Professor D.Sci. Judit KosáryNutritional biochemistry of the vitamins and secondary metabolites (2016-2)

(Lectures for students with advanced knowledge of biochemistry and food chemistry)

The lectures present the known biochemical functions of the vitamins and the possible explanations for the effects of their deficiency or excess. The biosynthesis of vitamins and secondary metabolites used in food industry is also discussed. All of vitamins and other agents are detailed in point of their:

biosynthesis, metabolism, metabolic functions, deficiency and excess, nutritional status, uses in food industry, pharmacological uses.

Topics:1. Definition and nomenclature of vitamins, their nutritional status and non-nutritional uses. 2. Precursors of reagents for biochemical reactions (molecules with coenzyme function) (water-soluble vitamins):

a) Precursors of coenzymes of oxydoreductases : niacin, riboflavin, ascorbic acid.b) Precursors of coenzymes of transferases:

1. C1 transfer: biotin, folic acid, cyanocobalamin,2. C2 transfer: thiamin, pantothenic acid,3. transfer of other groups: pyridoxine.

c) Compounds of doubtful vitamin status: taurine, carnitine, choline, inositol.3. Vitamins of other functions – vitamin lipids (fat-soluble vitamins):

1. retinol and -carotene,2. cholecalciferol and its vitamers,3. tocopherols,4. phylloquinone and its vitamers,

4. Definition and types of secondary metabolites. Secondary metabolites used in food industry (e.g. flavour agents of spices, pigments, antioxidants, etc.).

The lectures also present information about the biochemical functions and the metabolism of some important types of secondary metabolites, which are of different molecules of the living organisms, and they are more or less needed for their functioning. Secondary metabolites are synthesized from the different intermediates of biomolecules. The presented types of secondary metabolites are coenzymes, regulating (e.g. hormones), attracting (e.g. the sweet sucrose, the fruit esters as scent agents etc.) and repelling agents (e.g. alkaloids and toxins).

Literature: Bender, D.A.: Nutritional biochemistry of vitamins. Cambridge University Press Cambridge New York Port Chester Melbourne Sydney 1992; Luckner, M.: Secondary metabolism in microorganisms, plants and animals Springer Verlag Berlin Heidelberg New York London Paris Tokyo Hong Kong 1990.

Nutritional biochemistry of the vitamins and secondary metabolites Professor D.Sci. Judit Kosáry

The vitamins are a disparate group of organic compounds whose only common feature is that they are essential (cannot be synthesized inside) and required in small amount for the normal functioning of higher animals and the human body, therefore they must be provided in nutrition. These compounds can be synthesized by plants and – some of them – by animals from intermediates of their primary metabolic pathways. According to their role vitamins have diverse functions in the human metabolism. Polar (“water-soluble”) vitamins are precursors of reagents so-called coenzymes in metabolic organic chemical reactions. The water-soluble vitamins are vitamin C and a series known as the vitamin B complex. Nonpolar (apolar) (“lipid-soluble or fat-soluble”) vitamins can be hormones, modulators or regulators and non-specific antioxidants in human metabolism.

In contrast with primary metabolism that is practically identical in all types of living organisms, secondary metabolism is a collection of a wide variety of biochemical pathways characteristic for only a few species of organisms and their distribution is different in specialized cells. Compounds formed in these reactions called secondary products. According to their biosynthetic pathways vitamins can be considered secondary metabolits but with a view to their formation in cell-division (cytogenesis) vitamins can be classified as primary metabolits.

In the course Nutritional biochemistry of the vitamins and secondary metabolites the vitamins are discussed in topics: biosynthesis; metabolism; metabolic functions; explain the effects of deficiency and excess; nutritional status; uses in food industry; pharmacological uses and scientific basis for Recommended Intakes. Topics for secondary metabolites used in food industry: flavour agents, pigments, antioxidants, etc.

1. Definition and nomenclature of vitamins. Their nutritional status and non-nutritional uses.

Essential compoundsA compound, that the human body cannot synthesize them from other compounds at the

level needed for normal growth, is called an essential compound. These materials must be obtained from food. The essential amino acids (Val, Leu, Ile, Phe, Lys, Thr, Trp, Met, Arg, His) are needed in a large quantity. Of other essential compounds we only need small quantities e.g. vitamins).

Definition of vitaminsThe vitamins are a disparate group of organic compounds whose only common feature is

that they are essential (cannot be synthesized inside) and required in small amount for the normal functioning of higher animals and the human body, therefore they must be provided in nutrition. These molecules serve nearly the same roles in all forms of life, but higher animals (and human body) have lost the ability to synthesize them. The original definition of vitamins based on their dietary essential, but now it is partly a historical category. Later it was proved that some of vitamins could be synthesized in the body (e.g. tocopherol and niacin). Nevertheless, for historical reasons these compounds are classified as vitamins.

At the beginning of the twentieth century the history of vitamins started by feeding experiments of Hopkins, who found that animals fed by known and characterised nutrients as

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fats, proteins, carbohydrates and mineral salt failed to grow without addition a small amount of milk. Hopkins suggested that milk contained ‘accessory growth factors’. The characteristic functional group of the first of ‘accesory food factors” isolated was an amine (thiamin). Therefore these kind of food factors were called by Funk (1912) ‘vitamin’ from the combination of the Latin expression of the word ‘life’ (vita) and the name of the functional group (amine). Later it came to light that the presence of an amine group is not essential forl of vitamins, besides the structures and functional groups of vitamins are highly various but the name ‘vitamin’ is preserved.

Nomenclature of vitaminsAt first vitamins had an apparently illoginal system of accepted trivial nomenclature then

after identification they got chemical names and short ‘accepted’ names, as well. The basis of the trivial system was on one hand the polar-apolar properties of the vitamin (water-soluble and fat-soluble) and the other hand the order (sequence) of isolation according to ABC. For example at first the name of all of water-soluble vitamins were planned as ‘vitamin B’ depending on the time of isolation e.g. B1, B2 or Bn. Parallel and false identifications made the system confused. In addition there were different chemical compounds with the same biological activity. Now it is known that they are different intermediers of the final active compound therefore they are called vitamers, e.g. retinol and -carotene or nicotinic acid and nicotinamide (niacin) or cholecalciferol and ergocalciferol or phylloquinone and menaquinone.

Table 1. Alphabetical name; accepted name; principal fuctions and deficiency diseases of vitamins

(On the basis of Nutritional biochemistry of the vitamins by D.A. Bender)Alphabetical name Accepted name(s)

(and vitamer precursors)

Principal functions Deficiency diseases

A retinol (and its precursor: -carotene)

visual pigments,cell differentiation

night blindness, xenophthalmia, keratomalacia

B1 thiamin (aneurine) precursor of coenzyme TPP and role in neurotransmission

beriberi

B2 riboflavin precursor of redox coenzyme FAD

no distingtive signs

(B3) niacin (nicotinamide and its precursor: nicotinic acid)

precursor of redox coenzymes NAD+

and NADP+

pellagra

(B5) pantothenic acid precursor of coenzyme A

burning foot syndrome

B6 pyridoxin precursor of coenzyme PAL and role in steroid action

convulsions, metabolic disturbances

(B10) folic acid precursor of coenzyme THF

megaloblastic anaemia

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B12 cobalamine precursor of coenzyme cobalamide

anaemia

C ascorbic acid redox coenzyme with oxygen comsuption and anti-oxidant

scurvy

D cholecalciferol (calciol)

calcium homeostasis rickets, osteomalacia

E -tocopherol (and its precurors: -tocopherol and -tocopherol)

anti-oxidant (haemolytic anaemia of newborn)

H biotin coenzyme rare - skin lesionsK phylloquinon (K1)

and metaquinon (K2) (from common precursor)

carboxylation of glutamate in postsynthetic modification of proteins, role in blood coagulation

bleeding disorders

Table 2. Alphabetical name; accepted name; name of biologically active derivative and metabolic function

(On the basis of Nutritional biochemistry of the vitamins by D.A. Bender)Alphabetical name Accepted name(s)

(and vitamer precursors)

Name of biologically active derivative

Metabolic function

A retinol (and its precursor: -carotene)

11-Z-retinal, the light absorbing group of visual pigments

photoisomerisation of 11-Z-retinal to all-E-retinal in retina

B1 thiamin (aneurine) thiamin pyrophosphate (TPP)

pyruvate dehydrogenase multienzyme complex

B2 riboflavin flavinadenine dinucleotide (FAD)

oxidative degradation of biomolecules by radical mechanism

B3 niacin (nicotinamide and its precursor: nicotinic acid)

nicotinamide adenine dinucleotide (NAD+) and nicotinamide adenine dinucleotide phosphate (NADP+)

oxidative degradation of biomolecules by ionic mechanism (NAD+) and reductive biosynthetic

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processes (NADPH)B5 pantothenic acid coenzyme A central macroerg

intermediate both in biosyntheses and degradation processes

B6 pyridoxin pyridoxalphosphate (PAL)

elimination of amino group in degradation of amino acids, amine binding function in active sites of enzymes

B10 folic acid tetrahydrofolic acid (THF)

transfer of C1

fragment (e.g. methyl group) on biomolecules

B12 cobalamin cobalamide alkyl rearrangementC L-ascorbic acid L-ascorbic acid coenzyme in

oxidation by oxygen and protection against oxidative damage together with -tocopherol

D cholecalciferol (calciol)

calcitriol, a hormon of the metabolism calcium and phosphorous in bones

induction of osteocalcin, a calcium binding protein in bone

E -tocopherol (and its precurors: -tocopherol and -tocopherol)

-tocopherol protection against oxidative damage together with L-ascorbic acid

H biotin biotin rare - skin lesionsK phylloquinon (K1)

and metaquinon (K2) (from common precursor)

phylloquinon (K1) and metaquinon (K2) (from common precursor)

carboxylation of glutamate in postsynthetic modification of proteins

Table 3. Alphabetical name of parallel and false vitamin identifications(On the basis of Nutritional biochemistry of the vitamins by D.A. Bender)

Name of vitamin StoryB3 Assigned to a compound which was probably pantothenic acid,

nowadays is used for nicotinamide or nicotinic acid (niacin).B4 Assigned to a mixture of arginine, glycine and cysteine, later

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assigned to AMP.B5 Assigned to a compound which mighth be pyridoxine or nicotinic

acid, now used for pantothenic acid.B7 Never assigned.B8 Never assigned.B9 Earlier used for pantothenic acid.B10 Assigned to a compound which mighth be a mixture of folic acid

and thiamin, now used for folic acid.B11 Assigned to a compound which mighth be a mixture of folic acid

and thiamin.B13 Assigned to orotic acid, that is not vitamin.B14 Assigned to an unknown substance in urine which increases

proliferation of bone warrow in culture.B15 Assigned to pangamic acid, its vitamin function was not

established.B16 Never assigned.B17 Assigned to a cyanogen glycoside (amygdalin or laetrile) with no

established vitamin functionBc Obsolete name for folic acidBp Assigned to ‘anti-perosis’ factor for chikens which can be replaced

by cholin and manganeseBT Assigned to carnitine which is a grow factor for the meal worm, but

not a vitamin.Bw Assigned to a factor which was probably biotinBx Obsolete name for 4-aminobenzoc acid, a precursor of folic acid,

which is not vitamin.F Essential fatty acids (linolic acid and linoleic acid) which are not

classified as vitamins.G Obsolete name for riboflavin.H3 Assigned to gerovital which was later identified as novocaine. Not

recognised as a vitamin.L Factor in yeast claimed to promote lactation but it was not

established as a vitamin.M Obsolete name for folic acidP Pharmacologically active bioflavonoids with no vitamin function.

PP Obsolete name for niacin (pellagra-preventing vitamin).T Assigned to a mixture of folic acid, cyanocobalamin and and

nucleotides.U Assigned to methylsulphonium salt of methionine which may have

pharmacological acions, but it is not a vitamin

Nutritional status of vitamins – Determination of essentialityBefore declaring a compound as a vitamin it must be shown to be a dietary essential. That

means its elimination from the diet results in a more or less clearly defined deficiency disease that can be cured or prevented by adding of this compound to the diet. Only the fact of a

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pharmacological action or curing a disease is a necessary condition but not a sufficient evidence to classify a compound as a vitamin.

