STARCH AND STARCH TECHNOLOGY.

STARCH AND STARCH TECHNOLOGY.

STARCH is a polysaccharide material found in cereal grains of wheat, corn and rice, and in root and stem tubers of cassava, yam and potato.  As a polysaccharide polymer starch is composed of a large number of glucose monosaccharide units.

Starch is the storage form of carbohydrates in plant seed endosperm and tubers where it exists as granules.  Each granule usually contains millions of amylose molecules and a lower number of millions of amylopectin molecules.  Amylose molecules are always smaller than amylopectin molecules.

Starch is also metabolized for energy in plants and animals, and is used to produce a large number of industrial products.

AMYLOSE AND AMYLOPECTIN OF STARCH.

Starch is composed of two fractions – amylose and amylopectin. Natural starches (i.e unmodified starches) are mixtures of amylose (10-20%) and amylopectin (80-90%).
AMYLOSE forms a colloidal dispersion in hot water whereas amylopectin is completely insoluble in it.  A colloid is a type of chemical mixture in which one substance (amylose) is dispersed evenly throughout another (hot water).
A colloidal system (i.e, a colloidal solution or colloidal suspension) consists of two separate phases: a dispersed phase (or internal phase) and a continuous phase (or dispersion medium).
Colloids may be translucent because of the Tyndall effect, which is the scattering of light by particles in the colloid, or they may be opaque.
Amylose consists of long chains of glucose units connected by alpha (α) acetal linkage.  All of the monomer units are α -D-glucose, and all the α acetal links connect carbon no. 1 (C1) of one glucose to carbon no. 4 (C4) of the next glucose.
C1 is called an anomeric carbon because it can either have the α configuration of attached groups or the beta (β) configuration.  C1 is an acetal carbon because it has two ether oxygens attached to it.

The α configuration is when the ether oxygen on the C1 is on the opposite side of the ring as the C6.  In the chair structure this results in a downward projection. This is the same as the -OH in a hemiacetal.  Thus a glycosidic bond, which joins the hemiacetal group of a carbohydrate molecule to the hydroxyl of another group, and which may or may not be another carbohydrate, is an acetal bond.

Amylose characteristically forms a helix (a spiral that looks like a coiled spring). 
Amylose is specifically responsible for the formation of a deep blue colour of starch with iodine.  This is because iodine molecules can penetrate into the amylose coil.
Amylose molecule (beween 300 – 3000 molecules of glucose)

AMYLOPECTIN is formed by α (1 – 4) acetal bonds between α –D-glucose units, as in amylose, but additionally there are branches formed by α (1 – 6) acetal linkages between glucose molecules.  Amylopectin has 12-20 glucose units between the branches.
Amylopectin molecule (several thousands)

CLASSIFICATION OF STARCHES
There are basically two types of starches:  native starches and modified starches.
NATIVE STARCHES
Native or natural starches are produced by simply separating naturally occurring starch from grain or root crop such as rice, maize, wheat, potato or cassava.  Native starch products retain the original structure and characteristics as in the crop.  They are used for the purpose of food texturizing and thickening, in pharmaceuticals and as an industrial raw material.
Native starches are insoluble in cold water, and they can swell to different degrees, depending on the temperature applied. They have good thickening, gelling, adhesion and moisture retention properties.

MODIFIED STARCHES

Any process that brings about change in any of the properties of native starch is known as modification of starch, and the product is called modified starch.  This is often done in order to obtain some industrial benefits that are impossible with native starches. Physical, chemical and enzymatic methods may be used to modify starch.  These procedures bring about changes in the physical and chemical properties of starch.  Modifications may involve changing the form or structure of the granule, or changing the shape and composition of the amylose and amylopectin molecules of starch.  Modified starches are used in food, pharmaceutical, paper pulp and textile industries.

When starch is modified, any of the following properties can be altered, depending on the specific modification procedure: 

1.                 thickening.

2.                 gelatinization.

3.                 water retention.

4.                 palatability.

5.                 adhesion.

6.                 opacity.

PRODUCTION OF STARCH FROM CASSAVA.

Starch is the main component of cassava, with only a small amount of secondary substances such as protein and lipid.

Starch is used to produce diverse products such as food, textiles, adhesives, beverages, paper, building materials, and pharmaceuticals. Cassava starch has high paste viscosity, and high freeze-thaw stability, which makes it very desirable in many industries.

The procedure for cassava starch production is summarized as follows and illustrated in the chart below:

1.                 Cassava tubers are peeled and washed.

2.                 They are milled in a machine into a paste.

3.                 The paste is put in a sieve-bag (shasha bag) and about 3 to 4 times volume of water is added.

4.                 It is vigorously stirred, and the bag pressed to squeeze out liquid from it into containers.  Of course starch will also go out with this liquid into the containers.

5.                 The containers are allowed to stand for about 1 to 2 hours to allow starch to settle as a thick hard white deposit at the bottom.

6.                 The liquid on top is decanted, while the starch deposit is sun-dried, weighed and packaged.

About 25% starch yield may be obtained from cassava tubers.

STEPS IN THE PRODUCTION OF CASSAVA STARCH:

USES OF CASSAVA STARCH. 

1.                 Production of medical and industrial alcohol.

2.                 It used as food especially in Nigeria.  It is also used in bakery. Modified starch i.e. starch derivatives are used for texturing, thickening, binding, and stabilizing food products such as canned foods, frozen foods, and salad dressings.

3.                 It is used in confectionery as jellys and gums.

4.                 It is used to make monosodium glutamate valued for enhancing food flavour in Asia.  Example is Ajinomoto.

