Lecture notes 2014

MICRONUTRIENTS
Micronutrients are essential compounds required by the human body throughout life in
relatively small quantities to perform a wide range of biochemical functions. They include
vitamins, minerals, and biologically active phytochemicals.

VITAMINS

Vitamins are essential nutrients with a wide range of biochemical functions. They function
as antioxidants, precursors of coenzymes, which are drivers of numerous biochemical
reactions. The distinguishing feature of the vitamins is that they generally cannot be
synthesized by mammalian cells and, therefore, must be supplied in the diet in small
quantities.

Vitamins are categorized into two distinct types: water soluble vitamins (WSV) and fat
soluble vitamins (FSV) depending on their solubility properties. FSVs include vitamins A, D, E
and K; and water soluble vitamins which include Vitamins C, B1, B2, B3, B5, B6, B12, folic
acid and biotin. Water-soluble vitamins have limited retention in the body and therefore
need regular supply through diet. Fat-soluble vitamins are stored in the liver and fatty
tissues, allowing accumulation when consumed in high amounts over time
Property WSVs FSVs
Solubility Soluble in water but insoluble in Soluble in lipids but insoluble in
lipids. water.
Absorption Directly absorbed into the blood. First into the lymph, and then
blood.
Transport Travel freely. Many require lipoprotein carriers.
Storage Circulate freely in water filled parts Stored in cells associated with fats.
of the body and mainly not stored.
Excretion The kidney detects and removes Less readily excreted; tend to
excess in urine. remain in fat storage sites.
Toxicity No toxicity arises from excess intake. Can develop toxicity.
Requirements Needed in frequent doses/intake. Needed in periodic doses.
Examples Vitamin C, B1, B2, B3, B5, B6, B12, A, D, E and K.
folic acid, biotin, lipoic acid.

© Richard L Maseruka
1 Biochemistry department
 Lecture notes 2014

Terminologies

Cofactor: A cofactor is a chemical component required by the enzyme for its activity. It may
be organic (also known as co-enzyme) or a metallic ion.

Coenzyme: This is a heat stable, low molecular weight, non-protein, organic molecule, that
binds to an apoenzyme to make it active.

Metallic activators; inorganic ions specially required by an enzyme for its activity e.g. Na +,
Mg2+, Mn2+, Co4+, Ca2+, Zn2+, Fe2+ etc.

An apoenzyme is an inactive protein part of an enzyme.

Prosthetic group: This is either a coenzyme or metallic ion bound covalently to an enzyme
protein to make it active. They include FAD, FMN, lipoic acid and biotin.

WSVs

1. Ascorbic Acid (Vitamin C)

Sources: Citrus fruits and their juices, raw or minimally cooked vegetables.

Active form: ascorbate acid itself.

Functions: The main function of ascorbate is as a reducing agent in a number of different

reactions, most importantly the hydroxylation of proline residues in collagen which
strengthens the collagen. Vitamin C is, therefore, required for the maintenance of normal
connective tissue as well as for wound healing since synthesis of connective tissue is the first
event in wound tissue remodeling. Vitamin C also is necessary for bone remodeling due to
the presence of collagen in the organic matrix of bones.

Deficiency: Leads to the disease scurvy characterized by easily bruised skin, muscle fatigue,
soft swollen gums, decreased wound healing and hemorrhaging, osteoporosis, and anemia.
Vitamin C is readily absorbed and so the primary cause of vitamin C deficiency is poor diet
and/or an increased requirement. The primary physiological state leading to an increased
requirement for vitamin C is severe stress (or trauma). This is due to a rapid depletion in the
adrenal stores of the vitamin.

© Richard L Maseruka
2 Biochemistry department
 Lecture notes 2014
2. Thiamin (Vitamin B1)

Sources: Whole grain cereals, legumes, pork, and enriched grain products.

Active form: Thiamin pyrophosphate (TPP), formed by reaction of vitamin with ATP.

Storage: Has limited storage and the liver stores can be depleted in 12-14 days.

Functions: TPP is required as an absolute cofactor for the pyruvate and α-ketoglutarate
dehydrogenase catalyzed reactions as well as the transketolase catalyzed reactions of the
pentose phosphate pathway.

Deficiency: A deficiency in thiamin intake leads to a severely reduced capacity of cells to

generate energy as a result of its role in these reactions.

The dietary requirement for thiamin is proportional to the caloric intake of the diet. If the
carbohydrate content of the diet is excessive then an increase in thiamin intake will be
required.

