In the past years, clinical, epidemiology and molecular studies have reported close relation between essential fatty acids and health. Essential fatty acids are vital as supplement to our health as they cannot be synthesized by human body and are required for some important biological processes. So, they need to be included in our diet.
These essential fatty acids are mainly long chain polyunsaturated fatty acid (PUFA), which is high number of double bonds chain with multiple unsaturated sites. The first double bond from the methyl terminal is a unique functional group that shows distinct important biological properties of PUFA and distinguishes PUFA from the other fatty acids. PUFA may be divided into two groups, which are alpha-linolenic acid, anÂ n-3 PUFA (omega-3) andÂ linoleic acid, an n-6 PUFA (omega-6) (Deckelbaum et al., 2006). The term omega is refer to the terminal carbon from the functional carboxylic acid group (-COOH). The structure of n-3 PUFAs and n-6 PUFAs is illustrated in figure 1. The first double bond of n-3 PUFAs is located at the third carbon molecule (C3) whereas the first double bond of n-6 PUFAs is located at the sixth carbon molecule (C6).
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Figure 1: Omega-3 and Omega-6 (Colussi et al., 2007)
Alpha-linolenic acid is just the precursor molecule of the n-3 PUFAs. Thus, dietary n-3 PUFAs need to undergoes conversion to eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in the endoplasmic reticulum. The EPA and DHA are then utilizing by body to give rise to various physiological effects. Alpha-linolenic acid is elongated and desaturated into EPA and then into DHA (Colussi et al., 2007). This is important as EPA and DHA are two key bioactive component or biological modulators that express the effects of n-3 PUFAs (Deckelbaum et al., 2006). On the other hand, linoleic acid, which is a precursor molecule as well, is converted into arachidonic acid in the endoplasmic reticulum (Colussi et al., 2007).
Figure 2: n-3 PUFAs and n-6 PUFAs metabolism (Colussi et al., 2007).
3.2 Evidences and importance of n-3 PUFAs and n-6 PUFAs
Daily consumption of n-3 PUFAs and n-6 PUFAs has been proved to show significant effects on human health. American Heart Association has demonstrated that Sourthern Europe recorded low rate of cardiovascular diseases compare to other countries in the world. Interestingly, populations involved tend to have higher plasma high-density lipoproteins (HDL). High level of HDL is benefit to reduce the risk of cardiovascular diseases. Based on the research, the scientists found that this is due to the role of Mediterranean diet, which contains high percentage of n-3 PUFAs and n-6 PUFAs in prevention of cardiovascular diseases (Eletto et al., 2005).
Apart from that, populations in Eskimo especially in the Greenland also tend to have higher level of plasma HDL, lower cholesterol level, triglyceride and lower level of low-density lipoproteins (LDL). This is due to their diet that involved high quantity of fish. Based on the survey, they consumed about 400g of fish per day (Guil-Guerrero, 2007). Besides that, lowest rate of death from cardiovascular diseases is recorded in Okinawa, Japan. The amount of fish consumption of local people recorded over 200g per day (Kagawa et al., 1982).
Based on the cases above, there is no doubt to conclude that fish is the main source of n-3 PUFAs and n-6 PUFAs. Another phenomenon is also observed, where consumption of fish may directly affect the lipid metabolisms. This is because the lowering in the risk of cardiovascular diseases is mainly contributed from healthy lipid and cholesterol level in the body. Based on American Heart Association, a person is considered healthy if his/her plasma lipid level is maintained below 150mg/dL (miligrams per deciliter of blood). Therefore, the evidences above show the importance of daily diet with high percentage of PUFAs and their role in prevention of cardiovascular diseases.
In this case, several hypothesis can be made. First, PUFAs may enhance the oxidation of fatty acid and inhibits the synthesis of fatty acid. Secondly, the removal and excretion of cholesterol may be induced as well. However, in the 19th century, the research carried out show only very brief results in terms of lipid level and so on. The exact pathway and targeting site involved are not well determined. This has limits further application of PUFAs in clinical usage. Thus, the effect of PUFAs on lipid metabolism will be reviewed in this report in the narrower and details view. In addition, the biotechnology nowadays makes the research in narrower view to be possible.
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3.3 Comparison of n-3 PUFAs and n-6 PUFAs
Among the PUFAs, the n-3 PUFAs are known as potent PUFA to reduce the risk of cardiovascular diseases and have significant roles on the brain development, inflammatory responses and prevention of cancer. Based on Seo and colleagues, the n-3 PUFAs show positive effects to health through alteration of inflammatory responses and related protein expression, incorporation into phospholipids membrane, regulation of enzymes associated to various signalling pathways and direct affect the gene to alter the gene expression (Seo et al., 2005). It is believed that these pathways are highly interactive and n-3 PUFAs act through multiple coordinated mechanisms.
On the other hand, n-6 PUFAs shows slightly opposite effects compared to the effects shown by n-3 PUFAs. In 1998, Gerster discovered that n-6 PUFAs tend to inhibit the synthesis of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in most of the cases. In addition, n-6 PUFAs are shown to prevent the incorporation of EPA and DHA into the phospholipids membrane (Gerster, 1998). As mentioned by Seo and co-workers, DHA and EPA need to incorporate into the phospholipids membrane in order to express their effects. However, presences of n-6 PUFAs inhibit this pathway and prevent EPA and DHA to show their impact in optimizing the body health. Thus, n-3 PUFAs demonstrate more beneficial effects compared to the n-6 PUFAs.
