eChapters

For the first time, isotope dilution analysis was introduced by Hevesy and Hafer in 1934, but it was not until 1940, the usefulness of isotope dilution technique was revived by Rittenberg and Foster (1940). There are three general types of isotope dilution methods. These are (a) direct (b) inverse and (c) double isotope dilution. In general isotope dilution methods may be used for the following type of studies.

    (a)    In isolation purification and identification of unknown intermediate in a chain of metabolic reactions.

    (b)     To obtain evidence of synthesis (incorporation), and precursor-product relationships between known compounds.

    (c)    As an analytical tool in the assay of known compounds. In isotope dilution techniques, the labelled intermediate, which is being diluted by unlabelled carrier, is a known compound.

We may study the nutrient metabolism in a better way as explained in the following summarized form in Table 1.

Ionization Chambers for Gases

Generally these type of instruments are known as dose-rate meter (e.g., the so called “cutie-pie”). Radiation intensity (i.e., a constant stream of particles or photons) gives rise to a continuous series of pulses, and if these are allowed to merge, they form a weak electric current, which may be amplified and registered by an electronic circuit. The final scale reading will then be a measure of the energy dissipated in the following chamber per unit of time by the ionizing particles or photons. In gas-flow counters, the radioactive samples are placed inside the detector which will be transfused by a gas at atmospheric pressure. This instrument counts particles of low energy, such as the b particles from 14C, effectively (Window-less counting).

Activation : The process of causing a substance to become artificially radioactive by subjecting it to bombardment by neutrons or other particles.

Activation Analysis : Any analytical procedure permitting the detection and measurement of trace quantities of elements following their exposure to a flux of neutrons.

Activity :  The strength of a radioactive source. In absolute units, it relates to the number of radioactive atoms decaying per unit of time. It is also a synonym for radioactivity. Absolute activity is usually expressed in curies or millicuries.

Alpha Particle : A particle which is identical to the helium nucleus, consisting of two protons and two neutrons. It carries a positive charge of 2.

Alpha Rays: A stream of helium nuclei. The helium nucleus has a mass number of 4 and an atomic number of 2. If consists of two protons and two neutrons.

The term “laboratory” refers to the room in the building specifically equipped for the chemical manipulation of radioisotope. The word “shall” is used to indicate procedure which are mandatory.

    1.    No unnessary materials are to be brought into the laboratory. Eating drinking, smoking and the using cosmetic in the laboratory are specifically forbidden.

    2.    Pipetting or the performance of any similar operation by mouth suction is prohibited.

    3.    Laboratory protective clothing (gloves, smocks, etc.) shall be left in the laboratory; gloves shall be removed before using the counting equipment.

    4.    Before leaving the laboratory, the hands shall be washed first, then checked with a beta-gamma survey meter. Contamination remaining after thorough washing shall be reported to the instructor.

    5.    If, in the course of work, personal contamination is suspected, a survey with a suitable instrument shall be made immediately, to be followed by the required cleansing. Routine precautionary surveys should be made at intervals.

    6.    No person shall work with active materials while having abrasions in the skin on the hands without using rubber gloves.

The term “laboratory” refers to the room in the building specifically equipped for the chemical manipulation of radioisotope. The word “shall” is used to indicate procedure which are mandatory.

    1.    No unnessary materials are to be brought into the laboratory. Eating drinking, smoking and the using cosmetic in the laboratory are specifically forbidden.

    2.    Pipetting or the performance of any similar operation by mouth suction is prohibited.

    3.    Laboratory protective clothing (gloves, smocks, etc.) shall be left in the laboratory; gloves shall be removed before using the counting equipment.

    4.    Before leaving the laboratory, the hands shall be washed first, then checked with a beta-gamma survey meter. Contamination remaining after thorough washing shall be reported to the instructor.

    5.    If, in the course of work, personal contamination is suspected, a survey with a suitable instrument shall be made immediately, to be followed by the required cleansing. Routine precautionary surveys should be made at intervals.

    6.    No person shall work with active materials while having abrasions in the skin on the hands without using rubber gloves.