Clinical Significance of Thiamin Deficiency

The earliest symptoms include constipation, appetite suppression, nausea as well as mental
depression, and fatigue. Chronic thiamin deficiency leads to more severe neurological
symptoms including ataxia, mental confusion and loss of eye coordination. Other clinical
symptoms of prolonged thiamin deficiency are related to cardiovascular and musculature
defects.

Beriberi, is the result of a diet that is carbohydrate rich and thiamin deficient. An additional
thiamin deficiency related disease is known as Wernicke-Korsakoff syndrome. This
syndrome is most commonly found in chronic alcoholics due to their poor dietetic lifestyles
and results in loss of memory and rhythmical motion of the eyeballs.

Biochemical assessment: Indicators used to estimate thiamin requirements are urinary

excretion, erythrocyte transketolase activity coefficient, erythrocyte thiamin, blood
pyruvate and lactate, and neurologic changes. The excretion rate of the vitamin and its
metabolites reflects intake, and the validity of the assessment of thiamin nutritional status
is improved with load test. Erythrocyte transketolase activity co-efficient reflects TPP levels
and can indicate rare genetic defects. Erythrocyte thiamin is mainly a direct measure of TPP
but also is a measure of thiamin and thiamin monophosphate by high performance liquid
chromatography (HPLC) separation.

Thiamin status has been assessed by measuring urinary thiamin excretion under basal

© Richard L Maseruka
3 Biochemistry department
 Lecture notes 2014
conditions or after thiamin loading, transketolase activity, and free and phosphorylated
forms in blood or serum. The erythrocyte transketolase assay, in which an activity
coefficient based on a TPP stimulation of the basal level is given, continues to be a main
functional indicator

3. Riboflavin (Vitamin B2)

Sources: Milk, eggs, liver, kidney, heart, yeast, germinating cereals, and many vegetables.

Active forms: Flavin Adenine Dinucleotide (FAD) and Flavin Mono Nucleotide (FMN).
Riboflavin is converted to FMN in the intestinal mucosa and to FAD in the liver.

Requirements: Its requirement is related to protein use and increases during growth,
pregnancy, lactation and wound healing.

Functions: The enzymes that require FMN or FAD as cofactors are termed flavoproteins.
These are involved in a wide range of redox reactions, e.g. succinate dehydrogenase. During
the course of the enzymatic reactions involving the flavoproteins, the reduced forms of FMN
and FAD are formed (FMNH2 and FADH2, respectively).

Clinical Significances of riboflavin Deficiency

Riboflavin deficiencies are rare because it is wide spread in nature. However, Riboflavin
deficiency is often seen in chronic alcoholics due to their poor dietetic habits.

Symptoms of riboflavin deficiency include:

• Glossitis (swollen, painful and mangenta coloured tongue)

• Seborrheic dermatitis (rough and scaly skin)

• Angular stomatis (fissures at angles of the mouth)

• Cheilosis (Cracked and swollen lips)

• Photophobia

• Corneal vascularisation (growth of blood vessels in the cornea)

• Riboflavin decomposes when exposed to visible light. This characteristic can lead to
riboflavin deficiencies in newborns treated for hyperbilirubinemia by phototherapy.

© Richard L Maseruka
4 Biochemistry department
 Lecture notes 2014

4. Niacin (Vitamin B3)

Sources: Dietary sources of niacin include cereals, legumes and meats. However, about 10%
of the niacin requirement in humans is formed in the body during catabolism of tryptophan.

Active form: Niacin exists in several forms such as Nicotinic Acid and Nicotinamide. The
active forms are nicotinamide adenine dinucleotide (NAD+) and nicotinamide adenine
dinucleotide phosphate (NADP+).

Functions: Niacin is required for the synthesis of the active forms of vitamin B3, NAD+ and
NADP+. Both NAD+ and NADP+ function as cofactors for numerous dehydrogenase, e.g.,
lactate and malate dehydrogenases.

Clinical Significances of Niacin and Nicotinic Acid deficiency: A diet deficient in niacin
leads to glossitis of the tongue, dermatitis, weight loss, diarrhea, depression and dementia.
The severe symptoms, depression, dermatitis and diarrhea, are associated with the
condition known as pellagra. Several physiological conditions (e.g. Hartnup disease and
malignant carcinoid syndrome) as well as certain drug therapies (e.g. isoniazid) can lead to
niacin deficiency.

