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

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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 SRILANKA

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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

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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.

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CONTENTS

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

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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.

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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.

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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.

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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

Adetailed 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

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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.

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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

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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}0_{C, for 3 months at -20}0_C.

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.

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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 0_C.

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 0_C

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 0_{C. (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 0_{C (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) (02)

)

(03) (04)

Albumin standard 40 g/l (ml) 0.5 1.0 1.5 2.0

Sodium chloride/Sodium azide solution (ml) 1.5 1.0 0.5 0.0

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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 : 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

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

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3. AMYLASE

Amylases are a group of hydrolases that split complex carbohydrates constituted of α-D-glucose units linked through carbon atoms 1 and 4 located on adjacent α-D-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.

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 2- 12 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.

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

3-5 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 0_{C for a week (free}

of bacterial contamination).

3.5 APPARATUS AND CHEMICALS APPARATUS:

Hot plate or Bunsen burner Water bath at 37 0_{C, pH meter}

Visible Spectrometer wavelength at 660nm or Colorimeter with red filter Ilford No: 608 (680 nm)

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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

ortho-phosphate (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 0_{C 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 0_{C 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 0_C.

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 0_C.

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 0_{C 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 0_{C 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

Amylase activity U/L = B-T x 1470 B

B = absorbance of reagent blank

T = absorbance of test

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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

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4. ALKALINE PHOSPHATASE

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 non-specific 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.

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 - 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.

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 0_C

pH meter

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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 hexahydrate-

Disodium 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 0_C.

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 -250_{C. MgCl}₂_.6H₂_{O 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 0_{C. We have observed that this solution is stable}

even at room temperature at 20-30 0_C.

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 0_C.

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

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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.

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 0_{C 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 0_{C 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.

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) : 20- 90 U/L

Females (age 15-60 years) : 20-90 U/L

Children (age 1-12 years) : up to 350 U/L

During the growth spurt of puberty : 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

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5. ASPARTATE AMINO TRANSFERASE

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.

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 12}th_days;

activities then gradually decrease, reaching normal levels by the 3rd_{to 5}th_{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

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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.

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

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 0_{C over 1-3 days. Specimen is best stored frozen if they are to be kept}

more than 3-4 days

pH meter

Visible spectrometer wavelength at 505 nm or Colorimeter with green filter Ilford No 604 (520 nm)

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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 0_C.

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 - 250_{C, which can be achieved by air condition system. However we have observed that}

this solution is stable in our room temperature (20-30 0_{C) 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 0_C.

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 0_{C. 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.

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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 0_C.

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.

Amount of pyruvate formed = (A Test-A Sample Blank) x 4x1x1000

(µmol/min/litre) 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 0_C)

Calculated pyruvate (µmol/min/litre)

AST result (U/L at 37 0_C)

AST result (U/L at 37 0_C) 2 4 28 52 4 6 30 56 6 10 32 60 8 12 34 64 10 15 36 69 12 19 38 73 14 23 40 77 16 27 42 81 18 31 44 85 20 35 46 92 22 40 48 98 23 42 50 106 24 44 52 114 26 48 54 125

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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

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18

6. ALANINE AMINO TRANSFERASE

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.

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 0_{C. Minimal loss of}

activity occurs at 0-4 0_{C over 1-3 days. Specimen is best stored frozen if they are to be kept}

more than 3-4 days

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 0_C.

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 0_{C. we have observed this solution is}

even stable at our room temperature 20-30 0_C

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.

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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 0_C.

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 0_{C. We have observed that}

this solution is stable at our room temperature (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 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.

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 water(reagent blank) or 100 µl pyruvate standard (standard) mix and incubate at 37 0_C

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.

Amount of pyruvate formed = (A Test-A Sample Blank) x 4x1x1000

(µmol/min/litre) 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

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Use the table below to convert the amount of pyruvate into U/L (expressed as µmol/minute/litre at 37 0_C)

ALT result (U/L at 37 0_C)

ALT result (U/L at 37 0_C) 2 2 54 42 4 4 56 44 6 4 58 46 8 5 60 47 10 7 62 49 12 7 64 53 14 9 66 55 16 11 68 56 18 13 70 60 20 13 72 62 22 15 74 64 23 15 76 66 24 16 78 67 26 16 80 69 28 18 82 71 30 20 84 73 32 22 86 76 34 24 88 80 36 25 90 84 38 27 92 87 40 29 94 91 42 31 96 95 44 33 98 98 46 35 100 102 48 36 102 109 50 38 52 40

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

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7. BILIRUBIN

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.

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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.

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.

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