PALACKÝ UNIVERSITY

FACULTY OF MEDICINE

Z. Dvořák, J. Vičar, S. Dvořáčková

BIOCHEMISTRY Laboratory classes

OLOMOUC 2006

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1 METHODS OF CHROMATOGRAPHY 1.1 Thin layer chromatography of amino acids. 1.2 Separation of paprika carotenoids by column adsorption chromatography on

aluminium oxide. 1.3 Test for purine and pyrimidine bases by thin layer chromatography 1.4 Gel chromatography of hemolyzed blood. 1.5 Deionization of calcium chloride solution on ion exchangers 1.6 Preparative chromatography of azo-dyes on thin layer of silica gel 2 FUNDAMENTALS OF PHOTOMETRIC ANALYSIS 3 AMINO ACIDS AND PROTEINS 3.1 Chemical reactions of amino acids and proteins. 3.2 Separation of acidic, neutral and basic amino acids by electrophoresis. 3.3 Determination of isoelectric points of histidine. 3.4 Determination of isoelectric point of casein 3.5 Isolation of albumins and globulins by fraction salting out. 3.6 Determination of total serum proteins by biuret reaction 3.7 Serum protein electrophoresis on cellulose acetate membrane and agarose gel 3.8 Determination of urea in serum 4 SACCHARIDES 4.1 Chemical reactions of saccharides 4.2 Polarimetric observations of D-glucose mutarotation 4.3 Polarimetric determination of glucose in urine 4.4 Determination of glucose in blood and urine by reagent kit OXOCHROMGLUKOSA 4.5 Oral glucose tolerance test (oGTT) 4.6 Determination of glucose and ketones by diagnostic test strips. 5 LIPIDS 5.1 Chemical reactions of lipids. 5.2 Determination of the iodine number of lipids. 5.3 Determination of total cholesterol in blood serum 5.4 Plasma lipoproteins analyses 5.5 Determination of triacylglycerols in blood serum 6 NUCLEIC ACIDS 6.1 Isolation and degradation of nucleoproteins from bakers'yeast 6.2 Demonstration of dehydrogenase activity of xanthinoxidase 6.3 Determination of uric acid in blood serum 7 TOXICOLOGY AND ANALYSIS OF DRUGS 7.1 Determination of salicylic acid in Acylpyrine 7.2 Quantitative determination of salicylates by photometry. 7.3 Identification of levomepromazine and its metabolites in biological materials by thin

layer chromatography. 7.4 Determination of nitrates in biological material by ion selective electrode 7.5 Determination of vitamin C in fruit juices. 7.6 Isolation and demonstration of alkaloids from Chelidonium majus 8 ENZYMES 8.1 Demonstration of enzymatic character of peroxidase reaction 8.2 Demonstration of substrate specificity of amylase and sucrose 8.3 Inhibition of enzymatic activity - inhibition of catalase by cyanide 8.4 Determination of pH optimum for trypsin 8.5 Determination of optimum temperature for salivary amylase 8.6 Determination of Michaelis constant of alkaline phosphatase

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8.7 Determination of aspartate aminotransferase (AST) activity in serum 8.8 Determination of lactate dehydrogenase (LD) activity in serum 8.9 Determination of the activity of α-hydroxybutyrate dehydrogenase (α-HBD) in serum 8.10 Determination of creatine kinase (CK) activity 9 TETRAPYRROLES 9.1 Determination of hemoglobin in blood 9.2 Determination of total bilirubin in serum 9.3 Test for blood in feaces (test for occult bleeding) 9.4 Gallstones analysis 10 URINALYSIS 10.1 Physical analysis of urine 10.2 Chemical analysis of urine 10.3 Microscopic urinalysis 10.4 Chemical analysis of renal calculi (urolithiasis) 11 CREATININE CLEARANCE AND GASTRIC SECRETION 11.1 Determination of creatinine in blood serum and urine and creatinine clearance

calculation 11.2 Determination of gastric output

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1. METHODS OF CHROMATOGRAPHY General principle: Chromatographic methods are used to separate and to detect the individual components of mixtures. In all chromatographic separations, molecules are partitioned between a stationary (solid or liquid) and a mobile (gas or liquid) phase. The separation depends on the differential affinity of the components for one or the other phase. Types of chromatography: According to the separation process: - Adsorption chromatography - Partition chromatography - Ion exchange chromatography - Gel filtration chromatography According to the technique used: - Column chromatography - Thin layer chromatography - Paper chromatography According to the purpose: - Analytical chromatography - identification of compounds in a mixture - Preparative chromatography - isolation of compounds from a mixture

Adsoption Chromatography In its simplest, classical form, adsorption chromatography represents the separation of substances during the filtration of a solution of those substances through a column of finely powdered adsorbent in a glass tube. The substances are adsorbed at the top of the column and a suitable solvent or solvent mixture is let to flow through the column. The substances which have more affinity for the solvent (i.e. those that which have lesser tendency to be adsorbed) move more rapidly down the column. If the substances are pigments, the separation is readily visualized and coloured bands are seen to move down the column. If the substances are colourless, a series of the filtrate fractions from the column is collected and the separated substances are detected in the fractions by various methods. The relative affinity of a substance for the adsorbent is a function of its chemical constitution, the nature of the solvent, and the nature of the adsorbent. Solid adsorbents commonly used are aluminium oxide, silica gel, charcoal, cellulose, and starch. Among the solvents used there are petroleum ether, hexane, toluene, ether, dichloromethane, various alcohols, ketones and others. Another type of adsorption chromatography is affinity chromatography. This technique is based on specific interaction between e.g. antibody and antigen or enzyme and substrate. Affinity chromatography is used for isolation of enzymes, antibodies, etc. Partition chromatography In partition chromatography, the separation is achieved by differential migration of substances resulting from different distribution between two immiscible solvents. One solvent, the stationary phase (anchored-fixed on carrier), is successively washed with a second phase, the mobile phase, in such a manner that the substances partition between the two phases. The extent of the separation can be roughly predicted from the partition coefficient, KD, of the

substances:

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B

AD c

cK =

cA ..concentration of a compound in solvent A at equilibrium

cB ..concentration of a compound in solvent B at equilibrium

Carriers commonly used are chromatographic paper, silica gel, cellulose, polyamide, dextrane polymers. Stationary phase is usually water, mobile phase are organic solvents. Ion Exchange Chromatography Ion exchange chromatography utilizes the differential affinity of charged molecules in solution for other charged substances (ion exchangers). An ion exchanger consists of an insoluble matrix (synthetic resins, polysaccharides etc.) to which charged groups are covalently bound. The charged groups are associated with mobile counter-ions. These counter-ions can be reversibly exchanged with other ions of the same charge. It is possible to have both positively and negatively charged ion exchangers. Positively charged ion exchangers have negatively charged counter-ions (anions) available for exchange and so they are called anion exchangers. Functional groups of anion exchangers are – N+R3, -N+R2- etc. Negatively charged exchangers have positively charged counter-ions (cations) available for exchange and so they are termed cation exchanger. Functional groups of cation exchanger are –COO-, -SO3-, etc.

General description of the function of cation and anion exchangers is given below (Z indicates the charged group of the exchanger).

Cation exchanger: Na+ + H+Z- ---> H+ + Na+Z

-

Anion exchanger: Cl- + Z+OH

- ---> OH- + Z+Cl

-

The capacity of an ion exchanger is a quantitative measure of its ability to take up exchangeable counter-ions. The total capacity is the amount of charged and potentially charged groups per gram of dry ion exchanger. The separation in ion exchange chromatography is obtained by reversible adsorption. Most ion exchange experiments are performed in two stages. The first stage is sample application and adsorption. Unbound substances can be washed out from the exchanger. In the second stage, substances are eluted from the column and separated from each other. The separation is obtained since different substances have different affinities for the ion exchanger due to the differences in their charge. These affinities can be affected by varying conditions such as ionic strength and pH. The differences in charge properties of biological compounds are often considerable, and since ion exchange chromatography is capable of separating species with very small differences in properties, such as two proteins differing by only one amino acid, it is a very powerful separation technique indeed.

Gel Filtration Chromatography This technique involves use of dextran gel, column of this sort conveniently separate large from small molecules regardless of their chemical natures. Molecules larger than the largest pores of the swollen gel particles, i.e. above the exclusion limit, cannot penetrate the gel particles and therefore they pass through the bed in the liquid phase outside the particles. They are thus eluted first. Smaller molecules, however, penetrate the gel particles to a varying extent depending on their size and shape. Molecules are therefore eluted from a gel in order of decreasing molecular size. The cut off between what is large and what is small is determined

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by the arbitrary choice of the packing material, granules of differing porosities being available. Thin layer chromatography (TLC) and column chromatography These methods are performed by all type of chromatography. They are used for isolation or identification of compounds from mixtures. By TLC, the distance travelled by each compound from the start (base line) related to the solvent front is defined as the RF

b

aRF =

a - distance from base line travelled by a compound; b - distance from base line travelled by a solvent. The RF value is characteristic of the particular compound measured under specified conditions-solvent system, temperature, and the sorbent.

start

front

a b

0.2

0.0

0.6

0.4

1.0

0.8

Rf

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1.1. Thin layer chromatography of amino acids.

Principle: Amino acids are divided by partition chromatography through a thin layer of cellulose or similar inert material (silufol) supported on a glass or plastic plate. The detection of amino acids is performed by ninhydrin solution. Reagents: Standard solutions of amino acids (glutamine acid, glycine, lysine) Mixture of unknown amino acids Silufol Elution system: ethanol - water (7:3) Ninhydrin (30 mol.l-1) in ethanol Procedure: Mark four different points of start on the silufol foil (8x15cm) in regular distances between each other (approx. 15cm) and 2cm far from the lower margine. Using the glass capillaries apply the samples of standards and analyzed mixture. Let the foil develop in the chromatographical chamber together with the elution mixture. After the developing procedure is over, dry out the foil, spray it with the ninhydrin solution, let it dry out again and warm it up in the drying room for 15 minutes on the temperature of 100°C.

b

aRF =

Evaluation:

Calculate the RF values in component standards and after the comparison with RF values of

spots (stains) found in the mixture determine their constitution. 1.2. Separation of paprika carotenoids by column adsorption chromatography on aluminium oxide. Principle: The separation is done by adsorption chromatography. Reagents: Powdered red paprika Aluminium oxide Petroleum ether:dichloromethane, 9:1 Petroleum ether:dichloromethane, 7:3 Dichloromethane Method: Preparation of the paprika extract: Stirr 1 g of red paprika in an Erlenmayer flask with 5 ml of petroleum ether:ether (9:1), mixture, extract for 10 minuts and filter. Preparation of the column. Fix the chromatography column in vertical position, insert a piece of cotton into the bottom of the column. The eluate petroleum ether:dichloromethane (9:1) is poured into the column , and the necessary amount (depends of column heigth) of adsorbent is added. The column is tapped from all sides so that the packed column might is homogenous

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and without air bubbles. After the adsorbent has settled perfectly, the surface is covered with a filter paper disc. The level of the solvent should never drop below the adsorbent surface. Separation and elution. Carefully apply the extract of paprika on the top of the column (without whirling the adsorbent). Then start the elution of the column with petroleum ether:dichloromethane (9:1), continue with petroleum ether: dichloromethane (7:3). In the course of the elution, the substances of the mixture are separated according to their polarity. Evaluation: The values of adsorption coeficients of carotenoids increase in the order: α-carotene, β-carotene, γ-carotene, lycopene, xanthophyll. Which carotenoid passes most rapidly through the column?

1.3. Test for purine and pyrimidine bases by thin layer chromatography Principle: Individual pyrimidine and purine bases are identified in the yeast hydrolysate prepared in previous task (5.1). The bases are identified by thin-layer chromatography on ready-to-use silica plates ("Silufol UV 254") containing a fluorescent dye. The inclusion of a fluorescent dye into silica can be used to detect substances which quench its fluorescence and so result in dark zones when the chromatogram is viewed under ultraviolet light. Reagents: Ammonia solution (25 %). Adenine solution (10 g/l) (reference). Solvent for elution: H2O. "Silufol UV 254" ready-to-use plates (containing a fluorescence dye). Procedure: 2 ml of RNA hydrolysate is alkalized with ammonia to pH 9-9.5 (indicator paper) and diluted with 4 ml of water. The diluted hydrolysate and adenine reference solution are then applied on "Silufol" plate by a capillary under constant drying by a hair dryer. The chromatogram is developed with water, dried, and observed under UV-lamp at 254 nm. The bases are visible as violet spots on green fluorescent background. Evaluation: The spots on the chromatogram are marked with a pencil. The result is copied to the protocol. Calculate the RF values. Try to identify individual bases comparing your RF values with the known ones: adenine 0.37, guanine 0.40, uracil 0.63, thymine 0.67. 1.4. Gel chromatography of hemolyzed blood. Principle: High-molecular substances of blood (proteins) are separated from the small molecules (for example phosphate) by the gel chromatography on Sephadex column. Small molecules diffuse into swelled up gel, they are delayed, large molecules flow around the gel and thus are eluted first from the column.

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Reagents: Haemolysed blood (25 times diluted) Sephadex G-25 Physiological solution (0.15 mol.l-1 NaCl) Sulphosalicylic acid (1 mol.l-1) Molybdanum solution Hydrochinone Procedure: On Sephadex column (approx. 1x20cm), previously washed through by physiological solution, apply 1ml of diluted filtered blood. The column is washed through with the physiological solution in approx. 2 ml/min. rate. Observe the progress (advancement) of the haemoglobin zone and after it reaches the lower part of the column, start to pick up 1ml of component fractions to the test tubes. Find out the presence of proteins in each fraction by the reaction with sulphosalicylic acid (a coagulum of denatured protein appears) and the presence of phosphates by the reaction with molybdanum solution (a yellow ammonium molybdatephosphate is formed; after hydrochinone is added a molybden blue is formed). After the chromatography is over, using the physiological solution wash through the column until the reaction on proteins and phosphates is negative.

Evaluation: Record the obtained results from the test to the table (mark the positive reaction by a cross) and give reasons.

1.5. Deionization of calcium chloride solution on ion exchangers Principle: Diluted solution of calcareous chloride loses Ca2+ ions after it has run through the catex column in H+- cycle. Also when the identical solution runs through the second column which is including anex in OH- cycle, the ions Cl- are removed from the CaCl2 solution. Deionized water leaks out from the column. Reagents: 4 mol.l-1 HCl 1 mol.l-1 NaOH 0.03 mol.l-1 CaCl2 0.1 mol.l-1 AgNO3 0.25 mol.l-1 (COONa)2 Procedure:

1. Bringing catex into H+- cycle and anex into OH-- cycle: Through the catex column let run 30 ml of HCl (4 mol.l-1), through the anex column 30 ml of NaOH (mol.l-1). Both columns wash through with distillated water (approx. 100 ml) until the neutral reaction of eluate is reached. 2. Removing Ca2+ ions: In the solution CaCl2 give the evidence of Ca2+ ions presence by the reaction with sodium oxalate, Cl- ions by the reaction with AgNO3. Measure the pH value using the universal indicator pH paper and also measure the conductivity by the conductometer.

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Apply 30 ml of CaCl2 on the catex column and let it gently leak into the flask. In eluate perform again the reactions on Ca2+ and Cl- ions, measure pH and conductivity. 3. Removing Cl- ions: Apply the eluate from catex on the anex column and let it gently leak into the flask. In the obtained eluate perform again the reactions leading to ions, measure pH and conductivity. Warning: The air cannot penetrate into the columns with resins during the procedure. Thus be sure and have the column covered with a layer of liquid (3-5 mm is the minimum). Evaluation: Record in the protocol the equations representing the ion exchange in the component columns. In the table synoptically line up the results of reactions performed, of measured pH values and of conductivity in the starting (initial) CaCl2 solution and in both eluates. Give reasons. 1.6. Preparative chromatography of azo-dyes on thin layer of silica gel Principle: Thin layer of silica gel is used for the separation and isolation of azo-dyes Methyl Yellow (4-dimethylaminoazobenzene) and Sudan Red (2-hydroxynaphtalene-1-azo-1°-benzene-4°-azobenzene) from their mixture. Chromatography on silica gel is based on the principle of adsorption and partition chromatography. Silica gel is polar adsorbent which adsorbs polar substances from non-polar solvents. Reagents: Thin layer of silica gel fixed with gypsum binder Solvent (dichloromethane:ethyl acetate:formic acid, 5:4:1) Mixture of azo-dyes (Methyl Yellow + Sudan Red) dissolved in dichloromethane Dichloromethane Procedure: Before applying samples, the of start line is slightly marked with a pencil at the distance of 20 mm from the lower margin of the sheet, 15 mm from the side margins. The mixture is applied with a capillary on the whole start line. Then the sheet is placed in a chromatographic chamber containing the solvent. When the solvent front reaches the necessary distance, the sheet is taken out of the chamber and dried. The zones of azo-dyes are collected with a spatula into Erlenmayer flasks, and both azo-dyes are extracted with dichloromethane. The isolated Sudan Red, Methyl Yellow and original mixture are applied on s sheet of Silufol, mobile phase is the mixture described above or toluene. Evaluation: Calculate the RF value of each azo-dye. Compare the RF value of each azo-dye with RF value of azo-dyes in the original mixture.

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2. FUNDAMENTALS OF PHOTOMETRIC ANALYSIS MOLECULAR ABSORPTIOMETRY Photometric measurements provide the basic for the majority of quantitative methods in biochemistry and are related to the amount of radiation absorbed rather than the nature of such radiation. BEER-LAMBERT RELATIONSHIP Lambert's law states that the proportion of radiant energy absorbed by a substance is independent of the intensity of the incident radiation. Beer's law states that the absorption of radiant energy is proportional to the total number of molecules in the light path. �Beer's law describes the basic relationship between the concentration of the absorbing substance and the measured value of absorbed radiation. Lambert's law is of major significance in the manner in which measurements are made and the fact that a given sample always absorbs the same proportion of the incident radiation regardless of its intensity greatly simplifies the design of instruments. It is not possible to measure directly the amount of radiation absorbed by a substance and it is usually determined by measuring the difference in intensity between the radiation falling on the sample (incident radiation, I0) and the residual radiation which finally emerges from the sample (transmitted radiation, I). Using such measurements, the Beer-Lambert law can be expressed as an equation: log10 I/I 0 = εεεε....c.l where c is the concentration of the substance in gram molecules per litre, l is the light path in centimetres and ε is known as the molar absorption coefficient for the substance and is expressed in litres per mole per centimetre (l mol-1 cm-1). The values for I and I0 cannot be measured in absolute terms and the measurements are most conveniently made by expressing I as a percentage of I0. This value is known as the percentage trasmittance (T) and only shows a linear relationship with the concentration of the test substance if the logarithm of its reciprocal is used. It is therefore more convenient to report the measurements initially as this logarithmic function of I and I0, a parameter which is known as absorbance (A)

Percentage transmittance (T) = I/I0 x 100 A = log10 100/T DEVIATIONS FROM THE BEER-LAMBERT LAW The Beer-Lambert equation expresses the linear relationship between the concentration of the sample and the absorbance values recorded. However, the relationship is only an experimental one and not a fundamental law of nature and as a result the linearity is only true under certain limiting conditions. The basic assumption that the difference between incident and transmitted radiation is a measure of the absorbed radiation is not completely true because incident radiation may not appear in the transmitted form for other reasons besides absorption. A certain amount of radiation will be reflected from the surface of the sample holder, usually a glass or plastic cell, or absorbed by the material of which the cell is composed. The sample may also be dissolved in a solvent which itself may also absorb or reflect radiation.