Biochemical assessment: Indicators used to estimate niacin requirements are urinary

excretion, plasma concentrations of metabolites, and erythrocyte pyridine nucleotides. The
excretion rate of metabolites, mainly N'-methyl-nicotinamide and its 2- and 4-pyridones,
reflects intake. Concentrations of metabolites, especially 2-pyridone, are measured in
plasma after a load test. Erythrocyte pyridine nucleotides measure NAD concentration
changes.

5. Pantothenic Acid (Vitamin B5)

Sources: Widely distributed in whole grains, cereals, legumes, and meat.

Active form: Coenzyme A (CoA), Acyl carrier protein (ACP)

Functions: Pantothenate is required for synthesis of coenzyme A (CoA) and is a component

of the acyl carrier protein (ACP) domain of fatty acid synthase. Pantothenate is, therefore
required for the metabolism of carbohydrate via the TCA cycle and all fats and proteins. At
least 70 enzymes have been identified as requiring CoA or ACP derivatives for their function.

© Richard L Maseruka
5 Biochemistry department
 Lecture notes 2014
Deficiency: It is extremely rare due to its widespread distribution in whole grain cereals,
legumes and meat. Symptoms of pantothenate deficiency are difficult to assess since they
are subtle and resemble those of other B vitamin deficiencies.

Biochemical assessment: Indicators used to estimate pantothenate requirements are

urinary excretion and blood levels. Excretion rate reflects intake. Whole blood, which
contains vitamin and pantothenate-containing metabolites, has a general correlation with
intake.

6. Vitamin B6 (Pyridoxal, Pyridoxamine And Pyridoxine)

Sources: Meat, fish, poultry, whole grains, cereals, and certain vegetables.

Active form: Pyridoxal phosphate (PLP). All the three compounds are efficiently converted
to the biologically active form PLP by the ATP requiring enzyme, pyridoxal kinase.

Functions: PLP functions as a cofactor in enzymes involved in transamination reactions

required for the synthesis and catabolism of the amino acids as well as in glycogenolysis as a
cofactor for glycogen phosphorylase.

The requirement for vitamin B6 in the diet is proportional to the level of protein
consumption. During pregnancy and lactation the requirement for vitamin B6 increases.

Deficiencies of vitamin B6 are rare and usually are related to an overall deficiency of all the
B-complex vitamins.

Biochemical assessment: Indicators used to estimate vitamin B6 requirements are PLP,

urinary excretion, erythrocyte aminotransferases activity coefficients, tryptophan
catabolites, erythrocyte and whole blood PLP, and plasma homo-cysteine.

7. Biotin

Sources: Biotin is found in numerous foods and also is synthesized by intestinal bacteria and
as such deficiencies of the vitamin are rare.

Active form: Biotin is not modified bu must be covalently bonded to the enzymes that
require it as a prosthetic group.

Functions: Biotin is the cofactor required of enzymes that are involved in carboxylation

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6 Biochemistry department
 Lecture notes 2014
reactions, e.g. acetyl-CoA carboxylase and pyruvate carboxylase.

Deficiencies are generally seen only after long antibiotic therapies which deplete the
intestinal fauna or following excessive consumption of raw eggs. The latter is due to the
affinity of the egg white protein (avidin) for biotin preventing intestinal absorption of the
biotin.

Biochemical assessment: Indicators used to estimate biotin requirements are urinary

excretion and 3-hydroxyisovalerate excretion. The excretion rate of the vitamin and its
metabolites in urine is assessed by avidin-based radioimmunoassay with HPLC.

8. Cobalamin (Vitamin B12)

Sources: Vitamin B12 is synthesized exclusively by microorganisms and is found in the liver
of animals bound to protein as methycobalamin or 5'-deoxyadenosylcobalamin. The vitamin
must be hydrolyzed from protein (in the stomach by gastric acids or the intestines by trypsin
digestion), in order to be active. The vitamin is then bound by intrinsic factor, a protein
secreted by parietal cells of the stomach, and carried to the ileum where it is absorbed.

Active form: Methyl cobalamin.

Functions: There are only two clinically significant reactions in the body that require
vitamin B12 as a cofactor. During the catabolism of fatty acids with an odd number of carbon
atoms and the amino acids valine, isoleucine and threonine. The second reaction requiring
vitamin B12 catalyzes the conversion of homocysteine to methionine and is catalyzed by
methionine synthase. This reaction results in the transfer of the methyl group from
N5-methyltetrahydrofolate to hydroxycobalamin generating tetrahydrofolate (THF) and
methylcobalamin during the process of the conversion.

