In the cardiovascular system

John W.Karanian and Norman Salem

INTRODUCTION

The role of fatty acids in cardiovascular disease

Much evidence has accumulated from various lines of inquiry that indicates that the dietary fatty acid intake and lipid acyl composition of tissues is a determinant of many of the chronic diseases prominent in the Western World, notably cardiovascular disease. It has been appreciated since the 1950s that when dietary polyunsaturated fat intake is increased, a decrease in the total serum and lipoprotein cholesterol levels results [1]. Bang and Dyerberg, in their study of the Greenland Eskimos, a group in which CVD had an extremely low incidence, made the ground breaking inference that the high dietary intake of the long chain polyunsaturated fatty acids, eicosapentanoate and docosahexaenoate was responsible for this protection from disease [2], More recently, several epidemiological studies have reported a relationship between dietary n-3 polyunsaturates and the risk of CVD [3-8], For example, Dolechek et al. found an inverse relationship between alpha-linolenate and mortality from CVD, all CVD and on all cause mortality, however no relationships were found for linoleate [3], They also observed an inverse relationship of fish oil fats with coronary heart disease, CVD, and all cause mortality. In agreement with these findings was the study by Siscovick et al. who observed that a level of fish fats equivalent to about one meal a week was associated with a 50% reduction in the risk of primary cardiac arrest [4], The n-3 fatty acids have also been the subject oflarge secondary prevention trials. In the LYON Heart Study, deLorgeril et al. found that a diet enriched with alpha-linoleate was more effective than other diets in use for this purpose for the secondary prevention of coronary events and death [9], Burr et al. in the DART trial, found a 29% reduction in overall mortality after 2 years when a large group of men advised to consume fish twice a week after surviving a heart attack [10], Singh et al. reported a decline in coronary heart disease events in patients with suspected myocardial infarctions after one year of consuming either 2 g oflong chain n-3 fatty acids or2.9g of alpha-linolenic acid per day [11], A very large trial (GISSI-Prevenzionne) of 11,324 patients demonstrated that 850 mg of eicosapentaenoate/docosahexanoate per day led to a 20% reduction in total mortality over a3.5 year follow-up period in patients with a history of CVD [12], McLennan et al. found that fish oil fed marmoset monkeys were resistant to cardiac arrhythmias [13], Billman et al. subsequently showed that dogs infused with a fish oil emulsion had a remarkable resistance to cardiac arrhythmias induced by compression of the left circumflex artery [14], Kang and Leaf, in a series of publications (for review, see [15]), demonstrated that DHA had the highest efficacy in heart and neuronal cells in causing a reduction in electrical excitability; this is the basis for the protective effects of long chain polyunsaturates against arrhythmias. Lands et al. proposed the unifying concept that dietary supplementation with n-3 fatty acids leads to loss of cellular arachidonate and increases in eicosapentanoate and docosahexaenoate [16], This then leads to a diminished eicosanoid response upon cellular stimulation due to the decreased efficacy of the n-3 eicosanoid analogues to the arachidonate-derived eicosanoids. Lands et al. suggested that the percentage of plasma phospholipid arachidonate is related to thromboxane generation and platelet activation and thus to the risk of cardiovascular deaths [16], Harris, in his review, suggested that increased long chain n-3 fatty acid intake leads to decreases in plasma triglyceride levels due to decreased hepatic synthesis and secretion [17], A recent meta-analysis ofl7 studies involving over 46,000 men and 10,000 women suggested that the plasma triglycerides level is a risk factor for CVD that is independent of HDL-cholesterol [18], In addition to the aforementioned studies, Knapp et al. found an anti-hypertensive effect of fish oil [19] and Christensen et al. a beneficial effect on heart rate variability [20], It should be clear then that the balance of dietary fatty acids and the resulting tissue fatty acid composition are critical determinants of cardiovascular function and the predisposition to disease.

Fatty acid nomenclature

Essential fatty acids (EFA) are often defined metabolically; that is, they are the fatty acids that cannot be produced by de novo synthesis by a mammal. Both linoleic acid (LA) and alpha-linolenic acid (LNA) are the key essential fatty acids for mammals and must be supplied preformed in the diet. Plants have the enzymes necessary for total synthesis of these fats and provide for their origin in the food chain [21 ].

Palmitic acid (16:0) Oleic acid (18:1n9)

Linoleic acid {1B:2nG} Arachidonlc acid (20;4nG)

Linplenic aCid [1fl:3n3J Eicos&petaenoic acid (2Q;&ri3)

DocosahexaeriCMC acid (£2:6n3J

Figure 16.1 The structures of some essential and non-essential fatty acids commonly found in mammalian cells.