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incident (I0) = absorbed + transmitted (I) + other losses For this reason the radiation transmitted by a blank sample is measured and taken to be the effective incident radiation. This blank should be identical to the test sample in all aspects except the presence of the test substance: blank reading = I0 - other losses Hence: absorbed = blank - transmitted (I) An important factor which influences the Beer-Lambert relationship is the wavelength range of the incident radiation. Ideally the radiation should only provide a specific unit of energy and not a range of energy levels. Such specific radiation is known as monochromatic radiation and can only be produced under very restricted conditions. The best that can often be achieved is radiation with a very limited range of wavelengths. If the incident radiation contains wavelengths which will not be absorbed by the test substance, the difference between the intensities of incident and transmitted radiation will not be proportional to the concentration of the test substance. In such cases a non-linear relationship will exist. This problem is particularly relevant to instruments which use simple glass filters rather than monochromating systems such as prisms or diffraction gratings. Alterations which may occur in the molecular nature of the sample due to changes in concentration may also result in deviation from the Beer-Lambert relationship. Molecules may tend to associate with one another when the concentration is high or, conversely, complexes may tend to dissociate in low concentrations. Both types of change may possibly affect the absorption characteristics of the compound and result in non-linear graphs. QUANTITATIVE MEASUREMENTS The measurement of the intensity of radiation is indirect, involving the generation and measurement of an electric current, and it is necessary to standardise instruments before test readings are made. The method of standardisation is in principle the same for all instruments but does vary in practice depending upon the design of a particular instrument. The basic procedure involves setting the minimum and maximum conditions of transmitted radiation and adjusting the metering system to give appropriate readings. To set maximum transmittance a blank sample is used and the instrument is adjusted to give either a reading of 100% transmittance or zero absorbance. Zero transmittance is set when all light to the detector is cut off using an opaque shutter and the meter is adjusted to give transmittance reading of zero. In order to achieve maximum sensitivity from the method, the absorbance measurements for any given concentration should be as great as possible and preferably should be made at the absorption maximum. Absolute methods It is possible to measure the absorbance of a sample of a known compound at its absorption maximum and to calculate the actual concentration of the compound in the sample using a known value for the molar absorption coefficient ε (often obtainable from published spectral tables). In practice, however, this is not always possible due to variety of reasons. If the sample is a mixture of several compounds, the measured absorbance value will be the cumulative effect of all the substances which absorb at the selected wavelength. In addition the precise value of the molar absorption coefficient depends to some extent on the particular

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instrument used and ideally values for the coefficient should be determined rather than accepted from the literature. Comparative methods If the use of molar absorption coefficient is inappropriate then it may be sufficient to use a single reference solution of known concentration (known as a standard or calibrator solution) and to compare the test absorbance with that of this standard. The principle of quantification is exactly the same as before except that the molar absorption coefficient is eliminated from the calculation by measuring the absorbance of both the test and standard solutions at the same wavelength and comparing their absorbance values. A standard solution of concentration cs gives an absorbance value of As while the test solution gives an absorbance of At. Hence: As = εεεεcsl and At = εεεεctl Therefore As/At = εεεεcsl/ εεεεctl = cs/ct and the concentration of the test ct = At/As x cs The use of a single standard in this way assumes that the Beer-Lambert relationship is valid over the absorbance range measured and again it is necessary to confirm the relationship before using the method routinely. The analysis of a range of known concentrations of the test substance is necessary to validate the Beer-Lambert relationship. If the plot of absorbance values against concentration results in a straight line then either of the two previously outlined methods may be used. If, however, the resulting graph shows a curve instead of a straight line then the implication is that the actual value for the molar absorption coefficient is dependent to some extent upon the concentration of the compound and as a result invalidates both methods. In such circumstances it is essential that quantification is done by using a graphical plot (calibration curve).

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3. AMINO ACIDS AND PROTEINS 3.1. Chemical reactions of amino acids and proteins. Tasks: Colourful reactions of amino acids and proteins Precipitation reactions of amino acids and proteins Colourful reactions of amino acids and proteins Various reactions can be used: a) General reactions - demonstrate the presence of peptide bond, free amino carboxyl and

amino groups b) Specific reactions - demonstrate the characteristic functional groups of component amino

acids a) General reactions: NINHYDRIN REACTION Principle: The most common assay for total amino acids is the ninhydrin methods. Ninhydrin (triketohydrindene) reacts with all free amino groups of proteins, peptides and amino acids to yeild mostly a purple product. The amino acids proline and hydroxyproline give a yellow product; arginine gives an orange product. Ninhydrin reaction is routinely used for detection and quantitative determination of amino acids (quantitative determination and detection during the chromatography). Reagents: Protein solution (egg white dissolved in physiological solution in ratio 1:30) Ninhydrin (0.5 g/l in ethanol) Procedure: Add a few drops of the ninhydrin reagent to the protein solution, and warm up to the boiling point. A violet-blue colour appears. BIURET TEST Principle: Compounds containing two or more peptide bounds (for example, proteins) give a characteristic purple colour when treated with dilute copper (II) sulphate in alkaline solution. The colour of the compounds depends on the size of the molecule; proteins give a violet, peptides a red colour. The colour is apparently due to the coordination of the cooper atom and four nitrogen atoms, two from each of two peptide chains (see figure below). The test is used for quantitative determination of proteins. Reagents: Protein solution 1-M NaOH CuSO4 solution {0.5% (w/w)} Procedure:

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Use NaOH solution to alkalize about 1 ml of a protein solution, and add a few drops of a very dilute solution of CuSO4. A purple colour appears. b) Specific colourful reaction XANTHOPROTEIN REACTION Principle: Aromatic amino acids tyrosine and tryptophane can react with concentrated nitric acid to give yellow nitro compounds, which are given general name, xanthoproteic acid (phenylalanine is not nitrated under the given conditions) The yellow colour of nitro compounds turns to orange on alkalization. Reagents: Protein solution HNO3 {65% (w/w)} 1-M NaOH Procedure: Add 1 ml of concentrated HNO3 to 2 ml of protein solution in tube. A white precipitate is formed. After warming up, it turns yellow and dissolves in most cases. After cooling, make the mixture alkaline by excess of NaOH, the yellow colour deepens into orange. MILLON'S TEST Principle: Tyrosine reacts with Millon’s reagents to give red coloured mercuric complex of nitro compounds. Reagents: Protein solution Millon's reagent (50 g of mercury + 50 ml of HNO3 in 100 ml solution) Procedure: Add a few drops of Millon's reagent into 1 ml of protein solution, and warm up to the boiling point. The white precipitate turns into a red colour. REACTION WITH EHRLICH'S REAGENT Principle: Tryptophane condenses with aldehydes in the presence of the strong acids and red-violet solutions are formed. Reagents: Protein solution Ehrlich's reagent (20 g of dimethyl-p-aminobenzaldehyde in 1 l of 5% hydrochloric acid) Procedure: Add a few drops of Ehrlich's reagent into 1 ml of protein solution in a tube, mix well. Then incline the tube, slowly and carefully allow approximately 1 ml of concentrated H2SO4 to flow down the side of the tube so that is forms a layer beneath the aqueous solution. A purple ring develops at the interface between both layers.

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DETECTION OF SULPHURIC AMINO ACIDS Principle: Anion S2- is formed by alkaline hydrolysis of proteins containing –SH or –S-S- group. It gives black coloured PbS, by reaction with Pb(II) salt. Cystine, cysteine (both free and bound in proteins or peptides) are detected by this reaction, not methionine which is a dialkylsulphide. Reagents: Protein solution NaOH (1 mol/l) Lead (II) acetate (1 mol/l) Procedure: Mix 2 ml of protein solution, 2 ml of sodium hydroxide and a few drops of lead (II) acetate in a test tube. Boil the mixture carefully. The solution will turn brown or black, or a black precipitate will be formed. SAKAGUCHI REACTION Principle: Guanidine groups of arginine develop an intense red colour when treated with 1-naphthol. Reagents: Protein solution 1-naphthol in ethanol (1 mol.l-1) Sodium bromide (NaBr) (1 mol.l-1) Procedure: Using the distilled water dissolve 0.5 ml of the protein solution in order to get quadruple amount of the mixture. Add 2-3 drops of 1-naphthol in ethanol and approximately 0.5 ml of sodium bromide. A red colour appears. REACTIONS WITH DIAZO-REAGENTS Principle: Histidine and tyrosine couple with diazo reagents and orange-red azocompounds are formed. Reagents: Protein solution Diazo I (0.5 g sulfanilic acid + 1.5 ml of hydrochloric acid in 100 ml solution) Diazo II (sodium nitrite 20 g/l), sodium carbonate (1 mol/l) Procedure: Add a few drops of sodium nitrate (diazo II) and 2 ml of protein solution to 1 ml of sulfanilic acid solution in HCl (diazo I). After a careful alkalization by sodium carbonate and warming up, the final product shows an orange-red colour.

Precipitation reactions of amino acids and proteins Principle: The precipitation of proteins may occur either by denaturation or by salting out procedure. The solubility of most proteins in aqueous solutions can be attributed to hydrophilic interactions between polar molecules of water and ionized groups of proteins molecules.

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Reagents which change the dielectric constant or the ionic strength of an aqueous solution would, therefore, be expected to influence the solution of proteins. Nearly all proteins precipitate reversibility in a medium with very high ionic strength, e.g. after increasing the salt concentration by addition of great amounts of certain salts such as ammonium sulphate - salting out effect. The similar precipitating effects occur also by action of some alcohols or acetone. The dilution (the decrease of ionic strength or the concentration of a less polar solvent) can contribute to obtaining the colloid solution of a protein again.

Any change in the structure of a protein (secondary and tertiary structures) from its native state is called denaturation. One feature of denaturation is precipitation. Denaturation may be caused by physical (by elevated temperature) and chemical (action of strong acids, cations of heavy metals etc.) effects. These processes are mostly irreversible. Reagents: Protein solution Acetic acid Nitric acid (65% (w/w)) Sulphosalicylic acid (200 g/l) Trichloracetic acid (100 g/l) Lead acetate (1 mol/l) Copper sulfate (1 mol/l) Sodium hydroxide (1 mol/l) Ethanol Procedures: Salting out efect Add stepwise small amounts of finely ground sodium chloride the protein solution in a test tube until the protein precipitate develops. On diluting with water, the precipitate dissolves again. Protein precipitation with ethanol Add stepwise small volumes of ethanol (stir well after each dose) to the protein solution. Turbidity arises and fine flocks of the precipitated protein appear later. When dilute with water, they dissolve again.

Precipitation of proteins after their thermal denaturation Warm up 1 ml of protein solution in a tube to the boiling point. If the protein concentration is not high enough, precipitation does not usually occur. Observe the influence of adding a drop of dilute acetic acid (isoelectric precipitation). Precipitation of proteins with concentrated mineral acids Nitric acid: Overlayer the protein solution over to 1 ml of concentrated nitric acid placed in a test tube by careful pouring in on the inclined wall of the test tube. A white disc of precipitated, denatured protein is formed at the interface between the two liquids (nitric acid test). Sulphosalicylic acid: Add a few drops of sulphosalicylic acid to 2 ml of the protein solution. A white turbidity or precipitate develops. (The methods is used to detect proteins in urine). Trichloroacetic acid: Add a few drops of trichloroacetic acid to 2 ml of the protein solution. The denatured protein is formed. (This acid is often used for the deproteinization of solution).

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Precipitation of proteins with cations of heavy metals Add a few drops of lead(II) acetate to 1 ml of the protein solution; also a few drops of sodium hydroxide may be added. Observe the formation of a precipitate. 3.2. Separation of acidic, neutral and basic amino acids by electrophoresis. Principle: Electrophoretic methods make possible the separation of molecules or particles bearing electric charges, mostly ampholytes or ions. If their mixture is subjected to the action of an electric field, molecules or particles start moving. Their mobility is dependent on charge, size and shape of molecules, on environmental conditions and on the strength of electric field. Charge of the molecule (particle) is dependent on the extend ionization, on pH, and on ionic strength of the environment. For example, at pH 5.7, glutamic acid possesses a net negative charge and migrates toward the anode; histidine possesses a net positive charge and migrates toward the cathode; the neutral glycine will remain at the start line. Electrophoresis may be carried out on a single sheet or strip of paper. The amino acids are then detected by ninhydrin reaction. Reagents: Solution of glycine, lysine, glutamine acid (20 g/l) Solution of picric acid (20 g/l) Buffer pH 5.7 (5ml of CH3COOH + 20 ml of pyridine in 2 l solution) Ninhydrin in ethanol (5 g/l) Procedure: Electrophoresis is performed on paper strip38 x 11 cm. Position of the starting line is 16 cm from the narrower edge. Sample of amino acid and picric acid are applied as a small bands. Paper strip is moistened in a buffer pH 5.7., quickly blotted, without pressure, on filter paper, and stretched on the frame. The same buffer is also used in electrode chambers of the electrophoresis apparatus. Electrophoretic separation proceeds at 700 V for 45 minutes. After the separation, the paper is dried, sprayed with ninhydrin reagent and warmed up to 100 °C. Evaluation: Explain the different position of amino acids on the electrophoreograme. 3.3. Determination of isoelectric points of histidine. Principle: The titration curve of histidine shows three steps that correspond to three values of pKa (the ionisation of carboxyl group, imidazolium group, ammonium group, resp. In the region of basic pH (between pKa2 and pka3 in case of histidine) the molecule contains only ionised H3N

+ and COO- groups. Although this molecule has both positive and negative charges, its net charge is zero. An ampholyte in this state is called a zwitterions. At one particular value in this pH region, the average charge on histidine is zero. This is called the isoelectric point (pI). Reagents: L-Histidine dichloride (0.05 mol.l-1) NaOH (1 mol.l-1)

19

Procedure: Pipette 50 ml of L-histidine dichloride (0.05 mol.l-1) into plastic beaker. Then add 1 M NaOH from the micro-burette in following doses: 4x0.5 ml; 24x0.25 ml; 2x1.0 ml. After each addition, measure the pH. Titration reaction chart:

pKa1 pKa2 pKa3

++

HN

N+

CH2 CH COOH

H

NH3+

++ -

HN

N+

CH2 CH COO-

H

NH3+

+-

HN

N

CH2 CH COO-

NH3+

-

HN

N

CH2 CH COO-

NH2

Calculation of pI:

( )3a22

1apKpKpI +=

Evaluation: Construct the titration curve, determine the values of the equivalence points and the values of pKa1,pKa2,pKa3 and calculate pI. 3.4. Determination of isoelectric point of casein Principle: Side chains of amino acids in the molecule of protein considerably affect physico-chemical properties of the protein. Behavior of the protein in solution is determined by number and type of side chains of amino acids on the surface of the protein that can have various charges. The presence of both types of charges (i.e. positive and negative) on the surface of protein implies that molecule of protein can behave as amphion. Surface charge of protein is important for formation of solvation sphere made of molecules of water and ions of salts that stabilize molecule of the protein in solution. The charge of acidic and basic amino acids residues on the surface of protein is determined by primary structure of the protein. This charge depends on pH. With increasing pH decreases protonization of basic amino acids and increases dissociation of acidic residues of amino acids. At certain pH the number of protonized and dissociated amino acids residues is balanced; this pH corresponds to isoelectric point of the protein. The value of isoelectric point ranges between pH 2 - 10. When the overall charge of the molecule of protein is zero then salvation sphere is minimal. Under these conditions, the molecule of protein has low stability and the addition of urea or non-polar solvent to solution may disturb hydrogen and hydrophobic bonds (destruction of quaternary, tertiary and secondary structures). Consequently, protein is precipitated and the process can be either reversible or irreversible. Reagents: 0.1 mol/L sodium acetate 1.0 mol/L acetic acid 0.1 mol/L acetic acid 0.4% solution of casein (0.2 g of casein is dissolved in 5 mL of solution 1 and complete with water up to 50 mL).

20

Procedure: Prepare the solutions in 6 tubes according to the table and mix them well. Add casein to tubes and mix them well. After 5-10 min turbidity will develop. Estimate the intensity of turbidity (-;+;++;+++). Measure absorbance at 550 nm (water as blanc). Return the mixture back to tube, add 2 mL of ethanol and mix well. Estimate turbidity again (-;+;++;+++). Tube # 1 2 3 4 5 6 Reagent (volume; mL) 0,1 mol/L sodium acetate 2,0 2,0 2,0 2,0 2,0 2,0 0,1 mol/L acetic acid 0,25 0,5 1,0 2,0 4,0 - 1,0 mol/L acetic acid 0 0 0 0 0 0,8 Distilled water 3,75 3,5 3,0 2,0 0 3,2 0.4% solution of casein 0.1 0.1 0.1 0.1 0.1 0.1 pH in tube 5,6 5,3 5,0 4,7 4,4 4,1 A550 Turbidity (from + to +++) Ethanol 1 1 1 1 1 1 Determine the dependence of turbidity on pH in absence and presence of ethanol. Compare the results obtained by subjective evaluation and by measurement of absorbance 3.5. Isolation of albumins and globulins by fraction salting out.

Principle: Albumins and globulins can be isolated from solution by fraction salting out. Globulins are excluded from solution already by semi-saturated solution of ammonium sulfate, whereas albumins are excluded by saturated solution of ammonium sulfate. Reagents: blood serum saturated solution of ammonium sulfate (NH4)2SO4 (NH4)2SO4 (solid substance) solution 1 M – NaOH solution 0.1 M – CuSO4 Procedure: Mix up 5 mL of blood serum and 5 mL of saturated solution of ammonium sulfate (NH4)2SO4 in centrifugation tube. The serum globulins are excluded from solution. Centrifuge the tube (10 min; 2500 rpm). Sediment contains globulins. Transfer supernatant to another tube and keep adding solid ammonium sulfate to this solution until it is dissolved. The serum albumins are excluded from solution. Centrifuge tube again and remove carefully supernatant. Dissolve the sediments of serum globulins and albumins in 10 mL of water (each) and perform the biuret reaction to prove the presence of proteins. Evaluation: Explain the principle of salting out of serum proteins.

