Investigation of Lipid Oxidation in commonly consumed Fish oil supplements in Syrian market

 

Okba Hatem1*, Zeinab Sarem2

1Master's Student in Analytical and Food Chemistry Department, Faculty of Pharmacy, Tishreen University, Latakia, Syria.

2Head of Department of Analytical and Food Chemistry, Faculty of Pharmacy, Tishreen University, Latakia, Syria.

*Corresponding Author E-mail: okba.htm@gmail.com, zeinab.sarem@hotmail.com

 

ABSTRACT:

Fish oil supplements are a rich source of omega-3 polyunsaturated fatty acids (ω3-PUFA) which are important to maintain a good health. However, oxidation of these fatty acids might lead to deterioration of the quality of food and reverse their beneficial effects since oxidation products are implicated in variety of diseases. It might also be the underlying reason of the conflict outcomes of clinical studies that evaluated ω3-PUFA health benefits. We investigated the oxidative status of three brands (A, B, and C) of fish oil supplements from the Syrian marketplace by measuring peroxide value (PV), anisidine value (AV) and total oxidation value (TOTOX), and monitoring the oxidation progress for one year. PV and TOTOX of brands A and B were above the acceptable limits set by Global organization for EPA and DHA omega-3 (GOED) from the beginning of the study. AV of brand A exceeded the limit by the end of the study, while AV of brand B was unaccepted from the third month. On the other hand, values of brand C were in the allowed range for the whole period of our study. Only one brand met the criteria for oxidation levels set by GOED while the other two were highly oxidized. We recommend that oxidation levels of fish oil have to be evaluated prior to any clinical study.

 

KEYWORDS: lipid oxidation, fish oil, polyunsaturated fatty acids, Peroxide value, Anisidine value.

 

 

 

1. INTRODUCTION:

Interest in fish and fish oil has been increasing since the first observations of the positive correlation between consuming high-fish diets and low risk of cardiovascular diseases (CVD) among Greenland Eskimos [1]. Fish and fish oil benefits are attributed to their high content of omega-3 polyunsaturated fatty acids (ω3-PUFA), mainly eicosapentanoic acid (EPA) and docosahexanoic acid (DHA) [2]. Several studies have demonstrated the positive effects of these fatty acids on CVD, inflammation [3,4], triglyceridemia [5], and neurodegenerative diseases [6,7].

 

World Health Organization (WHO) recommends that people should consume 1-2 servings of fish per week [8].

 

On the other hand, children and pregnant or nursing women are advised by FDA to avoid eating certain kinds of fish and limit their consumption of other kinds in order to reduce their exposure to methylmercury that might accumulate in fish [9]. In terms of lowering triglyceride levels, patients need to reach a dose of 2-4 g/day of (EPA+DHA) which might be difficult to achieve through only fish consumption. For these reasons, fish oil supplements, which are mercury-free, might be the best option to obtain a controlled dose of ω3-PUFA [10]. Undoubtedly, fish oil became the number one selling supplement in the USA in 2012, with more than 18.8 million people taking it [11].

 

ω3-PUFA are highly prone to oxidation because of their high content of double bonds. Oxidation of ω3-PUFA occurs through auto oxidation, photo oxidation and enzymatic mechanisms, of which auto oxidation is the most abundant [12]. Auto oxidation of ω3-PUFA is the interaction between these fatty acids and the atmospheric oxygen, and it involves the production of free radicals through three phases: initiation, propagation and termination. The initiation phase is induced with an initiator, such as oxygen or heat, and it leads to the production of free radicals. In the propagation phase, fatty acids undergo addition of oxygen leading to the production of hydro peroxides, also known as primary oxidation products (POPs). Hydro peroxides are relatively instable and, when the oxidation progresses, they decompose to a group of compounds including aldehydes and kitones, known as secondary oxidation products (SOPs). The termination phase is the interaction between free radicals or between a free radical and an antioxidant to produce stable compounds ending the oxidation reaction [13-15].

