3.3	9 months	Rats	18	0%	22%	5%	94	-	0.9	-	10-min reperfusion
Isensee, 1994	Linseed	5.2	10 weeks	Rats	10	-	60%	40%	-	35%*	-	5.5*†	20-min ischemia
							-	60%		-		-	20-min reperfusion
	Fish oil	3.0	10 weeks	Rats	10	-	0%	10%	-	36%*	-	10*†	20-min ischemia
							-	40%		-		-	20-min reperfusion

VT = ventricular tachycardia; VF = ventricular fibrillation; VPB = ventricular premature beats; IS = infarct size/size of ischemia zone; AS = arrhythmia score (according to Curtis et. al.); TSR = length of time in normal sinus rhythm; min = minute

-

Not reported

*

estimated value from figures

†

p<0.05 between groups

¶

An increase in TSR is a desirable outcomes for antiarrhythmic effects

Two studies directly compared omega-3 long-chain PUFAs (EPA and DHA) to ALA (Table 3-13). Both studies found a non-significant reduction in the incidence of VT and VF in ischemia-induced or reperfusion-induced arrhythmias in rats fed fish oils compared to those fed soybean or linseed oils (rich in ALA) ^{23, 27}. Abeywardena et al. found that no deaths occurred in rats fed fish oil, while 11% of rats fed soybean oil died from ischemia-reperfusion-induced arrhythmias ²³. The results also showed that rats fed fish oil had fewer numbers of VPBs and less severe arrhythmias as indicated by arrhythmia score than did rats fed soybean oil. However, none of these results were statistically significant. In addition to the incidence of VT and VF, Isensee et al. ²⁷ also examined the infarct size and the length of time in normal sinus rhythm. The results showed no difference in infarct size between rats fed a fish oil diet and those fed a linseed oil diet after 20 minutes of ischemia. The length of time in normal sinus rhythm was almost 50% longer in rats fed fish oil compared to rats fed linseed oil.

Indirect comparisons between omega-3 long chain PUFAs (EPA and DHA) and ALA based on meta-analysis are described in the Discussion (Chapter 4).

Studies Comparing Pre-fed Omega-3 PUFAS to Saturated Fatty Acids

As shown in Table 3-1, we analyzed 5 studies that compared omega-3 PUFAs to saturated fatty acids. In each study, experimental and control oils were added to the animals' basic diets in equal amounts (see Evidence Table 1). Therefore, all comparisons reflect iso-caloric intake from fat. Fish oils and sardine or mackerel oils were used as the source of omega-3 PUFAs in the experimental groups, while controls were fed coconut, lard, sheep peri-renal fat, or butter. The dosages of EPA plus DHA were 0.6g/100g ³⁰, 2.9g/100g ³¹, 5.5g/100g ³² and 5%kcal ^{33, 34}.

Summary Table 3-14. Total Deaths in Ischemia-Reperfusion-Induced (more...)

Summary Table 3-14. Total Deaths in Ischemia-Reperfusion-Induced Arrhythmias: Comparison of Animals Fed Fish Oil (EPA+DHA) With Controls Fed Saturated Fats

Author, year	Omega-3 Arms	Dosage, g/100 g	Duration	Omega-3 Fatty Acids		Control		Experiment Protocols
				Event	Total	Event	Total
Rabbits
Chen, 1994	Fish oil	5.2 %kcal	2 weeks	3*	12	5*	14	10-min ischemia; 1-hr reperfusion
Chen, 1994	Fish oil	5.2 %kcal	2 weeks	6 †	14	8 †	15	1-hr ischemia; 4-hr reperfusion
Piglets
Hartog, 1987	Mackerel	0.6	16 weeks	1	7	0	6	5-min ischemia; 10-min reperfusion

*

Two deaths in each group occurred during reperfusion

†

50% deaths occurred during ischemia; 50% occurred during reperfusion

Effects on incidence of arrhythmia deaths. As shown in Table 3-14, deaths in ischemia-reperfusion-induced arrhythmias were monitored in 2 studies. In 1 study ³³, rabbits fed fish oil corresponding to a dose of EPA plus DHA of 5.2g/100g were compared to controls fed coconut oil. The animals were fed for 12 weeks before arrhythmias were induced. In one arm of the study, animals were subjected to 10 minutes of ischemia followed by one hour of reperfusion. Three deaths (25%) were observed among the 12 rabbits fed fish oil, compared to three deaths (36%) among the 14 rabbits fed coconut oil. Two of the deaths in both groups occurred during reperfusion. In another arm of the study, rabbits were subjected to 1 hour of ischemia followed by 4 hours of reperfusion. Six deaths (43%) were observed among the 14 rabbits fed fish oil, compared to 8 deaths (53%) in the 15 rabbits fed coconut oil. About 50% of the deaths occurred during ischemia and 50% occurred during reperfusion in both groups in this arm.