21

3.6. Determination of total serum proteins by biuret reaction

Principle:

Serum proteins react with the cupric cations in alkaline solution to form a purple violet complex. Concentration of serum proteins is determined using photometry.

Reagents:

Biuret reagent (solution of CuSO4 and potassium sodium tartrate and NaOH)

Protein standard solution (70 g.l-1)

Procedure:

Pipette the reagents into test tubes according to the following scheme:

Reagents (ml) sample standard blank

Serum 0.1 - -

Standard - 0.1 -

Distilled water - - 0.1

Biuret reagent 5.0 5.0 5.0

Mix well and after 30 min measure the absorbance at 546 nm against the blank.

Calculation:

Concentration of total serum proteins:

c (g.l-1) = (Asample/Astanard) x cstandard

Reference values:

fS - total proteins: 65 - 85 g.l-1

Evaluation:

Low values of total protein concentration – hypoproteinemia - may be due to the impairment of protein synthesis in liver diseases or in large tumour diseases.

High values of total protein concentration – hyperproteinemia - are rarely found. The cause is in the dehydration of the body, during chronic inflammation or in gammopathies.

22

3.7. Serum protein electrophoresis on cellulose acetate membrane and agarose gel

Introduction:

The laboratory evaluation of serum proteins can be carried out

in two basic ways: (i) Total proteins (proteinemia) or individual components can be

quantified specifically in order to derive information about some distinct disorder. (ii) Alternatively, serum proteins can be separated using electrophoresis. This analytical method is useful as a starting point or as a screening procedure.

Principle:

Serum proteins are separated using electrophoresis on cellulose acetate membrane or agarose gel. Protein migration is dependent on the charge and the size of protein molecules.

Reagents:

Barbital buffer pH 8.6 (5.5 g of sodium diethyl barbiturate and 0.25 g of citric acid in 150 ml of water)

Staining solution (1 g of Amino Black + 20 ml acetic acid + 180 ml methanol)

Destaining solution (150 ml of acetic acid and 1350 ml of methanol)

Acetic acid solution (3 %)

Acetate cellulose membrane

Agarose

Procedure:

(a) Electrophoresis on cellulose acetate membrane

Electrophoresis: The cellulose acetate membranes should be handled with tweezers.

To moisten them, one lays them on the buffer surface and immerses them about 5 min later, when they are fully saturated. Then they are quickly blotted, without pressure, on filter paper, and stretched on the glass plate. The serum sample is applied to strip using a special applicator. The strip is placed in a horizontal electrophoresis chamber so that the ends dip into the buffer solution, the position of serum sample is near the cathode. Electrophoresis is carried out at 250 V for 30 min at room temperature. After 30 min the current is switched off.

Staining: The strip is stained 5 min in the staining solution, and destained in three tanks containing destaining solutions, for 3 min in each tank. The strip is stretched on the glass

plate, and carefully dried at 60-70oC for 5 min.

(b) Electrophoresis on agarose gel

Electrophoresis: Add 0.24 g of agarose to 15 ml of barbital buffer,and boil in the water bath

until solution clears. After cooling to about 48oC (in a water bath at 48oC) this thin gel is poured onto a glass plate. The gel layer coating the plate is about 2 mm thick. The agar gel is

23

solidified in a wet chamber for 15 min. After solidification, the gel-coated glass plate is placed in a template, and the wells are punched out of the gel using vacuum and a hollow needle. These holes are filled with serum samples using a pipette. The plate is placed in the electrophoretic chamber (sample start is near to the cathode). Electrophoresis is carried out at 220 V for 30 min at room temperature.

Staining: The gel is fixed by covering with acetic acid solution for 30 min. It is then covered with filter paper and dried for several hours at room temperature, using a fan. The dried gel layer is stained for 10 min in the staining solution. Excess dye is removed by dipping the gel layer into acetic acid solution.

Evaluation:

Electrophoretic serum protein fractions are then compared to the electrophoreogramm of normal serum.

3.8. Determination of urea in serum

a) The Bio-La-Test reagent kit "UREA F"

Principle:

Urea forms with o-phtaldialdehyde in an acidic medium N-carbamoyl-1,3 dihydroxyisoindoline. This reacts with N-(1-naphthyl)ethylenediamine to form red coloured adduct suitable for photometric kinetic determination.

Reagents:

1. Solution I (o-phtaldialdehyd, 4.5 mmol/l + sulphuric acid, 4 mol/l)

2. Solution II (N-(1-naphtyl)ethylenediamine, 6.4 mmol/l)

3. Urea standard (15 mmol/l)

Procedure:

Mix 2.50 ml of solution I with 0.05 ml of sample and preincubate for 1 minute at room temperature. Start reaction with 0.50 ml of solution II and read absorbances A at 540 nm in a 1 cm cuvette 30 and 90 seconds after starting. Use the difference

dA = (A90- A30) of a serum (dA1), standard (dA2) and blank (dA3)

for the calculation. For blank use distilled water.

Calculation:

serum urea (mmol/l) = (dA1- dA2) / (dA2- dA3) x 15

Reference values:

fS serum urea: 1.7 - 8.3 mmol/l

24

b) The Merck reagent kit "UREA GRANUTEST 15 PLUS"

Principle:

Urea reacts in the presence of enzymes according to the following two partial equations:

UREASE

Urea + H2O + 2 H+ ---------> CO2 + 2 NH4+

GIDH

NH4+ + 2-oxoglutarate + 2 NADH -------> 2-glutamate + NAD+ + H2O

GIDH = Glutamatdehydrogenase

Reagents:

1. Reaction solution (2-oxoglutarate, 9mmol/l + NADH, 0.3 mmol/l + ADP 0.8 mmol/l + GIDH, 1.2 łkat/l + urease, 4.5 łkat/l)

2. Urea standard solution (13,3 mmol/l = 0.8 g/l)

Procedure:

Pipette into test tubes:

Ml Serum Standard Blank

Standard - 0.010 -

Serum 0.010 - -

Reaction solution 2.000 2.000 2.000

Mix, after 1 min measure absorbances A1 of the reagent blank value (A1B), standard (A1ST)

and serum (A1SE) at regular intervals.

Incubate at + 25oC and read off the A2B, A2ST and A2SE values each exactly 5 min after the

A1 measurement. Wavelength: 365 nm, light path: 1 cm.

AST = (A1- A2)ST - (A1- A2)B

ASE = (A1- A2)SE - (A1- A2)B

Calculation:

serum urea (mmol/l) = (ASE / AST ) x 13.3

(g/l) = (ASE / AST ) x 0.8

25

Reference values:

fS urea: 3.3 - 6.7 mmol/l (0.2 - 0.4 g/l)

The normal range of urea concentration depends on protein intake.

Evaluation:

Considerable elevations of urea in serum are caused very often by a kidney disease or damage.

26

4. SACCHARIDES 4.1. Chemical reactions of saccharides Principle: The presence of carbohydrate in a preparation may be detected by either the Molisch test or the anthrone test. Both tests are based on the hydrolysing and dehydrating action of concentrated H2SO4 on carbohydrates. In these tests the strong acid catalyses the hydrolysis of any glycosidic bond present in the sample and dehydration to furfural (pentoses) or hydroxymethyl furfural (hexoses) of the resulting monosaccharides. These furfurals then condense with α-naphtol (Molisch test) or anthrone to give a coloured product. The Molisch test is qualitative test for the presence of carbohydrate in a sample of unknown composition. Seliwanoff's test is a timed colour reaction that is specific for ketoses. In concentrated HCl solution, ketoses undergo dehydration to yield furfural derivatives more rapidly than aldoses. Further, most furfural derivatives will form complexes with resorcinol to yield colour. Consequently, the relative rates of colour development in a solution containing sugar, HCl, and resorcinol provide evidence for the aldose or ketose nature of the sugar in question. Bial's test is a colour reaction that is specific for pentoses. Under carefully controlled conditions of temperature, time, and HCl concentration pentoses are rapidly converted to furfural. In presence of ferric ion and orcinol (5-methylresorcinol), furfural condenses rapidly to yield a coloured product. The most common method for detecting the presence of free reducing groups in carbohydrate involves a capacity of the carbohydrate-containing sample to reduce Cu2+ in alkaline solution. As stated previously, the carbohydrate is oxidatively degraded in nonstoichiometric manner, with a corresponding reduction of the oxidizing agent. In spite of the nonspecific course of the reaction, it is found that when the conditions for the copper reduction are rigorously controlled, the amount of Cu2+ reduced to Cu+ is directly proportional to the amount of reducing sugar in the sample analysed. But equimolar amounts of different reducing sugars differ in the rate at which they will reduce Cu2+. The Cu+ formed in the reaction precipitates as the rust-coloured Cu2O (Fehling's test, Benedict's test). Fehling's test

and Benedict's tests are used for the detection of glucose in urine. Reagents: Saccharide solution (1 mol.l-1) - glucose, fructose, arabinose, maltose, sucrose) 1-Naphthol (50 g/l in ethanol) Conc. H2SO4 Selliwanoff's reagent (0.5 g of resorcinol + 30 ml of 36% hydrochloric acid in 100 ml of solution) Bial's reagent (1.25 g of orcinol + 1 g of FeCl3 in 500 ml of 7.7 M-HCl)

Fehling's solution I (4.3 % CuSO4)

Fehling's solution II (346 g of potassium sodium tartrate + 104 g of NaOH in 1 l solution) Benedict's reagent (86.5 g of trisodium citrate + 37 g of Na2CO3 + 5.6 g of CuSO4 in 500 ml

of water) Procedures: Molisch test - general test of saccharides Add a few drops of 1-naphthol solution to 2 mL of glucose solution in a tube and mix well. Then incline the tube; slowly and carefully allow approximately 1 mL of concentrated H2SO4

27

to flow down the side of the tube so that it forms a layer beneath the aqueous solution. A purple ring develops at the interface between both layers. Selliwanoff's test - detection of ketoses Mix 0.5 mL of fructose solution and 2 mL of Selliwanoff's reagent in a tube and warm up the

mixture slowly until the temperature 60-65oC is reached. A red colour indicates the presence of ketoses. Bial's test Place 1 mL of arabinose solution in a tube and add 2 mL of Bial's reagent, heat the solution gently and then cool down. A green colour indicates the presence of pentoses. Fehling's test Add equal portions (1 mL) of Fehling I and Fehling II solutions into a test tube, warm up for a while and then add 2 mL of saccharide solution into the mixture. Keep the mixture boiling for 1 - 2 minute. Benedict's test Add 5 mL of Benedict's reagent to 1 mL of saccharide solution and keep the mixture boiling for 2 minutes. Evaluation: Observe and record the results of the tests, and explain them on the basis of the structure of individual saccharides.

4.2. Polarimetric observations of D-glucose mutarotation Principle:

The specific rotations of the α and β anomers of the D-glucose are +112o and +19o, respectively. Crystalline D-glucose is α- D(+)-glucopyranose. When α anomer is dissolved in

water, the specific rotation changes with time until an equilibrium value of +53o is attained. This change, called mutarotation, results from the formation of an equilibrium mixture containing about one-third of α anomer and two thirds of β anomer. The changes of optical rotation are measured on polarimeter. Theory of Polarimetry The magnitude and direction of rotation of the plane of polarized light by a chiral compound is a specific physical property of a compound that may be used in its characterization. The measurement of this property is called polarimetry. When plane polarized light is passed through a solution of a pure compound, the degree to which the plane of light is rotated is found to be directly proportional to the number of chiral molecules in the solution through which the light passes. Therefore the rotation observed will depend on the nature of chiral compound, the concentration of the compound in solution, and the length of the light path through the solution. In addition, the observed rotation will vary with the wavelength of plane polarized light used and with the temperature. When these variables are controlled, one may relate, by the formula

[ ]cl

TD ×

= αα

28

degree of rotation α actually observed to characteristic capacity of the compound to rotate polarized light. Here "α" is the observed rotation, "c" is the concentration in g/ml, and "l" is the length of the light path in decimetres. The quantity [α]T is called the specific rotation at the temperature T and when D the D line of the sodium spectrum (589 nm) is used as the light source. This formula is frequently written as

[ ]cl

TD ×

×= 100αα

where "c" is the concentration in g/l instead of g/ml, and "l" is the length of the light path in meters. The specific rotation is thus the actual rotation imparted to a beam of plane-polarized light passing through 0.1 ml of the solution containing 1 g of optically active solute in 1 ml solution. Every optically active compound has a characteristic specific rotation, which may be used as a physical constant to distinguish it from similar compounds. The polarimeter contains two Nicol's prisms: the polariser and the analyser. Plane-polarized monochromatic light (in the polariser) is passed through a solution of optically active compounds to the analyser, where rotation is measured.

Scheme of a polarimeter:

Reagents: α-D-glucose Procedure: Fill up the polarimetric tube with distilled water and take care to avoid introducing air bubbles. Adjust the polarimeter to zero degree using water (the blank). Starting point for the determination is the average of several readings with the water blank. Weigh 4 g of D-glucose and dissolve it in about 40 ml water in a 50-ml volumetric flask. Fill the flask to the mark with distilled water. Mix the content, and immediately transfer the solution to the polarimetric tube. Determine the observed rotation in the polarimeter. Repeat the measurements in 5 minutes intervals for half an hour. The rest of glucose solution is then warmed up for 10 minutes at the temperature 60°C. After cooling, measure the optical rotation again. Evaluation: Making correction for the water blank if necessary, calculate the specific rotations in each time interval and after warming up. Construct the curve of dependence of specific rotation on time.

half-shade

prism

polariser polarimetric tube

analyser

objective

lens

eye

light

source

29

4.3. Polarimetric determination of glucose in urine Principle: Concentration of glucose in urine is determined polarimetrically. Reagents: Sulphosalycilic acid Charcoal

Procedure: Urine have to be clear, colourless and without proteins. Perform first the test for proteins. a) Negative result – add 1 spoon of charcoal to 20 mL of urine in a beaker, shake and filtrate. b) Positive test – add 20 mL of acetate buffer (pH 4.7) and 1 spoon of charcoal to 20 mL of urine in beaker and keep the mixture boiling for 1 minute. Filtrate and cool to 20°C. Fill up the polarimetric tube with distilled water and take care to avoid introducing air bubbles. Adjust polarimeter to zero degree using water – blank. Starting point for the determination is the average of several readings with water blank. Transfer the filtered urine to the polarimetric tube. Determine the optical rotation and calculate the concentration of urine glucose (g/L). Correction must be made for dilution of the sample with acetate buffer. Multiply by 2. Evaluation: Compare values of urine glucose by various methods.

4.4. Determination of glucose in blood and urine by reagent kit OXOCHROMGLUKOSA Principle: Glucose oxidase (GOD) catalyses oxidation of D-glucose by oxygen to D-glucono-1,5-lactone and hydrogen peroxide, or gluconate (in the presence of base). Hydrogen peroxide reacts with 3-methylphenol and 4-aminoantipyrine in the presence of peroxidase and a quinine diimine dye is formed. Material: Samples of serum and urine Reagents: - glucose reagent (phosphate buffer pH 8, 140 mmol/L; 3-methylphenol 10 mmol/L; 4-aminoantipyrine 1 mmol/L; glucose oxidase 166 µkat/L; peroxidase 16 µkat/L) - glucose standard solution 5 mmol/L Procedure: The sample of urine is diluted: 0.1 mL of urine + 1 mL of water. Pipette the reagents into test tubes:

30

Reagent (mL) serum urine standard blank Glucose reagent 2.00 2.00 2.00 2.00 Serum 0.02 - - - Diluted urine - 0.02 - - Glucose standard - - 0.02 - Distilled water - - - 0.02 Shake all test tubes and incubate for 30 minutes at room temperature or for 1 minutes at 37°C. Protect from exposure to light. Measure the absorbance (A) of serum or urine samples and the standard against blank at 498 nm within 40 minutes after incubation. Evaluation: Glucose concentration (mmol/L): csample = cstandard x D x (Asample / Astandard) where D is dilution of urine samples (equals 11) Reference values: Capillary blood fB-glucose) 3.3 – 5.6 mmol/L Plasma or serum (fP, fS-glucose) 4.2 – 6.1 mmol/L Urine (dU-glucose) 0 – 0.25 g (0 – 1.4 mmol) per day Higher values – hyperglycemia, glucosuria Lower values – hypoglycemia Hyperglycemia is characteristic for diabetes mellitus, the disorder of intermediate metabolism caused by insufficient regulation by insulin; i.e. disorder in insulin synthesis, antibodies to insulin, its breakdown, low ability of cells to respond to insulin stimulus. Glycosylated hemoglobin (HBA1) may also be used in the detection of diabetes. HBA1 is a result of non-enzymatic glycosylation of hemoglobin. The test provides an accurate index of the mean concentration of blood glucose during two to three preceding months, complementing so the more traditional ways of glucose control, such as glucose testing in urine and blood. 4.5. Oral glucose tolerance test (oGTT) Principle: Determination of blood glucose following an oral glucose load is the classical method for discovering glucose intolerance. The oGTT should be performed in all patients with high fasting blood glucose or glucosuria. It may also be of diagnostic value in patients with hypoglycaemia. High dose of glucose are given to the patient orally and the changes in blood glucose concentration are monitored. Material: Capillary blood

31

Reagents: -Bio-La-Test reagent kit “OXOCHROMGLUCOSA” - glucose standard solution (5 mmol/L) Procedure: After an overnight fasting, patient is given glucose in a dose of 75 g, dissolved in 300 mL of water. Solution should be ingested in less than 5 minutes. Capillary blood samples are drawn in the fasting state, and 1 h, and 2 h after oral glucose load. Blood samples are collected into plastic tube containing heparin, and they are centrifuged immediately (1000 rpm / 5 min). Blood plasma (supernatant) is used for determination. Evaluation: cP-glucose (mmol/L) 0 h 1 h 2 h Normal glucose tolerance < 6.1 < 12 < 9 Impaired glucose tolerance < 7 < 12 9 - 12 Diabetes mellitus ≥ 6.1 ≥ 12 ≥ 12 4.6. Determination of glucose and ketones by diagnostic test strips. Principle: Diagnostic test strip have test areas (indicator zone) attached to a white plastic strip. Glucose indication zone (in Glukophan or Diaphan) contains enzymes glucose oxidase and peroxidase with a special chromogen which forms red-coloured products in the presence of glucose. The ketone indicator zone (Glukophan) is impregnated with an alkaline buffer and nitroprusside giving with acetoacetic acid or acetone a violet colour the intensity of which is proportional to the ketone concentration. Procedure: Dip the strip into the urine for 3 seconds and withdraw it immediately. Wipe off the excess urine on the vessel edge. Glucose zone – after 3 minutes compare the colour with the colour chart. Ketone zone - after 1 minute compare the colour with corresponding colour chart. The test can record the presence of glucose from approx. 2 mmol/L, creating distinct orange colour. Higher concentrations of urinary ascorbic acid can lead to false results. Diagnostic test strips for determination of blood glucose, which are used on the glucometer, are based on the same principle.