Requirements and recommendationsAs vitamins are present in foods and in body fluids or tissues in very small amounts (of

the order mol, nmol, even pmol/kg), furthermore they can be exist in multiple chemical forms (sometimes in biologically unavailable forms), their analysis and the estimation of the useable vitamin content demand a combination of several chemical and biological methods. With the modern techniques – e.g. radio-ligand binding assays (radio-immunassay) and hplc techniques – individual chemical forms of most of the vitamins can be determined with great precision and specificity. Microbiological and biological assays can be essential to determine the relative biological activity of different vitamers.

There are several official recommendations for vitamin consumption. On the basis of population examined recommended intakes are various in different countries. All around the world accepted lists are Recommended Dietary Allowance (RDA) for an adult man aged between 25-50 for a day from the 1989 US Tables (National Research Council/National Academy of Sciences, 1989) and Reference Nutrient Intake (RNI) for an adult man aged between 19-50 for a day from the 1991 UK Tables (Department of Health/Ministry of Agriculture, Fisheries and Food, 1991).

Table 4. Recommended intakes of vitamins(On the basis of Nutritional biochemistry of the vitamins by D.A. Bender)

Accepted name(s) (and vitamer precursors)

RDA (USA) RNI (UK)

retinol (and its precursor: -carotene)

1 mg 0.7 mg

thiamin (aneurine) 1.5 mg 1.0 mgriboflavin 1.7 mg 1.4 mg

niacin (nicotinamide and its precursor: nicotinic acid)

19 mg 17 mg

pantothenic acid 4-7 mg (average intake)

4-7 mg (average intake)

pyridoxin 2.0 1.4folic acid 0.2 mg 0.2 mgcobalamin 2.0 g 1.5 g

L-ascorbic acid 60 mg 40 mgcholecalciferol (calciol) 5 g 10 g (for house-

bound elderly-tocopherol (and its precurors: -

tocopherol and -tocopherol)10 mg 7 mg (it depends on

intake of poly-unsaturated fatty

acids)biotin 30-200 g (average

intake)30-200 g (average

intake)phylloquinon (K1) and metaquinon

(K2) (from common precursor)80 g 70 g

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Non-nutritional uses of vitaminsRecommended intakes of vitamins for people the maitenance of normal health and

metabolic integrity and the prevention of deficiency. There are suggestions about the benefit of vitamins (and other nutritional supplements) intake higher than nutritional requirements. In some cases no scientific foundations exist and these hypotheses are based only on their pharmacological action in relatively high intake and speculations. But there are evidences from medical and laboratory studies that some vitamins may have health benefit in relatively high doses. For example folic acid supplements in pregnancy is a protective factor against tube defects and higher intakes of antioxidants (L-ascorbic acid, tocopherols and -carotene) can reduce probability of cancer and cardiovascular diseases. Nevertheless, several vitamins are acutely or chronically toxic in high excess (e.g. retinol, cholecalciferol, niacin and pyridoxin).

Vitamin deficiency (avitaminosis and hypovitaminosis)Vitamin deficiency is a lack of one or more vitamins in humans (or other living

organisms). In the case of hypovitaminosis the level of a vitamin is lower than the recommended level. In the case of hypervitaminosis the level of a vitamin is higher than recommended level. There are five vitamins those are associated with a pandemic deficiency disease: niacin (vitamin B3) – pellagra, ascorbic acid (vitamin C) – scurvy, aneurine (vitamin B1) – beriberi, calciferol (vitamin D) – rickets, retinol (vitamin A) – night blindness.

Iatrogenic hypovitaminosisThere are drugs with antivitamin actions causing called drug-induced malnutrition. For

example folics antagonists are used in cancer chemotherapy and some of antischizophrenic agents are riboflavin antagonists. For the normal health smoking persons need more of different vitamins than non-smoking ones.

The solubility of vitaminsThe molecules with apolar character (they can not form no H-bonds with water in most of

the cases because of their non-polarized bonds) are not soluble in water (fat-soluble molecules). The molecules with polar character (they have polarized bonds e.g. alcohols therefore they can form H-bonds with water molecules) can be soluble in water (water-soluble molecules).

Vitamins are classified as either water-soluble or fat-soluble. In humans there are 13 vitamins. Ordinary four vitamins (A, D, E and K) are labelled as fat-soluble vitamins but sometimes vitamins F are mentioned among them. The water-soluble vitamins are the different vitamins B, vitamin H and vitamin C. Water-soluble vitamins dissolve easily in water, and in general, are readily excreted from the body, to the degree that urinary output is a strong predictor of vitamin consumption. Because they are not readily stored, consistent daily intake is important. Many types of water-soluble vitamins are synthesized by bacteria. Water-soluble vitamins are coenzymes or the starting materials of coenzymes (they are the reagents in the biochemical reactions). Fat-soluble vitamins are absorbed through the intestinal tract with the help of lipids (fats). Because they are more likely to accumulate in the body, they are more likely to lead to hypervitaminosis than are water-soluble vitamins.

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Essential polar and apolar characters of simple functional groups

Fat-soluble vitaminsThere are two types of lipid (apolar – fatty soluble) biomolecules. Simple lipids cannot be

hydrolyzed by sodium hydroxide and complex lipids can be hydrolyzed by sodium hydroxide. Fat-soluble vitamins can be stored in the apolar part of cell membranes, sometimes this can cause hypervitaminosis.

The two main types of simple lipids are the fatty acids and the terpenes. Fatty acids (C16

and C18) are building blocks of complex lipids (neutral triglycerides and phospholipids). Saturated fatty acids are palmitate (CH3(CH2)14–COOH) and stearate (CH3(CH2)16–COOH). Unsaturated fatty acids are the unsaturated versions of stearate (C18): oleate, linoleate and linolenate. The essential linoleate (-6-fatty acid) and linolenate (-3-fatty acid) are known as PUFA (polyunsaturated fatty acids) or vitamins F. Polyunsaturates fatty acids can be found in unsaturated oils (oil of fishes, sunflower, flaxseed – linseed, etc.). Essential fatty acids play an important role in the life and death of cardiac cells.

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Formulas of oleate, linoleate and linolenate

Terpenes can be derived from isoprene (methylbutadiene) CH2=C(CH3)–CH=CH2 (C5H8). Monoterpenes contain two (C5H8)2 (C10), diterpenes four s (C5H8)4 (C20), triterpenes six (C5H8)6

(C30) and tetraterpenes eight isoprene units (C5H8)8 (C40). The branched end of isoprene is called the head (fej in Hungarian) and the other part is called the tail (láb in Hungarian). There are different variations for connecting the isoprene units. Most frequent are head-to-tail connections, while and tail-to-tail and head-to-head variations are rare. The formation and oxidative degradation of vitamins with terpene structure are similar to other kinds of terpenes.

H2C C CH CH2

CH3izoprén fej-fej

fej-láb láb-láb

fej láb

Az izoprén egységek kapcsolódási fajtáiDifferent variations of connecting isoprene units: head-to-head, head-to-tail and

tail-to-tail

Calciferols (vitamin D)The chain of the triterpene squalene can be cyclized to molecule cholesterol. The ring

system of cholesterol is called sterane skeleton (without methyl groups – gonane skeleton). Cholesterol can be the starting material of different kinds of steroids – among them sexual hormones. There are different vitamin D molecules – their differences are only in the number of methyl groups. For example ergocalciferol – vitamin D2 contains an extra methyl group (compared to cholecalciferol – vitamin D3) at the carbon atom next to the branched carbon atom and a double bond in the side chain. Vitamin D molecules formed from cholesterol by uv light play an important role in calcification of cartilage and bone. From vitamin D calcidiol is formed in the liver then calcitriol in the kidney by hydroxylation. Calcitriol is the biologically active form of vitamin D.

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The symptoms of vitamin D deficiency are: brittle and fragile bones (rickets), burning in mouth and throat, diarrhea, insomnia, irregular heartbeat, low blood calcium, myopia, nervousness, pale skin, poor metabolism, rickets, sensitivity to pain, soft bones and teeth, osteoporosis and osteopenia, and hypocalcemia. Vitamin D also enhances the immune system. Hypervitaminosis of vitamin D can cause similar symptoms in bones to hypovitaminosis. Vitamins D can be found in cod liver oil, liver, milk, egg, butter and cheese.

A B

C D

CH3

CH3

A B

C D

gonánváz szteránváz koleszterol

cikloalkánok

CH3 CH2

CH3

CH3 CH3

CH3

O

CH3

CH3

HO

CH3 CH3

CH3

limonén kámfor mentol

Néhány monterpén képlete

OHH

CH3

CH3

CH3

HO

CH3 CH3

CH3

A

C D h

hB

CH3

HO

CH3 CH3

CH3CH2

D3-vitamin (kolekalciferol)

CH3CH3 CH3 CH3

CH3 CH3CH3

CH3

CH3 CH3ox.

-jonon-karotin -jonon

CH2OHCH3CH3 CH3 CH3

CH3retinol (A-vitamin)

CH3CH3 CH3

CH3

CHO

CH3

11-cisz-retinál

A gonánváz, a szteránváz és a koleszterol képlete

Formula of gonane and sterane skeletons, and cholesterol

A B

C D

CH3

CH3

A B

C D

gonánváz szteránváz koleszterol

cikloalkánok

CH3 CH2

CH3

CH3 CH3

CH3

O

CH3

CH3

HO

CH3 CH3

CH3

limonén kámformentol

Néhány monoterpén képlete

OHH

CH3

CH3

CH3

HO

CH3 CH3

CH3

A

C D h

hB

CH3

HO

CH3 CH3

CH3

CH2

D3-vitamin (kolekalciferol)

CH3CH3 CH3 CH3

CH3 CH3CH3

CH3

CH3 CH3ox.

-jonon-karotin -jonon

CH2OHCH3CH3 CH3 CH3

CH3retinol (A-vitamin)

CH3CH3 CH3

CH3

CHO

CH3

11-cisz-retinál

A gonánváz, a szteránváz és a koleszterol képlete

A D3-vitamin keletkezése a koleszterolból

A legismertebb tetraterpén, -karotin és a belôle képzôdô diterpének képlete

Formation of vitamin D3 from cholesterol

Retinol (vitamin A)Tetraterpene carotenoides are organic pigments that are naturally occurring in the

chloroplasts and chromoplasts of plants. There are two classes of carotenoides: hydrocarbons i.e. carotenes and xanthophylls containing oxygen. Because of polyconjugated double bond system, carotenoids can absorb light energy for use in photosynthesis, and as antioxidants they protect chlorophyll from photodamage. Antioxidants can eliminate free radicals by reduction. In humans -carotene and other carotenoids can be converted to retinol (vitamin A) by an oxidative cleavage. Retinal synthesized from retinol by oxidation and isomerization is essential for vision.

Night blindness is one of the first signs of vitamin A deficiency because its derivative cisz-retinal has a major role in phototransduction. This is a process in which the light is converted into electrical signals in the retina of the eye. Vitamin A deficiency also contributes to maternal mortality and other poor outcomes in pregnancy and lactation. The hypervitaminosis of vitamin A can cause liver problems.

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According to a new research vitamin A stimulates producing of insulin that plays an important role in the regulation of glucose level. In this way vitamin A deficiency can be a risk factor in forming of type 2 diabetes mellitus.

Good retinol source is cod liver oil, and it can be formed by oxidation of their precursor forms carotenoids from plants (e.g. carrot and spinach), or by hydrolysis of retinyl esters from the liver and egg of animals.

A B

C D

CH3

CH3

A B

C D

gonánváz szteránváz koleszterol

cikloalkánok

CH3 CH2

CH3

CH3 CH3

CH3

O

CH3

CH3

HO

CH3 CH3

CH3

limonén kámfor mentol

Néhány monterpén képlete

OHH

CH3

CH3

CH3

HO

CH3 CH3

CH3

A

C D h

hB

CH3

HO

CH3 CH3

CH3

CH2

D3-vitamin (kolekalciferol)

CH3CH3 CH3 CH3

CH3 CH3CH3

CH3

CH3 CH3ox.

