Estimation of Blood Glucose

Practicals

Lab. Practical – 1 Blood Glucose Estimation

Lab. Practical – 2 Oral Glucose Tolerance

Lab. Practical – 3 Glycated Hemoglobin Estimation

Lab. Practical – 4 Measurment of Triglycerides

Lab. Practical – 5 Measurement of Total Cholesterol , HDL and LDL

Lab. Practical – 6 Renal Function Tests: BUN and Creatinine estimation

Lab. Practical – 7 Creatinine Clearance Estimation

Lab. Practical – 8 Liver Function Tests –1: Bilirubin ; Total and Direct

Lab. Practical – 9 Liver Function Tests –2: Enzymes; ALT, AST and GGT

Lab. Practical – 10 Albumin and Total Protein Estimation

Lab. Practical – 11 Bone profile Testes

Lab. Practical – 12 Cardiac profile testes

Lab. Practical 13 – 15 Tutorials Introduction To Applied Biochemistry

General Comments about testing

There are so many different methods used to analyze different chemical compounds that to state one method over another is unfair. Another issue is that your body’ chemistry changes throughout the day in response to external conditions such as exercise and internal conditions such as kidney function. This makes comparisons among various tests difficult to do. One method to lessen these variables is to try to have your tests done by the same laboratory so that comparisons of test values are possible. It is also beneficial then to have your tests drawn under the same conditions (fasting/non fasting, early morning/late afternoon, etc.) so that you can eliminate these interferences when you look at your results. Practices of Clinical chemistry

Estimation of Blood Glucose

Introduction:

The importance of testing the blood glucose level comes from the fact that the brain cells are very dependent on the extracellular glucose concentration for their energy supply; hypoglycemia is likely to impair cerebral functions as well as do the hyperglycemia especially of rapid onset, which can cause cerebral dysfunction by affecting extracellular osmolarity.

Objectives:

-To know the different methods for estimation of blood glucose -To know the precautions needed to get accurate results and better interpretation of glycemic status in relation to disease condition.

Methods: Many methods were developed to estimate the glucose level in body fluids among which the commonly used nowadays, the enzymatic methods. These methods can be summarized and categorized into

A) Reduction methods : These methods depend on the reductive property of glucose(aldose)

1-Ferriccyanide( Hoffman’s) method: using ferricyanide which is reduced by the glucose . Fe+++ Fe++ (color change from yellow to colorless solution that will diminish the absorbance measured photometerically )

2- Copper sulfate methods : Benedict: The reagent contains Na-citrate &Na carbonate with CuSO4. It gives color acc. To conc. of glucose (green-----yellow-----brown-----red). Fehling : using KOH &Na/K tartrate with CuSO4 Folin- Wu : Alkaline Cu SO4 +Phosphomolybdic acid molybdenum blue by reducing Cu2O CuO2

3- Smogi-Nelson method : using Arsenomolybdate

N.B. The reduction methods need alkaline medium &heat These methods are qualitative & semi-quantitative.

B) Aromatic amines method : O-toludine +glucose (aldhyde) heat &acidity glucosamine (colored )

C) Enzymatic methods: 1-Hexokinase methods(The reference method). With pre-deproteinization of sample or without. Glucose +ATP +HKADP+G6P G6P +NAD +G6PD 6 P-gluconolactone +NADH+H (measured at 340)

2- Glucose oxidase methods:

-Trinder’s (Enz.-Dye Colorimetric ) method: which is colorimetric either by spectrophotometer or refractrometer (refractrometeric methods either in a film form [kodak Ectachem] or a strip form [Dry chemistry] ). -Kinetic method: by measuring the increase in absorbance through increase in NADH+H

N.B. GOD/POD method can not used for detection of urine glucose because the urine contains interfering substances for peroxidase (POD) . - To use this method treatment of the urine sample either by Somogi Nelson filtrate or Ion Exchange Resin is taken before running . Also, using GOD/POD method in urine with modification like , Polarigraphic determination with post-reaction elimination of H2O2 by: ethanol & catalase or Iodide & molybdate.

3- Glucose Dehydrogenase Method:

Glucose +NAD GDH Gluconolactone +NADH+H (measured at 340)

Glucose Oxidase for blood glucose estimation (Experiment #1) PRINCIPLE OF THE METHOD Glucose oxidase (GOD) catalyses the oxidation of glucose to gluconic acid. The formed hydrogen peroxide (H2O2), is detected by a chromogenic oxygen acceptor, phenol-aminophenazone in the presence of peroxidase (POD): Principle: (Trinder’s method ) -D-glucose Mutarotase -D-glucose Glucose oxidase -D-glucose +H2O+O2 D-gluconic acid+H2O2

Peroxidase H2O2+ 4-aminophenazone+phenol Quinonemine +4 H2O The intensity of the color formed is proportional to the glucose concentration in the sample.

CLINICAL SIGNIFICANCE Glucose is a major source of energy for most cells of the body; insulin facilitates glucose entry into the cells. Diabetes is a disease manifested by hyperglycemia; patients with diabetes demonstrate an inability to produce insulin. Clinical diagnosis should not be made on a single test result; it should integrate clinical and other laboratory data.

PREPARATION Working reagent (WR): Ready for use

Signs of reagent deterioration: 1- Presence of particles and turbidity. 2- Blank absorbance (A) at 505 nm 0.10.

Requirements: Samples: -Blood samples Whole blood Serum Plasma (with Ca.oxalates/NaF), which is the preferred sample -Fresh urine by double void collection technique…….? -CSF collected in sterile clean container and to be done immediately or centrifuged to get cell free fluid.

Instrumentation: -Photometer adjusted on wavelength 540 nm -Cuvette (light path) 1 cm -Water bath at 37 ºC -Automatic pipettes, disposable test tubes , racks and disposable tips for the dispensers.

PROCEDURE 1 1. Assay conditions: Wavelength: ...... 505 nm (490-550) Cuvette: ...... 1 cm light path Temperature...... 37ºC / 15-25ºC 12. Adjust the instrument to zero with distilled water. 23. Pipette into a cuvette:

Blan 1.0 (WR (mL -- Standard ((µL -- Sample ((µL

14. Mix and incubate for 10 min at 37ºC or 15-20 min at room temperature (15- 25ºC).

15. Read the absorbance (A) of the samples and standard, against the Blank. The colour is stable for at least 30 minutes.

CALCULATIONS (A) Sample x 100 (Standard conc.) = mg/dL glucose in the sample (A) Standard

Conversion factor: mg/dL x 0.0555= mmol/L.

*Linearity of the test = 400 mg/dl (Samples give higher level must be retested with dilution by suitable buffer or dist. H2O)

Result: Abs. Of the Standard ~ 0.3 As the concentration of glucose standard = 100 mg/dl The Glucose concentration in the sample = 333 X Abs. Of the Sample

Normal Range: Blood glucose… Fasting= 70 - 110 mg/dl & 2 hrs. Postprandial = 110 - 140 mg/dl Urine glucose .. < detectable limit (Nil) CSF glucose ~ 60 - 90 mg/dl N.B. To express the result in mmol/L divide by 18 ( MW of Glucose =180) Interpretation:

I -Hypoglycemia : The patient considered critically hypoglycemic if:

Whole Blood glucose level < 40mg/dl Serum/Plasma glucose level < 45mg/dl

A- Well Fed State Hypoglycemia :

1- Excessive Insulin Release: a. Reactive Hypoglycemia b. Alimentary Hyperinsulinism c. Leucine Hypersensitivity

2- Inherited Enzyme Defect: a. Galactose -1- Phosphate b. Fructose -1- Phosphate

3- Fed Status Functional Hypoglycemia:

B- Fasting Hypoglycemia:

1-Organic Hypoglycemia: a-Pancreatic B-Cell disease/CA b-Non-Pancreatic Tumors c-Anterior Pituitary Hypo-function d-Adrenocortical Hypo-function e-Ingestion of Akee Fruit

2- Functional Fasting Hypoglycemia

:a- On specific hepatic enzyme deficiency ِِِِِِِِِِِِِِ

1- Genetic Deficiency or Delayed Maturation of Enzymes in Pre- mature Babies

2- Glycogen Storage Disease

b- Induced by Exogenous Agents:

1-Alcohol Intake

2-Excessive Insulin Administration

3-Excessive Sulfonylurea Administration

II - Hyperglycemia :

- Diabetes Mellitus - Hemochromatosis - Hypokalemia - Stress - Pheochromocytoma - Anesthesia - Pregnancy - Hyperthyroidism - Cushing disease - Hyperpituitarism (gigantism)

Discussion:

*Physiological & Biochemical Background: Glucose metabolism, Insulin action and other hormonal effects on glucose in the human body.

*Pathological & Disease Correlation: Diabetes Mellitus, Cushing disease ,Hyperthyroidism …..etc

Questions:

1- What is the basis of reduction methods for glucose estimation ? 2- Give short notes on Trinder’s method for glucose estimation. 3- When does a person considered hypoglycemic? 4- What are the types of hypoglycemia ? 5- Give an account on the principle of glucose oxidase method for glucose estimation.

