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The active form of vitamin A or retinol could be synthesized from β-carotenes (AACC 2000). Cereals do not contain reti-nol but some contain significant quantities of carotenes. These carotenes are efficiently converted to retinol in the intestinal mucosa and liver. One molecule of β-carotene is transformed into two identical retinol units because of the hydrolysis of the carotene by diooxygenase to yield two retinoaldehyde mol-ecules with the later transformation to retinol with a liver reduc-tase. Fat-soluble retinol units are transported from the intestinal epithelial cells in chylomicrons to the liver and then to the rest of the body bound to retinol-binding protein. The vitamin A is stored in the liver and gradually used by the body.

Most nutritionists agree that the second most prevalent global deficiency of micronutrients is vitamin A. Recent statistics indicate that, annually, more than half a million children become partially or totally blind due to chronic deficiency of vitamin A, whereas 13 million preschool chil-dren are affected by xerophthalmia (Onuma Okezie 1998).

Vitamin A deficiency occurs more frequently among peo-ple in developing countries such as Asia, Africa, and Latin

69 Determination of Chemical and Nutritional Properties of Cereal Grains and Their Products

America. Approximately one-fourth to one-half of preschool children show symptoms of vitamin A deficiency in these countries. As with most nutrient deficiencies, the lack of vita-min A is more prevalent among children between 1 and 5 years of age that live in poverty. Humans require vitamin A to sustain normal growth, resist infectious diseases, and to have normal vision. More specifically, the lack of vitamin A is observed in the eyes and condition of epithelial tissues associated with skin, gastrointestinal tract, respiratory, and urogenital systems which leads to dry skin, diarrhea, and pneumonia. Its deficiency also affects bone development, exacerbates anemia, and depresses the immune and nervous systems. The most common symptoms are xerophthalmia, hyperkeratinization, and keratomalacia characterized by the irreversible drying of the eye which leads to cornea degenera-tion and permanent blindness (Hoffmann 1972; Mason et al.

2001; Van dan Briel and Webb 2003).

The typical vitamin A deficiency starts when newborns rapidly deplete the stored hepatic vitamin A reserves that usually last 4 to 6 months. In developing countries, most infants are weaned with cereal-based gruels that, unfor-tunately, lack sufficient amounts of carotenes. The chronic deficiency of vitamin A leads to night blindness and a higher susceptibility to infectious diseases. The vitamin A require-ment increases in infants that present episodes of diseases such as measles, chicken pox, intestinal parasites, and calorie and protein malnutrition.

Both carotenes and vitamin A or retinol are frequently deter-mined because they should be declared in nutritional food labels.

These compounds are usually extracted with solvents and then quantified in a spectrophotometer or using HPLC systems.

A. Samples, Ingredients, and Reagents

• Test samples

• Aluminum oxide

• Chloroform (reagent grade)

• Ethanol

• Potassium hydroxide (KOH reagent grade)

• Acetic anhydride

• Vitamin A reference solution

• Cotton

• Antimony trichloride (SbCl3)

• Anhydrous sodium sulfate (Na2SO4)

• Carotene reference crystals

• Stopcock or extraction apparatus

• Glass tube

• Delivery pipette (10 mL)

• Stirrer

• Saponification reflux apparatus^†

• Desiccator

• Extraction apparatus (500 mL)

• Glass rod

• Volumetric flask (100 mL and 1 L)

• Chromatographic tubes (18 mm × 200 mm)

• Cuvettes

• Amber bottles

• Thermometer C. Procedure

1. Prepare de following reagents:

a. Aluminum oxide adsorbant. Hydrate to 5%

water by pouring measured amount of water into small glass-stoppered bottle. Distribute over walls, then add alumina and mix by shaking bottle until no lumps are observed.

Let stand to cool at least 2 hours before use and store in tightly closed bottle. Do not expose to air. Heat in muffle furnace at 750°C, cool, spread in thin layers over flat dishes in desiccators at 200 mL H2SO4 (spe-cific gravity of 1.35) for 48 hours equilibra-tion, and place adsorbant in tightly closed jars. Moisture content must be 4.5% to 5%.

b. Acetone in hexane solution (4% and 15%).

Dilute acetone with hexane.

c. Potassium hydroxide solution. Dissolve 50 g of reagent-grade KOH in water and dilute to 100 mL with water in a volumetric flask.

d. Antimony trichloride. Add CHCl3 to 200 g of SbCl3 to make 1 L. Warm and shake to dissolve and then cool. Add 30 mL of ace-tic anhydride. If solution is not clear, filter, centrifuge, or let settle, and decant. Store in stoppered amber bottles. The translucent solution should be stable for several months.

e. Vitamin A reference solution (obtained from USP Reference Standards, 12601 Twinbrook Pkwy, Rockville, MD).

f. Carotene reference crystals (from General Biochemicals). Crystals are 10% α-carotene, and 90% β-carotene in sealed 100 mg or 200 mg vials. Crystals should dissolve in hexane

*A lipless graduated cylinder (100 mL) fitted with a two-hole stopper.