(Method of Froesch and Renold, 1956; Froesch et al., 1957)
Enzymatic with Glucose-oxidase / Peroxidase

Introduction

Glucose oxidase enzyme specific for the oxidation of glucose, was discovered in a fungus preparation by Müller in 1928. This enzyme is present in bacteria and in moulds such as (Pencillium notatum), which promotes the oxidation of glucose (b-ring form) to gluconic acid with the production of an equivalent amount of hydrogen peroxide. Normal values for whole blood glucose oxidase are 60 to 90 mg/100 ml. Results by the enzyme methods are lower than by the best “true  sugar” reduction methods largely because of their specificity, but inhibition of glucose oxidase by uric acid may be a contributory factor. In suspected cases of diabetes, values below 120 mg per 100 ml call for further investigation, for example, by glucose tolerance test. Due to the presence of glycolytic enzymes in red cells, blood must be collected into an antiglycolytic/anti-coagulant preservative, if there is to be a delay of more than 1 to 2 h. in processing or estimation. A fluoride/oxalate mixture, containing 2 mg of fluoride per ml of blood, is suitable and the glucose content will then remain unchanged for upto 3 days at 4ºC.

Blood Cholesterol

(Colorimetric Method of Lieberman, 1885: Burchard 1889 Modified by Watson, 1960).

Introduction

According to Liebermann-Burchard reaction, treatment of chloesterol with acetic anhydride and concentrated sulphuric acid in a water-free environment, polymerized unsaturated hydrocarbon with an intense blue-green colour are produced. The values for cholesterol esters may be calculated by deducting free cholesterol from total cholesterol.

Introduction

(Titrimetric Method of  Cherry and crandall, 1932 Modified by Tietz and Fiereck, 1966).

In 1932, Cherry and Crandall employed olive oil as the substrate for pancreatic and serum lipase (LPS. Triacylglycerol lipase) determinations. In subsequent years this method was improved (Tietz et al., 1959; Tietz and Fereck, 1966: Tietz. 1972), reducing the original 24 h, period to 6 h and finally to 3 h.

Principle
This procedure is based on the hydrolysis of triglycerides in olive oil into fatty acids, diglycerides and to some small extent into monoglycerides and glycerol.

(Method of Bloor, 1928 Modified by Folch et al., 1957)
Gravimetric Method — Principle

Total lipids in plasma consist of neutral fats, phospholipids, chloesterol esters and free cholesterol. Minor components include non-esterified fatty acids (NEFA), phosphatides and sphingo myelin. Total lipids in serum or plasma are extracted using ethyl alcohol and petroleum  ether, separated and weighed after drying at 105ºC.

Procedure
Pipette 5 ml serum in a separating funnel, add 5 ml ethyl alcohol and 12.5 ml pertroleum ether. Shake the contents of funnel two times for 3 min. atleast. Keep the contents of funnel as such for one hour. Transfer the contents of funnel in the moisture cup, dry and weigh. Calculate the result by difference method using the following formula.

(Method of Joseph Mac Gee, 1959)
Principle

A simple and rapid enzymatic method for the quantitative estimation of total cis-methylene- interrupted polyenoic acids has been devised. Linoleic, linolenic and arachidonic acids, the more common acids of this group were used to calibrate the method. The potassium salts of the fatty acids are oxidised by atmospheric oxygen in the presence of the enzyme lipo-oxidase, and the absorption of the conjugated diene hydroperoxide is measured at 234 mµ. As little as 5 µg of linoleic acid can be quantitatively measured with good accuracy and precision. The total content of polyunsaturated fatty acids containing the cismethylene-interrupted diene structure of fats, oils, hydrogenated oils, fatty acids, esters blood plasma, microorganisms and plant seeds has been measured directly by this method.

Principle
(Method of Patterson 1963)

The albumin bound long-chain fatty acids of plasma are usually referred to as unesterified or non-esterified fatty acids (NEFA) to distinguish them from the long-chain fatty acids present in the esterified form in neutral fats and phospholipid. In the case of ruminant plasma, it is also important to distinguish these “free fatty acids” from the small molecular weight steam volatile fatty acids of dietary origin which are found in the free state as the carboxylate ion.

