fatty acids, phospholipids and zoosterols levels of the ...

E.I. Adeyeye et al. / IJAIR

ISSN: 2278-7844

FATTY ACIDS, PHOSPHOLIPIDS AND ZOOSTEROLS LEVELS OF THE MUSCLE, SKIN AND LIVER OF BUSHPIG (POTAMOCHOERUS LARVATUS): DIETARY IMPLICATIONS E.I. Adeyeye*, A.J. Adesina and A.A Aladegbemi Department of Chemistry, Faculty of Science, Ekiti State University, PMB 5363, Ado Ekiti, Nigeria *Correspondence to: Prof. E.I. Adeyeye, Ph.D, Department of Chemistry , Faculty of Science, Ekiti State University, PMB 5363, Ado-Ekiti, Nigeria, E-mail: [email protected] Telephone: +2348035782925

© 2013 IJAIR. ALL RIGHTS RESERVED

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ABSTRACT The levels of fatty acids, phospholipids and zoosterols were determined in the muscle, skin and liver of the bushpig. The bushpig samples were collected from bush hunters who killed them in the wild, oven dried to constant weight and homogenised with a grinder. The total lipid values ranged from 1.46-4.56g/100 g dry weight. The SFA levels ranged from 39.5 % -47.6 %, MUFA levels ranged from 33.5 % - 37.4 %, PUFA levels ranged from 18.9 %-23.1 % of total sample weight fatty acids. Also total unsaturated fat ranged from 52.4 % - 60.5 %, n-6/n-3 range was 5.45-10.8, PUFA/SFA range was 0.398-0.585 and MUFA/SFA was 0.704-0.947. Total phospholipids levels ranged from 293 mg/100 g356 mg/100 g with phosphatidylcholine predominating in all the samples. The only significant sterol observed was cholesterol with a range of 316 mg/100 g -383 mg/100 g. The trend in the total lipids was liver > skin > muscle. In fatty acids, skin was best concentrated in: PUFA, MUFA, total unsaturated fat, PUFA/SFA, MUFA/SFA, EPSI, AA/DGLA, LA/ALA and EPA/DHA whereas muscle was only best in SFA. In the phospholipids and sterols the trend was: liver > skin > muscle. In energy distribution, the trend was skin (144 kJ/100 g) > liver (125 kJ/100 g) > muscle (51.5 kJ/100 g). Samples were good sources of EFA, phospholipids, cholesterol but generally low in total lipids to cause dietary anxiety. Key words: Bushpig, muscle, skin, liver, lipids composition, nutritional impact

INTRODUCTION The bushpig [Potamochoerus larvatus (F. Cuvier, 1822)] (1) a member of the pig family, lives in forests, woodland, riverine vegetation and reed-beds in East and Southern Africa. Possibly introduced populations are also present in Madagascar and in Comoros (2, 3). Nigeria is described as one of the possible sources of the bushpig (4). Bushpigs are mainly nocturnal and are several subspecies, among them the southern bushpig (P. llarvatus koiropotamus), whose range includes much of Southern Africa, apart from arid regions such as the Karoo. Adult bushpigs stand 66 to 100 cm at the shoulder, and weigh from 55 to 150 kg (5). They resemble the domestic pig, and can be identified by their blunt, muscular snouts, small eyes, pointed, tufted ears and buckled toes. Their colour varies from reddish-brown to dark brown and becomes darker with age. Both sexes have a lighter-coloured mane which bristles when the animal becomes agitated. The upper parts of the face and ears are also lighter in colour. Their sharp tusks are not very long and are inconspicuous. Unlike the warthog, the bushpig runs with its tail down. Males (boars) are normally larger than females.


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The bushpig is closely related to the red river hog, Potamochoerus porcus, with which it interbreeds. The bushpig is distinguished by its less gaudy markings, hair, and larger size. Many pig populations display physical characteristics intermediate to both species. Bushpigs are quite social animals and are found in sounders of up to 12 members. A typical group will consist of a dominant male and a dominant female, with other females and juveniles accounting for the rest. Litters of three to four young are born in summer (September to November) after a gestation period of approximately four months. Bushpigs can be very aggressive, especially when they have young. They are omnivorous and their diet could include roots, crops or carrion, as well as new-born lambs. They grunt softly while foraging, and make a long, resonant growl as an alarm call. Still distributed over a wide range, the bushpig occurs from Ethiopia and Somalia to eastern and southern DR Congo and southwards to Cape and KwaZulu-Natal Province in South Africa (5), and Madagascar and the Comoros, probably after having been introduced (3). In Nigeria, the bushpig meat is cherished as an animal protein source in preference to the domesticated pig meat. However, there is paucity of information on the nutritional value of the bush pig lipids composition hence this study. Therefore, the lipids composition study of the muscle, skin and liver of the bushpig will improve information on the nutritional value of the animal and may also improve or even add information to the food composition table. MATERIAL AND METHODS 1. Collection of samples The muscle (with the skin) was taken from the thigh muscle of the hind leg and the liver of the bush pig were collected from the bush hunter who killed the animal (male) from the wild at Ado- Ekiti, Ekiti State, Nigeria. The samples were collected in the month of June, 2011. Samples were washed with distilled water and then wrapped separately in aluminium foil and frozen at -4 oC for 5 days before samples were prepared for analysis. The hairs of the skin were carefully removed after deeping the skin in hot water to recover the edible portion. The samples were cut into small pieces and dried at 95 oC until constant weight and ground into fine powder. 2. Determination of ether extract An aliquot (0.25 g) of each part was weighed in an extraction thimble and 200 ml of chloroform/methanol (2:1) was added. The covered porous thimble containing the sample was extracted for 5 h using a Soxhlet extractor. The extraction flask was removed from the heating mantle


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when it was almost free of solvent, oven dried for 1 h, cooled in a desiccator and the weight of dried oil was determined (6). Determinations were in duplicate. 3. Preparation of fatty acid methyl esters and analysis A 50 mg aliquot of the dried oil was saponified for 5 min at 95 oC with 3.4 ml of 0.5 MKOH in dry methanol. The mixture was neutralized by 0.7 MHCl and 3 ml of 14 % boron trifluoride in methanol was added. The mixture was heated for 5 min at 90 oC to achieve complete methylation. The fatty acid methyl esters were thrice extracted from the mixture with redistilled n-hexane and concentrated to ml for analysis. The fatty acid methyl esters were analysed using an HP 5890 gas chromatograph (GMI, Inc, Minnesota, USA) fitted with a flame – ionization detector (FID) linked to an HP ChemStation integrator. Nitrogen was used as the carrier gas with a flow rate of 20-60ml/min. The oven programme was: initial temperature at 60 oC ramping at 10 oC/min for 20 min, held for 4 min, with a second ramping at 15 oC/min for 4 min and held for 10 min. The injection temperature was 250 oC and the detector temperature was 320 oC. A capillary column 30 m x 0.25 mm was packed with a polar compound (HP INNOWAX) onto a diameter of 0.25 µm was used to separate the eaters. A spilt injection was used with a ratio of 20:1. The identification of fatty acid methyl esters (FAMEs) was performed by external standards submitted to the same processes of manipulation as the experimental samples. FAMEs were identified by comparison of their retention times with those of standard mixtures and their areas were automatically integrated using nonadecanoic acid methyl ester (C19:0) as internal standard (6). Determinations were in duplicate. 4. Zoosterol analysis Aliquots of the dried oil were added to screw capped test tubes. The samples were saponified at 95 oC for 30 min, using 3 ml of 10 % KOH in ethanol, to which 0.20 ml of benzene was added to ensure miscibility. Deionised water (3 ml) was added and 2 ml of hexane was used in extracting the nonsaponifiable materials. Three extractions, each with 2 ml of hexane, were carried out for 1 h, 30 min and 30 min respectively, to achieve complete extraction of the sterols. Hexane was concentrated to 1 ml for gas chromatographic analysis (6). Determinations were in duplicate. 5. Phospholipids analysis Using a modified method of Raheja et al (7), 0.01 g of the dried oil was added to test tubes. Any remaining solvent was removed by passing a stream of nitrogen gas over the oil. Then 0.40 ml of chloroform was added, followed by addition of 0.10 ml of the chromogenic solution. The tube was heated to 100 oC in a water bath for 1 min 20 sec, cooled to room temperature, 5 ml of hexane was added and the tube was shaken gently several times. After separation of the solvent and aqueous layers, the hexane layer was recovered and concentrated to 1.0 ml for analysis. Analysis was performed using the gas chromatograph packed with a polar capillary column 30 m x 0.25 mm packed with a polar compound (HP5) onto a diameter 0.25 µm. The oven programme was: initially at 50 oC, ramping at 10 oC/min for 20 min, held for 4 min, a second ramping at 15 oC/min for 4 min and held for 5 min. The injection temperature was 320 oC. As previously described, a split injection type was used © 2013 IJAIR. ALL RIGHTS RESERVED