ORAL GLUCOSE TOLERANCE TEST

Introduction:

On standard oral glucose dose, the response of the body regarding the absorption and metabolism of glucose said to be tolerant on meeting the normal elevation and return. Whereas abnormal and improper glucose metabolism is termed glucose intolerance. This used to diagnose diseases where the glucose metabolism is impaired as in Diabetes mellitus. Oral glucose tolerance test (OGTT) has been widely used as the golden standard for diagnosing diabetes mellitus in clinically doubtful cases. Lately, thought, the use of OGTT in primary care has been questioned for several reasons. It has low reproducibility and is very expensive. However, for the detection of diabetes in pregnant women, it is still recommended.

Objectives:

It is to practice the OGTT and knowing the uses and interpretation regarding the diagnostic benefits of this laboratory test.

Indications: 1- Borderline fasting blood sugar for >2 times (~ 110 – 125mg/dl) 2- Diagnosis of Gestational Diabetes (GDM) at 24 – 28 weeks of gestation especially for those have a family history of diabetes. 3- After delivery for those was suffering from GDM.

OGTT (Experiment # 2):

*Patient preparation (Perquisites) ;

Activity--Don't smoke or exercise strenuously for 8 hours before the test or during the test. Diet--Eat a high-carbohydrate diet (> 150 g/day) for 3 days, then fast for 10 to 12 hours before the test. Don't drink coffee or alcohol for 8 hours before the test. Drugs (medicines)-Inform the person performing the test to omit any medications listed, as under taking these drugs the test results may differ (contraceptives to be stopped one cycle before the performance of OGTT). The test must be performed at daytime (morning).

* General description of test

Test usually takes 3 hours but can last as long as 6 hours (extended OGTT). Drink water frequently during the test (the only allowed fluid to drink). The first blood sample and the first urine sample are collected between 7 A.M. and 9 A.M., after you have fasted for 12 hours. Operator gives a test load of glucose, usually 75 – 100 gram dextrose / 300 ml water, lemon flavored . Drink the entire solution in 5 minutes. Blood and urine samples are collected at 30 min., 60 min., 90 min.,120 min. and 3 hours and sometimes immediately after drinking oral glucose solution.

Dose of Oral Glucose: Dextrose: 1 – 1.75 g/kg. body wt. (for adults0 and not exceeds 100 g. It is to be dissolved in 250 – 300 ml lemon flavored water. Fortical : 113 ml completed to 300 ml water Lucozade: 350 ml. (ready to use)

*Samples: Blood samples ; fasting(basal) sample, 30min. after oral glucose load, 60min, 90min, 120min. (in extended OGTT another 2 samples will be taken at 2½hour and 3 hours). Urine samples ; first fasting urine and the hourly collected urine samples. Calculation: there are different methods to calculate and interpret the glucose levels (mg/dl)in OGTT:

Glucose sample Wilkerson Criteria Fajan-conn criteria Revised Summation Fasting > 130 1 point - 30 min. - - *If Σ of results 60 min. >190  ½ point >190  +1 (F + 60min. + 90 min. 90 min. - > 165  +1 + 120 min.) > 600 120 min. >140  ½ point > 140  +1 mg/dl = Diabetic 2 ½ hour >130  1 point - 2 – 3 point Diabetic 3  Diabetic *If Σ of results < 600 Calculation of ½ - 1 ½ point  1 – 2 Suspect = non diabetic Results Suspect Zero  Non Zero  Non diabetic diabetic

Results and Diagnosis: Glucose tolerance tests may lead to one of the following diagnoses:

Normal Response

A person is said to have a normal response when the 2-hour glucose level is less than or equal to 110 mg/dl, or following this normal levels.

Time Pregnancy Other Adults Child Fasting <100 <110 <130 30, 60 & 90 <200 <200 <200 minutes 120 minutes <145 <140 <140

Impaired Fasting Glucose

When a person has a fasting glucose equal to or greater than 110 and less than 126 mg/dl, they are said to have impaired fasting glucose. This is considered a risk factor for future diabetes, and will likely trigger another

Impaired Glucose Tolerance

A person is said to have impaired glucose tolerance when the 2-hour glucose results from the oral glucose tolerance test are greater than or equal to 140 but less than 200 mg/dl. This is also considered a risk factor for future diabetes. There has recently been discussion about lowering the upper value to 180 mg/dl to diagnose more mild diabetes to allow earlier intervention and hopefully prevention of diabetic complications.

Diabetes

A person has diabetes when oral glucose tolerance tests show that the blood glucose level at 2 hours is equal to or more than 200 mg/dl. This must be confirmed by a second test (any of the three) on another day. There has recently been discussion about lowering the upper value to 180 mg/dl to diagnose more people with mild diabetes to allow earlier intervention and hopefully prevention of diabetic complications.

Gestational Diabetes

A woman has gestational diabetes when she is pregnant and has any two of the following: a fasting plasma glucose of more than 105 mg/dl, a 1-hour glucose level of more than 190 mg/dl, a 2-hour glucose level of more than 165 mg/dl, or a 3-hour glucose level of more than 145 mg/dl.

Discussion:

Drugs may affect OGTT results Amphetamines. Arginine. Benzodiazepines. Beta-adrenergic blockers. Chlorthalidone. Clofibrate. Corticosteroids. Dextrothyroxine. Diazoxide. Epinephrine. Furosemide. Glucose I.V. Insulin. Lithium. MAO inhibitors. Nicotinic acid (large doses). Oral contraceptives (estrogen-progestogen combination). Oral hypoglycemics. Phenolphthalein. Phenothiazines. Phenytoin. Thiazide diuretics. Triamterene. Other factors that may affect test results Ethanol. Caffeine. Recent infection. Fever. Pregnancy. Acute illness. People over age 50 tend toward decreasing carbohydrate tolerance, which may cause conflicting results. Cushing's disease, hemochromo-cytosis, pheochromocytoma, injury to central nervous system, tumor of pancreas islet cells, malabsorption, Addison's disease, hypothyroidism, hypopituitarism. Reduced carbohydrate intake for several days before the test. Failure to follow dietary and exercise restrictions.

QUESTIONS:

a. What are the indications of OGTT ? b. What are the prerequisites of OGTT ? c. Draw a graph of a normal glucose tolerance. Glycated Hemoglobins

Introduction:

Glycohemoglobin (GHb, glycated hemoglobin, glycosylated hemoglobin) is a generic term for hemoglobin bound irreversibly (ketoamine form) to glucose. Often, the term is used to mean total glycated hemoglobin, and sometimes to mean hemoglobin A1c.

Total glycated hemoglobin (Total GHb) refers to all the glycated hemoglobins, including glycated hemoglobin variants. Total glycated hemoglobin is usually determined by affinity chromatography or immunassays.

Hemoglobin A1c (HbA1c) is the major subfraction of the glycated normal hemoglobin (HbA1). Determination of HbA1c is usually achieved by ion-exchange HPLC or gel electrophoresis.

Objectives:

It is to know the importance of glycated hemoglobin as a long term monitoring test which may be used to help controlling the treatment of diabetes mellitus.

Types of Glycated hemoglobins:

HbA1a1 = Hb + Fructose-1,6-bisphosphate (FBP)

HbA1a2 = Hb + Glucose-6-phosphate

HbA1b = Hb + Pyruvic acid

HbA1c = Hb + Glucose (N-terminal of β-chain)

HbA1d = Hb + Glucose ( internal a.a. of α/β- chain)

Using GHb :

Monitoring blood glucose is a key component of successful diabetes management. With the availability of self-monitoring and HbA1C testing, laboratory testing for fasting glucose and 2-hour post-75g glucose load should no longer be used routinely to assess glucose control. Laboratory measurement of glucose, however, may be useful to verify the accuracy of home glucose monitoring equipment or when there has been a loss of diabetic control.

HbA1C measurement provides a quantitative and reliable measure of glycemic status and control over an extended period of time, thereby complementing day- to-day monitoring. HbA1C levels are a better (and less expensive) measure of long- term glucose control than repeated fasting and p.c. glucose levels. Over the life of a red blood cell (which averages 120 days), a fraction of hemoglobin will become covalently bound to glucose and other sugar molecules. This reaction occurs non- enzymatically and at a rate which is proportional to the concentration of glucose in the blood. HbA1C is the largest single component of these glycated hemoglobins.

N.B. Blood Glucose level reflects the previous few hours glycemic state, glycated Albumin reflects 10 – 14 days glycemic state, while HbA1c reflects the longest (2- 3 months) glycemic state.

Methods:

There are currently four main techniques for determining glycated hemoglobins:

1. Cation-exchange chromatography - separates hemoglobins using HPLC based on net charge as a result of glycation; 2. Gel electrophoresis; 3. Affinity chromatography - separates total glycated hemoglobins by binding to solid-phase dihydroxyborate; 4. Immunoassay - based on binding to specific antibodies.

Experiment # 3: Estimation of HbA1c by using affinity chromatography column

Principle:

In a chromatography column, the hemoglobins in a hemolysed sample is bound by different affinity to dihydroxyborate. Elution of HbA1c is carried out by phosphate buffer, while the other hemoglobins separate (elute) after by sodium chloride solution.