Basically, the method of Dole (1959a) has been used to estimate plasma NEFA employed by Annison (1960), which is to evaporate the heptane extract of plasma to dryness before titration. This is conveniently done in a stream of nitrogen on a warm hot plate rather than by evaporation in vacua. In place of the 0.05 M- phosphate buffer (pH 6.0) of Annison or the 0.02 N-H₂SO₄ used by Dole (1956a), in the present method 0.02 N-HCI is used as an extraction medium. This is preferred because owing to its volatility, it is less likely to cause serious accidental contamination of the supernatant heptane layer.

Isolation of RNA from Rat Liver
(Method of Schmidt and Thannhauser, 1945)
Principle

Most of the RNA (ribouncleic acid) of cells is found in the nucleoli and microsomes.These fractions of cells may be isolated by differential centrifugation of cellular homogenates and thus provide an enriched starting material for RNA extraction. Such methods require high speed  centrifugation. An alternate procedure used in the present method is to disrupt the whole cells by homogenization and isolate the nucleic acid of entire homogenate. This procedure consists of the following sequential steps.

Two procedure are described for the determination of free gosssypol . The first for normal meals and the second for meals which have been chemically treated and so contain dianilinogossypol.

Reagents
 
    1.    Aqueous acetone:  7 parts acetone, 3 parts distilled water (V/V).
    2.    Aqueous acetone-aniline solution: To 700 ml acetone and 300 ml distilled water, add 0.5 ml redistilled aniline. Prepare solution daily.
    3.    Aqueous isopropyl alcohol solution: 8 parts isopropyl alcohol. 2 parts distilled water (V/V).
    4.    Aniline Distill reagent grade aniline over a small quantity of zinc, dust discarding the first and last 10 percent of the distillate. Store refrigerated in a brown glass stoppered bottle. Solution is stable for several months.

The method described here gives an approximate thioglucoside and isothiocyanates contents in rapeseed meal.

Reagents and Apparatus
    1.    Barium chloride (5% solution)
    2.    Volumetric flask, 600 ml
    3.    Steam bath

Method

To 10 g meal (defatted by soxhlet extraction) add 250 ml distilled water, hydrolyse at 54ºC for one hour and then boil for two hours, keeping volume constant. Filter, retaining filtrate and wash residue three times with 50 ml hot water. Add washings to initial filtrate and make up volume to 600 ml. Precipitate barium sulphate by heating and adding excess barium chloride solution. Leave on a steam bath for a few hours and then filter. Ash in muffle furnace and then weigh precipitate.

Introduction

Aflatoxins are a group of highly subsituted coumarine, of which aflatoxin B1 and G1 are  most  highly hepatotoxic (toxic to liver) to animals. The toxins are produced by some strains of Aspergillus flavus and other species of Aspergillus which develop in many food stuffs, particularly groundnuts. cottonseed, and their cake and flour. Protein concentrates meant for babies as well as adult are now a day  made from groundnut flour and the cakes are used as feed for poultry and livestock. It has been found that even fodgrains such as maize, rice and wheat are attacked by the fungus producing the toxins.

Nitrates and nitrites are known to have injurious effects on animals when excess amounts are ingested. Nitrites are much more toxic but nitrates are reduced to nitrites by bacterial action in the digestive tract

Nitrites (NO₂) are rarely found in plants  in appreciable amounts except under very unusual conditions such as severe disease infections; therefore, the results are usually reported in terms of nitrates (NO₃). The usual method is to determine the total nitrate and nitrite in terms of nitrate. However, the method can also be used to determine the nitrite content of a feed separately if a nitrite problem is suspected.

Principle

Cyanide is distilled from chloroform and water soultion into KOH solution, forming potassium cyanide. KCN is titrated with silver nitrate; 2 KCN + AgNO₃ = KCN. AgCN + KNO₃. An excess of AgNO₃ produces insoluble AgCN, which is the end point of titration.

Introduction

Substances found in many plants are generally related to one of the phenols, pyrogallol or catechol. Tannins are solids, usually extracted by hot water insoluble in ether, chloroform, carbon disulphide, benzene; soluble in alcohol ether mixture, alcohol, water and in ethyl acetate, possessing a bitter astringent taste.