32

5. LIPIDS 5.1. Chemical reactions of lipids.

Principle:

The alkaline hydrolysis (saponification) of lipids yields glycerol and soluble alkali salts of fatty acids (soaps). Ca2+ and Pb2+ salts of fatty acids are insoluble soaps. The double bonds of unsaturated fatty acids contained in lipids are able to add hydrogen or halogen. Cholesterol gives with sulphuric acid, in a water-free medium, unsaturated polymeric products of blue-green colour.

Reagents:

Oil; alcohol solution of KOH (1 mol/L in ethanol)

Solution of CaCl2 (50 g/L); solution of lead (II) acetate (50 g/L)

Sulphuric acid (1 mol/L); conc. sulphuric acid

Dichloromethane; ether; acetic acid anhydride; bromine water

Procedures:

Saponification of lipids

Mix 2 mL of oil and 10 mL of alcoholic solution of KOH in a beaker. Warm up and mix the solution, the saponification of lipids occurs after 10-20 minutes. Pour the mixture from the beaker into hot distilled water (50 mL) and the solution of alkali soap is formed.

Precipitation of soap by Ca2+and Pb2+ ions

Place 1 mL of alkali soap solution into a test tube and add a few drops of CaCl2 solution, a viscous precipitate of calcium salts of fatty acids arises. Similarly prepare a white precipitate of lead soap in a different test tube by adding lead (II) acetate to the alkali soap.

Precipitation of Ca2+ ions from tap water

Add gradually alkali soap solution to the 1 mL of tap water, an insoluble calcium soap is formed. The excess of alkali soap manifests by foam formation.

Detection of unsaturated fatty acids

Add ether and a few drops of oil into the test tube. Then add a few drops of bromine water and shake well. Observe and describe the gradual change of the colour.

Detection of cholesterol in lipids

Dissolve a few crystals of cholesterol in approx. 5 mL of dichlormethan, add 1 mL of acetic acid anhydride and a few drops of H2SO4. Perform the test in the hood, use dry test tubes, work carefully and use protecting shield.

33

Evaluation:

Observe and describe results of lipids reactions!

5.2. Determination of the iodine number of lipids.

Principle:

Iodine number is an indication of the quantity of unsaturated fatty acids present in a fat; it represents the number of grams of iodine absorbed by each 100 g of oils. It is a measure of number of double bonds presented in unsaturated fatty acids of oils. Iodine number is determined by indirect iodometry using Winkler's reagent.

Reaction of bromide and bromate in an acidic medium gives bromine, which is added on double bonds of unsaturated fatty acids. The excess of bromine releases an equivalent amount of iodine from the potassium iodide. The iodine concentration is then determined by titration with Na2S2O3. Starch gel is used as indicator.

To calculate the iodine number, it is necessary to find out the total amount of added bromine in Winkler's reagent in absence of fat. The procedure is the same. The difference in the amount of Na2S2O3 represents the amount of iodine equivalent to the bonding of bromine to double bonds of fatty acids.

Reagents:

Oil solution (1 g/20mL CH2Cl2)

Winkler's reagent (5.56g KBrO3, 200g KBr in 1000 mL of water)

HCl (100 g/L)

KI - substance

0,05M- Na2S2O3

starch gel

dichloromethane

Procedure:

Oil analysis:

Add 2 mL of analysed oil (100 mg of oil) and 4 mL of Winkler°s reagents and 2 mL of HCl solution. Then close the flask with a sealing and shake the liquid content well for a few minutes.

Add a small amount of potassium iodide to the mixture and shake it well. Iodine, which is released, is titrated by the solution of 0.05M-Na2S2O3. As soon as the solution starts losing its brown colour, add a starch gel as an indicator and perform the titration procedure until the solution is completely decolourised.

Blank test:

Blank test is performed in the same way, but 2 mL of the analysed oil is replaced by 2 mL of dichloromethane. Pipette 2ml of dichloromethane (without fat) into the flask, add 4 mL of Winkler's reagent and 2 mL of HCl. Shake the content well, add small amount of potassium iodide and perform titration by 0.05M-Na2S2O3.

34

Scheme of reactions:

5 Br- + BrO3- + 6 H+ → 3 Br2 + 3 H2O

- CH = CH - + Br2 → -CHBr – CHBr-

Br2 + 2 KI → 2 KBr + I2

I2 + 2 Na2S2O3 → 2 NaI + Na2S4O6

Calculation:

From Na2S2O3 used in the blank test (a), subtract the consumption of Na2S2O3 used during the oil analysis (b). The difference (x) corresponds to the amount of iodine eqivalent to bromine added on double bonds of fatty acids.

Iodine number = g of iodine / 100g of fat

1 mol/l Br2/2 = 1 mol/l I2/2 = 1 mol/l Na2S2O3

1 ml 0.05 mol/l Na2S2O3 = 126.9 x 0.05 = 6.34 mg of iodine

a ml - b ml = x ml

iodine number = x ml x 6.34 mg

Evaluation:

Compare iodine number of analyzed fat with the norm and consider the quality of fat.

Iodine numbers of some fats:

pork grease 49-64

sunflower oil 125-136

fish oil 150-180

olive oil 80-88

butter 26-38 5.3. Determination of total cholesterol in blood serum Principle: Esters of cholesterol are hydrolyzed by cholesterolesterase CHES to cholesterol and fatty acids. Cholesterol (from hydrolysis and the free one) is then oxidized by cholesteroloxidase CHOD to cholestenon and hydrogen peroxide, which is allows oxidative copulation of 4-aminoantipyrine with phenol by peroxidase POD. A red dye is formed by this reaction. Solutions: Working solution from diagnostic kit Bio-La-Test Oxochrom CHOLESTEROL (phosphate buffer pH 7.5, sodium cholate, 4-chlor-3-methylphenol, NaCl, 4-aminoantipyrin, CHES, CHOD, POD, detergent) Standard cholesterol solution (5.17 mmol/L)

35

Blood serum Procedure: Prepare 3 tubes according to the table: Tube: 1 2 3 Solutions Sample Standard Blank Working solution (µl) 2500 2500 2500 Standard solution (µl) - 20 - Blood serum (µl) 20 - - Shake well the content of the tubes and incubate 5 min at 37 °C. Let cool to room temperature and measure absorbance at 510 nm against blank. Calculation: Concentration of total cholesterol (mmol/L) = A serum/A standard * c standard Normal values: maximum 5.2 mmol/L 5.4. Plasma lipoproteins analyses Introduction: In the blood stream, lipids are transported in the form of complex particles called lipoproteins. Lipoproteins contain lipids (triacylglycerols, cholesterol and its esters, phospholipids, glycolipids) and proteins (apolipoproteins). The results of analytical ultracentrifugation have led to the following classification of lipoproteins: a) Chylomikrons – density < 0.95 g/mL; size 80-800 nm; lipid content (by weight): 99% (85-95% triacylglycerols, 1-2% cholesterol, 3-6% phospholipids). The protein apoB appears to be an essential component of chylomicrons. b) Very Low Density Lipoproteins (VLDL ) – density range 0.95 – 1.006 g/mL; size 28-75 nm; lipid content (by weight): 90% (50-60% triacylglycerols, 15% cholesterol esters, 10% cholesterol, 15-20% phospholipids). The apolipoproteins of VLDL are apoB, apoC and apoE. These lipoproteins are also termed pre-β-lipoproteins with the electrophoretic mobility of α2-globulins. c) Low Density Lipoproteins (LDL ) - density range 1.019 – 1.063 g/mL; size 21-25 nm; lipid content (by weight): 78% (10% triacylglycerols, 38% cholesterol esters, 10% cholesterol, 20% phospholipids). The apolipoprotein of LDL is apoB with only traces of apoC and apoA. These lipoproteins are also termed β-lipoproteins with the electrophoretic mobility of β-globulins. d) High Density Lipoproteins (HDL ) - density range 1.063 – 1.21 g/mL; size 9-12 nm; lipid content (by weight): 50% (3% triacylglycerols, 15-20% cholesterol esters, 5% cholesterol, 25-30% phospholipids). The apolipoprotein of HDL is apoA with small portion of apoC. These lipoproteins are also termed α-lipoproteins with the electrophoretic mobility of α1-globulins. Several variants of these families, as well as abnormal lipoproteins, occur in disease states. High values of lipoproteins – hyperlipoproteinemia (high level of LDL-cholesterol) are very significant risk factors in the development of coronary heart disease (CHD) subsequent to the onset of atherosclerosis. High values of HDL-cholesterol reduce this risk factor.

36

Frederickson et al. classify the familial hyperlipoproteinemias into five distinct types: Types of hyperlipoproteinemias: Type of hyperlipoproteinemia

Appearance of plasma

Electrophoretic pattern

Triglyceride level

Cholesterol level

Postheparin lipolytic activity

Type I Milky Chylomicrons present, other lipoproteins ↓ Pre-β often ↑

↑↑↑ ↑ ↓

Type II Clear (IIa) Slightly turbid (IIb)

β ↑↑↑ Pre-β ↑

Normal (IIa) or ↑

↑↑↑ Normal

Type III Turbid or clear

“broad β” present; pre-β ↑

↑ ↑↑ Normal

Type IV Turbid or clear

Pre-β ↑↑↑ ↑↑ ↑ Normal

Type V Turbid or milky

Chylomikrons present; pre-β ↑↑

↑↑ ↑ Normal or ↓

Plasma lipoprotein analysis: 1) Ultracentrifugation a) discontinuous gradient – plasma lipoprotein fractions are isolated accordingly to their density. Advantage = very good separation; disadvantage = expensive, time consuming. b) density gradient – a gradient is made up of solutions of different density in layers. Advantage = lipoprotein fraction isolation during one ultracentrifugation; disadvantage = special devise needed, time consuming (24-36 hours), expensive. 2) Chromatographic and electrophoretic methods a) electrophoresis on agarose gel; isoelectric focusation b) gel chromatography (with binding antibodies) c) ion chromatography d) high performance liquid chromatography 3) Precipitation methods a) Lipoprotein fractions are separated by precipitation with the help of different precipitation reagents, Advantage = speed (10 min – 2 h); cheap; useful for clinical screening analyses; disadvantage = not acceptable for apolipoprotein analyses. Calculation of lipids concentration 1) A serum contains 198 mg % of total cholesterol. Calculate the concentration in mol/L. Mr (cholesterol) = 386.64 2) Calculate the total serum cholesterol concentration. Absorbance of serum (0.350); absorbance of cholesterol standard solution (0.325); concentration of cholesterol standard solution (5.17 mmol/L)

37

3) a. 100 µL of cholesterol standard solution (200 mg %) and 400 µL of saline are mixed. Calculate quantity (µg) of cholesterol contained in 10, 20, 30, 40 µL of solution. Absorbances measured using standards 1-4 (cuvette 1 cm; total volume 1 mL) are:

No. µL of standard solution

µg of cholesterol Absorbance

1 10 0.065 2 20 0.127 3 30 0.196 4 40 0.260

b. serum is diluted with saline in ration 1:2. 20 µL of diluted serum is used for cholesterol determination and the absorbance was 0.200. Calculate: - quantity (µg) of cholesterol in 20 µL of diluted serum (use calibration graph) - cholesterol concentration in serum (mmol/L) 4) Calculate serum HDL-cholesterol concentration. Absorbance of serum (0.290); absorbance of HDL-cholesterol standard solution (0.270); concentration of HDL-cholesterol standard solution (1.034 mmol/L) 5) Calculate serum triacylglycerols (TAG) concentration. Absorbance of serum (0.210); absorbance of TAG standard solution (0.650); concentration of TAG standard solution (3.930 mmol/L) 6) Calculation of LDL-cholesterol (LDL-C): c (LDL-C) = c (CC) – c (HDL-C) – (c (TAG) / 2.18) c (CC) total cholesterol concentration c (HDL-C) HDL-cholesterol concentration c (TAG) triacylglycerols concentration Index of atherogenicity (IA) calculation: IA = c (CC) / c (HDL-C) Calculate c (LDL-C) and IA, evaluate the finding and estimate the type of hyperlipoproteinemia: a) c (CC) = 4.731 mmol/L c (HDL-C) = 1.249 mmol/L c (TAG) = 1.27 mmol/L b) c (CC) = 4.950 mmol/L c (HDL-C) = 0.65 mmol/L c (TAG) = 1.60 mmol/L c) c (CC) = 7.60 mmol/L c (HDL-C) = 0.95 mmol/L c (TAG) = 1.20 mmol/L

38

d) c (CC) = 4.95 mmol/L c (HDL-C) = 1.15 mmol/L c (TAG) = 1.60 mmol/L Determination of HDL-cholesterol Principle: β-lipoproteins (VLDL, LDL) are precipitated from blood serum by phosphowolframic acid and Mg2+ ions. The precipitate is removed by centrifugation and cholesterol is determined by the diagnostic kit Bio-La-Test Oxochrom CHOLESTEROL Solutions: Phosphowolframic reagent MgCl2 (2 mol/l) Working solution from diagnostic kit Bio-La-Test Oxochrom CHOLESTEROL Standard cholesterol solution (1.20 mmol/l) Blood serum Procedure: a) Isolation of HDL Into one centrifuge tube, pipette 1 ml of blood serum, 100 µ of phosphowolframic reagent and 25 µl of MgCl2 . Mix well, incubate 15 min at room temperature and centrifuge 30 min at 4000 RPM. The supernatant will be used for cholesterol determination in step b). b) determination of cholesterol in the supernatant containing HDL Prepare 3 tubes according to the table: Tube: 1 2 3 Solutions Sample Standard Blank Working solution (µl) 1000 1000 1000 Standard solution (µl) - 40 - Supernatant (µl) 40 - - Shake well the content of the tubes and incubate 5 min at 37 °C. Let cool to room temperature and measure absorbance at 510 nm against blank. Calculation: Concentration of HDL cholesterol (mmol/l) = A serum/A standard * c standard * 1,125 (1,125 is a correction for dilution during HDL isolation) Normal values: minimum 1.2 mmol/l Calculation of LDL-cholesterol concentration in blood serum c(LDL-chol) = c(total chol) – c (HDL-chol) – c(TAG)/2.18 Normal values: maximum 3.4 mmol/l

39

5.5. Determination of triacylglycerols in blood serum Principle: Triacylglycerols TAG are hydrolyzed by lipoproteins lipase LPL. The released glycerol is converted by glycerolkinase to glycerol-3-phosphate, which is than oxidized by glycerolphosphateoxidase GPO to dihydrogenphosphate and hydrogen peroxide, which allows oxidative copulation of 4-aminophenazone with N-ethyl-N-(3-sulphopropyl)-m-anisidine ESPAS) by peroxidase POD. A red dye is formed by this reaction. Solutions: Working solution from diagnostic kit Bio-La-Test Oxochrom TRIACYLGLYCEROLY (PIPES buffer, pH 7.0, ATP, 4-aminoantipyrin, ESPAS, GPO, GK, POD, LPL, detergent and stabilizers) Standard TAG solution (1.42 mmol/l) Blood serum Procedure: Prepare 3 tubes according to the table: Tube: 1 2 3 Solutions Sample Standard Blank Working solution (µl) 1500 1500 1500 Standard solution (µl) - 10 - Blood serum (µl) 10 - - Shake well the content of the tubes and incubate 10 min at 37 °C. Let cool to room temperature and measure absorbance at 510 nm against blank. Calculation: Concentration of total cholesterol in blood serum (mmol/l) = A serum/A standard * c standard Normal values: maximum 1.7 mmol/l

40

6. NUCLEIC ACIDS 6.1 Isolation and degradation of nucleoproteins from bakers'yeast Principle: In cells, nucleic acids are bound to proteins forming nucleoproteins. Nucleoproteins and ribonucleic acids can be isolated in a simplified way from bakers' yeast. Isolated substances are then degraded into purine or pyrimidine bases, pentose, and phosphoric acid, which can be detected by specific reactions. Reagents: Fresh bakers' yeast NaOH (1 mol/l) Ether Acetic acid, concentrated. Acetic acid, diluted. Hydrochloric acid, concentrated. Sulphuric acid, diluted. Ethanol. Tollens' reagent, freshly prepared (made by adding excess ammonia solution to a solution of silver(I) ions. It contains the [Ag(NH3)2]

+ ion in alkaline solution. The complexing of Ag+ by NH3 prevents precipitation of AgOH. 1 ml AgNO3 (1 mol/l) is precipitated by NaOH (1 mol/l), the brown precipitate (AgOH) is then dissolved in NH4OH. Do not store Tollens' reagent or the products after testing with it - danger of explosion. Ammonium molybdate (5 g (NH4) 2MoO4 in 100 ml of water and 35 ml conc. nitric acid. Hydrochinone (solid). Procedure: Isolation: Rub 10 g of yeast, 10 drops of ether, and 10 drops of water with sand in a mortar. Then add: 50 ml NaOH (cooled to 0-4°C, mix well, allow to stand in a refrigerator for 15-30 min, and neutralize by cool acetic acid (concentrated first, then diluted) to pH 6.0-6.5 (indicator paper). The suspension is then divided into two centrifuge tubes and centrifuged at 1000 g for 15 min. The combined supernatant is then acidified with HCl to pH 3-4 and, in a volumetric cylinder, a fourfold volume of ethanol is slowly added under stirring. The formed precipitate is then centrifuged. The sediment contains the raw ribonucleic acid. Hydrolysis: The sediment is mixed with 20 ml of diluted H2SO4, transferred into a flask and boiled on a water bath for 20-60 min. The mixture is cooled, centrifuged, and the supernatant is used for further tests. Tests for degradation products: a) Purine bases – 2 ml of the hydrolysate is alkalized to pH 8-9 with ammonia and a few

drops of Tollens' reagent is added. A white precipitate of silver salts of adenine and guanine is formed.

b) Pentoses - by the reaction with Bial's reagent (see saccharides).

41

c) Phosphoric acid - add slowly ammonium molybdate in HNO3 to 1 ml of the hydrolyzate. A yellow ammonium salt of phosphomolybdic acid is formed. On addition of hydrochinone, the colour turns to blue (reduction of Mo(VI) to blue Mo(V)).

