Vitamin Levels in Fresh and Smoke-Dried Body Parts of Clarias Gariepinus Depicting the Loss of Vitamins during Heat Processing

Fish Materials

Five pieces of matured Clarias gariepinus were harvested from a local fish vendor who manages the fish ponds located in Basiri quarters of Ado-Ekiti, Ekiti State, Nigeria in the month of December, 2017. After cropping the life fish samples were weighed giving an average of 1.40kg. The fresh samples were sacrificed and dissected to give three parts (head, muscle and liver). The harvested fish samples were later cleaned and transferred into a frozen container for transportation to the laboratory. The parts (head, muscle and liver) required for the analyses were carefully separated and stored in a freezer prior to sample preparation. The parts were later removed from the freezer, rinsed with deionized water, ground by agate mortar and later blended with an Excella Mixer blender.

The fishes were also smoked using African traditional method of smoking called charcoal smoking. The fishes were smoked for about 12 h at moderate to high temperature (80°C-110°C) during which turning over was done at interval of 30 minutes to achieve a uniform smoking. The smoked samples were further dried at low temperature overnight over the ember to ensure that the samples were properly dried. The smoking process involved placing a piece of cardboard over the fishes so as to trap the smoke directly on the fish. The fish parts (head, muscle and liver) were separated from the smoked fish and homogenized using a mortar and pestle before being blended with a blender. Determinations were made in duplicate.

The homogenized samples (smoked and fresh) were stored in glass bottles and kept in a refrigerator at less than 4°C for subsequent analyses. Samples were designated as FL, FM, FH, FT, DL, DM, DH, and DT where F = fresh, L = liver, M = muscle, H = head, D = dry and T = total.

Simultaneous Analysis of Fat and Water Soluble Vitamins

The samples were brought out of the less than 4°C compartment in the laboratory and placed on the bench to allow acclimatizing to the laboratory conditions.

Extraction of Water-Soluble Vitamins

The sample was grinded with the aid of the laboratory mortar and pestle. The accurately weighed 0.100g of ground sample was put into 100ml volumetric flasks and 80ml of deionized water added. After 15min of ultrasonic extraction, the water was added to the volumetric flask mark.

Extraction of Fat-Soluble Vitamins

An accurately weighed 0.125g of ground sample was added into 10ml volumetric flasks and 8ml of CH3OH-

CH2Cl2 (1:1, v/v) was added to the volumetric flask mark. The prepared solution was stored in the dark; and diluted if necessary. Prior to injection, the solutions were filtered through a 0.2µm filter (Millex-GN).

Optimized Chromatography Conditions

Water and fat soluble vitamins were separated simultaneously under the following optimized chromatography conditions combined with valve switching, double injection, envelope-injection and wavelength switching.

Column

Acclaim PA, 3µm, 120A, 3.0 x 150mm for fat soluble. Acclaim C18, 3µm, 120A, 3.0 x 150mm for water soluble.

25°C

Mobile phase

Injection Volume 10µl (Dionex, Technical Note 89) (www.dionex.com). Statistical Evaluation Data results on the pairwise groups of FL/DL, FM/DM, FH/DH and FT/DT were subjected to statistical correlation coefficient (rxy), regression coefficient (Rxy), coefficient of alienation (CA), index of forecasting efficiency (IFE), coefficient of determination or variance (rxy2). Other calculations were grand mean, standard deviation (SD) and coefficient of variation (CV %). The rxy was compared to the critical Table value to see if significant difference existed among each pair comparison results at r = 0.01 [6].

Vitamin Levels in Fresh/Dry Weight Basis Compared For Liver, Muscle and Head

The concentration (mg/100g) of vitamins on fresh and dry bases in the liver of Clarias gariepinus can be seen in Table 1. In the group of vitamins, both fat-soluble and water-soluble vitamins were determined. For the fat- soluble vitamins we have vitamins A, D, E and K; for the water-soluble ones we have the B-complex which were vitamins B1 (thiamine), B2 (riboflavin), B3 (niacin), B5 (pantothenic acid), B6 (pyridoxine), B9 (folic acid), B12 (cyanocobalamin) and vitamin C (ascorbic acid).