Tannic acid is Pentadigalloyl glucose, on hydrolysis yields digallic acid and glucose. Tannins are water soluble glucosides of polydepsides, polymeric ester of gallic acid. It is hydrolysed enzymatically and some efforts have, therefore, been made to preserve tannic, if it is to exert its activity in the duodenum. On the basis of physical or chemical principles which they employ, the methods of tannin analysis  can be grouped as follows.

Method I
Reagents
    1.    20% sodium carbonate.
    2.    Conc. hydrochloric acid.
    3.    4% sodium hydroxide
    4.    10% calcium chloride
    5.    Ammonium hydroxide
    6.    Potassium permanganate (0.05 N)

Procedure
Take 2-5 g of powdered sample in a beaker without spout, and add 40 ml of water and boil for half an hour. In case of faeces, add 40 ml of 4 percent NaOH instead of 40 ml of water to form silicates. Now boil, for half an hour with cold water flask over the beaker, then add 10 ml of 20 percent sodium carbonate solution and boil for  another half an hour. During these two cooking processes, the following changes take place.

Principle

Dried milled samples are extracted successively with diethyl ether, 0.5 percent ammonium oxalate solution, NH₂SO₄ and 72 percent sulphuric acid. Soluble carbohydrate in the ammonium oxalate extract determined with anthrone and hydrolysed pentosans and hexosans in the combined N H₂SO₄. Seventy two percent sulphuric acid extract are determined with aniline acetate and chromotropic acid respectively. The method gives a maximum variation of 4 percent from the mean of duplicates with each carbohydrate component. The residual material is ashed to obtain crude lignin and a nitrogen correction applied.

Soluble carbohydrate - this group, which includes sugars, oligosaccharides, and the fructosans of grasses, provides a source of nutrients  readily available to livestock.

Principle

Dried milled samples are extracted successively with diethyl ether, 0.5 percent ammonium oxalate solution, NH₂SO₄ and 72 percent sulphuric acid. Soluble carbohydrate in the ammonium oxalate extract determined with anthrone and hydrolysed pentosans and hexosans in the combined N H₂SO₄. Seventy two percent sulphuric acid extract are determined with aniline acetate and chromotropic acid respectively. The method gives a maximum variation of 4 percent from the mean of duplicates with each carbohydrate component. The residual material is ashed to obtain crude lignin and a nitrogen correction applied.

Soluble carbohydrate - this group, which includes sugars, oligosaccharides, and the fructosans of grasses, provides a source of nutrients  readily available to livestock.

Estimation of Cellulose in Feeds and Fodders
Principle

The feed well ground is digested with acetic acid which dissolves all other constituents like protein, disaccharides, fatty acids, etc., and cellulose and mineral contents are unaffected. The residue is made acid and alcohol free by washings. Alcohol removes water, benzene removes pigments and fat and at the end the sample is dried and ashed. The difference between the dried weight before ashing and weight after ashing gives cellulose content of the feed sample.

Principle

The residue from the cell walls determination is extracted with boiling 8 percent sulphuric acid for one hour to hydrolyse the hemicellulose without dissolving cellulose, lignin or acid insoluble ash.

Equipment
    1.    Refluxing apparatus.
    2.     Filtering apparatus: same as described for cell wall determination
    3.    Filtering crucibles: same as described for cell wall determination.

Reagents

    1.    Sulphuric acid (8%) : Standardize Reagent grade sulphuric acid to specific gravity of 1.052 at 20ºC.
    2.    Bromocresol green indicator solution: Same as used for lignin determination.

Principle

The residue remaining from the hemicellulose determination is digested with 72 percent sulphuric acid at room temperature. The lignin and acid insoluble  ash are retained in the filter crucible while the digested carbohydrate passes through. The lignin is separated from acid insoluble ash by ashing the residue.

Equipment

    1.    Acid digestion apparatus. A glass tray about 18 cm x 30 cm, 4 cm deep will hold twelve, 100 ml Griffen beakers. Another tray of the same size can be inverted over the beaker.
    2.    Filter crucibles. Same as described for cell wall determination.
    3.    Filtering apparatus. Same as described for cell wall determination.