-jonon-karotin -jonon

CH2OHCH3CH3 CH3 CH3

CH3retinol (A-vitamin)

CH3CH3 CH3

CH3

CHO

CH3

11-cisz-retinál

A gonánváz, a szteránváz és a koleszterol képlete

A D3-vitamin keletkezése a koleszterolból

A legismertebb tetraterpén, -karotin és a belôle képzôdô diterpének képlete

The conjugated double bond system in -carotene and its derivatives

Tocopherols (vitamin E)Vitamin E is a generic term for tocopherols and tocotrienols (they have three double

bonds in the side chain). Vitamin E is a family of α-, β-, γ-, and δ-tocopherols (they have methyl groups of different number in different positions) and corresponding four tocotrienols. Vitamin E is a fat-soluble antioxidant that stops the version of the molecule (containing a quinone structure) produced in this process may be recycled back to the active reduced form through reduction by other antioxidants, such as ascorbate (vitamin C) or retinol (vitamin A). Among them α-tocopherol has been most studied as it has the highest bioavailability.

Vitamin E deficiency causes neurological problems due to poor nerve conduction. It can also cause anaemia, due to oxidative damage to red blood cells. Its deficiency can cause enlarged prostate gland, gastrointestinal disease, dry or falling out hair, impotency, miscarriages, muscular wasting, muscle weakness, sterility. Vitamin E helps prevent cancer, cardiovascular disease, cataracts and reduces scarring from some wounds. Zinc and Vitamin E work together.

It is supposed that vitamin E in high doses for extended periods increases the risk of death. Vitamins E can be found in whole grains, seeds and vegetable oils, like sunflower, nuts and nut oils, like almonds and hazelnuts and some green leafy vegetables.

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O

Az E-vitaminok legismertebb fajtája, az -tokoferol

CH3

H3C

CH3

HO

CH3

CH3 CH3CH3

CH3

Formula of α-tocopherol

The types of tocopherols

Lipid peroxidation is a radical oxidative degradation process of polyunsaturated fatty acids (PUFA) as linolic acid and linoleic acid (linoleate and linolenate). Lipid peroxidation is a process mediated by the formation of free radicals at the methylene group(s) between the double

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bonds of these polyunsaturated fatty acid components of fats and oils, because the hydrogen of this methylene group is active. The formation of hydroperoxides (>CH–O–O–H), an important step in lipid peroxidation, is often a simple autoxidation, but an alternative enzymatic reaction catalyzed by lipoxygenases is also known. Decomposition of hydroperoxides by a radical mechanism is a complex process that leads to the formation of different short-chain ketones, aldehydes and carboxylic acids with an unpleasant odour or flavour and to dialdehydes. Besides the important role that lipid peroxidation plays in flavour deterioration and rancidity in food and food raw materials, there is also considerable interest in its role and the role of other free radical reactions in human diseases (e.g. arteriosclerosis, myocardial infarction and cancer). Lipid peroxidation can be prevented by the help of antioxidant vitamins, especially by tocopherols because of their apolar character.

Vitamin KVitamin K is a group of hydrophobic vitamins that are needed for the posttranslational

modification of certain proteins (e.g. protrombin), mostly required for blood coagulation but also involved in metabolism pathways in bone and other tissue. They are 2-methyl-1,4-naphthoquinone derivatives.

As vitamin K is an important factor of the cascade system of blood clotting, its deficiency can cause low platelet count in blood and poor blood clotting later on serious haemophilia. There is physiological and observational evidence that vitamin K plays a role in bone growth and the maintenance of bone density. Its deficiency can cause brittle or fragile bones. The efforts to delay the onset of osteoporosis by vitamin K supplementation have proven ineffective, but it is supposed that vitamin K can help to prevent osteoporosis. It is supposed that vitamin K, especially K2 plays an important role in the formation of osteocalcin that is one of the proteins in the bones. This protein is responsible for the infiltration of calcium into the bones. Vitamin K also converts glucose into glycogen for storage in the liver therefore its deficiency can cause a high glucose level in blood.

Vitamin K1 is also known as phylloquinone. vitamin K2 (menaquinone) (containing three double bond in side chain) is normally produced by bacteria in the large intestine, therefore dietary deficiency is extremely rare unless the intestines are heavily damaged, are unable to absorb the molecule, or are subject to decreased production by normal flora, as seen in broad spectrum antibiotic use. Vitamin K1 can be found in foods with plant original (in leafy green vegetables such as spinach and Brassica: e.g. cabbage, cauliflower, broccoli, and brussels sprouts) and vitamin K2 can be found in foods with animal original (e.g. liver). Bacteria in the large intestine can form vitamin K2 from vitamin K1.

CH3

O

O CH3 CH3 CH3CH3

CH3

Az K-vitaminok legismertebb fajtája, a K1-vitamin

Formula of vitamin K1 – phylloquinone

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Water-soluble vitaminsWater soluble vitamins are starting materials of most of the compounds with coenzyme

function. Oxidoreductases and transferases need reagents (compounds with coenzyme function) for the catalyzed reactions. Compounds with coenzyme function (henceforth they are called as coenzymes) are connected to enzymes either by secondary bonds (they are really coenzymes – they can be regenerated also in other reactions) or by covalent bonds (prosthetic groups – they can be regenerated only in their original place). Compounds with coenzyme function have two forms (unreacted and reacted) – only lipoic acid has three forms. The starting materials for coenzymes are water-soluble vitamins and in a few cases essential amino acids.

Coenzymes for oxidoreductasesIn primary metabolism oxidoreductases are always dehydrogenases, because the

reoxidation of reduced coenzymes is connected with the producing of energy in form of macroerg bonds in respiratory chain (mitochondrial electron transport chain of terminal oxidation). The mechanism of these oxidoreductase coenzymes can be ionic (hydrogen molecules are transported as hydride anions and protons) or radical (one hydrogen molecule is transported in form of two hydrogen atoms).

In the oxidative destroying processes of catabolism NAD+ (its starting material is nicotinamide i.e. vitamin B3) – its reduced form is (NADH+H+) (nicotinamide adenine dinucleotide) involve an ionic, while FAD (its starting material is riboflavine i.e. vitamin B2) – its reduced form is FADH2 (flavin adenine dinucteotide) and FMN – its reduced form is FMNH2

(flavin mononucleotide) a radical mechanism. FMN takes part only in terminal oxidation. In reductive biosyntheses of anabolism the coenzyme is (NADPH+H+) in both mechanisms. The difference between NAD+ NADP+ is the presence of a phosphoryl group on the C-2 hydroxyl group of ribose in NADP+. Flavin-containing coenzymes are always prosthetic groups. Nicotinic acid is one of the chelating compounds in glucose tolerance factor forming an organic chromium complex.

Niacin (vitamin B3)Niacin (also known as vitamin B3, nicotinic acid and vitamin PP) is water-soluble solid

pyridine derivative with a carboxyl group (COOH) at the 3-position. Other forms of vitamin B3 include the corresponding amide, nicotinamide ("niacinamide"), where the carboxyl group has been replaced by a carboxamide group (CONH2). The terms niacin, nicotinamide, and vitamin B3

are often used interchangeably to refer to any member of this family of compounds, since they have the same biochemical activity. Niacin cannot be directly converted to nicotinamide but both compounds could be converted to NAD+ and NADP+ in vivo (in the living organisms).

The name of niacin is a generic term for two vitamers nicotinic acid and nicotinamide. The essential compound is nicotinic acid that is the precursor of nicotinamide. This amide derivative is the precursor of NAD+ and NADP+. Nicotinic acid is the only vitamin that was discovered by chemical synthesis: by oxidation of nicotine in 1867. Its name comes from the name of tobacco alkaloid nicotin after Jean Nicot, French Ambassador who was one of the first to grow tobacco in Portugal in the 16th century. Its metabolic function was recognized when NAD+ and NADP+ was discovered in the first decades of the 20th century. There was confusion in the literature in connection with the name. The original compound name is connected to tobacco and smoking so it can be associated with an unhealthy activity. Therefore name ‘niacin’ was suggested. But in the North American usage name ‘niacin’ is specially nicotinic acid and

15

nicotinamide is known as ‘niacinamide’. Later it was proved that niacin could be synthesized in the body from tryptophan (if the level of pyridoxine – vitamin B6 is high enough), nevertheless, for historical reasons it is classified as a vitamin.

N

H

CONH2

N

HHCONH2

HH

+ H

max = 260 nm max = 260 és 340 nm

A nikotinamidot tartalmazó koenzimek redukálódási folyamata

The process of reduction of coenzymes containing a nicotinamide structure

N

N

N

N

NH2

2H (H + H )

R = H (NADH + H+ )

R = (NADPH + H+ )P

R = H (NAD+ ) nikotinamid-adenin-dinukleotid

R = (NADP+ )P

A NAD+ és NADP+ koenzimek

NH

O

O

NH

HOCH2O O

OH OH

H HHH

pszeudo uridin (C)

HN

NH

O

O

dihidro–uracil (DHU)

Néhány ritka nukleotid képlete

N

N

N

N

NH2

N

H HHH

OHOH

OCH2

CONH2

O

H H

CH2

H HHH

OROH

OO

N

H HHH

OHOH

OCH2

CONH2

O

H

CH2

H HHH

OROH

OO

O

P

P

O

P

P

H

Coenzymes NAD+ and NADPH+

Hydride anion from its reduced form (NADH+H+) is the reducing agent in reductive (energy-demanding) biosyntheses of biomolecules by both ionic and radical mechanism. Reduction (regeneration) of NADP+ is in catabolic pentose phosphate pathway (hexose phosphate shunt) that is an alternative oxidative degradation of glucose to pentoses and CO2. NAD+ and

16

NADP+ coenzymes are often referred to as the pyridine nucleotide coenzymes and their notation is NAD(P+). These coenzyme are often mentioned as NAD(P) without notation of their charge. During reduction by hydride anion C-4 of pyridine ring results in a saturated carbon (sp3 hybrid status) and these hydrogen atoms are above and below of the plane of heterocyclic ring.

As the aromatic ring systems both pyridine and purine ring of NAD+ or NADP+

coenzymes have an absorption peak at 260 nm. Because of the quinon-like structure of the partly saturated pyridine ring of their reduced form they have an absorption peak at 340 nm, as well. Therefore the activity of dehydrogenase enzymes using pyridine nucleotide coenzymes can be measured easily by spectrophotometric way. Their molar absorption coefficient is 6220 in water. Moreover, these coenzymes can be used also for enzymic concentration determination of different biomolecules. For example the glucose concentration of an aqueous solution can be measured by a system containing hexokinase, glucose-6-phosphate dehydrogenase and NADP+. Different diagnostic kits are available for medical analyses containing pyridine nucleotide coenzymes.

The absorption diagrams of NAD+ and (NADH+H+)

In maize the most of niacin content is biologically unavailable because it is bound to polysaccharides, polypeptides and glycopeptides with ester bond. The name of nicotinoyl esters of these macromolecules (ranging between 1500-17 000 D) is niacytin. It was found that less than 10% of the total niacin content of maize is biologically available as a result of hydrolysis by gastric acid. The treatment of cereals with alkali (e.g. soaking overnight in calcium hydroxide solution, as is the traditional method for the preparation tortillas in Mexico and baking with alkaline baking powder releases much of the nicotinic acid. This may explain why avitaminosis of niacin (pellagra) is has always been rare in Mexico despite the fact that maize is the most important food there. During roasting of whole grain maize, that is popcorn, the ammonia released from glutamine can form free nicotinamide by ammonolysis.

Vitamin B3 deficiency occurs mostly in developing countries but it can be found also in developed countries. The symptoms of the avitaminosis of vitamin B3 – pellagra are dry hair, eye sties, fatigue, insomnia, impaired growth, itching and burning eyes (high sensitivity for sunlight), loss of smell, dry skin, sinus trouble, weakness, diarrhea, eventually dementia. Because of the decreased immune system function, the cancer susceptibility is increased.

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Vitamin B3 can be found in fish and liver, cereals, green and yellow fruits and vegetables, Apricots, asparagus, beets, broccoli, butter, cantaloupe, carrots, cheese, garlic, green olives, milk products, mustard (fresh), papaya, parsley, peaches, prunes, red peppers, sweet potatoes, spinach, sweet potatoes, pumpkin and watercress.

In plants the starting materials of niacin are D-glicerinaldehyde-3-phosphate and L-aspartic acid. In animals and human persons the starting material is L-tryptophane. From intermediate dicarboxylic acid quinolinic acid NAD+ and NADP+) is synthesized in several steps in a cycle. Into this cycle can enter niacin (nicotinic acid or nicotinamide). During the degradation of nicotinic acid ammonia and different carboxylates are formed.

