DIAGNOSTIC IN CLINICAL

CHEMISTRY I
MKEB 2404

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

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 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

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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

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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 2,4-
dichlorophenol-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

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 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

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 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:

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 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:

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 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.

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