MATERIALS AND METHODS

Transcription

1 MATERIALS AND METHODS Place of study: This study was carried out at Department of Child Health, Institute of Medicine, Kathmandu. Subjects were taken either from out patient department or from emergency ward of Kanti Children's Hospital, Maharajgunj, Kathmandu. Type of study: Cross sectional study with control group. Time and duration: staring from October 1998 to September The study was conducted for a period of 12 months Pre-test before actual study: A pre-test was conducted among 20 children from both PEM and non-pem group was performed before the actual study and necessary changes in proforma was done. Sample Size Determination: calculated by the following formula. 88 The required sample size for the study was p - π Z = [π (1-π)/ n] Here, n = required sample number Z= two tailed Z score at selected confidence interval p-π = allowable discrepancy between population and sample proportion in % π = proportion of biochemical abnormality (e.g. hypoglycaemia) in population of PEM. Confidence interval was set at 95% for this, Z score is Value of p-π was set at 6%. In various studies mentioned in literature, the prevalence of hypoglycaemia is around 10%. So the value of π is equal to 0.1. Thus, 6

2 1.96 = [0.1 (1-.0.1)/n] Therefore, n = 96 One hundred and twenty patients were, therefore, selected for the study. Selection of Subjects: Total number of study subjects was 120. They were divided into two groups based on whether they are above or below the 80% of 50 th percentile of weight for height based on growth chart of National Centre of Health Statistics, USA (NCHS) Subjects were randomly selected from out patient department and ward. Total no. of subjects in PEM group (weight for height less than 80% of 50 th percentile) = 60; Male = 30 Female = 30 Total no. of subjects in non-pem group (weight for height more than 80% of 50 th percentile) = 60; Male = 30 Female = 30 Relevant history and physical examination based on proforma of data collection sheet was completed. Height or length was taken using non-stretchable steel tape and weight was taken from a weighing scale and the same was used for both groups. MUAC was measured using plastic tape using standard technique at the midpoint between olecranon process and acromion process. Specimen collection and storage: After completion of history and physical examination venous blood was taken without using a tourniquet. The blood sample was quickly centrifuged and sent directly to the laboratory for estimation of serum levels of glucose, sodium, potassium, protein, albumin and calcium. Inclusion Criterion: Children between the ages of 6 months and five years. Exclusion Criteria: Children who are being given intravenous fluids. Children of parents who refused to give consent for blood examination.

3 Statistical Analysis: and necessary statistical tests were done. The data was put on computer in epi-info-6 program Biochemical Methods: a. Estimation of Serum Glucose In individuals with a normal haematocrit, fasting whole blood glucose concentration is approximately 12 to 15% lower than plasma glucose. In most clinical laboratories, plasma or serum is used for majority of glucose determination, whereas most methods for self-monitoring of glucose use whole blood. During fasting, capillary blood glucose level is only about 2-5 mg % higher than that of venous blood and after a glucose load, however capillary blood glucose concentrations are mg % (mean 30 mg %) greater than concurrently drawn venous blood samples. Glucolysis decreases serum glucose by approximately 5-7 % in 1 hour (5 to 10 mg/dl) in normal uncentrifuged coagulated blood at room temperature. The rate of in vitro glycolysis is higher in presence of leucocytosis or bacterial contamination. Glycolysis can be inhibited and glucose stabilised for as long as 3 days at room temperature by adding sodium iodoacetate or sodium fluoride to the specimen. Fluoride ions prevent glycolysis by inhibiting enolase, an enzyme that requires Mg +2. Fluoride is also a weak anticoagulant because it binds calcium however clotting may occur after several hours and it is therefore advisable to use a combined fluoride oxalate mixture. It is probably not necessary to use a fluoridecontaining tube if within 60 minutes of blood collection, plasma is separated from cells or glucose is measured. Many analytical methods are used to measure blood glucose level. In the past analyses were often performed with relatively non- specific methods that resulted in falsely elevated values. In this study glucose oxidase method was used. Principle of Glucose oxidase method:

