SIGNIFICANCE, CHARACTERISTICS AND DETERMINATION OF VITAMIN C V. L. VUKAŠINOVIĆ-PEŠIĆ 1, N. Z. BLAGOJEVIĆ 2. Synopsis

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1 NATURA MONTENEGRINA, PODGORICA, 3, 2004, UDK (045)=111 SIGNIFICANCE, CHARACTERISTICS AND DETERMINATION OF VITAMIN C V. L. VUKAŠINOVIĆ-PEŠIĆ 1, N. Z. BLAGOJEVIĆ 2 Synopsis Vitamin C (ascorbic acid) is very widespread in nature: it can be found in fresh fruits and vegetables, it is added to different food, and it also can be found in a large number of pharmaceutical products. Because of its huge importance for normal function of human beings it is necessary to continously intake it by food. This paper points to the significance and characteristics of vitamin C and presents some new methods for its determination. Keywords: vitamin C, significance, characteristic, determination Sinopsis ZNAČAJ, OSOBINE I ODREĐIVANJE VITAMINA C Vitamin C (askorbinska kiselina) je jako rasprostranjen u prirodi: nalazi se u svježem voću i povrću, dodaje se mnogim prehrambenim proizvodima, a nalazi se i u velikom broju farmaceutskih proizvoda. Ima veliki značaj za normalno funkcionisanje organizma čovjeka, pa ga je potrebno konstantno unositi putem hrane. Biće naveden značaj i osobine vitamina C, kao i kratak pregled najnovijih metoda za određivanje njegovog sadržaja u velikom broju različitih uzoraka. Ključne riječi: vitamin C, značaj, karakteristike, određivanje 1,2 Faculty of Metallurgy and Technology, University of Montenegro, Cetinjski put b.b., Podgorica, Serbia and Montenegro

2 240 Natura Montenegrina, 3/2004 CHARACTERISTICS AND SIGNIFICANCE L-ascorbic acid (C 6 H 8 O 6 ) is the common name of Vitamin C. The chemical name is 2- oxo-l-threo-hexono-1,4-lactone-2,3-enediol. L-ascorbic and dehydroascorbic acid are the major dietary forms of vitamin C. Ascorbyl palmitate is used in commercial antioxidant preparations. All commercial forms of ascorbic acid except ascorbyl palmitate are soluble in water. L-ascorbic acid and its fatty acid esters are used as food additives, antioxidants, browning inhibitors, reducing agents, flavor stabilizers, dough modifiers and color stabilizers. Ascorbyl palmitate has been used for its greater lipid solubility in antioxidant preparations. Vitamin C was first isolated in 1928, by the Hungarian biochemist and Nobel Prize winner S z e n t - G y o r g y i. It is one of the important water soluble vitamins. The stability of aqueous solutions of vitamin C is dependent on the temperature, ultraviolet light, oxygen pressure, ph and catalysts such as copper (II) ions (K h a n and M a r t e l l, 1967; O g a t a et al., 1968; O g a t a and K o s u g i, 1969; B a k e r and L o w e, 1985). Most plants and animals synthesize ascorbic acid for their own requirement. However, apes and humans can not synthesize ascorbic acid due to the lack of an enzyme gulonolactone oxidase. Hence, ascorbic acid has to be supplemented mainly through fruits, vegetables and tablets. Vitamin C is very widely distributed in nature. It is found in citrus fruits, green peppers, red peppers, strawberries, tomatoes, broccoli, brussels sprouts, turnip and other leafy vegetables. Fish and milk also contain small amount of vitamin C. Ascorbic acid is one of the important and essential vitamins for human health. It is needed for many physiological functions in human biology. It helps in the metabolism of tyrosine, folic acid and tryptophan. It helps to lower blood cholesterol and contributes to the synthesis of the amino acids carnitine and catecholamine that regulate nervous system. It is needed for tissue growth and wound healing. It helps in the formation of neurotransmitters and increases the absorption of iron in the gut. Being an antioxidant, it protects the body from the harmful effects of free radicals and pollutants. In general vitamin C seems to operate as an anti-stress factor which enables the organism to adapt itself better to environmental influences. Mega doses of vitamin C are used in the treatment and prevention of large number of disorders like diabetes, cataracts, glaucoma, muscular degeneration, atherosclerosis, stroke, heart diseases and cancer. Deficiency of this vitamin can lead to anemia, scurvy, infections, bleeding gums, muscle degeneration, poor wound healing, atherosclerotic plaques, capillary hemorrhaging and neurotic disturbances. Toxicity normally does not occur. It is therefore important to include plenty of vitamin C-containing foods in one's daily diet. The daily requirement in human is difficult to establish. The current United States Government's National Academy of Sciences recommended daily allowance (RDA) for ascorbic acid ranges between mg/per day for adults.the Vitamin C Foundation recommends that every man, woman and child over the age of 3 consume at least 3 g (3000 mg) vitamin C daily in order to enjoy optimum health. The Linus Pauling Institute and the Levin group at the National Institutes of Health recommend 200 mg. Also it is better to take multiple and smaller doses of vitamin C throughout the day than one large dose. The requirement of the organism increases during pregnancy and lactation, in disease and in old age as well as in new born children who are artificially fed. Fresh fruits, vegetables and also synthetic tablets supplement the ascorbic acid requirement of the body. However, stress, smoking, infections and burns deplete the ascorbic