Procedure:

A1 X 100 Calculation: % HbA1c = /A1 + ( 4.75 X A2)

Reference ranges:

Degree of glucose control Total GHb Hb A1c Normal (non-diabetic) < 7% < 6% Near normoglycemic 7 to 8% 6 to 7% Diabetes Control and Compliance Trial (DCCT) Less than 7% therapeutic goal In good control 8 to 9% 7 to 8% Actions suggested 9 to 11% 8 to 9% Not in control > 11 % > 9%

The determination of a glycated hemoglobin level may assist in the initial diagnosis of diabetes, or it may be used to indicate the degree of long-term diabetic control in diabetic patients. The significance of a low glycated hemoglobin level has not been established.

Annual HbA1c < 1.1 times the upper limit of normal (8.8%), suggesting less likely occurring complications.

Annual HbA1c > 1.7 times the upper limit of normal (13.5%), suggesting more likely occurring complications.

Correlation with Mean Blood Glucose Levels

A single fasting blood glucose measurement only gives an indication of the patient's immediate past (last 1 to 2 hours) condition, and may not represent the true status of blood glucose regulation. In contrast, the level of glycated hemoglobin is directly related to the average glucose concentration over the life- span of the hemoglobin in the circulation.

Various formulae have been proposed to demonstrate the correlation between the mean blood glucose (MBG) and Hemoglobin A1c (HbA1c).

MBG mg/dl = (33.3 X HbA1c) - 86

Or, MBG mg/dl = 10 ( HbA1c +4 )

Discussion:

Causes of elevated HbA1c:

- Uncontrolled D.M. - ↑ HbF - ↑ Triglycerides - Lead toxicity - ↓ iron anemia - Splenectomy - CRF ± Hemodialysis

Causes of decreased HbA1c:

- Causes of ↓ RBCs life span ( hemolytic or hemorrhagic) - Hemodilution (e.g. pregnancy)

Questions: - Give the objectives of glycated hemoglobin estimation. - Write down different methods for the determination of glycated hemoglobin. - What is the principle for the determination of glycated hemoglobin by chromatography ? Lipid Profile

Introduction:

Some beneficial aspects of lipids include the following: energy course, function and structural components of cell membranes, and precursor compound to many important substances such as vitamin D and steroid (sex) hormones.

With evidence of a link between elevated lipids and atherosclerosis (also known as arteriosclerosis or atherothrombosis), there is increase interest from both the medical and lay community in the battery of tests commonly ordered as a lipid profile. Preparation for having blood collected for lipid testing should include a 12-14 hour overnight fast.

Objectives:

- The contribution of hypercholesterolemia to coronary heart disease (CHD) risk, including the importance of elevations in total cholesterol, LDL cholesterol, HDL cholesterol, ratio of total to HDL cholesterol.

- The classification of dyslipidemias, including who to screen, and how often

- The available diagnostic studies and their use, particularly determinations of HDL, LDL and total cholesterol, as well as the need to test for other cardiovascular risk factors .

Experiment # 4:Glycerol-Phosphate Oxidase method for Triglycerides

PRINCIPLE OF THE METHOD

Sample triglycerides incubated with lipoproteinlipase (LPL), liberate glycerol and free fatty acids. Glycerol is converted to glycerol-3-phosphate (G3P) and adenosine-5-diphosphate (ADP) by glycerol kinase and ATP. Glycerol-3-phosphate (G3P) is then converted by glycerol phosphate dehydrogenase (GPO) to dihydroxyacetone phosphate (DAP) and hydrogen peroxide (H2O2). In the last reaction, hydrogen peroxide (H2O2) reacts with 4-aminophenazone (4-AP) and p- chlorophenol in presence of peroxidase (POD) to give a red colored dye:

Principle:

lipase Triglycerides + H2O Glycerol + FFA

Glycerol + ATP glycerol kinase Glycerol-3-phosphate + ADP

Glycerol-3-phosphate + O2 Glycerol-Phosphate Oxidase DHAP + H2O2 H2O2 + 4- Aminoantipyrine + Chlorophenol peroxidase Quinoneimine

The intensity of the color formed is proportional to the triglycerides concentration in the sample.

CLINICAL SIGNIFICANCE

Triglycerides are fats that provide energy for the cell. Like cholesterol, they are delivered to the body’s cells by lipoproteins in the blood. A diet with a lot of saturated fats or carbohydrates will raise the triglyceride levels. The increases in serum triglycerides are relatively non-specific. For example liver dysfunction resulting from hepatitis, extra hepatic biliary obstruction or cirrhosis, diabetes mellitus is associated with the increase Clinical diagnosis should not be made on a single test result; it should integrate clinical and other laboratory data.

PREPARATION Working reagent (WR): Dissolve () the contents of one vial R 2 Enzymes into one bottle of R 1 Buffer. Working reagent (WR): Dissolve () the contents of one vial R 2 Enzymes in 10 mL of R 1 Buffer. Cap and mix gently to dissolve contents. WR stability: 6 weeks at 2-8ºC or 1 week at room temperature (15-25ºC).

SAMPLES Serum or heparinized or EDTA plasma1. Stability of the sample: 5 days at 2-8ºC .

PROCEDURE

1. Assay conditions: Wavelength: ...... 505 nm (490-550) Cuvette: ...... 1 cm light path Temperature ...... 37ºC / 15-25ºC 2. Adjust the instrument to zero with distilled water. 3. Pipette into a cuvette:

Standa Blank rd 1.0 1.0 (WR (mL 10 -- Standard ((µL -- -- (Sample (µL

4. Mix and incubate for 5 min. at 37ºC or 10 min. at room temperature. 5. Read the absorbance (A) of the samples and Standard, against the Blank. The colour is stable for at least 30 minutes. CALCULATIONS A Sample x 200 (Standard conc.) = mg/dL triglycerides in the sample A Standard Conversion factor: mg/dL x 0.0113= mmol/L.

Reference ranges:

Normal Fasting blood triglycerides = 60 – 160 mg/dl

It is considered normal as long as it is below 200 mg/dl

Discussion:

-Types of hyperlipidaemias

QUESTIONS:

a. Give the different methods for the determination of triglycerides. b. Write a short note on hypertriglyceridemia. c. Give the upper cut off value of triglyceride for a diagnosis of hypertriglyceridemia. DETERMINATION OF CHOLESTEROL:

INTRODUCTION:

Cholesterol is a waxy substances used in every cell membrane you have and as a base for several hormones. The recommended daily allowance for dietary cholesterol intake is 300 milligrams. Most cells have some capacity to synthesize cholesterol. The largest percentage of synthesized cholesterol is made in the liver. Cholesterol lowering medications prescribed by physicians inhibit the synthesis of cholesterol by the liver, thereby reducing the level in the blood stream.

OBJECTIVES:

The estimation of cholesterol along with other parameters of lipid profile is necessary for the classification and diagnosis of lipemias

PRINCIPLE OF THE METHOD The cholesterol present in the sample originates a coloured complex, according to the following reaction: The intensity of the color formed is proportional to the cholesterol concentration in the sample

Principle (Experiment #5):

Cholesterol esterase Cholesterol esters + H2O Cholesterol + FA

Cholesterol Oxidase Cholesterol + O2 Cholesterol-3-one + H2O2

Peroxidase H2O2 + 4-AAP + Phenol Quinonimine

CLINICAL SIGNIFICANCE Cholesterol is a fat-like substance that is found in all body cells. The liver makes all of the cholesterol the body needs to form cell membranes and to make certain hormones. The determination of serum cholesterol is one of the important tools in the diagnosis an classification of lipemia. High blood cholesterol is one of the major risk factors for heart disease5,6. Clinical diagnosis should not be made on a single test result; it should integrate clinical and other laboratory data.

PREPARATION Working reagent (WR): Dissolve () the contents of one vial R 2 Enzymes in one bottle of R 1 Buffer. Cap and mix gently to dissolve contents. (WR) is stable: 4 months at 2-8ºC or 40 days at 15-25ºC. Avoid direct sunlight. SAMPLES Serum or plasma1,2: Stability of the sample for 7 days at 2-8ºC or freezing at – 20ºC will keep samples stable for a few months. PROCEDURE 1. Assay conditions: Wavelength: ...... 505 nm (500-550) Cuvette: ...... 1 cm light path Temperature ...... 37ºC /15-25ºC 2. Adjust the instrument to zero with distilled water. 13. Pipette into a cuvette:

Samp Standa Blank le rd 1.0 1.0 1.0 (WR (mL -- 10 -- Standard ((µL 10 -- -- Sample ((µL

14. Mix and incubate for 5 min. at 37ºC or 10 min. at room temperature. 25. Read the absorbance (A) of the samples and Standard, against the Blank. The colour is stable for at least 60 minutes.

CALCULATIONS A (Sample) x 200 (Standard conc.) = mg/dL cholesterol in the sample A (Standard) Conversion factor: mg/dL x 0.0258= mmol/L.