6.2 Demonstration of dehydrogenase activity of xanthinoxidase Principle: Xanthinoxidase is a low specific dehydrogenase catalysing the oxidation of hypoxanthin via xanthin to uric acid and H2O2. It belongs to flavine enzymes with flavinadeninedinucleotide (FAD) as prosthetic group. Xanthinoxidase is contained e.g. in kidney or in milk. Formaldehyde can be used as a substrate in the model of xanthinoxidase action since this low specific enzyme oxidizes even aldehydes. Methylene blue serves as an acceptor of hydrogens provided by the reduced prosthetic group of the enzyme. On reduction, methylene blue forms colourless leuco form.The experiment is observed under anaerobic conditions (beneath a layer of paraffine oil). By mixing with air, hydrogen passes from the leuco base to oxygen and the reaction mixture turns back to blue. This reaction is used as a test for the native state of milk; in boiled or pasteurised milk the xanthinoxidase activity is absent.

Reagents: Fresh native milk. Boiled milk. Methylene blue (5 g/l). Paraffine oil. Formaldehyde (1 g/l). Procedure: Pipette 5 ml of native milk into a test tube and 5 ml of boiled milk into the second one. Add 0.5 ml of methylene blue solution and 0.5 ml of formaldehyde solution to both tubes, mix well and overlay with paraffine oil (about 1 ml). Write down the time and let both test tubes incubate at 37°C. Observe the difference in colour and determine the time necessary for decolourizing the tube with native milk. Then shake well both tubes to provide good contact with air. Observe and write down the observed colour changes. 6.3 Determination of uric acid in blood serum Introduction: Uric acid is formed mostly in the liver as final product of purine bases catabolism in man. The elevation of uric acid level in serum (hyperuricemia) reflects always a disturbance of purine

HH

OH

H

OHOH

HOH

O

H2O

H2O

FAD

FADH

Methylene blue

(reduced)

Methylene blue

(oxidized)

½ O2

H2O2

42

metabolism, either of genetic origin or a secondary disturbance provoked by a disease such as leukemia, myelom or kidney diseases. A long-term hyperuricemia may, owing to low solubility of uric acid at physiological pH, cause the gout. The gout is characterized by the deposition of uric acid salts in synovial liquid in the joints of the feet and hands (esp. in the big toe), in kidney and under the skin.

Principle: Uric acid is oxidized by atmospheric oxygen in the presence of uricase to allantoin, carbon dioxide and hydrogen peroxide. H2O2 in the presence of catalase oxidizes methanol to formaldehyde that condenses with acetylacetone and ammonium ions to 3,5-diacetyl-1,4-dihydrolutidine suitable for the photometry. Reagents: Solution I - phosphate buffer pH 7, peroxidase, uricase, 4-aminoantipyrine, potassium ferrocyanide, 4-chloro-3-methylphenol Solution II - phosphate buffer pH 7, 4-chloro-3-methylphenol Uric acid standard solution (357 µmol/l) Blood serum Procedure: Pipette into four test tubes according to the scheme: Test tube 1 (Sample) 2 (Standard) 3 (Control 1) 4 (Control 2) Solution 1 2 2 2 - Serum 0.1 - - - Standard solution - 0.1 - - Water - - 0.1 - Solution 2 - - - 2 Mix and incubate at 25°C for 10 min in dark. Measure the absorbance at 500 nm in a 1 cm cuvette against water.

Absorbance A1 A2 A3 A4

Uric acid concentration in serum is calculated:

357*)(

)mol.l(32

4311-

AA

AAAC

−+−

=µ

Reference values: fS - uric acid: males 200-420 µmol/l

females 140-340 µmol/l

Evaluation: Compare obtained values to the norm with respect to hyperuricemia.

43

7. TOXICOLOGY AND ANALYSIS OF DRUGS 7.1. Determination of salicylic acid in Acylpyrine Principle: Acetylsalicylic acid may partially hydrolyse to form salicylic and acetic acid. So, these substances are often present as admixtures to acetylsalicylic acid and there is a need for exact determination of the content of acetylsalicylic acid in pharmaceutical preparations. The analysis cannot be done by simple titration with NaOH as all three compounds in question are acids and form sodium salts. But only one of them, sodium acetylsalicylate, containing an ester group, can be hydrolysed with sodium hydroxide to form sodium acetate and sodium salicylate. This hydrolysis is suppressed in alcoholic solution. The determination is carried out by two titrations, the first one under conditions not favouring hydrolysis (room temperature, addition of ethanol), where sodium acetylsalicylate is formed. There after an excess of aqueous sodium hydroxide solution is added and sodium acetylsalicylate is hydrolysed at about 90°C. Sodium hydroxide, which was not consumed during hydrolysis, is then titrated by an acid in the second titration. If the consumption of sodium hydroxide during the titration of acetylsalicylic acid is the same as the amount of sodium hydroxide necessary for hydrolysis of sodium acetylsalicylate, the sample contains only pure acetylsalicylic acid. Higher consumption of sodium hydroxide during the first titration indicates the presence of salicylic and acetic acid in the sample.

Reagents: Tablet of Acylpyrine (declared content: 500 mg of acetylsalicylic acid) Ethanol (96%) 0.1 M-NaOH 0.05-M H2SO4 Phenolphthalein Procedure: Dissolve one tablet of Acylpyrine in 10 ml of ethanol in an Erlenmeyer flask, add 5 drops of phenolphthalein and titrate with 0.1 M-NaOH until pink colour. Then add 35 ml of 0.1 M-NaOH, insert a boiling stone, cover with a glass lid, and heat the mixture gently for 10 minutes using the Bunsen burner. After the mixture has cooled down, titrate remaining sodium hydroxide with sulphuric acid (0.05 mol/l) until the reaction mixture decolourises. Calculation: Determination of acetylsalicylic acid (C9H8O4, Mr 180.2) content: 1 ml of 0.1 M-NaOH corresponds to 1ml of 0.1 M-C9H8O4 that is 0.01802 grams of C9H8O4 = (35 - consumption of 0.05 M-H2SO4) x 0.01802 Evaluation: − Calculate the theoretical consumption of 0.1M-NaOH which reacts with 500 mg of

acetylsalicylic acid − Compare the experimental consumption of 0.1 M-NaOH during the first and the second

titration and explain the difference. − Calculate the weight of acetylsalicylic acid in one Acylpyrine tablet and express the result

in percent of declared content.

44

7.2 Quantitative determination of salicylates by photometry. Principle: Photometry can be used in toxicology for the determination of salicylates in biological material. The principle of this determination is a quantitative formation of colour complex salt of salicylic acid with ferric ions. The reagent contains not only ferric ions but also hydrochloric acid and mercuric chloride necessary for the removal of proteins that may be present in the sample. The concentration of salicylates in the examined sample is determined from the calibration curve. Reagents: Salicylic acid solution (c=?) Stock solution of salicylic acid (3.62 mmol.l-1) Reagent (40g Fe(NO3)3.9H2O + 40g HgCl2 + 120ml 1M-HCl in 1 litre of solution) Procedure: a) Preparation of standard solutions Prepare the series of standard solutions and one blank sample according to the following scheme.

Solution A B C D E Blank

Standard solution of salicylic acid

(ml)

0.2 0.4 0.6 0.8 1.0 -

Distilled water (ml) 0.8 0.6 0.4 0.2 - 1.0

Reagent (ml) 5 5 5 5 5 5

Concentration of salicylic acid

(mmol.l-1

)

0.72 1.45 2.17 2.9 3.62

b) Wavelength selection Measure the absorption spectrum of the formed complex salt in the region 430-700 nm using the solution D and a 1 cm cuvette. Adjust the wavelength on the photometer, insert the cuvette containing the blank sample, adjust the absorbance value to zero, insert the cuvette containing the sample (D), and measure the absorbance of the sample. Follow these steps after every change in wavelength (use 430, 450, 480, 500, 520, 530, 535, 540, 545, 550, 600, 650, 700 nm). Record absorbance values in a table and trace the absorption spectrum, i.e. the dependence of absorbance on wavelength. Read the wavelength of absorption maximum and use it later for the determination. c) Calibration curve Measure the absorbance of solutions A-E in 1 cm cuvette at 540 nm against the blank. Trace the dependence of absorbance values on salicylic acid concentration (including the beginning - zero concentration). d) Determination of salicylic acid concentration Using calibration curve:

45

Pipette 1 ml of salicylic acid solution of unknown concentration and add 5 ml of reagent. Measure the sample in 1 cm cuvette at 540 nm against the blank. Read the value of the concentration from the calibration curve. Using standard sample: To calculate the concentration of the test sample ct, use the formula below, where At is the absorbance of the sample with salicylic acid of unknown concentration and As is the absorbance of standard sample (for example B) of known concentration cs.

ST

STVZVZ A

CAC

.=

Evaluation: Compare the results obtained by both methods. 7.3. Identification of levomepromazine and its metabolites in biological materials by thin layer chromatography. Principle: Levomepromazine belongs to the group of toxicologically important substances called phenothiazines. It is an active constituent of several medicaments (Tisercine) that are used as psychopharmacs and neuroleptics. Maternal form of levomepromazine in gastric content and levomepromazine metabolites in urine are separated by partition chromatography on thin layer of silica gel. Detection is performed using Marquis reagent. Reagents: solution of levomepromazine solution containing levomepromazine and its metabolites extract obtained from gastric content extract obtained from urine mobile phase: ethylacetate-methanol-ammonia (34:4:2) Marquis reagent (90 mL of 96% sulfuric acid, 4 mL of 40% formaldehyde) Procedure: Draw carefully a line (START) on Silufol folia and mark three points on this line (cca 15 mm from each other). Apply on the start (using capillary) the solutions of the standard and extracts from gastric content and urine. Put the folia in chromatography chamber in the way that lower margin is immersed to mobile phase but the start line is above the level of the liquid. Let the mobile phase rise 3-5 cm from the upper margin of folia, then remove folia from the chamber and mark the position of the FRONT. Dry folia using hair dryer and then spray over the folia Marquis reagent (work in fume-hood). Levomepromazine and its metabolites react with Marquise reagent and blue-violet colored spots are developed. Mark these spots. Evaluation: Calculate Rf values for maternal drug and metabolites.

46

7.4. Determination of nitrates in biological material by ion selective electrode Introduction: Nitrates present in the food are reduced to toxic nitrites by micro flora of oral cavity and in case of some infections also by intestinal micro flora. This situation may be severe after the intake of high quantities of nitrates (more that 300 mg for adults). Moreover, high concentration of nitrates in drinking water is an indicator of possible bacterial contamination. Principle: According to Nernst equation, the electric potential of nitrate ion selective electrode (ISE) is a function of concentration of nitrates in solution. This potential is measured as electromotive force (EMF) of open circuit voltaic cell consisted of nitrate ISE and referent kalomel electrode. The relation between potential of ISE and concentration of nitrates (NO3-) is given by equation: E = E0 + (RT/nF)* 2.303 * log c This equation for nitrate ISE ( n = -1; T = 298 K) acquires following form: E = E0 – 0.059 * log[NO3-] The potential of ISE will decrease with increasing concentration of NO3- EMF is measured in the series of calibration solutions with known concentration of NO3-. Calibration plot (EMF vs log [NO3-]) must be constructed for determination of NO3- concentration. Reagents: Standard solution of KNO3 (concentration of NO3- = 1000 mg/L) Drinking water Vegetables Maceration solution (20 g Al2(SO4)3.18 H2O + 1 g Ag2SO4 in 1 liter of solution) Procedure: a) Construction of calibration plot: Prepare the solutions of KNO3 in concentrations of 100, 10, 5 and 1 mg/L by diluting standard solution of KNO3 (concentration of NO3- = 1000 mg/L). Immerse the electrodes (ISE and referent) to the beaker filled with these solutions and measure EMF. Construct calibration plot using the obtained values of EMF vs logarithm of concentration of NO3-. b) Content of nitrates in drinking water: Measure EMF in the sample of drinking water using nitrate ISE (see item a). Note: It is necessary to wash electrodes with distilled water between individual measurements. Calculate the concentration of nitrates in drinking water using calibration plot (see item a). c) Determination of nitrates in vegetables: 100 g of clean (washed with distilled water and dried) vegetables is homogenized by cutting and kitchen mixer with 50 mL of distilled water. Mix 15 g of homogenized vegetables with 30 mL of boiling (95°C) maceration solution in the beaker. Stir the mixture for several minutes with glass rod and then let the suspension sediment. Transfer supernatant to calibrated flask of 100 mL using piece of cellulose for filtration. Repeat maceration of vegetables three times and collect the supernatants in

47

calibrated flask of 100 mL as described above. Cool down the solution in calibrated flask in cold tap water and fill the flask up to total volume of 100 mL. The content of nitrates is measured as described in item b. Concentration of nitrates in vegetables s calculated as follows: cnitrates (mg/1 kg of vegetables) = cnitrates (mg/L) x 10 Evaluation: According to Czech hygienic and epidemiologic standards, the limit of nitrates in drinking water is 50 mg/L. In food, the highest concentration of nitrates is found in smoked food, meat in tin wraps and in some vegetables (in particular in root vegetables where the concentration of nitrates is above 1000 mg/kg). 7.5. Determination of vitamin C in fruit juices. Introduction: Determination of vitamins content has became routine practice in pharmaceutical industry, clinical biochemistry and food analyses. Vitamins are structurally diverse compounds; hence there is not one universal method for their determination. The principles of vitamins determination are based on their structural features and chemical properties: Principle: Determination of vitamin C is based on its ability to undergo oxidation where dehydroascorbic acid is formed as a product. This property is used in red-ox titration:

NO OH

Cl

Cl

+ N OH

HCl

Cl

HO

C

C

C

HC

C

CH2OH

HO

HO

HO

O

H

O

CH2OH

+

C

C

C

HC

CHO

O

H

O

O

O

Ascorbic acid reacts in acidic milieu with 2,6-dichloro-phenolindophenol (2,6-DPIP). 2,6-DPIP blue colored and its color becomes pink in acidic milieu. Its reduced form (leukoform) is colorless. The solution of 2,6-DPIP is decolorized in the course of titration and beyond titration stop the solution becomes pink. 2,6-DPIP is used as titration agent and indicator of titration stop at the same time. Oxidation of ascorbic acid can be also measured spectrophotometrically; the decrease of absorbance at 590 nm is measured. Reagents: 2% hydrochloric acid 1 mmol/L 2,6-DPIP in 30 mmol/L phosphate buffer, pH 7,0 Instruments: Grinding mortar, burette, titration flasks, balance, fruits and vegetables, juices.

48

Procedure: Preparation of homogenate of fruits or vegetables: Pulverize fruits or vegetables (cca 25 g) in grinding mortar with 2% HCl. Centrifuge resulting suspension (1 000 g; 3 min); transfer supernatant to 50 cm3 graduated flask; fill up to 50 cm3 with 2 HCl and mix well. Vitamin C tablet: Dissolve tablet in 2% HCl; transfer solution to 50 cm3 graduated flask; fill up to 50 cm3 with 2 HCl and mix well. For titration, pipette 0.5 mL of solution to titration flask, add cca 10 mL of distilled water and then titrate. Fruit juice: Dilute juice 10 x before titration; i.e. pipette 5 mL of juice to 50 cm3 graduated flask; fill up to 50 cm3 with 2 HCl and mix well. Titration: Pipette 5 mL of extract to titration flask and titrate with 2,6-DPIP until mixture becomes pink. In case of too low consumption of titration agent, repeat titration with larger volume of extract (e.g. 10 mL). Evaluation: Concentration of ascorbic acid in the samples is calculated as follows: vitamin C (mg/1 g fruits) = (Mr * cagent * Vagent * Vextract)/(Vtitr * m) vitamin C (mg/1 L juice) = (Mr * cagent * Vagent) * Dil/(V titr) cagent concentration of 2,6 DPIP (mmol/L) Vagent consumption of 2,6 DPIP for titration (L) Vextract total volume of extract (L) Vtitr volume of extract used for titration (L) m weight of fruits or vegetables used for the preparation of extract (g) Mr relative molecular weight of vitamin C (176,1 g/mol) Dil dilution of juice Conclusion: Determine the content of ascorbic acid in selected fruit, vegetable or juice. Calculate the amount of vitamin C in 100 g of fruit or vegetable or in 1 liter of juice. 7.6. Isolation and demonstration of alkaloids from Chelidonium majus Principle: Chelidonium majus contains more than 20 alkaloids, the most of them are biologically active. Major alkaloids are chelidonine, sanguinarine and chelerythrine. Alkaloids are extracted from the plant material by methanol. Their separation from other extracted substances is based on the fact that they are weak organic bases. Weak organic bases are soluble in nonpolar (organic) solvents; in acidic environment they form salts and as such they are soluble in polar solvents (i.e. water). Thus by repetitive distribution between two immiscible phases (i.e. water and chloroform) using changes in pH, it is possible to isolate and purify alkaloids.

49

Reagents: Chelidonium majus - dried drug Standards of alkaloids sanguinarine and chelerythrine Methanol Ether H2SO4 (0.5%) K2CO3 and Na2CO3 (solid substances) Elution system: toluene - methanol (9:1) Dragendorf reagent Procedure: 5 g of crushed dried drug is weighed into an extraction cartridge. The cartridge is placed into the Soxhlet apparatus and the extraction is performed for 4 hours using 300-350 ml of methanol in the distillation flask. Methanol is then evaporated using rotary vacuum evaporator. The evaporation residue is dissolved in 150 ml of 0.5% sulphuric acid. 25 ml of the solution is transferred into a separating funnel and extracted three times with 25 ml of ether. Ether extracts are poured into the ether waste bottle. Water phase, which contains alkaloids, is alkalised by solid Na2CO3 to pH 8. Then the solution is extracted three times with 25 ml of ether. Ether extracts are combined, dried with solid K2CO3 and filtered through cotton. The alkaloids are obtained by evaporation of ether solution. The alkaloids are identified by thin layer chromatography on Silufol, using solvent system toluene-methanol (9:1), detection by UV light or Dragendorf reagent, with the help of alkaloid standards. Evaluation: Calculate RF values of alkaloids of the extract and compare them with those of alkaloid standards.

50

8. ENZYMES 8.1. Demonstration of enzymatic character of peroxidase reaction Principle: Peroxidases are enzymes contained in plants and in some animal tissues. They serve as catalysts for dehydrogenation (oxidation) of substrates by H2O2 that is reduced to water: RH2 + H2O2 R + 2 H2O Peroxidases may have strict substrate specificity, but some of them catalyse the dehydrogenation of a broader spectrum of substrates. Their effect may be observed on substances (substrates) that give coloured products as the result of oxidation, such as tetramethylbenzidine or aminoantipyrine ("pyramidone"). Reagents: 1. Enzyme source: Grate a peeled potato on a plastic grater, rub thoroughly with sea sand in

a bowl and add approximately one volume of water. After all ingredients have been mixed, the obtained thick suspension is used.