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Riboflavin (vitamin B2)Riboflavin, also known as vitamin B2, is an easily absorbed micronutrient with a key role

in maintaining health in humans and animals. As it was mentioned it is the central component of the coenzymes FAD and FMN, and is therefore required by all flavoproteins. As such, vitamin B2 is required for a wide variety of cellular processes. Like the other B vitamins, it plays a key role in energy metabolism, and for the metabolism of fats, ketone bodies, carbohydrates, and proteins.

N

N

N

N

H

HA flavint tartalmazó koenzimek redukálódási folyamata

2H

The process of reduction of coenzymes containing flavines

The name "riboflavin" comes from "ribose" (the sugar which forms part of its structure, and flavin, the ring-moiety which imparts the yellow color to the oxidized molecule (from Latin flavus, "yellow"). The reduced form, which occurs in metabolism, is colorless. Riboflavin is best known visually as the vitamin which imparts the orange color to solid B-vitamin preparations, the yellow color to vitamin supplement solutions, and the unusual fluorescent yellow color to the urine of persons who supplement with high-dose B-complex preparations (no other vitamin imparts any color to urine).

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N

NNH

N

CH2

HCOH

HCOH

HCOH

H2C

H3C

H3C

O

O

N

N

N

N

NH2

CH2

H HHH

OHOH

O

2H

N

NNH

N

CH2

HCOH

HCOH

HCOH

H2C

H3C

H3C

O

O

O

H

H

N

N

N

N

NH2

O CH2

H HHH

OHOH

O

FAD (flavin-adenin-dinukleotid) FADH2

N

NNH

N

CH2

HCOH

HCOH

HCOH

H2C

H3C

H3C

O

O

O–R

PR =R = H (B2 vitamin) riboflavin

FMN (flavin mononukleotid)

A flavint tartalmazó koenzimek és prekurzor vitaminjuk

OP POO OP P

Flavin-containing coenzymes and their precursor vitamin

Riboflavin is continuously excreted in the urine of healthy individuals, making deficiency relatively common when dietary intake is insufficient. However, riboflavin deficiency is always accompanied by deficiency of other vitamins. A deficiency of riboflavin can be primary – poor vitamin sources in one's daily diet – or secondary, which may be a result of conditions that affect absorption in the intestine, the body not being able to use the vitamin, or an increase in the excretion of the vitamin from the body.

In humans, signs and symptoms of riboflavin deficiency include cracked and red lips, inflammation of the lining of mouth and tongue, mouth ulcers, cracks at the corners of the mouth, and a sore throat. A deficiency may also cause dry and scaling skin, fluid in the mucous membranes, and iron-deficiency anemia. The eyes may also become bloodshot, itchy, watery and sensitive to bright light. Riboflavin deficiency is classically associated with the oral-ocular-genital syndrome. Angular cheilitis, photophobia, and scrotal dermatitis are the classic remembered signs. Although the effects of long-term subclinical riboflavin deficiency are unknown, in children this deficiency results in reduced growth. Subclinical riboflavin deficiency has also been observed in women taking oral contraceptives, in the elderly, in people with eating disorders, and in disease states such as HIV, inflammatory bowel disease, diabetes and chronic heart disease. The fact that riboflavin deficiency does not immediately lead to gross clinical manifestations indicates that the systemic levels of this essential vitamin are tightly regulated.

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Vitamin B2 can be found in milk, cheese, leafy green vegetables, liver, kidneys, legumes, tomatoes, yeast, mushrooms, and almonds are good sources of vitamin B2, but exposure to light destroys riboflavin.

The starting material of riboflavin is GTP, the ribitol part of the molecule is comes from ribose of GTP. The dimethylbenzene fraction of the ring system is derived from the tetrolose phosphate (C4). From riboflavin one of the intermediate (5,6-dimethyl-benzimidazole) of vitamin B12 can be formed. The ring system of riboflavin can be illustrated in two different ways – with the pyrimidine ring on the left or the right side of the molecule.

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The biosynthesis of riboflavin

L-Ascorbic acid (ascorbate, vitamin C)

22

The coenzyme of direct oxygenases is ascorbic acid (ascorbate, vitamin C). It is a coenzyme in at least eight enzymatic reactions, including several collagen synthesis reactions that cause the most severe symptoms of scurvy when they are dysfunctional. In animals, these reactions are especially important in wound-healing and in preventing bleeding from capillaries. One of secondary structures of peptide chains is collagen structure. Collagen structure contains three of left-handed extended helical polypeptide chains rolled into a cable form of a right-handed helix. In tropocollagen units are Gly-Pro-Hyp triplets, hydroxyproline (Hyp) is synthesized by a direct oxidation of proline in peptide chain by means of L-ascorbate).

1/2 O2

(az aszkorbinsavközvetítésével)

Hyp részlet afehérjeláncban

NO

HOPro részlet afehérjeláncban

NO

A hidroxi-prolin képzôdése a peptidláncbanOxidation of proline to hydroxyproline in the peptide chain by L-ascorbate (vitamin C)

Scurvy is a disease resulting from a deficiency of vitamin C, which is required for the synthesis of collagen in humans. The chemical name for vitamin C, ascorbic acid, is derived from the Latin name of scurvy, scorbutus, which also provides the adjective scorbutic ("of, characterized by or having to do with scurvy"). Scurvy leads to the formation of spots on the skin, spongy gums, and bleeding from the mucous membranes. The spots are most abundant on the thighs and legs, and a person with the ailment looks pale, feels depressed, and is partially immobilized. In advanced scurvy there are open, suppurating wounds and loss of teeth.

In living organisms, ascorbate is also an antioxidant, since it protects the body against oxidative stress. Oxidative stress creates free radicals and antioxidants can eliminate free radicals by reduction. Ascorbic acid is well known for its antioxidant activity, acting as a reducing agent to reverse oxidation in liquids. When there are more free radicals (reactive oxygen species, ROS) in the human body than antioxidants, the condition is called oxidative stress, and has an impact on cardiovascular disease, hypertension, chronic inflammatory diseases, and diabetes as well as on critically ill patients and individuals with severe burns. There is some evidence that it reduces symptom severity but not incidence of the common cold. Vitamin C helps prevent infection, enhances immunology and can help prevent cancer.

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Formation, oxidation and stability of ascorbic acid

Almost all organisms can synthesize ascorbic acid; except some mammalian groups (among them monkeys, apes and human beings). Ascorbic acid can not be synthesized also by guinea pigs, capybaras, and some species of birds and fish. The starting material of the biosynthesis of ascorbic acid is UDP-glucose that is also the the starting material of different kinds of glucosides (e.g. sucrose). After its oxidation and hydrolysis D-glucuronic acid is formed that is oxidized to L-gulonic acid then after cyclization L-gulonolacton. The last step of ascorbate biosynthesis is the oxidation of L-gulonolacton by oxygen. Humans, some other primates, and guinea pigs are not able to synthesize L-gulonolactone oxidase because of a genetic mutation and are therefore unable to make ascorbic acid. Therefore ascorbic acid is essential for them and they require it in the diet.

While plants are generally a good source of vitamin C, the amount in foods of plant origin depends on the precise variety of the plant, soil condition, climate where it grew, length of time since it was picked, storage conditions, and method of preparation. The richest natural sources are fruits and vegetables, and of those, the Kakadu plum and rose hip contain the highest

24

concentration of the vitamin. It is also present in some cuts of meat, especially liver. There are also alternative routes in plants for ascorbate biosynthesis (e.g. L-galactose pathway).

O

HO

HOC

CH2OH

HHO

O

L-aszkorbinsav (C-vitamin)

ox

red

dehidro-aszkorbinsav (bomlékony)

O

O

OC

CH2OH

HHO

O

Az aszkorbinsav oxidált és redukált formája

Az ATP átadható csoportjai

N

N

N

N

NH2

O

OH OH

H HHH

P–O–P–O–P–CH2

Redox reactions of L-ascorbic acid

The oxidation product of ascorbic acid is dehydro-L-ascorbic acid in a reaction of radical mechanism. The balance of opening and closure reaction of the ring of lactons is known. But the product of the opening reaction of dehydroascorbic acid can not take part in a ring closure reaction. It can be oxidized to oxalic acid and tartarate. Therefore the ascorbate level of food raw materials with plant origin decreases during storage or producing foods.

Oxidative degradation of ascorbic acid to oxalic acid and tartarate

Other molecules with coenzyme function for oxidoreductasesThere are three other coenzymes of oxidoreductases but their starting materials are not

water soluble vitamins.Ubiquinone (coenzyme Q) (its starting material is tyrosine and its reduced form is

ubiquinol) is that kind of oxidoreductase coenzyme, which can work by both ionic and radical mechanism. This is an important participant of the mitochondrial electron transport in respiratory chain. The name of the human ubiquinone is CoQ10. Nowadays it is a popular food additive.

25

Redox reactions

of

ubiquinone

In various kinds of cytochrome proteins can play an important role in different kinds of electron transport. Their prosthetic group for electron transfer is heme group (by ferrous-ferric transformation). A heme group consists of an iron (Fe) ion (charged atom) held in a heterocyclic ring, known as a porphyrin. This porphyrin ring consists of four pyrrole molecules cyclically linked together with the iron ion bound in the centre. The iron ion, which is the site of oxygen binding, coordinates with the four nitrogen atoms in the centre of the ring, which all lie in one plane. It is mentioned that in hemoglobin transporting oxygen in blood the iron ion is always in ferrous form in heme because of the special connection between heme group and proteins. Heme is one of the starting materials of cobalamin (vitamin B12) and chorophylls. The starting material of the ring system is 5-aminolevulinate synthesized from different amino acids of different living organisms.

The structure of heme

Lipoic acid is a prosthetic group for both oxidoreductase and transferase function. That is connected to the -amino group of a lysine of the enzyme. It can be acetylated by active acetaldehyde with an oxidative reaction followed by an acyl transfer to coenzyme A. The details are given at vitamin B1 and B5. The precursor to lipoic acid, octanoic acid, is made via fatty acid biosynthesis.

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S S C CH3O

COOH

H

S S

COOH

H Hdihidro-liponsav

acetil-dihidro-liponsavA liponsav koenzim különbözô formái

S S

COOH

liponsav

Three forms of lipoic acid

Coenzymes for transferasesTransferases can catalyze several kinds of substitutions. The transferred groups can be

different carbon skeletons: C1 – CO2 (biotin that is vitamin H), only methyl group (SAM – S-adenosylmethionine, its starting material is methionine), methyl group, aldehyde group, etc. (THF – tetrahydrofolate, its starting material is folic acid i.e. vitamin B9); C2 – acetaldehyde (TPP – thiamine pyrophosphate, its staring material is aneurine, i.e. vitamin B1), acetyl group in a macroerg thiolester bond (coenzyme A, its starting material is panthotenic acid, i.e. vitamin B5); and other groups: phosphate group (ATP or other nucleoside triphosphate molecules), amino group (PAL – pyridoxal phosphate, its reacted form is PAM – pyridoxamine phosphate, and its starting material is pyridoxine i.e. vitamin B6).

Transfer of C1 components

Biotin (vitamin H, vitamin B7)

HN NHC

O

S CH2CH2 CH2

CH2 COOH

CO2

ATP ADP

NHC

O

N

COOHCH2CH2

CH2CH2S

HOOC

biotin(H-vitamin)

karboxi-biotin

A biotin keletkezése és formái

Carboxylation of biotin

Biotin is the coenzyme of carboxylation. From the name vitamin H the H represents ‘Haar und Haut’ – these are German words for hair and skin. The water-soluble vitamin is composed of an ureido (tetrahydroimidizalone) ring fused with a tetrahydrothiophene. Its side chain is a valeric acid (C5). Biotin is a coenzyme of the addition of a molecule CO2

(carboxylating agent) in gluconeogenesis, and also in the metabolism of fatty acids and leucine. The first step is the formation of an active CO2 complex by means of ATP followed by the

27

carboxylation of one of the nitrogen of biotin. Biotin binds very tightly to the tetrameric protein avidin deposited in the whites of the eggs of birds, reptiles and amphibians (e.g. frogs and toads).

Biotin is consumed from a wide range of food sources in the diet, however there are few particularly rich sources. Foods with relatively high biotin content include egg yolk, liver, and some vegetables. Biotin deficiency is relatively rare and mild, and can be addressed with supplementation. Such deficiency can be caused by the excessive consumption (e.g. 20 eggs in a day) of raw egg whites because of their high avidin content. Avidin can be deactivated by cooking, while the biotin remains intact. Symptoms of biotin deficiency are hair loss, conjunctivitis and dermatitis.

