Manual on Standard Operation Procedures,Sample Collection and Reference Ranges for Clinical Chemistry

MANUAL ON STANDARD OPERATION PROCEDURES, SAMPLE COLLECTION AND REFERENCE RANGES FOR

CLINICAL CHEMISTRY

WORLD HEALTH ORGANISATION, MINISTRY OF HEALTH AND THE DEPARTMENT OF BIOCHEMISTRY, MEDICAL RESEARCH INSTITUTE

SRI LANKA

MANUAL ON STANDARD OPERATION PROCEDURES, SAMPLE COLLECTION AND REFERENCE RANGES FOR

CLINICAL CHEMISTRY

Dr. Meliyanthi M. Gunatillaka Consultant Chemical Pathologist and Head, Department of Biochemistry Medical Research Institute, Colombo Ms. D. K. Daya Silva Superintendent Grade Medical Laboratory Technologist Medical Research Institute, Colombo Mr. M. Muhammed Hunais Medical Laboratory Technologist Medical Research Institute, Colombo

ISBN 978-955-1647-00-1 This document is NOT for sale. The document may, however, be freely reviewed, abstracted, reproduced or translated, in part or in whole for non commercial purposes.

CONTENTS

i

Contents ............................................................................................................................................i Acknowledgements.........................................................................................................................ii Preface.............................................................................................................................................iii General introduction .....................................................................................................................iv 1. Format of a technical procedure manual ............................................................................1 2. Albumin ..................................................................................................................................3 3. Amylase ...................................................................................................................................7 4. Alkaline phosphatase.......................................................................................................... 10 5. Aspartate amino transferase .............................................................................................. 13 6. Alanine amino transferase.................................................................................................. 18 7. Bilirubin................................................................................................................................ 21 8. Calcium ................................................................................................................................ 27 Calcium in urine .................................................................................................................. 31 9. Creatinine............................................................................................................................. 32 10. Urine Creatinine ............................................................................................................. 35 Creatinine clearance........................................................................................................ 36 11. Cholesterol ...................................................................................................................... 37 12. Glucose............................................................................................................................ 40 13. Inorganic phosphate ...................................................................................................... 46 14. Inorganic phosphate in urine ........................................................................................ 49 15. Total protein ................................................................................................................... 50 16. Urea.................................................................................................................................. 54 17. Uric acid .......................................................................................................................... 58 18. Urine uric acid................................................................................................................. 61 19. Electrolytes...................................................................................................................... 62 20. Urine sodium and potassium ........................................................................................ 67 21. Appendix 1 - Sample Collection and Transportation ............................................. 68 22. Appendix 2 - Diabetes mellitus ................................................................................. 71 23 Appendix 3 - Reference ranges.................................................................................. 73 References: .................................................................................................................................... 94

Department of Biochemistry, Medical Research Institute, Colombo-WHO Biennium 2004-2005

ii

ACKNOWLEDGEMENTS We would like to acknowledge the WHO representative to Sri Lanka, Dr. Kan Tun, for identifying the need for quality assurance in local laboratories and offering us the opportunity to publish this handbook. We thank the Director General of Health Services Dr. Athula Kahandaliyanage, the Deputy Director General (Planning) Dr. T. S. B. Tennekoon and the Deputy Director General (Education, Training and Research) Dr. Stanley De Silva, Deputy Director General (Laboratory Services) Dr. Ajith Mendis and Director Laboratory Services Dr. Jayasundara Bandara, for approval and facilitation of this project. We are grateful to the Director of the MRI Dr. G. S. S. K. Colombage and the Deputy Director of MRI Dr. Lulu Raschid, for all their support and encouragement in bringing this project to fruition. We appreciate the assistance of administrative staff of World Health Organisation and colleagues, resource persons, administrative staff of the Medical Research Institute and staff of the Department of Biochemistry.

Department of Biochemistry, Medical Research Institute, Colombo-WHO Biennium 2004-2005

iii

PREFACE Clinical laboratory services have become an important component of modern medicine. Clinical laboratories play a major role in the diagnosis, treatment, prognosis and monitoring of diseases. Quality assurance in laboratory services, aimed at improving reliability, efficiency and facilitating inter – laboratory comparability in testing is the backbone of quality health care delivery. The use of standard operating procedures in laboratory testing is one of the major factors in achieving quality. This manual provides guidelines on standard operating procedures, sample collection and reference ranges for routine biochemical methods. The publication aims at popularising the use of biochemical methods recommended by the world health organization, which may be adapted to local needs, based on the experience of the editors. It is hoped that this publication will be useful in achieving its objective of improving the quality of laboratory results using the available resources. .

Department of Biochemistry, Medical Research Institute, Colombo-WHO Biennium 2004-2005

iv GENERAL INTRODUCTION Standard Operating Procedure (SOP) manuals are required in the laboratory and are a key element in internal Quality Control within the laboratory. Hence, assuring quality health care, method manuals should include properly authenticated methods, or methods recommended by relevant professional organisations. A methods manual containing all methods and procedures needs to be authorised for use in the laboratory. The manuals shall be available in the appropriate work areas as bench copies and it is recommended that a master copy be maintained by the head of the unit. These manuals should be review at least annually, by the competent member of the technical staff and the head of the unit. The first consideration is the selection of the test methods best suited to full fill the function of the laboratory. Each test performed in the laboratory (for e.g. screening, routine clinical testing, reference laboratory service) must be evaluated and appropriate methods should be selected depending on the local needs and resources. Each method should be evaluated in terms of sensitivity, specificity, accuracy, simplicity, speed, reliability and economy. The methods that are included in this manual are based on world health organization recommended biochemical tests, adapted and evaluated by the editors taking into consideration the clinical and technical experience, quality control measures and the available resources. This standard operating procedure manual includes the routine biochemical tests based on manual spectrophotometric methods. The clinical significance of each analyte has been described in detail for the benefit of the users. The precautions to be followed are included for each analyte to maximize the performance. This is based largely on the experience of the editors. The manual contains the collection procedures of blood and urine samples. It includes the method of collection, selection of containers and preservatives, storage, transport and stability. There must be vigorous control of the procedures involved in the collection and identification of specimens to ensure the quality of the specimen to be examined in the laboratory. Copies of collection procedures should be available at all collection areas. The collection manual should be updated regularly as for laboratory procedure manual by the competent member of the technical staff. Specimen which do not conform to the requirements or are of inadequate quality for the test involved may be rejected with the consultation of the head of the unit and the sender informed. Appropriate techniques shall be used for the correct identification of specimens and release of results. Specimens shall be retained by the laboratory for a time appropriate to the nature and origin of the specimen. The manual includes a section for reference ranges. The reference ranges are given for many biochemical analytes considering the age and the sex. Standard adult and paediatric text books along with the information available on the internet were taken into consideration in accepting the relevant reference ranges. Department of Biochemistry, Medical Research Institute, Colombo-WHO Biennium 2004-2005

1

1.

FORMAT OF A TECHNICAL PROCEDURE MANUAL The following format for technical procedures should be adhered to as closely as possible and double spaced typing is recommended.

TITLE Name of test or procedure. INTRODUCTION

A brief, concise introduction of the analyte is recommended. (In this manual a detailed description is included for the benefit of the user to prepare their own local S.O.P) CLINICAL SIGNIFICANCE Give reasons as to why the test is usually requested and indicates the significance of low or high results using the reference PRINCIPLE A short but informative statement which describe the basis of the method. REFERENCE(S) A list of primary reference(s) as well as modifications.

SPECIMEN List the types of specimens which are appropriate and indicate stability limits if known GLASSWARE AND EQUIPMENT

A detailed description of required glassware and equipment should be provided. REAGENTS State concentration or use descriptive name where appropriate (e.g. Biuret reagent) and give detailed stepwise instructions for preparing each reagent. Specify chemical brand and grade where it is known to be critical. Unusual reagents/components should have source or company stated. Expiry of reagents should be specified. PROCEDURE Present a concise stepwise description of the method. CALCULATIONS Explain the use of formulae and indicate, briefly, the derivation of factors. UNITS Units used and abbreviations. REFERENCE RANGE Adult reference ranges for males and females and paediatric ranges where applicable. QUALITY CONTROL Quality control procedures, including documentation and methods of statistical analysis NOTES Elaborate on any particular points which may require further explanation

Department of Biochemistry, Medical Research Institute, Colombo-WHO Biennium 2004-2005

2

APPROVAL Each procedure to be dated and signed at the bottom of the final page by the appropriate senior authorised qualified staff. Any amendments should be authorised with date and signature. METHOD HISTORY This should be in a separate sheet and include date notations of method changes and review REPORTING Format and procedure of reporting, including procedures for urgent and especially clinically significant results, must be described. COPIES Any copies of method for bench use are to be made in full from the master copy and should include method history and approval sections SUPPLEMENTARY INFORMATION Certain information to be used at the bench to implement procedures may be extracted from the procedure manual. This information may include flow diagrams, index cards, and manufacturer product literature. Such supplementary information must be current and be referenced to the procedure manual by date, procedure and reviewer’s initials.

Department of Biochemistry, Medical Research Institute, Colombo-WHO Biennium 2004-2005

3

2.

ALBUMIN 2.1

INTRODUCTION

Albumin is the most abundant protein in human plasma from 20 weeks of gestation, representing 40-60% of the total protein. It is synthesized exclusively in the liver. The rate of synthesis is depended on protein intake and subject to feed back regulation by the plasma albumin level. The half life of albumin is estimated at 15-19 days. Traces of albumin can be found in almost all extra vascular body fluids. The loss of albumin via the glomerular filtrate is very small as almost all the albumin is reabsorbed by the proximal tubular cells. Albumin is catabolized in various tissues where it is taken up by cells by pinocytosis. Its constituent amino acids are released by intracellular proteolysis and returned to the body pool. Albumin has a molecular weight of approximately 66,000. Albumin is an anion at pH 7.4 with more than 200 negative charges per molecule. The chief biological functions of albumin are to transport and store a wide variety of ligands, to maintain the plasma osmotic pressure and to serve as a source of endogenous amino acids. The capacity of albumin to act as a binding protein is due to the large numbers of charges of each molecule as well as very large no of molecules available. Albumin binds nonpolar compounds such as bilirubin & long chain fatty acids. Albumin binds hormones such as thyroxine, triiodothyronin, cortisol and aldosterone, thus act as a reservoir in which these compounds are stored in inactive form but from which they are readily mobilised. Some 40% of serum calcium is bound to albumin. Many drugs such as phenylbutazone, warfarin and salicylates are also strongly bounded to albumin. Albumin concentration is the major determinant of plasma oncotic pressure, one of the factors that regulate partition of water between intra and extra vascular compartments.

2.2

CLINICAL SIGNIFICANCE



Hypoalbuminaemia is very common and may result due to the following factors o Impaired synthesis: Diminished protein intake or liver disease o Increased catabolism: Due to tissue damage and inflammation o Reduced absorption of amino acids: Malabsorption syndromes or malnutrition. o Protein loss in urine: Due to nephrotic syndrome,chronic glomerular nephritis, diabetes,or systemic lupus erythromatosis o Protein loss in faeces: Due to protein losing enteropathy o Protein loss through the skin: Burns o Altered distribution: Ascites(high pressure in the portal circulation drives albumin into the peritoneal fluid) Most severe hypoalbuminaemia is caused by protein loss by way of urine or faeces. When plasma albumin levels are less than 2.0 g/l oedema is usually present.  Hyperalbuminemia: is of little diagnostic significance except in dehydration. Albumin has more than 20 genetic variants, which are not associated with disease but which cause two bands or a single band in the albumin region on electrophoresis. The condition is called bisalbuminaemia. A transient form is sometimes caused by the intake of drugs. Congenital absence of albumin or analbuminaemia is asymptomatic except for occasional slight oedema.

2.3

PRINCIPLE OF THE METHOD

The requirements of a dye binding method for albumin include specific binding of the dye to albumin in the presence of other plasma or serum protein, high binding affinity between dye and albumin so that small changes in ionic strength, pH or the presence of competing

Department of Biochemistry, Medical Research Institute, Colombo-WHO Biennium 2004-2005

4

ligands do not break the dye-protein complex; a substantial shift in the absorption wavelength of the dye in the bound form so that it remains spectrally distinct from the free form present in excess, and absorption maximum for the bound form at a wavelength distinct from those at which Bilirubin and haemoglobin can interfere. Serum albumin and buffered BCG (Bromocresol green) are allowed to bind at pH 4.2, and absorption of the BCG/Albumin complex is determined spectrometrically at 632 nm (filter No 607) Albumin act as a cation to bind the anionic dye. Absorption reading taken within 30 seconds of mixing the serum and BCG avoids the problem of non specific reaction of BCG with globulin. The manual method described here for albumin by BCG is adaptable to automated analysis.

2.4

SPECIMEN TYPE, COLLECTION AND STORAGE

Serum is preferred. Fasting specimen is not an absolute requirement but it may be desirable because marked lipaemia interferes in the assay. Avoid, applying a tourniquet in specimen collection because haemoconcentration due to venous stasis increases the apparent concentration of albumin and other plasma proteins. Storage: Separate the serum within 2 hours of collection; separated serum in a tightly stoppered container is stable for 24 hours at room temperature 25-30 0 C, for 1 week at 2-8 0C, for 3 months at -20 0C. APPARATUS AND CHEMICALS APPARATUS: Visible spectrophotometer, wavelength 632 nm or Colorimeter, orange filter, Ilford 607 (600nm) pH meter GLASSWARE: Volumetric flasks (1 litre and 100 ml volumes) Micro pipette (20 µl) Graduated pipettes (10 ml in 0.1 ml) Beakers (5ml, 50ml, 500 ml and 1 litre) Amber colour reagent bottles (1 litre) Test tubes (125 mm x 16 mm) Measuring cylinders (1 litre) CHEMICALS: (All chemicals must be analytical grade) Bromocresol green sodium salt, also called BCG, water soluble Sodium azide caution: handle with care Sodium chloride Succinic acid Sodium hydroxide pellets Brij-35 (polyoxy7ethylene (23) lauryl ether) solid or solution 30% w/v Standard buffers for pH meter Bovine albumin or other available calibrator (fraction v powder) REAGENTS 1. Succinic acid solution 50 g/l: weigh out 1.0 g of Succinic acid, dissolve and make up to about 20 ml with distilled water. Prepare as required, discard after use. 2. Sodium hydroxide solution 10 g/l: Weigh out 1.0 g of sodium hydroxide in a glass beaker, dissolve and make up to 100 ml with distilled water. This solution is stable for several months at 20-25 C. Store in polypropylene bottle.

Department of Biochemistry, Medical Research Institute, Colombo-WHO Biennium 2004-2005

5

3. Brij-35 solution 250 g/l: Warm 25 g solid Brij-35 in a small volume of distilled water to dissolve and make up to 100 ml with distilled water or use the 30 % w/v Brij-35 solution. 4. Working dye solution: Dissolve 5.6 g of Succinic acid, 58 mg of Bromocresol green (Sodium salt) and 100 mg of sodium azide in about 900 ml of distilled water in a clean beaker. Add 1.0 g of sodium hydroxide and dissolve, and then add 2.5 ml of Brij-35 250 g/l solution. (If 30% Brij solution is used add 2.1 ml)Adjust to pH 4.2 using small volumes of sodium hydroxide solution or Succinic acid solution if necessary. Transfer slowly (avoid frothing) to 1 litre volumetric flask and make up to volume with distilled water, mix gently and transfer into a clean brown bottle. This solution is stable for several months at 2-8 0C. NOTE: The BCG working dye solution requires careful preparation. Some laboratories may find it economical to purchase this solution. There are several different BCG reagents available. Make sure you select a BCG reagent in Succinate buffer at pH 4.2. In 10 mm light path cuvette (cell) the working dye solution should have the following absorbance readings (zero the instrument with distilled water). Spectrometer

Colorimeter

Wavelength

Absorbance

Filter

Absorbance

430 nm

About 1.4

601

About 1.1

615 nm

About 0.25

607

About 0.2

5. Succinate buffer solution: Prepare in exactly the same way as the working dye solution but do not add any Bromocresol green. This solution is stable for several months at 2-8 0C 6. Albumin standard 40 g/l: Using a 5ml volumetric pipette dilute 5.0 ml of the bovine albumin standard (80 g/l) with 5.0 ml of sodium chloride/sodium azide solution to prepare an albumin standard containing 40 g/l. This standard is stable for 6 months at 2-8 0C. (Bovine albumin standard 80 g/l provide by the Department of Biochemistry, MRI) 7. Sodium chloride /Sodium azide solution: Weigh out 9.0 g of sodium chloride and 1.0 g of sodium azide, dissolve and make up to 1 litre with distilled water. This solution is stable indefinitely at 20-25 0C (room temperature) 2.5 PROCEDURE 1. Pipette 4.0 ml of working dye solution into test tubes. 2. Add 20 µl of standard or control or test sample, mix and measure the absorbance immediately (within 30 seconds). 3. Read the absorbance at 632 nm or filter No 607 after setting the instrument to zero absorbance with the working dye solution. PREPARATION OF CALIBRATION GRAPH Preparation of working albumin standard solutions: They are prepared by dilution of the albumin standard (40 g/l) in sodium chloride/sodium azide solution as follows: Working standard No

(01)

(03)

(04)

0.5

(02) )1.0

Albumin standard 40 g/l (ml)

1.5

2.0

Sodium chloride/Sodium azide solution (ml)

1.5

1.0

0.5

0.0

Concentration of working standards (g/l)

10

20

30

40

Department of Biochemistry, Medical Research Institute, Colombo-WHO Biennium 2004-2005

6

Working dye solution (ml) 4.0 4.0 4.0 4.0 Working standard No (20 µl) (01) (02) (03) (04) ) Mix each tube thoroughly Read the absorbance of each tube immediately at 632 nm (or filter No 607) after setting the instrument to zero with working dye solution. Plot the absorbance of each tube against the concentrations of working standards solution on the graph. The calibration graph should be linear up to 40 g/l. If the graph is linear then a single standard (40g/l) may be used for routine analysis but linearity should be confirmed for each new batch of working dye solution and at least once a month. The recommended method proposes the use of a bovine albumin solution as a calibrator. Alternative commercial bovine albumin solutions are available. 2.6 CALCULATION Albumin concentration = T/S x 40 g/l Where T=the absorbance of the test S=the absorbance of the standard NOTE: Always include a standard (40 g/l) in each and every batch of samples. QUALITY CONTROL OPTIMAL CONDITIONS VARIANCE should be attainable. ROUTINE CONDITIONS VARIANCE exceed 6%

: A coefficient of variation of around 3% : The value obtained for the RCV should not

REFERENCE VALUES New born : 25-50 g/l 1 Year : 35-50 g/l 2-3 Year : 36-50 g/l 4th Year and after: 37-50 g/l Adult : 30-45 g/l

2.7

LIMITATIONS

If a serum sample is extremely lipaemic a serum blank should be used. (Moderate lipaemia does not affect the results) The blank is prepared by adding 20 µl of sample to 4.0 ml of Succinate buffer solution. The absorbance of this blank, with distilled water as reference is subtracted from the test. Grossly haemolysed specimens are unsuitable for albumin determination. REFERENCES Ann.clin.Biochem.14 (1977)105-115 Tietz text book of clinical chemistry

Department of Biochemistry, Medical Research Institute, Colombo-WHO Biennium 2004-2005

7

3.

AMYLASE 3.1 INTRODUCTION Amylases are a group of hydrolases that split complex carbohydrates constituted of α-Dglucose units linked through carbon atoms 1 and 4 located on adjacent glucose residues. Both straight-chain (linear) polyglucans, such as amylose, and branched polyglucans, such as amylopectin and glycogen, are hydrolyzed, but at different rates. In the case of amylose, the enzyme splits the chains at alternate α-1, 4-hemiacetal (-C-O-C-) links, forming maltose and some residual glucose; maltose, glucose, and a residue of limit dextrins are formed if branched –chain polyglucans are used as substrate. The α-1, 6- linkages at the branch points are not attacked by the enzyme. Two types of amylases are recognized. Beta-amylase (e.g. plant and bacterial exoamylase) acts only at the terminal-reducing end of polyglucan chain; it splits off two glucose units (maltose) at a time. Animal amylases, including those present in human tissues, are α-amylases. They are also called endoamylases because they attack α-1, 4-linkages in a random manner anywhere along the polyglucan chain. Linear starch chains in helical form react with molecular iodine to form the well-known deep blue starch-iodine complex. The enzyme present in normal serum and urine is predominantly of pancreatic (p-type) and salivary gland (S-type) origin. These isoenzymes are products of two closely linked loci on chromosome 1. Each gene is allelic; thus there are 12 distinct phenotypes for the salivary isoenzyme and 6 for the pancreatic isoenzyme. 3.2 CLINICAL SIGNIFICANCE Assays of amylase activity in serum and urine are largely of use in the diagnosis of diseases of the pancreas. In acute pancreatitis a transient rise in serum amylase activity occurs in 212 hours of the onset; levels return to normal by the third or the fourth day. A four to six fold elevation in amylase activity above the reference limit is usual, with maximal levels attained in 12 to 72 hours. The magnitude of the elevation of serum enzyme activity is not related to the severity of pancreas involvement. However the greater the rise, greater the probability of acute pancreatitis. A significant amount of serum amylase is excreted in urine and therefore elevation of serum activity is reflected in the rise of urine amylase activity. Urine amylase as compared with serum amylase appears to be more frequently elevated, reaches higher levels and persist for longer periods. In quiescent chronic pancreatitis both serum and urine activity are usually subnormal. Acute pancreatitis is sometimes difficult to diagnose because it must be differentiated from other acute intra abdominal disorders and because an increase serum amylase activity may not necessary due to acute pancreatitis.

3.3

PRINCIPLE OF THE METHOD

In solution iodine reacts with starch to give an intense blue – violet complex. Amylase hydrolyses starch, forming maltose and other fragments which do not react with iodine. After incubation of serum with buffered starch solution, the amount of starch remaining is assayed by measuring the absorbance at 660 nm after the addition of iodine

3.4

SPECIMEN TYPE, COLLECTION AND STORAGE

3-5 ml of clotted blood in a clean dry bottle; avoid haemolysis; Separate serum as early as possible. Enzyme activity loss is negligible in sterile serum stored at 2-8 0C for a week (free of bacterial contamination). 3.5 APPARATUS AND CHEMICALS APPARATUS: Hot plate or Bunsen burner Water bath at 37 0C, pH meter Visible Spectrometer wavelength at 660nm or Colorimeter with red filter Ilford No: 608 (680 nm) Department of Biochemistry, Medical Research Institute, Colombo-WHO Biennium 2004-2005

8

GLASSWARE: Volumetric flasks (1litre volume) Automatic micro pipette 20 µl Graduated pipettes (1ml, 5ml, 10 ml in 0.1 ml) Beakers (50ml, 1 litre) Graduated cylinders (100 ml and 1 litre) Amber colour reagents bottles (100 ml and 1 litre)

CHEMICALS: Soluble starch pharmaceutical grade Potassium iodide AR Potassium iodate AR Disodium hydrogen orthophosphate (anhydrous) AR Sodium chloride AR Benzoic acid AR Hydrochloric acid (concentrated (37 % w/v) caution: highly corrosive) Buffers for pH meter

REAGENTS 1. Buffered starch substrate: Dissolve 26.6 g of anhydrous disodium hydrogen phosphate, 1.75 g of sodium chloride and 8.6 g of benzoic acid in about 500 ml of distilled water in a large beaker. Heat to boil. In a 50 ml beaker, mix separately 0.4 g of soluble starch in 10 ml of cold distilled water to form a paste. Add the paste with stirring to the boiling mixture, rinsing the beaker with distilled water. Continue to boil for one minute. Cool to room temperature, and transfer to volumetric flask and dilute to 1 litre with distilled water. This solution is stable for at least one year at 20- 25 0C and should have a pH of 6.9-7.1; the stability is monitored by noting the absorbance of the reagent blank with each set of tests. We recommend that the solution is stored at 4-8 0C in the refrigerator. Aliquot the required amount for daily use. If 1 litre substrate is excess then prepare 500 ml for use. 2. Stock iodine solution 50 mmol/l: Dissolve 3.57 g of potassium iodate and 45 g of potassium iodide in about 800 ml of water in a volumetric flask. Slowly and with mixing add 9.0 ml of concentrated hydrochloric acid. Dilute to 1 litre with distilled water. This solution should be stored in a dark bottle and is stable for a year at 4 -8 0C. 3. Working iodine solution: Dilute 10 ml of stock iodine solution with 90 ml distilled water in a graduated 100 ml volumetric flask. This solution should be stored in a dark bottle and is stable for 2 months at 2-8 0C. 3.6 PROCEDURE 1. Pipette 1.0 ml of buffered starch substrate into 150 x 16 mm test tubes. You will need 1 tube for each patient and control sample and 1 tube for a reagent blank. 2. Place all of the tubes in a water bath at 37 0C for 5 minutes to warm the contents. 3. Pipette 20 µl of patient’s or control serum into the bottom of the test tubes, mix and incubate at 37 0C for exactly 7 minutes and 30 seconds. (No serum is added to the reagent blank). 4. After 7 minutes and 30 seconds remove the test tubes from the water bath immediately add 1.0 ml of working iodine solution to each tube (samples and reagent blank) then add 8 ml of distilled water. 5. Mix the contents of each tube well then measure the absorbance without delay at 660 nm (red filter Ilford No. 608) setting the spectrometer to zero with distilled water. NOTE: Avoid contamination of the pipette with saliva

3.7

CALCULATION

Amylase activity U/L B T 1470

= B-T x 1470 B = absorbance of reagent blank = absorbance of test = factor to express values in U/L

Department of Biochemistry, Medical Research Institute, Colombo-WHO Biennium 2004-2005

9

If the result is greater than 735 U/L (i.e. there is no blue colour in tube T) then the sample must be diluted with saline (20 µl serum + 100 µl saline) and the analysis repeated using 20 µl of the diluted sample. The measured value must be multiplied by 6 to calculate the amylase activity of the sample to take into account the dilution factor. QUALITY CONTROL A quality control sample with a value in the range 200 – 700 U/L should be analysed with each batch of specimens. If single specimens are analysed a control specimen should always be included. OPTIMAL CONDITIONS VARIANCE: A coefficient of variation of around 6% should be attainable. ROUTINE CONDITIONS VARIANCE: This value should not exceed 12 % REFERENCE VALUES: Approximate reference values: 70 – 340 U/L REFERENCES WHO Manual LAB/86.3

Department of Biochemistry, Medical Research Institute, Colombo-WHO Biennium 2004-2005

10

4.