2. Pyramidone solution: solution of aminoantipyrine in ethanol (50 g.l-1). 3. H2O2 (hydrogen peroxide) (30 g.l-1). Procedure: Follow the data given in the scheme below:

Test tube No. 1 2 3 4 Potato suspension (ml) 3 3 3 - Water (ml) - - - 3

Tube No.3 is boiled for 2 min Pyramidone solution (ml) 1 1 1 1 H2O2 (ml) - 0.5 0.5 0.5

Agitate a observe colour development Evaluation: Test-tube 2represents a complete system, the results are negative in other tubes (1- absence of H2O2, 3 – enzyme denaturation by boiling, 4 - absence of enzyme). Notes: 1. It is necessary to use potato suspension and not the liquid above the sediment, otherwise

the results would be unconvincing. 2. If the suspension becomes thick during the denaturation procedure, fill in evaporated

water. 3. The colour in the supernatant or filtrate is more perceptible by observing against white

background.

51

8.2. Demonstration of substrate specificity of amylase and sucrose Principle: Amylase and sucrose hydrolytic break down glycosidic bonds. They have, however, different substrate specificity. Amylase splits α-1,4-glucopyranoside bonds of starch and related poly- or oligosaccharides yielding maltose. Sucrose splits N-fructofuranoside bonds of sucrose, yielding an equimolar mixture of glucose and fructose. To prove the substrate specificity of both enzymes consider the fact that none of both substrates exhibits reducing properties while the products of their hydrolysis (maltose, glucose) reduce Fehling's reagent. The proof of reducing substances in test tubes with corresponding couples enzyme-substrate and negative results in test tubes with an enzyme not corresponding to the substrate demonstrate the substrate specificity of amylase and sucrose. Reagents: 1. Enzyme sources:

a) Amylase: diluted saliva - take approximately 30 ml of distilled water into your mouth, after 5 minutes empty the mouth content into a beaker and filtrate through a sparse filter or cotton.

b) Sucrase: crush 2 g of yeast with sand in a mortar, add 50 ml of distilled water, mix well and centrifuge. Decant the supernatant, add 50 ml of water, mix well and centrifuge again. This procedure is then repeated once more. The soluble glycolytic enzymes, which would normally further metabolise glucose and fructose, are washed out from the yeast structure. Sucrose, bound to cell structures, remains. Finally, mix the sediment with approximately 30 ml of water and use the suspension for further investigation. 2. Substrates: a) Starch solution (10 g/l) in NaCl (0.05 mol.l-1)

b) Sucrose solution (0.3 mol.l-1)

3. Fehling's reagent (I and II), see Saccharides. Procedure: Follow the scheme below:

Test tube No. 1 2 3 4 Sucrose (ml) - - 5 5 Starch (ml) 5 5 - - Sucrase (ml) - 2 - 2 Amylase (ml) 2 - 2 -

Incubate at 37°C for 30 min. Use Fehling’s test for detection of reducing saccharides.

Evaluation: Fehling's reagent is reduced only in test tube 1 where amylase cleaves starch to maltose and in tube 4 where sucrose cleaves sucrose to glucose and fructose. Starch and sucrose were not cleaved in tubes 2 and 3.

52

8.3. Inhibition of enzymatic activity - inhibition of catalase by cyanide

Principle: Catalase is an oxidation-reduction enzyme contained in various animal tissues. It breaks down H2O2 to water and molecular oxygen (which escapes from the solution in the form of bubbles or forms a foam). Cyanides block the prosthetic group of catalase. Reagents: 1. Enzyme source: diluted blood (2 drops to 10 ml of water). 2. Substrate: Solution of H2O2 (30 g.l-1). 3. Potassium cyanide (0.3 mol.l-1). (BE CAREFUL - POISON!) Procedure: Pipette 2 ml of diluted blood into three test tubes. Let simmer and then cool down the second test tube; add 5 drops of potassium cyanide to the third one. Then add 1 ml of H2O2 to each test tube, shake well and observe the formation of oxygen (foam formation). Evaluation: The result of the experiment shows that catalase is inactivated not only by boiling but also by potassium cyanide. 8.4. Determination of pH optimum for trypsin Principle: Trypsin is an endopeptidase which hydrolyses peptide bonds of a protein to form peptide fragments that, in contrast to proteins, do not coagulate (precipitate) on addition of trichloroacetic acid. The amount of fragments formed is directly proportional to the rate of enzymatic reaction and is determined by biuret reaction after removing original, non-cleaved proteins by coagulation with trichloroacetic acid. The value of pH corresponding to maximum reaction rate is called pH optimum. Reagents: 1. Albumin (5 g.l-1) in physiological solution NaCl. 2. Britton-Robinson buffer:

component A: H3PO4, CH3COOH, H3BO3 (0.04 mol.l-1) component B: NaOH (0.2 mol.l-1).

3. Trypsin solution: 50 mg trypsin is dissolved in 20 ml of distilled water. 4. Trichloracetic acid (100 g.l-1). 5. NaOH (2 mol.l-1). 6. Biuret reagent: Dilute 45 g potassium-sodium tartrate, 5 g copper(II) sulfate and 5 g KI in

200 ml of NaOH (0.2 mol.l-1) and make up to 1 liter with NaOH (0.2 mol.l-1). Procedure: To the buffers in six test tubes, add albumin and trypsin and let the enzymatic reaction proceed. The reaction is stopped by trichloroacetic acid which also precipites non-cleaved proteins. After the precipitate has been removed by centrifugation, the solution is alkalized and the amount of peptide fragments is determined by biuret reaction. All steps are summarized in the following scheme.

53

Tube 1 2 3 4 5 6 (blank) pH 6.0 pH 7.0 pH 8.0 pH 9.0 pH 10.0 pH 8.0 B.R. buffer (ml) 2.5 2.5 2.5 2.5 2.5 2.5 Albumin (ml) 4

Allow to stand for approx. 5 min at 37°C, then add in half minute intervals Trypsin (ml) 0.5 0.5 0.5 0.5 0.5 - H2O (ml) - - - - - 0.5

Incubate for 30 min at 37°C, then add in half minute intervals TCA 2.0 2.0 2.0 2.0 2.0 2.0

Allow to stand for 5 min, centrifuge at 3000 g for 10 min (supernatant must be clear), then transfer the supernatant to the following set of tubes

Tube 1 2 3 4 5 6 (blank) Supernatant (ml) 2.0 2.0 2.0 2.0 2.0 2.0 NaOH (2 mol/l)(ml) 1.0 1.0 1.0 1.0 1.0 1.0 Biuret reagent (ml) 2.0 2.0 2.0 2.0 2.0 2.0 Stir, allow to stand for 10 min and measure the absorbance at 530 nm in a 1 cm cuvette

against the blank against the blank Evaluation: The measured absorbance values are directly proportional to the reaction rate. Determine the pH optimum from the dependence of absorbance on pH. Notes: 1. Attention! Do not mistake 0.2 mol/l NaOH (buffer component) for 2 mol/l NaOH (biuret reaction). 2. Review the principle of protein determination by biuret reaction. 8.5. Determination of optimum temperature for salivary amylase Principle: α-Amylase cleaves hydrolytic α-1,4-glycosidic bonds of starch to form smaller fragments up to maltose. The substrate for the determination of α-amylase activity is an insoluble, cross-linked starch derivative that contains a dye. The enzyme hydrolyses the substrate to yield smaller, coloured fragments, soluble in water. The colour intensity (measured by photometry) is directly proportional to the activity of α-amylase. Using the dependence of the activity on the temperature, the optimum temperature is determined graphically. Reagents: 1. Substrate: Blue insoluble starch derivative (diagnostic kit “activity of α-amylase”). 2. Solution of salivary amylase: Take approximately 30 ml of distilled water into your mouth, hold for 1 min and filtrate through cotton. 3. Stopping solution: dissolve 1 g sodium carbonate in 90 ml of water, add 10 ml acetone. Procedure: First, get ready water baths of required temperatures. According to the scheme, pipette the reaction components into thick-wall glass centrifuge tubes. Add the substrate and after the incubation, the reaction is terminated by the stopping solution. Photometry is carried out with a clear blue supernatant (or filtrate). The absorbance is directly proportional to α-amylase activity.

54

Test tube No. 1 2 3 4 5 Temperature (°C) 0 13-16 20-25 37 60-65 H2O 1 1 1 1 1 Amylase soln.(ml) 0.1 0.1 0.1 0.1 0.1

Allow to stand for approx. 5 min (pre-incubation), then add to each tube in 30 s intervals. Substrate susp. (ml) 1 1 1 1 1

Allow to stand without stirring for 15 min, then add in 30 s intervals. Stopping soln. (ml) 4 4 4 4 4

Stir well, after 5 min filtrate through a dense filter. The absorbance is measured at 620 nm In a 1 cm cuvette against water.

Evaluation: Plot the measured absorbance values against corresponding temperatures. The position of the maximum on the curve represents the optimum temperature for the activity of salivary α-amylase. Notes: 1. Water baths: Temperature 0°C: water with ice

13-16°C: tap water 20-25°C: water of laboratory temperature 37°C: temperature-controlled water bath 60-65°C: heated water or temperature-controlled water bath

It is necessary to measure the temperature before starting the incubation. 2. The water baths must be of a sufficient heat capacity in order to keep minimal temperature change during incubation, that means the volume should be at least 0.5-1.0 litre. 3. When pipette the substrate suspension prevent it from contamination by saliva. 4. When filtration is used instead of centrifugation, thick-wall tubes are not necessary. 8.6. Determination of Michaelis constant of alkaline phosphatase Introduction: Phosphatases are enzymes which cleave hydrolytically phosphoric acid esters. Alkaline phosphatase (ALP) takes part in the process of digestion and absorption from the gastrointestinal tract. It is present in high concentration in growing bone, in bile, and in the placenta. The level of serum ALP is elevated in osteoblastic bone diseases, hepatic duct or cholangiolar obstruction, and in hepatic diseases. The determination of ALP level helps to distinguish between hepatocellular (the level does not change) and obstructive (increased level) icterus. The level of ALP is physiologically increased in children (growing bones) and during pregnancy. To determine ALP activity, we follow the ALP catalysed hydrolysis of 4-nitrophenylphosphate in alkaline medium. In the course of the reaction, 4-nitrophenol is liberated, which is then determined photometrically. The course of an enzymatic reaction is expressed by the Michaelis-Menten equation:

[ ][ ]SKm

SVV

+= .max

55

The Michaelis constant represents a measure of affinity of an enzyme for a substrate.

Numerically, it corresponds to the substrate concentration at which the reaction rate V is half of its maximal value Vmax. It is expressed in mol.l-1 and depends on the pH and temperature. The lower the value of Michaelis constant, the higher the affinity of the enzyme for the substrate.

To determine Km it is necessary to measure the initial velocity of enzyme reaction at different substrate concentrations that are chosen to cover at least one order of magnitude. Temperature, pH and enzyme quantity must be kept constant. The values of Km and Vmax are most easily determined when the Michaelis-Menten equation is transformed in such a way that a linear graph is obtained. The most common way of doing so is to simply invert both sides of Michaelis-Menten equation to get a double reciprocal plot also called a Lineweaver-Burke plot:

V

1=

maxV

Km . [ ]S

1 +

max

1

V

A plot of 1/V versus 1/S gives a straight line with a slope of KM/Vmax. The intercept on the

vertical axis is 1/Vmax, the intercept on the horizontal one is -1/KM, see Figure 1.

Fig. 1. Lineweaver-Burke plot In order to get the values of the initial velocity (V) we observe the formation of the reaction product at different substrate concentrations using the same amount of enzyme. According to Beer-Lambert law, the quantity of the product is proportional to its absorbance. Hence, the change in quantity of the product for a period of time (i.e. reaction rate) is observed as a change in absorbance. Reagents: 1. Enzyme source: blood serum. 2. Substrate: 4-nitrophenylphosphate, disodium salt (91.5 mmol.l-1). 3. Inhibitor: EDTA in NaOH 1 mol.l-1. 4. N-methyl-D-glucamine buffer pH 10.1. 5. NaCl, MgCl2, concentrations cf. incubation solution.

56

The composition of the solution for incubation: N-methyl-D-glucamine buffer pH 10.1 0.35 mol.l-1, NaCl 70 mmol.l-1; MgCl2 0.50 mmol.l-1; 4-nitrophenylphosphate disodium salt 15 mmol.l-1 Procedure: Prepare substrate solutions of decreasing concentrations: Pipette 1.0 ml of redistilled water into five test tubes, then pipette 1.0 ml of substrate solution into the first one, mix well and transfer 1.0 ml of the content into the next test tube. Mix well and repeat this procedure until all five substrate solutions of concentrations given below are ready. Test tube No. Stock sol. 5 4 3 2 1 c (mmol.l-1) 91.5 45.75 22.87 11.44 5.72 2.86 Then prepare six pairs of test tubes and mark the substrate concentration on each tube. One test tube from each pair is marked S (sample) and the other one B (blank). Pipette 1.0 ml of the buffer into all twelve tubes and add 0.02 ml of the blood serum to those marked S. Place all tubes in a thermostat and let incubate at 37°C for 5 min. Using a stop-watch, start the reaction by pipetting 0.2 ml of substrate and finish it after exactly 15.0 min by adding 0.5 ml of inhibitor. Follow the time scheme below: Test tube No. 1 2 3 4 5 Stock sol. c (mmol.l-1) 2.86 5.72 11.44 22.87 45.75 91.5 Sample/Blank S B S B S B S B S B S B Start (min) 0 1 2 3 4 5 6 7 8 9 10 11 Stop (min) 15 16 17 18 19 20 21 22 23 24 25 26 Then add 0.02 ml of serum to each blank (B). Measure the absorbance at 405 nm against the blank within 30 min, proceeding from the lowest concentration upwards. Evaluation: Plot the results into the table (all concentrations are in mmol.l-1).

c c incub A405 1/cincub 1/A 2.86 0.47 5.72 0.94 11.44 1.88 22.87 3.77 45.75 7.5 91.5 15

c is original substrate concentration c incub is final substrate concentration in the incubation mixture (i.e. original concentration multiplied by dilution 0.2/1.22 =0.164) Evaluate graphically KM and Vmax and compare to literature values.

57

Notes: 1. It is necessary to use blood serum of a known ALP activity. If the ALP activity exceeds 2.5 µkat.l-1, the serum must be diluted. At this level of activity, the conversion of substrate will not exceed 5 per cent even with the lowest substrate concentration. 8.7. Determination of aspartate aminotransferase (AST) activity in serum Introduction: Enzyme activity and myocardial infarction. Aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase (LD), and creatine kinase (CK) are intracellular enzymes present in high concentrations in muscle, liver, and brain. Increase in concentration of these enzymes in the blood, manifested by increase in their activity, indicates necrosis or disease, especially of the tissues mentioned above. The activities of both aminotransferases (ALT, AST) are elevated after myocardial infarction or in acute infectious hepatitis or in cirrhosis of the liver. To diagnose these diseases: after the infarction, especially AST is elevated; in hepatitis ALT is usually more elevated than ALT; in cirrhosis the situation is reversed, AST is more elevated than ALT. Lactate dehydrogenase (LD) activity is elevated in all conditions accompanied by tissue necrosis, especially those involving acute injury of the heart, red cells, kidney, skeletal muscle, liver, lung, and skin. After myocardial infarction, there is a rise over 3-4 days followed by a slow decline during 5-7 days. The enzyme is also elevated during the acute phase of infectious hepatitis, but is seldom increased in chronic liver disease. Lactate dehydrogenase isoenzymes: LD consists of 5 isoenzymes separable by electrophoresis or chromatography, with different immunologic characteristics. Each LD isoenzyme is made of tetramers composed of subunits of two types - H (heart) and M (skeletal muscle). Isoenzymes are marked by numbers 1 (fastest moving in electrophoresis) to 5 (slowest moving). Isoenzymes 1 (HHHH) and 2 (HHHM) are present in high concentration in heart muscle, so elevation of their activity in serum manifests an infarction. Isoenzymes 4 (HMMM) and 5 (MMMM) are present especially in the liver and elevation of their activity is typical for acute hepatitis and also for acute muscle injury or muscular dystrophies. Creatine kinase (CK) is an enzyme present especially in skeletal and heart muscle and in the brain. After myocardial infarction the activity of CK increases rapidly (within 3-5 hours) and remains elevated shorter time (2-3 days) than does AST or LD (see the scheme). Principle: Determination of AST activity is based on the decrease in NADH according to reactions: AST 2-Oxoglutarate + L-Aspartate L-Glutamate + Oxaloacetate

MD Oxaloacetate + NADH + H+ L-Malate + NAD+ (MD = malate dehydrogenase) The enzyme activity corresponds to the decrease in NADH concentration, determined as the change (decrease) in NADH absorbance per minute. The wavelength of 365 nm was selected for NADH determination. At that wavelength, other components of the reaction mixture do not interfere.

58

Reagents: (a kit for AST determination) 1. Solution I containing: malate dehydrogenase, NADH, pyridoxal-5-phosphate, TRIS-buffer

pH 7.8, L-aspartate 2. Solution II containing: 2-oxoglutaric acid disodium salt Procedure: Pipette into a cuvette (cell): Solution I 1.0 ml Serum 0.1 ml Mix and after 10min add Solution II 0.1 ml Mix and after 30 s at laboratory temperature follow the absorbance at 365 nm in 1 min (exactly!) intervals for 3 min. When using photometer SPEKOL 11, filter must be set to the position marked ***** odstranění rozptýleného světla. Evaluation: Calculate the change in absorbance in 1 min (dA/dt). The molar absorption coefficient ε for NADH at 365 nm is 3.4x103 l.mol-1cm-1. The enzyme activity is expressed: fS - AST (µkat.l-1) = dA/dt . 58.82 (30°C, 365 nm) Explain the origin of the factor (58.82) in the equation above. Normal values: fS - AST: under 0.7 µkat.l-1 (30°C) Notes: 1. If the change in absorbance exceeds 0.1, it is necessary to repeat the analysis using a sample diluted ten times with the physiological solution. Correction must be made for dilution of the sample. 2. The analysis can be performed also at 37°C; normal values are then 0.22 - 0.87 µkat.l-1.