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having a split ratio of 20:1. Peaks were identified by comparison with the known standards. Determinations were in duplicate. 6. Quality assurance Standard chromatograms were prepared for sterols, phospholipids and fatty methyl esters which were then compared with respective analytical results; calibration curves were prepared for all the standard mixtures and correlation coefficient was determined for each fatty acid (35 number), sterol (7 number) and phospholipid (5 number). Correlation coefficient > 0.95 was considered acceptable. 7. Conversion of percentages of total fatty acids to fatty acids per 100 g of food At the data source and reference database levels, values for individual fatty acids are usually expressed as percentages of total fatty acids. At the user database levels, values per 100 g of food are required. A conversion factor derived from the proportion of the total lipid present as fatty acids is required (9) for converting percentages of total fatty acids to fatty acids per 100 g of food. Total lipid level was multiplied by a conversion factor of 0.953 to convert muscle and skin separately to total fatty acids and 0.741 to convert liver to total fatty acids (10). For fatty acids expressed in g per 100 g total fatty acids, precision is best limited to the 0.1 g/100 g level, trace being set at < 0.06 g/100 g of fatty acids (11). 8. Calculation of uncertainty interval percentage Further on quality assurance, the fatty acid values were subjected to the calculation of uncertainty interval percentage. Certified reference materials (CRMs) play a critical role in validating the accuracy of nutrient data. A range of food CRMs with assigned values and uncertainty intervals (UIs) for many nutrients are currently supplied by several organisations (12). The fatty acids evaluated in certified reference materials (CRMs) were: C16:0, C18:0, C16:1, C18:1, C18:2 (6, 12). CRM value was only available for cholesterol among the sterols but none for other sterols and phospholipids in the samples. The CRMs used here were from Wolf (13). The standard uncertainty intervals used were adopted from beef-pork blend (13). 9. Statistical analysis Statistical analysis (8) was carried out to determine the mean, standard deviation, coefficient of variation in per cent. Also calculated were the Chi-square (X2) values. The X2 was subjected to the table (critical) value at α 0.05 to see if significant differences existed in the values of fatty acids, sterols and phospholipids between the bushpig samples. RESULTS In Table 1, the crude fat varied between 1.46/100 g to 4.56 g/100 g. The values were slightly heterogenous with the coefficient of variation per cent (CV %) being 49.6. The total energy coming from the total fatty acids had a range of 51.5 to 144 kJ/100 g. © 2013 IJAIR. ALL RIGHTS RESERVED

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Table 2 contains the saturated fatty acid (SFA) and monounsaturated fatty acid (MUFA) values. The following acids under this section were not detected: C2:0, C3:0 whereas the following fatty acids (FAs) recorded 0.00 % of the total fatty acids: C4:0, C5:0 (only in muscle), C6:0 (in all samples), C8:0 (only in muscle), C10:0 (in all the samples) and C18:1 (trans-11) in muscle only. Low levels of C12:0, C24:0, C14:1 (cis-9), C24:1 (cis-15), C18:1 (trans-) and C18:1 (trans-9) with each of their values being less than 1.0 % of total fatty acids in all the samples. C22:0 was < 1.0 % in the liver, C16:1 (cis9) was < 1.0 % in the muscle whereas C18:1 (trans-11) was 0.00 % in the muscle but < 1.0 % in both skin and liver. The coefficient of variation per cent (CV %) was highest in C18:1 (trans-11) with a value of 89.5, lowest in C16:0 with a value of 2.40 % whereas many CV % values were 22.8. Among the SFA, C16:0 was the most concentrated in all the samples and had a range of 22.1-23.1 % total FA with CV % of 2.40. This was closely followed by C18:0 with values of 11.6-19.9 % and CV % of 27.7. For the two FAs the trend of concentration was skin (23.1 %) > muscle (22.9 %) > liver (22.1 %) for C16:0 and in C18:0: muscle (19.9 %) > liver (19.6 %)> skin (11.6 %). Among the C18:1 cis MUFA, C18:1 (cis -6) was the most concentrated with values of 14.1-16.4 % and CV % of 7.90 whereas C18:1 cis-9 followed with values of 12.7-17.0 % with higher CV % of 14.7. The best source of C18:1 cis-9 was in the skin (17.0 %). Total MUFA (cis) range was 32.9-36.1 % and CV % of 6.55. All the C18:1 trans levels were mostly lower than the C18:1 cis with higher levels of CV %. Total C18:1 trans was 0.65-1.36 % with CV % of 50.0. Table 2 was subjected to Statistical analysis with the results in the row columns being insignificantly different at α = 0.05. On the other hand all the vertical column results were significantly different among themselves at α = 0.05. It should be noted particularly for cases where there are more than two categories or groups that X2 cannot indicate or specify where significant difference lies, a situation similar to that found in ANOVA. However, post hoc tests that provide solution to the problem when encountered in ANOVA cannot be applied to Chi-square test. In case of X2-test, the category that contributes the highest proportion is declared as one that differs significantly from others (8). This meant that in the SFA, C16:0 was highest in all the samples whereas in the MUFA, C18:1cis-6 was highest in muscle, C18:1 cis-9 was highest in skin whereas C18:1cis-6 and C18:1 cis-9 shared the highest levels in the liver. In Table 3, PUFA n-6 and n-3 FA composition of the bush pig samples were depicted. Only C20:4 cis5, 8, 11, 14 had 0.00 % total fatty acid observed for it in the muscle whereas C22:6 cis-4, 7, 10, 13, 16, 19 was not detected in the liver. The most prominent FA was C18:2 cis-9, 12 in all the samples with values ranging from 9.27-12.3 % and closely followed by C20:4 cis -5, 8, 11, 14 in skin and liver (4.62-5.49 %). C18:3 cis-9, 12, 15 had a range of 0.856-1.37 %. Most CV % were 22.8 except in C18:2 cis-9, 12 (14.6 %), C20:4 cis-5, 8, 11, 14 and C22:6 cis-4, 7, 10, 13, 16, 19 (87.5 % each). In statistical analysis of the results in Table 2, all the row columns were not significantly different whereas in the vertical columns all the three samples were significantly different in their PUFA values with C18:2 cis-9, 12 contributing the highest value in each vertical column. In Table 4, the summary of the quality characteristics of the fatty acids profile is depicted. In SFA distribution, skin (39.5 %) < liver (45.8 %) < muscle (47.6 %); for MUFA distribution, muscle (33.5 %) < liver (35.2 %) < skin (37.4 %) and PUFA distribution, muscle (18.9 %) < liver (19.0 %) < skin © 2013 IJAIR. ALL RIGHTS RESERVED

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(23.1 %). The MUFA + PUFA which ranged from 52.4-60.5 % showed that the total fatty acids were mostly unsaturated. The n-6/n-3 range was 5.45-10.8, PUFA/SFA was slightly low at 0.398-0.585. The essential PUFA status index (EPSI) was fair at a range of 0.540-0.618; the AA/DGLA was good in muscle and skin at a range of 2.82-4.54. The precursors of eicosanoids ratio (LA/ALA) were good at a range of 9.75-12.1 and EPA/DHA was 0.528-0.532 but not calculated in the liver. Most CV % values in Table 4 were low at 0.45-50.0. Fatty acid per 100 g sample as food in the bushpig samples is shown in Table 5. The trend of contribution was as follows: SFA, 0.662-1.55 g/100 g; MUFA, 0.466-1.454 g/100 g; PUFA, 0.2630.898 g/100 g and MUFA + PUFA, 0.729-2.352 g/100 g. The energy contribution by each fatty acid group to the overall energy contribution is depicted in Table 6. Total energy contribution from the bushpig samples ranged between 51.5-144 kJ/100 g. A cursory look at these figures would show them to be equivalent to the fatty acid per cent. For examples: SFA energy was 24.5-57.3 kJ/100 g (39.5-47.6 %, see Tables 4 and 6); MUFA energy was 17.2-53.8 kJ/100 g (33.5-37.4 %, see Tables 4 and 6); PUFA energy was 9.73-33.2 kJ/100 g (18.9-23.1 %, see Tables 4 and 6); MUFA + PUFA energy was 27.0-87.0 kJ/100 g (52.4-60.5 %, see Tables 4 and 6). Table 7 shows the uncertainty interval per cent (UIP) for the fatty acids and cholesterol. Most of the literature Table UIP levels were correspondingly higher than the present results in all the bushpig samples. Phospholipids level (mg/100 g) of the samples are in Table 8. The overall values were generally high at 293-356 mg/100 g (dry weight) and CV % of 9.72 %. The statistical analysis of the results in Table 8 showed that no significant differences occurred in the values at row columns. On the other hand all the levels of the phospholipids for muscle, skin and liver were significantly different at each vertical column with phosphatidylcholine contributing the highest concentration in each vertical column. In Table 9, the zoosterol levels are shown. The cholesterol level predominantly dominated the zoosterol levels in all the samples. Cholesterol levels ranged between 316-383 mg/100 g while other sterols ranged between 3.25 x 10-4 – 8.86 x 10 -3 mg/100 g (muscle), 3.53 10 -4 -8.85 x10 -3 mg/100 g (skin) and 1.30 x 10 -4 – 8.85 x 10-3 mg/100 g (liver) with CV % range of 0.20-45.0. The statistical analysis showed that significant difference occurred in cholesterol row column and in the vertical columns, the sterol values were significantly different for muscle, skin and liver with cholesterol providing the highest concentration in each vertical column. DISCUSSION The crude fat levels of 1.46-4.56 g/100 g in Table 1 were found to be much lower than other animal protein sources found in literature. Some literature crude fat levels were: 67 % (beef fat), 72 % (lamb fat), 71 % (pork fat), duck meat and skin (43 %), calf liver (7 %), chicken, meat and skin (18 %) (14). The calculated energy from the crude fat gave values of 51.5-144 kJ/100 g. For somebody that © 2013 IJAIR. ALL RIGHTS RESERVED