ALKALINE PHOSPHATASE 4.1

INTRODUCTION

The alkaline phosphatases (ALP) are a group of glycoprotein enzymes that act as phosphotransferases by hydrolysing various types of monophosphate bonds at alkaline pH. ALP activity is found in virtually all tissues, particularly bone, liver, kidney, intestine, adrenal and placenta. The protein moieties comprise about 510 amino acid residues, to which is attached various amounts of carbohydrate and sialic acid. Tissue-specific posttranslational modifications occur to the carbohydrate content, leading to the formation of isoforms, e.g. bone, liver and kidney ALP, each of which contains the same tissue nonspecific protein. ALP is found attached to the outer lipid bilayer of cell membranes by a glycosyl-phosphatidylinositol group, in the case of liver ALP possibly as a tetramer of identical subunits; if released from cell membranes, ALP is dimeric. Liver ALP is located in the cell membranes of the hepatcoyte, and particularly in the outer layer of the cells adjacent to the bile canaliculi and also in the cells lining the sinusoids. Adult intestinal ALP lacks sialic acid and is found in the epithelial cells of the intestinal brush border. The placental enzyme is formed by the syncitiotrophoblast cells lining the microvilli that interface the placental and fetal blood circulations but the placental ALP does not cross into the fetal circulation.

4.2

CLINICAL SIGNIFICANCE

Serum ALP measurements are of particular interest in the investigation of two groups of conditions: hepatobiliary and bone disease associated with increased osteoblastic activity. The response of the liver to any form of biliary tree obstruction is to synthesize more ALP. The main site of new enzyme synthesis is the hepatocytes adjacent to the biliary canaliculi. Some of the newly formed enzyme enters the circulation to raise the enzyme level in serum. The elevation tends to be more marked (more than three-fold) in extrahepatic obstruction (e.g. by stone or by cancer of the head of the pancreas) than in intrahepatic obstruction and is greater the more complete the obstruction. Serum enzyme activities may reach 10 to 12 times the upper limit of normal, returning to normal on surgical removal of the obstruction. Inappropriate ALP synthesis is observed in some types of tumours. In children the ALP activity is due to the physiological response of osteoblasts during growth. ALP activity is pathologically high in children with rickets.

4.3

PRINCIPLE OF THE METHOD

4 - Nitophenylphosphate is hydrolysed by alkaline phosphatase at pH 10.3 at 37 0 C and 4 Nitrophenol is liberated. Alkali is added to stop the enzyme activity at the end of the timed incubation period and the increase in absorbance due to the 4-Nitrophenol released is measured at 410 nm.

4.4

SPECIMEN TYPE, COLLECTION AND STORAGE

Collect about 2-3 ml blood, separate serum as soon as possible, avoid haemolysis. Freshly collected serum sample should be kept at room temperature and assayed as soon as possible. If there is a delay in the assay, store the serum at -20 C. Sample should be completely thawed before the assay. Refrigeration of the serum at 40 C will increase the ALP activity.

4.5

APPARATUS AND CHEMICALS

APPARATUS: Water bath at 37 0C pH meter Spectrometer wavelength at 410 nm or Colorimeter with violet filter, Ilford 600 (410 nm) Department of Biochemistry, Medical Research Institute, Colombo-WHO Biennium 2004-2005

11

GLASSWARE: Volumetric flasks (100 ml and 1 litre volumes) Beakers (1 litre) Measuring cylinders (100 ml and 1 litre) Test tubes (125 mm x 16 mm or 150 mm x 16 mm) Volumetric pipettes (5 ml in 0.1 ml) Polyethylene reagent bottles (1 litre) Amber colour bottles Graduated pipettes (1ml, 5ml, 10ml)

Automatic pipette (50µl and 100µl) CHEMICALS: All chemicals must be analar grade

Standard buffer solutions for pH meter Hydrochloric acid (Concentrated (37% w/v); caution: highly corrosive) 2-Amino-2-methyl-1-propanol Magnesium chloride hexahydrateDisodium 4 – Nitrophenyl phosphate hexahydrate Sodium hydroxide pellets 4 - Nitrophenol

REAGENTS 1. AMP Buffer pH 10.3: Dissolve 78.5 g of 2-amino-2-methyl-1-propanol (CH3)2 C (NH2) CH2 OH in about 900 ml of distilled water. Adjust to pH 10.3 with concentrated hydrochloric acid (about 18 ml) and make up to 1 litre with distilled water. Store in an Amber colour reagent bottle. This solution is stable for 1 month at 20-25 0C. Method done at MRI: Use the chemical with the specifications of 95 % (CH3)2C (NH2)CH2OH with MW(89.14) and specific gravity of 0.93 g/ml (BDH). Add 84.4 ml of the above liquid to 800 ml of distilled water. 2. Magnesium chloride solution 1.5mmol/l: Dissolve 300 mg of magnesium chloride hexahydrate in water and make up to 1 litre. This solution is stable indefinitely at 20 250C. MgCl2.6H2O chemical and the solution (1.5mmol/l) are best stored in refrigerator. 3. Substrate solution 225 mmol/l in the magnesium chloride solution: Dissolve 83.5 mg of disodium 4 -Nitrophenyl phosphate hexahydrate (store the chemical in freezing compartment) in 1.0 ml of magnesium chloride solution as required. This solution is stable for one working day. It’s best to keep the solution in refrigerator since at room temperature colour development may occur. 4. Sodium hydroxide solution 250 mmol/l: Dissolve 10 g of sodium hydroxide in distilled water and make up to 1 litre. Store in a tightly Stopperd polyethylene bottle. This solution is stable indefinitely at 20-25 0C. We have observed that this solution is stable even at room temperature at 20-30 0C. 5. 4 – Nitrophenol stock solution 10.8 mmol/l: Weigh out 150 mg of 4 – Nitrophenol accurately in a beaker & transfer the chemical from beaker to a 100 ml volumetric flask using a funnel and wash any chemical remaining in the container into the volumetric flask with distilled water. Make up to the mark with distilled water. This solution is stable for about 6 months in an amber colour bottle at 4 0C. 6. 4 – Nitrophenol working solution 54 µmol/l: Pipette 0.5 ml of the 4 – Nitrophenol stock solution into a 100 ml volumetric flask, make up to 100 ml with sodium hydroxide solution (250mmol/l) Prepare this solution freshly before use. NOTE: The quality of the disodium 4 – Nitrophenyl phosphate should be checked to ensure that it does not contain excessive amounts of free 4 – Nitrophenol. Prepare sodium hydroxide solution (10mmol/l) by diluting 4.0 ml of sodium hydroxide solution (250mmol/l) to 100 ml with distilled water. Add 200 µl of substrate solution (reagent 3 above) to 3.8 ml of sodium hydroxide solution (10 mmol/l) Mix and measure the absorbance at 410 nm after setting the spectrometer to zero with sodium hydroxide solution (10 mmol/l). The quality of the substrate is satisfactory if its absorbance after dilution in sodium hydroxide solution is less than 0.25 (10 mm light path cuvette at room Department of Biochemistry, Medical Research Institute, Colombo-WHO Biennium 2004-2005

12

temperature and 410 nm) In a colorimetric the absorbance should be less than 0.12 (10mm light path and Ilford filter No 600) This procedure should be carried out when a new batch of chemical/bottle is introduced to the bench.

4.6

PROCEDURE

1. Pipette 1.4 ml of AMP buffer into sufficient test tubes for patients’ samples, controls and reagent blank and pre incubate in the water bath at 37 0C for about 5 minutes. 2. To each tube add 50 µl of serum, to the blank; add 50 µl of distilled water .Mix well. 3. To each tube in sequence add 100 µl of substrate solution at timed intervals. Mix well. 4. Incubate for exactly 15 minutes at 37 0C then add 4.0 ml of sodium hydroxide solution (250mmol/l) to each tube in sequence, maintaining timed intervals. Mix each tube and allow them to cool to room temperature. 5. Measure the absorbance of each test solution at 410 nm ( violet filter ,Ilford No :600) setting the spectrometer to zero with the blank 6. If the absorbance is greater than the absorbance of a 400 U/L standard, then repeat the procedures, but at step 4, incubate for exactly 5 minutes (instead of 15 minutes) then add 4.0 ml sodium hydroxide solution (250mmol/l) and complete the procedure described in steps 4 and 5. Multiply the activity by 3 before reporting the result. PREPARATION OF CALIBRATION GRAPH Tube No (1) (2) (3) (4) (5) (6) 4 – Nitrophenol solution 54 µmol/l (ml) 1 2 4 6 8 10 Sodium hydroxide 250 mmol/l (ml) 9 8 6 4 2 0 Activity (U/L) 40 80 160 240 320 400 Mix well and measure the absorbance of each tube at 410 nm (violet filter Ilford No 600) setting the spectrometer to zero with sodium hydroxide solution (250mmol/l) Plot the absorbance of each tube on the graph. Prepare a new calibration graph every 3 months.

4.7

CALCULATION

Read off the activities of alkaline phosphatase in the unknown and control samples from the calibration graph. Multiply by 3 if you used 5 minute incubation instead of 15 minutes incubation. QUALITY CONTROL At least two serum control specimens, having stated values in the range 20-350 U/L, one of which is unknown to the operator should be included with each batch of specimens. If single specimens are analysed a control specimen should always be included. OPTIMAL CONDITIONS VARIANCE: A coefficient of variation of around 10% should be attainable ROUTINE CONDITIONS VARIANCE: The value should be less than 20% APPROXIMATE REFERENCE VALUES Males (age 20 -60 years) Females (age 15-60 years) Children (age 1-12 years) During the growth spurt of puberty

: 20- 90 U/L : 20-90 U/L : up to 350 U/L : up to 500 U/L

REFERENCES LAB/86.3 A guide to diagnostic clinical chemistry By R.N. Walmsley and G.H. White -page 312 TIETZ TEXTBOOK OF Clinical chemistry page 831-832

Department of Biochemistry, Medical Research Institute, Colombo-WHO Biennium 2004-2005

13

5.

ASPARTATE AMINO TRANSFERASE 5.1

INTRODUCTION

Aspartate Amino Transferase & Alanine Amino Transferase The aminotransferases constitute a group of enzymes that catalyze the interconvertion of amino acids and α- oxo-acids by transfer of amino groups. Aspartate amino transferase (AST) and Alanine amino transferase (ALT) are the two enzymes that are of clinical significance. Distinct isoenzymes of AST are present respectively in cytoplasm and mytochondria of cells. The α- oxoglutarate/glutamate couple serves as one amino group acceptor and donor pair in all amino-transfer reactions; the specificity of the individual enzymes derives from the particular aminoacid that serves as the other donor of an amino group. AST

L-Aspartate + 2- Oxaglutarate

Oxaloacetate + L –Glutamate ALT

L-Alanine + 2- Oxaglutarate

Pyruate

+ L –Glutamate

The reactions are reversible, but the equilibria of the AST and ALT reactions favour formation of aspartate and alanine, respectively. Pyridoxal -5’ phosphate and its amino analogue, pyridoxamine-5’ –phosphate function as coenzymes in the amino transfer reactions. Transaminases are widely distributed in tissues. Both AST and ALT are normally present in human plasma, bile, cerebrospinal fluid and saliva, but none is found in urine unless a kidney lesion is present.

5.2

CLINICAL SIGNIFICANCE

In viral hepatitis and other forms of liver disease associated with hepatic necrosis, serum AST and ALT levels are elevated even before the clinical signs and symptoms of the disease (such has jaundice) appear. Levels for both enzymes may reach values as high as 100 times the upper reference limit, although 20- to 50- fold elevations are most frequently encountered. Peak values of transaminase activity occur between the 7th and 12th days; activities then gradually decrease, reaching normal levels by the 3rd to 5th week if recovery is uneventful. Alcoholic hepatitis has more modest elevations. In infectious hepatitis and other inflammatory conditions affecting the liver, ALT is characteristically as high as or higher than AST, and the ALT/AST ratio, which normally and in other conditions is less than 1, becomes greater than unity. The use of different assay methods for the two enzymes may alter the activities of the two enzymes relative to each other, and hence the numerical values of the ratio observed may differ between laboratories. Nevertheless, the principle that hepatitis is associated with comparable elevations of the two activities remains valid. The relatively similar elevations of AST and ALT in hepatitis have been attributed to the release of only the cytoplasmic isoenzyme of AST into the circulation from reversibly damaged parenchymal cells. When necrosis of the cells occurs, considerable amounts of mitochondrial AST are also released, depressing the ALT/AST ratio. The picture in toxic hepatitis is similar to that in infectious hepatitis, with very high ALT and AST activity being observed in severe cases. Elevations up to 20 times the upper reference limit may be encountered in infectious mononucleosis with liver involvement and somewhat lover values in intrahepatic cholestasis. Increased levels may also be observed in extrahepatic cholestatis, with levels tending to be higher the more chronic the obstruction. The aminotransferase levels observed in cirrhosis vary with the status of the cirrhotic process; they range from upper normal to some four to five times normal, with the level of AST activity higher than that of ALT activity. Elevations probably indicate continuing cellular necrosis. Five- to 10- fold elevations of both enzymes occur in patients with primary or metastatic carcinoma of the liver, with AST usually being higher than ALT, but levels are Department of Biochemistry, Medical Research Institute, Colombo-WHO Biennium 2004-2005

14

often normal in the early stages of malignant infiltration of the liver. Slight or moderate elevations of both AST and ALT activities may be observed after the intake of alcohol, in delirium tremens, and after administration of various drugs, such as opiates, salicylates, or ampicillin. Although serum levels of both AST and ALT become elevated whenever disease processes affect liver cell integrity. ALT is the more liver-specific enzyme. Serum elevations of ALT activity are rarely observed in conditions other than parenchymal liver disease. Moreover, elevations of ALT activity persist longer than do those of AST activity. Measurement of both AST and ALT has some value in distinguishing hepatitis from other parenchymal lesions. After myocardial infarction, increased AST activity appears in serum, as might be expected from the relatively high AST concentration in heart muscle. On average, serum levels do not become abnormal, however, until 6 to 8 h has elapsed after the onset of the chest pain. Abnormal AST levels are observed in more than 97% of cases of myocardial infarction when correctly timed blood specimens are analyzed. Peak values of AST activity are reached after 18 to 24 h, and the activity values fall within the normal range by the fourth of fifth day, provided no new infarct has occurred. The peak values of AST activity are roughly proportional to the extent of cardiac damage. Average increases are of the order of four to five times the upper limit of normal; levels of 10 to 15 times normal are frequently associated with fatal infarct. However, small elevations in serum levels do not necessarily indicate a favorable prognosis. ALT levels are within normal limits or are only marginally increased in uncomplicated myocardial infraction, because the concentration of ALT activity in heart muscle is only a fraction of that of AST activity. AST (and occasionally ALT) activity levels are increased in progressive muscular dystrophy and dermatonyositis, reaching levels up to 8 times normal; they are usually normal in other types of muscle diseases, especially in those of neurogenic origin. Pulmonary emboli can raise AST levels to two to thee times normal, and slight to moderate elevations (two to five times normal) are noted in acute pancreatitis, crushed muscle injuries, gangrene, and hemolytic disease.

5.3

PRINCIPLE OF THE METHOD

Aspartate aminotransferase (AST or SGOT) effects the conversion of alpha keto glutarate and aspartate to glutamate and oxaloacetate respectively, by amino group transfer. The oxaloacetate thus formed is coupled with 2, 4 - dinitrophenylhydrazine to produce a coloured complex whose absorbance in alkaline solution is measured at 505 nm

5.4

SPECIMEN TYPE, COLLECTION AND STORAGE

3-5 ml clotted blood, avoid haemolysis. Separate serum as soon as possible and perform the assay or keep in the refrigerator. It is stable up to 24 hours at 40 C. Minimal loss of activity occurs at 0-4 0C over 1-3 days. Specimen is best stored frozen if they are to be kept more than 3-4 days

5.5

APPARATUS AND CHEMICALS

APPARATUS: Water bath at 37 0C pH meter Visible spectrometer wavelength at 505 nm or Colorimeter with green filter Ilford No 604 (520 nm)

Department of Biochemistry, Medical Research Institute, Colombo-WHO Biennium 2004-2005

15

GLASSWARE: Volumetric flasks (100 ml and 1 litre volumes) Measuring cylinders (1 litre) Automatic micro pipette (100 µl) Graduated pipettes (1ml, 2 ml, 5ml, 10 ml in 0.1 ml) Test tubes (150 x16 mm), Beakers (5ml, 10ml, 100ml and 1 litre) Reagents bottles clear and amber coloured (250 ml and 1 litre)

Polypropylene bottle CHEMICALS: Disodium hydrogen ortho-phosphate-anhydrous, analytical grade Potassium dihydrogen phosphate-anhydrous, analytical grade Alpha – ketoglutaric acid -analar DL- aspartic acid -analar, Sodium hydroxide pellets analar 2, 4- dinitrophenylhydrazine-AR; caution: may explode violently when dry, Hydrochloric acid concentrated-AR (37% w/v), caution: highly corrosive Sodium pyruvate (analytical grade) REAGENTS 1. Phosphate buffer pH 7.4: Dissolve 11.9 g of disodium hydrogen phosphate (anhydrous) and 2.2 g of potassium dihydrogen phosphate (anhydrous) in distilled water and make up to 1 litre. Check the pH adjust to pH 7.4 if necessary using small amounts of the appropriate phosphate (e.g. if the pH is more than 7.4 add potassium dihydrogen phosphate). This solution is stable for about 2 months at 2-8 0C. 2. Sodium hydroxide solution 1 mol/l: Weigh 40 g of sodium hydroxide in beaker, slowly dissolve in distilled water, transfer into a volumetric flask and make up to 1 litre. Store in a tightly Stopperd polypropylene bottle. This solution is stable indefinitely at 20 250C, which can be achieved by air condition system. However we have observed that this solution is stable in our room temperature (20-30 0C) for about 2 months. 3. Buffered substrate reagent: Weigh 29.2 mg of alpha- ketoglutaric acid 2.66 g of DL-aspartic acid into a small beaker. Dissolve in 20 ml of sodium hydroxide solution (1 mol/l) then adjust to pH 7.4 with more sodium hydroxide solution. Transfer to a 100 ml volumetric flask and make up to 100 ml with phosphate buffer and mix well. Store the reagent frozen in screw capped bottles; the volume should be the amount need for a day’s analysis. 4. Hydrochloric acid 1 mol/l: Dilute 9 ml of hydrochloric acid (concentrated (37% w/v)) to 100 ml with distilled water. 5. Colour reagent 2,4 –dinitrophenylhydrazine 1 mmol/l: Dissolve the equivalent of 19.8 mg of dry 2,4-dinitrophenylhydrazine in 100 ml of hydrochloric acid ( 1mol/l)and transfer into a amber colour bottle; Note that the weight of 2, 4 – dinitrophenylehydrazine must be adjusted to take into account the water content. e.g. If the label reads as 33% by weight of water is added to ensure safety in transit the calculation is as follows.100/67 x 19.8 = 29.55 mg. The prepared solution is stable for 2 months at 2 8 0C. 6. Sodium hydroxide solution 400 mmol/l: Dissolve 16.0 g of sodium hydroxide in distilled water in a beaker and make up to 1 litre. Store in a tightly stoppered polypropylene reagent bottle. This solution is stable indefinitely at 20- 25 0C. We have observed that this solution is stable at our room temperature at 20-30 0 C for 2 months. 7. Pyruvate standard solution 4 mmol/l: Weigh out 44 mg of sodium pyruvate in a beaker, transfer into a 100 ml volumetric flask and make up to the mark with phosphate buffer. Mix well, divide into small portions (about 1ml) and store in the freezer compartment of the refrigerator. The standard solution is stable for 6 months in the freezer.

Department of Biochemistry, Medical Research Institute, Colombo-WHO Biennium 2004-2005

16

5.6

PROCEDURE

1. Two test tubes are required for each serum or control sample( one for the “Test” and one for a sample blank) one for a reagent blank and one for the standard 2. Transfer 0.5 ml buffered substrate to each tube and pre-incubate in the water bath (37 0 C) for 5 minutes 3. Add 100 µl of patient’s or control serum to the “Test” tubes or 100 µl distilled water(reagent blank) or 100 µl pyruvate standard (standard) mix and incubate at 37 0C. 4. After exactly 60 minutes add 0.5 ml colour reagent to each tube, mix and remove from the water bath. 5. Add 100 µl of patient’s or control serum to the sample blank tubes and mix well. 6. Leave for 20 minutes at room temperature and then add 5.0 ml of sodium hydroxide solution (400mmol/l) and mix thoroughly. 7. Leave the tubes at room temperature for at least 5 minutes, but not longer than 30 minutes, and then read the absorbance at 505 nm. Set the spectrometer to zero with the reagent blank. CALIBRATION In this method the amount of pyruvate formed is calculated by comparing the absorbance of the samples (Test- Blank absorbance) with that of the pyruvate standard (4mmol/l). However alpha - ketoglutarate also contributes to the absorbance and the change in absorbance is not linearly related to enzyme activity expressed in U/L. The table must therefore be used to convert the amount of pyruvate formed into U/L.

5.7

CALCULATION

Amount of pyruvate formed (µmol/min/litre)

= (A Test-A Sample Blank) x 4x1x1000 A Standard 60 = A Test- A Sample Blank x 66.7 A Standard

With the reagent blank set to zero the spectrophotometer at 505 nm A = absorbance Use the table below to convert the amount of pyruvate into U/L (expressed as µmol/minute/litre at 37 0C) Calculated pyruvate (µmol/min/litre) 2 4 6 8 10 12 14 16 18 20 22 23 24 26

AST result (U/L at Calculated pyruvate 37 0C) (µmol/min/litre) 4 28 6 30 10 32 12 34 15 36 19 38 23 40 27 42 31 44 35 46 40 48 42 50 44 52 48 54

AST result (U/L at 37 0C) 52 56 60 64 69 73 77 81 85 92 98 106 114 125

Department of Biochemistry, Medical Research Institute, Colombo-WHO Biennium 2004-2005

17

NOTE: When the activity of a sample exceeds 125 U/L the measurement should be

repeated with 10 minute incubation (instead of 60 minutes) and the results multiplied by 6. When the activity is greater than 750 U/L, the serum should be diluted 1 in 10 with sodium chloride solution (150mmol/l), an incubation time of 10 minutes should be used and the result multiplied by 60.

QUALITY CONTROL At least two serum control specimens, having stated values in the range 20-125 U/L, one of which is unknown to the operator, should be included with each batch of specimens. Even if single specimens are analysed a control specimen should always be included. OPTIMAL CONDITIONS VARIANCE: A coefficient of variation of around 8% should be attainable ROUTINE CONDITIONS VARIANCE: The value should not exceed 16% REFERENCE VALUES Approximate reference values: 4 - 42 U/L. REFERENCES Reitman, .S. & Frankel. S. (1957) Am. J. Clin.Pathol., 28, 56-63

Department of Biochemistry, Medical Research Institute, Colombo-WHO Biennium 2004-2005

18

6.