*Normal range: Desirable blood cholesterol level < 200 mg/dl Suspect to vascular and CHD 200 – 240 mg/dl High risk group for CHD > 240 mg/dl Risk evaluation:

Normal Less than 200 mg/dL Borderline mg/dL 200-239 High mg/dL and above 240 LDL –Cholesterol

PRINCIPLE OF THE METHOD Direct determination of serum LDLc (low-density lipoprotein cholesterol) levels without the need for any pre-treatment or centrifugation steps. The assay takes place in two steps.

1º Elimination of lipoprotein no-LDL

catalase H2O2 2 H2O + O2

2º Measurement of LDLc

Peroxidase H2O2 + 4-AAP + Phenol Quinonimine + 4 H2O2

The intensity of the color formed is proportional to the LDLc concentration in the sample.

CLINICAL SIGNIFICANCE The LDLc particle is lipoproteins that transport cholesterol to the cells. Often called “bad cholesterol” because high levels are risk factor for coronary heart disease and are associated with obesity, diabetes and nephrosis 1,5,6. Clinical diagnosis should not be made on a single test result; it should integrate clinical and other laboratory data.

PREPARATION - R 1 and R 2: Are ready to use. - HDLc/LDLc CAL: Dissolve the contents with 1 mL of distilled water. Cap vial and mix gently to dissolve contents. SAMPLES Serum : After sampling, the test should be performed without delay. Repeated freezing and thawing should be avoided. Stability of the sample: 7 days at 2-8ºC .

PROCEDURE . Assay conditions: Wavelength: ...... 600 (590-700) nm Cuvette: ...... 1 cm. light path Temperature ...... 37ºC 2. Adjust the instrument to zero with distilled water. 3. Pipette into a cuvette:

Blan Standar Sampl k d e R1(µL) 300 300 300 Standard ------4 ------(µL) - Sample (µL) ------4 -

4. Mix and incubate for 5 min. at 37ºC. 5. Add:

R2 100 100 100 (µL)

6. Mix and incubate for 5 min. at 37ºC. 7. Read the absorbance (A), against the Blank.

CALCULATIONS (A) Sample x Standard.conc. = mg/dL of LDLc in the sample (A) Standard Conversion factor: mg/dL x 0.02586

. REFERENCE VALUES Levels of the risk Desirable < 100 mg/dL Medium 130-160 mg/dL High > 160 mg/dL HDL cholesterol

PRINCIPLE OF THE METHOD The very low density (VLDL) and low density (LDL) lipoproteins from serum or plasma are precipitated by phosphotungstate in the presence of magnesium ions. After removed by centrifugation the clear supernatant containing high density lipoproteins (HDL) is used for the determination of HDL cholesterol

CLINICAL SIGNIFICANCE HDL particles carry cholesterol from the cells back to the liver. HDL is known as “good cholesterol” because high levels are thought to lower the risk of heart disease. A low HDL cholesterol levels, is considered a greater heart disease risk. Clinical diagnosis should not be made on a single test result; it should integrate clinical and other laboratory data.

Procedure :

PTA + B. sample RT incubation for 10 min Centrifugation for 10 min. at 4000 rpm Supernatant cholesterol + Chol.Oxidase reagent - HDL- Chol. Conc.

SAMPLES Serum or plasma1: Free of hemolysis. Removed from the blood clot as soon as possible. Stability : HDL Cholesterol is stable for 7 days at 2-8ºC .

PROCEDURE

Precipitation 11. Pipette into a centrifuge tube:

10 (R (µL 0 1. Sample 0 ((mL 12. Mix well; allow to stand for 10 min at room temperature. 23. Centrifuge at 4000 r.p.m. for 20 min or 2 min at 12000 r.p.m.. 34. Collect the supernatant and test HDLc.

Test Following the Cholesterol reagent instructions.

CALCULATIONS - With Factor: A505 nm Sample x 320 = mg/Dl HDLc in the sample. A546 nm Sample x 475 = mg/Dl HDLc in the sample

Calculation of LDL-cholesterol According to the Friedewald Formula:

LDL cholesterol = Total cholesterol - Triglycerides -HDL cholesterol 5

Questions: a. Write a short note on Hyperlipidemia. b. Which one of the two – HDL/LDL is more dangerous to health and give reason. c. Which diet can cause increase in HDL-Chol ?

Write the equation for HDL-cholesterol calculated from TG, Total Chol.& LDL.

Laboratory Renal Function Tests

I- Urea Estimation & Blood Urea Nitrogen (BUN)

Introduction:

Kidney problems are very common in clinical medicine. Essentially all seriously sick patients will need their kidney function evaluated during the course of their illnesses.

After history and physical exam are complete, the initial steps in checking patients' kidneys are performing the following tests: (1) urinalysis (2) serum creatinine (3) serum urea ("blood urea nitrogen", "BUN"). Next, you may check (4) ability to concentrate urine.

Both creatinine and BUN are included on the common chemical profiles. You can check the ability to concentrate urine using a hygrometer, refractrometer, or dipstick.

Objectives: Methods:

1- Chemical (direct) method:

Urea + Diacetyl monoxime(DAM) Diacetyl-Urea

Diacetyl-Urea + Fe3+ +acidic pH Yellow Diazine (measured at 540)

2-Enzymatic (indirect) method:

Urease Urea + H2O CO2+ NH3 pH indicator Yellow Orange ( HgI2 NH3 dye (dry +KI) chemistry) (at 540 nm)

Ammonia Kinetic detecting Multi- Conductivity electrode enzymatic difference between Berthlot’s method non-ionized urea and ionized ammonia

Experiment # 6 (Modified Berthlot’s Reaction):

PRINCIPLE OF THE METHOD Urea in the sample is hydrolized enzymatically into ammonia (NH4+) and carbon dioxide (CO2). Ammonia ions formed reacts with salicylate and hypochlorite (NaClO), in presence of the catalyst nitroprusside, to form a green indophenol: The intensity of the color formed is proportional to the urea concentration in the sample

CLINICAL SIGNIFICANCE Urea is the final result of the metabolism of proteins; it is formed in the liver from its destruction. Elevated urea can appear in blood (uremia) in: diets with excess of proteins, renal diseases, heart failure, gastrointestinal hemorrhage, dehydration or renal obstruction1,6,7. Clinical diagnosis should not be made on a single test result; it should integrate clinical and other laboratory data. Principle:

Urease Urea + H2O CO2+ NH3

NH3 + Na-salicylate + Na-hypochlorite +Na-nitoprusside Indophenol

PREPARATION - Working reagent (WR): Dissolve one tablet R 3 Enzymes in one bottle of R 1 Buffer. Cap and mix gently to dissolve contents. Stability: 4 weeks in the refrigerator (2-8ºC) or 7 days at room temperature (15- 25ºC). - R 2 NaClO is ready to use : SAMPLES 1- Serum or heparinized plasma: Do not use ammonium salts or fluoride as anticoagulants. 2- Urine: Dilute sample 1/50 in distilled water. Mix. Multiply results by 50 (dilution factor). Preserve urine samples at pH < 4. Urea is stable at 2-8ºC for 5 days;

PROCEDURE 11. Assay conditions: Wavelength: ...... 580 nm Cuvette: ...... 1 cm light path Temperature...... 37ºC / 15-25ºC 12. Adjust the instrument to zero with distilled water. 13. Pipette into a cuvette:

Blan k 1.0 (WR (mL -- Standard ((µL -- Sample ((µL

14. Mix and incubate 5 min at 37ºC or 10 min at room temperature (15-25ºC). 25. Pipette:

1.0 R 2 ((mL 16. Mix and incubate 5 min at 37ºC or 10 min at room temperature (15- 25ºC). 27. Read the absorbance (A) of the samples and calibrator, against the Blank. The colour is stable for at least 30 minutes at 15-25ºC.

CALCULATIONS (A) Sample x 50 (Standard conc.) = mg/dL urea in the sample (A) Standard 10 mg/L urea BUN divided by 0.466 = 21 mg/L urea = 0.36 mmol/L urea. Conversion factor: mg/dL x 0.1665 = mmol/L.

REFERENCE VALUES1 Serum : 15- 45 mg/dL (2.49-7.49 mmol/L) Urine : 20 - 35 gr/24 h. Blood Urea Nitrogen (BUN) 8 – 25 mg/dl

Interpretation:

Interpretation of the BUN is usually straightforward, though there are a few things to remember.

#Increased BUN is, by definition, azotemia. It is due either to increased protein catabolism or impaired kidney function.

*Increased protein catabolism results from:

. a really heavy protein meal (Kebda, El-Bek, etc.) . severe stress (myocardial infarction, high fever, etc.) . upper GI bleeding (blood being digested and absorbed)

*Impaired kidney function may be "prerenal", "renal", or "postrenal".

. Prerenal azotemia results from underperfusion of the kidney: dehydration, hemorrhage, shock, congestive heart failure . Renal azotemia has several familiar causes: acute tubular necrosis, chronic interstitial nephritis, glomerulonephritis, etc. . Postrenal azotemia results from obstruction of urinary flow: prostate trouble, stones, surgical mishaps, tumors

N.B. In acute renal failure, BUN increases around 20 mg/dL each day (*estimates vary; range of increase is 10-50 mg/dL daily).