3.4 Lipid metabolism genes regulated by n-3 PUFAs
The recent studies suggesting that n-3 PUFAs serve as important biological mediators of gene expression. Figure 3 illustrates different gene families that are regulated by the n-3 PUFAs. In this case, the genes modulated are mainly involved in the lipid metabolism, inflammation and energy utilization (Deckelbaum et al., 2006). Based on the current studies, the dietary n-3 PUFAs have intense effects on the transcription of hepatic genes that lead to the alternation in lipid metabolism (Deckelbaum et al., 2006).
In the study of Eletto and colleagues, outcome in the sense of lipid metabolism is emphasized. However, known genes induced and inhibited by experimental diets are found in their study (appendix 1) (Eletto et al., 2005). The genes that either are induced or inhibited in this study not only limits to genes involved in the lipid metabolism but as well as genes involved in other metabolism of the body.
This discovery provides alternatives and advantages for further study. In my opinion, the effect of n-3 PUFAs towards body may not happens in just a single biological pathway. This is because each of the biological pathway and metabolism within the human body are interrelated and affect one another. For example, the regulation of lipid metabolism may not just depend on the lipid level of the body, but may be affected by thermal regulation and hormonal homeostasis as well. Every single genes need to be take into consider but not just focusing on the genes relating to the lipid metabolism.
Figure 3: Genes regulated by n-3 PUFAs (Deckelbaum et al., 2006).
In the research regarding the effects of n-3 PUFAs on cardiovascular diseases, n-3 PUFAs are known to decrease the blood triglyceride concentration or serum lipid level and blood cholesterol. In most of the studies, fish oil is used as a source to examined the effects show by n-3 PUFAs. Lise Madsen and colleagues provided evidence of regulation of fatty acid synthesis by n-3 PUFAs through their study. In this research, the fatty acid synthesis of normal differentiated adipocytes was indicated to increase throughout the time. Interestingly, the synthesis of fatty acid was suppressed in the presence of n-3 PUFAs (Madsen et al., 2004).
Based on the studies reviewed in this report, the effect of n-3 PUFAs is mainly results from the combine effects of sterol regulatory element-binding proteins (SREBPs) and peroxisome poliferator-activator receptor (PPAR) in the liver (Hirako et al., 2009). These two components are transcription factors that are critical to modulate the expression of hepatic genes controlling lipid metabolism.
3.4.1 Sterol-regulatory-element binding proteins (SREBPs)
Studies evaluating expression of sterol-regulatory-element binding proteins (SREBPs) in response to dietary have provided strong evidence that SREBPs are involved in the fatty acid synthesis and cholesterol homeostasis. SREBPs are classified into three isoforms, which are SREBP-1a, SREBP-1c and SREBP-2a. The SREBP-1a and SREBP-1c are encoded from the same gene by alternative promoters, which involved in the regulation of genes related to lipogenesis. Thus, SREBP-1 is known as adipocytes differentiation and determination factors. In contrast, SREBP-2a regulates the genes involved in the cholesterol metabolism. These display the pluripotent effects of n-3 PUFAs on the gene expression (Deckelbaum et al., 2006).
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The inactive precursor form of SREBP is located in the endoplasmic reticulum membrane and form a complex with SREBP cleavage-activating protein (SCAP) (Hirako et al., 2009). This complex envelope in a hairpin structure and attached to the Insig proteins. The Insig proteins dissociate from the SREBP/SCAP complex and the complex is transfers to the Golgi body via vesicular transport for activation in responding to regulatory signals of low intracellular cholesterol level (Yang et al., 2002). In the Golgi body, SREBP detaches from the SCAP and it is activated by goes through a two-step proteolytic cleavage event by site-1 protease and site-2 protease (Hirako et al., 2009). The active amino-terminal fragment of SREBP (n-SREBP) which contains transcriptional activation factors and DNA binding domains is translocated into the nucleus and bind to sterol regulatory elements in the targeted gene promoter region to facilitate the transcription of SREBP targeted genes (figure 4). This mechanism is regulated by end-product feedback such as intracellular cholesterol (Adams et al., 2004).
Figure 4: Activation and synthesis of SREBPs (Yang et al., 2002).
18.104.22.168 SREBPs targeted genes regulated by n-3 PUFAs
Kim and colleagues successfully found the genes regulated by SREBPs in lipogenesis and cholesterol homeostasis. It is reported that SREBP-1a and SREBP-1c involved in the transcription of lipogenesis related genes and the identified genes in this case are fatty acid synthase (FAS), acetyl-CoA carboxylase (ACC) and stearoyl-CoA desaturase (SCD). The dietary fish oil down regulates these genes (Kim et al., 1999).
Down regulation of FAS may cause significant impact to fatty acid synthesis. This is because FAS is a multi enzyme complex that carries many essential enzymes, which are needed in elongation of fatty acid during fatty acid synthesis. These enzymes are Î²-ketoacetyl-ACP synthase, Î²-ketoacetyl-ACP reductase, Î²-hydroxyacyl-ACP dehydrase, and Enoyl ACP reductase (figure 5) (Nicholson, 2002). On the other hand, down regulation of ACC may inhibit fatty acid synthesis as well. In this case, ACC is an enzyme catalyzing the conversion and activation of acetyl-CoA to malonyl-CoA before the onset of fatty acid synthesis (figure 6). The malonyl-CoA is needed as initial compound for synthesis of fatty acid (Nicholson, 2002). Thus, inhibition of this reaction may directly inhibit the onset of fatty acid synthesis. SCD is another crucial enzyme to catalyze the conversion of saturated fatty acid to monounsaturated fatty acid. Therefore, down regulation of SCD may prevent formation of monounsaturated fatty acid, which in turn give rise to formation of triglycerides (Nakumara et al., 2004).