loss. The least concentrated fat-soluble vitamin was vitamin D with values of 1.13e-1(FL) to 8.30e-2(DL), loss of 2.96e-2mg/100g or 26.3%; however the highest loss in this group was K (third concentration) with values of 4.36e-1mg/100g to 2.32e-1mg/100g with a difference (loss) of 2.04e-1mg/100g (46.8%). The coefficient of variation (CV%) values among the fat-soluble vitamins ranged from 11.7-43.2 meaning the vitamin E that had the highest concentration had the least variation of 11.7% whereas vitamin K that exhibited the highest difference also exhibited the highest variation between FL and DL with a value of 43.2%. For the vitamin B-complex group, vitamin B3 had the highest concentration of 2.92mg/100g (FL) and 2.63mg/100g (DL) with a difference of 2.92e- 1mg/100g (9.99%). Other B-complex vitamins in the range of 0.10mg/100g (and slightly above) were B6, B2 and B5 whereas those in the range of 0.10 (and slightly above) were B1 and B12. Vitamin B9 had insignificant values of 1.76e-4mg/100g to 1.32e-4mg/100g with a change of 4.39e-5 (25.0%). The CV% values in the B- complex ranged from 7.44-21.8, this meant that heat effect was more drastic in the reduction of vitamin levels in the fat-soluble vitamins than in the water-soluble vitamins since CV% in vitamin C (water-soluble) of 16.6 was within the values of 7.44-21.8. The least concentrated vitamin in the liver was vitamin C with the values of 3.40e-6mg/100g and difference (loss) of 7.14e-7 or 21.0%. Humans, other primates, guinea pigs, and some bats, birds and fish lack a liver enzyme, L-gulono-γ- lactone oxidase, hence such animals cannot synthesize vitamin C in their liver [7]. The total concentration of vitamins in the liver of Clarias gariepinus followed these trends, fresh: 34.5 mg/100g > 29.1 mg/100g; loss of 5.37 In Table 2 we have the concentration values (fresh and dry) of the fish muscle. As in Table 1, vitamin E was the most concentrated vitamin in both fat- and water-soluble groups; however the vitamin E here was much less than in the liver with muscle values of 8.65mg/100g(FM) and 8.10mg/100g(DM) with a change of 5.50e-1mg/100g (6.36% loss). Vitamin D that was least concentrated in the fat-soluble vitamin group in Table 1 came significantly second here among the fat-soluble vitamins with values of 3.59-2.97mg/100g with a difference of 6.20e-1mg/100g (a percentage loss of 17.3 lower than in Table 1 of 26.3%). Vitamin K was third and A was least in the fat-soluble vitamins with respective values of 1.70e-1 to 1.58e- 1mg/100g (and loss of 1.19e-2, 7.01%) and 6.78e-3 to 6.39e-3mg/100g (and loss of 3.81e-4, 5.63%). The fat- soluble vitamins had CV% range of 4.10 -13.4 meaning that their values were relatively close. In the B-complex vitamins, these vitamins had values greater than 2.0mg/100g: B3, B6 and B1; within the range of 0.10mg/100g were B5 and B12 whereas B2 and B9 were in the range of 3.21e-2 to 5.09e-5. The percentage loss to smoking was generally low at values of 3.18-5.78 and CV% of 2.29-4.21. The vitamin C content here was much more appreciable than in Table 1; here the values were 2.07mg/100g (FM) and 1.86mg/100g (DM) with a change of 2.18e-1mg/100g and second highest overall percentage loss of 10.5 whereas vitamin D had the highest overall percentage loss (17.3) in Table 2. The total concentrations in Table 2 were 23.1mg/100g (fresh muscle) and 21.4mg/100g (dry muscle) with a difference of 1.75mg/100g (7.56% loss) and low CV% of 5.55.

mg/100g (15.6%) and CV% of 11.9 meaning that the fresh and dry concentration values were close.