ALANINE AMINO TRANSFERASE 6.1

PRINCIPLE OF THE METHOD

Alanine aminotransferase (ALT or SGPT) effects the conversion of alpha-ketoglutarate and Alanine to pyruvate. Pyruvate formed is coupled with 2, 4-dinitrophenylhydrazine to produce a coloured complex. The absorbance in alkaline solution is measured at 505 nm.

6.2

SPECIMEN TYPE, COLLECTION AND STORAGE

3-5 ml clotted blood, avoid haemolysis. Separate serum as soon as possible and perform the assay or keep in the refrigerator. It is stable up to 24 hours at 4 0C. Minimal loss of activity occurs at 0-4 0C over 1-3 days. Specimen is best stored frozen if they are to be kept more than 3-4 days

6.3

APPARATUS AND CHEMICALS

APPARATUS: Water bath at 37 0C pH meter Visible spectrometer wavelength at 505 nm or Colorimeter with green filter Ilford No 604 (520 nm) GLASSWARE: Volumetric flasks (100 ml and 1 litre volumes) Automatic micro pipette (100 µl) Graduated pipettes (1ml, 2ml, 5ml and 10 ml in 0.1 ml) Test tubes (150 x16 mm), Beakers (5ml, 50ml, 100ml and 1 litre) Reagents bottles amber coloured (250 ml and 1 litre)

Polypropylene bottle CHEMICALS: Disodium hydrogen phosphate-Anhydrous analytical grade Potassium dihydrogen phosphate-Anhydrous analytical grade Alpha ketoglutaric acid AR DL Alanine AR Sodium hydroxide pellets analytical grade 2, 4-dinitrophenyl hydrazine AR Hydrochloric acid AR Sodium pyruvate analytical grade REAGENTS 1. Phosphate buffer pH 7.4: Dissolve 11.9 g of disodium hydrogen phosphate (anhydrous) and 2.2 g of potassium dihydrogen phosphate (anhydrous) in distilled water and make up to 1 litre. Check the p H and adjust to pH 7.4 if necessary using small amounts of the appropriate phosphate (e.g. if the pH is more than 7.4 add potassium dihydrogen phosphate). This solution is stable for about 2 months at 2-8 0C. 2. Sodium hydroxide solution 1 mol/l: Slowly dissolve 40.0 g of sodium hydroxide in distilled water in a beaker and make up to 1 litre. Store in a tightly stopperd polypropylene bottle. This solution is stable indefinitely at 20-25 0C. we have observed this solution is even stable at our room temperature 20-30 0C 3. Buffered substrate reagent: Weigh out 3.56 g of DL Alanine in a beaker and weigh 30 mg of alpha ketoglutaric acid accurately in another small beaker. Transfer it to same beaker by dissolving in the phosphate buffer. Add 0.5 ml of sodium hydroxide (1mol/l) solution. Check the pH. The pH should be at 7.4.Make up to 100 ml with phosphate buffer and mix. Divide the prepared substrate into small volumes and store in the freezing compartment or in the freezer. Discard the remaining substrate after use. Do not freeze the remaining substrate again. Department of Biochemistry, Medical Research Institute, Colombo-WHO Biennium 2004-2005

19

4. Hydrochloric acid 1 mol/l: Dilute 9 ml of hydrochloric acid (concentrated (37% w/v)) to 100 ml with distilled water. 5. Colour reagent 2,4 –dinitrophenylhydrazine 1 mmol/l: Dissolve the equivalent of 19.8 mg of dry 2,4-dinitrophenylhydrazine in 100 ml of hydrochloric acid ( 1mol/l)and transfer into a amber colour bottle; Note that the weight of 2, 4 –dinitrophenylehydrazine must be adjusted to take into account the water content. e.g. If the label reads as 33% by weight of water is added to ensure safety in transit the calculation is as follows.100/67 x 19.8 = 29.55 mg. The prepared solution is stable for 2 months at 2-8 0C. 6. Sodium hydroxide solution 400 mmol/l: Dissolve 16.0 g of sodium hydroxide in distilled water in a beaker and make up to 1 litre. Store in a tightly stoppered polypropylene reagent bottle. This solution is stable indefinitely at 20- 25 0C. We have observed that this solution is stable at our room temperature (20-30 0C) for 2 months. 7. Pyruvate standard solution 4 mmol/l: Weigh out 44 mg of sodium pyruvate in a beaker, transfer into 100 ml volumetric flask and make up to the mark with phosphate buffer. Mix well, divide into small portions (about 1ml) and store in the freezer compartment of the refrigerator. The standard solution is stable for 6 months in the freezer.

6.4

PROCEDURE

1. Two test tubes are required for each serum or control sample( one for the “Test” and one for a sample blank) one for a reagent blank and one for the standard 2. Transfer 0.5 ml buffered substrate to each tube and pre-incubate in the water bath (37 0C) for 5 minutes 3. Add 100 µl of patient’s or control serum to the “Test” tubes or 100 µl water(reagent blank) or 100 µl pyruvate standard (standard) mix and incubate at 37 0C 4. After exactly 30 minutes add 0.5 ml colour reagent to each tube, mix and remove from the water bath. 5. Add 100 µl of patient’s or control serum to the sample blank tubes. 6. Leave for 20 minutes at room temperature and then add 5.0 ml of sodium hydroxide solution (400mmol/l) and mix thoroughly. 7. Leave the tubes at room temperature for at least 5 minutes, but not longer than 30 minutes, and then read the absorbance at 505 nm. Set the spectrometer to zero with the reagent blank. PREPARATION OF CALIBRATION GRAPH In this method the amount of pyruvate formed is calculated by comparing the absorbance of the samples (Test- Blank absorbance) with that of the pyruvate standard (4mmol/l). However alpha - ketoglutarate also contributes to the absorbance and the change in absorbance is not linearly related to enzyme activity expressed in U/L. The table must therefore be used to convert the amount of pyruvate formed into U/L.

6.5

CALCULATION

Amount of pyruvate formed (µmol/min/litre)

= (A Test-A Sample Blank) x 4x1x1000 A Standard 30 = A Test- A Sample Blank x 133 A Standard With the reagent blank set to zero the spectrophotometer at 505 nm A=absorbance

Department of Biochemistry, Medical Research Institute, Colombo-WHO Biennium 2004-2005

20

Use the table below to convert the amount of pyruvate into U/L (expressed as µmol/minute/litre at 37 0C) Calculated pyruvate (µmol/min/litre) 2 4 6 8 10 12 14 16 18 20 22 23 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52

ALT result (U/L at Calculated pyruvate 37 0C) (µmol/min/litre) 2 54 4 56 4 58 5 60 7 62 7 64 9 66 11 68 13 70 13 72 15 74 15 76 16 78 16 80 18 82 20 84 22 86 24 88 25 90 27 92 29 94 31 96 33 98 35 100 36 102 38 40

ALT result (U/L at 37 0C) 42 44 46 47 49 53 55 56 60 62 64 66 67 69 71 73 76 80 84 87 91 95 98 102 109

NOTE: When the activity of a sample exceeds 109 U/L the measurement should be repeated with 10 minute incubation (instead of 30 minutes) and the results multiplied by 3. When the activity is greater than 750 U/L, the serum should be diluted 1 in 10 with sodium chloride solution (150mmol/l), an incubation time of 10 minutes should be used and the result multiplied by 30.

QUALITY CONTROL At least two serum control specimens, having stated values in the range 20-109 U/L, one of which is unknown to the operator, should be included with each batch of specimens. Even if single specimens are analysed a control specimen should always be included REFERENCE VALUES Approximate reference values: up to 2-27 U/L. REFERENCES King and wooton- enzymology

Department of Biochemistry, Medical Research Institute, Colombo-WHO Biennium 2004-2005

21

7.

BILIRUBIN 7.1

INTRODUCTION

Haem, released from aged red cells and maturing cells during erythropoiesis, or from degraded haemoproteins is converted to biliverdin in the reticuloendothelial system. Biliverdin is reduced to bilirubin which is secreted into the plasma where it is transported to the liver reversibly bound to albumin. The hepatocytes take the bilirubin up from the plasma, conjugate it to glucuronic acid and excrete it in the bile. There are six steps in this process namely; production, transport to the liver, hepatocyte uptake, conjugation, biliary secretion, gut degradation and excretion. The total bilirubin produced is around 250-350 mg/day (4 – 6 mmol/day). 15% to 20% is derived from immature red cell and haemoproteins (early labelled fraction) whilst the remainder comes from senescent red cells (1g of haemoglobin produces 620µmol of bilirubin) Haem is converted to bilirubin in the reticuloendothelial system by two enzymes, haem oxygenase and biliverdin reductase. Haem oxygenase breakes the alpha – CH bridge of protoporphyrin IX to produce biliverdin IX α, which is then reduced to bilirubin IX α by biliverdin reductase. Some cleavage occurs at the β, γ and δ bridges, but insignificant amounts of these isomers are produced. Although bilirubin IX α has two polar propionic acid side chains it is poorly soluble in water because of intramolecular hydrogen bonding between the propionic acid residues and other parts of the molecule. This bonding may also account for the necessity for conjugation with glucuronic acid prior to biliary excretion. Bilirubin is transported to the liver reversibly bound to albumin. This protein has one high – affinity binding site and an additional low – affinity site which is activated at high concentrations of bilirubin. The amount of unbound bilirubin is low; approximately 4 nmol/l at a total plasma concentration of 20 µmol/L. Compounds such as free fatty acids, sulphonamides, salicylate and ampicillin will displace bilirubin from its binding sites. This species of bilirubin is called unconjugated bilirubin or indirect reacting bilirubin. The bilirubin – albumin complex dissociates in the liver and the bilirubin is transported across the hepatocyte membrane into the cell where it is reversibly bound to cytosolic proteins; one such protein being ligandin. The function of these proteins appears to be prevention of efflux of bilirubin from the cell. Bilirubin is conjugated in the endoplasmic reticulum with glucuronic acid and to a lesser extent with glucose and xylose. The enzyme responsible is UDP – glucuronosyltransferase which esterifies one or both propionic acid side chains to produce di – and monoglucuronides. In man the product is predominantly bilirubin diglucuronide with lesser amounts of the monoglucuronide. The bilirubin conjugates are secreted into the biliary passages by an active process which is yet to be clarified. This transport mechanism differs from that responsible for the biliary excretion of bile acids. In the gut the bacterial flora reduce the bilirubin conjugates to urobilinogens which are excreted in the faeces as such. Some of the urobilinogens are reabsorbed from the gut to be re – excreted by the liver (enterohepatic circulation); as these compounds are water soluble they will also be excreted by the kidney, but this is an unimportant excretory route. Two points worthy of note are that conjugated bilirubin is more prone to reductive processes than unconjugated bilirubin and that unconjugated bilirubin can be reabsorbed from the gut. Thus, if bacterial flora are absent (e.g.: neonate, antibiotic therapy) deconjugation of bilirubin glucuronides by intestinal mucosal ß – glucuronidase may occur and reabsorption of the unconjugated compound result in an increase in the plasma unconjugated bilirubin level. The total plasma bilirubin concentration in the normal subject is usually less than 20 µmol/L. The clinical sign of jaundice appears when the plasma total bilirubin rises beyond 40 µmol/L. When normal plasma is evaluated by high performance liquid chromatography techniques four bilirubin fractions alpha, beta, gamma and delta are obtained.

Department of Biochemistry, Medical Research Institute, Colombo-WHO Biennium 2004-2005

22

Alpha fraction This fraction is unconjugated bilirubin which is water insoluble and bound to albumin. It is also called indirect reacting bilirubin because the diazo reaction used to measure the plasma level occurs only after the addition of accelerators. It is the major bilirubin fraction in normal plasma (> 90%). It does not appear in the urine because of its attachment to albumin and can only be cleared by the liver. Beta and gamma fractions The beta fraction is composed of bilirubin monoglucuronide whilst diglucuronide constitutes the gamma fraction. These bilirubins (< 10% in normal plasma) are water soluble and will appear in the urine if present in the blood in excess. They are called direct reacting bilirubins as they react with the diazo reagent without the addition of accelerators. Delta fraction The delta fraction, usually referred to as ‘delta’ bilirubin – not to be confused with bilirubin IX delta –is an interesting compound with the following characteristics:  it is direct reacting  it is tightly bound to albumin  its plasma level is increased in diseases associated with high plasma levels of conjugated bilirubin It appears to be derived from conjugated bilirubin as it can be produced by incubating bilirubin glucuronides with plasma albumin. It is thought that the bilirubin migrates from the glucuronic acid residues and becomes covelantly bound to albumin. As noted above this fraction increases in conditions associated with chronic conjugated hyperbilirubinaemia (eg: cholestasis) and, as it is tightly bound to albumin, it has a t1/2 comparable to that of protein (20 days) .Thus, after formation delta bilirubin ‘hangs around ‘as it is not cleared by the liver or the kidney. The bile pigments that may be found in the urine are urobilinogen and conjugated bilirubin. Unconjugated Bilirubin is water insoluble and bound to albumin, and is thus not available for urinary excretion. The normal subject excretes 1 – 4 mg (2 – 7 µmol) of urobilinogen daily (derived from the enterohepatic circulation) Increased values occur in liver disease (inability to excrete the small amounts reabsorbed from the gut) and in haemolytic disease (increased bilirubin production). Decreased excretion occurs in bile duct obstruction. (cholestasis) Bilirubin appears in the urine when the plasma level of conjugated bilirubin rises as in hepatocellular disease and cholestasis.

7.2

CLINICAL SIGNIFICANCE

The earliest clinical manifestation of hepatobiliary disease is often jaundice, but jaundice need not necessarily indicate liver pathology (e.g.: haemolysis) and liver pathology can present without jaundice (e.g.: space-occupying lesions). However, it is convenient to classify liver disease in terms of jaundice and to this end it is helpful to divide hyperbilirubinaemia into three categories: Prehepatic: liver disease not present, Hepatic: hepatocellular disease, Post hepatic: cholestasis (obstruction). Pathological hyperbiliruminaemia in the neonate if not diagnosed and treated at the early stages may lead to the development of kernicterus with resultant permanent neurological dysfunction.

7.3

PRINCIPLE OF THE METHOD

Sulfanilic acid is diazotized by the nitrous acid produced from the reaction between sodium nitrite and hydrochloric acid. Both conjugated and unconjugated bilirubin reacts with diazotized sulfanilic acid (Diazo reagent) to produce azobilirubin. Caffeine is an accelerator by splitting the unconjugated Bilirubin protein complex and gives a rapid and complete conversion to azobilirubin. The pink acid azobilirubin is converted to blue azobilirubin by an alkaline tartrate reagent and the absorbance of the blue green solution is measured at 600 nm. Measurement of the azobilirubin in an alkaline medium removes Department of Biochemistry, Medical Research Institute, Colombo-WHO Biennium 2004-2005

23

turbidity and increases specificity. There is very little interference by other pigments at 600 nm wavelength. Conjugated Bilirubin is determined by diazotization at an acidic pH, only the conjugated forms of Bilirubin react with the diazo reagent in the absence of the accelerator caffeine benzoate. The reaction is stopped by the addition of ascorbic acid minimizes the effect of haemolysis.

7.4

SPECIMEN TYPE, COLLECTION AND STORAGE

Clear non haemolysed serum, collect 3-5 ml of blood into a clean dry container. A fasting specimen is preferred to avoid lipaemia. Haemolysis should be avoided because it produces false values. Specimens should be protected from direct exposure to either artificial light or sunlight as soon as they drawn because conjugated and unconjugated bilirubin is photosensitive. The sensitivity to light is temperature dependent. For optimal stability serum separation should be done as early as possible and assay should be carried out within 2 hours of sample collection. Store the serum in dark and at low temperature (2-8 0C) if any delay is encountered. By inclusion of a sample blank haemolysed samples can be assayed for total bilirubin. APPARATUS AND CHEMICALS APPARATUS: Spectrophotometer, wavelength at 600 nm or Colorimeter, orange filter, Ilford 607 GLASSWARE: Volumetric flasks (100 ml, 500 ml and 1 litre volumes) Beakers (5ml, 100 ml and 1 litre) Automatic micro pipettes (50 and 100 µl) Graduated pipettes (1ml and 10 ml in 0.1 ml) Test tubes (100 x 13mm) & Rubber bulb Reagent bottle, clear and amber coloured CHEMICALS: (ANALYTICAL GRADE) Bilirubin powder (or commercial Bilirubin standards) Caffeine Sodium benzoate Sodium acetate trihydrate Sulphanilic acid Hydrochloric acid, concentrated (37% w/v) caution: highly corrosive Sodium nitirite Sodium hydroxide pellets Potassium sodium tartrate tetra hydrate Ethylenediamine tetra – acetic acid disodium salt dihydrate Ascorbic acid Sodium carbonate anhydrous REAGENTS 1. Caffeine- benzoate reagent: Dissolve 93 g of sodium acetate trihydrate, 56 g of sodium benzoate and 1 g of disodium EDTA in approximately 500ml of distilled water. Add 38 g of caffeine. Dissolve and dilute to 1 litre in a volumetric flask. Mix well and filter. This solution is stable for at least 6 months at 20-25 0C (Room temperature) 2. Sulfaninlic acid reagent: Add 2.5 g of sulfanilic acid to about 200 ml of distilled water in a 500 ml volumetric flask. Using a rubber bulb carefully pipette 7.5 ml of concentrated hydrochloric acid into the flask. Dissolve and make up to 500 ml. This solution is stable for up to 6 months at 20-25 0C (Room temperature)

Department of Biochemistry, Medical Research Institute, Colombo-WHO Biennium 2004-2005

24

3. Sodium nitrite solution: Dissolve 500 mg of sodium nitrite in distilled water and make up to 100 ml. This solution should be stored at 2-8 0C in an amber coloured bottle and must be renewed every month. Prepare about 25ml or 50 ml. 4. Diazo reagent: Mix 4 ml of sulfanilic acid reagent with 0.1 ml of sodium nitrite solution, leave for 2 minutes, then use within 5 hours. Keep at 2-8 0C in an amber coloured bottle. 5. Alkaline tartrate reagent: Dissolve 75 g of sodium hydroxide and 350 g of potassium sodium tartrate in about 800 ml of distilled water. Transfer to a volumetric flask and make up to 1 litre. This reagent is stable for at least 6 months at 20- 25 0C. Store in a polypropylene bottle. 6. Hydrochloric acid 50 mmol/l: Using a rubber bulb, carefully pipette 4.2 ml of hydrochloric acid (concentrated) and dilute to 1 litre with distilled water, require only for conjugated Bilirubin method. 7. Ascorbic acid 40 g/l: Dissolve 0.2 g in 5 ml of water, this solution must be prepared daily. Required only for the conjugated Bilirubin method. 8. Sodium carbonate 100mmol/l: Weigh out 1.06 g of sodium carbonate anhydrous very accurately and transfer quantitatively to a 100 ml volumetric flask, containing about 50 ml distilled water. Mix well and make up to 100 ml with distilled water.(Prepare about 50 ml for use) 9. Sodium hydroxide 100 mmol/l: Weigh out rapidly 2.0 g of sodium hydroxide in a beaker, dissolve and make up to 500 ml with distilled water. ( prepare about 100 ml for use) 10. Tris buffer(0.1 mol/l) pH 7.4 : Tris(hydoxymethyl) amino methane 1.21 g, distilled water 80 ml, adjust pH 7.4 ±0.05, with hydrochloric acid 2 N, make up to 100 ml with distilled water stable for about 1 month at 4 0C 11. Bovine serum albumin (BSA) diluent 40 g/l: Weigh out 4 g of Bovine albumin powder in a beaker. Dissolve in the Tris buffer and transfer into 100 ml volumetric flask and make up to the mark with Tris buffer. This solution is stable for one week at 4 0C 12. Bilirubin standard 342µmol/l: Weigh out 20 mg of Bilirubin powder accurately in a small beaker or in a weighing bottle. Cover the beaker with aluminium foil or black paper to protect from sunlight. Add 2 ml of sodium carbonate solution and 1.5 ml of sodium hydroxide solution and dissolve it .This solution must be red and clear. This procedure must not be carried out in strong sunlight. Transfer the solution quantitatively to a 100 ml volumetric flask. (This should be protected from direct sunlight by wrapping around with an aluminium foil or with a black carbon paper). Make up to 100 ml with BSA diluent mix the solution carefully without forming froth. Divide into small volumes in clean bottles keep in a box to protect from light and store deep frozen. (-200C) Do not reuse the standard once it has been used.

7.6

PROCEDURE (TOTAL BILIRUBIN)

1. For the standard, patient specimen, control and their blanks (SB, TB, and CB) pipette 1.0 ml of caffeine benzoate reagent into each of two test tubes. 2. Add 100 µl of standard or patient or control serum to each pair of tubes 3. Add 0.5 ml of diazo reagent to the test, standard, control and 0.5 ml of sulfanilic acid to the blanks. 4. Mix well and let stand for 10 minutes at room temperature. 5. Add 1.0 ml of alkaline tartrate reagent to each tube and mix thoroughly 6. Read the absorbance at 600 nm (Ilford filter No: 607) immediately, setting the spectrometer to zero with distilled water.

7.61 1. 2. 3. 4.

PROCEDURE (CONJUGATED BILIRUBIN)

For each patient or control specimen label two tubes: one test one blank. Add 100 µl of serum to each tube. Add 1.0 ml of hydrochloric acid (50mmol/l) to each tube. Add 0.5 ml of diazo reagent to the tube marked test and 0.5 ml of sulfanilic acid to the tube marked blank, mix well. Department of Biochemistry, Medical Research Institute, Colombo-WHO Biennium 2004-2005

25

5. After 5 minutes add 50 µl of ascorbic acid solution to each tube. 6. Add 1.0 ml of alkaline tartrate reagent to each tube. Mix well and read the absorbance of each solution at 600 nm immediately, setting the spectrometer to zero absorbance with distilled water. PREPARATION OF CALIBRATION GRAPH Prepare Bilirubin working standard solutions by diluting the Bilirubin standard solution (342µmol/l) with BSA solution as shown in the table below. The working standard solution must be freshly prepared each time a calibration graph is made. Bilirubin working Standard

(1)

(2)

(3)

(4)

(5)

(6)

(7)

Bilirubin standard Solution 342µmol/l(ml)

0

0.5

1

1

2

3

4

Bovine serum Albumin (ml) (BSA solution)

4

9.5

9

3

2

1

0

Concentration of Bilirubin Working standard solution (µmol/l)

0

17.1

34.2

85.5

171

256.5

342

A calibration graph is prepared from the Bilirubin working standards using the volumes of standard and reagents described in the table below: Tube No Caffeine benzoate Reagent (ml) Bilirubin working Standard (µl)

1 (Blank)

2

3

4

5

6

7

1.0

1.0

1.0

1.0

1.0

1.0

1.0

100

100

100 100 100 100 100

Mix well, protect the tubes from light then add Diazo reagent (ml)

-

0.5

0.5

0.5

0.5

0.5

0.5

Mix well and allow the solutions to stand at room temperature for 10 minutes. Protect the tubes from light. Sulfanilic acid Reagent(ml)

0.5

-

-

-

-

-

-

Mix well Alkaline tartrate 1.0 1.0 1.0 1.0 1.0 1.0 1.0 Reagent (ml) Mix well. Read the absorbance of each tube at 600 nm after setting the spectrometer to zero with the blank (Tube No 1) Plot the absorbance of the each tube on the vertical axis against the concentrations in µmol/l of working standards on the horizontal axis o The calibration graph should be prepared whenever the new batch of reagents are prepared or any changes made in the spectrophotometers o Freshly prepare all reagents and use clean glassware o Measure the standards (each concentration) in duplicate o Check the calibration graph by measuring a quality control serum

Department of Biochemistry, Medical Research Institute, Colombo-WHO Biennium 2004-2005

26

NOTE:  All the tubes or racks used for the assay should be covered with black papers to protect from direct light 

Icteric serum can be diluted 1 in 2 or highly icteric serum can be diluted 1 in 4 with normal saline and multiply the result by the diluting factor



Carryover is minimized by measuring the absorbance of all the serum blank solutions first, followed by all the test solutions. The colour of the azobilirubin is stable for about 30 minutes. After 30 minutes turbidity may occur and the absorbance of the serum blank increases

7.7 CALCULATION If the calibration graph is linear, calculate the results using the following formula: Concentration of Bilirubin (µmol/l) = T – TB x 342 S – SB Where: T =Absorbance reading of sample or control TB =Absorbance reading of control or patient sample blank S =Absorbance reading of Bilirubin standard (342 µmol/l) SB =Absorbance reading of standard blank If the calibration graph is not linear, then results should be read from a calibration curve prepared using working standards 1, 2, 3, 5 and 6. QUALITY CONTROL At least two serum control specimens having stated values in the range 20 – 200 µmol/l, one of which should be unknown to the operator, should be included with each batch of specimens. If single specimens are analysed a control specimen should always be included. OPTIMAL CONDITIONS VARIANCE: A coefficient of variation of around 6% should be attainable. ROUTINE CONDITIONS VARIANCE: The value obtained for the RCV should not exceed 12% REFERENCE VALUES Total bilirubin in adults: 3 – 21 µmol /l Conversion from SI units into ‘old’ units: µmol/l x 0.0585=mg/dl

REFERENCES WHO manual LAB /86.3

Department of Biochemistry, Medical Research Institute, Colombo-WHO Biennium 2004-2005

27

8.