4 In this method the aldehyde group of β-d- glucose is oxidised by glucose oxidase to give gluconic acid and hydrogen peroxide. Glucose Oxidase β-d-glucose + H 2 O + O 2 Gluconic acid + H 2 O 2 Addition of enzyme peroxidase and a chromogenic oxygen acceptor such as O-dianisidine result in the formation of a coloured compound that can be measured. O-dianisidine + H 2 O 2 (Colourless compound) Peroxidase Oxidised O - dianisidine + H 2 O (Coloured compound) Glucose oxidase is highly specific for β-d- glucose. The second step involving peroxidase is much less specific than the glucose oxidase reaction. Various substances, such as uric acid, ascorbic acid, bilirubin, haemoglobin, tetracycline and glutathione, inhabit the reaction presumably by competing with the chromogen for H 2 O 2 producing lower values. 89 Materials used for estimation of blood glucose. Micropipette 20 µml & 1000 µml (Bioscience) Microtips small and large (Bioscience) Dispenser 1000 variable 1-10 ml. Volumetric flask 1 litre. Spectrophotometer Hitachi quartz cuvet. magnetic stirrer c magnet. Centrifuge Timer Reagents Enzymatic (GOD / POD ) Ranbaxy/ Randox Distilled water Anhydrous glucose (500 gm) Merck / BDH.

5 Washing solution Steps of measuring glucose oxidase method: Draw 2-3 ml of blood in a clean dry test tube. After 10 minutes centrifuge it at 3000 RPM for 3 minutes. Separate serum and within one hour perform glucose estimation. If it is not measured immediately after collection preserve the serum into a clean, dry test at 4 C. Procedure: Test Standard Blank Working Solution 3 ml 3 ml 3 ml Serum 0.02 ml - - Standard(Known concentration) ml Distilled water ml Mix and incubate at 37 C for 15 minutes or at room temperature for 30 minutes. Read the absorbance of the test and standard against blank at 505 nm. Calculation: Absorbance of Test Glucose concentration (mmol/l) = x Concentration of Standard Absorbance of Standard b. Estimation of Total Protein in Serum Biuret method was used to measure the level of total protein in serum: Principle: All proteins contain a large number of peptide bonds and Cu +2 ions in a moderately alkaline medium, a coloured chelate complex of unknown composition is formed between the Cu ++ ions and carbonyl ( -C=O) and imine (= N-H) groups of the peptide bonds. Analogous reaction takes place between the cupric ion and organic compound biuret (NH 2 - and therefore the reaction is

6 termed as the biruet reaction. The reaction takes place between the cupric ion and compound containing at least two NH 2 CO-, NH 2 CH 2 -, NH 2 CS-, and similar groups joined together directly, or through a carbon or nitrogen atom. Amino acids and dipeptides cannot give the reaction, but tri- and polypeptides and proteins react to give pink or reddish violet products. In this reaction, one copper ion is linked to between four and six nearby peptide linkages by co-ordinate bonds, the more protein present, the more peptide bonds available for reaction. The intensity of colour produced is proportional to the number of peptide bonds undergoing reaction. Thus the biuret reaction can be used as a basis for a simple and rapid colorimetric method for determining protein. 89 Procedure: Blank Standard Test Biuret reagent 5.0 ml. 5.0 ml. 5.0 ml. Protein Standard ml. - Serum ml. Mix well and let stand for minutes at room temperature and read. Set zero with reagent blank. Calculation OD of Test Total Protein (g%) = x concentration of Standard OD of Standard Reagent preparation 1. Dissolve following reagent in about 900 ml. of water one after the other as given Sodium - Potassium tartrate Copper sulphate (CuSo 4. 5H 2 O) Potassium iodide Sodium Hydroxide make up to 1000 ml. with water. = 9 grams. = 3.0 grams. = 5.0 grams. = 8 grams.