The structures of some of the most common fatty acids in biological organisms are presented in Figure 16.1. Fatty acids are characterized by their chain length, number of double bonds and the positions of their double bonds. A saturated fatty acid, like palmitate, contains no double bonds. An unsaturate like oleic acid, contains at least one double bond. A polyunsaturated fatty acid (PUFA), like linoleic acid contains two or more double bonds. Long chain polyunsaturates (LCP) are those fatty acids that are 20-carbons in length or more and contain multiple double bonds. Fatty acids are often abbreviated in the short-hand designation X:YnZ, where X is the number of carbons, Y is the number of double bonds, and Z represents the number of carbons counting from the methyl end of the molecule until the first double bond is encountered. Thus, linoleic acid is denoted as 18:2n6 and alpha-linolenic acid as 18:3n3. The principal LCPs in the n-6 and n-3 families are arachidonic acid (20:4n6, AA), and eicosapentaenoic acid (20:5n3, EPA) or docosahexaenoic acid (22:6n3, DHA), respectively (Figure 16.1). The fatty acids with the n-3 or n-6 structures are referred to as families because they are not metabolically inter-convertible in animals [21],

FATTY ACID COMPOSITION

Heart and smooth muscle

Significant effects of alcohol have been noted when 20-C and 22-C fatty acid distribution/ content has been determined in heart [22], vascular smooth muscle [23] and platelet [24,25], Reitz et al. [22] reported that both AA and DHA declined significantly in rat heart following alcohol in the drinking water for 1 month. Mice exposed to ethanol vapors forlOd showed an increase in 18:2n6 (LA) and a decrease in DHA [26], Acute inhalation was associated with a significant loss of heart DHA that was preventable by pretreatment with vitamin E. Cunnane et al. [27] have shown a loss in AA and DHA in hearts ofhamsters given alcohol in their drinking water for one year.

Blood components

Reductions in AA have also been observed in rodent red blood cells [28-31], platelets [25,32] and serum [33] after alcohol administration. However, these results have also been inconsistent, since others have reported no effect [34] or an increase in AA [35], Moreover, no effect of short-term alcohol administration on AA levels has been found in mouse red blood cells [35], Interestingly, these effects of alcohol may be dependent on the duration of exposure, since longer-term alcohol administration (i.e., >21 days) has been shown in rat red blood cells [36] to result in reductions in AA. Consistent with this idea, the concentration of DHA was reduced in mouse blood following shorter-term (i.e., 7 day) alcohol administration [37],

Horrobin and Manku [38] have observed losses in both the plasma and red blood cell (RBC) PE level of AA and DHA in alcoholics. Similarly, subjects with alcoholic liver disease show losses in RBC LA, AA and DHA [39], Glen et al. [40] reported a marked loss in all major RBC polyunsaturates with the exception of AA in a group of 123 alcoholics. Others have also noted significant declines in RBC AA and DHA in alcoholics [41,42], In addition, the plasma of cirrhotic patients, of which half had an alcohol-related etiology, shows a decline in AA and DHA [43], Withdrawing alcoholics also show less AA in certain platelet phospholipid pools such as phosphatidylcholine and phosphatidylinositol [24],

The diet as a source of fatty acids

It is well known that many alcoholics have a relatively poor diet (for general reviews, see [44-48]). For example, in a population of middle-aged Scottish men who consumed a mean alcohol intake of 66 g/d, it was shown that they had a lower intake of protein, fat, polyunsaturated fat and linoleic acid [49], When the alcohol intake increases, there is a decrease in the amount of energy derived from macronutrients [50], There is also a decrease in the intake and tissue concentration of many vitamins and minerals [51]. Alcoholism may also lead to nutritional deficiencies through decreased intestinal absorption, altered vitamin metabolism and reduced storage of ingested vitamins [52-54],

It may be hypothesized then, that a reduction in highly unsaturated PUFAs may also be found in the diets of alcoholics. As noted below, alcohol has direct effects on fatty acid anabolism and catabolism, and these may lead to a decrease in tissue PUFA levels. It may be surmised then that a reduction in dietary 18-carbon EFAs and their longer chain metabolites, as well as the antioxidant vitamins and minerals that help to protect them, will exacerbate the nutrient deficiencies caused by the direct actions of alcohol.