CALCIUM 8.1 INTRODUCTION Calcium is the fifth most common element and the most prevalent cation found in the body. An average human body contains approximately 1kg (24.95 mol) of calcium. Calcium is found in three main compartments: the skeleton, soft tissues, and the extra cellular fluid. The skeleton contains 99% of the body’s calcium, predominantly as extra cellular crystals of unknown structure with a composition approaching that of hydroxyapatite. Soft tissues and extra cellular fluid contain about 1% of the body’s calcium. In blood, virtually all of the calcium is in serum or plasma, which has a mean normal calcium concentration of approximately 9.5mg/dL (2.37 mmol/L). Calcium exists in three physiochemical states in plasma, of which approximately 50% is free or ionised. Another 40%is bound to plasma proteins, chiefly albumin. Because calcium binds to negatively charged or anionic sties on albumin, it’s binding is pH-dependant. Alkalosis leads to an increase in binding and a decrease in free calcium; conversely, acidosis leads to a decrease in binding and an increase in free calcium. For each 0.1-unit change in pH, approximately 0.2-mg/dL (0.05mmol/L) of inverse change occurs in the serum free calcium concentration. Approximately 20% of protein-bound calcium in serum is associated with globulins. In some patients with multiple myeloma, the high concentrations of serum globulin may bind sufficient calcium, about 10%, is complexed with small diffusible anions including bicarbonate, lactate, phosphate and citrate. Calcium can be redistributed among the three plasma pools, acutely of chronically, independently affecting the quantities of free calcium and total calcium in the serum. The skeleton is a major storehouse for providing calcium for the extra cellular and intracellular pool. Approximately 5g of calcium is rapidly available from the skeletal exchangeable pool, which is accessible for maintaining normal physiological functions. Intracellular calcium has many important physiological functions within the cells, including muscle contraction, hormone secretion, glycogen metabolism and cell division. Extra cellular calcium provides a source for maintenance of intracellular calcium. In addition, it has an important role in providing calcium ions for bone mineralization, coagulation cascade and maintaining plasma membrane potential. Calcium stabilises the plasma membranes and influences permeability and excitability. A decrease in serum free calcium concentration increases neuromuscular excitability and tetany. An elevated free calcium concentration results in reduced neuromuscular excitability.

8.2

CLINICAL SIGNIFICANCE

Hypercalcaemia is found commonly in clinical practice. It may be uncovered as a biochemical abnormality in an otherwise asymptomatic patient or in association with severe illness. Hypercalcaemia occurs when the flux of calcium into the extra cellular fluid is greater than the efflux of calcium out of this compartment. For example, when excessive resporption of bone mineral occurs in malignancy, hypercalciuria develops. When the capacity of the kidneys to excrete filtered calcium is exceeded, hypercalcaemia develops. Hypercalcaemia can be due to increased intestinal absorption of calcium (vitamin D intoxication), enhanced renal retention of calcium (thiazide diuretics), increased skeletal resorption (immobilization), or a combination of these mechanisms (primary hyper parathyroidism). The pathogenesis, clinical presentation, and differential diagnosis therefore vary widely. Hypocalcaemia is due to the reduction in either the albumin-bound or free fraction. Hypoalbuminemia is probably the most common cause of reduction in the concentration of total serum calcium. Common clinical conditions associated with low serum albumin concentrations include chronic liver disease, nephritic syndrome, congestive heart failure, and malnutrition. Chronic renal failure is also frequently associated with hypocalcaemia. Contributing reasons for the low calcium values are hyperphosphatemia, impaired synthesis of 1, 25(OH)2D due to inadequate renal mass, and skeletal resistance to the action of Parathyroid hormone. Magnesium deficiency is the other common clinical cause of hypocalcemia. Magnesium deficiency impairs PTH secretion as Department of Biochemistry, Medical Research Institute, Colombo-WHO Biennium 2004-2005

28

well as the action of PTH on bone and kidneys. Acute symptomatic hypocalcaemia may be noted in hospitalized patients for various reasons. Patients undergoing surgical treatment for hyperthyroidism or primary hyperparathyroidism of receiving therapy for haematological malignancies may have rapid remineralisation of bone (hungry bone syndrome) causing a drop in serum calcium. Acute hemorrhagic and edematous pancreaitis is frequently complicated by hypocalcaemia. Ostomalacia or rickets secondary to vitamin D deficiency may also be associated with hypocalcaemia. The hypocalcaemia may be due in part to impaired intestinal absorption of calcium. In addition, vitamin D deficiency renders the skeleton resistant to PTH and thereby limits calcium resorption from bone.

8.3

PRINCIPLE OF THE METHOD

Serum or plasma calcium is measured with o-cresolphthalein complexone reagent containing ethanediol which maintains a clear solution in the presence of proteins and suppresses the ionization of o-cresolphthalein complexone in the reagent. Interference by magnesium is eliminated by the inclusion of 8-hydroxyquinoline.

8.4

SPECIMEN TYPE, COLLECTION AND STORAGE

2-3 ml clotted blood collected into acid washed container. Do not apply the tourniquet, avoid haemolysis. Fasting specimen is preferable. Separate the serum from red cells as early as possible. Pyrex vials with Teflon lined screw caps are recommended for specimen storage. Specimen may stored at -20 0C for several weeks or months

8.5

APPARATUS AND CHEMICALS

APPARATUS: Spectrophotometer with wavelength 575 nm or colorimeter with yellow filter-Ilford 606(580nm), Safety bulb GLASSWARE: Volumetric flask class A (100ml, 500ml and 1 litre volumes) Automatic micro pipettes (50 µl) Volumetric pipettes (5 ml, 10ml) Graduated pipettes (1 ml, 2 ml, 5ml and 10 ml in 0.1ml) Beaker (5ml, 250 ml)

Measuring cylinder (50ml), Test tubes (125 x 16 mm) CHEMICALS: Hydrochloric acid concentrated (37% w/v); caution: highly corrosive O-cresolphthalein complexone-AR Ethanediol-AR 2-amino-2-methyl-1-propanol-AR 8-hydroxyquinoline-AR Calcium carbonate-AR NOTES: All glassware must be thoroughly cleaned then soaked overnight in hydrochloric acid (0.5mol/l) to remove traces of calcium then thoroughly rinsed with distilled or deionised water and finally dried before use. REAGENTS 1. Hydrochloric acid 0.5 mol/l: Adding the acid to the distilled water, dilute about 45 ml of hydrochloric acid (concentrated) to 1 litre with distilled water. Use this solution for soaking glassware as described above. 2. Stock CPC reagent: Add 38 ml ethanediol and 13 ml of 2- amino-2-methyl-1-propanol to a 500 ml volumetic flask containing about 400 ml of distilled water. Weigh out 15 mg of o-cresolphthalien complexone and add it to the volumetric flask, mix until the all the chemicals are completely dissolved, make up to the mark with distilled water and

Department of Biochemistry, Medical Research Institute, Colombo-WHO Biennium 2004-2005

29

transfer the reagent into a clean brown bottle. This solution is stable for 3 weeks at 4 -6 0C 3. Working CPC reagent: Weigh out 100 mg of 8-hydroxyquinoline and transfer into a 100 ml volumetric flask using small volumes of stock CPC solution. Add about 80 ml of stock CPC solution mix until the chemical is completely dissolved and make up to the volume to 100ml using stock CPC reagent. The 8-hydroxyquinoline dissolves quite slowly. This solution is stable for 1 week at 4-6 0C and should have and absorbance at 575 nm of about 0.2 when measured with the spectrometer set to zero with distilled water. An absorbance higher than 0.2 indicates either that the reagent has deteriorated or that it is contaminated with calcium (use of dirty container or cuvettes) 4. Calcium stock standard 25mmol/l: Dry about 300 mg of calcium carbonate in a dry container in an oven for 4 hours at 80-100 0C. Remove the container from the oven and immediately close it with a lid. When it has attained to room temperature weigh out exactly 250 mg and transfer to a 100 ml volumetric flask. Dissolve the calcium carbonate in a minimum volume of hydrochloric acid (concentrated) approximately 0.5 ml is required, and then make up to100 ml with distilled water. 5. Calcium working standard 2.5mmol/l: Using a volumetric pipette transfer 10.0 ml of the stock standard to a 100 ml volumetric flask. Make up to 100 ml with distilled water.

8.6

PROCEDURE

1. The analysis must be performed in duplicate. Transfer 50 µl of serum or plasma to each of two clean tubes, add 5.0 ml working CPC reagent to each tube using a 5 ml volumetric pipette and mix well. 2. Transfer 50 µl of working calcium standard (2.5mmol/l) to each of 2 clean tubes; add 5.0 ml working CPC reagent to each tube using 5 ml volumetric pipette and mix well. 3. Transfer 50 µl of distilled water to a clean tube, add 5.0 ml working CPC reagent, Mix well. This is the reagent blank. 4. Set the spectrometer to zero at 575 nm with distilled water and measure the absorbance of the reagent blank which should be about 0.2. 5. Measure the absorbance of the standards (2.5mmol/l) and serum sample If the absorbance of duplicate readings varies by more than 0.015 then precision is unsatisfactory. Check that the test tubes and pipettes are clean. Check the precision of the 50 µl volumetric pipette. Use a 5.0 ml volumetric pipette for the working CPC reagent. Check that the spectrometer cuvettes (cells) are clean. PREPARATION OF CALIBRATION GRAPH The calibration graph must be prepared in order to confirm the linearity of the method and should be checked monthly. The calibration graph should not be used for calculating patients’ results. Prepare the calibration graph standards from the calcium stock standard as described in the table below in clean test tubes as accurately as possible using 1, 2 and 10 ml graduated pipettes Tube Number

1

2

3

4

5

Calcium stock standard ( 25mmol/l) ml

0.2

0.7

1.0

1.2

1.5

Distilled water (ml)

9.8

9.3

9.0

8.8

8.5

Calcium concentration (mmol/l)

0.5

1.75

2.5

3.0

3.75

Department of Biochemistry, Medical Research Institute, Colombo-WHO Biennium 2004-2005

30

Transfer 50 µl of distilled water into a clean test tube for the reagent blank and transfer 50 µl of each calibration graph standard to 5 other test tubes. Add 5.0 ml of working CPC reagent to each tube using a 5 ml volumetric pipette and safety bulb. Mix and measure the absorbance of each tube at 575 nm setting the spectrometer to zero with distilled water. Plot the absorbance of each tube on the vertical axis against the calcium concentration of the calibration graph standards in mmol/l on the horizontal axis. The purpose of preparing the calibration graph is to confirm the linearity of the method. If this is not linear beyond 3.0 mmol/l, then patients’ samples with calcium concentrations greater than 3.0 mmol/l should be diluted two-fold with distilled water before analysis. If the graph is linear up to 3.75 mmol/l then samples with calcium concentrations greater than 3.75 mmol/l should be diluted two-fold with distilled water before analysis.

8.7

CALCULATION

Calculate the results using the following formula: Concentration of calcium (mmol/l) = T-B x 2.5 S-B Where: T = Absorbance reading of sample or control S = Absorbance reading of working calcium standard (2.5 mmol/l) B = Absorbance reading of reagent blank If the sample or control result is above the linearity of the method then repeat the analysis after accurately diluting 200 µl of sample with 200 µl of distilled water in a clean tube. Use 50 µl of the diluted sample for the analysis. Remember to multiply the result by 2 to obtain the calcium concentration of the sample. QUALITY CONTROL At least two serum control specimens, having stated values in the range 2.00-2.90 mmol/l one of which is unknown to the operator, should be included with each batch of specimens. If single specimens are analysed a control specimen should always be included. OPTIMAL CONDITIONS VARIANCE should be attainable.

: A coefficient of variation of around 1.5%

ROUTINE CONDITIONS VARIANCE exceed 4%

: The value obtained for the RCV should not

REFERENCE VALUES The reference interval for healthy ambulant adults is 2.25-2.60 mmol/l. Conversion of SI units into “old” units: mmol/l x 4 = mg/dl. The reference values are only appropriate if the patient has a normal serum albumin concentration. If the patient has a low serum albumin then it may be helpful to report the albumin corrected calcium as well as the measured calcium. The albumin corrected calcium is calculated in this way: (40 – Patient’s albumin) + measured calcium = albumin corrected calcium 40 For example if the patient has an albumin of 20 g/l and a measured calcium of 1.90 mmol/l, then the albumin corrected calcium is 2.40 mmol/l (40-20) + 1.90 = 2.40 mmol/l 40 The albumin corrected calcium will be approximately 2.20-2.60 mmol/l if low measured calcium is a consequence of a low serum albumin.

Department of Biochemistry, Medical Research Institute, Colombo-WHO Biennium 2004-2005

31

NOTE: The estimation of calcium is difficult, particularly because of the possibility of contamination by calcium. It is essential that high quality chemicals are used and that the recommendations regarding cleaning of glassware are strictly followed. It may be found helpful to keep separate test tubes, pipettes etc, only for the analysis of calcium When preparing the reagents, be careful not to contaminate other chemicals or glassware with calcium carbonate. PRECAUTIONS  Use dry glass container which must be chemically cleaned and acid washed  Avoid use of plastic containers and use of rubber stoppers  Avoid use of acid etched glassware. It may lead either to calcium loss because adsorption of calcium on to the damage surface or to contamination with calcium because the etched areas cannot be cleaned thoroughly.

REFERENCES LAB/86.3

CALCIUM IN URINE SPECIMENS: a 24 hour urine collection in an acid washed 2.5 L bottle; 10 ml of 1 N HCl is added as the preservative. Acid washed beaker and a funnel should be issued from the laboratory along with collection bottle. PROCEDURE:  Mix the 24 hour urine collection and measure the total urine volume  Follow the procedure as for serum calcium analysis Urine samples with calcium concentration greater than 3.75 mmol/l should be diluted with distilled water before analysis. Multiply the results by the dilution factor CALCULATION: Concentration of calcium (mmol/l)

= T-B x 2.5 S-B

Concentration of calcium in 24 hour urine = T-B x 2.5 x (TV in litre) mmol/24 hour S-B Where: T = Absorbance reading of sample or control S = Absorbance reading of working calcium standard (2.5 mmol/l) B = Absorbance reading of reagent blank TV =24 hour urine total volume in litre In children concentration of urinary calcium= T-B x 2.5 x TV in litre x 1000 µmol/kg/24 hours S-B Body weight in kgs

REFERENCE VALUES: New Born Infants Older Children

: 0-17.5 µmol/kg/24 hours : up to 1000 µmol/kg/24 hours : up to100 µmol/kg/24 hours or 750-3750 µmol/24hours

Adults Calcium in diet Calcium free Low –average Average (800mg/day)

: 0.13-1.0 mmol/24 hours : 1.25-3.75 mmol/24 hours : 2.5-7.5 mmol/24 hours

Department of Biochemistry, Medical Research Institute, Colombo-WHO Biennium 2004-2005

32

9.

CREATININE 9.1

INTRODUCTION

Creatine is synthesized in the kidneys, liver and pancreas by two enzymatically mediated reactions. In the first, transamidination of arginine and glycine forms guanidinoacetic acid: the second methylation of guanidinoacetic acid occurs with S- adenosylmethionine as methyl donor. Creatine is then transported in blood to other organs such as muscle and brain, where it is phosphorylated to phosphocreatine a high energy compound. Interconversion of phosphocreatine and creatine is a particular feature of metabolic processes of muscle contration; some of the free creatine in muscle spontaneously converts to creatinine, its anhydride. Between 1 and 2% of muscle creatine is converted to creatinine daily. Because the amount of endogenous creatinine produced is propotional to muscle mass, the production varies with age and sex; non obese men excrete about 1.5 g/day, women 1.2 g/day. Daily excretion of creatinine can be 10% to 30% greater as a result of dietary intake of creatine and creatinine in meats. On the whole however, dietary fluctuations of creatinine intake cause only minor variation in daily creatinine excretion on the same individual. The excretion rate in one individual, in the absence of renal disease, is relatively constant and parallels endogenous production. Most of the interindividual variations of creatinine excretion in healthy persons are attributable in the main to age, sex, and lean body mass and intraindividual variation tends to be less than 15% from day to day.

9.2

CLINICAL SIGNIFICANCE

As creatinine is endogenously produced and released into body fluids at a constant rate and its plasma levels maintained within narrow limits, its clearance may be measured as an indicator of glomerular filtration rate. However, a small quantity of creatinine is reabsorbed by the tubules and a small quantity of creatinine appearing in the urine(7-10%)is due to tubular secretion. As a result creatinine clearance (if creatinine is measured with an accurate method) is approximately 7% greater than inulin clearance. Some methods for creatinine used in clinical laboratories are nonspecific, however, and thus this difference is often smaller. The creatinine clearance is performed by obtaining a 4-, 12- or 24-h urine specimen and also a blood specimen within the period of urine collection. The volume of the urine is measured, urine flow rate is calculated (millimetres per minute), and the assay for creatinine is performed on plasma and urine to obtain the concentration in milligrams per decilitre or per millilitre. Two factors influence measurement of creatinine clearance and thus its interpretation. First, the most common methods for measuring creatinine use the nonspecific alkaline picarate reaction, and thus noncreatinine chromogens in plasma increase the apparent plasma concentration by as much as 30% if serum values are less than 1.0mg/dL, and by approximately 10% is values exceed 1.0mg/dL. The percent increase is progressively less with higher creatinine concentrations. (Urine contains considerably fewer noncreatinine chromogens.) This overestimation of plasma creatinine concentration results in underestimation of creatinine clearance and partially offsets the apparent high clearance of creatinine that is due to tubular secretion. As a result, the endogenous creatinine clearance agrees closely with the inulin clearance throughout a substantial range of clearances. However, if accurate methods are used for assay of plasma creatinine, the GFR estimated by creatinine clearance may not correlate with the GFR estimated by inulin clearance. Secondly, GFR measured by creatinine clearance and GFR measured by inulin clearance in the same patient progressively diverge as renal failure progresses and plasma creatinine level rises. The greater apparent GFR found by creatinine clearance may be due to an increase in tubular secretory activity for creatinine when plasma levels increase much above normal and to the relatively smaller contribution of noncreatinine chromogens in the nonspecific assay of plasma creatinine. In clinical practice, it is now accepted that, by the time patients have lose one half to two thirds their normal renal function, as demonstrated by creatinine clearance, it is more reliable and

Department of Biochemistry, Medical Research Institute, Colombo-WHO Biennium 2004-2005

33

prudent to monitor their subsequent renal function and response to therapeutic initiatives by using radioisotopic markers of glomerular filtration and renal plasma flow.

9.3

PRINCIPLE OF THE METHOD

Protein free filtrate is mixed with an alkaline picrate solution which forms a yellow –red complex with Creatinine. The absorbance of the complex is measured at 500 nm.

9.4

SPECIMEN TYPE, COLLECTION AND STORAGE

Non haemolysed serum; collect about 4 - 5 ml blood into a clean dry bottle. Avoid haemolysis. Separate the serum as early as possible from the cells within 12 hours of collection. Serum is stable at 2-8 0C up to 24 hours Referral: Send about 1.0 ml serum kept cool to reach destinations within 18 hours

9.5

APPARATUS AND CHEMICALS

APPARATUS: Spectrophotometer at wavelength 500 nm or Colorimeter with blue green filter, Ilford 603) GLASSWARE: Volumetric flask (100 ml, 500 ml and 1litre volumes) Automatic micro pipettes (50µl, 100µl, 500 µl and 1000µl-5000µl) Graduated pipettes (5ml, 10 ml in 0.1 ml) Beakers (100ml, 500 ml) Test tubes (125 mm x 16 mm)

Conical centrifuge tubes 15 ml

CHEMICALS: Sodium hydroxide pellets Picric acid; Note: water is added to picric acid to ensure safety in transit Creatinine anhydrous -AR Hydrochloric acid concentrated (37% w/v) caution: highly corrosive Sulphuric acid-AR Sodium tungstate dihydrate Polyvinyl alcohol

Reagent bottles, clear and amber coloured (500 ml), Rubber bulb

REAGENTS 1. Saturated picric acid solution: Picric acid is supplied as a moist chemical. Weigh out the equivalent of 7 g of picric acid i.e. mix well the bottle and weigh out about 10.5 – 11 g if your picric acid container states that 50 % by weight of water has been added. Add 11 g of moist picric acid to 500 ml distilled water, stir for several hours to ensure that a saturated solution is produced. Transfer in to a brown bottle. This solution is stable indefinitely at room temperature. Note: the amount of picric acid weighed out will depend on the water content of the chemical 2. Picric acid 0.036 mol/l: Measure 705 ml of saturated picric acid solution in to volumetric flask and make up to 1 litre with distilled water store in a brown bottle. This solution is stable indefinitely 3. Acid tungstate solution: Weigh out 11.1 g of sodium tungstate dihydrate (9.8 g anhydrous salt) and dissolve in about 300 ml of distilled water in a 1 litre volumetric flask. Dissolve 1 g of polyvinyl alcohol in about 100 ml of distilled water with heating (do not boil) Allow to cool to room temperature then transfer into the volumetric flask containing sodium tungstate. Measure 2.1 ml of concentrated sulphuric acid in to 300 ml of distilled water in a beaker mix well. Add this solution also-in to the same volumetric flask containing tungstate solution. Dilute to 1 litre with distilled water when the solution is cool to room temperature. Store in a brown bottle. 4. Sodium hydroxide solution 1.4mol/l: Dissolve 56 g of sodium hydroxide in distilled water and dilute to 1 litre, store in a polypropylene bottle.

Department of Biochemistry, Medical Research Institute, Colombo-WHO Biennium 2004-2005

34

5. Hydrochloric acid 0.1mol/l: Carefully pipette 9.0 ml hydrochloric acid concentrated in to a volumetric flask containing distilled water, dilute to one litre with distilled water. 6. Creatinine standard 1.32mmol/l: Keep about 200 mg of Creatinine for overnight in a desiccator. Weigh out 149 mg of Creatinine anhydrous and dissolve in a small volume of hydrochloric acid (0.1mol/l) in a small beaker, transfer quantitatively to a one litre volumetric flask and make to 1 litre with hydrochloric acid. Store in a brown bottle

9.6

PROCEDURE

1. Label two conical centrifuge tubes one for the quality control serum and one for the patient’s sample. Pipette 4.0 ml of Acid tungstate reagent into each tube. 2. Pipette 500 µl of quality control serum or patients’ serum to the appropriate tubes. Mix vigorously for about 10 seconds then centrifuge for about 10 minutes to obtain a clear supernatant. 3. Transfer 3.0 ml of clear supernatant in to test tubes for each quality control serum or patient’s serum. For blank 3.0 ml of distilled water, for the standard pipette 3.0 ml of distilled water and 50 µl of Creatinine standard solution. 4. To each tube add 1.0 ml of picric acid solution (0.036 mol/l) mix well 5. Add 0.5 ml sodium hydroxide solutions (1.4mol/l) to the first tube, mix well at 30 second intervals add sodium hydroxide solution to remaining tubes. 6. Exactly 15 minutes after addition of sodium hydroxide read the absorbance against the reagent blank at 500 nm. Read absorbance of the tubes in sequence maintaining 30 seconds intervals between readings

9.7

CALCULATION

Dilution of standard in the assay Concentration of the stock standard In the assay condition Dilution of serum in the assay Serum Creatinine concentration

: 0.05ml STD + 3.0 ml of distilled water = 3/0.05 = 60 = 1.32 mmol/l (1320 µmol/l) =1.32/60 =0.022 mmol/l =1 in 9 =Abs of Test x Con of Std x Dilution of serum Abs of Std =T/S x 0.022 x 9 =T/S x 0.198 =T/S x 200 µmol/l

QUALITY CONTROL OPTIMAL CONDITIONS VARIANCE: A coefficient of variation of around 4% should be attainable. ROUTINE CONDITIONS VARIANCE: This value should not exceed 8% REFERENCE VALUES: For an adult 71-133 µmol/l Children under 12 years 20-80 µmol/l

9.8

LIMITATIONS

Proteins give positive Jaffe reaction therefore  The supernatant fluid should be clear  Pipetting of 3 ml of supernatant should be done carefully without disturbing the precipitate.  Sodium hydroxide solution should be added in timed intervals  Absorbance reading should be measured exactly 15 minutes after addition of Sodium hydroxide  Interfering substances reacting like Creatinine are acetone, acetoacetate, pyruvate and some cephalosporin antibiotics contribute to total colour production  Blood constituents such as glucose, ascorbate histidine and adrenaline may cause disturbances to the colour development.