Limitation of Weende Method

The partition of carbohydrate into fiber (CF) and nitrogen free extract (NFE) is presumed to represent a separation of less digestible starch and sugars. However, in 20 to 30 percent feeds listed by Morrison, the NFE is less digestible than the CF. This comes about for several reasons, the most important of which is the CF method (successive boiling with dilute sulphuric acid and sodium hydroxide) does not recover all the fiber and large portions of fibrous constituents  are extracted into the NFE. The most  important of these fractions are lignin and hemicellulose are dissolved by both acid and alkali. The basic error of the NFE concept is the assumption that if constituents are soluble they are digestible. Lignin, the rigid component of wood, not only is indigestible but lowers the digestibility of substances with which it is associated.

Another portion of the indigestible part of NFE arises from an artifact in the calculation of faecal NFE, where it is presumed that faecal nitrogen is protein (N x 6.25). Actually, 80-90 percent faecal nitrogen is NPN and is composed of bacterial residue in which the ratio of organic matter to nitrogen is about 1:4. Thus, part of the faecal NFE arises by an underestimation of the organic matter associated with faecal nitrogen.

Introduction

As per statement of Dr. Henneberg of Weende Experiment Station (Göttingen, Germany) the carbohydrates are divided into two groups; crude fiber and nitrogen free extract. The nitrogen free extract, which  comprises the sugars, starch and a large part of the material classed as hemicellulose, is determined by difference. It is represented by the figure obtained when the sum of the water, ash, protein, fat and crude fiber of a feed is subtracted from 100. Since the figure is determined by difference instead of directly, it includes the cumulative errors of the other determinations and thus is not an exact value.

Introduction

Wet ashing is suitable for the determination of Ca, Cu, Fe, Mg, Mn, K, Na, Se and Zn in biological samples and may be applicable to the determination of other elements as well.

The organic matter of the sample is oxidized with concentrated nitric acid and perchloric acids. The acids are partially removed by volatilization and the solube mineral constituents remain dissolved in nitric acid. Any silica present is dehydrated and made insoluble.

Introduction

Wet ashing is suitable for the determination of Ca, Cu, Fe, Mg, Mn, K, Na, Se and Zn in biological samples and may be applicable to the determination of other elements as well.

The organic matter of the sample is oxidized with concentrated nitric acid and perchloric acids. The acids are partially removed by volatilization and the solube mineral constituents remain dissolved in nitric acid. Any silica present is dehydrated and made insoluble.

Definition
The residue of incineration at 550-600ºC is the crude ash.

Reagent
Dilute hydrochloric acid -approximately 5 N prepared from concentrated hydrochloric acid. Dissolve 445 ml conc. hydrochloric acid (about 36 percent HCl) in one litre distilled water. 

Procedure for Total Ash Estimation
Take a vitreosil crucible in oven at 100ºC for six hours, remove and keep it in a desiccator and weigh up to constant weight. About 5-10 g of sample is taken in the weighted vitreosil crucible. The material in the crucible is charred on a low flame and the crucible is then kept in muffle furnace and temperature is allowed to raise to 600ºC and kept constant for two hours. It is removed on cooling and kept in a desiccator  and weight of crucible is taken.

1. Ammonia Nitrogen by Distillation Method
Method I (For Animal Feeds)
Reagents
    1.    Mangnesium oxide (carbonate free)
    2.    Standard sodium hydroxide 0.25 N
    3.    Standard sulphuric acid 0.5 N
    4.    Methyl red indicator

Weigh accurately 2 to 4 g  of the prepared sample. Shake it with water and filter. Wash the residue thoroughly with water. Transfer the filtrate to the distillation flask and dilute to about 200 ml with water. Add about 5 g of the magnesium oxide, Connect the flask to the condenser by means of the connecting bulb tube and distill about 100 ml of liquid into the receiver containing standard sulphuric acid and methyl red indicator solution, Titrate the contents of the receiver with the standard sodium hydroxide solution. Carry out a blank determination using all reagents in the same quantities but without the material to be tested.

