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VITAMIN AND TRACE ELEMENTS
Water soluble: vitamin c and B complex Fat soluble: Vit A, D, E, and K Vitamin A: Retinol and 3-dehydroretinol Precursors of vitamin A = carotenes, found in yellow and green parts of plants; especially abundant in carrots Active vitamin A formed by hydrolysis of β-carotene in intestinal mucosa Retinol transport: bound to α-globulin retinol-binding protein Stored in animal tissues, particularly liver Rhodopsin is destroyed in bright light Partial regeneration in the dark(rhodopsin - consists of protein opsin + Vitamin A) Vitamin A vital to maintain retinal levels Absorbance peak at 325 nm for retinol and 351 nm for 3-dehydroretinol Decreased levels seen in chronic infection, fever, hepatic disease, and many disorders that involve lipid metabolism Clinical effect of vit A deficiency: o Night blindness due to rhodopsin deficiency o Dry, squamous metaplasia of epithelial surfaces Hypervitaminosis A: o Vitamin A in large doses is toxic o Overdosage commonly due to excessive vitamin use without medical advice o Acute poisoning/intoxication – reported in Arctic regions as a result of consuming polar bear liver Colorimetric methods: Chromogenic reagent reacts with retinol to produce a blue colour Neeld – pearson procedure: collect fasting blood specimen o Specimen must be free from haemolysis and protected from light – method is sensitive to light o Extract vitamin A and β-carotene with petroleum ether o Read absorbance at 450 nm ⇒ amount of β-carotene o Evaporate and resuspend with triflouroacetic acid o Read at 620 nm o Correct reading for β-carotene 1|Page
Spectrophotometric method: The sample is irradiated with UV light and its absorbance is measured = proportional to vitamin A content in the sample HPLC method: o Normal and reverse phase column and UV detector at 280 nm o Hexane used to separate retinol from other substances which absorb radiant energy at equal or similar wavelengths to retinol
Vitamin B: Thiamine (B1), riboflavin (B2), nicotinamide (niacin), pyridoxine (B6), folate / folic acid (pteroylglutamate), B12 complex (cobalamins), biotin, pantothenic acid Many synthesized by colonic bacteria Thiamine, folate, vitamin B12 ⇒ actively absorbed from intestinal tract; The rest diffuse passively through intestinal mucosal wall Most act as enzyme cofactors Thiamine: o Component of thiamine pyrophosphate: essential cofactor for decarboxylation – conversion of pyruvate → acetyl CoA o Cannot be synthesized by body – from diet o Deficiency can seen in alcoholism, anorexia nervosa o Beri – beri (Anorexia, emaciation, neurologic lesions, cardiac failure) = aggravated by high carbohydrate diet ≈ dietary carbohydrate glycolysis pyruvate o Detection by microbiological method, chemical conversion, HPLC with UV detection, biochemical tests o Most useful for assessing thiamine status is measurement of whole blood or erythrocyte transketolase o Fluorometric methods: Treatment of thiamine with an oxidizing agent (ferricyanide or hydrogen peroxide) to form a flourescent compound (thiochrome) Riboflavin: source from diet o Flavine mononucleotide (FMN) o Flavine adenine dinucleotide (FAD) o FMN and FAD = reversible electron carriers in biological oxidation systems o Deficiency = ariboflavinosis o Fluorometric: Measuring characteristic yellowish green riboflavine fluorescence o Microbiological: Lactobacillus casei o Growth of this riboflavine organism correlates with the amount of vitamin in the sample 2|Page