3 241 VUKAŠINOVIĆ-PEŠIĆ V, & BLAGOJEVIĆ, N: Determination of Vitamin C acid reserves in the body and demands higher doses of ascorbic acid supplementation. Based on available biochemical, clinical and epidemiological studies, the current RDA for ascorbic acid is suggested to be mg/day to achieve cellular saturation and optimum risk reduction of heart diseases, stroke and cancer in healthy individuals (C a r r and F r e i, 1999). In view of its antioxidant property, ascorbic acid and its derivatives are widely used as preservatives in food industry. Many health benefits have been attributed to ascorbic acid namely antioxidant, anti-atherogenic and anti-carcinogenic activity. Lately some of these beneficial effects of ascorbic acid are contradicted. Thus, although ascorbic acid was discovered in the 17 th century, the role of this important vitamin in human health and disease still remains a mystery in view of many beneficial claims and contradictions (N a i d u, 2003). DETERMINATION Determination of ascorbic acid in vitamin and natural products is of great importance and several analytical tehniques have been proposed for it, such as titrimetry (L i e b m a n and A y e r s, 1945; K u m - T a t t and L e o n g, 1964; P a n d e y, 1982), spectrophotometry (S a r k a r and S a r k a r, 1994; T u t e m et al., 1992; Q u r e s h i et al., 1991), spectrofluorimetry (P e i n a d o et al., 1987), polarography (E s t e v e et al., 1995), HPLC (I w a s e and O n o, 1994; L y k k e s f e l d t et al., 1995; D a o o d et al., 1994) and chemiluminiscence (A l w a r t h a n, 1993). Of all of these techniques, spectrophotometry is, perhaps, the most commonly used. Ascorbic acid has a strong absorption at 265 nm. Spectrophotometric determination of ascorbic acid by direct measurement of its absorption in the ultraviolet region is very difficult as ascorbic acid is usually found in solution containing substances also absorbing in this region. In order to overcome the problem of interferences in the UV region from these substances, reagents have been used which have specific colour reactions with ascorbic acid. The most widely used spectrophotometric method for the determination of vitamin C have been based on one of the three classical methods. The first one is based on the reaction of the titrimetric method using 2.6-dichlorophenolindophenol adapted for colorimetric determination by means of an automatized system (G a r r y and O w e n, 1967; O w e n et al., 1974; G a r r y et al., 1974). The second method is that of Roe and Kuether (R o e and K u e t h e r, 1943), which relies on the oxidation of ascorbic acid to dehydroascorbic acid which reacts with 2.4-dinitrophenylhydrazine, in a concentrated sulphuric acid medium, to form coloured osayones and then determined spectrophotometrically. The latter method is based on the deep blue colour which is produced when ascobic acid is reacted with diazonium compounds (S c h m a l l et al., 1953; H a s h m i et al., 1973). However, these techniques are unsuitable for coloured samples which interfere with the detection of the end point. Also, titrimetric methods suffer the disadvantage of using a large amount of sample and undergoing interference from many redox reagents with the determination. The fluorimetric method is too consuming. For the chromatographic methods, the use of expensive equipment and the demand for more operator attention prevent their application in small industrial laboratories where only a few analyses are performed each day. The polarographic method requires long analysis time and suffers interferences from electrochemical impurities present in the food sample. In contrast to conventional spectrophotometry, derivative spectrophotometry (DS) enables the elimination of background absorption by the corresponding order of derivation, regardless of whether it was caused by irrelevant absorption or light scattering by turbid