True protein nitrogen is also termed a Albuminoid nitrogen in animal feeds and fodders and human foods. If material (Such as seeds, seed residue or oil cake) is rich in alkaline phosphates, 1-2 ml of 10 percent solution of potash alum is added to decompose them. If this is not done Cu3 (PO4) and free alkali may be formed and the protein-copper precipitate may partially dissolve in the alkaline liquid.

Principle

The nitrogen of protein and other compounds is transformed into ammonium sulphate by acid digestion with boiling sulphuric acid. the acid digest is cooled, dilute with water, and made strongly basic with sodium hydroxide. The released ammonia is distilled into a boric acid solution or standard sulphuric acid solution. When boric acid is used to collect ammonia then is the titrated with standard sulphuric acid or standard hydrochloric acid. When standard sulphuric acid is used to collect ammonia then it is titrated with standard sodium hydroxide solution.

Apparatus and Reagents

    1.    Soxhlet extraction asembly. This consists of 3 parts (a) condenser at the top, (b) the soxhlet or extractor in the middle, and (c) the receiver flask at the bottom. The 3 parts are assembled by means of their grounds glass interchangeable joints (Fig. 1).

    2.    Extraction thimble.

    3.    Petroleum ether (B.P. 40ºC to 60ºC)

    4.    Constant temperatue bath or six heater hot plate with temperature controlling device.

Calorimetry

The process of measuring  the amount of heat generated by the combustion of a substance is known as calorimetry. The first successful calorimeter using oxygen under pressure for the combustion of substance in a closed vessel was devised by Berthelot in 1881. Subsequent development by Mahler in 1992, Atwater in 1899, and Parr in 1912, served to improve and to reduce the cost of the original Berthelot apparatus by variations in mechanical details.

The dry matter content of feed samples and other materials is expressed on three aspects as fed, partially dry, and dry.

As fed refers to the feed as it is consumed by the animal, the term as collected is used for materials which are not usually fed to the animal, that is, urine, faeces, etc. If the analysis on a sample is affected by partial drying the analyses are made on the as fed or as collected sample. Similar terms: Air dry, that is hay: as received: fresh; green and wet.

Partially dry, reports to a sample of “as fed” or “as collected” material that has been dried in an oven (usually with forced air) at a temperature usually about 60ºC of freeze-dried and has been equilibrated with the air; the sample after these processes would usually contain more than 88 percent dry matter (12 percent moisture); some materials are prepared in the way so that they may be sampled, chemically analysed and stored. This analysis is referred to as “partial dry matter” percent of “as fed” or “as collected” sample. The partially dry sample must be analysed for dry matter (determined in an oven at 105ºC) to correct subsequent chemical analyses of the samples to a “dry” basis. This analysis is referred to as “dry , percent of partial dry sample similar term - “Air dry” is also used.

Dry matter is generally defined as the constant  weight a sample  attains when heated at 100ºC (occasionally 150ºC). This definition , suitable for inert materials such as sand, has three main lacunae when applied to biological materials.

    1.    In these materials, water is present in various states ranging from extraneous moisture  as rain or dew, through water present in cell sap, water present in various physico-chemical and chemical combinations.

    2.    Biological materials contain active respiratory enzymic systems which continue to function during the early stages of drying; in fact, as the material heats up, activity will be enhanced until it is stopped either  by denaturation of the enzymes or by desiccation. Besides altering the chemical composition of the material, such activity will  lead to loss of dry matter.

    3.    Most biological materials contain organic  compounds which are volatile at 100ºC and which are, therefore, lost on drying. This leads to an under-estimation of dry matter content, for example, in the case of faeces, silage and molasses.

A biological sample received in the laboratory should be processed as below:

Fresh Green Sample: During metabolism trial period it is always advisable to take a sample of fresh green fodder daily so as to  avoid chemical composition variation from place-to-place in a field. Take about two and half kilo sample from the bulk, cut the whole plant including the stem and stalks with a pair of scissors into small pieces and mix thoroughly. Weigh about 100g accurately in the aluminium tray 23156cm approximately for estimation of moisture. It is always recommended to weigh the sample immediately in the laboratory so as to avoid the losses of moisture.