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requires 2500 daily calories and 15 % coming from fat oil consumption, this translates to 41.6 g of fat per day. From the present report, a person for optimum weight loss, may reduce the overall fat/oil consumption by eating bushpig meat. The crude fat levels in the bushpig showed that each would supply different levels of energy with a low level of CV % (45.7). All the short-chain fatty acids in the samples were either not detected or 0.00 % of total fatty acids. Medium-chain fatty acids have 8 to 12 carbon atoms and are common in butterfat and the tropical oils. In the present samples only C12:0 was present in minor quantities in the samples (0.179-0.799 %), Table 2. Like the short-chain fatty acids, these fats have antimicrobial properties; are absorbed directly for quick energy; for this reason, they are less likely to cause weight gain than olive oil or commercial vegetable oils (15); and contribute to the health of the immune system. Long-chain fatty acids have from 14 to 18 carbon atoms and can be either saturated, monounsaturated or polyunsaturated. Myristic acid (C14:0) is a ubiquitous component of lipids in most living organisms, but usually at level of 1-2 % only. In the present samples C14:0 ranged from 1.10-1.33 %, Table 2. However, it is more abundant in cow milk fat, some fish oils and in those seed oils enriched in medium-chain fatty acids (e.g. coconut and palm kernel). In Oreochromis niloticus C14:0 formed 6.59 % FA in the skin and 4.19 % in the muscle (16) whereas it was 1.12 % (skin) and 1.05 % (muscle) of Tongue sole fish (17). This fatty acid is found very specifically in certain proteolipids, where it is linked via an amide bond to an N-terminal glycine residue, and is essential to the function of the protein components. Palmitic acid (C16:0) is usually considered the most abundant SFA in nature, and it is found in appreciable amounts in the lipids of animals, plants and lower organisms. It comprises 20-30 % of the lipids in most animal tissues, and it is present in amounts that vary from 10 to 40 % in seed oils. The present results are in conformity with C16:0 range in animal lipids with present C16:0 range of 22.1-23.1 % being the highest concentrated SFA. Stearic acid (C18:0) is the second most abundant SFA in nature, and again it is found in the lipids of most living organisms. In these samples C18:0 occupied the second highest position (11.6 -19.9 %) in the SFA group. In lipids of some commercial importance, it occurs in the highest concentrations in ruminant fats (milk fat and tallow) or in vegetable oils such as cocoa butter, and in industrially hydrogenated fats. It can comprise 80 % of the total fatty acids in gangliosides. The other SFA present in minor level was behenic acid (C22:0), a member of the very-long-chain fatty acids. The total SFA (Table 4) of 39.5-47.6 % could easily compare favourably with literature values; they are: 43 % (beef fat), 50 % (lamb fat), 37 % (pork fat), 33 % (chicken, meat and skin), 27 % (duck, meat and skin), 30 % (calf liver) (14). Oleic acid [9c-18:1 or 18:1 (n-9)] is by far the most abundant monoenoic fatty acid in plant and animal tissue, both in structural lipids and in depot fats. It comprised a level range of 12.7-17.0 % FA being the highest of the cis-MUFA. Olive oil contains up to 78 % of oleic acid, and it is believed to have especially valuable nutritional properties as part of the Mediterranean diet. It has a number of important biological properties, both in the free and esterified form. Oleic acid is the biosynthetic precursor of a family of fatty acids with the (n-9) terminal structure and with chain-lengths of 20-24 or more. Petroselinic acid (6c-18:1) occurs up to a level of 50 % or more in seed oils of the umbelliferae © 2013 IJAIR. ALL RIGHTS RESERVED

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family, including carrot, parsley and coriander. In the present report, petroselinic acid occupied the second highest position in the cis-18:1 FA with values ranging from 14.1-16.4 % and CV % of 7.90. All the C18: trans- MUFA values were all lower than 1.00 % in each of the samples. In nearly all higher organisms, including many bacteria, yeasts, algae, plants and animals, double bonds are introduced into fatty acids by an aerobic mechanism that utilizes preformed fatty acids as the substrate. Molecular oxygen and a reduced pyridine nucleotide (NADH or NADPH) are required cofactors. Thus in animals and yeasts, the coenzyme A ester of octadecanoic (stearic) acid is converted directly to oleoyl-CoA by a concerted removal of hydrogen atoms from carbons 9 and 10 (Dstereochemistry in each instance). The stearoyl-CoA desaturase system is in the endoplasmic reticulum membrane with the active centre exposed to the cytosol, and consists of three proteins, cytochrome b5 reductase, cytochrome b5, and the desaturase, which contains two atoms of iron at the active site. Fe2+

Fe3+

octadecanoyl-CoA +O2

oleoyl-CoA + H2O stearoyl-CoA desaturase

Membrane-bound enzymes are notoriously difficult to purify, but the evidence suggests that the yeast ∆9 desaturase consists of two membrane spanning regions with the bulk of the protein protruding into the cytosol. The enzyme has much in common with hydroxylases and contains eight essential histidine residues that coordinate with the di-iron centre at the active site. The cytochrome b5 component is fused to the desaturate and is believed to facilitate electron transfer from NADH reductase to the catalytic di-iron core. Palmitoileate is synthesised from palmitate by a similar mechanism. Subsequently, oleate can be chain elongated by two carbon atoms to give longer-chain fatty acids of the (n-9) family, while palmitoleate is the precursor of the (n-7) family of fatty acids. In mammalian systems the elongases are known to be distinct enzymes that differ from those involved in the production of longer-chain polyunsaturated fatty acids. Alpha- and beta-oxidation can also occur to give shorter chain components of the two families. 9-18:1

11-20:1

13-22:1

15-24:1

18:1(n-9)

20:1(n-9)

22:1(n-9)

24:1(n-9)

9-16:1

11-18:1

13-20:1

15-22:1

16:1(n-7)

18:1(n-7)

20:1(n-7)

22:1(n-7)

etc.

etc.