4 242 Natura Montenegrina, 3/2004 solutions and suspensions (P o p o v i ć et al., 2000). DS has so far been applied mainly for determination of vitamin C content in pharmaceuticals (A b d e l - H a m i d et al., 1985; Aburjai et al., 2000; I n e s T o r a l et al., 2001). Özgür and Sungur (Ö z g ü r and S u n g u r, 1995) employed third order DS to meashure vitamin C levels in kiwi, grapefruit and parsley. A second-order derivative UV spectrophotometric method was used for determination of vitamin C content in a variety of natural samples (P f e n d t et al., 2003). The method is based on the measurement of a peak-baseline amplitude in the second derivative of the ascorbic acid spectrum at nm. The following corresponding regression equation was obtained within the concentration range 2x10-5 to 1x10-4 M ascorbic acid ( mg/cm 3 ascorbic acid) in 1.0 M HCl solution: 2 D =2.420x10 4 c+0.025; the correlation coefficient was and the detection limit 4.2x10-6 M. The results obtained by analysing 21 different species of fruits, vegetables and juices indicated a possibility for a more extensive application of this method. Further investigation confirmed that this method is also suitable for the other samples of fruits and juices even if they are intensively coloured (B l a g o j e v i ć et al., submitted). This method is rapid, simple and it requires no pretreatment of the analysed material, which makes it suitable for routine analysis. REFERENCES Abdel-Hamid, M. E.; Barary, M. H.; Hassan, E. M.; Elsayed, M. A. 1985: Analyst 110: Aburjai, T.; Amro, B. J.; Aledeh, K.; Abuirjele, M.; Al-Khalil, S. 2000: Pharmazie 55: Alwarthan, A. A. 1993: Analyst 118, 639. Baker, W. L.; Lowe, T. 1985: Analyst 110, Blagojević, N. Z.; Vukašinović -Pešić, V. L.; Zejnilović, R. M. submitted Carr, A. C.; Frei, B : Am. J. Clin. Nutr 69 (6): Daood, H. G.; Biacs, P. A.; Dakar, M. A., Hajdu, F. J : Chromatogr. Sci 32, 481. Esteve, M. J.; farre, R.; Frigola, A. 1995: Fresenius' J. Anal. Chem. 351, 804. Garry, P. J.; Owen, G. M. 1967: Automation in Analytical Chemistry-Technicon Symposia, 1, 507. Garry, P. J.; Owen, G. M.; Lashley, D. W.; Ford, P. C. 1974: Clin. Biochem. 7, 131. Hashmi, M. H.; Shakid, M. A.; Akhtar, M.; Chguhtai, N. A. 1973: Mikrochim. Acta 1973, 901. Ines Toral, M. J.; Nelson, L.; Richter, P.; Tassara, A.; Tapia, A. E.; Rodrigues, C. J. 2001: Assoc. Anal. Communities Int 84:

5 243 VUKAŠINOVIĆ-PEŠIĆ V, & BLAGOJEVIĆ, N: Determination of Vitamin C Iwase, H.; Ono, I. J. 1994: Chromatogr., Biomed. Appl. 655, 195. Khan, M. M. T.; Martell, A. E. J. 1967: Am. Chem. Soc., 89, Kum-Tatt, L.; Leong, P. C. 1964: Analyst, 89, 674. Liebman, H.; Ayers, A. D. Analyst 1945, 70, 411. Lykkesfeldt, J., Loft, S.; Poulsen, H. E. 1995: Anal. Biochem. 229, 329. Naidu, K. A. 2003: Nutrition Journal Ogata, Y.; Kosugi, Y.; Morimoto, T. 196: Tetrahedron 24, Ogata, Y.; Kosugi, Y. 1969: Tetrahedron 25, Owen, G. M.; Kram, K. M.; Garry, P. J.; Lowe, J. E.; Lubin, A. H. 1974: Pediatrics 53, Part II. Özgür, M. Ö.; Sungur, S. 1995: Talanta,, 42: P a n d e y, N. K. 1982: Anal. Chem. 54, 793. Peinado, J.; Toribio, F.; Pérez-Banditi, D. 1987: Analyst, 112, 775. Pfendt, L. B.; Vukašinović, V. L.; Blagojević, N. Z.; Radojević, M. P. 2003: Eur. Food. Res. Technol. 217: Popović, G. V.; Pfendt, L. B.; Stefanović, V. M. 2000: J. Serb. Chem. Soc., 65: Qureshi, S. Z.; Saeed, A.; Haque, S.; Khan, M. A. 1991: Talanta 38, 637. Roe, J. H.; Kuether, C. A. 1943: J. Biol. Chem. 147, 399. Sarkar, M.; Sarkar, A. R. 1994: Analusis 22, 155. Schmall, M.; Piefer, C. W.; Wollish, E. G. 1953: Anal. Chem. 25, Tutem, E.; Ulkuseven, B.; Apak, R. 1992: Anal. Lett 25, 471.

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DR.PAWAN TOSHNIWAL ASSISTANT PROFESSOR BIOCHEMISTRY,

DR.PAWAN TOSHNIWAL ASSISTANT PROFESSOR BIOCHEMISTRY, CURIOSITY???? WHAT? WHERE? WHY? DISORDES? CURIOSITY = ANSWER TO FIND WHAT ARE VITAMINS AND THERE REQUIREMENTS? WHERE THEY ARE SYNTHESISED, ABSORPED,