5.                 It is used as sweetener.

6.                 It is used to make glues or adhesives.

7.                 It is used for production of tablets in pharmaceutical industries as binders or fillers.

8.                 It is used to manufacture plywood.

9.                 It is used in the paper making process.

10.            It is used in the textile industry.

PRODUCTION OF GLUCOSE SYRUP FROM STARCH.

Glucose syrup is a concentrated aqueous solution of glucose (dextrose sugar), maltose and other nutritive saccharides. Although it is found in nature in grapes, it is produced and used in industry to make bakery products, pharmaceuticals and liquors.

The process for making glucose syrup from cassava consists of gelatinization, liquefaction, saccharification, and purification, which are described and illustrated below:
Gelatinization
At room temperature, the microscopic granules of natural starch are insoluble in water. But if slurry of starch is heated above 60^oC, the granules swell and rupture. This is called gelatinization of starch, which increases the viscosity of starch.  Gelatinized starch is next reacted with the enzyme amylase to hydrolyze starch. Actually gelatinization of starch and its partial hydrolysis with heat-stable amylase can be carried out in practise as a one-step process called liquefaction. The partially degraded starch chains are called dextrins.
Liquefaction
Starch slurry is made with 30-35% dry starch solids and its pH is adjusted to 6.0-6.4. Calcium, which helps to stabilize the enzyme, is added as calcium hydroxide or calcium chloride.  A heat-stable α-amylase is mixed into the slurry, which is then quickly heated to 100 ^oC and held at this temperature for 10 min before cooling it to 90 ^oC. This is maintained for 1-3 h to further hydrolyze the starch.
Dextrins produced at the end of liquefaction have a dextrose equivalent (DE) between 8 and 15, and DE, which is the total reducing sugar in the syrup expressed as dextrose on a dry weight basis determines the physical properties of the syrup.
Saccharification
After liquefaction, pH is reduced to 4.2 – 4.5 and the solution is cooled to 60 ^oC. An α glucomylase is added immediately. Reaction time for saccharification is usually between a day or two depending on the amount of enzyme added. Glucoamylase releases single glucose monomers from the ends of dextrin molecules. Syrups of 95% glucose are often manufactured in this way.

PRODUCTION OF GLUCOSE SYRUP.

REFERENCES

INTERNET:  www.elmhurst.edu/~chm/.../547starch.html

INTERNET: http://www1.lsbu.ac.uk/water/hysta.html
INTERNET: en.wikipedia.org/wiki/Colloid 
INTERNET: cheng.cam.ac.uk/research/groups/.../Starchstructure.html
INTERNET: en.wikipedia.org/wiki/Glycosidic bond

INTERNET: http://www.cassavabiz.org/postharvest/starch03.htm

INTERNET: http://www.cassavabiz.org/postharvest/gsyrup01.htm

ASSIGNMENTS

1.                 What is starch?  Discuss.

2.                 Compare and contrast amylose and amylopectin components of starch.

3.                 Differentiate between an hemiacetal bond and an acetal bond in a starch molecule.

4.                 What are native starches?

5.                 What are modified starches?

6.                 Outline the uses of cassava starch in various industries.

7.                 Describe the processes involved in starch production from cassava.

8.                 Describe the processes involved in glucose syrup production from starch.

9.                 Outline the changes that can be introduced when starch is modified.

10.            Compare cassava starch with starches from other natural sources.

11.            Compare and contrast amylose and amylopectin components of starch.

12.            What are modified starches?

13.            Describe the processes involved in glucose syrup production from starch.

DRUG METABOLISM

DRUG METABOLISM

DEFINITIONS:

XENOBIOTIC:  This is a compound foreign to the physiology of a living organism.  Drugs are almost all xenobiotics.  Other examples are pesticides and environmentally distributed chemicals in general.

a

DETOXIFICATION:  This refers to decrease in biological activity of a drug after it has been metabolized in the body.

TOXIFICATION:  This refers to increase in biological activity of a drug after it has been metabolized in the body.  By this we understand that sometimes metabolism of a drug can result in increase in its activity.  However, in this lecture focus will be on metabolic detoxification of drugs.

BIOTRANSFORMATION:  This is another term for the metabolism of a drug that results in its transformation from a harmful substance to a less harmful product that can easily be excreted from the body.

RENAL EXCRETION AND BIOTRANSFORMATION OF DRUGS.

Biotransformation of drugs leads to termination or alteration of their biologic activity, otherwise most drugs would have a prolonged duration of action.

This is important especially for those drugs that are lipophilic (fat-soluble, or water-insoluble).  They cannot therefore ionize, or rather ionize only partially, at physiologic pH.

For some few drugs however, which are hydrophilic, ionisable compounds, their biologic activity is normally terminated simply by renal excretion (i.e, by being excreted through the urine).

For lipophilic, unionized compounds, because they bind strongly to plasma proteins, they are not easily filtered at the glomerulus of the kidney.  Even though they are filtered, the lipophilic nature of renal tubular membranes makes it easy for them to be reabsorbed into the blood stream.  Consequently, for these compounds (most drugs), termination of biologic activity is not directly by renal excretion, but by metabolic biotransformation.

Lipophilic xenobiotics are usually transformed to more polar and hence readily excretable products, which are less active or even inactive compared to the parent drug.

Note however, that some metabolic biotransformation products have enhanced activity or toxic properties such as mutagenicity (inducing heritable alteration of DNA), teratogenicity (producing birth defects) and carcinogenicity (causing uncontrolled growth of cells and tissues).

LIVER AS THE MAJOR ORGAN OF DRUG METABOLISM.

Although several organs such as gastrointestinal tract, the lungs, the skin, and the kidneys, play important roles in the metabolism of drugs, the liver is the major site of drug detoxification for the following reasons:

1.                 The liver is a large organ.

2.                 It is the first organ perfused by chemical compounds absorbed in the gastrointestinal tract.

3.                 There are very high levels of most drug-metabolizing enzyme systems compared to other organs.

4.                 The liver is well endowed with the smooth endoplasmic reticulum where detoxification occurs specifically.