Pregnant women tend to have high risk of biotin deficiency. Research has shown that nearly half of pregnant women have an abnormal increase of 3-hydroxyisovaleric acid, which reflects reduced status of biotin. Numbers of studies reported that this possible biotin deficiency during the pregnancy may cause infants' congenital malformations such as cleft palate (this is a split on the roof of the mouth). It is suggested that Cot Death (Sudden Infant Death Syndrome) may be due to a marginal biotin deficiency. It is supposed that in the case of a modest metabolic stress (e.g. a mild fever) causes a higher requirement of gluconeogenesis. In the case of low biotin concentration this can cause acute hypoglycaemia.

Biotin is synthesized from alanine and pimeoyl CoA (HOOC-(CH2)5-CO-CoA) in several steps. Sulfur atom derives from a methionine in the last step of the synthesis.

Folic acid (vitamin B9)

CHCOOH

CH2

CH2

COHNCH2–NHN

NN

HN

H2N

O H

HCOOH

C1

123

45 6

784-aminobenzoesav

Glu

C1: CHO CH3

CH2OH

CHCOOH

CH2

CH2

COHNCH2–NHN

NN

HN

H2N

O

COOHfolsav (B10 vitamin)

A C1 részleteket szállító koenzim és prekoenzim vitaminja

CH2

tetrahidro-folsav (THF)

Transfer coenzyme – C1 – THF

Tetrahydrofolic acid (tetrahydrofolate, THF) is the coenzyme of transferases catalyzing the transfer of different C1 fragments –methyl, aldehyde, methylene and hydroxymethyl groups. Folic acid (also known as folate, folacin or vitamin B9) is a typical example for the confusion in

28

the nomination of vitamin B types. Originally pantothenic acid was called vitamin B9. At that time the name of folic acid was vitamin B10. Now 4-aminobenzoic acid (para-aminobenzoic acid, PABA), a part of folic acid is vitamin B10, and folic acid is named vitamin B9.

29

Biosynthesis of folic acid

Folic acid is itself not biologically active, but its biological importance is due to tetrahydrofolate and other derivatives after the reduction of folic acid to dihydrofolic acid in the liver. The name of folic acid and its derivatives comes from the Latin word folium (which means leaf). Leafy vegetables are a principal source, although, in Western diets, fortified cereals and bread may be a larger dietary source. Folic acid contains pterin ring system.

Folic acid can be found in beans, beef, bran, barley, brown rice, cheese, chicken, dates, green leafy vegetables, lamb, lentils, liver, milk, oranges, organ meats (like liver), split peas, pork, root vegetables (like carrots), salmon, tuna, whole grains, whole wheat and yeast.

The starting material of folic acid is GTP from that dihydropteroic acid is synthesised after several steps. This intermediate reacts with glutamic acid to give dihydrofolic acid that is reduced to THF. Folic acid that is essential for mammalians. THF is synthesised from vitamin B9

in two consecutive reducing steps. The deficiency symptoms of folic acids are sore tongue, B12 deficiency, depression or

anxiety, fatigue, and birth defects in pregnant women. Folic acid is needed for energy production, protein metabolism, the formation of red blood cells and it vital for normal growth and development.

THF is the most important methylating agent in the living organisms. It is involved in the synthesis of dTMP (2′-deoxythymidine-5′-phosphate) from dUMP (2′-deoxyuridine-5′-phosphate). It is a substrate for an important reaction that involves vitamin B12 and it is necessary for the synthesis of DNA, and so required for all dividing cells. Therefore THF can cause birth defects in pregnant women. Methylene-THF is formed from THF and a C1 donor ( formaldehyde, serine or glycine). Methyl-THF can be formed from methylene THF by a reduction with (NADPH+H+). Vitamin B12 is the only acceptor of methyl-THF. And homocystein is also only acceptor for methyl-B12. This reaction results in methionine and catalyzed by homocysteine methyltransferase. This is important because a defect in homocysteine methyltransferase or a deficiency of B12 can lead to a methyl-trap of THF and a subsequent deficiency. Thus, a deficiency in B12 can generate a large pool of methyl-THF that is unable to undergo reactions and will mimic folate deficiency.

30

CHCOOH

CH2

CH2

S

CH3

H2N

+

O

OH OH

H HHH

–O–CH2

N

N

N

N

NH2

PPi Pi

Met ATP

N

N

N

N

NH2

O

OH OH

H HHH

CH2H3C

H2N

S

CH2

CH2

COOHCH

S-adenozil-metionin (SAM)

CH3

N

N

N

N

NH2

O

OH OH

H HHH

CH2

H2N

S

CH2

CH2

COOHCH

S-adenozil-homocisztein (SAH)

O PP PO

A SAM keletkezése és különbözô formáiDemethylation process of methionine to homocysteine

From methionine and ATP S-adenosyl methionine (SAM) is formed. SAM is a coenzyme of ionic methylation of hydroxy and amino groups. When SAM looses the methyl cation S-adenosyl homocysteine is formed. After its hydrolysis to homocysteine and adenosine methionine can be regenerated by the reaction of homocysteine and methyl-THF. Homocysteine is the starting material of cysteine in animals and humans. The intermediate of this biosynthesis is cystathionine. The energy of this synthesis is from the macroerg bond of succinyl-CoA. The formation of cysteine is connected with an intermolecular oxidation (on homocysteine part cystathionine) and reduction (between sulphur and the side chain of homocysteine) followed by the hydrolysis of imino-acid resulting in cysteine and -ketobutyrate. In this way in animals and humans cysteine is synthesized from methionine. In plants methionine is synthesized from homocysteine that is formed by another degradation of cystathionine made from homoserine and cysteine. This degradation results in homocysteine and pyruvate.

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Forming of cysteine from homocysteine in animals and humans

32

In the case of some trouble in sulphur containing amino acid (about 20-30% of people) can increase the concentration of homocysteine. A high level of blood serum homocysteine "homocysteinemia" is a powerful risk factor for cardiovascular disease. Homocysteine in high concentration can take part in auto-oxidation and react with reactive oxygen intermediates. This can cause damage in endothelial cells and a higher risk to form a thrombus. Elevated levels of homocysteine can increase the possibility of fractures in elderly persons. Homocysteine does not affect bone density. It is supposed that it can influence the structure of collagen.

As it was mentioned the 4-aminobenzoic acid (para-aminobenzoic acid, PABA) part of folic acid is considered as a vitamin (vitamin B10). It is sometimes referred as vitamin Bx. PABA is an intermediate in the bacterial synthesis of folic acid. Some bacteria in the human intestinal tract such as Escherichia coli require PABA. Humans require folate since we lack the enzymes to convert PABA to folate. Therefore, in humans, PABA is not a vitamin and is considered non-essential. Despite the lack of any recognized syndromes of PABA deficiency in humans, many claims of benefit are made by commercial suppliers of PABA as a nutritional supplement. Benefit is claimed for fatigue, irritability, depression, weeping eczema (moist eczema), scleroderma (premature hardening of skin), patchy pigment loss in skin (vitiligo), and premature grey hair. Oral supplements of PABA can make the skin less sensitive to sun damage. PABA is largely non-toxic, but allergic reactions can occur. PABA is formed in the metabolism of certain ester local anesthetics, and many allergic reactions to local anesthetics are the result of reactions to PABA.

The similarity between 4-aminobenzoic acid in amide bond (H2N–C6H5–CONH–) and sulfonamide (H2N–C6H5–SO2NH–) containing drugs is an excellent example for the importance of bioisosteres. According to the chemical definition the bioisostere is a compound resulting from the exchange of an atom or of a group of atoms with another, broadly similar, atom or group of atoms. The objective of a bioisosteric replacement is to create a new compound with similar biological properties to the parent compound. The bioisosteric replacement may be physicochemically or topologically based.

33

In medicinal chemistry, bioisosteres are substituents or groups with similar physical or chemical properties that impart similar biological properties to a chemical compound. In drug design, the purpose of exchanging one bioisostere for another is to enhance the desired biological or physical properties of a compound without making significant changes in chemical structure. The main use of this term and it techniques are related to pharmaceutical sciences.

Bioisosteres can connect to an acceptor or a substrate binding site in competition with the original substrate, but in most of the cases this complex formed can not execute the function of the complex with the original substrate, therefore an antagonism or inhibition can be found. Sulphonamide drugs are structurally similar to PABA, and their antibacterial activity is due to their ability to interfere with the conversion of PABA to folic acid by the enzyme dihydropteroate synthetase. From the sulphonamide analogue of dihydrofolic acid THF can not be synthesized. In this way bacterial growth is limited through folic acid deficiency without effect on human cells. At the time of the Second World War sulphonamides saved of the numberless American soldiers from the bacterial infections. Later on bacteria became resistant to sulphonamides and penicillin derivatives pushed out their medical use. Nowadays a combination of sulphonamides and trimethoprim (a good inhibitor of dihydrofolate reductase) is used because of their synergism. The synergism is an interaction of discrete agencies (as industrial firms), agents (as drugs), or conditions such that the total effect is greater than the sum of the individual effects.

Cobalamine (vitamin B12)Vitamin B12 consists of a class of chemically-related compounds (vitamers) with vitamin

activity. It contains the biochemically rare element cobalt. Biosynthesis of the basic structure of the vitamin in nature is only accomplished by simple organisms such as some bacteria and algae, but conversion between different forms of the vitamin can be accomplished in the human body. A common synthetic form of vitamin B12 is cyanocobalamin. This synthetic material is used in vitamin combinations because of its stability and lower cost. In the body it is converted to the physiological forms methylcobalamin and adenosylcobalamin instead of cyanide group. Hydroxycobalamin produced by bacteria is also can be used. The starting materials of vitamin B12 are heme group and one of the intermediates (5,6-dimethyl-benzimidazole) of riboflavin biosynthesis.

The transfer coenzyme of alkyl groups is adenosylcobalamin (5'-deoxyadenosylcobalamin), that is the coenzyme of methylmalonyl coenzyme A mutase. It takes part in the oxidative degradation of fatty acids having an odd number of carbon atoms (these are minor species). In this case the end product of the -oxidation of fatty acids is one molecule of propionyl CoA instead of acetyl-CoA. This molecule is converted to succinyl CoA (an intermediate of citric acid cycle) in two steps (carboxylation followed by an intramolecular rearrangement). This process is catalysed by methylmalonyl coenzyme A mutase. Methylcobalamine is the coenzyme of the change of a methyl group between methyl-THF and homocysteine catalyzed by5-methyltetrahydrofolate-homocysteine methyltransferase.

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N

C

NCH

N

C

N

CoCN

H3CH3C

H3C H3C

CH3

CH3

CH3

H3CH2NOCCH2

H2NOCCH2 CH2CONH2

CH2CH2CONH2

CH2CH2CONH2

H2NOCCH2O=C–CH2CH2

CH3NH

CH2–CH–O

CH3P

O

O O

A B

CD

N

N CH3

CH3

OH

HOCH2 OA B12 vitamin képlete

Cyanocobalamin

As it was mentioned earlier the action of together folic acid and cobalamin is needed for methylating reactions therefore some of deficiency symptoms are common. Symptoms of deficiency of vitamin B12 are appetite loss, diminished reflex responses, fatigue, irritability, memory impairment, mental depression and confusion, nervousness, pernicious anemia (megaloblastic anaemia – because of the inhibition of DNA synthesis in red blood cells), unpleasant body odor, walking and speaking difficulties, weakness in arms and legs. A deficiency can cause problems with digestion, absorption of food, metabolism of carbohydrates and fats, nerves, fertility, growth and development. There can also be hallucinations, memory loss, eye disorders, and anemia. A vitamin B12 deficiency can indicate there is a problem with absorption (common in people with digestive disorders).

The sources of vitamin B12 are all animal sources: beef, blue cheese, cheese, clams, crab, fish, eggs, herring, kidney, liver, mackerel, milk and milk products, pork, seafood and tofu, therefore vega (vegetarian) persons need supplement of vitamin B12. In the case of a long lasting alcoholism vitamin B12 absorption can be decreased from the gastrointestinal tract.