Clinical Significances of B12 Deficiency: The liver can store up to six years worth of vitamin
B12, hence deficiencies in this vitamin are rare. Pernicious anemia is a megaloblastic anemia
resulting from vitamin B12 deficiency that develops as a result a lack of intrinsic factor in
the stomach leading to malabsorption of the vitamin. The anemia results from impaired DNA
synthesis due to a block in purine and thymidine biosynthesis. The block in nucleotide
biosynthesis is a consequence of the effect of vitamin B12 on folate metabolism.

Assessment of vitamin B12 status: Traditionally it was thought that low vitamin B12 status
was accompanied by a low serum or plasma vitamin B12 level. Recently, it’s been suggested
that a proportion of people with normal vitamin B12 levels are in fact vitamin B12 deficient.

© Richard L Maseruka
7 Biochemistry department
 Lecture notes 2014
It is also suggested that elevation of plasma homo-cysteine and plasma Methylmalonic Acid
(MMA) are more sensitive indicators of vitamin B12 status.

9. Folic Acid

Sources: Yeasts and leafy vegetables and animal liver. Folate is easily destroyed by cooking.

Active form: Tetrahydrofolate (THF).

Function: The coenzyme THF carries and transfers various forms of one carbon units during
biosynthetic reactions. The one carbon units are either methyl, methylene, methenyl,
formyl or formimino groups. These one carbon transfer reactions are required in the
biosynthesis of serine, methionine, glycine, choline and the purine nucleotides and dTMP.
positions 7 & 8 carry hydrogens in
dihydrofolate (DHF)
positions 5-8 carry hydrogens in
tetrahydrofolate (THF)

© Richard L Maseruka
8 Biochemistry department
 Lecture notes 2014

Clinical Significance of Folate Deficiency: Folate deficiency results in complications nearly

identical to those described for vitamin B 12 deficiency. The most pronounced effect of folate
deficiency on cellular processes is upon DNA synthesis, which leads to cell cycle arrest in
S-phase of rapidly proliferating cells, in particular hematopoietic cells. The result is
megaloblastic anemia as for vitamin B12 deficiency. The inability to synthesize DNA during
erythrocyte maturation leads to abnormally large erythrocytes termed macrocytic anemia.

Folate deficiencies are rare due to the adequate presence of folate in food. Poor dietary
habits as those of chronic alcoholics can lead to folate deficiency. The predominant causes
of folate deficiency in non-alcoholics are impaired absorption or metabolism or an increased
demand for the vitamin. The predominant condition requiring an increase in the daily intake
of folate is pregnancy. This is due to an increased number of rapidly proliferating cells
present in the blood. The need for folate will nearly double by the third trimester of
pregnancy

© Richard L Maseruka
9 Biochemistry department
 Lecture notes 2014
SUMMARY OF THE B-COMPLEX VITAMINS

NAME COENZYME ENZYME FUNCTIONS

Thiamine, B1 TPP pyruvate dehydrogenase & Carbohydrate metabolism

transketolase

Riboflavin, B2 FAD & FMN Succinate dehydrogenase Redox reaction

Niacin, B3 NAD+ & NADP+ Lactate & malate Redox reaction

dehydrogenase

Pantothenic ACOA 70 groups of enzymes AcetylCoA & ACP synthesis;

acid, B5 Carbohydrate metabolism
& all fat metabolism

Pyridoxal, B6 PLP Pyridoxal kinase Deamination

Decarboxylase Decarboxilation

Transaminase Transamination

Biotin Biotin prosthetic ACOA carboxylase Carboxylation

group
Pyruvate carboxylase

Cobalamin,B12 DNA synthesis

Folic acid Tetrahydrofolate Very many enzymes Transfer 1 cabon units

/THF during biosynthesis of
pyrimidines and purines

© Richard L Maseruka
10 Biochemistry department
 Lecture notes 2014

FSVs

1. Vitamin A

Vitamin A consists of three biologically active molecules, retinol, retinal and retinoic acid.

Each of these compounds are derived from the plant precursor molecule, β-carotene.
Beta-carotene, which consists of two molecules of retinal linked at their aldehyde ends, is
also referred to as the provitamin form of vitamin A.

Ingested β -carotene is cleaved in the lumen of the intestine to yield retinal. Retinal is
reduced to retinol by retinaldehyde reductase.

Vision and the Role of Vitamin A

Photoreception in the eye is the function of two specialized cell types located in the retina;
the rod and cone cells. Both rod and cone cells contain a photoreceptor pigment in their
membranes. The photosensitive compound of most mammalian eyes is a protein called opsin
to which is covalently coupled an aldehyde of vitamin A.