FIRST-PASS METABOLISM.

Several drugs that are administered orally show limited levels of bioavailability in the parts of the body where its action is required.  This often limits the therapeutic effectiveness of these drugs. 

The reason for this is known as the first-pass effect (first-pass metabolism).  First-pass metabolism is due to extensive metabolism in the liver of many drugs that are absorbed intact (without metabolism) from the small intestine and transported first through the hepatic portal system to the liver.  Examples of such drugs are isoproterenol and morphine. 

Morphine.

Additionally, extensive intestinal metabolism of some orally administered drugs can also contribute to the first-pass effect.  Examples of such drugs are chlorpromazine and clonazepam.

For some drugs, alternative routes of administration other than oral may be employed in order to achieve therapeutically effective levels in the blood.

BIOTRANSFORMATION OF DRUGS

Although metabolic transformation of drugs can occur in the intestinal lumen due to bacterial activity, or in the villi of the intestinal wall, most biotransformations occur between absorption of the drug into blood circulation and its renal excretion.  Nevertheless all biotransformation of drugs in the body occur as either phase I reactions or phase II reactions or both.

PHASE I REACTIONS.

These reactions are also called nonsynthetic reactions.  In phase I reaction a drug is converted to a more polar metabolite by introducing or unmasking a functional group, such as hydroxyl (– OH), amino (- NH2), sulphydryl (- SH), carboxylic acid (- COOH) groups, etc.  These reactions may occur as oxidation, reduction, hydrolysis, cyclization, decyclization, or as carboxylation, etc.

Oxidation enzymes include:

1.                 Cytochrome P450 monooxygenase system.

2.                 Alcohol dehydrogenase.

3.                 Aldehyde dehydrogenase.

4.                 Flavin-containing monooxygenase system.

5.                 Monoamine oxidase.

Reduction enzymes include:

1.                 NADPH-cytochrome P450 reductase.

2.                 Reduced (ferrous) cytochrome P450.

Hydrolysis enzymes include:

1.                 Esterases and amidases.

2.                 Epoxide hydrolase.

STEPS IN MICROSOMAL DRUG OXIDATIONS

Microsomal drug oxidations require cytochrome P450 (so named because it is a hemoprotein which in its reduced (ferrous Fe2⁺) form, binds carbon monoxide to give a ferrocarbonyl compound that absorbs maximally in the visible region of the electromagnetic spectrum at 450nm), cytochrome P450 reductase (an enzyme that contains flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD)), NADPH, and molecular oxygen (O₂).  Four steps are involved in the cycle:

1.                 Oxidized, i.e Fe3⁺ cytochrome P450, combines with a drug substrate to form a complex.

2.                 NADPH donates an electron to cytochrome P450 reductase, which in turn reduces the oxidized cytochrome P450 – drug complex.

3.                 Another electron is introduced from NADPH through the same cytochrome P450 reductase, which serves to reduce O₂ to form an “activated oxygen”-cytochrome P450-substrate complex.

4.                 This complex then transfers “activated” oxygen to the drug substrate to form the oxidized product.

Phase I metabolites may be readily excreted at this stage, haven been made sufficiently polar.  Examples of drugs that are metabolized in this way include paracetamol, steroids and phenothiazines.

Paracetamol (para-acetyl-amino-phenol, also called acetaminophen)

PHASE II REACTIONS.

However, many of the phase I products are not eliminated rapidly, and hence undergo a subsequent reaction in which an endogenous substrate such as glucuronic acid, acetic acid or an amino acid combines with the newly introduced functional group to form a highly polar conjugate that is readily excreted in the urine.  Hence phase II reactions are also called conjugation reactions.

Conjugation or coupling reactions require high-energy compounds, and specific transfer enzymes known as the transferases (located in microsomes (endoplasmic reticulum) and the cytosol.  Transferases catalyze either the conjugation of an activated endogenous compound (e.g, uridine 5^’-diphosphate (UDP)derivative of glucuronic acid, i.e, UDP-glucuronic acid) with a drug containing phenol; or the coupling of an activated drug (e.g, S-CoA derivative of benzoic acid, i.e, benzoyl S-CoA,) with an endogenous substrate such as the amino acid glycine.

Phase II conjugation enzymes include:

1.                 Glutathione S-transferases

2.                 UDP-glucuronosyl transferases

3.                 N-acetyltransferases

4.                 Sulfotransferases

5.                 Amino acid N-acyl transferases

FACTORS THAT AFFECT DRUG METABOLISM.

1.                 Induction of drug metabolizing enzymes

2.                 Inhibition of drug metabolizing enzymes

3.                 Physiological factors such as age, individual variation, nutrition and differences in sex

4.                 Pathological factors such as liver, kidney, heart disease, and endocrine dysfunction (such as malfunctions of pituitary, gonads and thyroid).

REFERENCES.

Bhatia S.C (2006), Environmental Chemistry, CBS Publishers and Distributors, India, pg 421 - 423.

INTERNET:  http://en.wikipedia.org/wiki/drug metabolism.

INTERNET:  Common household gadgets, cleaners, and medications are increasing the incidence of autism and cancer,  http://cancerpreventiontheory.info/benzene_in_pain_relievers.htm, 27th January, 2010.

INTERNET:  The role of chemistry in history, http://itech.dickinson.edu/chemistry/?cat=107, 27^th January, 2010.

ASSIGNMENT.

1.                 Define and explain the following terms:  xenobiotics, detoxification, toxification and biotransformation.