Figure 5: Elongation phase of fatty acids (Nicholson, 2002).
Figure 6: Activation of acetyl-CoA (Nicholson, 2002).
While, the identified genes of SREBP-2 that involved in the cholesterol synthesis are HMG-CoA-reducing enzyme, HMG-CoA synthetic enzyme and low-density lipoprotein-receptor (LDL-receptor) (Kim et al., 1999). Down regulation of HMG-CoA-reducing enzyme and HMG-CoA synthetic enzyme, directly inhibit the synthesis of cholesterol. In addition, decrease in LDL-receptor that mediates theÂ endocytosisÂ of cholesterol-richÂ LDL is beneficial to reduce cardiovascular diseases especially atherosclerosis.
In 2009, the study of Hirako and colleagues has further support the study of Kim and colleagues. The effects of n-3 PUFAs on lipid metabolism are examined with experimental dietary 2% cholesterol (w/w) and 20% or 50% energy fish oil. The safflower is used as control to make comparison with fish oil. This study reported that the expression level of SREBPs was significantly suppressed with the supplement of dietary fish oil. In this research, the expression level of transcription factor SREBPs and SREBP target genes were established clearly (Hirako et al., 2009).
Based on result table, the expression level of transcription factors such as SREBP-1c and SREBP-2 are decreased in the FO group compared to the SO group. On the other hand, the Insig proteins also show significant decrease in the FO group compared to the SO group. Insig-1 and Insig-2 are proteins in the endoplasmic reticulum that play key roles in lipid and cholesterol homeostasis through inhibiting excessive synthesis of lipid and cholesterol (Lee et al., 2006). The lipid and cholesterol level are regulated as Insig proteins are responsible for the activation and expression of SREBPs. When level of Insig proteins are reduced, expression of SREBPs may be decreased as well. As the result, synthesis of cholesterol and fatty acids may directly reduce. This is important to prove further the statement by Yang and colleagues about the involvement of Insig proteins in the activation of SREBPs. The expression of SREBP-1c target genes, FAS and SCD, show similar decreasing trend with study of Kim and colleagues. The suppression of these genes would eventually inhibit the fatty acids synthesis process in the body (Hirako et al., 2009). Apart from that, the expression of SREBP-2a target genes in FO groups, HMG-CoA reductase and LDL-receptor, were found to be reduced as well. The low transcription level of these genes would cause the reduction in the serum cholesterol. Thus, n-3 PUFA is also beneficial to health by decreasing the serum cholesterol (Hirako et al., 2009).
In the studies of Hirako and colleagues, as well as Eletto and colleagues, the technique of real-time polymerase chain reaction (RT-PCR) is applied to measure the expression level of hepatic genes in the liver (Hirako et al., 2009). However, Eletto and colleagues used both of the RT-PCR and microarray analysis in order to confirm the resulting gene array.
The study of Nakatani and co-workers is again support the previous studies by showing the similar trend of results. In their study, the mice were fed with experimental fish oil in a range from 0% to 60% in order to clarify the effect of fish oil on lipid metabolism. It is reported that the level of SREBP-1 protein is decreased by 50% with 10% of fish oil feeding. Besides that, the genes expression involved in the synthesis of fatty acid such as fatty acid synthase (FAS) and acetyl-CoA carboxylase (ACC) are markely down regulated in this study (Nakatani et al., 2003). This result is consistent with the previous studies.
Interestingly, Xu and co-workers demonstrated that n-3 PUFAs down regulating the expression of SREBP-1 through accelerating transcription decay. It was reported that decay rate of both SREBP-1a and -1c was enhanced in the presence of n-3 PUFAs. The results showed that the half-life of both SREBP-1a and -1c mRNA was significantly shorten by 50%. The half-life of SREBP-1c was reduced from 10.0 to 4.6 h whereas half-life of SREBP-1a was reduced from 11.6 to 7.6 h, respectively (Xu et al., 2001).
In addition, the decay process of SREBP-1c was found to be greater than SREBP-1a. They carried out another research in order to confirm the site and stage of SREBP decaying are occurred. In this case, the hepatocytes are treated with cycloheximide which is a protein translational inhibitor. As the result, the decaying of SREBP-1 is prevented although n-3 PUFAs is consumed (Xu et al., 2001). This suggested that decaying of SREBP-1 is occurred in the protein form. Thus, mRNAs of SREBP-1 need to enter into translation process before they are degraded by the n-3 PUFAs. However, the genes regulated by SREBP-1 are not been down regulated in this way. The expressions of these genes are inhibited during transcription stage. The mechanism of how the genes regulated by SREBP-1 are inhibited will be discussed in the next section (22.214.171.124).
In conclusion, the modulation of gene expression is achieved by dietary n-3 PUFAs in order to reduce lipid level in the body. Dietary n-3 PUFAs reduce the level of lipid in the body by down regulating the SREBPs target genes encoded for fatty acid synthesis and cholesterol metabolism. In short, n-3 PUFA can be considered as inhibitor of SREBPs. In the case of fatty acid synthesis, fatty acid synthase (FAS), acetyl-CoA carboxylase (ACC) and stearoyl-CoA desaturase (SCD) are down regulated. The HMG-CoA-reducing enzyme, HMG-CoA synthetic enzyme and low-density lipoprotein-receptor are down regulated in the case of cholesterol homeostasis. Consequently, the health can be optimized by lowering of both lipid and cholesterol level. For this reason, fish oil is now becomes a potential dietary for prevention and treatment of cardiovascular diseases.