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Petroselinic acid (6-18:1) in seed oils of the Umbelliferae is synthesised by an enzyme that removes hydrogens from position 4 of palmitate, before the resulting 4-16:1 is elongated by two carbon atoms. 16:0 desaturation 4-16:1 elongation 6-18:1 (petroselinic acid). The relative proportion of MUFA/SFA is an important aspect of phospholipid compositions and changes to this ratio have been claimed to have effects on such disease states as cardiovascular conditions and cancer. For example, they have been shown to have cyto-protective actions in pancreatic β-cells. MUFA/SFA values in the bushpig ranged from 0.704-0.947 (Table 4) which could be regarded as being good ratios. cis-Monoenoic acids have desirable physical properties for membrane lipids in that they are liquid at body temperature, yet are relatively resistant to oxidation. They are now recognised by nutritionists as being beneficial in the human diet. Current nutritional thinking appears to be that dietary trans-monoenoic fatty acids, both from ruminant fats and from industrial hydrogenation processes, should be considered as potentially harmful and in the same light as SFA. However trans-monoenoic acid in the bushpig was low at total level of 0.651.36 % (Table 4) and may not constitute any health hazard. In Table 3, we have four FAs in the group of long- chain whereas we have seven FAs in the group of very-long-chain FAs. The two essential fatty acids (EFAs) are C18:2 cis-9, 12 and C18:3 cis-9, 12, 15 with respective values of 9.27-12.3 % and 0.856-1.37 %. Another important long- chain fatty acid is gamma-linolenic acid (GLA). It formed a level of 0.792-1.20 % in the bushpig. It is found in evening primrose, borage and black currant oils. The body makes GLA out of omega-6 linoleic acid and uses it in the production of substances called prostaglandins, localized tissue hormones that regulate many processes at the cellular level. Eicosadienoic acid [C20:2 cis-11, 14 or 20:2(n-6) all- cis-11, 14eicosadienoic acid] or homo-gamma-linoleic acid is an uncommon naturally occurring PUFA. It is not enriched in any particular tissue, it is rare in all lipid classes. Dietary sources include herring and menhadenoils, cattle liver, swine brain lipid, shark oil (18). Homo-γ-LA had levels of 0.146-0.235 % in the bushpig total FAs. The homo-γ-LA inhibits the binding of [3H]-LTB4 to pig neutrophil membrane with a Ki of 3 µm. The levels of C18:2cis –9, trans-11 ranged from 0.437-0.698 % as seen in Table 3. These levels were much lower than their corresponding LA (9.27-12.3 %) (Table 3). In Table 2 vaccenic acid levels ranged from 0.00 %-0.805 %. Conjugated linoleic acids make up a group of polyunsaturated FAs found in meat and milk from ruminant animals and exist as a general mixture of conjugated isomers of LA. Of the many isomers identified, the cis-9, trans-11 CLA isomer (also referred to as rumenic acid or RA) accounts for up to 80-90 % of the total CLA in ruminant products (19). Naturally occurring CLAs originate from two sources: bacterial isomerization and/or biohydrogenation of trans- fatty acids in the adipose tissue and mammary glands (20). Microbial biohydrogenation of LA and alpha linolenic acid (aLA) by an anaerobic rumen bacterium, Butyrivibrio fibrisolvens is highly dependent on rumen pH (21). Grain consumption decreases rumen pH, reducing B. fibrisolvens activity, conversely grass-based diets provide for a more favourable rumen environment for subsequent © 2013 IJAIR. ALL RIGHTS RESERVED

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bacterial synthesis (22). Rumen pH may help to explain the apparent differences in CLA content between grain and grass-finished meat products. De novo synthesis of CLA from 11t-c 18:1 TVA has been documented in rodents, dairy cows and humans. Studies suggest a linear increase in CLA synthesis as the TVA content of the diet increased in human subjects (23). The rate of conversion of TVA to CLA has been estimated to range from 5-12 % in rodents to 19-30 % in humans (23). True dietary intake of CLA should therefore consider native 9c11t-C18:2 (actual CLA) as well as the 11tC18:1 (potential CLA) content of foods (24). The very low levels of both 11t-C18:1 and 9c 11t-C18:2 gave concrete evidence that bushpig is not a ruminant animal. The relative values of PUFA in all the samples made them averagely important in diet. The eicosanoids help regulate blood clot formation, blood pressure, blood lipid (including cholesterol) concentration, the immune response, the inflammation response to injury and infection and many other body functions (25). A deficiency of n-6 fatty acids in the diet leads to skin lesions. A deficiency of n-3 FAs leads to subtle neurological and visual problems. Deficiencies in PUFA produce growth retardation, reproductive failure, skin abnormalities and kidney and liver disorders. However, people are rarely deficient in those FAs (26). The relative amounts of PUFA and SFA in oils is important in nutrition and health. The ratio of PUFA/SFA (P/S ratio) is therefore important in determining the detrimental effects of dietary fats. The higher the P/S ratio the more nutritionally useful is the oil. This is because the severity of atherosclerosis is closely associated with the proportion of the total energy supplied by SFA and PUFA (27). The present PUFA/SFA varied between 0.398-0.585 (Table 4). A minimum value of PUFA/SFA ratio recommended is 0.45 (28). The n-6 and n-3 FAs have critical roles in the membrane structure (29) and as precursors of eicosanoids, which are potent and highly reactive compounds. Since they compete for the same enzymes and have different biological roles, the balance between the n-6 and n-3 FAs in the diet can be of considerable importance (30). From several in vivo and in vitro studies with different animal species it is well known that aLA, LA and oleic acid (18:1 n-9) compete for the same ∆-6 desaturase in the metabolic cascade. Dietary studies on rats and other animals have shown that aLA is a strong suppressor of n-6 FA metabolism, whereas 10 times as much LA is required to give an equal suppression of n-3 metabolism (31). In vitro studies on rat liver micosomes have confirmed that the n-6 and n-3 substrate competition occurs at several steps in the microsomal pathway (32). Information from above and other literature sources showed that the ratio of n-6 to n-3 in the diet should be between 5:1 and 10:1 (30) or 4-10 g of n-6 FAs to 1.0 g of n-3 FAs (33). The UK Department of Health recommends an ideal ratio of n-6/n/3 of 4.0 at maximum (28). Values higher than the maximum value are harmful to health and may promote cardiovascular diseases (34). The present n-6/n-3 values ranged from 5.45-10.8 (Table 4). As LA is almost always present in foods, it tends to be relatively more abundant in animal tissues. This is supported in the present report as follows: C18:2 (n-6) ranged as 9.27-12.3 % whereas C18:3 (n-3) ranged as 0.856-1.37 % leading to LA/aLA of 9.75-12.1. In turn, these FAs are the biosynthetic precursors in animal systems of C20 and C22 PUFAs, with 3-6 double bonds, via sequential desaturation and chain-elongation steps (desaturases in animal tissues can only insert a double bond on the carboxyl side of an existing double bond) (35). © 2013 IJAIR. ALL RIGHTS RESERVED

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A high ratio between AA and DGLA, as an indicator of ∆-5 desaturase activity, in the skeletal muscle phospholipids has been related to good insulin sensitivity; the AA/DGLA in muscle and skin of the bushpig ranged from 2.82-4.54 (Table 4) which is good enough. For the assessment of the essential PUFA status of an individual, the total amount of the various EFA and PUFA in plasma or erythrocyte phospholipids is a useful indicator (36). The following are further used as additional status markers to reliably assess the functional PUFA status (37). The best known marker is Mead acid (C20:3n-9). The synthesis of this fatty acid is promoted if there are insufficient concentrations of LA and aLA to meet the need for the synthesis of long-chain PUFA. EPA and DHA inhibit Mead acid synthesis; the presence of Mead acid indicates a general shortage of all essential PUFA. Our result had a ratio of EPA/DHA in muscle and skin as 0.528-0.532 and no Mead acid was produced. Another suitable indicator of essential PUFA status is the essential PUFA status index (EPSI), which is the ratio between all essential PUFA (the sum of all n-3 and n-6 FAs) and all nonessential unsaturated FAs (the sum of all n-7 and n-9 FAs). The higher the EPSI status indexes the better the essential PUFA status. Our present results had values of EPSI range of 0.540-0.618 which were all above average. Finally, if there is a functional shortage of DHA, the body starts to synthesize the most comparable long-chain PUFA of the n-6 family, osbond acid (22:5n-6). Therefore, under steady state conditions, the ratio between DHA and osbond acid is a reliable indicator of the functional DHA status (38). Hence the PUFA in the bushpig samples cannot cause functional distress. Literature results for MUFA were: beef fat (48 %), lamb fat (39 %), pork fat (41 %), chicken, meat and skin (42 %), duck, meat and skin (54 %); their corresponding PUFA were: beef fat (4 %), lamb fat (5 %), pork fat (15 %), chicken, meat and skin (19 %), duck, meat and skin (12 %), calf, liver (26 %) (14). All the bushpig MUFA levels are favourably comparable to the literature values shown above as the MUFA bushpig levels were 33.537.4 % (Table 4). On the other hand all the PUFA levels in the bushpig samples were either higher or very close to the literature PUFA levels shown above since the samples had levels of 18.9-23.1 % (Table 4). The C18:2 levels from the literature were (in %): rabbit, lean (13.5), brain, sheep (0.4), liver: ox (7.4), sheep (5.0), pig (14.7), calf (15.0) (39) which are highly comparable with the bushpig results at 9.27-12.3 %. From literature for C18:3, we had (in %): rabbit, lean (0.7), brain, sheep (-), liver: ox (2.5), sheep (3.8), pig (0.5), calf (1.4) (9); these results are highly comparable to the bushpig C18:3 results of 0.856-1.37 %. The fatty acid per 100 g sample as food in bushpig is shown in Table 5. From Table 1 total fatty acids in the samples ranged from 1.39-3.89 g/100 g. Table 5 showed that all these values were accounted for in the fatty acid as food. The SFA contributed 0.662-1.55 g/100 g as food, MUFA contributed 0.4661.45 g/100 g as food, PUFA contributed 0.263-0.898 g/100 g as food and MUFA+PUFA contributed 0.729-2.352 g/100 g as food. The PUFA content of some selected foods for LA are (mg/100 g): beef (muscles only), 80; calf’s kidney, 61; chicken (breast), 980; chicken (leg), 370; horse meat (average), 160; pork (muscles only), 110; turkey (breast), 180; turkey (leg), 750; veal (muscles only), 197. For aLA (mg/100 g): calf’s kidney, 61; chicken (breast), 2.7; chicken (leg), 10; horse meat (average), 260; pork (muscles only), 25 and veal (muscles only) 9.1 (39). The present levels of LA and aLA are in good relationship with the literature values having values of 163-478 mg/100 g (linoleic acid) and 1945 mg/100 g (linolenic acid). © 2013 IJAIR. ALL RIGHTS RESERVED