2.                 Compare and contrast lipophilic drugs and hydrophilic drugs in relation to their detoxification.

3.                 Discuss renal excretion and metabolic biotransformation of drugs.

4.                 Why do you think the liver is the major organ of detoxification of drugs in the body?

5.                 What do you understand by “First-pass metabolism”?

6.                 What factors do you militate against bioavailability and therapeutic effectiveness of orally administered drugs.

7.                 Discuss phase I reactions of drug biotransformation.

8.                 Discuss phase II reactions of drug biotransformation.

9.                 Outline the steps involved in microsomal oxidation of drugs.

10.            Elaborate on factors that affect drug metabolism.

11.            What contributions do orally administered drugs that are metabolized in the GIT make to the first-pass effect?

12.            What contributions do orally administered drugs that are absorbed intact from the GIT into general circulation make to the first-pass effect.

13.            Discusss Mutagenicity, Teratogenicity and Carcinogenicity in relation to drug metabolism.

TOXINS AND TOXICS

TOXINS AND TOXICS

DEFINITIONS

TOXIN:

Technically, a toxin is a poisonous substance produced by living organisms.
By this definition, man-made substances produced artificially are not considered to be toxins.
Toxins are different from biological agents such as bacteria, viruses etc. According to the International Committee of the Red Cross definition of toxins, toxins are chemical agents and not biological agents: "Toxins are poisonous products of organisms; unlike biological agents, they are inanimate and not capable of reproducing themselves."
Toxins are usually small molecules, peptides, or proteins that are poisonous to organisms when they interact with cellular components such as macromolecules, enzymes or receptors. Toxins differ in their severity, ranging from minor (eg ant sting) to acute (e.g bee sting) to deadly (e.g botulinum toxin).

EXOTOXINS:

These are toxins excreted by an organism.

ENDOTOXINS:

These are toxins released only when an organism such as bacteria is lysed.

TOXOID:

This is a weakened or suppressed toxin.

VENOMS:

These are toxins in the sense of use by some types of animals.

BIOTOXINS:

This word is used to emphasize the biological origin of a toxin. Biotoxins are produced spiders, snakes, scorpions, jellyfish and wasps for the purpose of predation. Biotoxins are also produced ants, bees, termites, honeybees and some frogs for the purpose of defense.

TOXICANT:

This is a toxic substance that is not produced by living organisms. They are not biologically produced from living organisms, but are artificially produced. They are synthetic products, and can also be called xenobiotics.
Plural for toxicant is toxics.

CATEGORIES OF TOXINS.

There are three broad categories of toxins. The term “toxin” is however used in this context in the non-technical sense to include any chemical substance that is injurious or detrimental to a living organism. Here it includes both toxins and toxics.

A) ENVIRONMENTAL TOXINS:

These are toxins that are produced artificially by human beings. They are environmental pollutants of land, water and air. They are also called xenobiotics because they are foreign and synthetic. Examples include:
1. Benzene
2. Benzoic acid
3. Phenobarbitones
4. Chlorinated hydrocarbons such as the chlorobenzoates
5. Xylenes
6. Nicotine
7. Pesticides etc

BENZENE

BENZOIC ACID

DIQUAT (organo-nitrogen pesticide) (NOTE:  insert N+ at the base of the Benzene structures)

PARAQUAT (NOTE:  insert N+ at the ends of the Benzene structures)

B) FOOD TOXINS: 

These are toxins found in food stuff. They are:
1. Chemical food toxins.
2. Biological food toxins.

CHEMICAL FOOD TOXINS.

These include the following:

1. COAL TAR: All artificial color and flavor additives in soft drinks and cosmetics are produced from coal tar. Coal tar dyes give foods bright colour and strong flavour. They are very carcinogenic.

2. ARTIFICIAL SWEETENERS: Cyclamates and saccharin, artificial sweeteners, can cause cancer of the gastrointestinal tract.

3. NITROSAMINES: Nitrosamines are produced in the body from three possible sources:
a) From the ingestion of chemical preservatives and colour enhancers.
b) From the nitrites and nitrates that are added to meat during processing.
c) From the nitrites and nitrates from chemical fertilizers which get into water bodies through runoff water and from thence into living organisms.
Nitrosamines are highly carcinogenic, causing cancer of the liver, stomach, brain, bladder and kidneys.

4. ADDITIVES LIKE BUTYLATED HYDROXYLTOLUENE (BHT), BUTYLATED HYDROXYLANISOLE (BHA), MONOSODIUM GLUTAMATE (MSG) AND DIETHYLSTILBESTROL (DES): BHT and BHA are added to foods as antioxidants in order to preserve the fat in them. MSG is added to food as a taste enhancer. DES is added especially to meat in the USA as artificial sex hormone.These are all carcinogenic poisons.

5. ALUMINUM: Aluminium used as a cookware (i.e., as a cooking utensil) is a kind of food additive, which is poisonous to the body. Small particles of aluminium enter the food as it is being cooked. It has been banned in Sweden. It is safer to use stainless steel for cooking.

6. CHEMICALS SUCH AS HEXACHLOROPHENE, BENZIDINES, ANILINE DYES, NAPHTHALENE, ASBESTOS AND PESTICIDES: These may produce free radicals in the body and thus become strongly carcinogenic to bladder, kidney and lungs.

7. HEAVY METALS SUCH AS NICKEL, LEAD, MERCURY, AND CADMIUM: These could accumulate over a long period of time in the body and become very poisonous.

BIOLOGICAL FOOD TOXINS:

This includes:
1. Food poisoning by toxic plants and animals.
2. Food poisoning by bacteria.
3. Food poisoning by moulds.

FOOD POISONING BY TOXIC PLANTS AND ANIMALS.

PLANTS:

1. Certain mushrooms are very toxic, especially when a poisonous mushroom is mistaken for a non-poisonous one. Some are deadly.