126.96.36.199 Possible pathways of SREBPs regulation by n-3 PUFAs
In the earlier years, the mechanism by which n-3 PUFAs regulate the SREBPs is not yet been identified. However, there is a hypothesis made by Johnson and colleagues in 2003. Based on them, the inhibition of SREBPs by n-3 PUFA is related to both of the physical and biochemical effects. These scientists created an experiment by using large and small unilamellar vesicles as a model for plasma and intracellular membranes. The experiment showed decreases in the affinity of cholesterol for phospholipids membrane by addition of n-3 PUFAs. In this case, the transfer of cholesterol from cholesterol-rich region such as plasma membrane to cholesterol-poor region such as endoplasmic reticulum is triggered. This condition leads to decreases in the transportation of cholesterol out of endoplasmic reticulum to the Golgi body that in turn reduces the activation process of the SREBPs. Thus, the expression of SREBPs related genes is down-regulated (Johnson et al., 2003).
Worgall and colleagues also postulated another possible mechanism in 2002. Based on their studies, the level of SREBPs was found to be affected by the cellular composition of membranes. There is a compound known as sphingomyelin in the cellular plasma membrane. These scientists found out that the addition of n-3 PUFAs would hydrolyze the sphingomyelin to ceramide. Theoretically, sphingomyelin has higher affinity towards cholesterol than other phospholipids in the plasma membrane. Thus, the hydrolysis of sphingomyelin to ceramide would eventually reduce the affinity of plasma membrane towards the free cholesterol. This affects the SREBPs related gene transcription and cholesterol homeostasis in two pathways. First, lower amounts of sphingomyelin decreased the solubilized free cholesterol which results in a decreased in SREBP mediated gene transcription. Secondly, the ceramide produced is an effective inhibitor of SREBPs itself. As a result, the activation process of SREBPs is affected by sphingomyelin metabolism (Worgall et al., 2002).
The mechanisms stated above illustrating the down regulation of cholesterol level by n-3 PUFAs through inhibition of SREBPs. These mechanisms are mainly regulated through the feedback homeostasis system. In the presence of n-3 PUFAs, a cascade of inhibitory reaction is triggered to reduce the level of cholesterol. This is beneficial to understand the effects of fish oil in reducing the risk of cardiovascular diseases. However, the pathway of SREBPs in down regulating of lipid level is not yet determined. Thus, further investigation is necessary to identify the pathway of SREBPs in reducing lipid level.
3.4.2 Peroxisome proliferator-activated receptors (PPARs)
PUFAs have unique coordination on the hepatic lipid metabolism through transcriptional factors called peroxisome proliferator-activated receptors (PPARs). PPARs are receptor proteins present on the nuclear membrane, which function as transcription factors in regulating the expression of genes. There are three isoforms of PPAR being indicated, which are alpha, beta, and delta. These isoforms are distributed mainly in the liver, muscles, adipose tissue and bloodstream. Each of this isoform is accountable for distinct function in the lipid homeostasis (Cho, 2008).
It is widely reported that high n-3 PUFA intake are essential to regulate the expression of PPARs. In this case, PPARs induce the expression of genes involved in mitochondrial and peroxisomal fatty acid oxidation (Î²-oxidation). The PPAR-Î± involve in Î²-oxidation in the liver and muscular tissue while PPAR-Î´ only involved in the Î²-oxidation in the muscular tissue. Therefore, PPARs are critical for regulating the expression of crucial genes that are responsible for lipid homeostasis and PPAR-Î± is the major isoform that contributes to the regulation of overall Î²-oxidation.
188.8.131.52 PPAR-Î± targeted hepatic genes regulated by n-3 PUFAs
Hirako and co-workers found the genes targeted by PPAR-Î± as well, which were acyl-CoA synthetase (ACS), acyl-CoA dehydrogenase (ACD), and acyl-CoA oxidase (ACO) (Hirako et al., 2009). Each of these genes plays a key role in the Î²-oxidation. Based on the study, these genes are up regulated with the intake of dietary fish oil.
In the Î²-oxidation, ACS catalyzes the condensation of fatty acids with CoA, with simultaneous hydrolysis of ATP to AMP and pyrophosphates. This reaction activates fatty acids for the Î²-oxidation (Nelson et al., 2005). Apart from that, ACD is a major enzyme in the first step of Î²-oxidation that catalyzes the oxidation of fatty acyl-CoA whereas ACO oxidizes CoA derivatives of fatty acids, which is the rate-determining step of Î²-oxidation (Nakajima et al., 2002).
In addition, Nakatani and co-workers were carried out the research by set the experimental diet in a range from 10% to 60% of fish oil, which contain abundant of n-3 PUFAs. The genes they studied included acyl-CoA oxidase (ACO), acyl-CoA dehydrogenase (ACD), acyl-CoA synthetase (ACS), and carnitine palmitoyltransferase. Similarly, the expressions of these genes were up regulated proportional to the amount of dietary fish oil. It is found that the effect shown by fish oil is dose-dependent as higher amount of fish oil may show effect that is more significant. These results are consistent with the previous results. In this case, another gene, carnitine palmitoyltransferase is found to be induced as well. Inducing of this gene is linear with the theory. This is because carnitine palmitoyltransferase is an essential enzyme to transport long chain fatty acids into mitochondrial matrix for oxidation (Guo et al., 2005).