Table 3 has the vitamin concentration values of C. gariepinus head. As in Tables 1 and 2, vitamin E had the highest concentration values but much lower than in Table 2 but much, much lower than in Table 1. Vitamin E here had values of 6.92mg/100g (FH) and 6.75mg/100g (DH) with a low value change of 1.70e-1mg/100g (2.45% loss) and CV% of 1.76. Vitamin D [2.67(FH)-2.56(DH) mg/100g] was the second most concentrated among the fat-soluble vitamins in the head. Difference in vitamin D was 1.11e-1mg/100g (4.14% loss) and low CV% of 2.99. As in Table 2, vitamin K was third here and A was fourth here with respective values of 1.21e-1 to 8.65e-2mg/100g (3.46e-2mg/100g difference and 28.6%loss) with CV% of 23.6 and 5.34e-3 to 5.14e-3mg/100g (1.95e-4mg/100g difference and 3.65% loss) with low CV% of 2.63. As in Table 2, vitamin B1 was the highest concentrated vitamin among the B-complex vitamins. Here, it has values range of 2.24mg/100g (FH)-1.94mg/100g (DH), change value of 3.00e-1 with a percentage loss of 13.4 and low CV% of 10.2. Vitamins B6 and B1 had concentration values greater than 2.00mg/100g whereas B3 had value greater than 1.00mg/100g with vitamins B2, B9, B5 and B12 being in the concentration bracket of 1.2e-1 to 4.48e- 5mg/100g (FH) with corresponding 1.14e-1 to 4.28e- 5mg/100g (FH). Vitamin C in Table 3 had values of 1.79mg/100g (FH) and 1.64mg/100g (DH) having a difference of 1.44e-1mg/100g (a loss of 8.08%) and CV% of 5.95. The vitamin C values here were lower than the values in Table 2 but much higher than the values in Table 1. Generally the CV% values were at a range of 1.76-23.6. Total head vitamins were 18.4mg/100g (FH) and 17.5mg/100g (DH), a change of 9.72e-1mg/100g (5.27%) and low CV% of 3.83.

Table 4 compared the vitamin levels from the fresh samples of Clarias gariepinus liver, muscle and head. The glaring variations in the fresh samples were conspicuously demonstrated in the CV% values. The two least varied CV% values were recorded in vitamin B3 (22.9) and total vitamins (32.6). Other CV% values ranged between 69.9 in vitamin K to 173 in vitamin A meaning that fat-soluble vitamins were the most varied overall. On individual vitamin concentrations, the followings were

Table 4: Concentration (mg/100g) of vitamins in the fresh liver, fresh muscle and fresh head samples of Clarias gariepinus compared. Fresh liver, fresh muscle and fresh head were used to calculate mean, SD and CV%, In Table 5 the counterpart results of the C. gariepinus vitamins were shown on dry weight basis. The dry weight results had their CV% values as varied as observed in Table 4. The CV% range was 18.5-173. Least varied again here as in Table 4 were B3 (18.5%) and total vitamins (26.2%). As in Table 4, vitamin A had CV% of 173. Vitamins of significant concentrations in the dry samples were (mg/100g dry weight): B3 (6.55), B6 (4.99), C (3.50),

Vitamins E and C are considered antioxidants owing to their ability to reduce the stress response in fish [8]. Vitamin C is essential in many metabolic processes including collagen synthesis (tissue repair), protection of cell membranes, metal absorption and detoxication of xenobiotics. In addition, vitamin C is considered an intra- and intercellular reducing agent. It is known that the concentration of ascorbic acid affects the mixed-function oxidases activity that is involved in the metabolism of xenobiotics [9]. Vitamin C content of pike perch, common carp and European catfish was identified to be 1.91mg/100g, 1.14mg/100g and 2.15mg/100g respectively [10]. The vitamin C content determined by Lall & Parazo [11] in white fish muscle had values of 1.0- 5.1mg/100g; reports from seafood of Pacific Northwest were 0.3mg/100g [12]. Vitamin C in the muscle of the sample was 2.07mg/100g (fresh) and 1.86mg/100g (dry) which fell within the values above. Total vitamin C contents were 3.86mg/100g (fresh) and 3.50mg/100g (dry). However, these values fell short of the daily intake of 60-100mg.