Department of Biochemistry, Medical Research Institute, Colombo-WHO Biennium 2004-2005

35

10.

URINE CREATININE 10.1

SPECIMEN TYPE, COLLECTION AND STORAGE

24 hours collection of urine: empty the bladder completely and discard the sample, note the time, from that time onward collect the sample into a beaker and transfer (use a funnel) into an amber colour bottle (2.5 L) with preservative till 24th hour, keep the bottle in the refrigerator during collection period. A sample of blood (3-4 ml) should be drawn during the collection period of urine. If the bottle is inadequate, collect the excess urine into a chemically clean bottle and store in the refrigerator until it is dispatched to the laboratory.

10.2

PRINCIPLE OF THE METHOD

After dilution, the urine is mixed with picric acid and sodium hydroxide solution which forms a yellow red complex with Creatinine. The absorbance of the complex is measured at 500 nm.

10.3

APPARATUS AND CHEMICALS

Same as for serum Creatinine estimation. PRESERVATIVE 1 Normal hydrochloric acid (1 N)-10 ml or Thymol in Isopropanol (100g/l)-5 ml

10.4

PROCEDURES

1. Mix the 24 hours urine collection. Measure the total urine volume. 2. Pipette 1 ml of urine into a 100 ml volumetric flask. Make up to 100 ml with distilled water. Mix well (1:100 dilution) 3. Three test tubes are required. One for the Test, one for a reagent blank, and one for the standard 4. Transfer 3.0 ml of diluted urine into the Test. 3.0 ml of distilled water into the reagent blank. 3 ml of distilled water and 0.1 ml of standard solution into the standard. 5. To each tube add 1.0 ml picric acid solution (0.036mol/l). Mix well. 6. Add 0.5 ml sodium hydroxide solution (1.4mol/l) to the first tube, mix well at 30 seconds intervals and add sodium hydroxide solution to the remaining tubes. 7. Exactly 15 minutes after adding sodium hydroxide read the absorbance against the reagent blank at 500 nm. Read the tubes in sequence maintaining 30 seconds intervals between readings.

10.5

CALCULATION

Same method as serum Creatinine but 3 ml of diluted urine (1:100 with distilled water) is used instead of filtrate No protein precipitation is needed  Concentration of standard = 1.32 mmol/l (1320 µmol/l)  Standard dilution is = 1 in 30 (i.e. 0.1 ml standard diluted with 3.0 ml of distilled water) 

∴standard concentration



urine dilution is



∴ urine Creatinine



24 hour Urine Creatinine (mmol/24 hour)

= 1.32 mmol/l = 0.044 mmol/l (in assay condition) 30 = 1: 100 = Test x 0.044 x 100 mmol/l Standard = Test(T) x 4.4 x Total volume in litre Standard(S)

CHILDREN 

Urine Creatinine

(µmol/kg/24 hours)

= T x 4.4 x Total volume in litre x 1000

S

x

Body weight in kgs

REFERENCE VALUES: For an adult 8.84 to 17.6 mmol/24 hours Department of Biochemistry, Medical Research Institute, Colombo-WHO Biennium 2004-2005

36

Children under 12 years 44 -354 µmol/kg/24 hours

CREATININE CLEARANCE 

     

The following details of the patients are required for calculation of Creatinine Clearance  Height in cm  Body weight in kg(BW)  Age  Total volume of urine collection over timed period ( 24 hours) Blood sample should be collected during the 24 hours urine collection Serum Creatinine and 24 hours urine Creatinine should be performed for calculation of Creatinine clearance Creatinine Clearance =UV/P U-Urine Creatinine in mmol/l V-Rate of urine flow in ml/minute=24 hours urine volume in ml 24 x 60 P-Serum Creatinine in mmol/l

Conversion of urinary Creatinine excretion in mmol/24 hours to mmol/l Concentration of urine Creatinine in mmol/l=Concentration of urinary Creatinine mmol/24 hours Total volume in litre Creatinine Clearance=Concentration of urine Creatinine in mmol/l x 24 hour urine volume in ml (ml/minute) Concentration of serum Creatinine in mmol/l 24 x 60

Clearance varies with body size and is proportional to the body area (A) where this varies much from the normal in adults and in all cases in children. The determined clearance is corrected to a standard surface area of 1.73 m2 by multiplying 1.73/A. The A [Body surface area (in m2) can be calculated using a nomogram. From height (in cm) and weight (in kg)] Corrected Creatinine Clearance= Creatinine clearance x 1.73 ml/minute/1.73 m2

A REFERENCE VALUES: Refer appendix 3 REFERENCES WHO manual Varley’s practical clinical Biochemistry sixth edition

Department of Biochemistry, Medical Research Institute, Colombo-WHO Biennium 2004-2005

37

11.

CHOLESTEROL 11.1

INTRODUCTION

Although every living organism examined has been found to contain sterols, cholesterol is found almost exclusively in animals and humans, in which it is also the main sterol.Virtually all cells and body fluids contain some cholesterol.Like other sterols,cholesterol is a solid alcohol of high molecular weight and possesses the tetracyclic perhydrocyclopentanophenanthrene skeleton. The molecule contains 27 carbon atoms.Cholesterol is the initial starting point in many metabolic pathways. These include vitamin D synthesis, steroid hormone synthesis, and bile acid metabolism. Cholesterol is presented to the intestinal wall from three sources: the diet, bile and intestinal secretions and cells. Animal products, especially meat, egg yolk, seafood, and whole-fat dairy products, provide the bulk of dietary cholesterol. Cholesterol intake varies considerably according to the dietary intake of animal products. A similar amount of cholesterol is present in the gut from biliary secretion and the turnover of mucosal cells. Practically all cholesterol in the intestine is present in the unesterified (free) form. Esterified cholesterol in the diet is rapidly hydrolyzed in the intestine to free cholesterol and free fatty acids (FFA) by cholesterol esterase in pancreatic and small intestinal secretions. Although portion of the body’s cholesterol is derived from dietary intake, most tissue and plasma cholesterol is synthesized endogenously by the liver and other tissues from simpler molecules, particularly acetate. Once synthesized cholesterol is released into the circulation for transport in combination with specific apoproteins, the apolipoproteins, in complexes known as lipoproteins. Minimal cholesterol esterification occurs within the liver before its release, and cholesterol is mainly esterified within the vascular compartment. Esterification is important because it serves to enhance the lipid carrying capacity of the lipoproteins. The reaction is catalyzed by the enzymes lecithin-cholesterol acyltransferase (LCAT) in the plasma and acylcholesterol acyltransferase (ACAT) intracellularly.The intracellular ACAT pathway is the major pathway in the liver, intestine, adrenal cortex, and probably in the arterial wall. Once cholesterol enters the cell, the esters are hydrolyzed by the action of specific esterases and enters into specific metabolic pathways. Cholesterol reaching the liver is either secreted unchanged into bile or metabolized to bile acids. Approximately one third of the daily production of cholesterol is catabolized into bile acids. The first step in the bile acid synthesis involves the rate limiting step, 7 alpha hydroxylation. Two bile acids, cholic and chenodeoxycholic, constitute the primary bile acids.They are conjugated with either glycine or taurine and enter the bile canaliculi. Some of the bile acids are deconjugated and converted by bacteria in the intestine to secondary bile acids. Cholic acid is converted to deoxycholic acid, and chenodeoxycholic acid is metabolized to lithocholic acid.Virtually all bile acids except lithocholic are reabsorbed in the lower third of the ileum and returned to the liver via the portal vein, thus completing the enterohepatic circulation.

11.2

CLINICAL SIGNIFICANCE

High prevalence of atherosclerosis and ischaemic heart disease is seen where dietary fat intake is relatively high. High plasma levels of LDL, IDL and possibly VLDL are associated with an increased risk of premature atherosclerosis and ischaemic heart disease. This relationship appears to be a continuous (curvilinear) one, i.e. there is no threshold above which risk abruptly appears. Plasma HDL cholesterol concentration is a negative

Department of Biochemistry, Medical Research Institute, Colombo-WHO Biennium 2004-2005

38

risk factor, so that high levels appear to protect against ischaemic heart disease and low levels are associated with an increased risk of ischaemic heart disease.

11.3

PRINCIPLE OF THE METHOD

The cholesterol is determined after enzymatic hydrolysis and oxidation. The indicator quinoneimine is formed from hydrogen peroxide and 4 – aminophenazone in the presence of phenol and peroxidase.

11.4 



PATIENT PREPARATION

No change in dietary habits for at least 3 weeks 12 hours fasting is preferred but not an absolute requirement

BLOOD DRAWING TECHNIQUE  Patient should be seated.  Blood is drawn preferably with out a tourniquet.  Patient should be subjected to minimum stress during blood drawing.

11.5

SPECIMEN TYPE, COLLECTION AND STORAGE

12 hours fasting; 2-3 ml clotted blood; blood drawn with out a tourniquet; avoid haemolysis. Serum should be separated from cells as early as possible. Specimen preferably be analysed on the day of collection. Serum is stable for 4 days at 4 0C, for 3 months at -20 0C and for many years at -70 0C. As referral sample send about 0.5 ml clear serum, kept cool, to reach destination within 24 hours.

11.6

APPARATUS AND CHEMICALS

APPARATUS: Spectrophotometer at wavelength 500 nm Water bath at 37 0C Vortex mixer Automatic micropipette 10 µl Automatic pipette 1000µl GLASSWARE: Test tubes 100mm x 13mm Semi micro cuvette (capacity =1ml) REAGENTS: There are various brands of commercially available reagents and standards for cholesterol estimation. Evaluate the kits using quality control samples (low, high and normal values) and consider the following to select the commercially available kits 1. Performance of the test 2. Reagent stability 3. Expiry date 4. Feasibility of test procedure 5. Interfering substances 6. cost

11.7

PROCEDURE

Following procedure is based on commercially available reagent kits. Test procedure may differ among various products. Therefore strict to the test procedure provided with reagent kits 1. label tubes for Blank, Standard, Quality control and test 2. Add 10 µl of distilled water to Blank , 10 µl of standard solution to Standard, 10µl of QC sample to Quality control and 10µl of patients serum to test Department of Biochemistry, Medical Research Institute, Colombo-WHO Biennium 2004-2005

39

3. Add 1000 µl of cholesterol reagent to all the tubes 4. Mix well and incubate all the tubes at 37 0C for 5 minutes in water bath 5. Mix well, zero the spectrophotometer with reagent blank and read the absorbance at 500 nm

11.8

CALCULATION

Concentration = Absorbance of test x Standard concentration (mmol/l) Absorbance of standard QUALITY CONTROL Include Quality Control sample for every batch of tests. OPTIMAL CONDITIONS VARIANCE should be attainable.

: A coefficient of variation of around 7%

ROUTINE CONDITIONS VARIANCE

: This value should not exceed 14%

REFERENCE VALUES: Refer appendix 3

11.9    

LIMITATION

The test is linear up to a cholesterol concentration of 750 mg/dl (19.3mmol/l). Dilute samples with a higher cholesterol concentration 1+2 with physiological saline (0.9 %) and repeat the determination. Multiply the result by 3. Haemoglobin up to 200 mg/dl does not interfere with the test Bilirubin >5mg/dl and ascorbic acid>10 mg/dl interfere the test This limitation varies with kits.

PRECAUTIONS  Allow the samples, standard, QC and reagents to attain room temperature  Mix the thawed sample well.  Do not include more than 20 samples for a batch to maintain good quality  Verify the temperature of the water bath (at 37 0C) before the commencement of the test.  As the final volume would be 1 ml, use appropriate tubes. (You may encounter difficulties in pipetting with long tubes.)  Wipe outside of the pipette tip using a piece of gauze  Add serum to the bottom of the tube  Mix well in each step  Any colour change of the blank should be compared with previous blank readings REFERENCES Leaflets of commercially available kits

OTHER METHODS Liebermann –Burchard method Specific enzymatic methods (as described above) have replaced the chemical methods based on Libermann- Burchard and Salkowski reactions.

Department of Biochemistry, Medical Research Institute, Colombo-WHO Biennium 2004-2005

40

12.

GLUCOSE 12.1

INTRODUCTION

Glucose is the primary energy source for the human body. It is derived from the breakdown of carbohydrates in the diet (grains, starchy vegetables and legumes) and in the body stores (glycogen), as well as by endogenous synthesis from protein or the glycerol moiety of triglycerides. When energy intake exceeds expenditure, the excess is converted to fat and glycogen for storage in adipose tissue and liver or muscle respectively. When energy expenditure exceeds caloric intake, endogenous glucose formation occurs from the breakdown of carbohydrate stores and from non carbohydrate sources. (Amino acids, lactate, and glycerol) The glucose level in blood is maintained within a fairly narrow range under diverse conditions (feeding, prolonged fasting or severe exercise) by regulatory hormones. These include insulin, which decreases blood glucose, and the counter regulatory hormones (glucagon, cortisol, noradrenaline and growth hormone) which increase blood glucose levels.

12.2

CLINICAL SIGNIFICANCE

Diabetes mellitus is a group of metabolic disorders of carbohydrate metabolism in which glucose is underutilized, producing hyperglycemia. Some patients may develop acute life threatening hyperglycemic episodes, such as ketoacidosis or hyperosmolar coma. As the disease progresses the patients are at increased risk of developing specific complications including retinopathy leading to blindness, renal failure, neuropathy(nerve damage),and atherosclerosis.The last may result in stroke, gangrene or coronary disease. (Please refer the article in annexure for current World Health Organization recommended criteria for diagnosis of Diabetes Mellitus.) Hypoglycemia is a blood glucose concentration below the fasting range, but it is difficult to define specific limits. No symptoms are specific for hypoglycemia. A rapid decrease in plasma glucose to hypoglycemic levels usually triggers a sympathetic response, with the release of nor adrenaline, which produces classical signs and symptoms of hypoglycemia: weakness. Sweating, nausea, rapid pulse, lightheadedness and hunger. The brain is totally dependent on blood glucose, and very low levels of plasma glucose(less than 20 or 30 mg /dl cause severe central nervous system dysfunction. Neonatal blood glucose concentrations are much lower than adults and decline shortly after birth when live glycogen stores are depleted. Glucose levels as low as 30 mg/dl in a term infant and 20mg/dl in a premature infant may occur without any clinical evidence of hypoglycemia. The more common causes in the neonatal period include prematurity , maternal diabetes and maternal toxemia. These are usually transient. Hypoglycemia with onset in early infancy is usually less transitory and may be due to inborn errors of metabolism or ketotic hypoglycemia, which usually develop after fasting or febrile illness.

12.3

PRINCIPLE OF THE METHOD

The aldehyde group of β - D – Glucose present in the plasma is oxidized by the enzyme Glucose oxidase to gluconic acid with liberation of hydrogen peroxide. The hydrogen peroxide is converted to water and molecular oxygen by the enzyme peroxidase. In the presence of an oxygen acceptor or 4 - aminophenazone together with phenol, a pink colour is formed which is measured at 510 nm. SPECIMEN CONTAINERS

Department of Biochemistry, Medical Research Institute, Colombo-WHO Biennium 2004-2005

  

41

Blood containers should be leak proof and be easy to close and open without contaminating the fingers. Screw capped bottle with a rubber liner (Bijou bottles) are satisfactory. Bottles should be washed with a detergent, rinsed in several changes of clean water, rinsed in distilled water and dried well.

ANTICOAGULANT AND PRESERVATIVE  4 mg of a mixture of potassium oxalate and sodium fluoride in the ratio of 3:1 is sufficient to collect 1 ml of blood.  A solution can be prepared so that 0.1 ml contains 3 mg of potassium oxalate and 1mg of sodium fluoride. PREPARATION OF BLOOD SUGAR BOTTLES  Weigh out 3 g of potassium oxalate and 1 g of sodium fluoride separately into beakers  Dissolve the chemicals well and transfer into a 100 ml volumetric flask, mix well and make up to 100ml with distilled water.  Store the solution in a bottle at room temperature  To collect 1 ml of blood add 0.1 ml of the prepared solution into bijou bottle and dry in the oven at 60 0C  Allow to cool; stopper and label the bottles, the amount of blood is to be collected should be mentioned

12.4  

SPECIMEN TYPE, COLLECTION AND STORAGE

1 ml blood collected into blood sugar bottle Haemolysis free plasma

FASTING SPECIMEN  For adults the fasting time is usually 10-12 hours.  For children the fasting time is 6 hours unless longer time is indicated. POST PRANDIAL SPECIMEN  Blood collected 2 hours after a meal RANDOM SPECIMEN  Blood sample collected at any time regardless of food intake STABILITY  Glucose stabilized up to 24 hour at room temperature when collected in an oxalate and fluoride mixture.  Plasma should be separated soon after collection preferably within 1 hour  Separated plasma should not contain RBC or Leucocytes BLOOD COLLECTION VENOUS BLOOD  Avoid an intravenous (IV) infusion arm  Do not shake the blood but gently mix with the anticoagulant.(to prevent haemolysis)  Exact amount of an anticoagulant and blood should be mixed since sodium fluoride inhibits the action of glucose oxidase and peroxidase in the assay.

12.5

APPARATUS AND CHEMICALS

APPARATUS: Analytical balance accurately calibrated Oven, temperature at 100 0C Water bath, temperature at 37 0C Department of Biochemistry, Medical Research Institute, Colombo-WHO Biennium 2004-2005

42

Visible Spectrophotometer with 510 nm Automatic micro pipettes (100 µl) GLASSWARE: Volumetric flask (100 ml A grade and 500 ml volumes) Graduated pipettes (0.5ml, 1 ml, 2 ml in 0.1 ml) Petri dish, watch glass or beaker Pasteur pipette Test tubes (100 mm x 13mm) Reagent bottles clear and amber coloured Rubber bulb

CHEMICALS: Benzoic acid 4 - amino phenazone/4 - amino anti pyrine-AR Glucose oxidase lyophilized powder from Aspergillus Peroxidase lyophilized powder Phenol crystals-AR Tween 20 D-Glucose anhydrous-AR Disodium hydrogen phosphate dihydrate – AR (Na2HPO4.2H2O) Potassium dihydrogen phosphateAR (KH2PO4) Sodium azide AR

REAGENTS 1. Benzoic acid solution 1g/l: Weigh 1g of benzoic acid and transfer it to a 1 litre volumetric flask. Add about 800 ml of distilled water and mix to dissolve the chemical completely. Make up to 1 litre mark with distilled water and mix well. Transfer to a clean bottle, label the bottle and store at room temperature. The solution is stable indefinitely. Benzoic acid does not dissolve easily (Distilled water at 50 – 70 0C can be used – Monica) 2. Stock glucose standard solution 1 g %( 55.55 mmol/l)  Use dry and clean glassware  Weigh 1.3 g of D-Glucose anhydrous (analytical grade) into a watch glass or Petri dish or into a beaker. Spread the chemical over the bottom of the container and keep in an oven at 60- 80 0C for 4 hours.  Allow to cool in a dessiccator and weigh out 1 g of dried glucose accurately  Transfer the chemical from the weighing container to a volumetric flask using a funnel. Wash any chemical remaining in the container into the volumetric flask with the benzoic acid solution (1 g/l). Always use a funnel to transfer the chemical or solutions from any container to a flask  Half fill the volumetric flask with benzoic acid solution and mix until the chemical is completely dissolved. Make the solution up to 100 ml with benzoic acid solution. Make sure the bottom of the meniscus of the fluid is on the graduation mark when viewed at eye level  Use a Pasteur pipette to add the final volume of the benzoic acid solution to the flask.  Mix the solution well by inverting the flask for several times. Rinse the bottle with small quantity of the standard solution, transfer in to the bottle and put the date of the preparation on the label  The standard is stable for three months at 2- 8 0C NOTE: The glucose standard solution should be kept at room temperature for 24 hours to α - β forms to reach in equilibrium after preparation (H&W- 6ht edition) 3. Working glucose standard 5.55 mmol/l:  Allow the stock glucose solution to attain room temperature.  Pipette accurately 10 ml of stock glucose solution using a volumetric pipette A grade ( bulb pipette)  Carefully dispense into a 100 ml volumetric flask A grade

Department of Biochemistry, Medical Research Institute, Colombo-WHO Biennium 2004-2005

43



Make up to the 100 ml mark with benzoic acid solution (1 g/l) use a Pasteur pipette to add the final volume of the benzoic acid solution to the flask. Make sure the bottom of the meniscus of the fluid is on the graduation mark when viewed at eye level. Stopper and mix the solution well by inverting the flask several times.  Rinse a clean dry bottle with small quantity of standard solution and transfer the solution into the bottle. Store in the refrigerator at 2- 80 C. This solution is stable for three months at 2- 80 C 4. Phosphate buffer 100 mmol/l pH 7.0 : Disodium hydrogen phosphate dihydrate [Na2HPO4.2H2O] 12.95 g Anhydrous Potassium dihydrogen phosphate [KH2PO4 ] 4.95 g Sodium azide [NaN3 ] 0.50 g Distilled water to 1 litre  Measure about 800 ml of distilled water into a 1 litre volumetric flask  Weigh out chemicals and add one by one in the order into the flask. Mix to dissolve the chemicals  Check that the pH is 7.0 ± 0.05 with a pH meter, make up to 1 litre mark with distilled water and mix well.  Transfer the reagent to a clean bottle and label. The reagent is stable for 3-4 months at 2-8 0C 5. Colour reagent(100ml) 4 – Amino phenazone 16 mg Glucose oxidase 1800 units Peroxidase 100 units Phenol 105 mg Tween 20 50 µl Phosphate buffer to 100 ml To prepare 500 ml of colour reagent: i. Glucose oxidase (GOD): Available as lyophilised powder and as liquid form. Different products are found in different definitions for units of activity of glucose oxidase. Read the label for the activity. E.g. 250 units/mg Therefore the amount of GOD powder to be weighed to contain 1800 units of GOD is 1800/250=7.2 mg To prepare 500 ml of colour reagent 7.2 x 5 =36 mg is required, weigh out 36 mg of GOD lyophilized powder accurately in a small beaker and dissolve in 10 ml of phosphate buffer carefully. ii. Peroxidase (POD): Read the label for the activity. E.g. 63 units/mg Therefore the amount of peroxidase powder to be weighed to contain 100 units of POD is 100/63 = 1.58 mg To prepare 500 ml colour reagent 1.58 x 5 = 7.9 mg is required; weigh out 7.9 mg of POD in a small beaker and dissolve in about 10 ml of phosphate buffer. iii. Transfer about 400 ml of phosphate buffer into a 500 ml volumetric flask iv. Add the glucose oxidase solution into the flask using a funnel. Rinse out the beaker into the flask with a little of the phosphate buffer to make sure all the GOD is transferred to the flask v. Add the POD solution into the flask described as above ( iv) vi. Weigh out 80 mg of 4 – aminophenazone in a small beaker and transfer into the flask with rinsing with the phosphate buffer. vii. Weigh rapidly 525 mg of crystalline phenol in a beaker. Transfer into the flask carefully using the funnel. Rinse the beaker into the flask with phosphate buffer and mix well. NOTE: Phenol is highly corrosive, toxic and hygroscopic chemical. Therefore handle it with great care. To avoid damaging the balance pan, always remove the beaker when adding or subtracting the chemical. Make sure the stock bottle of phenol is tightly stoppered after use. Department of Biochemistry, Medical Research Institute, Colombo-WHO Biennium 2004-2005

44

viii. ix.

12.6

Measure 250 µl (0.25 ml) of Tween 20 and add into the flask. Make up to the 500 ml of mark with phosphate buffer, stopper and mix well. Transfer to a clean brown bottle, label and store at 2-8 C. The reagent is stable for about one month.