In another study ³⁰, 13 piglets were fed either 9%w/w lard fat (n=6) or 4.5%w/w mackerel oil plus 4.5%w/w lard fat (n=7) for 16 weeks. The corresponding dose of EPA plus DHA in the group fed mackerel oil plus lard fat was 0.6g/100g. Defibrillation was unsuccessful in one piglet from the mackerel plus lard oil group. This piglet died of ventricular asystole during the fifth reperfusion.

Summary Table 3-15. Ventricular Tachycardia in Ischemia-Reperfusion-Induced (more...)

Summary Table 3-15. Ventricular Tachycardia in Ischemia-Reperfusion-Induced Arrhythmias: Comparison of Animals Fed Fish Oil With Controls Fed Saturated Fats

Author, year	Omega-3 Arms	Dosage, g/100 g	Duration	Omega-3 Fatty Acids		Control		Experiment Protocols
				Event	Total	Event	Total
Rats
Pepe, 1996	Fish oil	5.2	16 weeks	7 †	20	14 †	20	15-min ischemia; 10-min reperfusion
Piglets
Hartog, 1987	Mackerel	0.6	16 weeks	2*	7	1*	6	5-min ischemia; 10-min reperfusion

1

All events occurred during ischemia procedure

†

Some events occurred during ischemia; some occurred during reperfusion

Effects on incidence of ventricular tachycardia. As shown in Table 3-15, 2 studies examined the incidence of VT in ischemia-reperfusion-induced arrhythmias. One of these studies was in rats ³⁵, and 1 was in piglets ³⁰. In the rat study, 7 (35%) of the 20 rats fed fish oil developed VT, compared to 14 (70%) of the 20 rats fed sheep peri-renal fat (P<.05).

In the piglet study, the incidence of VT was 29% (n=7) and 17% (n=6) in piglets fed mackerel oil and lard fat, respectively. All VT events occurred during the ischemia procedure.

Summary Table 3-16. Ventricular Tachycardia in Ischemia-Reperfusion-Induced (more...)

Summary Table 3-16. Ventricular Tachycardia in Ischemia-Reperfusion-Induced Arrhythmias: Comparison of Animals Fed Fish Oil With Controls Fed Saturated Fats

Author, year	Omega-3 Arms	Dosage, g/100 g	Duration	Omega-3 Fatty Acids		Control		Experiment Protocols
				Event	Total	Event	Total
Rats
Pepe, 1996	Fish oil	5.2 %kcal	16 weeks	0	20	16	20	15-min ischemia; 10-min reperfusion
Yang, 1993	Fish oil	5.4 %kcal	5 days	3 *	8	7*	9	15-min ischemia; 10-min reperfusion
Piglets
Hartog, 1987	Mackerel	0.6	16 weeks	3 †	7	0	6	5-min ischemia; 10-min reperfusion

*

VT (%) or VF (%). All events occurred during reperfusion

†

Some events occurred during ischemia; some occurred during reperfusion

Effects on incidence of ventricular fibrillation. As shown in Table 3-16, 3 studies examined the incidence of VF in ischemia-reperfusion-induced arrhythmias. Two of these studies were in rats ^{34, 35} and 1 was in piglets ³⁰. Both rat studies found a significantly reduced incidence of VF in ischemia-reperfusion-induced arrhythmias among rats fed fish oil compared to rats fed saturated fats.

In the piglet study, the incidence of VF was 43% (n=7) in piglets fed mackerel oil, while no piglet fed lard fat developed VF in ischemia-reperfusion-induced arrhythmias. In the same study, programmed electrical stimulation was performed to induce VF in another 20 piglets, 10 in the mackerel-oil group and 10 in the control group. The incidence of VF was not reported, but VFTs were measured in control condition and during 15 minutes of ischemia. The threshold current for VF induction was reduced in all dietary groups during ischemia but remained significantly higher in the mackerel-oil-fed group than in the saturated-fat-fed group.

Summary Table 3-17. Ventricular Premature Beats in (more...)