o Growth response measured by measuring turbidity Niacin (B3): o Nicotinamide formed in the body from nicotinic acid o Humans can synthesize some nicotinic acid from tryptophan o NAD+ - nicotinamide adenine dinucleotide o NADP+ -nicotinamide adenine dinucleotide phosphate o NAD+ and NADP+ ⇒ cofactors in oxidation-reduction reactions o Deficiency can cause pellagra o Measure metabolites of niacin excretion (done by HPLC): N(1)methylnicotinamide and N(1)-methyl-3-carboxamide-6-pyridone o Colorimetric assay: Assay semi quantitatively with sulfanilic acid → yields a yellow colour o Microbiological (most sensitive) - Lactobacillus plantarum o Growth of organism correlates with the amount of vitamin in the sample o The growth response measured by measuring turbidity Vitamin B6: 3 forms o Pyridoxine (pyridoxol) Balancing of hormonal changes in women Assisting the immune system Growth of new cells Processing and metabolism of proteins, fats and carbohydrates Balancing of sodium and potassium o Pyridoxal – aldehyde o Pyridoxamine – amine o Widely distributed in food; dietary deficiency rare o Decomposed by UV light o In women ⇒ pre-menstrual fluid retention, severe period pains, emotional PMS symptoms o Deficiency: may cause roughening of skin, peripheral neuropathy, sore tongue o Fluorometric assay: After condensation with fluorophore o HPLC: Pyridoxic acid (level drops during deficiency) o Microbiological assay using Saccharomyces uvarum which measures free B6 Folic acid: o Present in green vegetables and some meats o Easily destroyed during cooking →→ dietary deficiency o Absorption through small intestine o Active form = tetrahydrofolate ⇒ important in purine and pyrimidine synthesis 3|Page
o Deficiency: common in intestinal malabsorption syndrome, megaloblastic anemia o Microbiological assay: Streptococcus faetalis, Lactobacillus casei Liberate free biotin by proteolytic digestion Add aliquot to biotin deficient medium inoculated with test organism Derive calibration curve from growth with calibrators containing known amounts of biotin Vitamin B12: cobalamins o Found in animal products but not green vegetables o Dietary deficiency rare o Absorbed in terminal ileum, combined with intrinsic factor derived from gastric parietal cells o Vitamin B12 can only be absorbed when it has formed a complex with the intrinsic factor o Deficiency → megaloblastic anaemia o Microbiologically: Lactobacillus leichmannii Panthothenic acid: o Pantothenate ⇒ component of coenzyme A (CoA) o CoA – essential for fat and carbohydrate metabolism o Microbiological assays using Saccharomyces carlbergensis and Lactobacillus plantarum
Vitamin C: ascorbate Found in fruits and vegetables Cannot be synthesized by the body Easily and irreversibly oxidized and loses its biological activity in presence of oxygen, catalyzed by heat Function in hydrogen carrier, wound healing and normal collagen formation Deficiency: scurvy: hemorrhaging at the gums, large ecchymosed (bruising under the skin), poor wound healing, anemia due to impaired erythropoietin Quantization: rely on reductive properties of ascorbic acid Reduction of 2,4-dinitrophenylhydrazine to hydrazone and 2,4dichlorophenol-indophenol to its colourless form The principle of this method is titration with dichlorophenolindophenol (or phenol-indo-2:6-dichlorophenol, also known as DCPIP) Ascorbic acid reacts with DCPIP – changing the color from blue → colorless Fluorometric: Oxidation of ascorbic acid to dehydroascorbic acid which reacts with phenylene diamineto to produce a fluorescent compound 4|Page
Intensity is proportional to the vitamin concentration
Vitamin D: Derived from, cholecalciferol (vitamin D3), ergocalciferol (vitamin D2) 3 types: Vitamin D, 25-hydroxy vitamin D, 1,25-dihydroxy vitamin D Can be used to separate the 3 types chromatographically using alumina and silica columns White, odourless crystals that are soluble in fats and organic solvents The absorption maximum of both in hexane is at 264.5 nm Deficiency in bone and mineral disorders 3 steps in quantitation: o Extraction: Frees and partially purifies the metabolite that is almost completely associated with D transport protein (DBP) and albumin o Purification: Separates vitamin D metabolites, lipids and interfering substances o Quantitation: using RIA or UV absorption Vitamin E: Deficiency rare because of easy availability in food Excess can be harmful Known as a potent antioxidant Newborn infants are deficient in vitamin E – which may be associated with haemolytic anaemia Deficiency in adults may be due to prolonged and severe fat malabsorption →→ neurological symptoms Viscous oil at room temperature Can be measured spectrophotometrically Vitamin K: Also known as the “clotting vitamin” ⇒ without vitamin K, blood would not clot Available in diet as green leafy vegetables, beans Also synthesized by bacteria that line the gastrointestinal tract, in the ileum Deficient →→ increased propensity to bleeding and bruising Cofactor needed for synthesis of prothrombin, factor 7, 9, 10 and protein C and S. Warfarin is sometimes prescribed as a "blood thinner" because it is an effective vitamin K antagonist Destroyed by alkaline solution and reducing agents and sensitive to UV light 5|Page
Determination requires 3 steps: extraction, chromatographic separation and quantitation
Steatorrhoea: Impaired fat absorption Intestinal malabsorption: fat digestion normal but impaired absorption of products of digestion Pancreatic steatorrhoea – absorptive capacity normal but fat cannot be digested because of deficiency of digestive enzymes TRACE ELEMENTS Elements that required for life Absence cause severe malnutrition and cause death
Iron: Interacts reversibly with oxygen and in electron transfer reactions Required as the functional ion in the porphyrin ring of heme in hemoglobin, myoglobin, catalase, peroxidase and cytochromes Deficiency relatively common: blood loss during menstrual bleeding Determination of liver irons is used to diagnose hemochromatosis Lower hair iron noted in patients with inflammatory bowel syndrome and liver cancer Zinc: Non toxic, essential for normal growth and development, wound healing. Regulation of zinc absorption through to be controlled by amount of metal free albumin Zinc absorption decreased in presence of high dietary phosphate, excessive calcium Actively absorbed from gut into epithelial cells – stored as mucosal methallothionen Increased zinc intake depresses copper absorption Deficiency: failure to grow, skin rashes, impaired cell mediated immunity, failure of sexual maturation, poor wound healing, growth retardation Plasma levels decrease after meals, during acute infections, liver disease, malignant tumor, permicious anemia and late pregnancy Acrodermatitis enteropathica: genetic disorders of zinc metabolism (zinc deficiency) Inhalation of zinc oxide fumes can cause nausea and headaches Zinc chloride fumes is highly corrosive to skin Copper: 6|Page
Plays key role in formation of red blood cells and maintenance of normal brain function Wilson’s disease: copper accumulates in the liver – progressive liver damage Deposition of copper in cornea – kayser Fleisher rings Menkes disease (kinky hair syndrome) – rare copper deficiency caused by x chromosomal defect in copper absorption from intestinal mucosa to the blood Chronic copper poisoning – liver disease
Cobalt: Necessary for the activity and function of cobalamin (vit B12) Stored in RBC, with small amount in kidney, liver and pancreas Excess cobalt can cause enlargement of thyroid gland Iodine: Used in hormone production Deficiency can cause enlargement of thyroid glands (goiter) Molybdenum: Cofactor for enzyme xanthine oxidase (purine degradation pathway) High molybdenum intakes can result in copper deficiency Low molybdenum intake can cause copper toxicity Selenium: Overdose can cause toxicity: hair loss and liver disease High selenite intake in children cause stunting of growth Deficiency can cause Keshan disease (myocardiopathy) and Kaschin back disease (endemic degenerative osteoarthropathy) Lab assessment of trace elements: Reinsch test: o Initial indicator for presence of trace elements o Used when suspecting poisoning o Presence was confirmed by absorption / emission on spectroscopy and x-rays Marsh test: o Very sensitive in detection of arsenic and was use in forensic toxicology Battery of test: body tissue, body fluid, metalloenzyme assay. Contamination of samples: 7|Page
Primary problem (external contamination): rubber, wood, paper product, metal surface, dust, skin, dandruff and hair Washing by use soak in nitric acid and rinse thoroughly Use disposables for sample collection.