Additional Role of Retinol

Retinol also functions in the synthesis of certain glycoproteins and mucopolysaccharides

necessary for mucous production and normal growth regulation.

Clinical Significances of Vitamin A Deficiency

Vitamin A is stored in the liver and deficiency of the vitamin occurs only after prolonged lack
of dietary intake. The earliest symptoms of vitamin A deficiency are night blindness.
Additional early symptoms include follicular hyperkeratinosis, increased susceptibility to
infection and cancer and anemia equivalent to iron deficient anemia. Prolonged lack of
vitamin A leads to deterioration of the eye tissue through progressive keratinization of the
cornea, a condition known as xerophthalmia.

The increased risk of cancer in vitamin deficiency is thought to be the result of depletion in
β-carotene. Beta-carotene is a very effective antioxidant and is suspected to reduce the risk
of cancers known to be initiated by the production of free radicals. However, caution needs
to be taken when increasing the intake of any of the lipid soluble vitamins. Excess
accumulation of vitamin A in the liver can lead to toxicity which manifests as bone pain,
nausea and diarrhea.
© Richard L Maseruka
11 Biochemistry department
 Lecture notes 2014
2. Vitamin D

Vitamin D is a steroid hormone whose biologically active form of the hormone is

1,25-dihydroxy vitamin D3 (1,25-(OH)2D3, also termed calcitriol). Calcitriol functions
primarily to regulate calcium and phosphorous homeostasis. Calcitriol functions together
with parathyroid hormone (PTH) and calcitonin to regulate serum calcium and phosphorous
levels. PTH is released in response to low serum calcium and induces the production of
calcitriol. In contrast, reduced levels of PTH stimulate synthesis of the inactive
24,25-(OH)2D3. In the intestinal epithelium, calcitriol functions as a steroid hormone in
inducing the expression of calbindin, a protein involved in intestinal calcium absorption.
When plasma calcium levels fall the major sites of action of calcitriol and PTH are bone
where they stimulate bone resorption and the kidneys where they inhibit calcium excretion
by stimulating reabsorption by the distal tubules. The role of calcitonin in calcium
homeostasis is to decrease elevated serum calcium levels by inhibiting bone resorption.
Clinical Significance of Vitamin D Deficiency

The main symptom of vitamin D deficiency in children is rickets and in adults is osteomalacia.
Rickets is characterized improper mineralization during the development of the bones
resulting in soft bones. Osteomalacia is characterized by demineralization of previously
formed bone leading to increased softness and susceptibility to fracture.

3. Vitamin E

Vitamin E is a mixture of several related compounds known as tocopherols. Vitamin E is

absorbed from the intestines. The major site of vitamin E storage is in adipose tissue.

The major function of vitamin E is to act as a natural antioxidant by scavenging free radicals
and molecular oxygen.
Clinical significances of Vitamin E Deficiency

No major disease states have been found to be associated with vitamin E deficiency due to
adequate levels in the average diet. The major symptom of vitamin E deficiency in humans is
an increase in red blood cell fragility. Since vitamin E is a fat soluble vitamin and is absorbed
from the intestines, any fat malabsorption diseases can lead to deficiencies in vitamin E
intake. Neurological disorders have been associated with vitamin E deficiencies associated
with fat malabsorptive disorders. Polyunsaturated fatty acids tend to form free radicals
upon exposure to oxygen and this may lead to an increased risk of certain cancers.

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12 Biochemistry department
 Lecture notes 2014
4. Vitamin K

The K vitamins exist naturally as K1 (phylloquinone) in green vegetables and K2

(menaquinone) produced by intestinal bacteria and K3 is synthetic menadione. When
administered, vitamin K3 is alkylated to K2.

The major function of the K vitamins is in the maintenance of normal levels of the blood
clotting proteins.

Clinical significance of Vitamin K Deficiency

Absorption of vitamin K takes place in the intestines, only in the presence of bile salts and
other lipids. Therefore, fat malabsorptive diseases can result in vitamin K deficiency. Since
the vitamin K2 form is synthesized by intestinal bacteria, deficiency of the vitamin in adults
is rare. However, long term antibiotic treatment can lead to deficiency in adults. The
intestine of newborn infants is sterile, therefore, vitamin K deficiency in infants is possible if
lacking from the early diet. The primary symptom of a deficiency in infants is a hemorrhagic
syndrome.

© Richard L Maseruka
13 Biochemistry department