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BIOLOGICAL VALUES & ESTIMATION OF ENZYMES & CARBOHYDRATES

ANALYTIAL CHEMISTRY

BIOLOGICAL VALUES
Biological value (BV) is a measure of the proportion of absorbed proteins from a food which becomes incorporated into the proteins of the organism's body. It summarises how readily the broken down protein can be used in protein synthesis in the cells of the organism. Proteins are the major source of nitrogen in food, unlike carbohydrates and fats. This method assumes protein is the only source of nitrogen and measures the proportion of this nitrogen absorbed by the body which is then excreted. The remainder must have been incorporated into the proteins of the organisms body. A ratio of nitrogen incorporated into the body over nitrogen absorbed gives a measure of protein 'usability' - the BV. Unlike some measures of protein usability, biological value does not take into account how readily the protein can be digested and absorbed (largely by the small intestine). This is reflected in the experimental methods used to determine BV. BV uses two similar scales: 1. The true percentage utilization (usually shown with a percent symbol). 2. The percentage utilization relative to a readily utilizable protein source, often egg (usually shown as unit less). These two values will be similar but not identical. The BV of a food varies greatly, and depends on a wide variety of factors. In particular the BV value of a food varies depending on its preparation and the recent diet of the organism. This makes reliable determination of BV difficult and of limited use fasting prior to testing is universally required in order to make the values reliable. BV is commonly used in nutrition science in many mammalian organisms, and is a relevant measure in humans. It is a popular guideline in bodybuilding in protein choice For accurate determination of BV: 1. The test organism must only consume the protein or mixture of proteins of interest (the test diet). 2. The test diet must contain no non-protein sources of nitrogen. 3. The test diet must be of suitable content and quantity to avoid use of the protein primarily as an energy source.

There are two scales on which BV is measured; percentage utilization and relative utilization. By convention percentage BV has a percent sign (%) suffix and relative BV has no unit. Percentage utilization Biological value is determined based on this formula. BV = ( Nr / Na ) * 100 Where: Na = nitrogen absorbed in proteins on the test diet Nr = nitrogen incorporated into the body on the test diet However direct measurement of Nr is essentially impossible. It will typically be measured indirectly from nitrogen excretion in urine. Faecal excretion of nitrogen must also be taken into account - this part of the ingested protein is not absorbed by the body and so not included in the calculation of BV. An estimate is used of the amount of the urinary and faecal nitrogen excretion not coming from ingested nitrogen. This may be done by substituting a proteinfree diet and observing nitrogen excretion in urine or faeces, but the accuracy of this method of estimation of the amount of nitrogen excretion not coming from ingested nitrogen on a protein-containing diet has been questioned. BV = ( ( Ni - Ne(f) - Ne(u) ) / (Ni - Ne(f)) ) * 100 Where: Ni = nitrogen intake in proteins on the test diet Ne(f) = (nitrogen excreted in faeces whilst on the test diet) - (nitrogen excreted in faeces not from ingested nitrogen) Ne(u) = (nitrogen excreted in urine whilst on the test diet) - (nitrogen excreted in urine not from ingested nitrogen) This can take any value from 0 to 100, though reported BV could be out of this range if the estimates of nitrogen excretion from non-ingested sources are inaccurate, such as could happen if the endogenous secretion changes with protein intake. A BV of 100% indicates complete utilization of a dietary protein, i.e. 100% of the protein ingested and absorbed is incorporated into proteins into the body. The value of 100% is an absolute maximum, no more than 100% of the protein ingested can be utilized (in the equation above Ne(u) and Ne(f) cannot go negative, setting 100% as the maximum BV).

Relative utilization Due to experimental limitations BV is often measured relative to an easily utilizable protein. Normally egg protein is assumed to be the most readily utilizable protein and given a BV of 100. For example: Two tests of BV are carried out on the same person; one with the test protein source and one with a reference protein (egg protein). relative BV = ( BV(test) / BV(egg) ) * 100 Where: BV(test) = percentage BV of the test diet for that individual BV(egg) = percentage BV of the reference (egg) diet for that individual This is not restricted to values of less than 100. The percentage BV of egg protein is only 93.7% which allows other proteins with true percentage BV between 93.7% and 100% to take a relative BV of over 100. For example, whey protein takes a relative BV of 104, while its percentage BV is under 100%. Conversion Providing it is known which protein measurements were made relative to it is simple to convert from relative BV to percentage BV: BV(relative) = ( BV(percentage) / BV(reference) ) * 100 BV(percentage) = ( BV(relative) / 100 ) * BV(reference) Where: BV(relative) = relative BV of the test protein BV(reference) = percentage BV of reference protein (typically egg: 93.7%). BV(percentage) = percentage BV of the test protein While this conversion is simple it is not strictly valid due to the differences between the experimental methods. It is, however, suitable for use as a guideline.