Chemical changes occur during oven-drying because of the direct effect of high temperature, or of enzymic or bacterial changes during the early stages of drying, or of loss of volatile consitituents. Feeds containing molasses should be ground in a mortar.

Dried materials should be ground to pass 1 mm sieve. Samples expected to moisture during the treatments are usually predried. If wet samples are used directly in the assay, sufficient large quantity has to weighted out. In the first stage, roughages about 250-1000g are used, somewhat dependent upon homogeneity and moisture content of the wet material. These samples are weighted into trays and dried in draft oven at 60-70ºC. The dry samples are then again weighed, determined the moisture content. The trays are then left to equilibrate in air at room temperature for two days. before a second reweighing of the trays with samples. Then the sample is milled and filled into airtight containers and labelled as dry samples.

General Views about Weende’s System of Analysis

A team of scientists under the leardship of Henneberg and Stohmann of (University of Göttingen, Germany) developed proximate principles analysis system in the sixties of last century at Weende’s Experimental Station (Germany), located at the University of Göttingen. Main components of different fractions in the proximate analysis of foods are moisture, ash, crude protein, ether extract, crude fiber, and nitrogen-free-extract. These workers emphasized that carbohydrates could be grouped into: (1) the starches and the sugars, and (2) coarse fibrous fractions. Later on an insoluble residue after boiling the fat free food sample first with dilute acid and then with dilute alkali was obtained based on the acid stomach digestion and the subsequent alkaline intestinal digestion of consumed food. At the end of digestion insoluble organic residue was denoted as crude fiber.

The Metric System

At present metric system is followed to calculate final results in analysis work. This system is quite simple in nature as well as in principle. The units of the metric system are: (1) the  metre (linear measure), (2) the litre (measure of capacity), and (3) the gram (measure of mass.)

One litre is the volume of pure water at 4ºC and 760 mm pressure which weigh 1 kilogram (kg). One litre equals 1000.027 cubic centimetres (Cm3) for most work, the units “millilitre”, “cubic centimetre” and used interchangeably.

At present there are three different temperature scales in common use. These are: the Fahrenheit (F), used chiefly in English-speaking countries. the centigrade (C), used universally in scientific work and the Absolute (T) or Kelvin (K) scale, differing from the centigrade only in the position of the zero point, its divisions being of the same size. its zero point is -273ºC. The kelvin scale is used especially in dealing with very high or very low temperature and in computations involving the laws of gases. The following formulae can be used for converting one scale to another:

Normal Solution in Analytical Chemistry

A normal solution is that one that contains one equivalent weight expressed  in grams (one gram equivalent weight) to be dissolved substance per litre of solution, or one gram-milliequivalent weight per millilitre.

In the case of acid a 1N solution contains 1.008 gram of replaceable hydrogen per litre of solution. A 1N solution of a base is one that contains 17.008 of hydroxyl per litre. A 1N solution of a precipitating agent contains a weight of precipitating ion equivalent to 1.008 hydrogen. The quantity of pure reagent necessary per litre of 1N solution of a precipitating solution is calculated by dividing the gram molecular weight by the valency of the precipitating ion. In case of normal solution of oxidising and reducing agents, the amount is calculated by dividing the gram molecular weight by the total valency change in the ion concerned.

Appendix - 1

Differential Diagnosis of Poisons based on Clinical Signs

Chemical warfare involves using the toxic properties of chemical substances to kill, injure or incapacitate an enemy.

Chemical warfare is different from the use of conventional weapons or nuclear weapons because the destructive effects of chemical weapons are not primarily due to any explosive force. The offensive use of living organisms (such as anthrax) is considered to be biological warfare rather than chemical warfare; the use of nonliving toxic products produced by living organisms (e.g., toxins such as botulinum toxin, ricin, or saxitoxin) is considered chemical warfare under the provisions of the Chemical Weapons Convention. Under this Convention, any toxic chemical, regardless of its origin, is considered as a chemical weapon unless it is used for purposes that are not prohibited (an important legal definition, known as the General Purpose Criterion)

Definition 

Radiation poisoning, also called “radiation sickness” or a “creeping dose” is a form of damage to organ tissue due to excessive exposure to  radiation. The term is generally used to refer to acute problems caused by a large dosage of radiation in a short period, though this also has occurred with long term exposure to low level radiation. Many of the symptoms of radiation poisoning occur as ionizing radiation interferes with cell division.