PROCEDURE

1. Label sufficient test tubes for the batch including standard (S) Quality controls (C1,C2) and patients samples (1,2,3, etc) 2. pipette into the appropriately labelled 13 x 100 mm tubes as follows S1 S2 C1, C2 1, 2, 3 Distilled water (ml) 1.9 1.8 1.9 1.9 Glucose standard 5.5 mmol/l (µl) 100 200 Quality control/Patient’s plasma (µl) 100 100 Mix well 3. Label a second set of tubes including reagent blank (B), standard (S1, S2), Quality controls (C1, C2 ) and patient’s samples (1, 2, 3 ) 4. Pipette into the tubes as follows. Blank S1 S2 C1, C2 1, 2, 3 Distilled water (µl) 100 Diluted standards (µl)

-

100

100

-

-

Diluted patient’s sample(µl)

-

-

-

100

100

Colour reagents(ml)

1.2 1.2 1.2 1.2 1.2 5. Mix all tubes well, incubate at 37 in a water bath for 15 minutes. 6. Shake tubes two or three times during this period to ensure adequate aeration 7. Remove from the water bath, cool to room temperature and read the absorbance in a spectrophotometer at 510 nm. Set the instrument to zero with the reagent blank (B). 8. Perform the standards in duplicate for greater accuracy and precision. 9. Calculate the results in mmol/l and check the quality control results. NOTE: This procedure is designed for spectrophotometers that require a minimum volume of reaction mixture in the cuvette of 1 ml. Economical use of reagents is possible with this protocol, thus the cost per test can be kept to the minimum. However if the laboratory employs a spectrophotometer requiring a large volume of the reaction mixture for measurement, viz 5 ml, it is advisable to increase the volume of all reagents mentioned under step 4 proportionately. 0C

PREPARATION OF CALIBRATION GRAPH The calibration graph must be prepared in order to confirm the linearity of the method and should be checked whenever a new batch of reagents are introduced or any change in the spectrophotometer is being made. Prepare the calibration graph standards from the Glucose working standard 5.55 mmol/l as described in the table below in clean tubes (13 x 100 mm) as accurately as possible. S1 S2 S3 S4 S5 S6 Glucose working standard 5.55mmol/l (µl) 50 100 200 300 400 500 Distilled water (ml) 1.95 1.9 1.8 1.7 1.6 1.5 Glucose concentration mmol/l 2.77 5.55 11.1 16.6 22.2 27.7 Mix well Follow the procedure 12.6 and draw a calibration graph by plotting the absorbance values of standards against the concentration of standards. The points should be linear and the graph should pass through the origin Since the procedure for standard tube S2 and test is identical, the standard S2 will represent a concentration of 5.55 mmol/l. the glucose concentration represent by other standard tubes are S1=2.77, S3=11.1, S4=16.6, S5=22.2, S6=27.7 mmol/l Department of Biochemistry, Medical Research Institute, Colombo-WHO Biennium 2004-2005

45

12.7

CALCULATION

When the calibration graph is linear one of the standards used to prepare the calibration graph should be included in each batch of tests. The Beer & Lambert formula can be used to calculate the concentration of unknown samples. Concentration of Glucose in mmol/l = Test /Standard x Concentration QUALITY CONTROL Include QC sample for each batch of tests OPTIMAL CONDITIONS VARIANCE: A coefficient of variation of around 3 % should be attainable. ROUTINE CONDITIONS VARIANCE: This value should not exceed 6 % REFERENCE VALUES Random plasma Glucose level Fasting plasma Glucose level

12.8  

≤7.8 mmol/l 3.3-6.1 mmol/l

LIMITATION

Any sample that gives a glucose value >25 mmol/l should be diluted 1:2 with 0.9 % Sodium chloride solution and the correct value obtained by multiplying the result by 3. At high plasma levels of uric acid, glutathione and Bilirubin may interfere with the assay by causing a decrease in glucose values. Ascorbic acid will decrease glucose values by retarding colour development. Do not report results from specimens with suspected interference. Inform the requesting physician of the problem.

PROBLEMS AND PRECAUTIONS  In preparation of sugar bottle add correct volume of anticoagulant/fluoride mixture as excess NaF may lead to falsely low glucose levels.  As this is an enzymatic method the factors that control enzymatic reactions should be kept under control. ( pH of buffer at 7.0 , temperature at 37 0C and the period of incubation)  No colour development or low colour development may be due to o Expired colour reagent o Unsuitable or expired glucose oxidase or any other chemicals o Check the incubation time and temperature of the water bath o Pipetting errors  As the final volume is 1.3 ml, use a semi micro cuvette (1 ml). Do not use macro cuvettes (2 ml) as it will cause errors in absorbance readings.

12.9

OTHER METHODS 1.

O-Toluidine

COPPER REDUCTION (NOT RECOMMENDED DUE TO NON SPECIFICITY) 2. 3. 4. 5.

Phosphomolybdate (Folin wu) Arsenomolybdate (Somogyi-Nelson) Neocuproine Alkaline fericyanide method

ENZYMATIC 6. 7. 8.

Hexo kinase (HK) Glucose oxidase (GOD)-oxygen consumption Glucose dehydrogenase

REFERENCES Trinder,p. (1969).Annals of Clin.Biochem.6:24-27 Barham D and Trinder P. (1972). Analyst 97:142-145 Department of Biochemistry, Medical Research Institute, Colombo-WHO Biennium 2004-2005

46

13.

INORGANIC PHOSPHATE 13.1

INTRODUCTION

Phosphorus in the form of inorganic or organic phosphate is an important and widely distributed element in the human body. An adult human has approximately 600g (19.4 mol) of phosphate expressed as phosphorus, of which about 85% is in the skeleton and the rest principally in soft tissues. In the soft tissues most phosphate is cellular. Although both inorganic and organic phosphate are present in cells, most is organic and incorporated into nucleic acids, phospholipids and high energy compounds involved in cellular integrity and metabolism. Serum phosphate has a diurnal variation. It is higher in the afternoon and evening. The serum phosphate level is dependent on meals and variation in the secretion of hormones such as parathyroid hormone. Serum calcium and phosphate levels are regulated by the kidneys. The most important intracellular function of phosphate is the high energy bond in adenosine triphosphate. These energy sources maintain many physiological functions such as muscle contractility, neurological functions and electrolyte transport. Phosphate is a constituent of cyclic adenine and guanine nucleotides as well as nicotinamide adenine dinucleotide phosphate, which is important in many enzyme systems. It is an element in phospholipid cell membranes, nucleic acids and phosphoproteins. It is also involved in the regulation of intermediary metabolism of proteins, fats and carbohydrates, as well as in gene transcription and cell growth. Extra cellular phosphate maintains the critical intracellular concentration and provides substrate for bone mineralization. The skeleton serves as a store house for phosphate. The cellular demands for metabolic function in bone cells are similar to those in other cells.

13.2

CLINICAL SIGNIFICANCE

Hypophosphataemia is defined as the concentration of inorganic phosphate in the serum below the normal reference interval. Hypophosphatemia does not necessarily imply intracellular phosphate depletion. Hypophosphataemia may be present when cellular levels are normal, and cellular phosphate depletion may exist when serum concentrations are normal or even high. Phosphate depletion may have four general causes    

Intra cellular shift (A high carbohydrate diet stimulates insulin secretion, increasing the transport of glucose and phosphate into the cell.) Renal Phosphate wasting (Any cause of excessive parathyroid hormone secretion may result in hypophosphataemia due to phosphaturia Decreased intestinal phosphate absorption (Increased loss due to vomiting, diarrhoea and phosphate binding antacids: Decreased absorption in malabsorption syndromes.) Cellular phosphate loss (Acidosis results in catabolism of organic compounds within the cell so that inorganic phosphate shifts into the plasma and excreted in the urine.

Hyperphosphatemia is usually due to acute or chronic renal failure because the kidneys fail to excrete the amount taken in the diet. Lack of Parathyroid hormone and increased growth hormone causes increased tubular reabsorption of phosphate resulting in increased phosphate levels in blood. Increased phosphate intake, increased extra cellular phosphate load in acidosis and any cause leading to cell lysis causes hyperphostaemia

Department of Biochemistry, Medical Research Institute, Colombo-WHO Biennium 2004-2005

47

13.3 PRINCIPLE OF THE METHOD The filtrate obtained after removing proteins by means of trichloro acetic acid is treated with an acid molybdate reagent which reacts with inorganic phosphate to form phosphomolybdic acid. The molybdenum of the phosphomolybdic acid is reduced by means of 1-amino-2- naphthol-4-sulphonic acid to give a blue compound which is measured colorimetrically at 700 nm.

13.4

SPECIMEN TYPE, COLLECTION AND STORAGE

Clear, non haemolysed serum is suitable, collect about 4-5 ml blood without haemolysis into a clean container. Ideally specimens should be obtained without the tourniquet from a recumbent fasting patient. Serum should be separated from erythrocytes as soon as possible within 1 hour of collection, as organic phosphate present in erythrocytes are hydrolysed with formation of inorganic phosphate causing the serum concentration to rise. Hydrolysis proceeds more rapidly at room temperature and at 37 0C. Haemolysed samples are unsuitable because erythrocyte contain high concentration of organic phosphates which can be hydrolysed to inorganic phosphate during storage.

13.5

APPARATUS AND CHEMICALS

APPARATUS: Centrifuge Spectrophotometer Automatic pipette (500 µl, 1000 µl) GLASSWARE: Volumetric flasks (100 ml, 200ml, 500 ml) Beakers (5ml, 200 ml, 500ml) Graduated pipette (0.5 ml, 1ml, 5ml, 10 ml) Conical centrifuge tubes 15 ml Test tubes (16 x 150 mm)

CHEMICALS: Ammonium molybdate-AR 1-Amino-2-napthol-4-sulphonic acid Perchloric acid AR Sodium metabisulphite-AR (store in refrigerator) Sodium sulphite-AR (store in refrigerator) Potassium dihydrogen phosphate KH2 PO4 - AR

Trichloro acetic acid - AR REAGENTS 1. Reducing agent: Dissolve 12 g of sodium Meta bisulphite and 2.4 g of sodium sulphite in about 80 ml of distilled water. Add 0.2 g of 1-amino-2-naphthol-4-sulphonic acid. Dissolve and dilute to 100 ml in volumetric flask. Store in the refrigerator in a brown bottle. The solution keeps up to 4 weeks. It is better prepare fresh reagent ( about 10 ml) 2. Trichloroacetic acid 10 %: Dissolve 10 g of Trichloroacetic acid in distilled water and make up to 100 ml with distilled water. Store in the refrigerator. 3. Perchloric acid AR 4. Ammonium Molybdate 5%: Dissolve 5 g of Ammonium molybdate in distilled water and make up to 100 ml with distilled water. Store at room temperature. Discard the solution when a precipitate appears. 5. Stock phosphate standard solution 32mmol/l: Keep about 2.5 g of pure potassium dihydrogen phosphate in a dessicator to dry, weigh exactly 2.194 g of potassium dihydrogen phosphate and transfer into a 500 ml flask. Dissolve in distilled water and make up to 500 ml with distilled water. Store at room temperature. 6. Working phosphate standard solution 0.128 mmol/l: Dilute 2 ml of stock phosphate standard to 500 ml with distilled water. Store at room temperature Department of Biochemistry, Medical Research Institute, Colombo-WHO Biennium 2004-2005

48

13.6

PROCEDURE

1. Label sufficient centrifuge tubes for quality control (C) and patient’s samples (1, 2, 3) 2. Pipette into tubes as follows C

1, 2, 3

Trichloroacetic acid 10 % (ml)

9.0

9.0

Quality control serum (ml)

1.0

-

-

1.0

Patient’s sample (ml)

3. Mix well and leave for about 10 minutes and centrifuge ( 2500-3000 rpm) for about 10 minutes 4. Label a set of test tubes including reagent blank (B) Standard (S) Quality control (C1, C2) and patient’s samples (1, 2, 3 …) 5. Pipette into the tubes as follows

Trichloro acetic acid 10% (ml) Working standard solution (ml) Supernatants from tubes above(ml) Perchloric acid (ml) Ammonium Molybdate 5% (ml) Reducing reagent (ml)

B 5.0 0.4 0.4 0.2

S 5.0 0.4 0.4 0.2

C 5.0 0.4 0.4 0.2

1, 2, 3 5.0 0.4 0.4 0.2

6. Mix the tubes after each addition of reagents. 7. Leave at room temperature for 10 minutes 8. Read the absorbance at 700 nm , set the instrument to zero with tube B NOTE: For inadequate specimen use appropriate volumes of serum and reagents.

The supernatant should be clear. 13.7

CALCULATION

Concentration of standard is 0.128 mmol/l, serum dilution is 1:10 ∴Concentration of phosphorus

= =

REFERENCE VALUES

13.8

T x 0.128 x 10 S T x 1.28 mmol/l S

: For an adult 0.80 -1.44 mmol/l

LIMITATION

Glucose phosphate, CPK and other organic phosphates may also be hydrolyzed by assay conditions, resulting in overestimation of inorganic specimens. PRECAUTIONS  Glass ware should be properly cleaned and rinsed because phosphate is a common component of many detergents  Discard Ammonium Molybdate solution when a precipitation formed at the bottom of the container  Recommend to prepare only 10 ml of reducing agent at a time as the stability of the reagent is one month.

Department of Biochemistry, Medical Research Institute, Colombo-WHO Biennium 2004-2005

49

14.

INORGANIC PHOSPHATE IN URINE 14.1

INTRODUCTION

Urinary phosphate excretion varies with age,muscle mass,renal function,parathyroid hormone level,the time of the day and diet.

14.2

PRINCIPLE OF THE METHOD

Urine is diluted with distilled water and mixed with an acid molybdate reagent which reacts with inorganic phosphate to form phosphomolybdic acid. The molybdenum of the phosphomolybdic acid is reduced by means of 1-amino-2-naphthol-4-sulphonic acid to give a blue coloured complex which is measured at 700nm spectrophotometrically.

14.3

SPECIMEN TYPE, COLLECTION AND STORAGE

A 24 hours collection of urine is need. 10 ml of 1 N HCl is added as the preservative APPARATUS, CHEMICALS, GLASSWARE AND REAGENTS ARE AS FOR SERUM PHOSPHATE ESTIMATION

14.4

PROCEDURE

1. Mix the 24 hours urine collection 2. Measure the total volume of urine 3. Pipette 1 ml of urine into a 100 ml volumetric flask. Make up to 100 ml with distilled water. 4. Label 3 test tubes for Blank (B), Standard (S) and Test (T) 5. Pipette 5 ml of distilled water into the tube B, 5 ml of working standard into the tube S and 5 ml of diluted urine into the tube T 6. Add 0.4 ml of Perchloric acid into each tube, Mix well 7. Then add 0.4 ml of Ammonium Molybdate 5% solution to each tube and mix well 8. Add 0.2 ml of reducing agent to each tube, Mix well and leave for 10 minutes at room temperature 9. Read the absorbance at 700 nm, setting the spectrophotometer to zero with blank solution

14.5

CALCULATION

Concentration of the Standard Urine phosphate

24 hour urine phosphate

= 0.128 mmol/l = T x 0.128 x 100 mmol/l S = T x 12.8 mmol/l S = T x 12.8 x V (24 hour urine volume in litre) mmol/24 hrs S

In children urinary phosphate excretion=T x 12.8 x Total volume in litre (mmol/kg/24hrs) S x Body weight in kg REFERENCES VALUES: For an adult : 16-48 mmol/24 hours For children : Refer appendix 3

Department of Biochemistry, Medical Research Institute, Colombo-WHO Biennium 2004-2005

50

15.

TOTAL PROTEIN 15.1

INTRODUCTION

The term ‘ plasma proteins’ describes the very large number of complex molecules that share a common primary structure ; but have an enormous diversity of function .Many of the plasma proteins are classified according to function, e.g. enzymes, clotting factors, acute phase proteins, immunoglobulins, complement components, protease inhibitors, apolipoproteins and transport proteins. Normal adult plasma contains 60 -80 g/L of total protein,the major contributor being albumin and the rest due to globulins. GLOBULINS Electrophoresis of normal serum performed on a carrier such as cellulose acetate reveals four major non-albumin bands staining for protein/lipoprotein, designated by mobility as α1, α2 β, γ Band

Concentration (g/L)

Major components

α1-globulin

2-5

α1- antitrypsin, Apolipoprotein

α2 –globulins

4-10

Caeruloplasmin, α2 macroglobulins, Haptoglobins

β-globulin

γ-globulins

6-12

8-16

Transferrin β-lipoproteins

Immunoglobulins

15.2 CLINICAL SIGNIFICANCE The two general causes of alterations of serum total protein are a change in the volume of plasma water and a change in the concentration of one or more of the specific proteins in the plasma. Decrease in the volume of plasma water(haemoconcentration) is reflected as relative hyperproteinemia: concentrations of all the individual plasma proteins are increased to the same degree. Hyperproteinemia is noted in dehydration due to inadequate water intake or to excessive water loss as in severe vomiting, diarrhoea or diabetic acidosis. Hemodilution(increase in plasma water volume) is reflected as relative hypoproteinemia; concentrations of all the individual plasma proteins are decreased to the same degree. Hemodilution occurs with water intoxication or salt retention syndromes, during massive intravenous infusions and physiologically a recumbent position is assumed. A recumbent position decreases total protein concentration by 0.3 to 0.5 g/dL. Of the individual serum proteins, albumin is present in such high concentrations that low levels of this protein alone may cause hypoproteinemia. Such hypoproteinemia is common and has many causes as described earlier. Mild hyperproteinemia may be caused by an increase in the concentration of specific proteins normally present in relatively low concentrations as for example in acute phase proteins and polyclonal immunoglobulins as a result of infection.Marked hyperproteinemia may be caused by high levels of the monoclonal immunoglobulins produced in multiple myeloma and other malignant paraproteinemias.

Department of Biochemistry, Medical Research Institute, Colombo-WHO Biennium 2004-2005

51

15.3

PRINCIPLE OF THE METHOD

Serum proteins form a violet- blue complex with copper ions in alkaline solution. The absorbance of the complex is measured at 540 nm. A method using a sample blank is recommended in order to avoid errors due to turbidity.

15.4

SPECIMEN TYPE, COLLECTION AND STORAGE

3 ml clotted blood collected into dry clean glass bottle without applying a tourniquet, avoid haemolysis, fasting specimen is desired to decrease lipaemia. Separate the serum from cells as early as possible. Serum is stable at 4 0C

15.5

APPARATUS AND CHEMICALS

APPARATUS: Spectrophotometer at wavelength 540 nm or colorimeter with yellow-green filter, Ilford 605(550 nm) Automatic micro pipettes (50 µl) GLASSWARE: Volumetric flasks (500 ml and 1 litre volumes) Graduated pipette (5 ml in 0.1 ml) Measuring cylinders (100ml and 500 ml) Test tubes (150 mm x16 mm) Beaker (250 ml) Polyethylene reagent bottles (I litre) CHEMICALS: Sodium chloride-AR Sodium azide-AR caution: handle with care Sodium hydroxide pellets-AR Copper sulphate pentahydrate-AR Potassium sodium tartrate tetrahydrate-AR Potassium iodide-AR Albumin bovine, a fraction v powder is suitable REAGENTS 1. Sodium hydroxide solution 6 mol/l: Dissolve 120 g of sodium hydroxide little by little in about 400 ml of distilled water. After cooling dilute to 500 ml. Store in a tightly- closed polyethylene bottle. This solution is stable indefinitely at 20 -25 0C(room temperature) 2. Biuret reagent: Dissolve 3.0 g of copper sulphate in about 500 ml of distilled water. Add 9.0 g of potassium sodium tartrate and 5.0 g of potassium iodide. When they have completely dissolved, add 100 ml of sodium hydroxide solution (6 mol/l) and make up to 1 litre. The solution is stable indefinitely at 20 -25 0C(room temperature) store in a tightly closed polyethylene bottle 3. Blank Biuret reagent: Dissolve 9.0 g of potassium sodium tartrate and 5.0 g of potassium iodide in distilled water. Add 100 ml of sodium hydroxide solution (6mol/l) and make up to 1 litre with distilled water. When kept in a tightly-stoppered polyethylene bottle this solution is stable indefinitely at 20 -25 0C(room temperature) 4. Sodium chloride/Sodium azide solution: Weigh out 9.0 g of sodium chloride and 1.0 g of sodium azide, dissolve and make up to 1 litre with distilled water. This solution is stable indefinitely at 20 -25 0C (room temperature) 5. Protein standard 80 g/l: Weigh out about 4.3 g of bovine albumin powder and dry it in the oven at about 60 C for 8-10 hours. After drying weigh out exactly 4.0 g of dried Department of Biochemistry, Medical Research Institute, Colombo-WHO Biennium 2004-2005

52

bovine albumin powder. Float the powder on the surface of about 30 ml of sodium chloride/sodium azide solution in a small beaker. When the albumin has dissolved transfer the solution into a 50 ml volumetric flask (pour slowly down the side of the flask to avoid frothing). Rinse the beaker with small volumes of sodium chloride/sodium azide solution and make the final volume to 50 ml. This solution is stable for 6 months at 2-8 0C. Store in a clean sterile bottle.

15.6

PROCEDURE

1. For the standard and each patient or control sample, pipette into test tubes 2.5 ml of Biuret reagent (standard and test) and 2.5 ml of blank Biuret reagent (standard blank and test blank) 2. Add 50 µl of standard (80 g/l) or samples to each pair of tubes. 3. A reagent blank is set up for each batch and contains 2.5 ml of Biuret reagent and 50 µl of water. 4. Mix each tube and allow them to stand at room temperature for 30 minutes or at 370 C for 10 minutes. NOTE: the same temperature must be used for standards and samples. 5. Measure the absorbance at 540 nm (yellow-green filter, Ilford No 605) setting the spectrometer to zero with blank Biuret reagent. First read the absorbance of the sample blanks, then the reagent blank, and then the tests. 6. If results are greater than 100 g/l then repeat the analysis using 20 µl of sample. Multiply the result by 2.5 to obtain the protein concentration. CALIBRATION Although the measurement of total protein is simple, results of external quality assessment programmes indicate that many laboratories have difficulty in producing accurate without sample blank correction. The recommended method proposes the use of a bovine albumin solution as a calibrator. One alternative is to use lyophyilised serum calibrators. However many of these show significant turbidity when they are reconstituted and sometimes it is difficult to know whether the contribution that the turbidity makes to the absorbance at 540 nm has been subtracted in the calculation of the total protein value. When possible use a calibrator with a value assigned by a total protein method that incorporates a sample blank. A sample blank for the standard (SB) is not necessary if a solution of bovine albumin is used. A second alternative is to use an out – of-date of human serum albumin from a blood transfusion or pharmacy department. Use the stated concentration of albumin for the total protein value. One bottle will last for many months at 4-6 0C if small volumes (5-10 ml) are withdrawn as required using a sterile syringe. A calibration curve must be prepared as described below to check the linearity of the spectrometer. If it is linear, then a single standard can be used routinely as described under “Technique” .The calibration curve should be repeated at least once a month. PREPARATION OF CALIBRATION GRAPH USING BOVINE ALBUMIN Prepare a calibration graph to confirm that the method is linear with your spectrometer to at least 80g/l. Provided that it is linear, a single standard (80 g/l) can then be used with each batch of patients’ samples. Working standard No

1

2

3

4

Protein standard 80 g/l (ml)

0.25

0.50

0.75

1.0

Sodium chloride/sodium azide solution (ml)

0.75

0.50

0.25

0

Department of Biochemistry, Medical Research Institute, Colombo-WHO Biennium 2004-2005

53

Concentration of working standard solution (g/l)

20

40

60

80

Label five test tubes as follows: reagent blank (RB), Working standard: No.1 (20 g/l); No.2 (40 g/l); No.3 (60g/l); No.4 (80g/l); Pipette 2.5 ml of Biuret reagent into each tube; add 50 µl of distilled water to the reagent blank, and 50 µl of each working standard solution into the corresponding tubes. Mix and leave to stand at room temperature for 30 minutes or at 37 0C for 10 minutes. Read the absorbance at 540 nm after setting the instrument to zero with the reagent blank. Prepare the calibration graph by plotting the absorbance against the protein concentration for each tube.

15.7

CALCULATION

S= A standard - A standard blank – A reagent blank T= A sample - A sample blank- A reagent blank The serum protein concentration of sample=T/S x 80 g/l Where 80 is the concentration of the standard (g/l) Conversion from SI to “old” units g/l x 0.1=g/100 ml QUALITY CONTROL At least two quality control specimens, having stated values in the range 40-80 g/l, one of which is unknown to the operator, should be included with each batch of specimens. If single specimens are analysed a control specimen should always be included. OPTIMAL CONDITIONS VARIANCE: A coefficient of variation of around 2% should be attainable. ROUTINE CONDITIONS VARIANCE: The value obtained for the RCV should not exceed 4% REFERENCE VALUES Approximate reference values for an adult 60-80 g/L

REFERENCES Doumas, B.T. (1975) Clin. Chem, 21, 1159-1166.

Department of Biochemistry, Medical Research Institute, Colombo-WHO Biennium 2004-2005

54

16.

UREA DIACETYLE MONOXIME METHOD

16.1

INTRODUCTION

Urea is the major nitrogen containing metabolic product of protein catabolism in humans, accounting for more than 75% of the non protein nitrogen eventually excreted. The biosynthesis of urea from amino nitrogen-derived ammonia is carried out exclusively by hepatic enzymes of the urea cycle. More than 90% of urea is excreted through the kidneys, with losses through the gastrointestinal tract and skin accounting for most of the remaining minor fraction. Urea is neither actively reabsorbed nor secreted by the tubules but is filtered freely by the glomeruli. In a normal kidney, 40 to 70% of the highly diffusible urea moves passively out of the renal tubule and into the interstitium, ultimately to re-enter plasma. The back diffusion of urea is also dependent on urine flow rate; with more entering the interstitium in slow-flow states. Urea production is dependent on several non renal variables such as diet and hepatic synthesis.