Transfer of C2 components

Thiamine (aneurine, vitamin B1)The name of thiamine comes from thio-vitamin that is sulphur-containing vitamin. The name of aneurin comes from one of deficiency symptoms (detrimental neurological effects). Its phosphate derivatives are involved in many cellular processes. The coenzyme derived from vitamin B1 is thiamine pyrophosphate (TPP), a coenzyme of the transfer of acetaldehyde in the catabolism of sugars (coenzyme of pyruvate dehydrogenase multienzyme complex) and the metabolism of amino acids. In yeast, TPP is also required in the first step of alcoholic fermentation. The name of TPP substituted with acetaldehyde is active acetaldehyde.

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P

N

NCH2 N

CHS

NH2

H3C

H3C CH2CH2O O P

tiamin-pirofoszfát (TPP)

N

NCH2 N

CHS

NH2

H3C

H3C CH2CH2OH

tiamin (aneurin) B1-vitamin

Az acetaldehidet szállító koenzim és prekurzor vitaminja

HN NHC

O

S CH2CH2 CH2

CH2 COOH

CO2

ATP ADP

NHC

O

N

COOHCH2CH2

CH2CH2S

HOOC

biotin(H-vitamin)

karboxi-biotin

A biotin keletkezése és formái

P O P

N

NCH2 N

CS

NH2

H3C

H3C CH2CH2O

CH3CH

OH"aktív acetaldehid"

Transfer coenzyme – acetaldehyde – TPP

The reaction catalyzed by pyruvate dehydrogenase

Active acetaldehyde seems to be a secondary alcohol but really it can react as a carbonyl compound (it can be oxidized to acyl group), because the carbon atom between N+ and S is a reactive carbon atom with acidic (dissociable) proton. During the decarboxylation of pyruvate

36

the molecule reacts as an acetaldehyde and the properties of the product is similar to a glucoside (its hydroxyl group is a glycosidic hydroxyl). This kind of structure is called C-glycoside.

Acetaldehyde in free form can not be originated from the reaction of pyruvate and TPP. It can be synthesized mostly from ethanol by oxidation followed by its oxidation to acetic acid in the liver catalyzed by enzyme alcohol dehydrogenase. In contrast to acetic acid acetaldehyde is a toxic molecule. The elevated acetaldehyde concentration in blood is responsible for the symptoms of discomfort feeling after heavy drinking (hangover). It is a probable carcinogen. In the liver of heavy drinkers a defect in the biosynthesis of alcohol dehydrogenase can be found causing permanent drunkenness.

Thiamine is synthesized only in bacteria, fungi, and plants. The thiazole and pyrimidine moieties are synthesized separately and then they are assembled to thiamine-phosphate. The exact biosynthetic pathways can be slightly different in different organisms. The starting material of pyrimidine moiety is a 5-aminoimidazole derivative that is an intermediate of purine biosynthesis. There are three types of precursors of the thiazole moiety: an amino acid (glycine or tyrosine), some sugar derivative and cysteine.

Insufficient intake in birds produces a characteristic polyneuritis. In mammals thiamine deficiency results in a disease called beriberi affecting the peripheral nervous system (polyneuritis) and/or the cardiovascular system, with fatal outcome without thiamine. In less

37

severe deficiency there are non-specific symptoms include tiredness, weight loss, irritability and confusion.

Thiamine is found in a wide variety of foods at high concentrations. Yeast and pork are the best sources, but cereal grains are rich in thiamine, as well. The whole grains contain more thiamine than refined grains, as thiamine is found mostly in the inner layers of the grain and in the germ (which are removed during the refining process). It is known that the rich people in ancient Japan or China were more susceptible to beriberi than poor ones because they consumed husked rice.

Pantothenic acid (vitamin B5)The name of pantothenic acid means from everywhere in Greek (pantothen) because small

quantities of pantothenic acid are found in nearly every food, and high amounts are in whole-grain cereals, legumes, eggs and meat. Pantothenic acid is an ingredient in some hair and skin care products. Pantothenic acid is one of the starting materials of coenzyme A (CoA) (this is the only coenzyme that served the original nomenclature that was similar to the nomenclature of vitamins)

Koenzim-A

ciszteamin

-alanin

O

O OH

H HHH

CH2O

N

N

N

N

NH2

P

2,4-dihidroxi-3,3-dimetil-vajsav P

N

N

N

N

NH2

O

O OH

H HHH

CH2O

CH3–CO–SKoAacetil koenzim A"aktív ecetsav"

CH2OH

C

CH–OH

CH3CH3

C=ONH

CH2

CH2

COOH pantoténsav(régen B9-vitamin)(újabban B5 vitamin)

NH3C

HO CH2OH

CH2OH

NH3C

HO CH2O–

CHO

P

NH3C

HO CH2O–

CH2NH2

P

piridoxin(B6-vitamin)

piridoxál-foszfát (PAL)

piridoxamin-foszfát (PAM)

Az aminocsoportot szállító koenzim és prekurzor vitaminja

OP PO

CH2

C

CH–OH

CH3CH3

C=ONH

CH2

CH2

C=ONH

CH2

CH2

SH

O

CH2

C

CH–OH

CH3CH3

C=ONH

CH2

CH2

C=ONH

CH2

CH2

S–C–CH3

O

PP O

A koenzim-A különbözô formái

Transfer coenzyme – acetyl group – coenzyme A

The acyl derivatives coenzyme A containing macroerg bond can transfer acyl group in the biochemical processes. Acetyl-CoA is not only a transfer component for a C2 fragment but it is the common metabolite during the oxidative degradation of different biomolecules entering to citric acid cycle to produce CO2 and reduced coenzymes. During the regeneration (reoxidation)

38

of these reduced coenzymes in terminal oxidation (respiratory chain) energy can be produced by forming macroerg bonds of ATP. CoA is also important in the biosynthesis of many important compounds such as fatty acids, cholesterol, and acetylcholine.

Pantothenic acid deficiency is exceptionally rare and has not been thoroughly studied. Symptoms of deficiency are similar to other vitamin B deficiencies. There is impaired energy production, due to low CoA levels, which could cause symptoms of irritability, tiredness and apathy. Acetylcholine takes play an important role in the function of nervous system (it is one of the neurotransmitters), therefore neurological symptoms can also appear in deficiency of pantothenic acid. In the case of low pantothenic acid concentration hypoglycemia or an increased sensitivity to insulin also can be found. It can be in connection with the change in acylation capacity.

As it was mentioned earlier small quantities of pantothenic acid are found in most foods. The major food source of pantothenic acid is in meats, although the concentration found in food animals' muscles is only about half that in humans' muscles. Whole grains are another good source of the vitamin, but milling often removes much of the pantothenic acid, as it is found in the outer layers of whole grains. Vegetables, such as broccoli and avocados, also have an abundance of the acid.

The biosynthesis of -ketoisovaleric acid that is the starting material of pantothenic acid

The starting material of Co-A is pantothenic acid. Pantothenic acid is the amide between pantoate (2,4-dihydroxy-3,3-dimethylbutyric acid) and -alanine. Pantoate (pantoic acid) is synthesized from -ketoisovaleric acid. -Alanine is connected to pantoic acid by means of energy of a macroerg bond of ATP. that is phosphorylated than a peptide bond with cysteine (with the energy of a macroerg bond of ATP) is formed. Cysteine part of this molecule looses a molecule of CO2 forming a cysteamine part (4’-phosphopantethein). This molecule reacts with ATP followed by the phosphorylation of 3’-hydroxyl group of the ADP part of the molecule with another ATP.

39

40

Transfer of other groupsFor the transfer of phosphate group phosphorylating agents (NTP, especially ATP) are

used. The coenzyme of transfer of amino group is pyridoxal phosphate (PAL) synthesized from pyridoxine (vitamin B6)

Pyridoxine (vitamin B6)Pyridoxine is the starting material of the biosynthesis of pyridoxal phosphate. It is not

normally found in plants. This vitamin is made by certain bacteria. Some vegetarians may get adequate pyridoxine simply from eating plants that have traces of soil (like potato skins). Most people get their supply of this vitamin from either milk or meat products.

NH3C

HO CH2OH

CH2OH

NH3C

HO CH2O–

CHO

P

NH3C

HO CH2O–

CH2NH2

P

piridoxin(B6-vitamin)

piridoxál-foszfát (PAL)

piridoxamin-foszfát (PAM)

Az aminocsoportot szállító koenzim és prekurzor vitaminja

Transfer coenzyme – amino group – PAL-PAM and its starting material pyridoxine

benzaldehid(mandulaillat)

benzaldehid-ciánhidrin

CH

CN

O genciobiózgenciobióz

A keserûmandula szaganyaga

CH3C

HO

acetaldehid

H2NCH

COOH

R+

-aminosav

a fenntiek szerint

ANCH3 CH N C

COOH

HR

HCH3 CH N

R

COOHC

HCH3 CH N C

COOH

R

CH3 CH2 NR

COOHC

ketimin

H2O

H2O

CH3CH2 NH2 +COOH

CR

O

etil-amin -keto-karbonsavAldimin – ketimin tautomer átalakulás

H C N C

O

H

+C

H O–H

CN

amigdalin

H2O

H2O

Aldimine-ketimine tautomerism illustrated on acetaldehyde molecule

Pyridoxal phosphate (PAL or PLP) containing an aldehyde group is a prosthetic group of some enzymes of amino acid biochemistry. It takes part in all transamination reactions, and in some decarboxylation and deamination reactions. In transamination reactions it can transfer amino group of amino acids by an aldimine-ketimine tautomerism. This tautomerism is illustrated on acetaldehyde instead of PAL. The name of amine containing coenzyme version is pyridoxamine phosphate (PAM).

The pyridoxine deficiency can cause different problems in amino acid metabolism. Earlier pellagra was attributed to pyridoxine deficiency. Later on it came to light that pellagra is

41

caused by niacin deficiency. In the case low L-tryptophane and pyridoxine level the biosynthesis of niacin is not enough. There are biogenic amines playing different important roles in the nervous system. Cholamine: H2N–CH2CH2–OH is formed from serine, and its methylated derivative is choline: (CH3)3N–CH2CH2–OH. Acetylcholine is one of the neurotransmitters in nervous system. Pyridoxine deficiency can cause low acetylcholine level.

Formation of biogenic amines

There is another important biogenic amine GABA formed from glutamic acid. Its official name is 4-aminobutyric acid, earlier -aminobutyric acid. Its short name GABA is from the latter name. GABA is the most important inhibitory neurotransmitter in the mammalian central nervous system. It can moderate the sensitivity of neurons in the nervous system. Since GABA does not penetrate the blood brain barrier, GABA is synthesized in the brain from glutamic acid by means of PAL via a metabolic pathway called GABA shunt. Therefore it is supposed that the reason of the familiar pyridoxine-dependent epilepsy of babies is in connection with pyridoxine deficiency.

glutaminsav (Glu)

H2NCH

COOH

CH2

CH2

COOH

-amino-vajsav (GABA)

CH2

CH2

COOH

CH2

H2NCO2

A GABA keletkezése a glutaminsavbólFormation of GABA from glutamic acid

It was mentioned earlier pyridoxine is not synthesized in plants. The starting materials of pyridoxine derivatives are dihydroxyacetone phosphate from glycolysis and acetaldehyde then glycerinaldehyde-3-phosphate. The nitrogen of pyridine is from a glutamine.

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Prenatal vitamins – Foetus-saving vitaminsIt was known earlier that there are important vitamins, especially folic acid that can

prevent some structural birth defects as neural tube defect, anencephaly and spina bifida. Dr. Czeizel, a Hungarian professor suggested that a combination of three vitamins (folic acid, vitamin B12 and pyridoxine) can give a general prevention for foetus (except genetic damages): not only for problems mentioned above but against congenital abnormalities of urinary tract, mainly obstructive defects, and cardiovascular malformations, mainly conotruncal defects, including ventricular septal defect. In addition, there was a trend in the reduction of isolated limb deficiencies. There is a special bread is in the trade in USA with foetus-saving vitamins for pregnant women. Later on it came to light that this bread prevents different cardiovascular diseases (e.g. reduces the number of heart attacks) in the adult people, especially in middle-aged men, as well. Among others folic acid and vitamin B12 can help the permanent thymidine biosynthesis by methylation of uridine; and the combination of these three vitamins can eliminate the dangerous homocysteine concentration.