 

Variety of methods have been developed to measure lipid oxidation, including measuring POPs by peroxide value (PV) and SOPs by anisidine value (AV). These two values are combined in one equation to evaluate the overall oxidation through total oxidation value (TOTOX). Global organization for EPA and DHA omega-3 (GOED) set the levels - shown in table-1 - by which a fish oil supplement is accepted throughout the stated life time of the product (shelf-life). [16].

 

Table-1: limits of PV, AV, and TOTOX set by GOED

Value

Limit

PV

5 meq /kg

AV

20

TOTOX

26

 

Oxidation of ω3-PUFA has deleterious effects on food characteristics and health. Hydro peroxides consume the endogenous antioxidants that are present in the food [17]. They also react with proteins which results in changing the functionality of these proteins and reduces the nutritional value of amino acids [18]. SOPs are responsible for the rancidity of food since they include volatile compounds that produces off-aromas and off-flavors [19]. Concerning effects on health, Awada et al. revealed that feeding mice with oxidized ω3-PUFA diet led to increasing levels of plasma inflammatory markers [20]. Another study showed that feeding pregnant rats with oxidized fish oil was associated with an important increase in the odds of newborn mortality compared to groups fed with unoxidized fish oil, suggesting that oxidation products were the underlying reason of the low survival rates [21]. Thus, evaluation of lipid oxidation levels in food and food supplements is vital. The aim of this study was to evaluate lipid oxidation levels in some fish oil supplements purchased from Syrian pharmacies, and to monitor the progress of oxidation through a one-year period.

 

2. MATERIALS AND METHODS:

Chemicals:

Glacial acetic acid, sodium thiosulphate and chloroform (Hemedia Laboratory, India). Potassium iodide (Labcheme, India). n-hexane (Merck, Germany). p-Anisidine (Sigma-Aldrich, Germany).

 

Sample collection:

Samples of three brands of fish oil soft-gelatin capsules close to their production date were purchased from three local pharmacies. Two batches of brands A and B, and one batch of brand C were analyzed.

 

Oil mass was measured according to [22], 10 capsules were weighed (m1), pierced, washed with hexane and left until they dried. The empty capsules were then re-weighed (m2) and the oil mass was calculated as: m1-m2.

 

Peroxide Value (PV):

Determination of PV, expressed as melliequevalent of O2/kg oil (meq/kg), was conducted according to the official method of AOAC [23]. A sample of the previously obtained fish oil (approximately 1g) was weighed into a 100 ml Erlenmeyer flask and dissolved in 6 ml of acetic acid and chloroform mixture (3:2 v:v). The next step was the addition of 0.1ml of saturated potassium iodide followed by stirring the flask for one minute. At this point, 6ml of distilled water was added and titration was performed with 0.01M sodium thiosulfate solution until the yellow color was almost gone. Then, 0.1ml of starch solution 0.1% was used as an indicator and titration was completed by sodium thiosulfate solution until the blue color disappeared. A blank experience was conducted, replacing the oil with distilled water. PV was calculated according to the following equation: PV = S* M* 1000/m

 

where S is the volume of sodium thiosulfate corrected to the blank, M is the molarity of sodium thiosulfate, and m is the sample weight.

 

Anisidine Value:

AV is a measurement of SOPs, mainly aldehydes. American Oil Chemists' Society (AOCS) defines AV as ''100 times the optical density measured at 350 nm in a 1 cm cuvette of a solution containing 1g of the oil in 100 ml of a mixture of solvent and reagent'' [24]. AV was measured according to [25]. A sample of previously obtained fish oil (approximately 0.25g) was weighed into a 25ml volumetric flask, dissolved and made up to volume with n-hexane.

 

The absorbance (A1) of this solution was measured against n-hexane at 350 nm. The next step was to pipette 5 ml of the oil solution to a test tube A and 5 ml n-hexane to a test tube B. Next, 1ml of p-anisidine in glacial acetic acid (0.25% w/v) was added to each tube. The test tubes were shaken and left in dark for 10 minutes. The absorbance (A2) of the content of tube A was measured against tube B. AV was calculated according to the following equation: AV = 25 *(1.2 A2 – A1)/sample weight

 

where A1 is the absorbance of oil in n-hexane corrected to the absorbance of n-hexane, A2 is the absorbance of oil in n-hexane with p-anisidine corrected to the absorbance of n-hexane with p-anisidine.