Vitamin B12 (cyanocobalamin) is found mainly in fish, meat, poultry and dairy products. Individuals following vegetarian diets are at risk for developing Cbl deficiency owing to suboptimal intake as vitamin B12 (Cbl) is essential for the synthesis of nucleic acids, erythrocytes and in the maintenance of myelin, deficiency may result in a variety of symptoms. Some of these symptoms may be severe whilst others may be irreversible [13]. Human requirements are extremely small, ca/µg daily. In the samples wet weight values ranged from 10µg/100g - 455µg/100g whereas the dry weight values ranged from 8.39µg/100g -429µg/100g; the highest concentration in both wet and dry samples came from the muscle: 455µg/100g (wet) and 429µg/100g (dry). These values far outstripped the daily human requirement of 1µg.

The entry of calcium and phosphorus into the digestive tract is positively enhanced by the presence of vitamin D [14]. That means that vitamin D is the key to getting calcium and phosphorus to enter the blood stream via the gut. Low levels of vitamin D can result in insufficient levels of both calcium and phosphorus. Therefore, it is not surprising that low levels of vitamin D can also be associated with soft, brittle or deformed bones. Maintaining proper levels of vitamin D has been associated with the prevention of softening and weakening of bones in children (known as rickets) as well as softening of the bones (known as osteomalacia) in adults. Vitamin D and calcium also help protect older adults from reduction in bone mass and thinning of the bone which predisposes the bones to break at the slightest movement known as osteoporosis [15]. People avoiding the sun and those who suffer from milk allergies, or adhere to a strict vegan diet may be at risk for vitamin D deficiency. In the present results, vitamin D in fresh fish body parts ranged from 1.13e-1 to 3.59mg/100g giving a total of 6.37mg/100g FT whereas in the dry samples, it ranged from 8.30e-2 to 2.97mg/100g giving a total value of 5.61mg/100gDT. In each physical state, the muscle predominated. The values here were greater (except in the liver) than the reports in commercially important fish species of Lakshadweep Archipelago, India (10 different fish species were involved) having range values of 0.37- 0.77mg/100g [16]. The vitamin D requirement for optimum health is 400IU/d, regardless of age. Since 1µg of vitamin D is equivalent to 40IU of vitamin D, it meant that 400IU/d will be equivalent to 10µg of vitamin D per day. From the results above, all the C. gariepinus body parts were good sources of vitamin D.

Vitamin B1 (thiamine) in the fresh samples ranged from 7.54e-2 to 2.98mg/100g and total of 5.29mg/100g FT whereas the dry samples had values of 5.69e-2 to 2.84mg/100g and total of 4.83mg/100g DT. In mammals, including humans, the organs with high thiamine concentration (µg/g of moist tissue) are the heart (2.8- 7.9), kidney (2.4-4.8), liver (2.0-7.6), and brain (1.4-4.4); lesser amounts are found in the spleen, lungs, adrenals and muscle. Normal blood contains ca 90ng/ml which may vary considerably [17]. A value below 40ng/ml may be indicative of thiamine deficiency. Further literature values were in (mg/100g): pike perch (0.04), common carp (0.08) and European catfish (0.08) [10]. Souci, et al. [18] reported that vitamin B1 level for some marine and freshwater fish species were 0.02-0.2 mg/100g. In the 10 fish species of Lakshadweep Sea, vitamin B1 ranged from 0.39-0.61mg/100g [16].

Vitamin B2 ranged in the fresh samples as 2.81e-2 to 3.37e-1 and total value of 3.97e-1 with corresponding dry sample results of 2.70e-2 to 2.47e-1mg/100g and total of 3.04e-1mg/100g. Vitamin B2 in pike perch, common carp and European catfish had values of (mg/100g): 0.01, 0.04 and 0.03 respectively [10]. whereas in the 10 species of Lakshadweep Archipelago India, B2 varied from 0.13- 0.86mg/100g [6]. In the report for some marine and freshwater fish species by Souci, et al. [18], vitamin B2 in mackerel was 0.36mg/100g, herring (0.22mg/100g), European eel (0.32mg/100g), rainbow trout (0.08mg/100g) and carp (0.053mg/100g). An adult requires ca 1.5 -3.0mg riboflavin (vitamin B2) daily.