1 5 10 15

days

CK

LD

AST

59

3. The described method using dA/dt is called a kinetic method of enzyme activity determination. Kinetic methods are automatically performed on modern clinical photometers today. Thus, only a factor related to ε (molar absorption coefficient) is input into the photometer and the enzyme activity is displayed automatically. 8.8. Determination of lactate dehydrogenase (LD) activity in serum Principle: The decrease in NADH, as measured by the change in absorbance at 365 nm, is proportional to the activity of LD in the reaction:

LD Pyruvate + NADH + H+ L-Lactate + NAD+

The decrease in absorbance is proportional to the velocity of the reaction and thus proportional to the enzyme activity in the sample. Reagents: (a kit for LD determination) 1. Solution I containing: NADH, TRIS-buffer pH 7.5 2. Solution II containing: sodium pyruvate Both solutions have to be equilibrated at 25°C prior to use. Procedure: Pipette into a 1 cm cuvette (cell): Solution I 1.0 ml Serum 0.02 ml Solution II 0.05 ml Immediately after the addition of NADH mix thoroughly and follow the decrease in absorbance at 365 nm against water. Read the absorbance when switching on the stop-watch. The decrease in absorbance is read after the first, the second, and the third minute (exactly). An average absorbance decrease dA/dt is then calculated. Evaluation: The molar absorption coefficient ε for NADH at 365 nm is 3.4x103 l.mol-1cm-1. The LD activity is then calculated: The enzyme activity is expressed: fS - LD (µkat.l-1) = dA/dt .159.4 (25°C, 365 nm) Explain the origin of the factor (58.82) in the equation above. The result is compared with the value displayed automatically (see Note 3 in Task 15.1). Normal values: fS - LD: 1.4 - 4.1 µkat.l-1 (25°C) Note: If the decrease in absorbance is greater than 0.05/min, it is necessary to repeat the analysis using the serum diluted 5-10 times with the physiological solution. Correction must be made for dilution of the sample.

60

8.9. Determination of the activity of αααα-hydroxybutyrate dehydrogenase (αααα-HBD) in serum Principle: α-Hydroxybutyrate dehydrogenase (α-HBD) is an LD isoenzyme marked as LD 1 (see introduction to this chapter). Since its substrate specificity is somewhat different from that of remaining LD isoenzymes, it is possible to determine its activity in the presence of other LD isoenzymes. The determination is based on the decrease in NADH according to the reaction:

α-HBD α-Oxobutyrate + NADH + H+ α-Hydroxybutyrate + NAD+

The decrease in NADH absorbance (measured at 365 nm) is proportional to the velocity of the reaction and thus proportional to enzyme activity in the sample. Reagents: (a kit for α-HBD determination) 1. Solution I containing: α-oxobutyrate, phosphate-buffer pH 7.5 2. Solution II containing:NADH Procedure: Pipette into a 1 cm cuvette (cell): Solution I 1.5 ml Serum 0.05 ml Solution II 0.02 ml Immediately after the addition of NADH mix thoroughly and follow the decrease in absorbance at 365 nm against water. Read the absorbance when switching on the stop-watch. The decrease in absorbance is read after the first, the second, and the third minute (exactly). An average absorbance decrease dA/dt is then calculated. Evaluation: The enzyme activity is expressed: α-HBD (µkat.l-1) = dA/dt . 159.4 (25°C, 365 nm) Normal values: fS - α-HBD: under 2.38 µkat.l-1 (25°C) Notes: 1. If the decrease in absorbance is greater than 0.05/min, it is necessary to repeat the analysis with the serum diluted 5-10 times with the physiological solution. Correction must be made for dilution of the sample. 2. In a clinical laboratory, fS - α-HBD in serum is usually determined simultaneously with LD. Normally, α-HBD makes 0.6Ť to 0.8 of total LD. After myocardial infarction, α-HBD is markedly elevated and the values of the coefficient α-HBD/LD exceed 0.9. On the contrary, the coefficient smaller than 0.6 manifests usually a liver disease accompanied by releasing isoenzymes LD 4 and LD 5, see introduction to this chapter.

61

8.10. Determination of creatine kinase (CK) activity Principle: Creatine kinase catalyses transformation of creatine into creatine phosphate. Phosphate, liberated afterwards by acid hydrolysis, is determined photometrically after deproteinization in the form of the complex molybdatovanadatophosphoric acid (yellow). Reagents: (a kit for CK determination) 1. Solution I containing: imidazol, magnesium acetate, D-glucose, EGTA, N-acetylcysteine, NAD, ADP, AMP, diadenosine pentaphosphate and mixture of enzymes: hexokinase (HK), glucose-6-phosphatedehydrogenase (G-6-DP) 2. Solution II containing: creatine phosphate Procedure: Pipette into a tube: Solution I 2 ml Serum 0.1 ml Mix and after 5 min at laboratory temperature add Solution II 0.2 ml Mix and 90 s after the addition of solution II the decrease in absorbance in 1 cm cuvette at 365 nm against water is measured. Read the absorbance when switching on the stop-watch. The decrease in absorbance is read after the first, the second, and the third minute (exactly). An average absorbance decrease dA/dt is then calculated. Evaluation: The molar absorption coefficient ε for NADH at 365 nm is 3.4 x 103 l.mol-1cm-1. The LD activity is then calculated: CK (µkat.l-1)= dA/dt .109.5 (25°C, 365 nm) Normal values: fS - CK: man under 2.0 µkat.l-1 (37°C) woman under 1.7 µkat. l-1 (37°C) fS - CK : under 0.33 µkat.l-1 (37°C)

62

9. TETRAPYRROLES

9.1. Determination of hemoglobin in blood

Principle:

Hemoglobin (Hb) is converted to methemoglobin by the action of ferricyanide. Methemoglobin reacts with cyanide to form cyanmethemoglobin. The absorbance of this derivative at 540 nm is used for quantitation. Molar absorption coefficient of

cyanmethemoglobin is 44 000 liter mol-1cm-1. Molecular weight of hemoglobin is 68 000.

Material:

1. Heparinized blood

Reagents:

1. Drabkin°s reagent (1.0 g of NaHCO3 + 50 mg of potassium cyanide + 200 mg of potassium

ferricyanide in 1 liter of water).

Procedure:

Place 5 ml of Drabkin°s reagent and 20 łl of blood into a test tube and shake. After 10 min measure the absorbance A at 540 nm in a 1 cm cuvette against Drabkins reagent.

Evaluation:

Calculation of hemoglobin concentratoin cHb:

cHb (mol/l) = (A / 44) x (5.02 / 0.02) = A x 5.7

ŃHb (g/l) = (A / 44) x (5.02 / 0.02) x 68 = A x 387.9

Reference values:

The hemoglobin concentration in blood ranges from 1.8 to 2.5 mmol/l (120 - 170 g/l).

Increased hemoglobin levels in the blood are found in polycythemia or dehydration.

Decreased hemoglobin levels are symptomatic of anemias or hemopoesis.

9.2. Determination of total bilirubin in serum

Introduction:

Two types of bilirubin occur in serum. Unconjugated bilirubin is transported in the blood stream as a bilirubin-albumin complex. This bilirubin is water-insoluble. Conjugated bilirubin

63

is formed in the hepatic cells. Albumin is separated from the complex, and bilirubin conjugated with glucuronic acid. The product, bilirubin diglucosiduronate is water-soluble, and is secreted from the hepatic cells to bile.

Principle:

Bilirubin in the serum reacts with diazotized sulphanilic acid to form a coloured azobilirubin and its absorbance is measured. Conjugated bilirubin reacts readily ("direct bilirubin").

Uncojugated bilirubin reacts very slowly, and therefore the addition of accelerators (caffein, alcohol, benzoate) is necessary ("indirect bilirubin").

Material:

Serum sample

Reagents:

1. Reagent (sulphanilic acid, 30 mmol/l in HCl, 0.15 mmol/l)

2. Buffer (potassium-sodium tartrate, 0.93 mol/l in NaOH, 1.9 mol/l)

3. Accelerator (caffeine, 0.26 mol/l and sodium benzoate, 0.52 mol/l)

4. Sodium nitrite (25 mmol/l)

5. Saline (0.15 mol/l)

Procedure:

Pipette into test tubes:

Sample Blank

Reagents ml

Reagent 0.2 0.2

Sodium nitrite 1 drop 1 drop

Accelerator 1.0 1.0

Saline - 0.2

Sérum 0.2 -

Mix and add after 10 min

Buffer 1.0 1.0

Mix and after 5 min measure the absorbance against the blank at 605 nm in a 1 cm cuvette.

Evaluation:

The calibration graph is used for the determination of total bilirubin

concentration in serum.

64

Reference values:

Reference ranges of total bilirubin for infants over 1 month and adults: fS total bilirubin up to 17 łmol/l; fS conjugated bilirubin up to 5 łmol/l

Hyperbilirubinemia in newborn (as much as 135 łmol/l of unconjugated bilirubin in the second day of life) but increased serum bilirubin level is quickly normalized.

The hyperbilirubinemia, high levels of some enzymes, and the presence of bile pigments are typical for different jaundice (icterus):

JAUNDICE BLOOD URINE FAECES

(ICTERUS) Bilirubin level Colour

conj. unconj. ALT ALP BIL UBG

Prehepatic - ↑ - - - + normal

(hemolytic)

Hepatic ↑ ↑ ↑ - + + acholic

Posthepatic ↑ - - ↑ + - acholic

(obstructive) (white)

BIL = bilirubin UBG = urobilinogen↑↓

9.3. Test for blood in feaces (test for occult bleeding)

Principle:

The test is based on the peroxidase-like activity of haemoglobin which catalyzes the non enzymatic dehydrogenation of chromogenous organic substrate H2R (aminophenazone) by a

peroxide.

Hb

H2O2 + H2R ----> 2 H2O + R

Material:

Sample of stool

Reagents:

Kits KRYPTO-HAEM SSW or CK TEST:

65

1. Test plate

2. Reagent solution (acetic acid, H2O2, aminophenazone)

3. Control sample

Procedure:

Apply stool sample to the window No. 1 and control sample to

the window No. 2. After the samples have dried, apply reagent

to reverse sides of windows.

Evaluation:

A positive result gives a blue-green colour.

9.4. Gallstones analysis

Introduction:

Gallstones formation: Stones tend to form when the quantity of cholesterol in the bile is excessive, when qualitative changes in its constituents render the bile less efficient in maintaining cholesterol in micellar solution. Precipitation may tend to occur at night, when bile is concentrated in the gallbladder.

Types of gallstones:

1. cholesterol gallstones - frequent occurrence, contain largely cholesterol

2. pigment gallstones, black (carbonate gallstones) - high content of calcium carbonate and calcium phosphate, also include cholesterol and undissolved bilirubin polymers

3. pigment gallstones, brown (bilirubin gallstones) - infrequent, mainly contain bilirubin and its calcium salts, also cholesterol and fatty acids

4. mixed gallstones - most frequent, contain cholesterol, calcium salts of bilirubin, calcium carbonate.

All gallstones include bile acids and their taurin derivatives.

Principle:

The gallstones are dissolved and analysed. Cholesterol test is based on the Liebermann-Burchard reaction i.e., colour reaction with acetic anhydride in the presence of conc. sulphuric acid. Bilirubin reacts with diazonium salts, forming coloured azo derivatives.

Material:

Gallstones

66

Reagents:

1. Dichloromethane

2. Conc. sulphuric acid

3. Acetic anhydride

4. Diazo I solution (0.5 g of sulphanilic acid + 1.5 ml of conc.HCl

in 100 ml of water)

5. Diazo II solution (potassium nitrite, 20 g/l)

6. Sodium carbonate, saturated solution

Procedure:

A piece (0.2 g) of gallstone is powered in a mortar and dissolved in 5 ml of dichloromethane

in a water bath at 60oC. Warm solution is filtrated through cotton wool. The filtrate is used for testing.

Cholesterol test:

Add 0.5 ml of acetic anhydride and few drops of conc. H2SO4 to 2 ml of filtrate in a test tube.

Perform the test in the hood, use dry test tubes, work carefully and use protecting shield.

Bilirubin test:

Evaporate dichloromethane from the filtrate (2 ml) in the water bath. Dissolve the residue in 2 ml of sodium carbonate and add 3 ml of mixture of diazo solutions (5 ml of diazo I + 5 drops of diazo II).

Evaluation:

Observe and describe results of both tests.

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10. URINALYSIS Qualitative tests can be performed on fresh urine (the first morning specimens are preferred since they have the maximum concentration of all constituents). Quantitative determinations are usually made with reference to a fixed collection period. An adequate 24-hours specimen is prepared as follows: the first urine voided by the patient in the morning is discarded. All subsequent voidings for the next 24 hours, including the first one of the next morning, are collected in a vessel stored in a refrigerator. The urine is mixed and its volume measured. Depending on the assay procedure, either the entire sample or only a fraction of it is sent to the laboratory. Bacterial growth in the urine can be prevented by addition of thymol or formalin.

10.1. Physical analysis of urine

Urine colours

Normal urine is of light yellow to yellow colour.The urine colour is dependent on pathological processes but in many instances the colour is due to the presence of a drug (or its metabolites) or food. Bile pigments give the fresh urine a brown, dark yellow or orange colour, turning green after standing in the air. Pink to red urine may be due to blood. Melanogens or methemoglobin colour the urine dark brown to black.

Spume

Normal urine spumes a little. White spume is found in proteinuria, and yellow spume turning to green is due to the presence of bilirubin.

Odour

The odour of fresh urine is characteristic. After standing, the smell changes to an ammonia odour. A very characteristic smell is detected in ketonuria (acetone). The ingestion of drugs or

poisons impart a characteristic odours, as well.

pH reaction

Normal urine pH is about 6.0. With time the pH becomes alkaline and ammonia is evolved due to hydrolysis of urea. After vegetal food pH is about 8. An accurate measurement of pH is achieved using a pH-meter. For routine urinalysis, pH indicator papers are appropriate.

Turbidity

Fresh voided urine immediately after passing should be transparent, any turbidity of urine still warm is considered as a pathological finding. The nature of the turbidity can be ivestigated most simply by testing solubility of urine in HCl, acetic acid, or NaOH. Urine turbidity is dissolved after:

a) warming - uric acid salts

b) adding acetic acid - carbonate or phosphate

c) adding HCl solution - oxalate, leucine, tyrosine, xanthine

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d) adding NaOH - uric acid

Density and osmolality of urine.

The urine density values in healthy subjects are between 1,015 and 1,020 g/l (osmolality between 500 and 850 mmol/kg). Both values depend on the concentration of dissolved solids and vary with the solute load to be excreted, as well as with the urine volume (e.g. perspiration, pain). The measurement of urine density (or osmolality) is useful in the clinical test of urine concentrating capacity, which is an important function of renal tubules. High values of s.g. can be found in excessive glucosuria and proteinuria. Density is measured using a urinometer.

Volume of urine

The urine volume varies between 500 and 3,000 ml per day (diuresis). Increased urine volume, polyuria is found in diabetes mellitus, and in diabetes insipidus - the lack of antidiuretic hormone ADH (10-20 l daily). Decrease urine volume, oliguria (400-500 ml per day) is a sign of severe impairment of renal function. Total blockage of urine excretion, anuria (less then 50 ml daily) is a very serious symptom.

10.2 Chemical analysis of urine

Tests for proteinuria - proteins in urine

The protein concentration does not exceed 0.1 g/l in the healthy adult, if the usual urine volume is formed. Proteinuria is considered when the protein concentration is above 0.1 - 0.2 g/l. Albuminuria is most frequent. Globulin proteins or other proteins e.g. Bence-Jones proteins are seldom present.

a) Sulphosalicylic acid test

Principle:

Sulphosalicylic acid denatures and precipitates proteins. The precipitate is insoluble in an excess of reagent or after heating. The sulphosalicylic acid test is sensitive and useful for to preliminary screening. The urine sample should be clear, any turbidity must be removed by filtration. If not successful, the result of test is evaluated by comparing the unfiltered sample with the filtered one.

Reagents:

Sulphosalicylic acid (20 %)

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Procedure:

Add 5-10 drops of sulphosalicylic acid to 2 ml of urine and mix. A turbidity or precipitate is formed depending on the protein concentration

turbidity: protein g/l

opalescent 0.05 - 0.1

light 0.1 - 0.25

milk opaque (without floccules) 0.25 - 1.0

milk opaque (with floccules) 2.0 - 1.0

precipitate 4.0 and more

Interference:

False positive values: presence of sulphonamides, peroral antidiabetic drugs (tolbutamide), calcium salts, high doses of penicillin, para-amino salicylic acid, high concentrations of uric acid.

b) Diagnostic strip Albuphan

Principle:

Diagnostic test strips have test areas (indicator zones) attached to a white plastic strip. The areas contain an acid-base indicator, which shows the so-called protein error. The indicator colour is changed in the presence of proteins.

Procedure:

Dip the strip into the fresh urine for 3 seconds and withdraw it immediately. Wipe off the excess urine on the vessel edge. After 1 min compare the test area colour with the colour chart on the container.

Interference:

False positive results: strongly alkaline urine, the urine of patients who are given drugs derived from quinine or quinoline.

Evaluation:

If the test of urine proteins is positive (proteinuria), the tests for hemoglobin and a microscopic examination of the urine sediment must be done. In indicated cases, daily urine protein excretion, electrophoresis, immunofixation must be performed. Proteinuria indicates a severe diseases or toxic injury to the kidney. Proteinuria may be due to inflammation of the lower urinary tract, and also (local excretion of immunoglobulins or small, incidental heamorrhages), the so-called postrenal proteinuria. Some glycoproteins from the urinary tract epithelium (previously called mucin) and/or polypeptides from the enzyme degradation of proteins (previously called albumose) may be present in the urine. These substances

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precipitate after acidification at room temperature and a part of precipitate dissolves by heating. Bence-Jones protein (plasmocytoma) - slightly acidified urine is heated, and turbidity

is formed at 50-60oC. When the temperature is higher, the turbidity disappears. After cooling the turbidity is formed again.

Tests for glucosuria - glucose in urine

a) Benedicts test

Principle:

Glucose reduces Cu2+ (citrate complex Cu2+- Benedict's reagent) to Cu+. The Cu+ formed in the reaction precipitates as rust-coloured Cu2O and glucose is oxidized to gluconic acid).

Reagents:

Benedict's reagent (86,5 g trisodium citrate + 37 g Na2CO3 + 5.6 g CuSO4 in 500 mL of

water)

Procedure:

Add 5 ml of Benedict's reagent to 1 ml of urine and keep the mixture boiling for 2 minutes.

Interference:

False negative result: high concentration of ascorbic acid, sodium gentisate, and alkaptonuria.

Evaluation:

Glucosuria occurs either when high hyperglucosemia is present (in diabetes mellitus, in healthy subjects after an exceptionally high intake of glucose) or at the normal levels of plasma glucose when the resorbing capacity of the tubular mechanism is impaired (renal glucosuria - from renal disorder).

b) Diagnostic strips Glucophan and Diaphan

Principle:

Diagnostic test strips have test areas (indicator zone) attached to a white plastic strip. Glucose indication zone (in Glukophan or Diaphan) contains enzymes glucose oxidase and peroxidase with a special chromogen which forms red products in the presence of glucose.