16.2

CLINICAL SIGNIFICANCE

A wide variety of renal diseases with different permutations of glomerular, tubular, interstitial or vascular damage can cause an increase in plasma urea concentration. The usefulness of urea as an independent indicator of renal function is limited by the variability of its blood levels as a result of non renal factors. Mild dehydration, high protein diet, the increased protein catabolism, muscle wasting as in starvation, reabsorption of blood proteins after a gastrointestinal haemorrhage, treatment with cortisol, and decreased perfusion of kidneys may cause increased blood urea that is called pre renal ureamia. Impaired perfusion may be due to decreased cardiac output or shock secondary to blood loss or other causes. Urea values above 20 mmol/L always indicate intrinsic renal disease. However the plasma urea usually does not rise until the glomerular filtration rate falls to less than 50%.

16.3

PRINCIPLE OF THE METHOD

Proteins in whole blood, plasma or serum are precipitated with trichloroacetic acid. The urea in the supernatant reacts with diacetyle monoxime in the presence of thiosemicarbazide and cadmium ions under acid conditions. The absorbance of the resulting rose-purple solution is measured at 530 nm.

16.4

SPECIMEN TYPE, COLLECTION AND STORAGE

3 ml of clotted blood collected into clean dry bottle; avoid haemolysis. Separate serum from cells as early as possible. Serum urea is stable for 24 hours at room temperature (250C), for 7 days at 2-60C, for longer duration (2-3 months) when frozen. As a referral sample send about 0.5 ml clear serum, kept cool, to reach correct destination within 24 hours.

16.5

APPARATUS AND CHEMICALS

APPARATUS: Water bath or heating block (temperature range 10- 110 0C) Spectrophotometer wavelength 530 nm Colorimeter green filter, Ilford 604 (520nm) Rack for boiling tubes Department of Biochemistry, Medical Research Institute, Colombo-WHO Biennium 2004-2005

55

Automatic micro pipettes (50 and 100 µl) GLASSWARE: Amber coloured reagent bottles (500 ml volume) Graduated pipettes (5 and 10 ml in 0.1 ml) Graduated cylinders (50 ml, 100 ml and 500 ml) Volumetric flasks (50 and 100 ml and 500 ml volumes) Glass stoppered boiling tubes (160 x 18 mm) Conical centrifuge tubes 15ml CHEMICALS: Benzoic acid-AR Cadmium sulphate (as 3CdSO4.8H2O)-AR Diacetyle monoxime-AR Orthophosphoric acid (85% w/v)-AR caution: corrosive, handle with care Sulphuric acid, concentrated (95 – 97 % w/v)-AR caution: corrosive, handle with care Thiosemicarbazide-AR Trichloroacetic acid-AR caution: corrosive, handle with care Urea-AR REAGENTS 1. Acid reagent : Add about 200 ml of distilled water to a 500 ml volumetric flask. Keep the flask in basin containing water and then add slowly 44 ml of sulphuric (concentrated) acid and 66 ml of orthophosphoric acid. Cool the solution to room temperature but do not use ice water as a cooling bath. Add 50 mg thiosemicarbazide and dissolve, then add 1.6 g cadmium sulphate and dissolve, add 1.5 ml of the urea working standard solution (2.5mmol/l). Make up to 500 ml with distilled water. Transfer to an amber coloured bottle. This reagent is stable for at least six months at 2- 8 0C. NOTE: The presence of a small amount of urea in the reagent improves the linearity of the calibration curve. The cadmium sulphate improves the stability of the final coloured product. 2. Diacetyl monoxime reagent: Weigh out 2.0 g of diacetyl monoxime, dissolve in distilled water and dilute to 500 ml with distilled water in a volumetric flask. This solution is stable for at least six months at 2-8 0C 3. Colour reagent: Use a graduated cylinder and mix 50 ml of acid reagent with 50 ml of diacetyl monoxime reagent in a small bottle. This amount is sufficient for 33 reactions. This reagent must be prepared daily, therefore, the volume made up should depend on the number of reactions anticipated; 3 ml is required for each reaction. 4. Benzoic acid solution 1 g/l: Weigh out 0.5 g of benzoic acid and transfer to a 500 ml volumetric flask. Add distilled water, mix well to dissolve and make up to 500 ml with distilled water. This solution is stable for several months at 20-250C (room temperature) 5. Trichloroacetic acid solution 50 g/l: Weigh out 25 g of trichloroacetic acid in a beaker, dissolve, transfer into 500 ml volumetric flask and make up to 500 ml with distilled water. This solution is stable for several months at 20-25 0C. We recommend storing the solution in the refrigerator. 6. Stock urea solution 125 mmol/l: Weigh out 1g of urea –AR in a beaker and keep in the dessicator overnight. Weigh out 750 mg urea from the dessicator and transfer into a 100 ml volumetric flask. Add 50 ml of benzoic acid solution; dissolve and dilute to 100 ml with benzoic acid solution. This reagent is stable for several months at 2-80 C 7. Working urea standards: Prepare working urea standards in 50 ml volumetric flasks according to the table below;

Department of Biochemistry, Medical Research Institute, Colombo-WHO Biennium 2004-2005

56

Working standard No

1

2

3

4

5

6

Stock urea standard (ml)

1

2

3

4

6

8

Benzoic acid solution Concentration of working standard Solution (mmol/l)

Up to 50 ml for each 2.5

5.0

7.5

10

15

20

These standards are stable for several months at 2-80 C PREPARATION OF CALIBRATION GRAPH In this method the formation of the coloured product depends on the composition of the colour reagent and the period of heating at 100 0C. Small variations may occur from day to day and it is therefore essential to check the calibration each time that patients’ samples are analysed. When you are familiar with the shape of the calibration graph on your spectrometer you may find that you can omit some of the standards, e.g. prepare your daily calibration using for example the 5, 10 and 20 mmol/l standards, or use the 10 mmol/l standards should be prepared when the acid reagent and diacetyl monoxime reagent are renewed to check that the reagents are correct. Follow the procedure described under “Procedure”. Plot the absorbance of each tube on the vertical axis against the concentration of the working urea standard solutions in mmol on the horizontal axis.

16.6

PROCEDURE

1. Pipette 0.5 ml of trichloroacetic acid solution using a rubber bulb into centrifuge tubes for each standard, control serum and patient’s sample. Add 50 µl of standard, control serum or patient’s sample to the appropriate tube, mix and leave at room temperature for 5 minutes, then centrifuge to obtain a clear supernatant. 2. Label another set of tubes (18 x 160mm) and pipette 3.0 ml of colour reagent into test tubes for each standard, blank, control serum and sample. 3. Add 100 µl of trichloroacetic acid solution to the blank tube, and 100 µl of supernatant form the standard, control or samples to the appropriate tube. 4. Mix well and heat at 100 0C for 15 minutes exactly. 5. Cool the tubes to room temperature in a bowl of water (about 5 min) mix and read the absorbance at 530 nm (green filter, Ilford No 604) first read the absorbance of the blank against distilled water and note down the reading, then set to zero with the blank and read the standards and unknowns. The absorbance measurements should be made as soon as possible and not more than 30 minutes after the end of step 4.

16.7

CALCULATION

Read the results from the calibration curve or use the following formula if your calibration graph is linear. Concentration of urea in mmol/l = T x C S Where: T = Absorbance reading of patient’s test S = Absorbance reading working standard C = Concentration of working standard (10 mmol/l) If the result is greater than 20mmol/l, dilute 50 µl of supernatant from that sample with 100 µl of trichloroacetic acid solution. Mix well. Repeat the analysis using 3 ml of colour reagent and 100 µl of the diluted supernatant. Remember to multiply the result by 3 to obtain the urea concentration in the patient’s sample. Department of Biochemistry, Medical Research Institute, Colombo-WHO Biennium 2004-2005

57

QUALITY CONTROL At least two serum control specimens, having stated values in the range 3-20 mmol/l, one of which is unknown to the operator, should be included with each batch of specimens. If single specimens are analysed, a control specimen should always be included. OPTIMAL CONDITIONS VARIANCE: A coefficient of variation of around 3% should be attainable. ROUTINE CONDITIONS VARIANCE: The value obtained for the RCV should not exceed 6% REFERENCE VALUES Approximate reference intervals for “healthy” ambulant adults: 2.5-6.6 mmol/l Conversion of SI units into “old” units: mmol/l x 6 = mg/100 ml Blood urea reference values are method dependable. Blood urea estimation by urease method usually gives higher values NOTE: The method recommended uses the reagents described in the above references. However a protein precipitant has been included because the direct method (No protein precipitation) gave results on patients’ samples which were significantly higher than other methods.

16.8

LIMITATION

Cadmium sulphate has been included as recommended by Wybenga et al. In the absence of cadmium ions, the absorbance decreased by about 7 % in 30 minutes; when the colour reagent is prepared as described, then the absorbance decreased by about 4% in 30 minutes. Provided that the batch size is kept small, so that the absorbance readings can be made over a period of a few minutes, the presence or absence of cadmium ions has little, if any effect on the results. REFERENCES WHO Manual LAB/86.3

Department of Biochemistry, Medical Research Institute, Colombo-WHO Biennium 2004-2005

58

17.

URIC ACID PHOSPHOTUNGSTATE REDUCTION –CARAWAY METHOD

17.1

INTRODUCTION

Uric acid is the end product of purine metabolism (adenine,guanine) in the human. On a normal diet some 5-6 mmol of urate is produced daily.Of this amount about 3-4 mmol is produced from purines synthesized in the body (de novo synthesis), whilst the remaining 1-2 mmol are contributed by dietary purine.Urate, the end product of purine base degradation, is formed from xanthine by the enzyme xanthine oxidase. Around 25-30% of the 5-6mmol of urate produced daily is eliminated via the gastrointestinal tract, where it is degraded by bacterial uricases. The remainder is excreted by the kidney. In renal failure the intestinal excretion can be markedly increased. Some 98% of the filtered urate is reabsorbed in the proximal tubule. The 2% that escapes reabsorption contributes around 20% of the total excreted, proximal tubular secretion accounting for the remainder. Minor reabsorption also occurs in the distal nephron Renal clearance is influenced by various drugs and metabolic products.

17.2

CLINICAL SIGNIFICANCE

Hyperuricemia is most commonly defined by serum or plasma uric acid concentrations greater than 7.0 mg/dL (0.42 mmol/L) in men or greater than 6.0 mg/dl (0.36 mmol/l) in women (if specific methods are used to measure the uric acid). Asymptomatic hyperuricemia is frequently detected through biochemical screening; Long term follow up of asymptomatic hyperuricemic patients is undertaken because many are at risk for renal disease that may develop as a result of hyperuricemia or hyperuricosuria; few of these patients ever develop the clinical syndrome of gout. Gout occurs when monosodium urate precipitates from supersaturated body fluids;the deposits of urate are responsible for the clinical signs and symptoms.Gouty arthritis may be associated with urate crystals in joint fluid as well as with deposits of crystals(tophi) in tissues surrounding the joint. The deposits may occur in soft tissues as well , and wherever they occur they elicit an intense inflammatory response consisting of polymorphonuclear leukocytes and macrophages.Renal disease associated with hyperuricemia may take one or more of several forms: (1)gouty nephropathy with urate deposition in renal parenchyma,(2)acute intratubular deposition of urate crystals, and (3) urate nephrolithiasis Hyperuricemia is also attributable to primary defects of enzymes in the pathways of purine metabolism.

17.3

PRINCIPLE OF THE METHOD

Serum proteins are precipitated with acid tungstate and a clear supernatant is obtained after centrifugation. A portion of the supernatant is added to alkaline phosphotungstate. Phosphotungstate reagent oxidizes the urate to allantoin and itself reduced to tungsten blue which is measured by its absorbance at 700 nm

17.4

SPECIMEN TYPE, COLLECTION AND STORAGE

4ml of clotted blood, collected into clean dry bottle, avoid haemolysis. Separate the serum from cells as early as possible. Uric acid in serum is stable for 48-72 hours at room temperature (250C), for 3-7 days at 4-6 0C and for 6-7 months when frozen. As a referral sample send about 1 ml of clear serum, kept cool, to reach the correct destination within 24 hours

17.5

APPARATUS AND CHEMICALS

APPARATUS: Water bath at 25 0C or Basin with water temperature maintained at 250C Automatic micro pipette 500 µl Quick fit complete assembly (round bottom flask and condenser) for refluxing Department of Biochemistry, Medical Research Institute, Colombo-WHO Biennium 2004-2005

59

GLASSWARE: Conical centrifuge tubes (15ml) Test tubes 16 x 150 mm Volumetric flask 100 ml, 200 ml and 1 L Graduated pipettes 1 ml, 5 ml and 10 ml CHEMICALS: Molybdate free sodium tungstate dihydrate-AR Orthophosphoric acid-AR Sodium carbonate-AR Sulphuric acid-AR Uric acid –AR Formaldehyde 40% solution REAGENTS 1. Stock Phosphotungstic acid reagent: Weigh out 50 g of molybdate free sodium tungstate dihydrate or 44.5 g anhydrous salt and dissolve in about 400 ml of distilled water in refluxing flask. Add 40 ml of Orthophosphoric acid (concentrated). Mix well and reflux gently for 2 hours. Allow the solution to cool. Then transfer with rinsing to a 500 ml volumetric flask. Make up to 500 ml with distilled water. Mix well. Transfer this solution into a clean dry brown bottle. This solution is stable in the refrigerator for about one year. 2. Working Phosphotungstic acid reagent: Dilute 10 ml of stock phosphotungstic acid reagent to 100 ml with distilled water. Transfer in to a clean, dry brown bottle. Stable in the Refrigerator for two weeks. 3. Sodium carbonate 10g/dl: Weigh 10 g of Sodium carbonate anhydrous, dissolve in distilled water and make up to 100 ml with distilled water. Store in a poly propylene bottle. 4. Sodium tungstate 10g/dl: Weigh out 10 g Sodium tungstate dihydrate, transfer into a 100ml volumetric flask, dissolve and make up to 100 ml with distilled water. Store in a brown bottle. 5. Sulphuric acid (2/3 N) 0.34mol/l: Add slowly 3.7 ml of sulphuric acid concentrated in to 150 ml distilled water in a beaker. Cool and stir well. Transfer in to a 200 ml volumetric flask and make up to 200 ml distilled water. Transfer in to a brown bottle. 6. Acid tungstate reagent: To 80 ml of distilled water while mixing add 5 ml of sodium tungstate solution, 0.05 ml of phosphoric acid (concentrated), 5 ml of 2/3 N sulphuric acid solution. Transfer in to a brown bottle. Keep at room temperature. 7. Stock Uric acid standard solution 1.5mmol/l: Keep about 300 mg of uric acid analytical grade chemical in a desiccator overnight. Weigh out 252.2 mg of uric acid and transfer in to a one litre volumetric flask. Weigh out 150 mg of Lithium carbonate; dissolve in about 50 ml of distilled water. Filter, heat the filtrate to 60 0C. Add this warm solution to the volumetric flask containing uric acid. Mix until the uric acid completely dissolved. Allow the flask to cool. Then add 20 ml formaldehyde 40 % solution. Add 500 ml of distilled water .Then add slowly 25 ml of sulphuric acid solution. (Prepared by adding 1 ml of sulphuric acid (concentrated) to 35 ml of water) Make up to one litre with distilled water. Mix and store in a brown bottle. This solution is stable for about one year at 4- 6 0C. 8. Working uric acid standard: Allow the stock uric acid standard solution to attain the room temperature. Pipette 2 ml of stock standard solution in to 100 ml volumetric flask. Make up to 100 ml with distilled water. This standard is equivalent to 300 µmol/l under the assay conditions employed. Solution is stable for one week at 4-6 0C. 9. Working Uric acid standard series for calibration: Prepare working standard series by quantitative transfer of 1ml, 2ml, 3ml and 4 ml of stock solution to each of four 100 ml volumetric flasks. Dilute to 100 ml with distilled water. Mix well. These standards are equivalent to 150µmol/l, 300µmol/l, 450µmol/l and 600µmol/l under the employed assay condition.

Department of Biochemistry, Medical Research Institute, Colombo-WHO Biennium 2004-2005

60

17.6

PROCEDURE

1. Pipette 4.5 ml of acid tungstate regent in to centrifuge tubes for each quality control sample and patient’s sample. 2. Pipette 0.5 ml of quality control serum or patients sample to the appropriate tube with mixing. 3. Leave at room temperature for 10 minutes and centrifuge for 10 minutes at 3000 rpm to obtain a clear supernatant. 4. Label a set of test tubes for each blank, standard, quality control sample and patient’s samples. 5. Pipette 2.5ml of distilled water to the tube marked blank, 2.5ml of working standard to the tube marked Standard, and 2.5 ml supernatants from the quality control sample or patients’ samples to the appropriate tube. Keep all the tubes in a water bath at 250C. 6. Add 0.5 ml of Sodium carbonate solution to all tubes, mix well and allow standing for 10 min at 25 0C. 7. Then add 0.5 ml of working phosphotungstic acid reagent to all tubes. Mix immediately. Allow to stand at 250C for 30 min. 8. Measure the absorbance at 700 nm, setting the spectrophotometer to zero with blank solution. PREPARATION OF CALIBRATION GRAPH Run the uric acid working standards series together with the regent blank exactly in the same manner as describe under technique. Plot the absorbance values of standards against the concentration of standards. The points should be linear and the graph should pass through the origin.

17.7

CALCULATION

Calculate the uric acid concentration in the patient’s specimens from the absorbance of the standard as follows Concentration of Uric acid in µmol/l= T/S x C=T/S x 30 x 10=T/S x 300 µmol/l Where T= Absorbance of patient’s test S=Absorbance of working standard C=Concentration of working standard (30µmol/l) Serum dilution 1:10

QUALITY CONTROL

Include Quality control sera for each batch of test REFERENCE VALUES

17.8

: For an adult 120 – 360 µmol/l

LIMITATION

Haemolysed sera interfere with the measurement of uric acid. Supernatants should be clear as turbidity may interfere with colour development

Department of Biochemistry, Medical Research Institute, Colombo-WHO Biennium 2004-2005

61

18.

URINE URIC ACID Method similar to used for serum uric acid can be applied to urine uric acid estimation

18.1

SPECIMEN TYPE, COLLECTION AND STORAGE

Collect 24 hour urine into a clean, dry, sterile 2.5 L bottle and keep in the refrigerator during the collection.

18.2

PROCEDURE

1. Mix the urine collection well. Measure the total volume. Warm an aliquot of urine for a few minutes to 600C to dissolve any urates in the deposit. Add 1ml of urine in to a 100ml volumetric flask. Make up to 100ml with distilled water. Mix well. (Dilution 1 in 100) 2. Pipette 2.5 ml of distilled water to the tube marked B for blank , 2.5 ml of working standard to the tube marked S as standard and pipette 2.5 ml of diluted urine in to the tube marked T for test. Keep all the tubes in a water bath at 250C 3. Carry out the same procedure as for serum uric acid estimation from step 6.

18.3

CALCULATION

Calculate urine uric acid concentration in the sample using the absorbance of the standard as follows. Urine uric acid concentration µmol/l = T/S x C Where T = Absorbance of patients test S = Absorbance of working standard C = Concentration of working standard Urine dilution 1:100 Urine Uric acid µmol/l Urine Uric acid (mmol/l) 24 hour urine uric acid

REFERENCE VALUES

= T/S x 30 x 100 (dilution factor=100) = T/S x 30 x 100 1000 =T/S x 30 x100 x 24 hour urine volume in litre (mmol/24 hour) 1000 : 1.48 - 4.43 mmol/24hours

Department of Biochemistry, Medical Research Institute, Colombo-WHO Biennium 2004-2005

62

19.

ELECTROLYTES DETERMINATION OF SERUM SODIUM AND POTASSIUM BY FLAME PHOTOMETRY [FLAME EMISSION SPECTROSCOPY]

19.1

INTRODUCTION

SODIUM In a normal adult the total body sodium is about 55mmol/kg of body weight; about 30% is tightly bound in the crystalline structure of bone and thus is nonexchangeable. Thus only 40 mEq/kg is exchangeable among the various compartments and accessible to measurements. The exchangeable sodium is distributed primarily in the extra cellular space. About 97% to 98% of the exchangeable sodium is found in the extra cellular water space and only 2% to 3% in the intracellular water space. Approximately 16% of exchangeable sodium is in plasma, 41% is in interstitial fluid (ISF) that is readily accessible to the plasma compartment, 17% is in ISF of dense connective tissue and cartilage, 20% is in ISF of bone and 3% to 4% in the transcellular water compartment. Total bone sodium (exchangeable and nonexchangeable) accounts for the 40% to 45% of the total body sodium. The amount of sodium in the body is a reflection of the balance between sodium intake and output. Sodium intake depends on the quantity and type of food intake. Under normal conditions, the average adults takes in about 50 – 200 mmol of sodium/ day. Sodium output occurs through three primary routes; the gastrointestinal tract, the skin and the urine. Under normal circumstances loss of sodium through the GIT is very small. Faecal water excretion is only 100- 200 ml/day for a normal adult and faecal sodium excretion only 1 to 2 mmol/ day. However, one should bear in mind that although losses of water and electrolytes are normally small, the total volume of GIT fluid secreted is large, averaging about 8 L/day. Almost all this volume is normally reabsorbed. However, with impaired GIT reabsorption, loss of water and electrolytes are large. Thus with severe diarrhoea or with gastric or intestinal drainage tubes, sodium loss via the GIT may exceed 100mmol/ day. The sodium content of sweat averages about 50 mmol/ L but is somewhat variable. The sweat sodium concentration is decreased by aldosterone and increased in cystic fibrosis. The rate of sweat production is highly variable, increasing in hot environments, during exercise, and with fever. Under extreme conditions sweat production can exceed 5 L/ day, accounting for a loss of more than 250 mmol of sodium .Under normal conditions, in a cool environment; sodium losses from the skin are small. With extensive burns or exudative skin lesions there is great loss of sodium and water. Major route of sodium excretion is through the kidney. Furthermore, urinary excretion of sodium is carefully regulated to maintain body sodium homeostasis, which in turn is critical to control of extra cellular volume. Sodium is freely filtered by the glomerulus. Approximately 70% of the filtered sodium is reabsorbed by the proximal tubule, about 15% by the loop of henle, 5% by the distal tubule, 5% by the cortical collecting tubule, and another 5% by the medullary collecting duct; thus normally less than 1% of the filtered sodium is excreted. POTASSIUM Approximately 98% of the total body potassium is found in the intracellular water space (ICW),reaching a concentration there of about 150 to 160 mmol/ l .In the extra cellular water space ,the concentration of potassium is only 3.5 to 5 mmol/l . Total body potassium in an adult male is about 50 mmol/kg of body weight and is influenced by age, sex, and very importantly muscle mass, since most of the body’s potassium is contained in muscleThe amount of potassium in the body is a reflection of the balance between potassium intake and output. Potassium intake depends on the quantity and type of food intake. Under normal conditions the average adult takes in about 50 to 100 mmol/ day, about the same amount as sodium. Potassium output occurs through three primary routes; the gastrointestinal tract, the skin and the urine. Department of Biochemistry, Medical Research Institute, Colombo-WHO Biennium 2004-2005

63

Under normal circumstances loss of potassium through the GIT is very small, amounting to less than 5mmol/day for an adult. The concentration of potassium in the sweat is less than that of sodium, and so potassium losses via the skin are usually small. The major means of potassium excretion is by the kidney. The kidney is capable of regulating the excretion of potassium to maintain body potassium homeostasis.

19.2

CLINICAL SIGNIFICANCE

SODIUM Hyponatremia occurs when there is a greater excess of extra cellular water than of sodium or a greater deficit of sodium than water. The symptoms of hyponatremia depend on the cause, magnitude, and rate of fall in serum sodium. With acute, pronounced hyponatremia caused by water intoxication, nausea, vomiting, seizures, and coma may occur. Symptoms are less fulminant with chronic hyponatremia caused by salt depletion. With progressively severe degrees of chronic hyponatremia, constant thirst, muscle cramps, nausea, vomiting, weakness, lethargy, and finally delirium and impaired consciousness may occur. Hypernatremia occurs when there is greater deficit of extra cellular water than of sodium. Greater excess of sodium than of water rarely occurs. Hypernatremia usually occurs as a chronic process secondary to loss of water in excess of sodium. Symptoms are therefore those of dehydration. POTASSIUM POTASSIUM EXCESS Potassium accumulates in the body when the intake of potassium exceeds output because of some abnormality of the potassium homeostatic mechanism. It should be noted that under most conditions the normal kidney is capable of excreting a great deal of potassium, and a high potassium intake leads to potassium retention only when kidney function is compromised. POTASSIUM DEPLETION This occurs when potassium output exceeds intake. Only small amount of potassium is loss in the faeces under normal conditions. GIT loss of potassium during diarrhoea and drainage of GIT secretions can be large. Alkalosis results in the total body potassium depletion. With alkalosis, potassium moves from the extra cellular compartment to the intra cellular space. In the cells of the distal nephrone of the kidney, this increase in intra cellular potassium stimulates potassium secretion and therefore increases renal excretion of potassium.