The clinical name for “radiation sickness” is acute radiation syndrome . A short exposure can result in acute radiation syndrome; chronic radiation syndrome requires a prolonged high level of exposure. A chronic radiation syndrome does exist but is very uncommon; 

Pollution is the introduction of contaminants into an environment that causes instability, disorder, harm or discomfort to the physical systems or living organisms they are in.  
Pollution can take the form of chemical substances, or energy, such as noise, heat, or light energy.

Pollutants, the elements of pollution, can be foreign substances or energies, or naturally occurring; when naturally occurring, they are considered contaminants when they exceed natural levels. Sometimes the term pollution is extended to include any substance when it occurs at such unnaturally high concentration within a system that it endangers the stability of that system. For example, water is innocuous and essential for life, and yet at very high concentration, it could be considered a pollutant.

Introduction 

A toxin (Greek: ôïîéêüí, toxikon, lit. (poison) for use on arrows) is a poisonous substance produced by living cells or organisms that is active at very low concentrations. Toxins can be small molecules, peptides, or proteins and are capable of causing disease on contact or absorption with body tissues by interacting with biological macromolecules such as enzymes or cellular receptors. Toxins vary greatly in their severity, ranging from usually minor and acute (as in a bee sting) to almost immediately deadly (as in botulinum toxin)

A toxin is simply a poison that can affect the body by internal or external means. A toxin can be a chemical which occurs naturally or in synthetic form. More than 120,000 human-made chemicals have been introduced into the environment, in one form or another, and this number continues to grow each year at a phenomenal rate. At the same time, microbial toxins, being influenced by the vast numbers of chemicals, are mutating beyond belief.

Introduction

The word mycotoxin is derived from myco, meaning mold and toxin, a poison. A great number of mold metabolites have been identified as mycotoxins. Thus mycotoxins are secondary fungal metabolites (i.e., metabolites not essential to the normal growth and reproduction of the fungus) which cause pathologic, physiologic, and/or biochemical lesions in other species (may include animals, plants, other microbes, etc.).

Mycotoxicosis - Poisoning of a biological system by a mycotoxin.

Mycotoxins  are  defined as toxic metabolites released by moulds under certain conditions conducive for their growth. Acute or chronic toxicoses can result from exposure to feed or bedding contaminated with toxins that may be produced during growth of various saprophytic or phytopathogenic fungi or molds on cereals, hay, straw, pastures, or any other fodder. A few principles characterize mycotoxic diseases:

Introduction 

In the thirst of modernization and industrialization man has contributed pollution to the life and ecology of plants, animals and microbes. Increased demand for food and fiber has lead to the chemicalization of agriculture we have reached to such a stage that modern agriculture is dependent on high yielding varieties, which can only be grown under the influence of fertilizers and pesticides. Pesticides are the man made chemicals which are being used to produce enough cheap food. In India, 90,000 MT of technical grade pesticides are used annually to control pests and plant diseases. The pesticides are classified as insecticides, fungicides, weedicides, herbicides, nematodicides and rodenticides; of which insecticides constitutes 77% of the total pesticides used in different agricultural and animal husbandry practices and in public health operations.

Introduction

Plants are the main source of nutrition to farm animals for their maintenance and production.  But, under certain conditions, plants intoxicate farm animal life due to the presence of some deleterious substances called phytotoxins. Such plants include a variety of fodder plants (Jowar, wheat straw, Lucerne), grasses (Millet grass), ornamental or fencing plants (Crotons, Nerium sps) and weeds.

(Tribulis) in the farm premises.
Generally, farm animals avoid such plants and weeds due to their unpleasant taste and offensive odor but during fodder scarcity caused by drought, floods or overgrazing, animals consume these plants. In addition, animals introduced into new areas, stall fed animals also sometimes lose their sense of discrimination and ingest such plants containing phytotoxins.