In short, both of the studies concluded that expression of ACS, ACD, ACO and carnitine palmitoyltransferase are regulated by transcription factor PPAR-Î±. Dietary fish oil induces the transcription of these genes through PPAR-Î± activation. Therefore, up regulation of these genes directly stimulate the rate of Î²-oxidation.
184.108.40.206 Possible pathways of PPARs regulation by n-3 PUFAs
The possible mechanisms of PPARs are determined in recent years. In 2005, Gerson and co-workers were discovered that dietary n-3 PUFAs incorporated into the membrane phospholipids to alter lipid metabolism (figure 7). The membrane fluidity and permeability are changed when n-3 PUFAs incorporated into membrane phospholipids (Gerson et al., 2005). These changes may directly influence the expression of important enzymes involve in the Î²-oxidation. The exact enzymes regulated are not determined in this study. However, Guo and co-workers found one crucial enzyme that was regulated through this mechanism, which is carnitine palmitoyltransferase. This enzyme is crucial in mediating the transport of long chain fatty acid across the mitochondrial membrane for further oxidation (Guo et al., 2005). This further proved the possibility of this pathway in the case of PPARs.
Figure 7: Incorporation of EPA and DHA into membrane phospholipids (Gerson et al., 2005).
Besides, Feige and co-workers propose another possible mechanism. Based on them, PPARs function as transcription factors through ligand-activated signaling pathway. The EPA and DHA present in the fish oil act as natural ligand of the PPARs, which is main transcription factors of lipid metabolism (figure 8). When fish oil is taken, EPA and DHA bind to the PPAR on the nucleus membrane. This binding causes the dimerization of PPAR with retinoid-X-receptor (RXR) in the nucleus. This complex is then binds to the promoter region to activate the transcription of the targeted genes. This promoter region is known as peroxisome proliferator response elements (PPREs). Eventually, the genes involved in the Î²-oxidation are expressed. Interestingly, several crucial genes regulated through this mechanism are identified such as carnitine palmitoyltransferase and hydroxyacyl dehydrogenase (Feige et al., 2006). Inducing of carnitine palmitoyltransferase may speed up the transportation of long chain fatty acids into mitochondrial matrix for oxidation whereas hydroxyacyl dehydrogenase may increase the oxidation process of fatty acids. Therefore, intake of fish oil may directly up regulating the expression of these enzymes, which in turn induces the rate of Î²-oxidation. As the result, the lipid level can be reduced.
Figure 8: EPA and DHA act as natural ligands for PPARs (Feige et al., 2006).
In my opinion, both the mechanisms discussed show strong evidence to support the effectiveness of n-3 PUFAs in reducing the lipid level through PPARs. Since PPARs are receptor proteins, it is very reasonable for EPA and DHA to act as the ligand for PPARs. This supports the earlier studies that stated EPA and DHA are biological regulators that show the effects of fish oil.
3.5 Sources of n-3 PUFAs
Since human beings need to obtain PUFAs from external source, it is important to identify which kind of diet that can provide sufficient of PUFAs. There is no doubt that fish is the main dietary constituent of PUFAs and shows significant nutritional value. The greatest PUFAs that can be found in the fish are n-3 PUFAs, especially DHA and EPA. Besides being the major source of n-3 PUFAs, fish is found to supply high value of proteins, vitamins, and minerals, which bring many beneficial effects to human beings. However, availability of essential n-3 PUFAs from fish is depends on several factors such as species, habitat, water temperature, type of food, seasonal and so on (Belda and Pourchet-Campos, 1991).
Firstly, fish which are permanently found in the cold water habitat may contain higher percentage of n-3 PUFAs. Wang and co-workers further support the theory of water temperature variations in affecting the percentage of n-3 PUFAs in the fish. They reported that the fish in the cold water tend to contain higher amount of n-3 PUFAs than tropical fish. The research also demonstrated that freshwater fish contain lesser amount of n-3 PUFAs compare to sea water fish (Wang et al., 1990). Thus, the major fish that best to consider are salmon, sardines, tuna, mackerel, and herring. Based on the research done by American Heart Association, salmon is the fish type that can provide highest percentage of n-3 PUFAs, which up to 2.6 grams (American Heart Association, 2004).
Over the years, there is an argument in whether wild or farmed salmon is able to provide higher value of n-3 PUFAs. This is because farmed salmon are tending to grew larger than wild salmon. American Heart Association demonstrated that consumer only needs to consume 1.5 to 2.5oz of farmed salmon in order to give 1 gram of n-3 PUFAs whereas 2 to 3.5oz of salmon need to be consumed in order to give 1 gram of n-3 PUFAs grams (American Heart Association, 2004).
Apart from that, fish which are fed on the phytoplankton may provide the greatest source of PUFAs especially n-3 PUFAs to the fish. Phytoplankton is well known to contain high percentage of essential fatty acids. Due to these reasons, fish is frequently chosen as to be the better source of n-3 PUFAs compare to other seafood. Generally, salmon (2.6g) is the fish type that contains highest percentage of n-3 PUFAs, followed by herring (2.224g), anchovy (2.096g), sablefish (2.003g), whitefish (1.821g), tuna (1.664g), and sardine (1.458g) (American Heart Association, 2004).