Summary Table 3-17. Ventricular Premature Beats in Ischemia-Reperfusion-Induced Arrhythmias: Comparison of Animals Fed Fish Oil With Controls Fed Saturated Fats

Author, Year	Omega-3 Arms	Dosage, g/100 g	Duration	Animals	Total N	VPB 1	Experiment Protocols
Ischemia-Induced Arrhythimas
Chen, 19942	Fish oil	5.2 %kcal	2 weeks	Rabbits	22	-50%	10-min ischemia
						-35%	1-hr ischemia
Hartog, 1987	Mackerel	0.6	16 weeks	Piglets	13	+53%	5-min ischemia
Pepe, 1996	Fish oil	5.2 %kcal	5 days	Rats	40	-73%*	15-min ischemia
Reperfusion-Induced Arrhythimas
Chen, 19942	Fish oil	5.2 %kcal	2 weeks	Rabbits	22	0%	10-min ischemia; 1-hr reperfusion
						-25%	1-hr ischemia; 4-hr reperfusion
Hartog, 1987	Mackerel	0.6	16 weeks	Piglets	13	-65%*	15-min ischemia; 10-min reperfusion

VPB = ventricular premature beat

*

P<0.05

1

See Chapter 2: Methods for the effects expressed as percent change

2

Study results were biased by excluding more subjects who died from

Effects on ventricular premature beats. As shown in Table 3-17, 3 studies examined the number of VPBs in schemia-reperfusion-induced arrhythmias. One of these studies was in rabbits ³³, 1 was in piglets ³⁰, and 1 was in rats ³⁵. In the rat study, the number of VPBs during ischemia was significantly reduced among rats fed fish oil compared to rats fed sheep-perirenal fat. In the piglet study, the incidence of VPBs during ischemia did not differ between the groups. However, during reperfusion the piglets fed mackerel oil had significantly fewer VPBs compared to those fed lard fat. In the rabbit study, there were no significant differences in the incidence of VPBs between rabbits fed fish oil and those fed coconut oil during ischemia or reperfusion. However, rabbits that died from arrhythmias were excluded from the analyses, and more rabbits died in the control group than in the experimental group. Thus, the true effects were underestimated.

Effects on arrhythmia scores or severity of arrhythmias. None of the studies that compared the arrhythmic effects of omega-3 PUFAs and saturated fatty acids reported arrhythmia scores as an outcome.

Effects on infarct size. None of the studies that compared the arrhythmic effects of omega-3 PUFAs and saturated fatty acids reported infarct size as an outcome.

Effects on length of time in sinus rhythm. None of the studies that compared the arrhythmic effects of omega-3 PUFAs and saturated fatty acids reported length of time in sinus rhythm as an outcome.

Studies Comparing Pre-fed Omega-3 PUFAS to No Treatment Controls

A total of 4 studies were included in this analysis (Table 3-1). As shown in Evidence Table 1, omega-3 PUFA oils were added to the diet of animals in the experimental groups, while controls were maintained on basic diets. As a result, energy intake from fat was higher in the experimental groups than in the control groups. Dogs were used in all studies. MaxEPA, menhaden oil, or EPA esters were used as the source of omega-3 PUFAs in the experimental groups, while controls were fed standard dog chows (Oriental Yeast Co. or Friskies^® Dinner). The dose of EPA plus DHA was 3.3%kcal ³⁶ and 1.0g/100g ³⁷ in the two fish-oil studies. The dose of EPA was 1.0g/100g in both of the EPA-ester studies ^{38, 39}.

Summary Table 3-18. Total Deaths in Induced Arrhythmias: (more...)

Summary Table 3-18. Total Deaths in Induced Arrhythmias: Comparison of Dogs Fed EPA and/or DHA With No Treatment Controls

Author, Year	Omega-3 Arms	Dosage, g/100 g	Duration	Omega-3 Fatty Acids		Control		Experiment Protocols
				Event	Total	Event	Total
Culp, 1980	Menhaden	3.3 %kcal	5~7 weeks	3	10	5	17	Coronary artery thrombosis induced by electrical stimulation
Otsuji, 1993	EPA ester	1.0	8 weeks	0	10	5	15	Coronary artery ligation (or ischemia)

Effects on incidence of arrhythmia deaths. Two studies ^{36, 39} that evaluated the incidence of arrhythmia deaths (Table 3-18) compared dogs fed EPA and/or DHA to no treatment controls. In one study, no significant difference was found in the incidence of sudden death after induced-coronary thrombosis. ³⁶ The other study found no deaths due to VF in the 10 dogs fed 1.0g/100g EPA ester, although five VF deaths (33%) occurred in the 15 untreated control dogs (P<.05). ³⁹.