2. Red kidney beans is toxic when eaten raw or when it is not properly cooked. The substance haemagglutinin must be boiled for about 10 minutes to inactivate it. It causes nausea, vomiting and diarrhea.

3. Cyanic acid in improperly processed cassava is poisonous. It is only careful fermentation and processing of cassava that can eliminate its cyanic acid load. Cyanic acid blocks enzymes of the respiratory chain.

4. Ricin is a toxin found in the castor bean plant.

ANIMALS:

1. Puffer fish poisoning.
The intestines of puffer fish (sphaeroides rubripes) contain the toxin tetrodotoxin.

The poisonous part has to be carefully removed before the fish can be eaten. The fish is an ingredient of a Japanese delicacy. This toxin acts by binding to Na+ channels (protein channels) of neurons, and thus preventing normal action potential. This blocks signals from nerves to muscles. In this way it causes paralysis and death.

2. Paralytic shellfish poisoning.

Shellfish such as oysters, mussels or clams that have fed on the phytoplankton, the marine dinoflagellate Gonyaulax become toxic because they accumulate the toxin saxitoxin produced by the plankton in their muscles. The shellfish itself is not sensitive to the toxin, but organisms higher up the food chain that consume it are harmed by a mode of action similar to that of tetrodotoxin.

FOOD POISONING BY BACTERIA.

This may be:
1. Food borne infection: The organism itself is the causative agent of the infection e.g Listeria monocytogenes and salmonella sp. They may cause gastrointestinal tract (GIT) disorders.

2. Food borne intoxication: This is caused by ingestion of toxins of microorganisms. The toxins of staphylococcus aureus and clostridium botulinum are heat stable even though the microorgainsm itself is already killed by heat. The toxin of clostridium botulinum is a protease that prevents the release of neurotransmitter at the synapse of two neurons. It leads ultimately to death.

3. Food borne disease: The organism does not grow in the food, but simply use it as a means of transfer from host to host, e.g faecal-oral route such as salmonella typhi.

FOOD POISONING BY MOULDS.

Toxins of moulds are called mycotoxins. Some mycotoxins are carcinogenic while some others attack specific organs and tissues. An example is aflatoxin found in groundnut infected by Aspergillus flavus. Aflatoxin can also be found in other foods like fresh beef, milk, beer or cocoa.

OTHER NON-FOOD SOURCES OF TOXINS.

1. The venom of the black mamba snake contains the toxin dendrotoxin which hinders K+ channels.

2. The venom of the rattlesnake contains the toxin hemotoxin, which targets and destroys red blood cells.

3. Necrotoxins, which cause necrosis (i.e. death) in the cells they come in contact with are produced in the venom of the puff adder (Bitis arietans).

4. Neurotoxins, which attack the nervous systems of animals, are produced in the venom of Elapid snakes, most scorpions, widow spiders and the cone snail.

5. Apitoxins are produced by the honey bee in its venom.

6. Other reptile/snake venom toxins are bungarotoxin, cobrotoxin, calciseptine, taicatoxin, calcicludine, cardiotoxin III.

7. Amphibian toxins include Allopumiliotoxin 267A, Batrachotoxin, Bufotoxins (Arenobufagin, Bufotalin, Bufotenin), Cinobufagin, Epibatidine, Histrionicotoxin Tarichatoxin.

8. Curare is a substance used as an arrow head poison. The active component is the toxin tubocurarine.

DETOXIFICATION OF ENVIRONMENTAL TOXICS.

Toxics are not of use to the organism, (infact they are harmful) and their absorption is not always prevented; hence it is necessary to neutralize reactive groups in these compounds and to expedite their excretion by active transport.
The metabolism of toxins is biphasic:
1. This involves the introduction of functional groups to the toxin, such as epoxides, sulphydryl (-SH), hydroxylamine (-NHOH), hydroxyl (-OH), carboxyl (-COOH) groups etc. The reactions are epoxidation, reduction, oxidation, hydrolysis etc. Sometimes a reaction may occur to expose reactive functional groups in the toxic. For example, in the detoxification of benzene, a hydroxyl group may be introduced in an oxidation reaction to form phenol. The purpose of this reaction step generally is to make the xenobiotic, especially the lipophilic (i.e., water insoluble) substances, water soluble in order to facilitate their excretion through the urine

2. The second step involves the conjugation of the phase I product with an endogenous substance in order to further enhance its water solubility and excretion via the urine. For example, phenol may be conjugated with glucouronic acid to form the easily excretable phenyl β-D-glucouronide.
Sometimes, however, the metabolism of a xenobiotic need not be biphasic. It may be monophasic, involving only conjugation. For example, the metabolism of benzoic acid involves direct conjugation with glycine or glucouronic acid to form hippuric acid or benzoyl glucouronide respectively.

ASSIGNMENT.

1. Differentiate between toxins and toxics.

2. Differentiate between technical usage and non-technical usage of the term toxin.

3. Discuss toxins of plant origin.

4. Discuss toxins of animal origin.

5. Discuss toxins of bacterial origin.

6. Discuss a known toxin of mould origin.

7. Explain the various ways snake venoms act.

8. What is biphasic metabolism of environmental toxins.

9. What is monophasic metabolism of environmental toxins.

10. Explain the following terms: toxin, toxicant, toxoid and biotoxin.

11. What are chemical food toxins?

12. Draw the structures of diquat, benzoic acid and tetrodotoxin.

13. List examples of environmental toxins.

ASSIGNMENT 1: BIOPHYSICAL CHEMISTRY

ASSIGNMENTS

1. What roles do water and pH play in the structural and functional stability of biomolecules.

2. Discuss the homeostatic regulation of water and pH in the body fluids of organisms.

3. What is acidosis? Discuss.

4. Discuss water under the following headings:
a) Structure of water
b) Water as a dipole

5. What is the pH value of 2.0 x 10-6 mol/L KOH

6. Derive the Henderson-Hasselbalch equation for a weakly ionizing acid (HA)

7. What is the pH of a solution whose OH- concentration is 4.0 x 10-4 mol/L

8. What is the pH of a solution whose H+ ion concentration is 3.2 x 10-4 mol/L

9. Discuss Sorenson’s definition of pH.

10. Explain why and how water forms hydrogen bonds.

BIOPHYSICAL CHEMISTRY (STH 303)