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Energy contribution (kJ/100 g) of the bushpig samples is shown in Table 6. Total energy contribution from the samples ranged from 51.5-144 kJ/100 g. For optimum weight loss, reduce your overall fat/oil consumption to a sensible level. A level of 15-20 % of your total calories should come from fat-and the majority of that should be essential fatty acids. To determine how many grams of fats this translates into, you multiply your total daily calories by 15 % (20 % for the high-end of the range) and then divide the result by 9, which is the number of calories in a gram of fat. Here is an example: 2500 daily calories x 0.15 = 375.375/9 = 41.6 or 42 grams of total fat per day-the bulk of which should be EFAs. It is known that 20 % energy from fat is consistent with good health. With 41.6 g of total fat per day, the bushpig that contain 1.39-3.89 g/100 g fatty acids will only be able to be a minor source of energy to its consumer. Table 7 shows the uncertainty interval per cent (UIP) for the fatty acids and cholesterol. Most of the literature UIP levels were correspondingly higher than the present results in all the samples. However values of UIP for C16:1 were all greater than the critical UIP where samples UIP ranged from 6.4819.5 whereas the critical value was 2.58. Beef –pork blend UIP information was used in comparing to the bushpig values. The fact that the majority of the sample UIP values were less than the critical values attested to the quality of the analytical determinations. Also the correlation determined for all the standards: fatty acids, phospholipids and sterols, all had values ranging as follows: 0.998330.99997 (fatty acids), 0.99909-0.99999 (phospholipids) and 0.99920-0.99994 (sterols); all the correlation values were greater than 0.95 which is the critical correlation for acceptance of these types of analytical results. Both the correlation values and the UIP values attested to the quality of the determinations. In Table 8 are the various types of phospholipids in the samples. Phospholipids are not essential nutrients: they are just another lipid and, as such, contribute 9 kcalories per gram of energy. The total phospholipids level ranged from 293-356 mg/100 g showing the bushpig to be a good source of phospholipids. The major phospholipids in all samples was phosphatidylcholine (lecithin) which ranged from 207-256 mg/100 g. Lecithin is usually the most abundant phospholipid in animals and plants, often amounting to almost 50 % of the total, and as such it is the key building block of membrane bilayers. This observation is true for lecithin values in these results. Phosphatidylcholines (PC) are a class of phospholipids that incorporate choline as a headgroup. They are a major component of biological membranes and can be easily obtained from a variety of readily available sources such as egg yolk or soy beans from which they are mechanically extracted or chemically extracted using hexane. They are also a member of the lecithin group of yellow-brownish fatty substances occurring in animal and plant tissues. Phosphatidylcholines are such a major component of lecithin that in some contexts the terms are sometimes used as synonyms. However, lecithin extract consists of a mixture of phosphatidylcholine and other compounds. It is also used along with sodium taurocholate for stimulating fed-and fasted-state biorelevant media in dissolution studies of highly-lipophilic drugs. Phosphatidylcholine is more commonly found in the exoplasmic or outer leaflet of a cell membrane. It is thought to be transported between membranes within the cell by phosphatidylcholine transfer protein (PCTP) (40). Phosphatidylcholine also plays a role in membrane-mediated cell signalling and © 2013 IJAIR. ALL RIGHTS RESERVED

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PCTP activation of other enzymes (41). At birth and throughout infancy, phosphatidylcholine concentrations are high (as high as 90 % of the cell membrane), but it is slowly depleted throughout the course of life, and may drop to as low as 10 % of the cellular membrane in the elderly. As is such, some researchers in the fields of health and nutrition have begun to recommend daily supplementation of phosphatidylcholine as a way of slowing down senescence (42) and improving brain functioning and memory capacity (43). In addition to the increased calorie burden of a diet rich in fats like phosphatidylcholine, a recent report has linked the microbial catabolites of phosphatidylcholine with increased atherosclerosis through the production of choline, trimethylamine oxide and betaine (44). Cephalin (phosphatidylethanolamine, PE) was the second largest concentrated entity in all the samples. Cephalin is found in all living cells, although in human physiology it is found particularly in nervous tissue such as the white matter of brain, nerves, neural tissue and in spinal cord (16). Lysophosphatidylcholine was in the third position in the level of concentration (4.56-4.86 mg/100 g). Phosphoinositides (PI) (P1, P2, P3) play important role in lipid signalling, cell signalling and membrane trafficking (16). The PI was of minor concentration in the samples (3.72-4.24 mg/100 g). Partial hydrolysis of lecithin with removal of only one fatty acid yields a lysophosphatidylcholine (45). An example of alterations in enzymic activity related to association of a membrane-bound protein with lipid is that of phenylalanine hydroxylase, which catalyzes the conversion of phenylalanine to tyrosine. The activity of this enzyme, which is attached to the endosplasmic reticulum, is enhanced fifty fold in the presence of lysophosphatidylcholine, with which it is probably complexed in the hepatic cell (46). Phosphatidylserine (Ptd-L-Ser or PS) was the least concentrated in the samples (9.85 x 10 -1 – 2.38 mg/100 g). It is usually kept on the inner leaflet, the cytosolic side, of cell membranes by an enzyme called flippase. When a cell undergoes apoptotic cell death, PS is no longer restricted to the cytosolic part of the membrane, but becomes exposed on the surface of the cell. PS has been demonstrated to speed up recovery, prevent muscle soreness, improve well-being, and might possess ergogenic properties in athletes involved in cycling, weight training and endurance running. PS supplementation promotes a desirable hormonal balance for athletes and might attenuate the physiological deterioration that accompanies overtraining and/or overstretching (46). In recent studies, PS has been shown to enhance mood in cohort of young people during mental stress and to improve accuracy during tee-off by increasing the stress resistance of golfers (47). The US Food and Drug Administration (USFDA) had stated that consumption of PS may reduce the risk of dementia and cognitive dysfunction in elderly persons (16). PS can be found in meat, but most abundant in the brain and innards such as liver and kidney. PS results in the bushpig samples were lower than the value in beef (69), pork (57), European pilchard (sardine) of 16.0 mg/100 g (47). In Table 9 we have the sterols content. Only cholesterol had levels greater than 0.1 mg/100 g in all the samples. Actually the value of cholesterol ranged from 316-383 mg/100 g. Cholesterol is a highmolecular-weight alcohol that is manufactured in the liver and in most human body cells. Like SFA, the cholesterol we make and consume plays many vital roles. Along with SFA, cholesterol in the cell membrane gives our cells necessary stiffness and stability. This is why serum cholesterol levels may go down temporarily when we replace SFA with PUFA oils in the diet (48). Cholesterol acts as a precursor to vital corticosteroids, hormones that help us deal with stress and protect the body against © 2013 IJAIR. ALL RIGHTS RESERVED