Insert chromatographic tube through one hole and glass tube to vacuum through the other. A water aspirator is satisfactory for vacuum.

† Any refluxing apparatus with ground-glass joint water-cooled condenser, and amber flask of 300 mL to 500 mL heated with boiling water or steam.

70 Cereal Grains: Laboratory Reference and Procedures Manual without residue and have a characteristic

spectrophotometric curve.

2. Prepare the adsorption column as follows:

a. Place a small amount of cotton at the bot-tom of the chromatographic tube and pack with alumina adsorbent mixture, adding in several portions, tamping each lightly with a blunt rod, to a depth of 7 cm. Keep column under suction during packing. Add a 0.5-cm layer of powdered anhydrous Na2SO4 on top of the column, level and pack lightly.

b. Check for recovery of vitamin A as follows:

saponify 0.1 g of USP vitamin A refer-ence solution plus 2 g of fresh cottonseed oil. Extract with hexane. Mix solution of 50 μg to 100 μg of carotene and 100 USP units of saponified vitamin A. Dilute to 15 mL. Wash column with 20 mL of hexane and adjust elution rate to two drops per sec-ond. Before the top of the column runs dry, add vitamin A with 15% acetone in hexane (30 mL should be enough). Inspect last few milliliters of this eluate that is suitable for vitamin A fluorescence and continue until no fluorescence is observed. Evaporate suit-able aliquot to dryness under nitrogen, add 1 mL of CHCl3 and determine vitamin A levels. Compare results with aliquot of saponified vitamin A in hexane that was not chromatographed.

3. Determination

a. Mill or grind the test sample to pass a 20-mesh screen. Accurately weigh sample containing approximately 400 to 800 units of vitamin A. Add 1 g of fresh cottonseed oil to premixes or concentrates with low fat contents. Add a volume of ethanol that is three times the sample weight (in grams), and swirl until particles are wet. Then, add a volume of 50% KOH equal to the sample weight and mix.

b. Reflux 30 minutes at a rate of two drops per second. Swirl occasionally during saponi-fication to prevent lumping. Cool to room temperature under running water. Add a volume of distilled water that is twice the sample weight.

c. Extract three times with hexane, first using a volume of hexane that is two to three times the sample weight and two thirds as much for subsequent extractions.

d. Combine all hexane extracts into one sepa-rator. Pour 100 mL of cool water into the separator and drain when the layers sepa-rate, retaining any emulsion in the hexane layer. Repeat washings with 100-mL por-tions of water, with shaking, until solution is

colorless to phenolphthalein. If emulsions cause difficulty, use 10% ethanol-water wash containing 0.1% HCl on the third wash. After the final wash, separate water as completely as possible. Swirl separator, let solution stand for 5 minutes and drain any water collected in the bottom. Add 10 g of Na2SO4 to the separator and shake to dry the hexane solution. Pour solution care-fully from the top of the separator through a small piece of cotton into the appropri-ate volume flask. Rinse separator and cot-ton with hexane and make to volume with hexane.

4. Chromatography

a. Chromatograph aliquot of 10 mL to 15 mL hexane extract containing approximately 100 units of vitamin A (preferably not less than 60 units). If necessary, concentrate portion of hexane extract under vacuum to obtain a sufficient concentration of vitamin A. In no case should more than 25 mL of the solution be chromatographed.

b. Pack column as explained above, wash with 20 mL of hexane and add extract containing vitamin A just before the top of the column runs dry. Eluate at a rate of two drops per second. Elute carotene and then vitamin A as above.

c. Dilute carotene and vitamin elutes to vol-ume for colorimetry. For vitamin A, 50 mL is convenient. An aliquot of 10 mL may be taken for carotene determinations.

5. Preparation of standard curves

a. Vitamin A standard curve. Accurately weigh 100 mg of vitamin A reference solu-tion and transfer to a volumetric flask.

Dilute to volume with CHCl3. Use solution as soon as possible (within 8 hours). Use amber glassware. Make a series of dilu-tions of the vitamin A solution with CHCl3

so that 1-mL aliquots treated similar to the samples give transmissions of 20% to 85%.

Plot absorbance against units of vitamin A.