WATER, PH AND BUFFER SYSTEM


1.1     INTRODUCTION


Water is so important in living organisms that it constitutes a large percentage of both the intracellular and extracellular fluids, (above 70%) Water is essential for solubilizing and modifying the properties of polar biomolecules such as nucleic acids, proteins, and carbohydrates. This it does by forming hydrogen bonds with their polar functional groups such as the carboxylic (-COOH), amino (-NH₂), hydroxyl, (OH), aldehyde (-CHO) and carbonyl (-C=O ) functional groups. These hydrogen bond interactions bring about conformational changes of these biomolecules in the aqueous environment, and these changes are essential to their functionality and the life process as a whole.


Again the structural and functional stability of biomolecules depend on the pH of the intracellular or extracellular fluids. The dissociation behaviour of the functional groups of biomolecules depends on the pH of the medium. And this is turn affects the rate of the enzyme-catalyzed  reactions in the cells. The extracellular fluids, e.g blood also depend on the pH to function in transporting nutrients, metabolic waste products, gases and hormones round the body.

Thus there is a pH optimum for optimal biologic function of both the intracellular and extracellular constituents of living organisms, that needs to be maintained. For the blood the pH for optimum activity is 7.4, and for the stomach gastric juice it is between 2.0 and 3.0. The maintenance of pH in living organisms at a constant optimum value is carried out by the buffer systems. In the blood, one of the most important of the blood buffers is the carbonic acid buffer system.    

1.2        HOMEOSTASIS 

This is the maintenance of the composition of the internal environment of

living things. It ensures that the right internal environment is preserved for the

right functioning of living organisms. Homeostatic regulation of the internal

environment ensures among other things that there is a balanced distribution

of water in the body, and that the appropriate pH of body fluids is maintained.

The regulation of water balance in the body depends on the following factors:

i.                   Hypothalamus (the part of the brain that regulates thirst).

ii.                Antidiuretic hormone (ADH), vasopressin.

ANTIDIURETIC HORMONE (ADH)

Vasopressin in also called antidiuretic hormone. It increases water

reabsorption in the kidney 

ii.                Ability of the kidneys to retain or excrete water  

iii.              Evaporative losses of water due to respiration and perspiration. 

Depletion or excess of water in the body is often accompanied by depletion or

excess of sodium. Thus anything that leads to sodium depletion for instance,

may invariably lead to water depletion. Water depletion may result from

decreased intake as in coma, or increased loss as in severe sweating, diarrhea

in infants and cholera. Excess water may occur through increased intake as in

excessive administration of intravenous fluids, and decreased excretion as in

severe renal failure.

Maintenance of extracellular fluid between pH 7.35 and 7.45, and of which the 

bicarbonate buffer system plays a key role, is essential for health. 

Disturbances of acid – base balance are known (diagnosed) in the clinical

laboratory by measuring the pH of arterial blood and the CO₂ content of

venous blood. Acid – base imbalance may either lead to acidosis (i.e. when the

blood pH is less than 7.35) or alkalosis (i.e. when the blood pH is greater than

7.45).

The causes of acidosis include diabetic ketoacidosis and lactic acidosis; and

causes of alkalosis include the vomiting of acidic gastric contents.

Homeostasis is a regulatory mechanism within a living organism that helps to

prevent water imbalance and acid-base disturbances. 

ACIDOSIS 


In (untreated) diabetes acetyl CoA accumulates resulting in the formation and accumulation of ketone bodies in the liver, which are acetone, acetoacetate and D-β- hydroxybutyrate. The utilization of these substances by extra-hepatic tissues such as the heart, skeletal  muscle, kidney and the brain falls short of the rate of their accumulation in the blood, thus resulting in the net accumulation of these compounds in the blood. This lowers the pH of the blood causing the condition called acidosis. This type is called diabetic ketoacidosis.






Thus the above shows that untreated diabetes and starvation are conditions that promote gluconeogenesis, slows the TCA cycle by drawing off oxaloacetate, and enhances the conversion of acetyl CoA to ketone bodies  




WATER IS THE IDEAL SOLVENT IN LIVING THINGS


Water is a slightly skewed (distorted) tetrahedral molecule.  The tetrahedral structure is a three dimensional structure composed of an apex or centre and four base corners.  The angle between any two corners is usually 109.5 degrees.  However, the three dimensional structure of a molecule of water is an irregular tetrahedron with oxygen at its centre.  The two bonds formed with hydrogen atoms are directed toward two corners of the tetrahedron, while the unshared electrons on the two sp³ – hybridized orbitals of oxygen occupy the two remaining corners.  The angle between the two hydrogen atoms is slightly less than the normal tetrahedral angle.  It is 105 degrees (fig 1.1)

Fig 1.1:  The tetrahedral structure of water.

 Diagram

Key: i) the Bohr atomic structure of oxygen atom forming bonds with two hydrogen atoms.

Xx – are the two sp³ – hybridized orbital unshared electrons.

i.                   The skewed tetrahedron of water.

Water molecule is a dipole.