Factors which affect BV The determination of BV is carefully designed to accurately measure some aspects of protein usage whilst eliminating variation from other aspects. When using the test (or considering BV values) care must be taken to ensure the variable of interest is quantified by BV. Factors which affect BV can be grouped into properties of the protein source and properties of the species or individual consuming the protein. Properties of the protein source Three major properties of a protein source affect its BV:

Amino acid composition, and the limiting amino acid, which is usually lysine Preparation (cooking) Vitamin and mineral content

Amino acid composition is the principal effect. All proteins are made up of combinations of the 21 biological amino acids. Some of these can be synthesised or converted in the body, whereas others cannot and must be ingested in the diet. These are known as essential amino acids (EAAs), of which there are 9 in humans. The number of EAAs varies according to species (see below). EAAs missing from the diet prevent the synthesis of proteins that require them. If a protein source is missing critical EAAs, then its biological value will be low as the missing EAAs form a bottleneck in protein synthesis. For example, if a hypothetical muscle protein requires phenylalanine (an essential amino acid), then this must be provided in the diet for the muscle protein to be produced. If the current protein source in the diet has no phenylalanine in it the muscle protein cannot be produced, giving a low usability and BV of the protein source. In a related way if amino acids are missing from the protein source which are particularly slow or energy consuming to synthesise this can result in a low BV. Methods of food preparation also have an impact on availability of amino acids in a food source. Some of food preparation may damage or destroy some EAAs, reducing the BV of the protein source. Many vitamins and minerals are vital for the correct function of cells in the test organism. If critical minerals or vitamins are missing from the protein source this can result in a massively lowered BV. Many BV tests artificially add vitamins and minerals (for example in yeast extract) to prevent this.

Advantages and disadvantages BV provides a good measure of the usability of proteins in a diet and also plays a valuable role in detection of some metabolic diseases. BV is, however, a scientific variable determined under very strict and unnatural conditions. It is not a test designed to evaluate the usability of proteins whilst an organism is in everyday life indeed the BV of a diet will vary greatly depending on age, weight, health, sex, recent diet, current metabolism, etc. of the organism. In addition BV of the same food varies significantly species to species. Given these limitations BV is still relevant to everyday diet to some extent. No matter the individual or their conditions a protein source with high BV, such as egg, will always be more easily used than a protein source with low BV. Common foodstuffs and their values:

Whey Protein: 96 Whole Soy Bean: 96 Human milk: 95 Chicken egg: 94 Soybean milk: 91 Cow milk: 90 Cheese: 84 Rice: 83 Defatted soy flour: 81 Fish: 76 Beef: 74.3 Immature bean: 65 Full-fat soy flour: 64 Soybean curd: 64 Whole wheat: 64 White flour: 41

Common foodstuffs and their values:


Whey protein concentrate: 104 Whole egg: 100 Cow milk: 91 Beef: 80 Casein: 77 Soy: 74 Wheat gluten: 64

ENZYMES Enzymes are biological catalysts. They increase the rate of chemical reaction taking place within the cells without themselves suffering any overall change. The reactants of enzyme-catalysed reactions are termed as substrate & each enzyme is quite specific in character, acting on a particular substrate to produce a particular product or products.They are proteins.Many of them are Conjugated protein group. CLASSIFICATION OF ENZYMES Enzymes are divided into three types such as : A. Diastatic : Enzymes that bring about the conversion of starch to CO2 & H2O soluble sugars. These are further classified as : I. Oxido-Reductase : Catalyses Oxidation-Reduction reactions. II. Transferase : Transefers a Chemical group from one molecule to another. III. Hydroxylase : Catalyses hydroxylation reaction. IV. Lyases : Catalyses the reaction in which addition of group near double bond of molecule takes place. V. Synthetase : Catalyses Condensation of two molecules. VI. Isomerase : Catalyses intramolecular rearrangement.

B. Protolytic : Proteins that converts Protein & Peptides to less soluble Prptones & Amino Acid. C. Thromboplastics : Enzymes that prevents the Co-agulation Effects of variables on Enzyme Activity I. Temp. : As increases from 100C to 270C enzyme activity increases. II. pH : Enzyme activity increases with in pH range of 5 to 7. III. Concentration : Enzyme activity increases as concentration of enzyme increases.

S.I unit of Enzyme Activity is Katal. Amount of enzyme which will result in the conversion of 1 mol. Of substrate to product in 1 Second. Kinetics of Enzyme Catalysed reactin is determined by Michalis Menten equation.

Here, represents the maximum rate achieved by the system, at maximum (saturating) substrate concentrations. The Michaelis constant is the substrate concentration at which the reaction rate is half of . Biochemical reactions involving a single substrate are often assumed to follow MichaelisMenten kinetics, without regard to the model's underlying assumptions. Mechanism of Enzyme Activity : Binding of rectants to enzyme surface & one of the function of enzyme is to reach to the reactants to each other.This idea was suggested by Fischer as Lock & Key Hypothesis.The enzyme being lock & Substrate the key & although it explaind the idea of the specificity of enzyme.