Determine the slope of the line.

b. Carotene standard curve. Prepare series of dilutions of α- and β-carotene reference crystals in hexane. Plot absorbance against the amount (in micrograms) of carotene and determine the slope of the line.

c. Determination of correction factor for yel-low pigment in vitamin A eluate. Correct for pigment if present in more than mere trace amounts. Make correction by saponi-fication and extraction of sample from yel-low maize. Chromatograph and save the 15% acetone-in-hexane fraction. Determine

71 Determination of Chemical and Nutritional Properties of Cereal Grains and Their Products

concentration of the yellow pigment in this fraction by comparison with carotene calibration. Transfer pigment to CHCl3

and make a series of dilutions covering the range of concentrations of yellow pigment in sample solutions in the 1 mL CHCl3 on which the vitamin A color is read. Obtain correction factor for reaction of this pig-ment in vitamin A determination.

6. Colorimetry

a. Carotene. Prepare standard curve as directed previously. Determine the concen-tration of carotene using a 440 nm filter or wavelength in a spectrophotometer.

b. Vitamin A. Transfer 10 mL of vitamin A eluate to spectrophotometer cuvette and read yellow color as carotene. If this is present in more than trace amounts, make corrections as directed in the next section.

Evaporate solvent under vacuum and heat (60–65°C) in a hot water bath. Dissolve residue in 1 mL of CHCl3 solution. Adjust spectrophotometer to 620 nm and 100%

transmittance, using 1 mL CHCl3 and 10 mL SbCl3 solution. Place tube containing 1 mL of CHCl3 solution of vitamin A in the colorimeter and add SbCl3 reagent rapidly.

Take maximum reading within 3 to 5 sec-onds. Convert readings to units of vitamin A by referring to previously prepared stan-dard curves. Correct value for vitamin A lost in analysis by adding a known amount of vitamin A to the sample or to the blank.

7. Calculate the amount of vitamin A or carotenes using the following equations:

Vitamin A, USP units/g = (units of vitamin A per milliliter/

recovery factor) × (dilution factor/sample weight, in grams).

β-carotene, μg/g = (μg carotene per milliliter × dilution factor)/(sample weight, in grams).

2.9.2 analysisoF b-CoMPlex VitaMins

Historically, humans have traditionally experienced deficien-cies of B vitamins. Cereal grains are considered an adequate source of these B vitamins, except B12 or cyanocobalamin.

The most common deficiencies are due to the lack of thia-mine (B1), niacin (B3), folic acid, and cyanocobalamin (B12).

Thiamine or B1 has been historically recognized as the main cause of beriberi. Thiamine exists in free and bound forms (thiamine diphosphate and protein-phosphate-thiamine com-plex). The bound forms are split in the gastrointestinal tract.

The absorbed thiamine acts as a coenzyme in energy metabo-lism mainly in the conversion of glucose to fats. In addition, it has high implications in the functioning of peripheral nerves, brain, and muscles. Thiamine deficiency causes weakness,

lack of appetite, constipation, depression, and in severe cases, cardiac insufficiency. It is one of the major causes of death among Asian babies.

Riboflavin or B2 functions as part of the enzymes called flavoproteins (FMN and FAD), which are critically impor-tant in respiration and cell metabolism. They play a major role with thiamine and niacin in oxidation-reduction reac-tions. The deficiency of this vitamin is characterized by pho-tophobia, angular lip stomatitis, dermatitis, and swelling of the tongue.

The discovery of the role of niacin was the result of humanities’ struggle against pellagra (pelle = skin and agra = sour). Pellagra spread with the dissemination and cultivation of maize and its use in human diet. Pellagra was endemic in Europe and Africa, and reached epidemic proportions in the United States during the Civil War. Today, pellagra is less prevalent thanks to the cereal-enrichment programs enacted in many nations. Africa is the only continent is which pella-gra is still a public health problem. The symptoms of pellapella-gra known as the 3D disease, are diarrhea, dermatitis, dementia, and in severe cases, death. In addition, pellagra causes apathy, confusion, anorexia, glossitis, and gastritis. Metabolically, this vitamin is a constituent of two important coenzymes (NAD and NADP) which are necessary for cell respiration and car-bohydrate, protein, and fat metabolism. These enzymes are involved in the release of metabolic energy.

Folic acid (pteroylmonoglutamic acid or PGA) exists in different forms in nature. These forms are changed to at least five active coenzymes that are critically important for the for-mation of purines and pyrimidines needed for the synthesis of DNA and RNA, the formation of hemoglobin, the intercon-version of amino acids such as homocysteine to methionine, and the synthesis of choline from ethanolamine. Vitamins B12, B6, and C are essential for the activity of folacin as coenzymes in many metabolic processes. In practical terms, folic acid is required for cell division and reproduction and prevents neu-ral tube defects in newborns and cardiovascular diseases in adults. Folacin and vitamin B12 lower the levels of homocys-teine, resulting in a cardiovascular protective role.

2.9.2.1 analysis of thiamine (Fluorometric