The niacin (vitamin B3) levels in the samples were 1.90-2.92mg/100g and totals of 6.97mg/100gFT whereas the dry counterparts varied from 1.84 -2.63mg/100g and totals of 6.55mg/100gDT. The term niacin has been used to encompass the active forms of this vitamin, nicotinic acid and nicotinamide; however, estimates of niacin requirements take into account preformed niacin as well as that obtained as niacin equivalent in the body from tryptophan (Trp) metabolism. Hence it was estimated that when 60mg of Trp is consumed by an adult, enough of Trp is oxidised to produce 1.0mg of niacin [19]. The best estimate of the average requirement for an adult is 15- 20mg per day. The recommended dietary allowance (RDA) for adults, expressed as vitamin B3 equivalents is 6.6mg per 4184 kJ (1000kcal, food calories) and not less than 13mg for an intake of less than 8368 kJ (2000kcal, food calories); one (1) NE is equivalent to 1.0mg niacin (or 60mg Trp) [19]. The Trp levels in the present report (not yet published) were (g/100g) fresh: LF (2.12), MF (2.10) and HF (1.70); for dry: LS (2.16), MS (2.02) and HS (1.88). This meant that the samples would be both good sources of vitamin B3 either directly or indirectly. B3 values in the 10 Indian fish species had values that ranged from 0.19-0.87mg/100g [16].

The concentrations of vitamin B6 were in the range of 1.58e-1 to 2.87mg/100g and total of 5.26mg/100g FT whereas the dry values were 1.41e-1 to 2.75mg/100g and 4.99mg/100g DT. Vitamin B6 is a generic name used for pyridoxine, pyridoxal and pyridoxamine, the co-enzyme forms of which are pyrodixal phosphate and pyridoxamine phosphate [19]. Vitamin B6 is needed in the synthesis of DNA bases; it is a co-enzyme in the biosynthesis of thymedine. A dietary vitamin B6 deficiency or an increase in the thymedine requirement at a critical time during cell division could result in initial cell mutations that develop into a tumor [20]. The RDAs were based on a ratio of 0.02mg of vitamnin B6 per gramme of protein consumed. From this estimate, the vitamin B6 required to satisfy the protein composition (assuming as being main protein source) from the body parts would be for fresh: 0.02 x 19.5 (=0.39mg) liver, 0.02 x 20.0 (=0.40mg) muscle and 0.02 x 20.5 (=0.41mg) head; for dry : 0.02 x 56.5 (=1.13mg) liver, 0.02 x 60.7 (=1.21mg) muscle and 0.02 x 58.6 (=1.17mg) head. Most human diets provide 3-10mg daily derived from a variety of natural foods. For adult patients on total parenteral nutrition 15mg is an adequate daily dose and 1mg daily is sufficient for infants [21]. Vitamin B6 values in the fishes of Lakshadweep Sea ranged from 0.11-0.91mg/100g [10].

People need vitamin B5 to synthesize and metabolize fats, proteins and co-enzyme A. Some of the important functions of B5 are: converting food into glucose; synthesizing cholesterol; forming sex and stress-related hormones and forming red blood cells. Deficiency of vitamin B5 is extremely rare in people; however, clinical trials have shown that deficiency may lead to: tiredness, depression, hypoglycaemia and can cause an increased sensitivity to insulin. Recommended daily intake ranged from 1.7mg per day to 7mg per day depending on age status [22]. Vitamin B5 in the samples ranged from 1.21e- 1 to 4.38e-1mg/100g and total of 6.96e-1mg/100g FT; in dry samples we have 1.14e-1 to 3.26e-1mg/100g and total of 5.70e-1mg/100g DT which were generally close to the values of 0.18-0.81mg/100g observed in 10 fish species of Lakshadweep Sea [16].