Effects on incidence of ventricular tachycardia and/or ventricular fibrillation. The incidence of VT and/or VF in induced arrhythmias was evaluated in a study of 30 dogs. ³⁸. Fifteen dogs were fed standard dog chow plus 1.0g/100g EPA ester for 8 weeks. Fifteen untreated control dogs were fed standard dog chow for 8 weeks. An ischemia-induced arrhythmia model was used in 10 experimental dogs and 10 controls. A digitalis-induced arrhythmia model was used in 5 dogs from each group. A fatal dose of digoxin (0.025 mg/kg/min) was administrated intravenously over a 60-second period immediately after coronary artery ligation.

There was no difference in the incidence of VF in ischemia between the groups. Two dogs in each group (20% vs. 20%) developed VF within 3 hours after coronary ligation. All 10 dogs that underwent digitalis-induced arrhythmias developed VT or VF. However, the VT or VF did not occur until at least 25 minutes after the administration of digoxin in the dogs fed EPA ester, while the events occurred about 10 to 15 minutes within administration of digoxin in the untreated control dogs.

Summary Table 3-19. Ventricular Premature Beats/Complex, (more...)

Summary Table 3-19. Ventricular Premature Beats/Complex, Infarct Size, Arrhythmia Score and Areas at Risk of Arrhythmias: Comparison of Dogs Fed EPA and/or DHA With No Treatment Controls

Author, Year	Omega-3 Arms	Dosage, g/100 g	Duration	Total N	Arrhythima Outcomes 1				Experimental Protocols
					VPB	AS 3	IS	ARAr
Kinoshita, 1994	EPA ester	1.0	8 weeks	20	-44%*	-55% †	-	-	3-hr ischemia
Culp, 1980	Menhaden	3.3 %kcal	5~7 weeks	27	Decreased	-	-52%	-	Electrical stimulation
Otsuji, 19932	EPA ester	1.0	8 weeks	20	-	-	-40%†	NS	Ischemia
Oskarsson, 1993	MaxEPA	1.0	6 weeks	22	-	-	-55%*	NS	90-min ischemia; 30-min reperfusion

VPB = ventricular premature beats/complex; IS = infarct size/size of ischemia zone; AS = arrhythima score (according to Curtis et al, 1987); TSR =length of time in normal sinus rhythm; ARAr = areas at risk of arrhythmias; ISH = ischemia

-

Not reported NS = no significant difference compared to controls

*

P<0.05 compared to controls

†

p<0.01 compared to controls

1

See Chapter 2: Methods for the effects expressed as percent change

2

Study results were biased by excluding more subjects who died from arrhythmias in the control group

Effects on ventricular premature beats. As shown in Table 3-19, 2 studies ^{36, 38} examined the number of VPBs in induced arrhythmias. Both studies found that dogs fed EPA and/or DHA had fewer VPBs compared with untreated controls.

Effects on arrhythmia scores or severity of arrhythmias. One study ³⁸ evaluated the arrhythmia score in ischemia-induced arrhythmias (Table 3-19), and found that the arrhythmia score obtained within 3 hours after coronary ligation was significantly reduced by EPA-supplementation. Dogs fed EPA esters for 8 weeks had significantly less severe ischemia-induced arrhythmias than the no treatment controls (P<.01).

Effects on infarct size. Infarct size, or size of the ischemic region, was evaluated in 3 studies (Table 3-19) ^{36, 37, 39}. All 3 studies showed that dogs fed EPA and/or DHA had a decrease in the infarct size in either electrical-stimulation-induced or ischemia-reperfusion-induced arrhythmias compared to untreated controls. However, areas at risk of arrhythmias were not significantly different between groups.

Effects on length of time in sinus rhythm. None of the studies that compared the arrhythmic effects of omega-3 PUFAs vs. untreated controls reported length of time in sinus rhythm as an outcome.

Whole-Animal Isolated Organ and Cell Studies

Summary Table 3-24. Effects of Omega-3 Fatty Acids (more...)