#Decreased BUN

. Lack of protein (celiac disease, some patients with nephrotic syndrome) . Severe liver disease (end-stage cirrhosis, yellow atrophy, really bad hepatitis, halothane or acetaminophen toxicity, enzyme defects) . Overhydration (iatrogenic, psychogenic water-drinking)

Discussion: -Physiological & Biochemical Background: -Pathological & Disease Correlation:

Questions:

- Write the different methods for estimation of blood urea? - Calculate BUN on estimation of blood urea. - Mention causes of hyperazotemia. II- Plasma Creatinine Estimation

Introduction:

Creatinine is the end product of muscle metabolism. It is excreted through the kidneys and changes in creatinine are an early indicator of kidney disease as well as being seen in severe muscle damage or wasting diseases or with many medications such as antibiotics. this test can be performed on specimens drawn from patients in either the fasting or non fasting state.

Methods:

1- Direct Chemical methods: a) Jaffe’ method : See the principle and procedure (Experiment ) b) DNB method (used in dry chemistry:

Creatinine +Dinitrobenzoic acid +alkaline pH purplish rose

2- Indirect Enzymatic methods: a) Deaminase method (One enzyme step method):

Creatinine Deaminase methyl hydantoin + NH3

(detected by different techniques)

b) Amidohydrolase method ( multi-enzymatic method):

- Creatinine Creatinine. Amidohydrolase Creatine

- Creatine Creatine kinase Creatine -p

- Creatine-p +ATP Creatine +ADP

- ADP + P-enol pyrovate (PEP) ATP +Pyruvate

- Pyruvate + NADH+H+ LDH Lactate + NAD (with diminished absorbance at 340 nm)

PRINCIPLE OF THE METHOD The assay is based on the reaction of creatinine with sodium picrate as described by Jaffé. Creatinine reacts with alkaline picrate forming a red complex. The time interval chosen for measurements avoids interferences from other serum constituents. The intensity of the color formed is proportional to the creatinine concentration in the sample. Principle (Jaffe’ Method):

Creatinine + Picric acid + alkaline pH 2,4,6 trinitrophenol (Janovski’s complex) measured at 520 nm

CLINICAL SIGNIFICANCE Creatinine is the result of the degradation of the creatine, component of muscles, it can be transformed into ATP, that is a source of high energy for the cells. The creatinine production depends on the modification of the muscular mass, and it varies little and the levels usually are very stable. Is excreted by the kidneys. With progressive renal insufficiency there is retention in blood of urea, creatinine and uric acid. Elevate creatinine level may be indicative of renal insufficiency1,4,5. Clinical diagnosis should not be made on a single test result; it should integrate clinical and other laboratory data.

PREPARATION Working reagent (WR): Mix equal volumes of R 1 Picric Reagent and R 2 Alkaline reagent. The working reagent is stable for 10 days at 15-25ºC.

Signs of reagent deterioration: 1- Presence of particles and turbidity. 2- Blank absorbance (A) at 492 nm 1.80.

SAMPLES 1- Serum or heparinized plasma. Creatinine stability: 24 hours at 2-8ºC. 1- Urine: Dilute sample 1/50 with distilled water. Mix. Multiply results by 50 (dilution factor); Creatinine stability: 7 days at 2-8ºC.

PROCEDURE 11. Assay conditions: Wavelength: ...... 492 nm (490-510) Cuvette: ...... 1 cm. light path Temperature...... 37ºC / 15-25ºC 12. Adjust the instrument to zero with distilled water. 23. Pipette into a cuvette:

Standa Blan k 1.0 (WR (mL -- Standard ((µL -- Sample ((µL

1 4. Mix and start stopwatch. 25. Read the absorbance (A1) after 30 seconds and after 90 seconds (A2) of the sample addition. 36. Calculate: A= A2 – A1.

CALCULATIONS Δ A Sample – Δ A Blank x 2 (Standard conc.) = mg/dL of creatinine in the sample ΔA Standard – ΔABlank

Conversion factor: mg/dL x 88.4 = µmol/L. l

REFERENCE VALUES

Serum or plasma mg/dL = (61.8 – 123.7)µ mol/L 1,4 - 0,7 Male mg/dL = (53.0 – 97.2 ) µmol/L 1,1 - 0,6 Female Urine: 15-25 mg/Kg/24 h 10 Male mg/Kg/24 h = 71– 177 µmol/Kg/24 h 18 – 8 Female

Interpretation: Causes of renal failure

Discussion: Physiological & Biochemical Background: Other Renal Function Tests: Tutorial

Creatinine clearance : is widely used to approximate glomerular filtration. You need a timed urine sample and a blood sample.

The clearance of a substance is the volume of plasma "cleared" of that substance per unit time.

Clearance = (conc. in urine) x (urine volume)

(conc. in plasma) X time of urine collection (min.).

In deciding how to "time" your collection, remember that you don't really need to collect urine for a full 24 hours. One group got more reliable results by a controlled collection over 4 hours, monitoring body position (kept them lying down) and hydration with body surface area measurement.

Creatinine clearance is not a perfect measure of GFR, because some is not filtered and some is secreted into the proximal tubule. These fractions tend to cancel each other out in health, but when GFR drops below 30 mL/min, tubular secretion approaches or even exceeds the amount filtered at the glomerulus.

*Also, lots and lots of red meat (protein and creatinine-rich) can lead to overestimates (maybe 30%) in GFR in renal failure patients.

Reference range for creatinine clearance is 90-120 mL/min for young adults; values tend to fall by around 0.5 mL/year over age 20, worse for hypertensives .

*Formulas to adjust "normal" for body surface area have been devised, etc. For kids, a height/creatinine ratio of 2.1 or less is normal. GFR for adults can be estimated by various formulas; try 1.12 x Creatinine Clearence - 20.6. Another formula to correct the clearance according to body surface area is {Corrected Cr.Cl. = Cr.Cl. X (1.7/ body surface area)}

*Whether or not "corrections" are applied, creatinine clearance is a pretty good estimate of glomerular filtration rate except at very low values, when tubular secretion of creatinine become proportionately greater.

DETERMINATION OF URIC ACID

INTRODUCTION:

Uric acid is apurine compound that circulates in plasma as sodium urate and is excreted by kidney. It is derived from the break down of nucleic acids that are ingested or come from the destruction of tissue cells; it is also synthesized in the body from simple compounds as shown in figure.

OBJECTIVES:

 To know the uric acid level in the body  To diagnose a case of Hyperuricemia (Gouts)

METHODS:

 Chemical Method (Phosphotungestic acid Method?)  Enzymatic Method

PRINCIPLE { Enzymatic Colorimetric (Uricase Method)}: Uric acid is oxidized by uricase to allantoine and hydrogen peroxide (2H2O2), which under the influence of POD, 4–aminophenazone (4-AP) and 2-4 Dichlorophenol sulfonate (DCPS) forms a red quinoneimine compound: Uric acid + 2H2O + O2 Uricase Allantoine + CO2 + 2H2O2

2H2O2 + 4-AP + DCPS POD Quinoneimine+ 4H2O

The intensity of the red color formed is proportional to the uric acid concentration in the sample.

CLINICAL SIGNIFICANCE Uric acid and its salts are end products of the purine metabolism. With progressive renal insufficiency, there is retention in blood of urea, creatinine and uric acid. Elevate uric acid level may be indicative of renal insufficiency and is commonly associated with gout Clinical diagnosis should not be made on a single test result; it should integrate clinical and other laboratory data.

PREPARATION Working reagent (WR): Dissolve the contents of one vial R 2 Enzymes in one bottle R 1 Buffer. Cap and mix gently to dissolve contents. (WR) is stable after reconstitution 1 month at 2-8ºC or 10 days at room temperature. SAMPLES - Serum or plasma: Stability 3-5 days at 2-8ºC or 6 months at –20ºC. - Urine (24 h)1: Stability 4 days at 15-25ºC, pH >8. Dilute sample 1/50 in distilled water. Mix. Multiply results by 50 (dilution factor); If urine is cloudy; warm the specimen to 60ºC for 10 min to dissolve precipitated urates and uric acid. Do not refrigerate.

PROCEDURE 1. Assay conditions: Wavelength: ...... 520 nm (490-550) Cuvette: ...... 1 cm light path Temperature ...... 37ºC / 15-25ºC 2. Adjust the instrument to zero with distilled water. 3. Pipette into a cuvette:

Blank Standar Sampl d e WR (ml) 1.0 1.0 1.0 Standard ------25 ------(µL) Sample (µL) ------25

4. Mix and incubate for 5 min at 37ºC or 10 min at 15-25ºC. 5. Read the absorbance (A) of the samples and Standard, against the Blank. The colour is stable for at least 30 minutes.