 

Total Oxidation value:

TOTOX is a measurement of the entire oxidation process, and it gives a better picture of the overall quality of the sample by combining the primary and secondary oxidation. TOTOX was calculated according to the following equation: TOTOX= 2PV + AV [26].

 

All the tests were performed in triplicate in 4 points: at the beginning of the study, in 3 months, in 6 months, and in 12 months.

 

Statistical analysis:

The data obtained in this study were performed using Excel 2013. The results of the analyses are presented as Mean ± STD of three triplicate measurements.

 

Differences were analyzed for statistical significance using Student's t test. Statistical significance was assumed at P < 0.05.

 

3. RESULTS AND DISCUSSION:

Oil Mass:

Results of oil mass of capsules are shown in table-2.

 

Table-2: Measured and claimed oil mass. Results are expressed as mean ± STD, n=3

Brand

Oil mass (g) (Mean±STD)

Label value (g)

A

0.24±0.01

0.25

B

0.24±0.02

0.25

C

0.99±0.03

1.0

No significant differences were found between measured values of oil mass and claimed values on the label of each brand.

 

Determination of PV, AV, and TOTOX:

Figure-1, Figure-2, and Figure-3 show the results obtained for PV, AV, and TOTOX, respectively.

 

Figure-1: PV of the three brands

 

 

Figure-2: AV of the three brands

 

Figure-3: TOTOX of the three brands

 

 

Table-3: PV, AV, and TOTOX of the three brands in 4 time points. Values are expressed as mean ± STD, n=3, a,b,c: significant differences in the same row, a: significant difference from baseline (0 point), b: significant difference between 3 and 6 time points, c: significant difference between 6 and 12 time points

 

 

Time (months)

 

 

0

3

6

12

Brand A

PV (meq/kg)

23.65± 0.74

32.49±1.02

34.01±0.68

27.85±0.74

AV

13.58±0.38

14.61±0.28

14.73±0.17

21.48±0.38

TOTOX

60.88±1.46

79.58±1.96

82.74±1.31

77.17±1.46

Brand B

PV (meq/kg)

27.7±0.97

36.27±0.82

36.57±0.9

31.24±0.66

AV

17.23±0.43

20.25±0.69

23.53±0.57

23.82±0.49

TOTOX

72.62±2.05

92.78±2.14

96.67±2.04

86.3±1.05

Brand C

PV (meq/kg)

2.87±0.24

3.06±0.39

3.02±0.43

4.39±0.67

AV

6.85±0.23

6.93±0.14

7.07±0.1

7.08±0.1

TOTOX

12.66±0.46

13.04±0.72

13.1±0.91

15.85±1.37

 

 

Detailed data are summarized in Table-3

PV of brands A and B were (23.65±0.74meq/kg) and (27.7±0.97meq/kg), respectively, exceeding the limit set by GOED (5meq/kg) from the beginning of the study, then they witnessed a statistically significant increase in the next two points (3 months and 6 months). The values went down significantly reaching (27.85±0.74) for brand A and (31.24±0.66) for brand B by the end of the study.

 

As for AV of brand A, it was acceptable (13.58±0.38) in the beginning, and it rose in the following phases that it was above the acceptable value (20) by the end of the period. On the other hand, AV of brand B was (17.23±0.43) in the first phase, but it exceeded the allowed range from the third month peaking at (23.82±0.49) in the last point of the study.

 

With respect to TOTOX values of brands A and B, they were above the limit of 26 from the beginning, continued to increase significantly until they reached a peak of (82.74±1.31) for brand A and (96.67±2.04) for brand B in the sixth month, finished by a significant decrease in the twelfth month.