7 2. Protein standard = 8.0g of bovine albumin in 100 ml. of isotonic sodium chloride. c. Estimation of Sodium and Potassium in Serum Flame emission photometry was used to estimate the sodium and potassium in serum. Principle: Atoms of many metallic elements, when given sufficient energy such as that supplied by a hot flame will emit this energy at wavelengths characteristics for the element. A specific amount or quantum of thermal energy is absorbed by an orbital electron. The electrons, being unstable in this high energy (excited) state, release their excess energy as photons of a particular wavelength as they change from the excited to their previous or ground state. If the energy is dissipated as light, the light may consist of one or more than one energy level and therefore of different wavelengths. These line spectra are characteristic for each element. Sodium, for example, emits energy primarily at 589 nm along with other much less intense emissions. The wavelengths to be measurement of an element depends upon the selection of a live of sufficient intensity to provide adequate sensitivity as well as freedom from other interfering lines at or near the selected wavelengths. Under constant and controlled conditions, the light intensity of the characteristic wavelength produced by each of the atoms is directly proportional to the number of atoms that are emitting energy, which in turn is directly proportional to the concentration of the substance of interest in the sample. Thus, flame photometry lends itself well to direct concentration measurement of some metals notably Sodium, Potassium and Lithium. 89 Reagent : 1. Lithium containing diluting solution. 2. Standard containing meq/l Na ( gm/l NaCi) and K 5 m Eq/L ( gm/l KCI) Procedure :

8 Three test tubes blank, standard and test are filled with 5 ml diluting solution. add 50 µl standard in standard tube and50 µl serum into test tube. Sip the blank to set zero on operating flame photometer, and then standardise with standard solution and then measure the test specimen. Tubes Blank Standard Test Diluting Solution (ml) Standard (µl) Sample (µl) Mix in verten mixture before sipping. d. Estimation of Calcium Estimation of Calcium was done by O-cresophthalein complexone method :- Principle : Calcium forms a coloured product with O-cresolphthalein complexone, which is measured at 575 nm. The reagent contains Ethanol that maintains a clean solution in the presence of proteins and also surpresses the ionisation of O- cresolphahalein complexone. The 8-hydroxyquinoline binds magnesium which otherwise would cause interference. 89

9 Procedure: Blank Standard Test O-cresolphathalein complexone 2.5 ml. 2.5 ml. 2.5 ml. Base reagent 2.5 ml. 2.5 ml. 2.5 ml. Calcium standard ml. - Serum ml. Mix well and read the absorbance at 570 nm., after 5 minutes, set zero with blank. Reagent Preparation: mg of O-cresophthlein complexone is added in 1.0 ml of cone hydrochloric acid. When completely dissolved the solution appears opalescent. Transfer this solution to 1000 ml volumetric flask by the help of 50 ml dimethyl suplhoxide. Add 2.5 g of 8-hydroxyquinoline and again wash down the neck of the bottle with 50 ml dimethyl sulphoxide when completely dissolved then only add water to make 1000 ml. 2. Base reagent: Dissolve 500g of potassium cyanide in water and add 40 ml diethylamine and dilute to make 1000 ml with water. Store in a polythene bottle. 3. Stock Calcium standard: Add 1.25g of calcium carbonate (AR grade) in 100 ml. water followed by 7 ml. conc. hydrochloric acid and make up volume to 1000 ml. (calcium carbonate must be dried C for 24 hours before use) 4. Working : Dilute 20 stoke Calcium to 100 ml. with water.

10 e. Estimation of Albumin method: Estimation of albumin was done by Bromocresol Green (Brom CG) Principle: Due to the cationic property of Albumin it binds with anionic Brom CG dye at ph lower than its isoelectric point, pi (i.e. ph 4.7). The yellow colour of the Brom CG reagent in changed into green complex when it binds with the albumin. Its maximum absorbance is 629 nm. 89 Brij - 35, an anionic detergent, is added to buffer and Brom CG solution to reduce the blank absorbance to prevent turbidity maximum in zero minutes so, it should be measured within few seconds. Procedure: Blank Standard Test B.C.G. 4.0 ml. 4.0 ml. 4.0 ml. Albumin Standard ml. - Serum ml. Mix well and read immediately. Set zero with blank. Calculation OD of Test Albumin (g%) = x concentration of Standard OD of Standard Reagent preparation Bromocresol green mg. Succinic and gm. Sodium chloride mg. Brij - 35 (35%) - 8 ml. Sodium hydroxide g.

11 Water to make ml. Check ph 4.2 adjust if necessary with succinic acid or sodium hydroxide. Albumin standard = Bovine albumin 5 g / 100 ml. in 0.85 g% sodium chloride.