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heart disease and cancer; and to the sex hormones like androgen, testosterone, estrogen and progesterone. Cholesterol is a precursor to vitamin D, a very important fat-soluble vitamin needed for healthy bones and nervous system, proper growth, mineral metabolism, muscle tone, insulin production, reproduction and immune system function. The bile salts are made from cholesterol. Bile is vital for digestion and assimilation of fats in the diet. Recent research shows that cholesterol acts as an antioxidant (49). This is the likely explanation for the fact that cholesterol levels go up with age. As an antioxidant, cholesterol protects us against free radical damage that leads to heart disease and cancer. Cholesterol is needed for proper function of serotonin receptors in the brain (50). Serotonin is in the body’s natural “feel good” chemical low cholesterol levels which have been linked to aggressive and violent behaviour, depression and suicidal tendencies. Mother’s milk is especially rich in cholesterol and contains a special enzyme that helps the baby utilise the nutrient. Babies and children need cholesterol-rich foods throughout their growing years to ensure proper development of the brain and nervous system. Dietary cholesterol plays an important role in maintaining the health of the intestinal wall (51). This is why low-cholesterol vegetarian diets can lead to leaky gut syndrome and other intestinal disorders. Cholesterol levels in literature from many animal protein sources were much comparable to the present results. Values in mg/100 g were: fish (50-60), egg yolk (1260), meat and poultry (60-120), brain (2000-3000), liver (300-350) (14); others were rabbit, lean (71), brain, sheep (2200), liver: ox (270), sheep (430), pig (260) and calf (370) (9). Most authorities, but not all, recommend a reduction in dietary cholesterol to around 300 mg or less per day (14); all the bushpig samples were higher than this value having levels of 316-383 mg/100 g. CONCLUSIONS Bushpig parts (muscle, skin, liver) are sources of low level of total fats whish are mostly unsaturated which might not promote cardiovascular diseases. The fatty acids are also high in the essential fatty acids. The samples were good sources of phospholipids and contained cholesterol as the only major sterol. For people on low fat animal protein source, the bushpig is recommended as their animal protein source. REFERENCES 1. Seydack, A. Potamochoerus larvatus. In: IUCN 2008. IUCN Red List of Threatened Species. Downloaded on 5 April 2009. 2. Wilson, Don E., Reeder, DeeAnn M., Mammal Species of the World. 3rd ed., Johns Hopkins University Press, 2 vols, Baltimore, 2005. 3. Wilson, D. E., Reeder, D.M., Mammal Species of the World. 2nd ed., Smithsonian Institution Press, Washington DC, 1993. 4. Vercammen, P., Seydack, A. W. H., Oliver, W.L. R., The bush pigs (Potamochoerus porcus and P. larvatus. In: Oliver, W.L.R. (ed), Pigs, Peccaries and Hippos: Status Survey and Action Plan. IUCN, Gland, Switzerland, pp 93-101, 1993. © 2013 IJAIR. ALL RIGHTS RESERVED

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5. Kingdon, J., The Kingdon Guide to African Mammals. Academic Press, London, 1977. 6. AOAC, Official Methods of Analysis. 18th ed., Association of Analytical Chemists, Washington DC, 2005. 7. Raheja, R.K., Kaur, C., Singh, A., Bhatia, I.S., New colorimetric method for the quantitative estimation of phospholipids without acid digestion. J Lipid Res, 14: 695-697, 1973. 8. Oloyo, R.A., Fundamentals of Research Methodology for Social and Applied Sciences. ROA Educational Press, Ilaro, Nigeria, 2001. 9. Paul, A.A., Southgate, D.A.T., McCance and Widdowson’s The Composition of Foods. 4th ed., HMSO, London, 1978. 10. Anderson, B.A., Comprehensive evaluation of fatty acids in foods. VII. Pork products. J Am Diet Assoc, 69: 44-49, 1976. 11. Greenfield, H., Southgate, D.A.T., Food Composition Data, Production, Management and Use. FAO, Rome, 2003. 12. Phillips, K.M., Wolf, W.R., Patterson, K.Y., Sharpless, K.E., Amanna, K.R., Holden, J.M., Summary of reference materials for the determination of the nutrient composition of foods. Accred Qual Assur, 12:126-133, 2007. 13. Sullivan, D.M., Carpenter, D.E., Methods of Analysis for Nutrition Labelling. AOAC International, Arlington VA, 1993. 14. Bender, A., Meat and Meat Products in Human Nutrition in Developing Countries. FAO Nutrition Paper 53. FAO, Rome 1992. 15. Portillo, M.P.J., Serra, F., Simon, E., del Barrio, A.S., Palou, A., Energy restriction with highfat diet enriched with coconut oil gives higher UCPI and lower white fat in rats. Int P Obes Relat Metab Discord, 22(10): 974-979, 1998. 16. Adeyeye, E.I., Levels of fatty acids, phospholipids and sterols in the skin and muscle of Tilapia (Oreochromis niloticus) fish. La Rivista Italiana Delle Sostanze Grasse- vol LXXXVIIIGennaio/Marzo: 46-55, 2011. 17. Adeyeye, E.I., Owokoniran, S., Popoola, F.E., Akinyeye, R.O., Fatty acids, phospholipids and sterols levels of skin and muscle of Tongue sole fish. Pak J Sci Ind Res Ser A: Phy. Sci., 54(3): 140-148, 2011. 18. Yagaloff, K.A., Franco, L., Suniko, B., Essential fatty acids are antagonists of the leukotriene B4 receptor. Prostaglandins Leukotriene Essential Fatty Acids, 52: 293-297, 1995. 19. Nuernberg, K., Nuernberg, G., Ender, K., Lorenz, S., Winkler, K., Rickert, R., Steinhart, H., Omega-3 fatty acids and conjugated linoleic acids of longissimus muscle in beef cattle. European Journal of Lipid Science Technology, 104: 463-471, 2002. 20. Griinari, J.M., Corl, B.A., Lacy, S.H., Chouinard, P.Y., Nurmella, K.V., Bauman, D.E., Conjugated linoleic acid is synthesized endogenously in lactating dairy cows by delta-9 desaturase. J. Nutr, 130: 2285-2291, 2000. 21. Pariza, M.W., Park, Y., Cook, M.E., Mechanisms of action of conjugated linoleic acid: evidence and speculation. Proceedings for the Society of Experimental Biolgy and Medicine, 32: 853-858, 2000. © 2013 IJAIR. ALL RIGHTS RESERVED

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22. Bessa, R.J.B., Santos-Silva, J., Ribeiro, J.M. R., Portugal, A.V., Reticulo-rumen biohydrogenation and the enrichment of ruminant edible products with linoleic acid conjugated isomers. Livestock Production Science, 63: 201-211, 2000. 23. Turpeinen, A.M., Mautanen, M., Aro, A., Saminen, I., Basu, S., Palmquist, D.L., Bioconversion of vaccenic acid to conjugated linoleic acid in humans. Am J Clin Nutr, 76, 504510, 2002. 24. Adlof, R.O., Duval, S., Emken, E.A., Biosynthesis of conjugated linoleic acid in humans. Lipids, 35: 131-135, 2000. 25. Whitney, E.N., Cataldo, C.B., Rolfes, S.R., Understanding Normal and Clinical Nutrition. 4th ed., West Publishing Company, New York, 1994. 26. Tapiero, H., Nguyen, Ba G., Couvreur, P., Tew, K. D., Polyunsaturated fatty acids (PUFA) and eicosanoids in human health and pathologies. Biomedicine and Pharmacotherapy, 56: 215-222, 2002. 27. Honatra, G., Dietary fats and arterial thrombosis. Haemostasis, 2: 21-52, 1974. 28. HMSO, Nutritional aspects of cardiovascular disease (report on health and social subjects No. 46). HMSO, London, 1994. 29. Kinsella, J.E., Possible mechanisms underlying the effects of n-3 polyunsaturated fatty acids. Omega-3 News, 5: 1-5, 1990. 30. WHO/FAO, Fats and Oil in Human Nutrition. Report of a Joint Expert Consultation, FAO Food and Nutrition Paper 57. WHO/FAO, Rome, 1994. 31. Holman, R.T., The slow discovery of the importance of omega-3 essential fatty acids in human health. J Nutr, 128: 427S-433S, 1998. 32. Mohrhauer, H., Christiansen, K., Gan M.V., Deubig, M., Holman, R.T., Chain elongation of linoleic acid and its inhibition by other fatty acids in vitro. J Biol Chem, 242: 4507-4517, 1967. 33. Canadian Government Publishing Center, Nutrition Recommendations: The Report of the Scientific Review Committee. Canadian Government Publishing Center, Ottawa, Canada, 1990. 34. Bhouri, A.M., Bouhlet, I., Chouba, L., Hammami, M., El Cafsi, M., Chaouch, A., Total lipid content, fatty acid and mineral compositions of muscles and liver in wild and farmed sea bass (Dicentrarchus labrax). Afr J Food Sci, 4(8): 522-530, 2010. 35. Berg, J. M., John, L., Typoczko, L., Lubert, S., Biochemistry. 6th ed., WA Freeman and Company, New York, 2007. 36. Hornstra, G., Essential fatty acids, pregnancy, and pregnancy complications: a roundtable discussion. In: Sinclair, A., Gibson, R. (eds), Essential Fatty Acids and Eicosanoids. American Oil Chemists’ Society, Champaign, pp 177-182, 1992. 37. Benatti, P., Peluso, G., Nicolai, R., Calvani, M., Polyunsaturated fatty acids: biochemical, nutritional and epigenetic properties. J Am Coll Nutr, 33(4): 281-302, 2004. 38. Neuringer, M., Connor, W.E., Lin, D.S., Barstad, L., Luck, S., Biochemical and functional effects of prenatal and postnatal omega-6 fatty acid deficiency on retina and brain in rhesus monkeys. Proc Natl Acad Sci USA, 83: 4021-4025, 1986. © 2013 IJAIR. ALL RIGHTS RESERVED