CALCULATIONS Serum or plasma (A) Samplex 6 (Standard conc.)= mg/dL uric acid in the sample (A) Standard Urine 24 h A) Samplex x 6 x vol. (dL) urine 24 h =mg/24 h uric acid Standard Conversion factor: mg/dL x 59.5=µ mol/L. REFERENCE VALUES: Serum or plasma: Women 2.5 - 6.8 mg/dL = 149 – 405 µ mol/L Men 3.6 - 7.7 mg/dL =214 – 458 µ mol/L Urine: 250 - 750 mg/24 h = 1.49 - 4.5 mmol/24 h

CLINICAL SIGNIFICANCE:

Hyperuricemia (Gout)

DISCUSSION: Causes of elevated uric acidemia

QUESTIONS:

1.Give the principle for the determination of serum uric acid by uricase method. 2. Write a short note on Uric acid metabolism. 3. Explain the hyperuricemia. PRERENAL VS. RENAL AZOTEMIA :

A very common clinical problem is to distinguish prerenal azotemia (due to shock, dehydration, CHF -- also "hepatorenal syndrome") from renal azotemia (acute tubular necrosis, "renal shutdown".)

Either could be the cause when a patient has been hypotensive and now is azotemic and oliguric. The management is different.

One older technique is to calculate the BUN/creatinine ratio. This is normally between 10 and 20.

Values over 20, suggest prerenal azotemia rather than acute tubular necrosis.

High values are also seen postrenal azotemia and upper GI bleeding.

In fact, a high BUN/creatinine ratio is a common finding, especially in the elderly, and a marker for ill-health .

Another approach is to measure sodium on a urine specimen.

In prerenal azotemia, urine sodium is low (the kidney responds to low blood flow by "trying to retain all the sodium it can.") In acute tubular necrosis, urine sodium is higher (the renal tubules are unable to concentrate or dilute the glomerular filtrate effectively.)

Urinary sodium under 20 mEq/L suggests prerenal azotemia (or hepatorenal syndrome, etc.); urinary sodium over 40 mEq/L suggests acute tubular necrosis.

*A further refinement, currently popular, is to measure the fractional excretion of filtered sodium, approximated by:

Values less than 1% indicate prerenal azotemia; values over 2% indicate acute tubular necrosis.

Several other factors can complicate the picture in such patients.

Diuretics will increase the excretion of filtered sodium, while secondary hyperaldosteronism (as in cirrhosis) will decrease sodium excretion.

In acute tubular necrosis due to myoglobinuria, sodium excretion is low (the tubules are plugged, not damaged.)

*Tip: If you obtain urine by squeezing a diaper or the absorptive balls you placed into the diaper, your estimate of urine creatinine will be low because these things absorb creatinine .

Urine Protein/creatinine ratio

Urine protein/creatinine ratio (UP/UCr) is used to calculate urine protein loss into the urine without a need for 24 hour urine collection. Compared to conventional 24 hour- urinary protein value, UP/UCr is less time consuming and less accurate.

Generally with proteinuria, UP/UCr is greater than 1.0.

 LESS FAMILIAR RENAL FUNCTION TESTS

1. Blood pH

Changes in acid-base balance is observed frequently in renal failure especially when advanced. 2. Lipids Hyperlipidaemia can occur with renal disease, such as nephrotic syndrome. Increased hepatic lipoprotein synthesis and hypoalbuminaemia is proposed in the pathogenesis.

3. Plasma protein

Generally the concentration of plasma protein is elevated due to dehydration but can be reduced in primary glomerular diseases such as glomerulonephritis and renal amyloidosis.

4. Amylase and lipase

Elevated plasma lipase and amylase levels can be observed in dogs with renal disease, because these two enzymes are eliminated by the kidneys.

5. Total Red blood cell

In chronic renal disease, non-regenerative anemia is commonly observed. It is mainly due to a reduced erythropoietin level secondary to the loss of renal parenchyma.Other causes of anemia in renal disease include haemorrhage, shorter the life span of erythrocytes and bone marrow depression.

6. N-acetyl-beta-D-glucosaminidase ("glucosaminidase", NAG) is a lysosomal enzyme (MW 140,000) found in serum and urine. Urinary NAG is a proposed marker for tubular disease, especially subtle industrial poisoning, acute pyelonephritis, early acute tubular necrosis, and early transplant rejection. (These functions are now largely taken over by beta-2 microglobulin).

7. Adenosine Deaminase Binding Protein is an enzyme from the brush borders of the proximal tubule. Like NAG, its presence in urine indicates tubular disease.

8. Alkaline phosphatase in urine comes from the proximal tubular brush border .

9. Beta-2 microglobulin (beta-2-m) is the short chain of the HLA class I proteins. In health, it is freely filtered by the glomerulus, and fully reabsorbed by the proximal tubule.

Serum beta-2-m has been suggested as a measure of glomerular filtration rate, similar to creatinine. Obviously this isn't a good idea for patients with tissue necrosis, lymphomas, etc. Urine beta-2-m has found widespread acceptance as an research tool. It appears if levels in the serum and glomerular filtrate exceed what the proximal tubule can reabsorb (more than 4.5 mg/L) or if there is renal tubular disease. It is very sensitive as an indicator of the latter.

10. Tubular functions: Urinary amino acids and maximum concentrating ability are sensitive screens for tubular damage. Lithium clearance is a researcher's way of estimating delivery to the distal tubule.

11. Isotope scans exist to compare the function of the kidneys. These may prove a valuable supplement to the intravenous pyelogram. More recently, the 12. color Doppler sonogram, which is cheap and portable, has proved even more useful than these scans in transplant patients. Most recent of all, there's a Tc99 scanner that monitors glomerular filtration minute by minute, suitable for the intensive care .

13. Positron emission tomography is the latest way of measuring renal blood flow.

SPECIFIC GRAVITY OF URINE

While not a "blood test", checking urine specific gravity provides very important information about tubular function and hydration.

People in our culture drink relatively little fluid. Thus "normal" people have fairly concentrated urine (SG greater than 1.010). Of course, the same is true of patients in prerenal azotemia (high urinary specific gravity, low or zero urinary sodium).

Patients with tubular disease ("renal azotemia", i.e., acute tubular necrosis, really bad bilateral pyelonephritis or interstitia nephritis, or on diuretics, or with end-stage kidney) will have isosthenuria.

Patients getting lots of fluid by IV, or with diabetes insipidus, or enthusiastic water-drinkers (asthmatics, crazies) will have low urine specific gravity.

Serum and Urine Osmolality

The term osmolality refers to the osmotic concentration of a fluid. The osmolality of serum, urine, or any other body fluid depends on the number of active ions or molecules in a solution. In laboratory reports, osmolality is expressed as "so many" milliosmoles per kilogram of water (mOsm/kg water). With a standard measurement of osmoles and of milliosmoles for clinical studies, the precise concentration of active solutes in the serum and urine can be calculated. Tests of both serum and urine osmolality can yield important information about a patient's ability to maintain a normal fluid balance status.

Sodium, blood urea nitrogen, and blood glucose levels are major factors in determining serum osmolality. In severe dehydration serum osmolality will be increased, as there is less water in proportion to solutes in the serum or blood. Urine osmolality, like specific gravity, is a measurement of the concentration of urine. Urine osmolality reflects the total number of osmotically active particles in the urine, without regard to the size or weight of the particles. Substances such as glucose, proteins, or dyes increase the urinary specific gravity. Therefore, urine osmolality is a more accurate measurement of urine concentration than specific gravity, and urine osmolality can be compared with the serum osmolality to obtain an accurate picture of a patient's fluid balance.

Reference values for osmolality:

 Serum osmolality: 282 - 295 mOsm/kg water; a serum osmolality of 285 mOsm correlates with a urine specific gravity of 1.010  Urine osmolality: extreme range of 50 - 1400 mOsm/kg water, but average is about 500 - 800 mOsm. After an overnight fast, the urine osmolality should be at least 3 times the serum osmolality

Increased serum and urine osmolality (hyperosmolality) levels are seen in:

 Renal disease  Congestive heart failure  Addison's disease  Dehydration  Diabetes insipidus  Hypercalcemia  Diabetes mellitus/hyperglycemia  Hypernatremia  Alcohol ingestion  Mannitol therapy  Azotemia

Decreased serum and urine osmolality (hypoosmolality) levels are seen in:

 Sodium loss due to diuretic use and a low salt diet  Hyponatremia  Adrenocortical insufficiency  SIADH  Excessive water replacement/overhydration/water intoxication

Panic values for serum osmolality are values of less than 240 mOsm or greater than 321 mOsm. A serum of osmolality of 384 mOsm produces stupor. If the serum osmolality rises over 400 mOsm, the patient may have grand mal seizures. Values greater than 420 mOsm are fatal.

When the serum osmolality is normal or increased, the kidneys are conserving water. As the serum osmolality rises, the urine osmolality should also rise. The higher the number of millosmoles in the urine, the more concentrated the urine; this is the expected physiological response to dehydration

This table shows the relationship between serum and urine osmolality and the clinical significance of laboratory values.

Serum Osmolality Urine Osmolality Clinical Significance Normal values: Normal values: 282-295mOsm 500-800mOsm Normal or increased Increased Fluid volume deficit Decreased Decreased Fluid volume excess Normal Decreased Increased fluid intake or diuretics Increased or normal Decreased (with no Kidneys unable to increase in fluid intake) concentrate urine or lack of ADH (diabetes insipidus) Decreased Increased SIADH

Urine Concentration tests:

An increase in plasma osmolarity stimulates ADH secretion by the posterior pituitary gland. ADH stimulates renal water resorption and increases urine SG. These tests are designed to identify concentrating defects in the kidney. They are indicated in animals that show polydipsia/polyuria (PD/PU) without azotaemia and dehydration and are contraindicated in dehydrated animals, pregnant animals or azotaemic animals with diluted urine.