The dipolar nature of water arises from its skewed tetrahedral structure, which permits the unequal distribution of electrons (negative charge) in the structure between the oxygen atom and the two hydrogen atoms. The higher electronegativity of the oxygen atom in relation to the hydrogen atoms also contribute to the unequal charge distribution in the structure. The shared electrons spend more time on the side of the oxygen atom that is opposite the two hydrogen atoms. In other words, the electron density is higher on this side than on the side directly facing the two hydrogen atoms. Consequently, oxygen being electron-rich is slightly negatively charged, while the electron – poor hydrogen atoms have slight positive charge.

Besides water there are other biomolecules that form dipoles due to unequal charge distribution in their structure. Examples are ammonia, alcohols, phospholipids, amino acids and nucleic acids.  

1.4    HYDROGEN BONDS IN WATER MOLECULES

The formation of hydrogen bonds in water to give rise to a macro-molecular arrangement or structure is a consequence of the dipolar nature of water. The ability of water dipolar nature to self-associate in both the solid and the liquid states make them exhibit macromolecular structure.

In water molecules a hydrogen bond is formed when electrostatic interaction occur between a hydrogen atom of one water dipole and the unshared electron pair of the oxygen atom of another water dipole.

The hydrogen bonds formed in liquid water are relatively transient in comparison to those of ice. Hydrogen bonds in liquid water have a half life for association – dissociation of about 1µs.  In both liquid water and ice one water dipole is usually associated with four other water dipoles . However, strictly speaking, association of a central water dipole with four other water dipoles by hydrogen bonding is typical of ice (4.0), and to a lesser extent, of liquid water (3.5)

In comparison to covalent bonds such as the O-H bond of water, the hydrogen bond O---H is weak. It requires 110kcal/mole to break the O-H bond of a water molecule, but 4.5kcal/mole to bread the O---H bond between water molecules.

The dipolar nature of water and its ability to form hydrogen bonds explains why it can readily dissolve organic molecules. Examples of organic compounds that can form hydrogen bonds with water are amines, esters, aldehydes, ketones and proteins.

Macromolecules such as proteins that are stabilized by intermolecular hydrogen bonds may exchange such hydrogen bonds for hydrogen bonds to water thus enhancing its solubility. As a result soluble proteins appear coated with a layer of water. 

1.5              DISSOCIATION OF WATER MOLECULES

Water dissociates slightly.

For the sake of simplicity the dissociation of water is represented as follows

If one wishes to come closer to reality, one may depict the dissociation of water as an intermolecular proton (H⁺) transfer between two water molecules, forming a hydronium ion (H₃o⁺) and a hydroxide ion (OH^-).

But again the reality is something still a little bit different. The transferred proton is actually associated with a cluster of water molecules and exists in solution not even as H₃o⁺ but as H₅o₂⁺ or H₇o₃⁺.

Thus in the simple equilibrium shown above the H⁺ is actually a highly hydrated ion.  In the simple equilibrium dissociation of water, it cannot be known at any given time (t) whether an individual hydrogen or oxygen is present as an ion (H⁺ or OH^-) or as part of a water molecule. At one micro moment it is an ion, and at another it is part of a molecule. The situation is very fluid and dynamic.  In a situation like this, at best, the status of a hydrogen or oxygen, whether as an individual entity or as part of a water molecule, can only be expressed as a statistical probability.

Actually, the probability of a hydrogen atom in pure water existing as a hydrogen ion is about 1.8x10^-9 (meaning that it has 1.8x10^-9 chance in one of being an ion). And the probability of the hydrogen atom being part of a water molecule is almost unity (1) (meaning that there is an almost one chance in one of finding the hydrogen atom as  part of a water molecule

The ionization of water can be mathematically represented as follows:

Where K is the dissociation constant, and the bracketed terms are the molar concentrations in mol/l of hydrogen ion, hydroxyl ion, and undissociated water molecules.

The value for the dissociation constant of water can be derived as follows.

The number of moles of a molecule can be expressed mathematically as

n = g/M

Where n = no of moles, g = measured weight (g) and M = molecular mass. So, 1  mole of water weighs 18g, and consequently 1000g (I litre) of water contains 1000/18moles

Pure water therefore has a molar concentration of 55.56 mol/l.

[H₂₀] = 55.56 mol/l

Since the probability that a hydrogen in pure water will exist as an H⁺ ion is 1.8x 10^-9, the molar concentration of  H⁺ ions [H⁺] (or of [OH^-]) in pure water is calculated by multiplying the probability by the molar concentration of water.

Ie, 55.56 mol/l H₂₀ has a probability of I therefore for a probability of 1.8x10^-9

Since water dissociates very slightly, the molar concentration of undissociated water (55.56 mol/l) remains approximately constant. The incorporation of this constant into the dissociation constant (K) provides a new constant called the ion product of water (K_w).

(K)[H2O] = [H+] [OH-]


Kw = (K)[H2O] = [H+] [OH-]

Alternatively,

       

     

1.5              PH AND THE H⁺ CONCENTRATION

Sorensen defined pH in 1909 as the negative logarithm of hydrogen ion concentration 

pH = -log[H+]

For pure water at 25⁰c, PH = - log 10^-7 = - (-7) = 7.0.

Low pH values mean high concentration of H⁺ ions and high acidity; but high pH values mean low concentration of H⁺ ions and low acidity.

It follows therefore that compounds that furnish a lot of hydrogen ions in solution are strongly acidic, while those that furnish low levels of H⁺ ions are weakly acidic. The quantity of H⁺ ions that a compound can release or donate in a solution depends on the degree of dissociation of that compound. Strong acids, for example, dissociate completely. Most inorganic acids are strong acids. Examples are hydrochloric acid (Hcl) and sulphuric acid (H₂S04)

Most organic acids dissociate slightly or partially and are thus weak acids.