19.3

PRINCIPLE OF THE METHOD

Using compressed air diluted serum is sprayed as a fine mist of droplets in to a non luminous flame. In the flame the elements in the compound are converted in to the atomic state. As the temperature rises due to the thermal energy of the flame, a small portion of these atoms excited and the electrons moves to higher energy level. When atoms excited they emit light in the form of a fixed wavelength to produce a spectrum. Light emitted from the thermally excited ions is directed to photo sensitive detector system. The amount of light emitted depends on the concentration of metallic ions present. The response compared with those obtained from standards.

19.4

SPECIMEN TYPE, COLLECTION AND STORAGE

Haemolysis free serum is suitable. Blood collected into clean, dry bottle or commercially available evacuated tubes or capillary blood collected in either micro tube or capillary tubes. Blood should not be collected from the arm receiving an electrolyte or intravenous infusion. Avoid muscle activity (clenching the fist) when collecting the blood sample as this can artificially increase the potassium values. Blood specimens should not chilled before separation of serum, false increase in potassium level occurs as a result of K+ leakage from erythrocytes and other cells.

Department of Biochemistry, Medical Research Institute, Colombo-WHO Biennium 2004-2005

64

Serum should be separated from cells immediately within 1 hour of collection at room temperature. Blood samples should not be centrifuged for longer periods. Grossly lipaemic serum samples are unsuitable for electrolyte estimation. Serum samples should be stored at 20C to 40C or frozen for delayed analysis.

19.5

APPARATUS AND CHEMICALS

APPARATUS: Analytical balance Flame photometer Automatic micro pipette 100µl GLASSWARE: Conical flasks 50 ml Graduated pipette 25 ml

Disposable sample cups (plastic) CHEMICALS: Sodium chloride (Analytical grade) Potassium chloride (Analytical grade) Corning diluent concentrated Deproteinising solution (Corning) LP gas

REQUIREMENTS (ENVIRONMENTAL CONDITIONS) TEMPERATURE : Operating 100C to 35 0C HUMIDITY : Operating 85% maximum at 350 C FUEL : High grade propane should be free of heavy hydrocarbon deposits and regulated at the cylinder to approximately 2.1 kg/cm2 (30 psig) AIR : A supply of clean air at a minimum pressure of 1 kg/cm2 (14 psig) at 6 litres /minute, as supplied by a Corning 850 Air Compressor. Maximum inlet pressure 2.1 kg/cm2 (30 psig).If condensation problems arises a ‘Corning 856 Air Compressor’ should be used, which has a water separator fitted. VOLTAGE : 90V to 127V or 198V to 264 V, 50/60Hz POWER : 20VA, 410 only REAGENTS All glassware used to prepare standard solutions should be chemically cleaned and finally rinsed with distilled water. Weigh out separately into two watch glasses or into two Petri dishes about 15 g of analytical grade Sodium chloride and about 1 g of Potassium chloride. Dry for 6 hours at 100 0C in an oven and allowed to cool to room temperature in a desiccator. 1. Stock Sodium solution 1000 mmol/l: To prepare 200ml, weigh accurately 11.7 g of dried Sodium chloride in a weighing bottle or in a beaker. Transfer it in to an ‘A grade’ 200 ml volumetric flask using a funnel. Wash in any chemical remaining in the weighing bottle or in the beaker in to the flask with a little amount of distilled water (glass distilled water) or deionised water. Dissolve in about 150 ml of distilled water and make up to 200 ml with distilled water. Use a pasture pipette or a wash bottle to add the final volume of distilled water to the flask. Mix the solution well by inverting the flask for several times. 2. Stock Potassium solution 100 mmol/l: Weigh accurately 0.746 g of dried Potassium chloride in a weighing bottle or beaker. Transfer it in to an ‘A grade’ 100 ml volumetric flask using a funnel. Wash any remaining in the weighing container in to the flask with distilled water. Dissolve in about 80 ml of distilled water. Make up to 100 ml with distilled water. Use a Pasture pipette or a wash bottle to add the final volume of distilled water to the flask. Mix the solution well by inverting the flask for several times. 3. Flame photometry deproteinising solution: The pack of deproteinising solution contains deproteinising base solutions and Sachet of catalyst. For use add the catalyst into base solution and mix thoroughly. This solution is stable for 4 weeks at 2-80 C. 4. Working Standard solution (Sodium-140 mmol/l, Potassium-5.0 mmol/l): Into a clean 500 ml ‘A grade’ volumetric flask, add 70 ml of stock solution-1(Stock sodium solution) and 25 ml of stock solution-2 (Stock potassium solution) Make up to 500 ml with good quality distilled water or deionised water. Use a pasture pipette or a wash bottle to add the final volume of distilled water to the flask. Mix the solution well by inverting the flask for several times. Rinse a Department of Biochemistry, Medical Research Institute, Colombo-WHO Biennium 2004-2005

65

polypropylene bottle with little of the prepared solution. Then transfer the standard solution into the bottle. Label and keep at room temperature. (Contamination with sodium may occur if a glass bottle is used) Stopper the bottle tightly to avoid evaporation. 5. Working diluent concentrate: Diluent concentrate recommended by the manufacturer should be used. For ‘Corning 410 C’ Corning diluent concentrate is used. Pipette 0.5 ml diluents concentrate in to clean 500 ml volumetric flask. Dilute up to 500 ml with good quality distilled water or deionised water. Store the working diluent concentrate in a polypropylene bottle. This solution is stable for 5 days at room temperature.

19.6

PROCEDURE

The details of the operation vary from one instrument to another. Following steps are related to ‘Corning 410’ clinical model flame photometer. 1. Sample dilution: Dilute each serum, quality control sample and working standard solution 1:200 with working diluent concentrate. Into 50 ml conical flasks pipette 19.9 ml of working diluent concentrate and 0.1 ml of working standard solution or quality control sample or patient’s serum and mix well. 2. Turn on the fuel supply at source 3. Depress the ‘power’ switch to switch on the instrument 410. The ‘power on’ LED will be illuminated, the air compressor will start an ignition cycle will commence. 4. If the flame on LED is not illuminated at the end of the ignition cycle, (Refer the operator’s manual available with the instrument) check that the air pressure gauge indicates a reading between 11 and 13 psig, if it does not, lower the air regulator locking ring and adjust the regulator for a reading of 12 psig on the air pressure gauge. Raise the locking ring to lock the air regulator adjuster. 5. Set the filter selector to the required position. Non luminous blue flame with distinct cones can be seen, if does not; adjust the fuel to get distinct blue cone flame. 6. Insert the Nebulizer inlet tube in a beaker containing approximately 100 ml of diluent and allow 15 minutes for the operating temperature to stabilize. This will ensure a stable burner temperature when solutions are aspirated, after the warm up period. 7. While aspirating the diluent, adjust the ‘blank’ control so that the display reads zero 8. Aspirate a pre diluted standard solution 9. Allow 20 seconds for a stable reading and then adjust ‘coarse’ and ‘fine’ controls for a convenient reading (if a 140 mmol/l Sodium standard is aspirated, set the display to 140) 10. Carefully adjust the ‘fuel’ control for a maximum reading on the display, ensuring that only small adjustments are made, with a pause of several seconds between adjustments. 11. Remove the standard solution, wait 10 seconds, then aspirate a blank solution of diluent for 20 seconds. Adjust the ‘blank’ control for a zero reading. Remove the blank solution and wait 10 seconds. 12. Repeat paragraphs 8, 9 and 11 until the blank reading is zero (within ± 0.2) and the calibration reading is within ± 1%. 13. Aspirate each of the unknown solutions for 20 seconds, then note the reading in mmol/l 14. Check the calibration frequently 15. When analyzing large batches of samples, recheck instrument calibration every 10 samples with a single standard solution. SHUT DOWN PROCEDURE 1. Aspirate deproteinising solution diluted 1 in 100 with deionised water for one minute 2. Aspirate diluent for 2 minutes 3. Turn off the fuel supply at source when the “flame on” LED is extinguished; switch off the “power” switch. NOTE:  Always use same batch of diluent for the blank, dilution of samples and standard, alternatively the corning dilutor can be used.

Department of Biochemistry, Medical Research Institute, Colombo-WHO Biennium 2004-2005



66

In any difficulty of obtaining a maximum sodium reading proceed as follows: Open the inspection flap and adjust the ‘fuel’ control until the flame just starts to lift off the burner. Then turn the ‘fuel’ control back, counter clockwise, until the cones of the flame are on the burner. Close the flap and proceed with paragraph 11.

PRECAUTIONS A diluent recommended by the manufacturer of instrument should be used. Deionised or high quality distilled water should be used to prepare the diluent. Deionised or distilled water must be free from contaminative elements (bacteria or moulds can cause inaccuracies by interrupting or blocking the flow of sample through the Nebulizer always use the same batch of diluent for the blank and the dilution of samples and standards.  Anticoagulants containing sodium or potassium salts must not be used. Use serum for measurement of sodium and potassium  Dilute the sera with care. Good quality calibrated pipette or a sensitive diluter must be used. Use the same pipette or dilution equipment for both standard and sample  Accuracy of the results depends on the accuracy and purity of the calibration standard. Always use accurately prepared standards.  Both the accuracy and precision of results depends on maintenance and adherence to operating instructions provided by the manufacturer. Careful cleaning of the atomizer-burner, cleanliness of sample containers, the aspirating systems, proper adjustment of flame size, (blue flame with distinct cone) aspiration rate, and geometry of the flame and uniform entry of atomized, diluted sample into the flame are also critical for accuracy and precision. Thermal equilibrium must be established before analysis of unknown samples. Warm up period is necessary because the initial evaporation of water in the flame decreases the temperature of the burner and the entire burner chamber.  Safety: Propane is highly inflammable and potentially explosive and commonly supplied as a liquid under pressure in a cylinder for use with the 410. Cylinder should never be subjected to heat or mechanical shock. Leakage of propane from tank, instrument fittings or from valves may be detected with the aid of soap solutions. 

Site conditions:  Never install the flame photometer beneath overhanging cupboards. There must be at least 1 metre of clear space above the 410 chimney  The environment must be clean and free from dust  The instrument must be placed on a strong, level work stop, free from vibration  Avoid the instrument to direct sunlight or draughts

QUALITY CONTROL At least two serum control specimens, having stated values in the range 120-150 mmol/l for sodium, 3.0-6.0 mmol/l for potassium, and one of which is unknown to the operator should be included with each batch of specimens. If single specimens are analysed a control specimen should always be included OPTIMAL CONDITIONS VARIANCE: A coefficient of variation of around 1% for sodium and 1.5% potassium should be attainable ROUTINE CONDITIONS VARIANCE: The value should not exceed 2% for sodium and 3% for potassium REFERENCE VALUES Serum Sodium : 136mmol/l - 146mmol/l Serum Potassium: 3.5mmol/l - 5.6mmol/l

Department of Biochemistry, Medical Research Institute, Colombo-WHO Biennium 2004-2005

67

19.7

LIMITATION

Haemolysed sera interfere with the measurement of Electrolytes which causes liberation of potassium from the red blood cells. REFERENCES Operator’s Manual Corning 410 LAB/86.3

20. 20.1

URINE SODIUM AND POTASSIUM

PRINCIPLE OF THE METHOD

Same as for serum sodium and potassium

20.2

SPECIMEN TYPE, COLLECTION AND STORAGE

Collect 24 hours urine (timed collection) into a dry, sterile, 2.5 litres brown bottle without addition of any preservatives 20.1

   

PROCEDURE Mix the 24 hour urine collection and measure the total volume using a clean measuring cylinder Perform the test as for serum electrolytes If the sodium level is low, dilute the neat sample 1:50 or 1:100 and divide the result by 4 or 2 respectively; dilution is depend on the level of the sodium If the potassium level is higher than 10 mmol/l then dilute the urine 1:500 or 1:1000 and multiply the result by 2.5 or 5 respectively. Dilution depends on the level of potassium

20.3

CALCULATION

Sodium (mmol/24 hour) Potassium (mmol/24 hour)

= mmol/l x 24 hour urine volume in litre = mmol/l x 24 hour urine volume in litre

REFERENCE VALUES Urine Sodium : 40-220 mmol/24 hour Urine Potassium : 25-150 mmol/24 hour REFERENCES Operator’s Manual Corning 410 LAB/86.3

Department of Biochemistry, Medical Research Institute, Colombo-WHO Biennium 2004-2005

68

21.

APPENDIX 1 - SAMPLE COLLECTION AND TRANSPORTATION SPECIMEN COLLECTION Collection of Blood Blood for analysis may be obtained from veins, arteries or capillaries. Venous blood is the specimen of choice and venipuncture is the method of for obtaining this specimen. Prior to the drawing of blood the phlebotomist should ensure the identity of the patient. The appropriate containers and required volumes should be estimated. All the containers should be labelled prior to the collection of blood. VENIPUNCTURE The phlebotomist should verify that the patient is fasting, if fasting be necessary to ensure medically useful results. The patient should be comfortably seated or supine (if sitting is not feasible.) and should be in that position for 20 minutes. The patients arm should be in a straight line from the shoulder to the wrist. An with an inserted intravenous line should be avoided,as should an arm with extensive scarring or haematoma at the intended collection site .If a woman has had a mastectomy arm veins on that side of the body should not be used since the surgery may have caused lymphostasis affecting the blood composition. The median cubital vein in the antecubital fossa is the preferred site for collecting venous blood in the adults. The area should be cleaned with alcohol swab.(Use benzylkonium chloride as the cleaning during determinations of plasma alcohol.)When the skin is cleaned a tourniquet is applied 10 tp 15 cms to obstruct the return of venous blood to the heart and to distend the veins.(A tourniquet should be 2.5 cms wide and 38-40 cms in length. The appropriate needle must also be selected. The larger the gauge size the smaller the bore. The usual choice for an adult with normal veins is gauge 21 and they should be sharp, sterile and without barbs. If blood is drawn for trace metals the needle should be stainless steel and free from contaminants.  In children butterfly needle with attached tubing is preferred. Appropriate volumes should be carefully estimated and the analytical methods at the laboratory should be able to accommodate small volumes of specimens.  Application of a tourniquet is not recommended when blood is drawn for serum proteins, calcium and lipid profile. When the flow of blood is obstructed by the tourniquet, the filtration pressure across the capillary walls is increased which causes fluid and low molecular weight compounds to pass through the capillary wall causing a relative hemoconcentration. The first drawn sample is the most representative of the composition of circulating blood. Therefore the first drawn blood specimen should be used for those tests pertinent to critical medical decisions.  Vigorous suction on a syringe during collection or forceful transfer from the syringe to the receiving vessel may cause haemolysis of blood.  Evacuated tubes are preferred to syringes because they are easy to use and less likelihood of contamination of their outside with blood. There are two main classes of blood tubes, those containing a serum separating material and without. Tubes with siliconised stoppers are preferred as there is less interference by silicone with test procedures. Tubes containing glycerin should not be used for lipid measurements. SKIN PUNCTURE If only a small volume of blood is required for a test skin puncture is performed and capillary blood is obtained. In an adult or grown child, blood may be obtained by puncturing the tip of a finger. When the skin is dry after cleaning with alcohol the tip of the finger is punctured by a sharp stab with a lancet.

Department of Biochemistry, Medical Research Institute, Colombo-WHO Biennium 2004-2005

   

69

To improve circulation of the blood, the finger may be warmed by the application of warm, wet wash cloth for 3 minutes prior to pricking. The first drop is wiped off and subsequent drops are transferred to the appropriate tubes. The blood may be collected into the capillary tubes by capillary attraction. In an infant the lateral and medial planter surface of the foot should be used for skin puncture. Blood collection on ambulatory patients from anywhere on the foot is avoided. For collection of blood specimens on filter paper for neonatal screening the skin is cleaned and punctured as described earlier. Then the filter paper is gently touched against a large drop of blood which is allowed to soak into the paper to fill the marked cycle. The filter paper should be air dried.

ARTERIAL PUNCTURE Arterial punctures require considerable skill and are usually performed by specially trained medical officers. Femoral artery is the preferred site. The needle and syringe should be flushed out with a heparin solution to ensure anticoagulation and to expel trapped air. If the collected specimen is intended for blood gas analysis the nozzle of the syringe containing the blood should be sealed and the syringe placed in ice water (melting ice) for immediate transport to the laboratory. Analysis should be performed within 15 minutes. Anticoagulants and Preservatives for blood  Heparin is the most widely used anticoagulant for clinical chemical analysis. It is available as sodium, potassium, lithium and ammonium salts. It acts as an antithrombin to prevent the transformation of prothrombin into thrombin and thus the formation of fibrin from fibrinogen.  The chelating agent ethylenediamenetetraacetic acid is useful for haematological examinations as it preserves the cellular components of blood. EDTA prevents coagulation by binding calcium which is essential for the clotting mechanism.  Sodium fluoride is usually considered as a preservative for blood glucose. It inhibits the action of enzyme enolase in the glycolytic pathway. The sodium fluoride and potassium oxalate should be in the 1: 3 concentrations. Excess of the preservative will cause inhibition of the glucose oxidase reagent resulting in low glucose values.  Oxalates of sodium, potassium, ammonium and lithium inhibit blood coagulation by forming insoluble complexes with calcium ions. During the preparation of tubes/bottles with oxalates the temperature of the drying oven should be carefully controlled to below 1000 C in order to avoid decomposition to carbonate which has no anticoagulant activity. URINE COLLECTION The type of urine sample to be collected is dictated by the tests to be performed.  A clean early morning fasting specimen is generally the most concentrated and thus is preferred for microscopic examination and for the detection of abnormal amounts of constituents such as proteins, amino acids and β chorionic gonadotrophins.  A double voided specimen is the urine excreted during a timed period following the complete emptying of the bladder. It is used for example to assess the glucose excretion during a glucose tolerance test.  Timed urine specimens are used to minimize the influence of short term biological variations. When specimens are to be collected over a specified period of time the patient’s close adherence to instructions is important. The bladder should be emptied at the time the collection is to begin, and this urine is discarded. Thereafter all urine should be collected until the end of the scheduled time. The urine should be collected in to a chemically clean container and should be transferred to the bottle using a funnel. Any excess urine should be collected into a separate refrigerated container. At the end of the collection period the last urine sample should be added into the bottle and dispatched to the laboratory with out delay. Other relevant information (weight Department of Biochemistry, Medical Research Institute, Colombo-WHO Biennium 2004-2005

70

and height of the patient) and a blood sample collected during the period of urine collection should be provided. Acid washed beaker and a funnel should be provided to the patient in urinary calcium estimations. Urine preservatives One of the most satisfactory forms of preservation of urine specimens is refrigeration; it is even more successful when combined with chemical preservation. HANDLING OF SPECIMENS FOR TESTING  Every specimen should be clearly labelled for proper identification. The minimum information on a label should include the patient’s name, location and identifying number, date and time of sample collection.  The specimen must be properly treated during the transport and from the time serum has been separated until it is analysed.  Plasma and serum should be separated from cells as soon as possible and certainly within 2 hours. If the specimens cannot be analysed at once the separated serum should be stored in the refrigerator in capped tubes both to maintain stability and prevent evaporation. If the analytes are unstable in the refrigerator, freeze the serum at -200 C or - 70 0C. At the time of analysis the specimen should be brought to the room temperature.  During the transportation of specimens to the referral laboratory extreme precautions should be made to ensure that the specimen reaches the destination in leak proof containers. The tube used for the holding of the specimen (primary tube) should be so constructed that the contents do not escape if the container is exposed to extremes of heat, cold and sunlight. The secondary container used to hold one or more specimen tubes must be constructed to prevent the tubes from knocking each other. For transportation of refrigerated specimens solid carbon dioxide may be used.  Various laws and regulations apply to the shipment of biological specimens.

Department of Biochemistry, Medical Research Institute, Colombo-WHO Biennium 2004-2005

71

22.

APPENDIX 2 – DIABETES MELLITUS Definition: A group of diseases characterized by elevated blood glucose level (hyperglycaemia) resulting from defects in insulin secretion, in insulin action or both. Classification  Type 1 Diabetes mellitus (Insulin dependent, juvenile D.M.) Immune mediated Autoimmune destruction of β cells of the pancreas Age of onset: childhood and adolescence; any age Antibodies against islet cells Idiopathic Asian /African: permanent low insulin 

Type 2 Diabetes mellitus Maturity onset; non insulin dependent Due to insulin insensitivity hyper secretion of insulin Relative insulin deficiency

characteristics Age of onset Genetic predisposition Antibodies to β cells Body habitus Plasma insulin C -peptide Metabolic feature

Type 1 35years high No Obese High

Insulin deficiency

Insulin insensitivity



Specific types of Diabetes Genetic defects of islet cell function Endocrinopathies Drug induced



Gestational D.M Any degree of clinical glucose intolerance with onset or first recognition during pregnancy.

Symptoms Polyuria Polydypsia Blurring of vision Weight loss Diagnostic Strategy for Diabetes (Refer annexure 1) W.H.O 2002 – pg 17 Corrections – Fasting plasma glucose and random plasma glucose Fasting plasma glucose without symptoms Fasting plasma glucose on 2 occasions Normal fasting plasma glucose : 3.3 - 6.1 mmol/L Impaired fasting glycaemia :> 6.1 7.0 mmol/L FPG = 7.0 mmol/l →repeat →7.0 mmol/L→DM Oral Glucose Tolerance Test Provide information on latent DM Department of Biochemistry, Medical Research Institute, Colombo-WHO Biennium 2004-2005

72

Is more sensitive than FPG Preparation of the patient Thee days carbohydrate rich diet and activity No medication on the day of the test 12 hour fast No smoking Glucose load: adults : 75g in 300-400 ml of water Children : 1.75 g/Kg up to 75g of glucose Plasma glucose sampling : 10 min before glucose load 120 min (2 hours) post glucose Urine glucose corresponding to the samples Evolution; Fasting plasma Glucose 6.1 -6.9 mmol/L (110 – 125mg/dl)

IFG

120min glucose

IGT

< 7.0 mmol/L (< 126mg/dl)

7.8 – 11.1m.mol/L (140- 199mg/dl)

Diabetes

>7.0m.mol/L (>126 mg/dl)

>11.1 mmol/L > 200mg/dl

OGTT is affected by  Metabolic stress( ↑ glucose secretion) Major surgery, M.I, drugs (steroids)  Malabsorption  Vomiting Gestational Diabetes Diagnosis; Fasting plasma glucose >7.0 mmol/L (126mg/dl) Random plasma glucose >11.1 mmol/L (200mg/dl) Lab diagnosis One step approach 75g glucose-OGTT Two step approach  First OGTT with 50g glucose; cut off value after 1 hour plasma glucose > 7.8 mmol/L (140 mg/dl)  Second OGTT with 75g of glucose load and evaluation as the standard OGTT Monitoring of Disease Maintain Plasma glucose level as close as possible to normal levels during the day.  

Use of the glucometer – only for monitoring not for the diagnosis

CALIBRATED glucometer

Department of Biochemistry, Medical Research Institute, Colombo-WHO Biennium 2004-2005

73

23

APPENDIX 3 - REFERENCE RANGES

Reference Ranges Analytes ACE (Angiotensin converting enzyme)

Sample 5 ml clotted blood / Lithium Heparin 1 ml Blood

Reference range Adult

30-100

Children 6m-4y Children 4-9y Children 9-13y Boys 13-18y Girls 13-18y

35-75 42-90 49-105 45-98 35-75

4 - 5 ml of blood into EDTA bottle ( Separated and frozen within 30 min) Avoid glass tubes as they adsorb ACTH Use pre-chilled polystyrene tubes

Cord Newborn Adult 8 h unrestricted activity 16 h supine

50-570 10-185

AFP Maternal

3-5 ml clotted blood

14 wks 15 wks 16 wks 17 wks 18 wks 19 wks 20 wks 21 wks 22 wks

AFP (Alpha Feto protein) Contact Department of Biochemistry (Endocrine) for daily/weekly Paediatric reference ranges

3-5 ml Clotted blood

Adult Newborns (may be higher if premature) Infants at 8 wk Infants at 20 wk Children

ACTH (Adrenocorticotropic Hormone )

Units U/L

Some common indication Sarcoid

ng/L

Pituitary function Adrenal function

µg/l

Neural tube defects Downs syndrome

< 10