 

This pattern of oxidation, considering reduction of PV while AV was increasing, is well known in the literature. This is because of the progression of oxidation by decomposing of hydroperoxides, which gives rise to SOPs [27], although more studies with more developed technologies (such as gas chromatography) is needed to be affirmative of this pattern.

 

Regarding the high oxidation levels of brands A and B, similar results were reported by Albert et al. who found out that most of the fish oil supplements in New Zealand exceeded the acceptable limits set by GOED. In their study, 36 brands of fish oil supplements were analyzed for PV, AV and TOTOX. Eighty-three percent of the products exceeded the recommended PV levels, 25% exceeded AV acceptable values and 50% were above the recommended limits of TOTOX [22]. Other researchers evaluated oxidation levels of 49 brands in Canada, and they reported that 17%, 41%, and 39% of the total analyzed samples exceeded PV, AV, and TOTOX, respectively [28].

 

Turning to the values of brand C, the initial PV was (2.87±0.24) and even though it witnessed a minor rise for the next points, it remained under the limit set by GOED. Similarly, AV and TOTOX were (6.85±0.23) and (12.66±0.46), respectively in the beginning, and they continued to rise for the following phases. However, they did not exceed the allowed ranges by the end of the study.

 

 

Bannenberg and colleagues tested 47 fish oil products in New Zealand of which 72%, 86%, and 77% complied with GOED limits of PV, AV, and TOTOX, respectively. Five of the tested fish oil products were within one year of shelf-life expiry, and were retested after one year. Concurring with the outcomes of our study TOTOX increased, almost reaching the limits of (26) [29].

 

Looking at the low levels of brand C comparing to other two, an important point to take into consideration is that although capsules of brand C were held in dark containers, capsules of brand A and B were put in transparent ones, exposing the capsules to light which can induce the oxidation process through photooxidation [30]. Moreover, fish oils are notably susceptible to oxidative deterioration during processing due to their high content of PUFA [13], so invalid processing of capsules of brands A and B might be the underlying reason of their high oxidation levels.

 

No data were written on brand A or brand B labels regarding their contents besides fish oil. Label of brand C, contrarily, stated that the product contains α-tocopherol (vitamin E), which is known to act as an effective antioxidant by scavenging free radicals [31]. Several studies have demonstrated the efficacy of adding tocopherols in lowering oxidation levels of fish oil [32, 33]. This draws attention to the importance of adding antioxidants during the production of fish oil supplements and evaluating the efficacy of these additives in protecting PUFA from oxidation.

 

The current study evaluated the oxidative status of fish oil supplements for three brands sold in Syria. Our study reveals that 2 out of 3 brands (brand A and brand B) are considered low-quality products in terms of oxidative stability according to GOED. In fact, this can be correlated to the price of each brand. Brand C is more expensive than the other two by more than four doubles. Different outcomes were reported by a study conducted to test oxidation of fish oil supplements in Australia. The study acknowledged that price did not correlate with oxidative status of the supplements [34].

 

Although several clinical studies were conducted to find out the positive effects of ω3-PUFA supplementation on health, some of these studies revealed no overall effect for these fatty acids on the risk of all-cause mortality, cardiac death, sudden death, myocardial infarction, or stroke [35]. The conflict outcomes might be explained by the oxidation levels of supplements used in these studies. Therefore, we highly recommend that oxidation levels of ω3-PUFA supplements must be evaluated prior to any clinical research.

 

4. CONCLUSION:

Fish oil consumption has grown in popularity due to its health benefits. However, consuming oxidized fish oil supplements might do the opposite, since oxidation products are harmful. This study tested the oxidative status of three brands of fish oil supplements sold in Syria. Two out of 3 are considered highly oxidized according to GOED, and only one brand met the criteria for oxidation limits. Fish oil supplements that contain vitamin E might be a better choice for consumers. The oxidative state of fish oil supplements must be stated before conducting clinical trials on them.

 

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Received on 22.05.2019           Modified on 18.06.2019

Accepted on 02.07.2019         © RJPT All right reserved

Research J. Pharm. and Tech. 2019; 12(11):5333-5337.

DOI: 10.5958/0974-360X.2019.00925.9