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39. Souci, S.V., Fachmann, W., Kraut, H., Food Composition and Nutrition Tables 1989/1990. Wissenschaftliche Verlagsgesellschaft mbH Stuttgart. 40. Wirtz, K.W., Phospholipid transfer proteins. Ann Rev Biochem, 60 (13): 73-99, 1991. 41. Kanno, K., Wu, M.K., Agate, D.A., Fanelli, B.K., Wagle, N., Scapa, E.F., Ukomadu, C., Cohen, D.E., Interacting proteins dictate function of the minimal START domain phosphatidylcholine transfer protein/StarD2. J Biol Chem, 282 (42): 30728-30736, 2007. 42. Mei-Chu, H., Koji, S., Riki, Y., Masao, S., Katsumi, I. Learning behaviour and cerebral protein kinase C, antioxidant status, lipid composition in senescene-accelerated mouse: influence of a phosphatidylcholine-vitamin B12 diet. Britsh Journal of Nutrition, 86: 163-171, 2001. 43. Chung, Shu-Ying, Tomoe, M., Eiko, U., Kayoko, U., Rieko, H., Noriko, Y., Tasnunobu, M., Toyohiko, K., Shigeru, Y., Administration of phosphatidylcholine increases brain acetylcholine concentration and improves memory in mice with dementia. The Journal of Nutrition, 125: 1484-1489, 1995. 44. Wang, Z., Klipfell, E., Bennett, B.J., Koeth, R., Levison, B.S., Duqar, B., Feldstein, A.E., Britt, E.B., Fu, X, Chung, Y.M., Wu, Y., Schauer, P., Smith, J.D., Allyee, H., Tang, W.H., DiDonato, J.A., Lusis, A.J., Hazen, S. L., Gut flora metabolism of phosphatidylcholine promotes cardiovascular disease. Nature, 472 (7341): 57-63, 2011. 45. White, A., Handler, P., Smith, E.L., Principles of Biochemisstry. 5th ed., McGraw-Hill Kogakusha Ltd., Tokyo, Japan, 1973. 46. Starks, M.A. Starks, S.L., Kingley, M., Purpura, M., Jager, R., The effects of phosphatidylserine on endocrine response to moderate intensity exercise. Journal of the International Society of Sports Nutrition, 5: 11, 2008. 47. Alter, T., More than you wanted to know about fats and oils. Sundance Natural Food Online retrieved 2006-08-31. 48. Jones, P.J., Regulation of cholesterol biosynthesis by diet in humans. The Am J Clin Nutr, 66(2): 438-446, 1997. 49. Cranton, E.M., Frackelton, J.P., Free radical pathology in age-associated diseases: treatment with EDTA chelation, nutrition and antioxidants. Journal of Holistic Medicine, Spring/Summer, 6(1): 6-37, 1984. 50. Engelberg, H., Low serum cholesterol and suicide. Lancet, March 21, 339: 727-728, 1992. 51. Alfin-Slater, R.B., Aftergood, L., Lipids, Modern Nutrition in Health and Disease. 6th ed., Lea and Febiger, Philadelphia, 1980.


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Table 1 :Crude fat and total fatty acid in g/100g of muscle, skin and liver of Bushpig Parameters

Muscle

Skin

Liver

Mean

Crude fat

1.46

4.08

4.56

3.37

a

1.39

3.89

3.38

51.5

144

125

Total fatty acids

Total energy (kJ/100g)

SD

CV%

1.67

49.6

2.89

1.32

45.7

107

48.9

45.7

a = muscle (1.46 x 0.593), skin (4.08 x 0.953), liver (4.56 x 0.741); SD = standard deviation; CV% = coefficient of variation per cent.


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Table 2: The saturated fatty acid (SFA) and monounsaturated fatty acid (MUFA) composition in % of total weight Fatty Acid

Muscle

Skin

Liver

Mean

SD

X2

CV%

Remark

SFA Acetic Propionic Butyric

Pentanoic Caproic Caprylic Capric Lauric Myristic Palmitic Stearic Arachidic Behenic Lignoceric

C2: 0 C3: 0 C4: 0 C5: 0 C6: 0 C8: 0 C10: 0 C12: 0 C14: 0 C16: 0 C18: 0 C20: 0 C22: 0 C24: 0

X2 Remark

0.00 0.00 0.00 0.00 0.00 0.179 1.10 22.9 19.9 1.66 1.52 0.189 227 *

0.00 0.00 0.00 0.00 0.799 0.589 1.11 1.33 23.1 22.1 11.6 19.6 1.41 1.04 1.29 0.956 0.160 0.118 200 223 *

*

0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.523 0.315 1.18 0.130 22.8 0.545 17.0 4.72 1.36 0.31 1.26 0.29 0.156 0.035 -

-

0.00 0.00 0.00 0.00 0.00 60.3 7.20 2.40 27.7 22.8 22.8 22.8 -

0.00 0.00 0.00 0.375 0.030 0.030 2.62 0.140 0.130 0.029 -

NS NS NS NS NS NS NS NS NS NS -

-

-


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MUFA(cis) C14:1(cis-9) 0.543 Palmitoleic C16:1(cis-9) 0.819 Petroselinic C18:1(cis-6) 16.4 Oleic C18:1(cis-9) 12.7 Gondoic C20:1(cis-11) 1.69 Erucic C22:1(cis-13) 0.525 Nervonic C24:1(cis-15) 0.19 MUFA (trans) t- petroselenic C18:1(trans-6) 0.60 Elaidic C18:1(trans-9) 0.053 vaccenic C18:1(trans-11) 0.00 X2 96.2 Myristoleic

Remark

-

*

0.461 0.340 2.47 2.17 14.1 15.1 17.0 15.1 1.44 1.06 0.446 0.329 0.160 0.118 0.506 0.045 0.805 95.3 *

0.373 0.034 0.594 95.9

0.448 1.81 15.2 14.9 1.40 0.434 0.156 0.491 0.045 0.467 -

*

-

0.102 0.880 1.20 2.19 0.318 0.098 0.035

22.8 48.4 7.90 14.7 22.8 22.8 22.8

0.111 0.010 0.417 -

1.81 NS 0.850 NS 0.187 NS 0.641 NS 0.145 NS 0.045 NS 0.016 NS

22.8 22.8 89.5 -

0.051 0.005 0.748 -

NS NS NS -

-

-

-

= not detected or not determined as the case may be; NS = not significantly different at α = 0.05; * = significantly different at α = 0.05; X2 = chi- square test.


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Table 3: Polyunsaturated fatty acid (PUFA) in % of total weight Fatty Acid Conjugated linoleic acid CLA Linoleic acid Gamma-linolenic acid (GLA) Eicosadienoic acid Dihomo-gamma-linolenic acid (DGLA) Arachidonic acid (AA) Docosadienoic acid Alpha-linolenic acid (ALA) Eicosatrienoic acid (ETE) Eicosapentaenoic acid (EPA ) Docosahexaenoic acid (DHA) X2 Remark

Muscle C18:2(trans-9,11) 0.698 C18:2(cis-9,12) 11.7 C18:3(cis-6,9,12) 1.20 C20:2(cis-11,14) 0.235 C20:3(cis-8,11,14) 1.93 C20:4(cis-5,8,11,14) 0.00 C22:2(cis-13,16) 0.188 C18:3(cis-9,12,15) 1.37 C20:3(cis-11,14,17) 1.01 C20:5(cis-5,8,11,14,17) 0.188 C22:6(cis-4,7,10,13,16,19) 0.356 66.06 *

Skin

Liver

0.593 12.3 1.02 0.199 1.64 4.62 0.160 1.163 0.860 0.160 0.301 62.87

0.437 9.27 0.792 0.146 1.21 5.49 0.118 0.856 0.634 0.118 17.97

*

*

SD

CV%

X2 Remark

0.576 11.1 1.00 0.193 1.59 3.37 0.156 1.31 0.835 0.156 0.219 -

0.131 1.62 0.205 0.044 0.363 2.63 0.035 0.257 0.190 0.035 0.192 -

22.8 14.6 20.4 22.8 22.8 87.5 22.8 22.8 22.8 22.8 87.5 -

0.059 NS 0.475 NS 0.084 NS 0.781 NS 0.166 NS 4.47 NS 0.117 NS 0.116 NS 0.086 NS 0.015 NS 0.335 NS -

-

-

-

Mean

-

-


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Table 4: Summary of quality characteristics of the fatty acids profile Fatty Acid

Muscle

Skin

Total PUFA n-6 Total PUFA n-3 Total PUFA (n-3 + n-6) MUFA total(cis) MUFA total(trans) MUFA total SFA total MUFA + PUFA n-6 / n-3 PUFA/SFA MUFA/SFA EPSI AA/DGLA LA/ALA EPA/DHA