Liver Function Tests (LFT)

Albumin estimation ======Introduction:

Albumin is made in the liver and is responsible for maintaining proper fluid balances. Too little albumin may result in fluids "leaking" out of the blood vessels into surrounding spaces such as the abdomen. Decreased amounts of albumin can occur when the liver is not making enough or if albumin is being lost through the kidneys. Increases in albumin do not occur naturally but can be seen in patients who had received albumin suspensions.

Methods

1-Precipitation method 2-Electrophoresis 3-Globulin Tryptophan content method 4-Immunochemical methods. 5-Dye binding methods

I) Precipitation method

*Use serum only *Not applied for automation *Used now for separation & manufacturing albumin .   *Precipitation is done by salting out of globulins &then albumin in the supernatant is measured using a protein estimation method.

II) Electrophoresis ======*Separation of Albumin from the major classes of protein in an electrical field & the staining %is obtained . Calculation of Albumin = % Albumin X Total Protein *Difficult method for Automation. *It is a quantitative method but tends to over estimate Albumin because albumin is the best binder of staining dyes &the band density of alb. scanned by densitometer.

III) Globulin Tryptophan content method ======*In this method Tryptophan content of the globulin is fist estimated as following; Glycoxylic acid +tryptophan (Globulin)Purple chromogen (measured at 540)  Calculation of Albumin = T.Protein - Globulin

IV) Immune chemical methods ======A)-Electro -immune-diffusion (EID): Considered the Reference method ,Quantitative &Manual. *Migration of protein fractions in an electrical field through a medium contains specific antibodies to albumin. The height of the Rocket precipitin line is correlated to albumin conc. *Used for serum only.

B)-Radial immune diffusion (RID):By measuring the diameter of precipitin ring between albumin &its antibodies incorporated in agarose gel. *Used for serum &CSF. Takes long time.

C)-Turbidimetry: The reaction between albumin and its specific antibodies form complexes ,that will decrease the light transmission through the reaction phase more than free albumin (antigen).

D)-Nephelometry:.

E)-Radio immune assay (RIA).

F)-Enzyme immune assay (ELISA)

PRINCIPLE OF THE METHOD Albumin in the presence of bromcresol green at a slightly acid pH, produces a colour change of the indicator from yellow-green to green-blue. The intensity of the color formed is proportional to the albumin concentration in the sample . CLINICAL SIGNIFICANCE One of the most important serum proteins produced in the liver is albumin. This molecule has an extraordinarily wide rage of functions, including nutrition, maintenance of oncotic pressure and transport of Ca++, bilirubin, free fatty acid, drugs and steroids. Variation in albumin levels indicate liver diseases, malnutrition, skin lesions such as dermatitis and burns or dehydration. Clinical diagnosis should not be made on a single test result; it should integrate clinical and other laboratory data.

PREPARATION Reagent and calibrator are ready to use

Signs of reagent deterioration: 1- Presence of particles and turbidity. 2- Blank absorbance (A) at 630 nm 0.40. SAMPLES Serum or plasma, free of hemolysis: Stability 1 month at 2-8ºC or 1 week at 15- 25ºC.

PROCEDURE 11. Assay conditions: Wavelength: ...... 630 nm (600-650) Cuvette: ...... 1 cm light path Temperature ...... 15-25ºC 12. Adjust the instrument to zero with distilled water. 23. Pipette into a cuvette:

Blan k 1.0 (R (mL -- Standard ((µL -- Sample ((µL

14. Mix and incubate for 10 min at room temperature (15-25ºC). 25. Read the absorbance (A) of the samples and Standard, against the Blank. The colour is stable 1 hour at room temperature.

CALCULATIONS (A) Sample x 5 (Standard conc.) = g/dL albumin in the sample (A) Standard Conversion factor: g/dL x 144.9 = µmol/L

REFERENCE VALUES

3.5 to 5.0 g/dL. Total protein estimation

Introduction:

A Total Protein can be done on either a fasting or non fasting specimen. It is usually done as a general screening assay since it is composed of two major fractions (albumin and globulin). Elevations or decreases in a total protein must be investigated to find out which of the two components is causing the problem. Since many of the next level tests may be reported as percentages or ratios, it is necessary to have the total protein rerun at the time these tests are performed. Overall, a general reference range is 5.0 - 8.0 gram/dL.. Since this is a stable assay, the range of variation is quite small. Acceptable variation is 1.0

If both the albumin and globulin are elevated, one possibility is dehydration or a slow down of blood flow. If both are decreased, the most common culprit is liver function. Since both albumin and globulin can be assayed individually, they are sometimes reported as an "AG ratio". (See albumin and globulin for specifics.)

Patients with Waldenström’s macroglobulinemia may have total proteins above 8.5. They should consider having tests performed on urine specimens as this will lessen the clotting problem found in the specimen but still provide adequate answers to the physician.

1-Ultraviolet absorption method 2-Specific gravity methods for T.P. a)Phillips or b)Lowry &Hunter 3-Refractrometry. 4-Kjeldahl nitrogen detection method a)Titration or b) kinetic 5-CuSO4 (Cu-Pr complex) Methods a)Lowry or b) Biuret

Normal (Reference)Ranges: - Ammonia (Plasma on Heparin)= 15-51 ug/dl - T.P Premature babies = begin from 3.6 g/dl Newborns = 4.6 -5.7 g/dl 7months -1yr. = 5.1 -7.3 g/dl 1-2yrs. = 5.7 -7.5 g/dl Adults = 6.0 - 8.0 g/dl - Exercise &Ambulatory 0.5 g/dl to T.P (by extravasation of proteins)

1- Ultraviolet Absorption:

270 –290 nm 200 -225 nm

*Used for Solutions rather than serum. *Using Quartz Cuvette (with no scratches)  On using serum , Dilute 1:1000 with NaCl 0.15 mol/L.  This method depends on Tryptophan &Tyrosine content of the protein. *Interference by free tyrosine,tryptophan, bil.&U.A.

2-Specific gravity method for Total proteins: a)Phillips et al.: *drops of serum are allowed to fall into "Universal"containers filled with CuSO4 soln. each of known sp.gr. (Stock soln.=159/L Sp.gr=1.1),then serial dilutions are made to get solns. of sp.gr between 1.015 1.035 At certain specific gravity a drop will not move neither up or down(=Sp.gr.of interest) Total protein(g/dl) = 365(Sp.gr.of Int. ▬ 1.007 ) *Can estimate T.P between 3.3 ▬10.3 g/dl

b)Column method (Lowry&Hunter): *Using only single gradient column of mixed organic liquids with CuSO4 jacket (maintaining constant temp.). *It requires only one drop,which will be hanged at certain gradient . *Like in Phillips method T.P can be calculated.

3-Refractometry method: *This method is based on the refraction of incident light by total dissolved solids. *A large drop of serum or urine is allowed to spread between slide &thin film and refracted rays make sharp line dividing the dark & light fields. *Can estimate T.P between 3.5 ▬11 g/dl 4-Kjeldahl Method: *The reference method *using protein free filtrate. *Depend on estimation of protein nitrogen.

Protein

H2SO4+Catalyst +Na-Molybdate

Alkaline PH

NH3 HCl(standard Sol.) NADPH +2oxoglutarate ( either)

(Titration)

Neutral PH NADP + glutamate (Nisselerization) (Monitor abs.change at 340 nm)

Concentration of total protein= detected nitrogen X100/16 = detected nitrogen X 6.25 *factor 6.25 is the result of 100/16 as each 100 g prt.16 g Nitrogen. * Corrected pr.conc. = (pr.N2-NPN) X 6.25

5-Alkaline CUSO4 soln.Methods:

Sample

NaOH + CuSO4.

Copper 6-peptide bond protein complex

Folin(Fenol)+ K&NaTartarate(=color stabilizer) Cio-Calteau(PTA+Ph-Molbdic a.)

Molybdinum blue + Violet color of Cu- Pr.Complex Tungesten blue(at 650- 750nm) (at 546nm)

LOWRY’s Method BIURET’s Method Sensitivity: 100 times > Biuret’s good for Pr. 2-12 g

Specificity: Less specific specific

No. of reagents: 2 Reagents One reagent

Drug Interference Dependence on Tryptophan&Tyrosine (salicylates,sulfa&tetracyclines) e.g Alb.=0.2 % Tryptophan by wt. Glob.=2% Tryptophan by wt.