Kishlowd proposed an alternative ie. Induced-Fit Hypothesis : This says that the binding of the substrate causes alternation in geometry of enzymes travelling in the correct orientation of appropriate groups both in the Sustrate & Enzyme.

Inhibition : Substance which decreases the rate of an enzyme catalysed reaction. Allostary : Allosteric enzymes show various activation & Inhibition effects which are competitive in nature & related to conformational chamges in structure of enzyme. Such allosteric enzymes are crucial in metabolic pathway & exerts control over whole sequence of allostaryy.The same allostary refers to the fact that Inhibition of enzymes is by substances which are not similar in shape to substrate.An allostaric enzymes in order to be catalytic active must be in the conformation which allow the binding of substrate. Its structure is normally flexible & may only stabilised by the building of other molecules.So,the building of an activator at activator site locks the enzyme in the form in which the substrate building site is available. The binding of an inhibitor at separate site inhibits causes distortion of both the substrate & binding site & activation site. Spectroscopic assay : Method : pipette the assay reaent (2.7 ml ) into clean dry glass cuvette & to attain room temp. Measure the absorbance of solution at 340nm. Add 0.2 ml sample & mix carefully. When absorbance shows either a slow but steady fall no fall, Initiate the reaction by adding 0.1ml o.5 ml Of alanine. Monitor the rection for atleast 5 mins. The rate of blank reaction & Total rection is determined graphically & reported as absorbance change/sec. Calculation : The enzyme activity in total/lit may be calculated from molar absorption coefficient for NADH at 340 nm. Monitoring of Technique : It is necessary to measure either the deflction of substrate or the accumulation of product.

Spectroscopic Method : It is very convenient & popular method for enzyme catalysed reaction due to its simplicity. Although only a small number of Substrate or products show absorption maxima in visible region of spectrum but a considerable number of them shows max. in U.v region.However in order to be able to measure the rate of substrate / product formation. It is necessary that these two components of reaction show different absorption characteristic.

CARBOHYDRATES
Polyhydroy Aldehydes or Ketones or such compounds which on hydrolysis gives Polyhydroxy aldehyde or ketones.

Souces : In plants produced by Photosynthesis. Significance : Animals eat the carbohydrats in the form of Starch or Cellulose / Sugars. They store it in liver in the form of glycogen which can be again broken down to glucose whenever required. Classification Of Carbohydrates : A. Monosaccharides : Basic unit of Carbohydrate. Divided in two parts : I. Aldoses : they are aldo trioses ,Aldo tetrase ,Aldo pentase ,Aldo hexase. II. Ketoses : they are Ketotriose,Tetrose,Pentose,Hexose.

B. Oligosaccharides : can be further hydrolysed.they are as follows : I. Diasaccharides II. Trisaccharides C. Pollysacchrides : Non-Crystaline.

ESTIMATION OF CARBOHYDRATES Molisch Test : Shows positive test for: All carbohydrates. Monosaccharides give a rapid positive test. Disaccharides and polysaccharides react slower. Reactions: The test reagent dehydrates pentoses to form furfural (top reaction) and dehydrates hexoses to form 5-hydroxymethyl furfural (bottom reaction). The furfurals further react with -naphthol present in the test reagent to produce a purple product (reaction not shown).

How to perform the test: Two ml of a sample solution is placed in a test tube. Two drops of the Molisch reagent (a solution of -napthol in 95% ethanol) is added. The solution is then poured slowly into a tube containing two ml of concentrated sulfuric acid so that two layers form.

A positive test is indicated by: The formation of a purple product at the interface of the two layers.

Benedicts Test Shows positive test for: Reducing sugars Reactions: Reducing sugars are oxidized by the copper ion in solution to form a carboxylic acid and a reddish precipitate of copper (I) oxide.

How to perform the test: One ml of a sample solution is placed in a test tube. Two ml of Benedict's reagent (a solution of sodium citrate and sodium carbonate mixed with a solution of copper sulfate) is added. The solution is then heated in a boiling water bath for three minutes. A positive test is indicated by: The formation of a reddish precipitate within three minutes.

a negative test (left) and a positive test (right)

Barfored Test Shows positive test for: Reducing monosaccharides Reactions: Reducing monosaccharides are oxidized by the copper ion in solution to form a carboxylic acid and a reddish precipitate of copper (I) oxide within three minutes. Reducing disaccharides undergo the same reaction, but do so at a slower rate.

How to perform the test: One ml of a sample solution is placed in a test tube. Three ml of Barfoed's reagent (a solution of cupric acetate and acetic acid) is added. The solution is then heated in a boiling water bath for three minutes. A positive test is indicated by: The formation of a reddish precipitate within three minutes.

a negative test (left) and a positive test (right)