FATTY ACID METABOLISM

Fatty acid elongation/desaturation

Fatty acids can be interconverted through enzymatic reactions occurring primarily in the endoplasmaic reticulum that lead to an extension of the chain length, termed elongation, or introduction of double bonds, termed desaturation. Desaturation and elongation are often a concerted sequence of reactions that leads to LA or LNA being metabolized to their LCP forms, e.g., AA and DHA, respectively. The general pathway for the elongation/desaturation of the n-3 and n-6 families of essential fatty acids is presented in Figure 16.2. It can readily be observed that LA is metabolized by desaturation to gamma-linolenic acid (18:3n6, GLA), elongated to dihommo-gamma-linolenic acid (20:3n6, DGLA) and then desaturated to AA, where metabolism often is terminated. However, metabolism may continue through elongation to docosatetraenoic acid (22:4n6, ETA), elongation to 24:4n6, desaturation to 24:5n6 and peroxisomal retroconversion to 22:5n6 (DPAn-6, [55]). Similarly, in the n-3 family, LNA is desaturated to 18:4n3, elongated to (blank weeded) 20:4n3, desaturated to 20:5n3 (EPA) and elongated to 22:5n3 (DPAn-3). The final desaturation reaction occurs in a manner analogous to that described above for the n-6 family as follows: 22:5n3 is elongated to 24:5n3, desaturated to 24:6n3 and then retroconverted to 22:6n3 (DHA).

Fatty acid metabolism in heart, blood cells and muscle

Although the primary site of essential fatty acid elongation/desaturation is in the liver and brain [56-58], organs and cells of the cardiovascular system may also participate in fatty acid metabolism. Heart cells in culture are known to possess the capability of performing elongation

ESSENTIAL FATTY ACID METABOLISM

M-3 FAMILY

FAMILY

{LNA. ftlpha-lirvjlenic acid) 10:3^3

1Bc4n3 20:4*3

(EPA. elcosapemaenoic acid) 20 ;5 r»3 ^ DPAn-3. decasapcrilacnosc acid) 22 ;5 r»3

24;5n3 24:6n3

(DHA, docosahExaenoic acid) 22:6rii3

(A 6-dcsahjfasi) (dongasc)

13:2n6 (LA. linolalc acid) 1 (GLA, gamma-linclEnic acid)

20:3n6 (DGLA, dihomnio-gamma-linalenicacid)

{A G-dE5aLura.&sj

(Elonyase)

(elongate)

2Û;4rt6 (AA. arachidonic acid)

24;4n6

{A fi-dEsaburate)

24;5n6

(relraconvErsion)

22 ;5 nd (DRfcn -6, docKapentaenoic acid)

Figure 16.2 Metabolism of the n-3 and n-6 families of essential fatty acids.

and desaturation reactions [59] and also have a high level of mRNA for delta-5 desaturase [58], Skeletal muscle and lung also contain mRNA for both delta-6 and delta-5 desaturase enzymes [57,58], Erythrocytes and plasma are probably devoid of this activity although whole blood has been shown to incorporate radioactivity from 14C-acetate into AA and DHA [60], Leukocytes and platelets are able to incorporate radioactivity from acetate into complex lipids and fatty acids [61,62], but this activity represents primarily chain elongation and not desaturation reactions [63], However, there is a report of apparent delta-6 and delta-5 desaturase activity in platelets [64], Although the cardiovascular system is not known to be a major site for EFA metabolism, the level of activity and mRNA for the key desaturases in the heart in particular indicates that it participates in fatty acid anabolism.

Effects of alcohol on essential fatty acid metabolism

As established in a section above, alcohol lowers the levels ofLCPs in many tissues. It has long been claimed that the mechanism underlying this change in fatty acid composition is the inhibitory action of alcohol on fatty acid desaturases [66], This interpretation was based on a series of in vitro experiments in which it was shown that the addition of ethanol to a tissue homogenate or subcellular fraction led to a decrease in a radioactive EFA substrate conversion to its more unsaturated form [51,66,67], For example, Nervi et al. demonstrated a decrease in both delta-6 and delta-5 desaturases in rat liver microsomes [68], Wang and Reitz found a reduction in the delta-9, delta-6 and delta-5 desaturases in liver microsomes after animals were given either acute or chronic exposure to ethanol [69], Nakamura et al. found a marked loss of delta-6 and delta-5 desaturase activity but no change in delta-9 desaturase activity in the minipig [70],