Procedure:

Dip the strip into the urine for 3 secondes and withdraw it immediately. Wipe off the excess urine on the vessel edge. Glucose zone - after 3 minutes compare the colour with the colour

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chart. The test can record the presence of glucose from approx. 2 mmol/l, creating distinct orange colour. Higher concentrations of urinary ascorbic acid can lead to false results.

Tests for ketonuria - ketone bodies in urine (i.e. acetoacetate,

3-hydroxybutyrate, and acetone)

Principle:

In an alkaline medium nitroprusside reacts with acetone or acetoacetic acid, and purple complex is produced (3-hydroxybutyrate does not react). The ketone indicator zone (Ketophan or Diaphan) is impregnated with an alkaline buffer and nitroprusside giving with acetoacetic acid or acetone a purple colour whose intensity is proportional to the ketone concentration.

Reagents:

Lestradets reagent (mixture of sodium nitroprusside, ammonium sulphate, and anhydrous disodium carbonate in the weight ratio 1:200:200)

Diagnostic strips Ketophan and Diaphan

a) Lestradets test

Procedure:

Place 2-3 spatulas of Lestradet°s on the test dish reagents and add a few drops of urine. Ketone bodies produce a purple colour that will fade during several minutes.

Interference:

False positive values: presence of acetylsalicylic acid and phenazone.

b) Diagnostic strips Ketophan and Diaphan

Procedure:

Ketone zone - after 1 minute compare the colour with corresponding colour chart.

Evaluation:

A sharp increase in ketone bodies formation is a result of the extended utilization of lipids (without providing enough glucose to metabolize them) - diabetes mellitus, during exhaustive physical activity, and prolonged starvation. In diabetic patients both ketonuria and glucosuria are the most common and apparent signs of the bad compensation of their metabolic disorder.

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Tests for hemoglobinuria - hemoglobin in urine

Principle:

The tests are based on the peroxidase-like activity which catalyzes the non enzymatic dehydrogenation of chromogenous organic substrate H2R (aminophenazone) by a peroxide.

Hb

H2O2 + H2R ----> 2 H2O + R

Reagents:

1. Pyramidone reagent - aminophenazone solution

(1-phenyl-2,3-dimethyl-4-dimethylamino-5 -pyrazolone)

2. Acetic acid

3. Hydrogen peroxide solution

4. Diagnostic strips Hemophan

Procedure:

a) Pyramidone test

Place 5 ml of pyramidone reagent and 2 ml of conc. acetic acid and 1 ml of H2O2 into test

tube. Add 2 ml of urine. Violet colour indicates the positive result.

b) Diagnostic strips Hemophan

The strip is dipped in fresh, mixed urine and removed immediately, excess liquid is wiped off on a flask edge. After 30 second the colour of the indicator area is compared with the colour chart on the container.

Interference:

False positive result: presence of Fe3+, iodide or strongly oxidizing reagents (disintfectans, chloramine).

Evaluation:

Hematuria - erythrocytes in urine results from:

a) the damaged ultrastructure of the filtration medium (glomerulonephritis, malignant hypertension nephrotic syndrom etc.), it is associated with distinct proteinuria.

b) a not very extensive haemorrhage in the kidney or in the lower urinary tract (injury, urolithiasis, inflammations or tumours), proteinuria might not be recognized.

Hemoglobinuria - results from intravascullar hemolysis (haemolytic compounds, transfusion from an unsuitable blood donor, etc.) that releases more hemoglobin into circulation than haptoglobin can pick up and the surplus free hemoglobin is then filtered through the glomerulus. Positive hemoglobin test without finding erythrocytes in urine sediment.

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Hemoglobinuria after crushing injuries of the muscle tissue often results in acute renal failure.

A positive hemoglobin test is always significant enough to warrant further investigation.

Tests for bilirubinuria - bilirubin in urine

Principle:

Bilirubin is oxidized by iodine to green coloured biliverdin, or is detected by reagent strips tests involving coupling of bilirubin to a stable diazonium salt to form a brown azo dye (Biliphan). Urine for bilirubin test has to be fresh, because of easy oxidation of bilirubin in air.

Reagents:

Iodine in ethanol (solution of 1 g iodine in 100 ml ethanol)

Diagnostic strips Biliphan

a) Iodine test

Place 2 ml of urine into a test tube, then incline the tube, slowly allow 2 ml of ethanolic solution of iodine to flow down the side of the tube so that it forms two separated layers. A green ring develops at the interface between both layers. In the case of high proteinuria, the ring is white.

b) Diagnostic strips Biliphan

Dip the strip in urine and remove it immediately. Wipe off the excess of urine on the vessel edge and after 30 second compare colour of test areas with corresponding colour charts on the container.

Evaluation:

The presence of conjugated bilirubin in urine indicates:

a) a defect in the ability of the hepatic cells to excrete it into the bile (hepatocellular hyperbilirubinemia)

b) a sign for mechanical blockage of the bile ducts (obstructive hyperbilirubinemia).

Urobilinogenuria - urobilinogen in urine

Principle:

Urobilinogens react with p-dimethylaminobenzaldehyde in strongly acidic medium to form red products - Ehrlichs test. For testing, fresh and cooled urine should be used and tested by

2 - 3 hours after voiding (urobilinogen easily oxidizes to urobilin in air).

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Reagents:

1. Ehrlich°s aldehyde reagent (p-dimethylaminobenzaldehyde, 135 mmol/l in HCl, 6 mol/l, sodium acetate (saturated solution)

2. Diagnostic strips UBG-phan

Procedure:

a) Ehrlichs reagent test

Add several drops of Ehrlich°s reagent to 5 ml of urine in a test tube. Compare it with the urine blank tube (without reagent). If the colour turns to red, " Ehrlich reacting" substances are present.

b) Diagnostic strips UBG-phan

Dip the strip into urine and remove immediately, wipe off the excess urine on the tube edge. After one minute compare the test area colour with the colour chart on the container.

Interference:

False positive results: The presence of nitrite, sulphonamide, formalin.

Evaluation:

A positive result indicates the overloading of the hepatic function (enormous physical activity, single heavy alcohol consumption, increased hemolysis) or any other hepatocelular damage (virus hepatitis, toxic substances, tumours of the liver).

Test for salicylate

Salicylate reacts with FeCl3, red product is formed. The same colour is given by acetoacetic

acid. As acetoacetic acid is destroyed by heating, we can distinguish salicylate from acetoacetic acid by heating the sample prior to FeCl3 addition. A positive test signifies then

the presence of salicylate.

Less frequent examinations

Test for nitrite, phenyl pyruvic acid, melanin, salicylate, sulphonamide, ascorbic acid etc.

10.3 Microscopic urinalysis

Urine sediment contains:

a) non visceral sediment (dependent on urine pH)

- crystals: oxalates, phosphates, urates, uric acid, tyrosine, leucine, cystine

- amorphous compounds: urate, phosphate, carbonate

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b) visceral sediment

- red blood cells - their forms depend on the hypertonic enviroment, and they are distinguished using polarization or interference microscopes (90 % of erytrocytes with impaired membranes "renal erytrocytes" - impaired glomeruli)

- leukocytes - small granular elements, after acidification the nucleus is visible.

- epithelial cells: - transitional epithelial cells from the bladder

- squamous epithelial cells from the lower genitourinary tract

- renal tubular cells - the sign for impaired kidney tubules

- casts (specific indicators of renal disease):

- hyaline casts (dehydration, hyperthermia, physical load)

- granular casts (impaired glomeruli)

- white cells casts (inflammation, pyelonephritis))

- red cells casts (glomerulonephritis)

- epithelial casts (tubular nephropatia)

- heme granular casts (glomerulal disease)

- waxy casts (high proteinuria, chronically diseased kidney)

c) microorganisms: - fungus, Trichomonas vaginalis, echinococcus

Procedure:

Routine microscopic urinalysis is done on a 5-10 ml aliquot of freshly voided urine. The sample is centrifuged at 3,000 rpm for 5 min and supernatant is discarded, leaving a button of sediment which is resuspended in 1 ml of residual urine in the tube. A drop is placed under a cover slip and examined with the high-power objective of a light microscope to semiquantitate cellular elements. A variety of techniques have been devised to improve quantitation. Hamburger°s sediment - urine is collected for 3 hours. 5 ml of urine centrifuged at 2 000 rev/min for 10 min. The supernatant is discarded to 0.5 ml of residual urine. The tube contents is mixed, Bürker chamber is filled up with the urine suspension, and urine elements are determined.

10.4 Chemical analysis of renal calculi (urolithiasis)

Renal calculi (a calculus, stone) form in the urinary tract when mineral salts precipitate over a mucoprotein core. Up to 60 % of the material in a stone is protein, the rest is made up of varying proportions of Ca, P, Mg, NH4, uric acid, occasionally cystine, and very rarely

xanthine. An obstruction to urine flow in the ureters can predispose to infection and stone formation.

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Main components of renal calculi:

Whewellite -calcium oxalate monohydrate

Weddelite -calcium oxalate dihydrate

Hydroxyapatite -calcium phosphate-hydroxide

Carbonateapatite -calcium phosphate-carbonate

Struvite -ammonium magnesium phosphate hexahydrate

Brushite -calcium hydrogen phosphate dihydrate

Uricite -uric acid (anhydrous)

-uric acid dihydrate

-ammonium hydrogen urate

-sodium hydrogen urate

-cystine

-cholesterol

Urostealit -fatty stone

Types of renal calculi and their causes

- calcium 90 % idiopathic, hyperparathyroidism, renal tubular acidosis, excess calcium ingestion

- uric acid 5-10 % gout, idiopathic

- cystin 1-2 % cystinuria

- oxalate 65 % rarely prime cause

Identification of urine calculi using physical techniques:

stereomicroscopy, IR-spectrophotometry, X-ray diffraction, termoanalysis, polarization microscopy.

Simplified chemical analysis of renal calculi: ANALYTICAL PROCEDURE

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Pulverized portion of the stone

effervescence

(CO2)

CARBONATE

(as component of

carbonateapatite)

insoluble

residues

positive

murexide

test

URIC ACID

URATE

+ diluted HCl

clear soln.

or filtrate

heat

alkalization

with NH3

clear soln.

CYSTINE

(test for cystine)

white

precipitate

Ca2+

+ conc. acetic acid

clear soln.

PHOSPHATE

crystalline

precipitate

OXALATE

in both cases test a new portion

for phosphate with molybdenate soln.

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11. CREATININE CLEARANCE AND GASTRIC SECRETION

11.1 Determination of creatinine in blood serum and urine and creatinine clearance calculation

Creatinine is formed as final product of metabolism of creatine in muscle. It is removed from the circulation almost entirely by the glomerular filtration action of the kidney.

Principle:

Picric acid reacts with creatinine in alkaline solutions to give a red-orange product. The change of absorption dA/dt is measured.

Material:

l. Blood serum

2. Urine - the urine specimen is diluted with distilled water

(1 ml of urine to 50 ml)

Reagents:

1. Picric acid (16.8 mmol/l)

2. Sodium hydroxide (0.75 mmol/l)

3. Creatinine standard solution (150 łmol/l)

Preparation of reagent solution: Picric acid and sodium hydroxide solutions are mixed in ratio 3:1; 30 minutes prior to use.

Procedure:

Pipette 1.6 ml of reagent solution into a cuvette, the reaction is started with 0.5 ml serum or urine or creatinine standard solution. The absorbances are measured 30 and 120 seconds after the start against the air.

Calculation:

serum absorbance dA1 = (A120 - A30)

urine absorbance dA2 = (A120 - A30)

standard absorbance dA3 = (A120 - A30)

serum creatinine concentration (µmol/l) = 150 x (dA1 / dA3)

urine creatinine concentration (µmol/l) = 150 x (dA2 / dA3) x 50

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Reference values:

fS - creatinine: 70 - 125 µmol/l for men; 50 - 105 µmol/l for women

dU - creatinine: 8 - 16 µmol/l for men per day; 5 - 13 µmol/l for women per day

Evaluation:

Urine creatinine concentration is proportional to the total muscle mass and depends to a great deal on the actual protein intake and on other factors. Creatinine concentration values are used for creatinine clearance calculation.

Creatinine clearance calculation

The renal function tests - glomerulal filtration rate (GFR), effective renal plasma flow (ERPF), and tubular functions are usually based on clearance tests. The renal clearance of substance X (inulin, creatinine, urea, glucose) represents a virtual volume (ml) of plasma from which substance X is completely cleared within one second. The clearance is obtained by dividing the amount of substance X excreted in the urine per second by corresponding plasma concentration. The clearance can be expressed in ml/s. The clearance is equal to the glomerulal filtration rate (GRF).

clearance (ml/s) (GFR) = (cu x Vu) / cs

cu ..concentration (mmol/l) of x in the urine

cs ..concentration (mmol/l) of x in serum (plasma)

Vu ..volume (ml) of urine (urine flow) per second

Creatinine is a convenient substance for the test of the glomerular filtration rate (GRF) because it is formed endogenously in muscle tissue and is liberated into the circulation in a very constant way, it is removed almost exclusively by glomerular filtration and is not reabsorbed. Inulin clearance is more accurate, but an intravenous infusion of inulin is required, so the test is uncomfortable for the patient. The measurement of creatinine clearance: Urine is collected over a specific period of time (4 hours or 24 hours), and the urine volume is measured. A venous blood sample is obtained at the midpoint of the urine collection period. The clearance is often corrected to a standard adult body surface area of

1.73 m2:

clearance (ml/l) = (cu x Vu x 1.73) / (cs x A)

A = the body surface area may be calculated from the body weight (w, in kilograms) and height (h, in metres) values according to the empirical formula

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A = 0.167 x (w x h)1/2

or found on the nomogram.

Approximately one fifth of the volume of plasma which circulates through the kidneys is filtred by the glomeruli resulting in some 170 liters of the ultrafiltrate per 24 hours in an adult.

The overwhelming part of this ultrafiltrate (e.g. water, electrolytes, glucose, aminoacids) is reabsorbed by the renal tubules. Most water is reabsorbed in the proximal tubule, however the final and regulated reabsorbtion takes place in the collecting tubule. The fraction of water that was reabsorbed from the glomerular filtrate, called the tubular reabsorption of water, R (H2O), is expressed:

R (H2O) = (GFR - Vu) / GFR = 1 - cs / cu

Fraction excretion of water E/F (H2O) is a fraction of water in the glomerular filtrate that was

not reabsorbed:

E/F (H2O) = Vu / GFR = cs / cu

Evaluation:

The value of creatinine clearance is dependent on the precision of plasma creatinine determination, quantitative urine collection, body surface area, age, sex, pathological creatininemia. In healthy subjects glomerular filtration ranges from about 1.25 to 2.5 ml/s in

adults between 20 and 60 years of age, corrected to standard surface area of 1.73 m2. Tubular reabsorption of water range: from about 0.985 to 0.997. A decrease of GRF value is a sign of insufficient renal function (e.g. organic impairment of the glomerulus, limited renal blood flow).

11.2 Determination of gastric output

The gastric secretion is known as gastric juice. It is clear, pale yellow fluid of 0.2 - 0.5 % HCl, with a pH of about 1.0 (1.3 - 1.8). The gastric juice is 97-99 % water. The remainder consists of mucin and inorganic salts, the digestive enzymes (pepsin and renin), and a lipase. In hypochlorhydria or achlorhydria the gastric juice contents only lactic acid.

Qualitative examination of gastric juice.

a) Measuring pH of gastric juice by use of indicator congored.

b) Test for lactic acid.

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Place 5 ml of phenol into a test tube and add 2 drops of FeCl3 solution. The violet colour is

formed (Uffelmanns reagent). Then add 2 ml of gastric juice, in the presence of lactic acid, the violet colour is changed to yellow.

Quantitative examination of gastric juice.

Chief cells and parietal cells secrete gastric juice in response to reflex stimulation or the action of gastrin. HCl is secreted in constant concentration ca 160 mmol/l.

Acid output is the amount of HCl secreted during the definite time interval. This value is one of important characteristics of the gastric mucosa function. The basal acid output (BAO) is the production of HCl determined after fasting, in basal conditions, the maximal acid output (MAO) is the production of HCl after intensive stimulation of parietal cells. The peak acid output (PAO) is the maximal level of the secretion after intensive stimulation. The actual acidity of the gastric juice, the pH value, depends ad- mittedly upon acid output, however it is also influenced by gastric juice volume and buffering action of other substances present, e.g. glycoproteins of mucus.

Principle:

The concentration of HCl in gastric juice is determined by titration. Indicator phenol red is used, colour change by pH 7.4 (blood pH).

Material:

Collection of gastric juice:

No food or drink should be ingested for 12 h before test. A thin tube is intubated into stomach, all the secretion already present is aspirated and discarded, which starts the first collection period. The basal secretion is collected for 30 minutes - fraction A. Then pentagastrin is administered subcutaneously as a parietal cells stimulant. Aspiration is continued for one hour in four 15-min fractions (1,2,3,4). The volume and pH of each fraction

is then measured (indicator papers). The concentration of H+ ions must be sufficient (pH < 3).

Reagents:

1. Sodium hydroxide (0.1 mol/l)

2. Phenol red solution in ethanol (1 g/l)

3. pH indicator paper

Procedure:

Pipette 10.0 ml of the sample into a titration flask, add 3 - 5 drops of phenol red indicator and titrate using 0.1 mol/l NaOH to the first red colour.

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Calculation:

1. Calculate the concentration (mmol/l) of HCl in each fraction

2. Calculate mmol HCl in each fraction

3. BAO calculation: output (mmol) of fraction A multiply by 2

4. MAO calculation: sum up outputs of fractions 1,2,3,4

5. PAO calculation: sum up two largest subsequent outputs and multiply by 2

The results are expressed in mmol/h (mmol per 1 hour)

Evaluation:

BAO: reference values 1-3 mmol HCl/h (women), 2-3 mmol HCl/h (men). Higher values are associated with duodenal ulcer and particularly with carcinoma of pancreatic G-cells, which produces excessive amount of gastrin (Zollinger-Ellison syndrome, gastrinoma). Lower values are found in patients with gastric carcinoma, possibly also with gastric ulcer.

MAO : reference values 10-17 mmol/h. There is a close correlation between the values MAO and PAO. Decreased values - hypoacidity, anacidity (achlorhydria, hypochlorhydria), atrophia of gastric mucosa, gastric carcinoma and pernicious anemia. Increased values -hyperacidity - often in duodenal ulcer and Zollinger-Ellison syndrom.