2. Secondary metabolites

Definition and classification of secondary metabolitesSecondary metabolism is the metabolism of different organic compounds in the living

organisms which are generally needed for their functioning. Unlike primary metabolites, the absence of secondary metabolites does not result in immediate death, but sooner or later it can cause more or less malfunction in the living organisms.

Secondary metabolites are synthesized from the different intermediates of biomolecules. Some of them are often restricted to a narrow set of species, especially in plants. The metabolism of secondary metabolites is not directly connected with the energy household of the living organism. Therefore their enzymes of oxidation-reduction reactions can use directly oxygen. These enzymes (oxygenases) often use other types of coenzymes e.g. L-ascorbic acid (vitamin C). The name ‘secondary metabolite’ is a generic term used for more than 30,000 different substances – most of them are plant secondary metabolites. In contrast to the primary metabolites secondary metabolites do not have nutrient characteristics for human beings. They are usually

43

found in very small amounts but have an effect on humans. Each plant family, genus, and species produces a characteristic mix of these chemicals, and they can sometimes be used as taxonomic characters in classifying plants. Secondary metabolites are produced by microbes, plants, fungi and animals, but not by all of them. Humans use some of these compounds as medicines, flavourings, or recreational drugs.

Secondary metabolites can be classified on the basis of their structure, their solubility in various solvents, the pathway by which they are synthesized (their starting materials) or their different biological functions. A simple classification of plant secondary metabolites includes three main groups: the terpenes (made from mevalonic acid, composed almost entirely of carbon and hydrogen), phenolics (made from simple sugars, containing benzene rings, hydrogen, and oxygen), and nitrogen-containing compounds (mostly alkaloids). The books about secondary metabolites this classification of are generally based on the starting materials.

According to their starting materials different types can be distinguished: carbohydrates (e.g. simple sugars), amino acids or their starting materials, acetyl coenzyme A or the intermediates of the metabolism of fatty acids or other intermediates. When the starting materials are one or more malonyl coenzyme A molecules, the name of secondary metabolites are ketides (oligoketides or polyketides).

The most important types of secondary metabolites are coenzymes (according to numerous scientist coenzymes and vitamins are closer to primary than secondary metabolism), regulating agents (e.g. hormones), attracting agents (e.g. the sweet sucrose, the fruit esters as scent agents etc.) and defensive (repelling) agents (e.g. alkaloids, toxins, antibiotics etc.).

AlkaloidsAlkaloids are a diverse group of low molecular weight, nitrogen-containing compounds

mostly derived from amino acids and found in about 20% of plant species. The most known alkaloids are plant-derived compounds but they can be produced by a large variety of other organisms (bacteria, fungi, animals), as well. They are part of a group of natural products (secondary metabolites). Alkaloids are playing a defensive role (repelling agents) in the plant against herbivores and pathogens. Due to their potent biological activity many of the approximately 12 000 known alkaloids are used as pharmaceuticals, stimulants, narcotics and poisons. Plant-derived alkaloids often have pharmacological effects and they are used as medications, as recreational drugs. Examples are the local anesthetic and stimulant cocaine, the stimulant caffeine, nicotine, the analgesic morphine, or the antimalarial drug quinine. Although alkaloids act on a diversity of metabolic systems in humans and other animals, they almost uniformly invoke a bitter taste.

The name ‘alkaloids’ is derived from the basic properties of some known derivatives. But this group also includes some related compounds with neutral and even weakly acidic properties. Also some synthetic compounds of similar structure are attributed to alkaloids. Beside carbon, hydrogen and nitrogen, molecules of alkaloids may contain sulfur and rarely chlorine, bromine or phosphorus.

The importance of alkaloids since the birth of human civilization is well illustrated by the drug opium, which is obtained from opium poppy (Papaver somniferum) and contains the analgesic morphine and numerous related alkaloids. Morphine is named after the Greek god Morpheus, the creator of sleep and dreams. In the epic story the Odyssey, opium is used as an ingredient in a wine-based drink called Nepenthes (Greek ne: not, penthos: sorrow) that was consumed by soldiers before combat to forget the horrors of battle. Socrates was one of the

44

founders of Western philosophy in ancient Greece. His most important contribution to Western was his dialectic method of inquiry. Because of political reasons he had to commit suicide by hemlock containing coniine. The Roman Emperor Nero murdered his stepbrother Britannicus with a mix of hemlock and opium. In most cultures, opium use was restricted to pain relief until the seventeenth century when recreational use of the drug began in China. The Opium Wars were fought between the British and Chinese to maintain free trade of the drug between the two countries. Opium remains the only commercial source for morphine and codeine.

The boundary between alkaloids and other nitrogen-containing natural compounds is not clear-cut. Compounds like amino acid peptides, proteins, nucleotides, nucleic acid, amines and antibiotics are usually not called alkaloids. Natural compounds containing nitrogen in the exocyclic position (mescaline, serotonin, dopamine, etc.) are usually attributed to amines rather than alkaloids. Some authors, however, consider alkaloids a special case of amines.

Compared with most other classes of natural compounds, alkaloids are characterized by a great structural diversity and there is no uniform classification of alkaloids. Historically, first classification methods combined alkaloids by the common natural source, e.g., a certain type of plants. This classification was justified by the lack of knowledge about the chemical structure of alkaloids and is now considered obsolete. More recent classifications are based on similarity of the carbon skeleton

Influence of alkaloids on nerve cellsAbout nervous system

Alongside the other components of the autonomic nervous system, the sympathetic nervous system aids in the control of most of the body's internal organs. The parasympathetic system generally works to promote maintenance of the body at rest. Phrenic nerve contains motor, sensory, and sympathetic nerve fibers. The neuron is the functional unit of the nervous system. Humans have about 100 billion neurons in their brain alone. Nerve cells (neuron) interact with other nerve cells at junctions called synapses. In the sympathetic nervous system the synapses between the short first (preganglionic neurons) and the long second (postganglionic neurons) neurons are called ganglions. The second synapses are between the second neurons and the peripheral target tissues. In the parasympathetic nervous system the neurons lead directly to the peripheral target tissues and the synapses are between the neurons and the target tissues.

The sympathetic nervous system

45

The plasma membrane of neurons, like all other cells, has an unequal distribution of ions and electrical charges between the two sides of the membrane. The outside of the membrane has a positive charge, inside has a negative charge. This charge difference is a resting potential and is measured in millivolts. Passage of ions across the cell membrane passes the electrical charge along the cell. The voltage potential is -65mV (millivolts) of a cell at rest, it is called resting potential. Resting potential results from differences between sodium and potassium positively charged ions and negatively charged ions in the cytoplasm. Sodium ions are more concentrated outside the membrane, while potassium ions are more concentrated inside the membrane. This imbalance is maintained by the active transport of ions to reset the membrane known as the sodium-potassium pump. The sodium-potassium pump maintains this unequal concentration by actively transporting ions against their concentration gradients.

The sodium-potassium pumpThe Na+/K+-ATPase helps maintain resting potential by an active transport. There are

several secondary membrane transport proteins, which import glucose, amino acids, and other nutrients into the cell using of the sodium gradient by symport. Later on these sodium ions are transported back to outside, to the intercellular region Na+/K+-ATPase by active transport. Inside of the cell three of sodium ions bind to the active site of this enzyme followed by a phosphorylation by ATP that causes a conformational change in the active site transporting sodium ions and the phosphorylated part to outside (eversion). This altered conformation is disadvantageous for sodium ions therefore they leave the enzyme followed by binding of two potassium ions. The presence of potassium ions causes a dephosphorylation followed by a conformational change in the active site again transporting potassium ions to inside (eversion again). This original conformation cannot bind potassium ions. After the releasing potassium ions the active site is ready for an active transport again.

There are similar pumps in the cell membrane. Ca2+-ATPase similarly can transport calcium ions out of the cells, but in this case the site of phosphorylation remains inside of the cell. Proton pump of the stomach works in the same way as Ca2+-ATPase does maintaining low pH value inside.

46

The proposed mechanism for sodium-potassium pump

Because of the connection a stimulating agent (e.g. the effect of adrenaline) to the receptor the permeability of the membrane of the preganglionic neuron changes, this process is called depolarization. On the effect of depolarization a transport of electron can be occurred in the preganglionic neuron until the presynaptic membrane of ganglion. The transmission of the signal though the synapse is by means of neurotransmitters. The neurotransmitter of the first synapse (ganglion) in the sympathetic nervous system and of the only synapse in the parasympathetic nervous system is acetylcholine; and the neurotransmitter of the second synapse in the sympathetic system is noradrenaline (norepinephrine).

Depolarization the postsynaptic membrane by increasing the conductance of sodium and potassium ions

The neurotransmitters are deliberated from their special storing places (vesicles) and through the synapse they connect to the receptor of the postsynaptic membrane causing a depolarization in the membrane of the second neuron in the sympathetic nervous system or of the peripheral target tissues in the parasympathetic nervous system. The neurotransmitter of the second synapse of between the first and the second neuron in the sympathetic nervous system is noradrenaline. The place on the postsynaptic membrane where neurotransmitter is connected is really the substrate binding site of the neurotransmitter on the receptor (cholinerg receptor for acetylcholine and adrenerg receptor for noradrenaline) for its degradation (hydrolysis for acetylcholine and oxidative deamination for noradrenaline). After degradation the residues of the neurotransmitter go back into the postsynaptic membrane for regeneration. In the case of receptors (active sites on the surface of membranes) the agents for activation are called agonists and the inhibitors are called antagonists.

The first synapse - the function the active site of cholinesteraseThe name of the substrate binding site of cholinesterase is anionic site because it can bind

the ammonium cation part of acetylcholine by ionic interactions. The connection of acetylcholine starts depolarization. The name of the catalytic site of cholinesterase is esterase site. The hydrolyzing agent of the ester group is the hydroxyl group of a serine activated by a strong

47

hydrogen bond by histidine. After the hydrolysis choline leaves the anionic site that stops depolarization. The acetylated serine in the esterase site is hydrolyzed by a water molecule.

The function of the active site of cholinesterase

After the return of choline and acetic acid to the presynaptic membrane the steps of the regeneration are the activation of acetic acid to acetyl-CoA using ATP and coenzyme A followed by the reaction between acetyl-CoA and chlorine then the last moment is the return of this regenerated acetylcholine to vesicles.

48

The biosynthesis and degradation of acetylcholine

The function of cholinesterase can be influenced in two steps: binding acetylcholine to the anionic site (no depolarization) and the leaving the active site (permanent depolarization). The anionic site can be inhibited by competitive inhibition of bioisostere molecules – e.g. alkaloids. Permanents depolarization can be caused by the phosphorylation of serine in the catalytic site by organic phosphate pesticides. Without ester hydrolysis acetylcholine can not leave the substrate binding site, this is the reason of permanent depolarization.

49

There are two different kinds of cholinerg receptors, they can be distinguished on the basis of their sensitivity to alkaloids nicotine (nicotinic acetylcholine receptors) and muscarine (muscarinic acetylcholine receptors). Nicotinic receptors are responsible for the initial fast depolarization of neurons. However, the subsequent hyperpolarization and slow depolarization of the postganglionic neuron from stimulation are actually mediated by muscarinic receptors, types M2 and M1 respectively. M1-type muscarinic acetylcholine receptors play a role in cognitive processing. In Alzheimer disease amyloid formation (insoluble fibrous protein aggregates sharing specific structural traits) may decrease the ability of these receptors to transmit signals leading to decrease cholinergic activity. M1-type muscarinic acetylcholine receptors can play a role in schizophrenia that is a mental disorder with hallucination and paranoid or bizarre delusions.

There are important M3 muscarinic receptor agonists those are used medically for a long time. Arecoline is an alkaloid containing pyridine ring that is present in Betel nut (Areca nut) (often wrapped in betel leaves are chewed in India). Pilocarpine containing imidazole ring is used in the treatment of chronic glaucoma. Glaucoma is a disease in which the optic nerve is damaged, leading to progressive, irreversible loss of vision. It is often, but not always, associated with increased pressure of the fluid in the eye.

Acetylcholine is the natural agonist of both kinds of receptors. It has two types of effects. The first type is termed muscarinic, which is the parasympathetic effect on the secretory exocrine glands, and on smooth and cardiac muscles through their corresponding receptors. The other type of its effect is termed nicotinic, which is on the skeletal (voluntary) muscles; it is not considered to be part of the peripheral autonomic nervous system.