Summary Table 3-24. Effects of Omega-3 Fatty Acids on Ion Currents In Whole Animal Isolated Organ and Cell Studies

Author, year	Animal Model [Type, Age, Sex]	Exposure Duration (weeks)	Comparison Groupa		Amount of Omega-3	Expt. Condition	Agent	INa	Ito	ICaL	IK	IKI	IKUR
			Omega-3 Fatty Acid (n)	Control (n)
RAT
Minarovic, 1998	ventricular myocyte, Young adult, male	2	FO (ND)	HF (ND)	100g/Kg/d	Ambient	None			NC Ac
										NC InAc
										NC A
Leifert, 2000	ventricular myocyte, Young adult, male	3	FO (17–28)	LARD (17–28)	29% Energy	Ambient	None	NC Ac	NC Ac
								NC InAc	NC InAc

INa=initial fast current; Ito=transient K + outward current or initial outward current; Ica.L=voltage dependent L-type Calcium current/inward current/calcium sparks; I K=delayed rectifier K + current; Iki=inward rectifier K + current; Ikur=ultra rapid K + current; Ac-activation parameter; In Ac = inactivation parameter; D = decrease; I = increase; NC = no change; ND=no data;

*

= p<0.05

**

= p<0.01;

***

= p<0.001

A =amplitude

Ac =activation parameter

FO = fish oil

InAc =inactivation parameter

HF =high fat

NC =no change

Table 2

Evidence Table. Whole-Animal Isolated Organ and Cell (more...)