More recently, Pawlosky et al. were able to develop methods for the in vivo determination of overall fatty acid metabolism using the deuterated precursors deuterated-LA and deuterated-LNA [71], This method does not measure single enzymatic steps but rather is the combination of elongation/desaturation rates coupled with the rates of transport and minus the catabolic processes at each stage; this is what is meant by overall accretion oflabeled metabolites. It should be recognized though that overall accretion ofLCPs is more closely related to the LCP composition of the tissue than is the measurement of desaturase activity in a liver homogenate or subcellular fraction in vitro. Pawlosky et al. found a stimulation of deuterated 18-carbon EFA incorporation into plasma AA and DHA in both cats [73] and rhesus monkeys [74] after chronic alcohol exposure. This is not consistent with the view that alcohol inhibits desaturases in vivo, in fact, a simpler explanation of the data may be that alcohol stimulates desaturases. Pawlosky et al. proposed the hypothesis that alcohol, through a peroxidative challenge, caused a marked increase in EFA catabolism [72-74]. This was evidenced, for example, in the rhesus work by a large increase in plasma hydroxy-nonenal and an increase in isoprostanes [74], The stimulation of EFA metabolism may thus be seen as an adaptive mechanism that attempts to maintain homeostasis in LCP composition during accelerated EFA catabolism. Clearly then, the rate of catabolism is dependent upon the degree of oxidative challenge from alcohol and this, in turn, is related to the dose, duration and frequency of alcohol consumption. In the rhesus studies ofPawlosky et al., the loss of organ essential fatty acids coupled with a diet low in EFAs led to the development ofliver fibrosis after 3 years of alcohol consumption [74], This is significant, since higher levels of alcohol consumption in non-human primates have not led to liver fibrosis in several studies (for review, see [75]).

In support of the view that alcohol stimulates EFA metabolism are our recent metabolic studies of alcoholics (Salem N., unpublished). Alcoholics showed marked increases in the amount and enrichment of deuterium in plasma DHA when given an oral dose of deuterated-LNA. This study was performed on alcoholics during alcohol withdrawal and so is not directly comparable to studies where alcohol is present in the circulation. Nevertheless, the expected decline in deuterium enrichment of DHA was not observed, providing no support for the 'alcohol inhibits desaturase' hypothesis. Taken together with the two large animal studies reported above, it is likely that an intense alcohol challenge, as is the case for alcohol abusers, leads to stimulation of both EFA anabolism and catabolism.

Effects of alcohol on essential fatty acid catabolism

In support of the hypothesis that alcohol stimulates lipid degradation/peroxidation is a growing literature. Lands et al. have recently reviewed the literature concerning the alcohol-induced decline in various vitamins and antioxidants [51]. Alcohol may interact with Cytochrome P450 2E1 and produce hydroxy radicals that can react with proteins, lipids and nucleic acids [76,77], Polyunsaturated fatty acids are susceptible to this attack and undergo reactions with molecular oxygen to generate hydroperoxy compounds as well as a variety of aldehydic compounds such as malonyldialdehyde (MDA) and the hydroxyalkenals, 4-hydroxynonenal (HNE) and 4-hydroxy-hexenal (HHE).

There have been several demonstrations that the in vivo level of aldehyde increases due to alcohol exposure [78-81], For example, the plasma concentrations of HNE and HHE were increased, and markedly so in the case ofHNE, in rhesus monkeys consuming alcohol [82], Domestic cats also show a four-fold increase in brain HNE after six months of daily alcohol exposure [72], In fetal liver mitochondria, HNE accumulates during alcohol exposure of the mother [83], MDA increases in the bloodstream of humans following alcohol consumption

[82], Aldehydes generated subsequent to alcohol consumption react in a covalent manner with proteins [8486], Aldehydes like HNE may interact synergistically with MDA in that the covalent reaction ofHNE with BSA increases several-fold in the presence ofMDA [87], Also, lipid peroxidation and aldehyde formation is associated with a decrease in levels of antioxidants. For example, the production ofMDA was linearly correlated with the alcohol-induced decrease in liver alpha-tocopherol and glutathione [88], Another useful marker for in vivo lipid peroxidation is the generation of isoprostanes. These are non-enzymatically produced from PUFAs [89], typically from AA, as opposed to the eicosanoids that are enzymatically produced by enzymes like cyclooxygenase and lipoxygenase. For example, the isoprostane, 8-epi-F2-alpha has been used as an index of free radical mediated injury [90] and in vivo lipid peroxidation [91], Isoprostane-like compounds have also been observed for DHA both in vitro [92] and in vivo in Alzheimer's brain [93], The F2-isoprostanes have also been observed in Alzheimer's brain [94], Alcohol consumption increases isoprostane concentration in rat and primate liver [95,96] and in rat plasma [97], Pawlosky et al. correlated the level of 8-isoprostane F2-alpha with the amount of alcohol that individual rhesus monkeys consumed when allowed to drink on an ad libitum basis [74], The alcohol consumption negatively correlated with plasma AA content indicating that AA catabolism was appreciable. Recently, several groups have reported increased isoprostanes in alcoholics [98-100],

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