In my opinion, wild salmon may provide greater percentage of n-3 PUFAs as wild salmon that live in the natural habitat are mainly consuming phytoplankton. This is because phytoplankton is the major plant type that can be found on the seafloor. On the other hand, most of the farmed salmon are fed with artificial diet. Although farmed salmon are larger, but the fat content in these salmon may not mainly compose of n-3 PUFAs. Higher fat contents cannot directly determine higher n-3 PUFAs content. Thus, wild salmon should give higher amount of n-3 PUFAs since phytoplankton is the source of n-3 PUFAs for wild salmon.
Interestingly, human milk is a very rich source of n-3 PUFAs. This is very useful for health and development of infant (Duchen and Bjorksten, 2001). Besides cardiovascular diseases, n-3 PUFAs show very significant effect in the developing of brain. In addition, human milk is natural source of nutrients which shows no harm to the infant. Nowadays, EPA and DHA become very important ingredients in the production of milk powder. This is important for milk powder to act as another source for infant besides human milk. So, n-3 PUFAs especially EPA and DHA are very helpful in the brain developing especially during early stage in the infant. However, the amount of n-3 PUFAs in the human milk depends strongly on the diet of mother before and during pregnancy. The proper diet should be consumed by the mother to maximize the amount of n-3 PUFAs in the breast milk produced. Hibbeln and co-workers demonstrated that vegetarian women or low consumption of seafood tend to show lower amount of n-3 PUFAs in the breast milk produced. Surprisingly, the amount of n-3 PUFAs varies before and after the childbirth. Hibbeln reported that amount of n-3 PUFAs may drops significantly three month after the childbirth (Hibbeln, 2002). In this case, it may be closely related to difference in hormone level for women during the pregnancy. Hormone secreted during pregnancy may directly stimulate the aggregating of n-3 PUFAs in the mammary glands to produce breast milk.
There are several sources for vegetarian as well. Firstly, algae are reported to be one of the plant sources that provide high amount of n-3 PUFAs in the form of DHA and EPA. Two common algae that are considered by Japan are Fucus vesiculosus and Chondrus crispus which contain 38.5% and 30.5% of n-3 PUFAs, respectively. However, algae are seldom consumed as daily diet besides Japan (Hunter, 1990).
Another great plant source of n-3 PUFAs is flax seeds. Flaxseed oil shows great percentage of n-3 PUFAs as high as 2.2 g/tbsp or 51.9% to 55.2% of fat content per seed (Vereshagin et al., 1965). Based on Vegetarian Society, a teaspoon of flaxseed oil give rise to 8.9g of total essential fatty acids in which 1.7g of n-6 PUFAs and 7.2g of n-3 PUFAs, respectively. This gives the ratio of 1:4 for n-6: n-3 (Vegetarian Society, 2010). In addition, flaxseed provides beneficial health value besides n-3 PUFAs such as fibre and lignin compound which can optimize the health. Due to this, flaxseed is widely used as commercial oil and developed into capsule to use as supplement in nowadays.
Apart from that, walnut oil is a good source as well which contains 1.4 g/tbsp. of n-3 PUFAs. The total proportion give rise to 10.7g of n-6 PUFAs and 2.5g of n-3 PUFAs in ratio of 4:1 for n-6: n-3 (Vegetarian Society, 2010). The essential plant sources of n-3 PUFAs are followed by canola oil (1.3 g/tbsp), soybean oil (0.9 g/tbsp), and olive oil (0.1 g/tbsp). The vegetable oils such as soybean, corn, sunflower and olive oil contain higher amount of n-6 PUFAs instead of n-3 PUFAs (Hunter, 1990).
However, it is recommended to take fish oil supplement as daily source n-3 PUFAs as consumption of n-3 PUFAs should base on amount of DHA and EPA. The EPA and DHA are the form of n-3 PUFAs that give the effects to human body. Fish oil supplement commonly contains 180mg of EPA and 120mg of DHA which best fulfill the requirement of body (American Heart Association, 2010). In addition, consumption of fish oil supplement is safer as fish may contain unwanted compounds such as methyl mercury and other contaminants. This is crucial to avoid mercury poisoning. Therefore, consumer needs to be aware of the risks of taking fish as source of n-3 PUFAs besides the benefits.
3.6 Ideal daily consumption amount of dietary n-3 PUFAs
Based on American Heart Association, one gram of n-3 PUFAs need to be consumed to fulfill the body's requirement of person without any illness. However, two to four grams of n-3 PUFAs are required for people who need the lowering of lipid level especially for those with mild hypertension (American Heart Association, 2010).
Apart from that, consumption of essential fatty acids gives best result when the ratio of n-6: n-3 is taking into consideration. The National Institute of Health reports that the ration of n-6: n-3 is best in 2: 1. In more accurate scale, a people need to consume about 4 grams of n-6 PUFAs and 2 grams of n-3 PUFAs per day (National Institute of Health, 2010). This is because dietary n-6 PUFAs are being metabolized into only one functional compound in the body which is arachidonic acid while n-3 PUFAs are being metabolized into two functional compounds which are DHA and EPA. Therefore, n-6 PUFAs are needed more than n-3 PUFAs. However, consumption of n-6 PUFAs needs to stick on the ratio and cannot be overdosed. This is because n-6 PUFAs tend to show more side effects as discussed in the earlier section.