Vitamin B9 (folate, folic acid, folacin) has a recommended adult daily intake in the U.S. of 400µg from foods or direct supplements. Folic acid is essential for the body to make DNA, RNA, metabolise amino acids, which are required for cell division. Not consuming enough folate can lead folate deficiency that may result into a type of anaemia in which low numbers of large red blood cells occur. In adults, normal total body folate is between 10 and 30 mg with blood levels of greater than 7nmol/L (3ng/ml) [23]. Low levels of folic acid in early pregnancy are believed to be cause of more than half of babies born with neural tube defects (NTDs), B9 values in the fish parts were 4.48e-5 to 1.76e-4 mg/100g and total of 2.72e- 4mg/100gFT; for dry we have values of 4.28e-5 to 1.32e- 4mg/100g and total of 2.23e-4mg/100gDT. These values translated to the following µg/100g levels: for fresh, we have 0.045-0.176µg/100g and total 0.272µg/100g FT; for dry, results would become 0.043-0.132µg/100g and total 0.223µg/100gDT. These values were much lower than in pike perch (12.13µg/100g), common carp (33.23µg/100g) and European catfish (12.23µg/100g) [10]. Lall & Parazo [11] reported that the average folic acid content of fish and shellfish was 0.5-10µg/100g in flesh. In the innards of male and female samples of Neopetrolisthes maculatus (a marine shellfish), values of folic acid ranges as 317µg/100g-419µg/100g(dry weight) [24]; in the flesh of the same shellfish, we have folic acid values of 407µg/100g-454µg/100g [25].

Vitamin A value was the second most concentrated vitamin in the liver but present in ultratrace levels in the muscle and the head. Fresh liver had level of 5.22mg/100g and dry was 4.40mg/100g; on the other hand, fresh muscle/head had values of 5.34e-3 to 6.78e-3 as mg/100g and dry was 5.14e-3 to 6.39e-3 mg/100g; for fresh muscle/head and dry muscle/head, values in µg/100g would be 5.34-6.78µg/100g and 5.14- 6.39mg/100g. It has been said that fat-soluble vitamins in the flesh of fish are affected by the level of fat [10]; this assertion is true in the present results. The crude fat content of muscle (10.4g/100g) whereas the head crude fat was 5.50g/100g; consistently vitamins A, D, E and K were higher in the muscle than in the head but all lower than in the liver. The flesh of the lean fish contains 7.5 to 15µg/100g vitamin A, while in the fatty species vitamin A ranges from 30 to about 1350 µg/100g [26]. In common carp we had 23.52µg/100g and 6.30µg/100g in European catfish [10]. The vitamin A contents in the head and muscle could be said to be medium and low but significantly high in the liver: 5220µg/100g (fresh) and 4400µg/100g (dry). In the 10 fish samples from India, vitamin A content varied from 0.19-0.72 mg/100g [16]. The normal daily requirement of vitamin A for adults is about 5000IU (or 1500 RE, retinal equivalent). One (1) IU of vitamin A = 0.3µg of retinal equivalent. Although vitamin A is known to play a vital role in general metabolism, the only biological function in which its action is clearly understood is vision. The amount of vitamin E present in any sample of food or tissue is quoted in tocopherol equivalents (TEs). Note that

1mg TE = 1mg of α-tocopherol = 1IU of tocopherol Vitamin E requirement is linked to that of PUFA. The requirement of vitamin E suggested by Indian Council for Medical Research [21] is 0.8mg/g of essential fatty acids. This roughly works out to 8-10mg tocopherol per day. The fish sample parts were high in vitamin E (α- tocopherol). In the fresh samples we had (mg/100g): FL (24.8) > FM (8.65) > FH (6.92) with total value of 40.4FT; in the dry we have; DL (21.0) > DM (8.10) > DH (6.75) with total value of 35.9DT. The samples were good sources of α-tocopherol. The per capita daily intake of α- tocopherol in the U.S.A. has been estimated from data on total purchases of food to be ca 15mg. The tocopherols are used as dietary supplements and in food technology as antioxidants (qv) to retard the development of rancidity in fatty materials. The vitamin E content in pike perch was 0.94 mg/100g, common carp had 0.46mg/100g and European catfish had 0.80mg/100g [10]. Values of vitamin K in the samples were as follows (mg/100g, µg/100g): FL (4.36e-l, 436), FM (1.70 e-l, 170), FT (1.2le-l, 121), total (7.28e-l, 728); DL (2.32e-l.232), DM (1.58e-l, 158), DH (8.65e-2, 86.5) and total (4.77e-l, 477). In the 10 fish species of Lakshadweep Archipelago in India, their vitamin K contents ranged from 0.19- 0.67mg/100g [16]. The Committee on Medical Aspects of Food Policy of the United Kingdom concluded that 1µg/kg/day of vitamin K is both safe and adequate for adults [21]. Hence the fish body parts were good sources of vitamin K.