Table 2

Evidence Table. Whole-Animal Isolated Organ and Cell Studies Part 1

Author, yr	Country Funding	Species Stage Sex	Exp-osure Duration (weeks)	Group [Sample Size]	Total Fat (omega-3 fatty acids)	Unit	SFA	MUFA	PUFA	ALA	EPA	DHA	Other omega-3 fatty acids
Benediktsdottir, 1988	Iceland U	Rats	16	Corn oil (CO) [ND]	10 %w	% total fatty acids	14.5	24.5	57.8	ND	0.0	0.0	0.03
		Adult
		Male		Cod-liver oil (CLO) [ND]	10 %w		22.0	47.0	27.2	ND	6.9	7.2	0.93
Black, 1989	Canada G	Rats	4	STD [6]		ND	ND	ND	ND	ND	ND	ND
		Adult
		Male		STD+FO [6]	0.5ml/kg/day	ND	ND	ND	ND	ND	ND	ND
Chemla, 1995	France G	Rats	4	N-3 [15]	15%w	%TFA	20.0	57.7	11.4	0.8	4.3	4.1
		Adult
		Male		N-6 [15]	15%w		19.8	58.9	20.7	0.5	0.0	0.0
Chen, 1994	Taiwan G	Rabbits	2	High cholesterol (HC) [11–15]	40 %kcal	% w/w	ND	ND	ND	ND	ND	ND
		Adult			40 %kcal (10 %kcal from fish oil)
		Male		HC+FO [11–12]			ND	ND	ND	ND	30.2	21.5
Croset, 1989a	USA G1	Mouse	2	STD [10]	0 %w	Mol%	11.8	31.7	56.1	0.0	ND	0.0
		Weanling		STD+0.4 %w/w DHAe[10]	10 %w/w		11.7	28.8	59.1	0.0	ND	0.1
		Male		STD+0.8 %w/w DHAe[10]	10%w/w		11.5	25.7	61.6	0.0	ND	0.2
				STD+ 4%w/w DHAe [10]	10 %w /w		5.3	9.3	85.3	0.0	ND	0.8
Croset, 1989b	USA	Mouse	2	OO+ALA e [6]	1.5+0.5%w	Mol%	27.6	44.5	27.9	20.5	0.2	0.1
		Weanling		OO+EPA e [6]	1.5+0.5%w		30.3	49.8	19.9	1.0	8.1	1.9
		Male		OO+DHA e [6]	1.5+0.5%w		27.5	47.3	25.1	0.5	0.9	16.5
Demaison, 1993	France G	Rats	8	SF [32]	100g/Kg	% TFA	11.8	16.2	71.7	0.2	ND	ND
		Weanling		LIN [29]	100g/kg		8.7	20.3	71.0	53.5	ND	ND
		Male
Gillis, 1992	Canada G	Rabbits	6	SAF (9)	10%w	%w	9.6	13.1	77.3	0.0	0.0	0.0	0.03
		Weanling		FO (9)	10%w		23.5	29.2	47.3	1.4	26.5	8.6	2.33
		ND
Gudmundsdottir, 1991	Iceland U	Rats	20	CO [5]	10%w	%w	13.6	24.6	58.6	2.6	0.0	0.0
		Adult		CLO (4)	10%w		18.1	51.0	27.7	0.0	7.1	8.1
		Male
		Rats	88	CO [5]	10%w	%w	13.6	24.6	58.6	2.6	0.0	0.0
		Aged		CLO (4)	10%w		18.1	51.0	27.7	0.0	7.1	8.1
		Male
Heard, 1992	USA U	Rats	4	SAF [18]	20%w	ND	ND	ND	ND	ND	ND	ND
		Adult		MenO+SAF [18]	19.5%+0.5%w		ND	ND	ND	ND	ND	ND
		Male
Honen, 2002	Australia G	Rats	3	Canola oil (6)	3ml/d	% TFA	6.2	60.0	33.8	12.1	0	0
		Adult		FO(6) G	3ml/d		1.7	15.8	77.7	0.5	48.0	26.2
		Male
Karmazyn, 1987	Canada G	Rats	12	STD [14]	10%w	ND	ND	ND	ND	ND	ND	ND
		Weanling		STD+Cod liver oil (CLO) [14]			ND	ND	ND	ND	ND	ND
		Male/ Female
Kinoshita, 1994	Japan U	Dogs	8	STD (15)	100mg/kg	mg/k	ND	ND	ND	ND	ND	ND
		Adult		STD+EPAe (15)		g/d	ND	ND	ND	ND	100	ND
		ND
Ku, 1997	Japan G	Rats	12	HC (5)	5.1%w	ND	ND	ND	ND	ND	ND	ND
		Aged		HC+EPA (5)	5.1%w (300mg/kg)		ND	ND	ND	ND	ND	ND
		Female		HC+DHA (5)	5.1%w (300mg/kg)		ND	ND	ND	ND	ND	ND
Lamers, 1988	Neth.; Italy G	Pigs	8	LARD [8]	9%w	%TFA	36	46	15	1	0	0
		Weanling
		Male/ Female		FO +LARD [8]	4.5% +4.5%w		32	40	11	1	8	5
Laustiola, 1986	Finland U	Rats	16	STD [20]		% TFA	26.2	23.6	49.7	5.3	1.3	2.7	0.23
		Weanling			10%na								2.74
		Male		STD+CLO [33]			22.8	46.5	28.6	1.7	6.4	8.2	0.73
													8.24
Leifert, 2000a	Australia G	Rats	3	LARD [6–8]	29% E (74kJ fat/d)	% w	58.0	39.4	2.6	0.7	0.1	0.0	0.03
		Young		FO [6–8] G5	29% E (74kJ fat/d)		27.3	28.2	44.6	1.1	24.3	12.1	2.33
		Adult
		Male
Leifert, 2001	Australia I+NP	Rats	3	SF (6)	17%w (10%w)	%w	36.4	55.1	8.5	1.2	0	0	0.03
		Adult		FO (6)	17%w (10%w)		18.6	44.0	37.4	0.9	17.8	8.9	1.73
		Male
Maixent, 1999	France G+NPl	Rats	8	STD [11]	0.5g of oil/kg	mg/g of oil	ND	ND	ND	ND	ND	ND
		Adult		STD+FO [10]			ND	ND	ND	ND	180	120
		Male
Minaro-vic, 1997	Slovak G	Rats	2	HF [10]	300g/kg	% w	47.0	39.7	13.3	ND	ND	ND
		Young		FO [10]	100g/kg		13.0	29.4	57.6	ND	ND	ND
		Adult
		Male
Pepe, 1999	USA U	Rats	6	N-6 (6)	15.6% w (11.7%w)	ND	ND	ND	ND	ND	ND	ND	ND
		Young		FO (5)	15.6%w (11.7%w)		ND	ND	ND	ND	ND	ND	ND
		Adult
		Male
Reig, 1993	Spain U	Rats	5	HF (20)	37%w	%TFA	36.7	40.0	19.4	2.2	0.0	0.0	0.03
		Young		HF+FO (20)	31%+6%w		30.0	33.0	37.1	3.4	4.6	3.4	1.03
		Adult
		Male
Swan-son, 1989	USA G	Mouse	2	SAF+CO (9)	12%w (2%+10%w)	%w	14.5	24.2	60.9	1.0	0.0	0.0	0.03
		Weanling		SAF+MenO (9)	12%w (2%+10%w)		28.5	26.1	44.7	1.8	12.9	9.1	2.03
		Male
Taffet, 1993	USA G	Rats	3	CO [11]	20%w	Mol %	14.3	26.3	59.3	0.0	0.0	ND
		Young		CO+MenO [12]	3%+17%w		39.9	28.3	31.9	1.3	16.5	ND
		Adult
		Female