Some minor side effects do occur following by overdose of fish oil. The most common problem is fishy aftertaste of fish oil. This is probably happening for some people who are not get used to consume diet with fish. Based on the survey done by Gruppo Italiano per lo Studio della Sopravvivenza nell'Infarto Miocardico (GISSI) prevention study, it is reported that consumers are likely to have this problem if take more than 3 grams of fish oil per day. Gastrointestinal disturbances and nausea are the second most reported side effects, which is about 4.9% in 41 people. Besides that, other side effects involved are clinical bleeding, and worsening glycemia. However, all the side effects mentioned above are unlikely to occur and do not really bring harm to human health. These side effects may only happen if the daily consumption of fish oil is more than 3 grams. In addition, not all of people may face these side effects as the population involved in this survey is very small, which are only 41 people.
4 The effects of dietary sesamin on lipid metabolism
In recent years, sesame seed has been widely used as nutritional supplement. It is scientifically proven to show various beneficial physiological effects such as maintaining healthy lipid level, enhancing detoxification activities in liver, neural protective, anti-oxidant, anti-hypertensive, anti-inflammatory, and anti-allergic. Most of the effects are contributed by sesamin, which is one of the most abundant lignans in the sesame seeds and oil. AÂ lignanÂ is a type of polyphenolic compound that can be extracted from some plants. The others minor lignans found in the sesame seeds and oil are episesamin, sesamolin and Î³-tocopherol (Ashakumary et al., 1999).
Although sesamin exerts many beneficial physiological effects, its potency in reducing lipid level has been emphasized by scientists nowadays. This is because lignans strongly influence lipid metabolism in human. In 1999, Ashakumary and co-workers firstly found that sesamin greatly increases the rate of hepatic Î²-oxidation. Takashi and co-workers as well as Jeng and his co-workers further prove this statement by identifying the genes involved.
4.1 PPAR-Î± targeted genes regulated by sesamin
In the study of Ide and co-workers, rats were fed with experimental diets containing various amount of sesamin, which were 0%, 0.2% and 0.4%. The hepatic enzymes involved in the Î²-oxidation were measured after 15 days. They were able to identify several genes involved in the Î²-oxidation, which are acyl-CoA oxidase (ACO), carnitine palmitoyltransferase, 3-Hydroxyacyl-CoA dehydrogenase, and 3-Ketoacyl-CoA thiolase. Based on the results, expression of these genes are markedly up regulated proportional to the increase in percentage of dietary sesamin (Ide et al., 2001). Therefore, the effect of sesamin is dose-dependent. Up regulation of these genes may directly increase the rate of Î²-oxidation. In the Î²-oxidation, ACO oxidizes CoA derivatives of fatty acids, which is the rate-determining step of Î²-oxidation (Nakajima et al., 2002). Carnitine palmitoyltransferase is another enzyme mediates the transport of long chain fatty acid across the mitochondrial membrane into the mitochondrial matrix for further oxidation (Guo et al., 2005). In addition, 3-Hydroxyacyl-CoA dehydrogenase, and 3-Ketoacyl-CoA thiolase are involved in the oxidation and cleavage of fatty acids during Î²-oxidation, respectively (Nakajima et al., 2002). Thus, suppression of these genes may lead to reduce in fatty acids synthesis.
On the other hand, Jeng and his co-workers carried out the research with 0%, 0.1%, 0.2% and 0.5% of experimental sesamin. Based on their results, sesamin greatly increased the expression of six hepatic fatty acid oxidation enzymes, including carnitine palmitoyltransferase, acyl-CoA dehydrogenase (ACD), acyl-CoA oxidase (ACO), 3-hydroxyacyl-CoA dehydrogenase, enoyl-CoA hydratase, and 3-ketoacyl-CoA thiolase. ACD is a major enzyme in the very first step of Î²-oxidation that catalyzes the oxidation of fatty acyl-CoA whereas enoyl-CoA hydratase catalyzes the hydration of 2-enoyl-CoA to 3-hydroxyacyl-CoA (Jeng et al., 2005). Out of six, there are four enzymes matched with the research carried by Takashi and co-workers. This is important to prove that sesamin is truly effective in regulating the expression of enzymes in Î²-oxidation. Apart from that, these genes are expressed more than 10 fold in the diet with 0.5% of sesamin. This is again consistent with previous study, which stated that effect of sesamin is dose-dependent.
In short, acyl-CoA oxidase (ACO), carnitine palmitoyltransferase, 3-Hydroxyacyl-CoA dehydrogenase, acyl-CoA dehydrogenase (ACD), enoyl-CoA hydratase, and 3-Ketoacyl-CoA thiolase are up regulated. These genes are up regulated through activation of PPAR-Î±. Therefore, up regulation of these genes directly increases the rate of Î²-oxidation.
4.1.1 Possible pathways of PPARs regulation by sesamin
At the same time, Ide and co-workers had demonstrated that the effects sesamin was showed through regulation of transcription factor PPAR-Î±. This is reasonable as the genes regulated in the studies above are targeted and controlled by PPAR-Î±.
Sesamin appears to be an active PPAR ligand and the most potent inducer of hepatic fatty acid oxidation among the various naturally occurring compounds so far reported. Sesamin is as said earlier is a naturally occuring lignan isolated from sesame seeds. A lignan is a molecule that combines with another ingredient functioning as an "activator." In the case of sesamin, it tends to bind to and activates PPAR-Î±, which is the major regulator of lipid metabolism. In the other words, sesamin itself is a PPAR-Î± agonist, in which the binding of sesamin to PPAR-Î± may induced the transcription rate.