16.00 2.93 18.9 32.9 0.65 33.5 47.6 52.4 5.45 0.398 0.704 0.564 2.82 9.75 0.528

20.60 2.49 23.1 36.1 1.36 37.4 39.5 60.5 8.28 0.585 0.947 0.618 4.54 12.1 0.532

Liver 17.43 1.61 19.0 34.2 1.00 35.2 45.8 54.2 10.8 0.415 0.769 0.540 -11.7 --

Mean

SD

CV%

18.3 3.25 2.71 0.311 20.3 2.40 34.5 2.26 1.00 0.502 35.5 2.76 44.3 4.25 55.7 4.25 8.18 2.68 0.466 0.103 0.807 0.126 0.574 0.040 3.68 1.22 11.2 1.26 0.53 0.00283

17.8 11.5 11.8 6.55 50.0 7.78 9.60 0.45 32.8 22.1 15.6 6.97 33.2 11.3 0.53

EPSI = essential PUFA status index, AA = arachidonic acid; DGLA = dihomo-gamma-linolenic acid; LA = linoleic acid; ALA- alpha- linolenic acid; EPA- eicosapentaenoic acid; DHA- docosahexaenoic acid. Mean, SD and CV% of AA/DGLA and EPA/DHA were based on muscle and skin only.

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Table 5: Fatty acids level (g) in the Bushpig per 100g muscle, skin and liver samples as food Fatty acid

Muscle

Skin

Liver

Mean

SD

CV%

C12:0 C14:0 C16:0 C18:0 C20:0 C22:0 C24:0 SFA C14:1(cis-9) C16:1(cis-9) C18:1(cis-6) C18:1(cis-9) C20:1(cis-11) C22:1(cis-13) C24:1(cis-15) MUFA (cis) C18:1(trans-6) C18:1(trans-9) C18:1(trans-11) MUFA (trans) MUFA (total) C18:2(trans-9,11) C18:2(cis-9,12) C18:3(cis-6,9,12) C20:2(cis-11,14) C20:3(cis-8,11,14) C20:4(cis-5,8,11,14) C22:2(cis-13,16) C18:3(cis-9,12,15) C20:3(cis-11,14,17) C20:5(cis-5,8,11,14,17) C20:6(cis-4,7,10,13,16,1 ) PUFA (n-6, total) PUFA (n-3, total) PUFA (n-6+n-3, total) MUFA+ PUFA (total) Total

0.002 0.015 0.319 0.277 0.023 0.021 0.003 0.662 0.008 0.011 0.228 0.177 0.024 0.007 0.003 0.458 0.008 0.0007 0.000 0.009 0.466 0.010 0.163 0.017 0.003 0.027 0.000 0.003 0.019 0.014 0.003 0.005 0.223 0.041 0.263 0.729 1.39

0.031 0.043 0.90 0.451 0.055 0.050 0.002 1.54 0.018 0.096 0.548 0.661 0.056 0.017 0.006 1.404 0.020 0.003 0.031 0.053 1.454 0.023 0.478 0.040 0.008 0.064 0.180 0.006 0.045 0.033 0.006 0.012 0.801 0.097 0.898 2.35 3.89

0.020 0.045 0747 0.662 0.035 0.032 0.0004 1.55 0.011 0.073 0.510 0.510 0.036 0.011 0.004 1.157 0.013 0.001 0.020 0.034 1.189 0.015 0.313 0.027 0.005 0.041 0.186 0.004 0.029 0.021 0.004 0.589 0.054 0.642 1.83 3.38

0.018 0.034 0655 0.463 0.038 0.034 0.0018 1.25 0.012 0.060 0.429 0.449 0.039 0.012 0.004 1.01 0.014 0.002 0.017 0.032 1.04 0.016 0.318 0.028 0.005 0.044 0.122 0.004 0.031 0.023 0.004 0.009 0.538 0.064 0.601 1.64 2.89

0.015 0.017 0.301 0.193 0.016 0.015 0.0013 0.510 0.005 0.044 0.175 0.248 0.016 0.005 0.002 0.491 0.006 0.001 0.016 0.022 0.511 0.007 0.158 0.011 0.003 0.019 0.106 0.002 0.013 0.010 0.002 0.005 0.292 0.029 0.319 0.828 1.32

81.3 49.3 46.0 41.6 42.5 43.1 72.9 40.8 42.8 72.3 40.8 58.2 41.5 41.9 38.2 48.6 43.1 62.5 92.4 69.0 49.2 41.0 49.5 41.2 50.3 42.5 86.6 38.2 42.3 41.8 38.2 55.0 54.3 45.8 53.2 50.5 45.7

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Table 6: Energy contribution (kJ/100g) of Bushpig Samples from their fatty acids Parameter

Muscle

Skin

Liver

Mean

SD

CV%

SFA

24.5(47.6%)

56.8(39.5%)

57.3(45.8%)

46.2(44.3%)

18.8

44.1

MUFA (cis)

16.9(32.9%)

51.9(36.1%)

42.8(34.2%)

37.2(34.4%)

18.2

48.8

MUFA (trans)

0.335(0.65%)

1.96(1.36%)

1.25(1.00%)

1.18(1.00%)

0.815 69.0

MUFA (total)

17.2(33.5%)

53.8(37.4%)

44.0(35.2%)

38.3(40.8%)

18.9

49.5

n-6 PUFA

8.24(16.0%)

29.6(20.6%)

21.8(17.4%)

19.9(18.0%)

10.8

54.3

n-3 PUFA

1.51(2.93%)

3.58(2.49%)

2.01(1.61%)

2.37(2.34%)

1.08

45.6

PUFA (total)

9.73(18.9%)

33.2(23.1%)

23.8(19.0%)

22.2(20.3%)

11.8

53.2

MUFA +PUFA

27.0(52.4%)

87.0(60.5%)

67.8(54.2%)

60.6(55.7%)

30.6

50.6

Total

51.5(100%)

144(100%)

125(100%)

107(100%)

48.9

45.7

Table 7: Uncertainty intervals as per cent of analytical results Parameter

a

C16:0

UIP (muscle)

UIP(skin)

UIP(liver)

25.0

1.31

1.30

1.36

C18:0

17.8

0.804

1.38

0.816

C16:1

2.58

19.5

6.48

7.37

C18:1n-6

40.0

-

-

-

-cis-6

-

2.20

2.55

2.38

-cis-9

-

2.83

2.12

2.38

7.08

-

-

-

-

1.45

1.38

1.83

134

1.49

1.25

1.23

C18:2 -cis Cholesterol a

UIP (critical)

UIP = uncertainty interval per cent.

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Table 8: The phospholipids content of muscle, skin and liver of Bushpig in mg/100g Phospholipids Phosphatidylethanolamine Phosphatidylcholine Phosphatidylserine Lysophosphatidylcholine Phosphatidylinsitol Total X2 Remark

Muscle 74.7 207 2.38 4.78 4.24 293 573

Skin 90.5 231 1.44 4.86 4.17 332 597

*

Liver 90.3 256 9.85x10-1 4.56 3.72 356 682

*

Mean 85.2 231 1.60 4.73 4.05 327 -

*

-

SD 9.05 24.7 0.710 0.158 0.284 31.8 -

CV% 10.6 10.6 44.3 3.30 7.00 9.72 -

-

-

X2 Remark 1.92 NS 5.29 NS 0.774 NS 0.0105 NS 0.036 NS -

-

Table 9: The zoosterols content of muscle, skin and liver of Bushpig in mg/100g Zoosterols Cholesterol Cholestanol Ergosterol Campesterol Stigmasterol Savenasterol Sitosterol Total

Muscle 316 4.34x10-4 3.25x10-4 5.05x10-3 1.66x10-3 8.86x10-3 6.40x10-3 316

X2

1938

Remark

*

Skin

Liver

Mean

377 383 358 -4 -4 4.58x10 2.71x10 3.88x10-4 3.53x10-4 1.30x10-4 2.69X10-4 5.05X10-3 5.03X10-3 5.04X10-3 1.67x10-3 1.65x10-3 1.66x10-3 8.85x10-3 8.85x10-3 8.85x10-3 6.40x10-3 6.40x10-3 6.40x10-3 377 383 359 2259 *

2297 *

90

-

SD

CV%

X2

Remark

37.2 10.4 7.73 NS -4 1.02x10 26.2 0.0067 NS 1.21x10-4 45.0 0.205 NS -6 -7 1.0X10 0.20 4.0x10 NS -6 1.30x10 0.80 2.0x10-7 NS 2.54x10-6 0.03 1.0x10-9 NS 1.58x10-6 0.03 1.0x10-9 NS 37.1 10.3 -

-

-

-

-

-

-

-