Examples are ethanoic acid and fatty acids

There are also strong bases and weak bases. Strong bases include potassium hydroxide (KOH) and sodium hydroxide (NaOH), but an example of a weak base is calcium hydroxide ca(OH)₂.

1.6              PH CALCULATIONS

Example I: what is the pH of a solution whose H⁺ ion concentration is 3.2x10^-4 mol/l

pH = -log[H+]

= -log (3.2 x 10-4) = -log3.2 + - log 10-4

= -log (3.2) - log (10-4)

= - 0.5 + 4.0 = 3.5

Example II: what is the pH of a solution whose OH^- concentration is 4.0 x 10^-4mol/l

POH = -log (OH-)

Kw = [H+][OH] = 10-14

log([H+][OH-]) = log10-14

log[H+] + log[OH-] = log10-14

 pH + pOH = 14

[OH] = 4.0 X 10-4

pOH = -log[OH-]

= -log(4.0) - log(10-4)

= -0.60 + 4.0

= 3.4

pH = 14 - pOH = 14 - 3.4 = 10.6

Example III: what is the pH value of 2.0x10^-6 mol/l KOH.

The OH^- arises from two sources – KOH and water 

i.          KOH --- K+ + OH-

ii.         H2O ---H+  + OH-

PH and POH are determined by the total [H⁺] concentration and [OH^-] concentration respectively. Hence both sources are considered.

Sine KOH is a strong base ionizing completely [OH^-]  = 2.0 x 10^-6 mol/l. [OH^-] from water = 1.0x10^-7 mol/l.

Total [OH^-] = 2.1 x 10^-6 mol/l

POH = -log(2.1 x 10-6)

POH = 5.7

PH = 14 - POH = 8.3

1.7              THE PHYSIOLOGIC SIGNIFICANCE OF WEAKLY ACIDIC FUNCTIONAL GROUPS

A lot of biochemical compounds possess functional groups that are weakly acidic or basic. Proteins possess carboxylic, amino and some other functional groups in their side chains that ionize in solution. Some metabolic intermediates possess the phosphoric acid group that ionizes strongly.

The dissociation behaviour of weakly acidic and weakly basic functional groups is very important to the structure and activity of metabolic intermediates. It is also very important to the separation and identification of biomolecules.

If the dissociation of a weak acid is represented as follows:

HA ---H+ + A-

HA (the protonated form of the acid) is the acid, and A^- (the unprotonated form) is the conjugate base. And similarly, A^- is the base and HA is the conjugate acid. For example, R-CH₂ – COOH is a weak acid and R-CH₂ – COO^- is the conjugate base.

                                                                                                               

                      

 GENERALLY,

PK = -log K

The table below shows the K and PK values for a monocarboxylic, a dicarboxylic and  a tricarboxylic acid

TABLE 1.0: DISSOCIATION CONSTANTS (K) AND PK VALUES FOR SOME CARBOXYLIC ACIDS

Acid	K	PK	Structure
Acetic	1.76x10-5	4.75	CH3COOH
Glutaric	1st: 4.58x10-5 2nd: 3.89x10-6	4.34 5.41	H          I H2N-C-COOH         (CH2)2           I          COOH
Citric	Ist: 8.40x10-4 2nd: 1.80x10-5 3rd: 4.00x10-6	3.08 4.74 5.40	H          I   H - C-COOH          I   H - C-COOH          I   H - C-COOH              I          H

THE LOWER THE PK VALUE OF A FUNCTIONAL GROUP THE HIGHER THE DEGREE OF DISSOCIATION. THEREFORE, THE STRONGER ACIDS HAVE LOWER PK VALUES

            A weak acid has a strong conjugate base, and similarly, a strong base is a weak acid. Therefore the relative strengths of bases are expressed in terms of the pK (pK_a) values of their conjugate acids. For example, the dissociation of the compound R-NH₃⁺ to R-NH₂ and H⁺ has a particular pK value.

           

           

 pK = -log K

           

          

  It is known that the pK value for this dissociation is the same as the pH value at which the concentration of the acid (R-NH₃⁺) equals that of the base (R-NH₂). That is, if (R-NH₂) = (R-NH₃⁺), then the equation above simplifies to:

            K = (H+)

            So that – log K = - log [H+]

            And thus, PK = PH.

            Therefore, stated in other words,

the pK of an acid group is that pH at which the protonated and unprotonated species are present at equal concentrations

The pk_a for an acid can be determined experimentally by titrating (adding) 0.5 equivalent of alkali with 1.0 equivalent of acid, and the resulting pH will be equal to the pk_a of the acid.

Consider a weakly dissociating acid (HA):

The concentration of the undissociated acid (HA) in equilibrium is very high compared to that of the ionic species. If half of HA in equilibrium is reacted with  equivalent base completely to furnish the conjugate base A^- of approximately the same concentration as that of the remaining HA in solution.

i)

     

ii) 

The pH of the equilibrium solution at this point is equal to the pK_a of dissociation of HA.

ASSIGNMENTS

1.                  What roles do water and pH play in the structural and functional stability of biomolecules.

2.                  Discuss the homeostatic regulation of water and pH in the body fluids of organisms.

3.                  What is acidosis?  Discuss.

4.                  Discuss water under the following headings:

a)      Structure of water

b)      Water as a dipole

5.                  What is the pH value of 2.0 x 10^-6 mol/L KOH

6.                  Derive the Henderson-Hasselbalch equation for a weakly ionizing acid (HA)

7.                  What is the pH of a solution whose OH^- concentration is 4.0 x 10^-4 mol/L

8.                  What is the pH of a solution whose H⁺ ion concentration is 3.2 x 10^-4 mol/L

9.                  Discuss Sorenson’s definition of pH.

10.              Explain why and how water forms hydrogen bonds.