4.2 SREBPs targeted genes regulated by sesamin
The research of Ide and co-workers also showed results of genes regulated by SREBP after consumption of experimental sesamin. The genes involved are acetyl-CoA carboxylase (ACO), and fatty acid synthase (FAS). The expressions of these genes were down regulated. As mentioned in the earlier part, down regulation of these genes may directly reduce the synthesis of fatty acids. The ACO is crucial to catalyze the conversion of acetyl-CoA to malonyl-CoA which is required as initial compound for fatty acid synthesis whereas FAS is multi enzyme complex that carries many essential enzymes that are involve in the elongation of fatty acid. Therefore, down regulation of these genes may significantly inhibit the whole process of fatty acid synthesis.
Besides that, the expression of SREBP-1 was reported to be suppressed in the presence of experimental sesamin. The suppression of SREBP-1 itself may influence both the fatty acid and cholesterol synthesis which are controlled by SREBP-1c and SREBP-1a, respectively. As the result, lipid level in the body can be reduced. Interestingly, the protein level of SREBPs in both precursor and mature form was found to be decreased as well. This indicates that dietary sesamin not only influences the expression of genes regulated by SREBPs, but also affects the synthesis and activation of SREBPs in the nucleus. Thus, sesamin is a useful inhibitor of SREBPs as sesamin reduces the activities of SREBPs through multi pathways.
However, the exact mechanism of how gene expression is regulated by SREBPs is not well determined. In my opinion, the mechanism involved in the case of sesamin may be similar to the mechanism shown by dietary fish oil. Since SREBPs are one of the major transcription factor involved in the regulation of lipid metabolism, the reduction in SREBPs may prevent the transcription of genes that are crucial in the synthesis of fatty acid. Both of fish oil and sesamin regulate the SREBPs targeting genes through either inducing or suppressing of SREBPs. Therefore, both the sesamin and fish oil are believed to cause physiological changes of lipid metabolism through the similar molecular pathway.
4.3 Comparison of fish oil and sesamin
Both of fish oil and sesamin show very significant effects to the lipid metabolism and effectively reduced the lipid level in the body. Both of fish oil and sesamin also show almost equally other beneficial effects such as anti-hypertension, anti-inflammation and so on. Fish oil shows the effects through n-3 PUFAs while sesamin shows the effects through abundant composition of lignans. Thus, it is interesting to determine which of these as better nutritional diets to show physiological effects on human health. Based on the study found, sesamin was reported to show the effects with just 0.1 to 0.2% of dietary levels. However, a diet needs to contain at least 3 to 10% of n-3 PUFAs in order to show the effects. In this case, sesamin shows the better effects in almost 10 times than n-3 PUFAs in reducing the lipid level.
In addition, sesamin shows large difference with n-3 PUFAs in the scale of time to see their effects. Based on the reviews done, n-3 PUFAs need to take at least 13 weeks to show their effects in lipid metabolism but sesamin only needs about two weeks' time to show the effects. This means that n-3 PUFAs need to use almost 7 folds of time compare to sesamin in giving physiological changes to human health. Based on these reasons, sesamin is a better choice as nutritional supplement when being compared to fish oil.
4.4 Combination of dietary fish oil and sesamin
Surprisingly, Ide and co-workers found that combination diet of fish oil and sesamin markedly increased the rate Î²-oxidation. Although diet with sesamin or fish oil alone were significantly increased the rate of Î²-oxidation, but a diet containing sesamin and fish oil in combination was up regulating all the genes involved in a synergistic manner. In addition, this experiment takes only 15 days to get the results although fish oil is involved (Ide et al., 2004). Based on the previous discussion, the experiment needs to take nearly 13 weeks to observe the effects shown. Therefore, sesamin may enhance the overall effects of fish oil when it is consumed together with fish oil. It is concluded that a diet containing both sesamin and fish oil markedly and synergistically enhanced hepatic Î²-oxidation by up regulating the gene expression involved compare to diet with either sesamin or fish oil alone.
In conclusion, both the fish oil and sesamin inhibit the lipogenesis via reduces in SREBPs and promoting fatty acid oxidation (Î²-oxidation) via activation of PPAR, respectively. The fish oil shows the effects through mainly n-3 PUFAs rather than other essential fatty acids while sesamin shows the effects through lignans involved. In the case of essential fatty acids, n-3 PUFAs give better results on enhancing the health when being compared to n-6 PUFAs. However, the combination of n-3 PUFAs and n-6 PUFAs diet in the ratio of 2:1 may best contributing to optimize the health especially in reducing the risk of cardiovascular diseases. In comparison to sesamin, fish oil shows lower efficiency as fish oil needs to take about 13 weeks to show the effects compare to two weeks by sesamin. The effectiveness of sesamin also more obvious as more consumption is required in the case of fish oil compare to sesamin. However, current study reported that diet with sesamin and fish oil in combination may markedly and synergistically reduce the lipid level compare to diet with either sesamin or fish oil alone.
Therefore, human health is closely related to daily diet consumed. It is proved that certain essential diets such as fish oil and sesamin can optimize the human health. In the future, research in the field of nutrigenomics should be continued in order to identify more nutritional diet that is beneficial to human beings. The mechanism of nutrition in influencing the biological activities in the body should be focused in the view of molecular. For example, the mechanism of SREBPs in regulating lipid metabolism need to be clarified to understand the preventive nutritional effects of fish oil as well as sesamin. This information may be useful for clinical and pharmacological use in the prevention of nutritionally related diseases.