Nicotine containing pyridine ring is the most important alkaloid of tobacco plant. Tobacco (Nicotiana tabacum), a native plant of the Americas and was in widespread use when Columbus arrived in the New World in 1492. Tobacco was sniffed, chewed, eaten, drunk, applied topically to kill parasites and used in eye drops and enemas. The act of smoking tobacco appears to have evolved from snuffing and is currently the most common means of administration. Tobacco was used ceremonially, medicinally and for social activities. Ironically, one of the first medicinal uses of tobacco was based on its purported anticancer properties.

Tobacco contains several structurally similar other pyridine alkaloids, as well. Nicotine acts on the nicotinic acetylcholine receptors, specifically the ganglion type nicotinic receptor and

50

one CNS (central nervous system) nicotinic receptor. In small concentrations, nicotine increases the activity of these receptors. Nicotine also has effects on a variety of other neurotransmitters through less direct mechanisms. Nicotine appears to enhance concentration and memory due to the increase of acetylcholine. Anxiety is reduced, the positive effects of dopamine on the brain (cognition, motivation) are extended and the sensitivity in brain reward systems is increased. The pyridine ring containing alkaloids are mostly synthesized from nicotinic acid.

Nicotine, muscarine, arecoline and pilocarpine

Smoking tobacco is a major cause of heart disease, stroke, peripheral vascular disease, chronic obstructive pulmonary disease, lung and other cancers, and various gastrointestinal disorders. Smoking can cause many other health problems including osteoporosis, impaired fertility, inflammatory bowel disease, diabetes and hypertension. Tobacco smoke contains a multitude of chemicals including polycyclic aromatic hydrocarbons; thus, nicotine is not solely responsible for these disorders. However, nicotine is one of the most biologically active chemicals in nature, binding to several different receptors and activating a number of key signal transduction pathways. Many of the physiological effects of nicotine, including addiction, are exerted by its action on nicotinic acetylcholine receptors. Nicotine modulates the phosphatidylinositol pathway and increases intracellular calcium levels, which are both universal

51

signalling components in physiological processes. The carcinogen effect of smoking is in connection with tar products of burning, but nicotine can promote the oxidative damage of reactive oxygen species.

Biosynthesis of nicotine

Coniine containing saturated pyridine ring (pyperidine ring) is the most important alkaloid of hemlock (Conium maculatum) that is native to Europe and western Asia. It contains seven structurally similar other piperidine alkaloids, as well. Including these piperidine alkaloids are synthesized from eight acetate units therefore they are polyketides. Coniine is a typical antagonist of nicotinic acetylcholine receptors by competitive inhibition. It induces a neuromuscular blockage later on paralyses the respiratory muscles.

Coniine

Muscarine containing tetrahydrofurane ring is an alkaloid of certain mushrooms. Muscarine acts as a selective agonist of the neurotransmitter acetylcholine on smooth muscles of the gastrointestinal tract, eye exocrine glands, and heart. It causes a strong activation of the peripheral parasympathetic nervous system that may end in convulsions and death. Muscarine poisoning is characterized by increased sweating and lacrimation within 15 to 30 minutes after ingestion of the mushroom. Death is rare, but may result from cardiac or respiratory failure in severe cases.

The starting material of alkaloids containing pyridine ring is nicotinic acid that is synthesized from L-tryptophane. Pyrrolidine part of nicotine is synthesized from N-methyl-’-pyrrolinium cation formed from L-ornithine. This cation is one of the starting materials of atropine that is a competitive antagonist of acetylcholine for the muscarinic acetylcholine receptors. Atropine is a racemic mixture of D- and L-hyoscyamine, with most of its

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physiological effects due to L-hyoscyamine. Its pharmacological effects are due to binding to muscarinic acetylcholine receptors. It is an antimuscarinic agent. Atropine effects on the vagus nerve (in Hungarian bolygóideg) of the parasympathetic nervous system on the heart. This effect decreases heart rate. As acetylcholine is the hormone of the waves of rhythmic (peristaltic) contraction move along the gut, atropine can decrease of the absorption of different poisons. Atropine is also an antagonist of the inhibitors of cholinesterase enzyme. Atropine can cause ventricular fibrillation, supraventricular or ventricular tachycardia, dizziness, nausea, blurred vision, loss of balance, dilated pupils, photophobia, dry mouth and potentially extreme confusion, dissociative hallucinations and excitation especially amongst the elderly people. Atropine is an alkaloid with a tropan skeleton from deadly nightshade (Atropa belladonna) and other plants of the family Solanaceae.

Scopolamine is an analogue of atropine containing an oxirane ring. It is less toxic as atropine is. It can be used as a depressant of the central nervous system, and was formerly used as a bedtime sedative. It can be used also against both major depressive disorder and depression due to bipolar disorder. Earlier it was supposed to be a truth serum.

In spite of their structure (it is a tropane alkaloid) cocaine can be in connection with noradrenaline, as well. Cocaine can be obtained from the leaves of the coca plant. It is a stimulant of the central nervous system, an appetite suppressant, and a local anesthetic. Specifically, it is a serotonin-noradrenaline-dopamine reuptake inhibitor, which mediates functionality of these neurotransmitters as an exogenous catecholamine transporter ligand.

Atropine (DL-hyoscyamine), scopolamine (L-hyoscine) and cocaine

Physostigmine (also known as eserine from éséré, West African name for the Calabar bean) is a parasympathomimetic, specifically, a reversible cholinesterase inhibitor alkaloid of the Calabar bean. It indirectly stimulates both nicotinic and muscarinic receptors. Its mechanism is to prevent the hydrolysis of acetylcholine by acetylcholinesterase at the transmitted sites of acetylcholine. This inhibition enhances the effect of acetylcholine, therefore it can be useful for the treatment of cholinergic disorders e.g. to improve the memory of Alzheimer’s patients due to its potent anticholinesterase activity. Earlier West Africa native population was used physostigmine as an ordeal. The innocent persons were killed by the treatment. The guilty ones

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survived the treatment, therefore they had to be killed. This was similar to ordeal by water in Europe in the Middle Ages. Neostigmine is a synthetic drug with the same action.

The second synapse – neurotransmitter is noradrenaline

The biosynthesis and degradation of noradrenaline

The second synapses are between the second neurons and the peripheral target tissues. The starting material of noradrenaline (norepinephrine) is L-tyrosine. Similarly to other secondary metabolites oxygenase (hydroxylase) enzymes using molecular oxygen play role in its biosynthesis. The product of the first step of the synthesis (catalyzed by tyrosine hydroxylase) is L-3,4-dihydroxyphenylalanine. Its short name L-DOPA (levodopa) is from its earlier name (dioxyphenylalanine). Dopa is the starting material of melanin in enzymatic browning of fruits and other parts in plants. Dopa, dopamine and noradrenaline are catecholamines containing both catechol (ortho-dihydroxybenzene) phenylethylamine structures. Catecholamines are sympathomimetic ‘fight-or-flight’ (fight or escape) hormones released by the adrenal glands in response to stress.

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The biosynthesis of papaverine and morphine

Noradrenaline is a catecholamine with multiple roles including as a hormone and a neurotransmitter. It increases blood pressure by its activation effect on adrenergic receptors. It can directly increase heart rate, trigger the release of glucose from energy stores, and increase blood flow to skeletal muscle. The degradation products of noradrenaline are different. One of the most important product is 3,4-dihydroxymandelic acid (an oxidised product of 3,4-dihydroxyphenyl glycolaldehyde that is the first oxidised product of the oxidative degradation of noradrenaline catalyzed by monoamine oxidase MAO).

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Papaverine

Many benzylisoquinoline alkaloids are used as pharmaceuticals due to their potent pharmacological activity, which is often an indication of the biological function of the approximately 2500 known members of this group. Their effectiveness suggests that these alkaloids function as herbivore deterrents (protection from infections by micro-organisms). Benzylisoquinoline alkaloids occur mainly in basal angiosperms including the Ranunculaceae, Papaveraceae, Berberidaceae, Fumariaceae,Menispermaceae and Magnoliaceae. The structure of these alkaloids always contains phenylethyl structure that suggests an agonist effect on the adrenerg receptors. These receptors are called opiate receptors.

A morphine, a codeine and heroin

Benzylisoquinoline alkaloid biosynthesis begins with decarboxylations, ortho-hydroxylations and deaminations that convert tyrosine to both dopamine and 4-hydroxyphenylacetaldehyde. The reaction between these two products results in the starting

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material of both papaverine (that is its polymethylated derivative) and morphine (after a special kind of cyclization). The methylation of one of hydroxyl groups of morphine results in codeine. Heroin is a diacetylated, synthetic derivative of morphine. These are opium alkaloids obtained from opium poppy (Papaver somniferum).

Papaverine is used primarily in the treatment of different kinds of spasm. Morphine is an analgesic and recreational drug. Because the possibility of drug-addiction its use is allowed only for cancer patients with terrible pains under control. Diacetylmorphine (heroin) is used as both an analgesic and a recreational drug. Frequent and regular administration is associated with tolerance and physical dependence, which can cause addiction. Internationally, diacetylmorphine is controlled under Schedules I and IV of the Single Convention on Narcotic Drugs. It is illegal to manufacture, possess, or sell diacetylmorphine without in almost all of countries. Drug-addition leads not only to degradation in the physical status of a person but in mental and moral status, as well.

Lisergic acid and its diethylamide (LSD)

Lysergic acid, also known as D-lysergic acid and (+)-lysergic acid, is a precursor for a wide range of ergoline alkaloids that are produced by the ergot fungus (Claviceps purpurea). There are ergoline alkaloids (e.g. ergotamine) those can contract blood vessels causing serious pains and necroses in both limbs and internal parts. This fungus can grow easily on rye in the case of cold and rainy weather. Ergoline alkaloids getting into flour and bread caused terrible illness and death. In the Middle ages it was thought that it is an epidemic caused by sexual sins. Amides of lysergic acid (lysergamides) are widely used as pharmaceuticals and as psychedelic drugs (increase of consciousness). Lysergic acid is usually produced by hydrolysis of lysergamides. Lysergic acid diethylamide, abbreviated (LSD) a semisynthetic psychedelic drug of the ergoline family. LSD is non-addictive and well known for its psychological effects which can include altered thinking processes, closed and open eye visuals, an altered sense of time and spiritual experiences. It is used mainly as a recreational drug and as an agent in psychedelic therapy. It is dangerous because it can cause a long lasting effect by modifying of the receptor.

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LSD can replace serotonin in the central nervous system. Serotonin can regulate sleep, mood and it has other important functions including cognitive function (e.g. memory and learning).

Serotonin

Literature1. Bender, D.A.: Nutritional biochemistry of the vitamins. Cambridge University Press

Cambridge New York Port Chester Melbourne Sydney 1992.2. Luckner, M.: Secondary metabolism in microorganisms, plants and animals. Springer-Verlag

Berlin Heigelberg New York London Paris Tokyo Hong Kong 1990.3. Stryer, L.: Biochemistry (3rd Edition) W.H. Freeman & Company New York 1988.4. Crozier, A., N. Clifford, M.N., Ashihara, H.: Plant Secondary Metabolites. Blackwell

Publishing Oxford 2006.

Topics in Nutritional biochemistry of the vitamins and secondary metabolites Assay questions – Two of these topics in short essay form in the exam.Exam 1-101. Definition and types of essential materials. Definition and types of secondary metabolites.

Classification of vitamins – the role of solubility.2. Precursors of coenzymes of oxydoreductases : niacin, riboflavin.3. Antioxidant vitamins (ascorbic acid and tocopherols) and lipid peroxidation.4. Precursors of coenzymes of transferases – C1 transfer: biotin, folic acid.5. Precursors of coenzymes of transferases – C1 transfer: biotin, cyanocobalamin.6. Precursors of coenzymes of transferases – C2 transfer: thiamin, pantothenic acid.7. Precursors of coenzymes of transferases – transfer of other groups: pyridoxine and its

connection with foetus-saving vitamins.8. Vitamin lipids (retinol and -carotene, cholecalciferol and its vitamers).9. Definition and types of secondary metabolites. Alkaloids and the nervous system. Cholinerg

systems.10. Definition and types of secondary metabolites. Alkaloids and the nervous system. Adrenerg

systems.

1+9, 4+10, 2+5, 3+7, 6+8.

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