PRINCIPLE OF THE METHOD Proteins give an intensive violet-blue complex with copper salts in an alkaline medium. Iodide is included as an antioxidant. The intensity of the color formed is proportional to the total protein concentration in the sample

CLINICAL SIGNIFICANCE The proteins are macromolecular organic compounds, widely distributed in the organism. They act like structural and transport elements. The proteins of the serum are divide in two fractions, albumin and globulins The determination of total proteins is useful in the detection of: - High protein levels caused by hemoconcentration like in the dehydrations or increase in the concentration of specific proteins. - Low protein level caused by hemodilution by an impared synthesis or loss (as by hemorrhage) or excessive protein catabolism. Clinical diagnosis should not be made on a single test result; it should integrate clinical and other laboratory data

PREPARATION The reagents are ready to use

Signs of reagent deterioration: 1- Presence of particles and turbidity. 2- Blank absorbance (A) at 540 nm 0.22. SAMPLES Serum or heparinized plasma: Stability of the sample: 1 month at refrigerator (2- 8ºC).

PROCEDURE 11. Assay conditions: Wavelength: ...... 540 (530-550) nm Cuvette: ...... 1 cm. light path Temperature ...... 37ºC / 15-25ºC 12. Adjust the instrument to zero with distilled water. 23. Pipette into a cuvette:

Blan Standar Sampl k d e R (mL) 1.0 1.0 1.0 Standard ------25 ------(µL) Sample(µL) ------25

14. Mix and incubate 5 min at 37ºC or 10 min at room temperature. 25. Read the absorbance (A) of the samples and Standard, against the Blank. The colour is stable for at least 30 minutes.

CALCULATIONS (A) Samplex 7 (Standard conc.)= g/dL of total protein in the sample (A) Standard

REFERENCE VALUES Adults: 6.6 – 8.3 g/dL Newborn: 5.2 – 9.1 g/dL

Bilirubin estimation

Introduction:

Bilirubin is the end product of red cell lysis and recycling of hemoglobin which is performed in the liver. The test quantifies two different forms of bilirubin, one is the final product while the other is an intermediate form. The build up of bilirubin in the blood stream is called jaundice and is a general sign of liver disease. Many medications, gall bladder disease as well as viruses such as infectious mononucleosis and hepatitis will have jaundice. Many infants are born with less than fully mature livers. As a consequence, for the first several days, they may show "neonatal jaundice" which is a build up of bilirubin in the blood stream. This should go away as the liver matures. Bilirubin determinations are used to study liver function and red cell metabolism

CLINICAL SIGNIFICANCE Bilirubin is a breakdown product of hemoglobin. It is transported from the spleen to the liver and excreted into bile. Hyperbilirubinemia results from the increase of bilirubin concentrations in plasma. Causes of hyperbilirubinemia: Total bilirubin (T): Increase hemolysis, genetic errors, neonatal jaundice, ineffective erythrpoiesis, and drugs. Direct bilirubin (D): Hepatic cholestasis, genetic errors, hepatocellular damage1,5,6. Clinical diagnosis should not be made on a single test result; it should integrate clinical and other laboratory data.

Methods for Bilirubin estimation ======

1- Direct Spectrophotometry:

*Restricted to newborn(< 28 days) up to 3 months, because their serum contains no Carotenes. *Measuring absorbency of Bilirubin in serum at 2 wave lengths 450 & 540 nm The difference in the absorbance represents bilirubin absorbance (A450 - A540 ) *That is because Hemoglobin reads the same at both W.L while bilirubin reads at 450 nm.

2- Direct Skin Bilirubinometer:

*Restricted also to newborns up to 3 months. *Needs calibration using:-Methyl Orange solution. Or -Filter Multilayered color glass. A450 - A 540 = Absorbance of bilirubin

3- Spectral shift change method: *Spectral shift through adding hydrophobic cationic polymer. *Used by KODAK ECTACAM only.

4-HPLC(High Purity Liquid Chromatography):

 Using Normal Silica Chromatography .  The Reference method.

5-Colometric methods (Experiment # 8): *The most commonly used methods.  Depend on reaction of bilirubin& Diazotized Sulfanilic acid (DSA)

MELLOY &EVELYN M. JENDRASSIL-GROF M.

*Accelerator: Methanol or Urea Caffeine &Na.Benzoate *PH : Neutral Alkaline(PH=12) *W.L : 660 560 nm *Color : Red purple red

Coloumetric Reaction: *Bil.Glucuronides + DSA Azobilirubin +H2O+Co2+Hcl (Direct Bilirubin) *Bil.Glucuronides +DSA +Accelerator Azobilirubin +H2O+Co2+Hcl(Total Bilirubin)

6-Bilirubin Oxidase Method :

*Specific for Bilirubin only. *Bil. +Bil. Oxidase Biliverdin (measured at 405 nm)

PRINCIPLE OF THE METHOD Bilirubin is converted to colored azobilirubin by diazotized sulfanilic acid and measured photometrically. Of the two fractions presents in serum, bilirubin-glucuromide and free bilirubin loosely bound to albumin, only the former reacts directly in aqueous solution (bilirubin direct), while free bilirubin requires solubilization with dimethylsulfoxide (DMSO) to react (bilirubin indirect). In the determination of indirect bilirubin the direct is also determined, the results correspond to total bilirubin. The intensity of the color formed is proportional to the bilirrubin concentration in the sample

PREPARATION All the reagents are ready to use

Signs of reagent deterioration: 1- Presence of particles and turbidity. 2- Color development in R 2.

Specimen Precautions: ======1- Serum or Plasma 2- Avoid Hemolysis 3- Avoid light exposure 4- Storage for 3 days in dark & refrigerator (for months if freezed at – 70˚ C) 5- Urine sample either Random or 24 hrs. not stored for >24 hrs.

PROCEDURE 11. Assay conditions: Wavelength: ...... … . . . . 555 nm (530-580) Cuvette: ...... … … . . .1 cm light path Temperature ...... 15-25ºC 12. Adjust the instrument to zero with distilled water. 23. Pipette into a cuvette:

Direct Blan Total Blan k B .k 1.5 1.5 -- -- R 1 (D) ((mL -- 1.5 1.5 R 2 (T) ((mL 50 -- 50 -- ( R 3 (µL 100 100 100 100 Sample

1 4. Mix and incubate exactly for 5 minutes at 15-25ºC. 25. Read the absorbance (A).

CALCULATIONS With Factor: ((A) Sample - (A) Sample Blank) x Factor* = mg/dL bilirubin in the sample : Theoretical factor: Bilirubin (T) = 19,1 ; Bilirubin (D) = 14 Conversion factor: mg/dL x 17.1 =µ mol/L.

REFERENCE VALUES

Bilirubin Total Up to 1.10 mg/dL=18.81 µmol/L Bilirubin Direct Up to 0.25 mg/dL=4.27µ mol/L

Questions:

- Write causes of jaundice - What are the commonest methods of estimating serum bilirubin in neonates? - Mention the different causes of elevated direct and indirect bilirubins.

Catalytic (Enzymatic) activities of Liver (ELFT)

1-Gamma Glutamyl Transferase (GGT)

Introduction: This enzyme used to metabolize materials in the kidney, liver, gall bladder, and pancreas. It is an exceptionally sensitive indicator of stress in these sites. As a consequence, variations in results may be quite common. Alcohol consumption (even a little) and many medications are the chief causes of these swings. This test is used to follow kidney, liver or pancreatic function

PRINCIPLE OF THE METHOD (Kinetic test (Szasz)

Gamma-glutamyl transferase (γ-GT) catalyses the transfer of γ-glutamyl group from γ-glutamyl-p-nitroanilide to acceptor glycylglycine, according to the following reaction: γ--L-Glutamyl-3-carboxy-4-nitroanilide + Glycylglycine γ-GT γ-L-Glutamyl-glycylglycine + 2-Nitro-5-aminobenzoic acid The rate of 2-nitro-5-aminobenzoic acid formation, measured photometrically, is proportional to the catalytic concentration of γ-GT present in the sample

CLINICAL SIGNIFICANCE Gamma-glutamyl transferase (γ-GT) is a cellular enzyme with wide tissue distribution in the body, primarily in the kidney, pancreas, liver and prostate. Measurements of gamma-glutamyl transferase (γ-GT) activity are used in the diagnosis and treatment of hepatobiliary diseases such biliary obstruction, cirrhosis or liver tumours Clinical diagnosis should not be made on a single test result; it should integrate clinical and other laboratory data.

PREPARATION Working reagent (WR): Dissolve one tablet of R 2 Substrate in one vial of R 1 Buffer. Cap and mix gently to dissolve contents. Stability: 21 days at 2-8ºC or 5 days at room temperature (15-25ºC).

Signs of reagent deterioration: 1- Presence of particles and turbidity. 2- Blank absorbance (A) at 405 nm ≥ 1.20.

SAMPLES Serum. γ−GT is stable for at least 3 days at 2-8ºC, 8 hours at 15-25ºC and 1 month at – 20ºC.

PROCEDURE 1. Assay conditions: Wavelength: ...... 405 nm Cuvette: ...... 1 cm light path Constant temperature ...... 25ºC /30ºC / 37ºC 2. Adjust the instrument to zero with distilled water or air. 3. Pipette into a cuvette: 14. Mix, wait for 1 minute. 25. Read initial absorbance (A) of the sample, start the stopwatch and read absorbances at 1 minute intervals thereafter for 3 minutes. 36. Calculate the difference between absorbances and the average absorbance differences per minute (∆A/min).

CALCULATIONS

Mean A= (ΔA)/min = (A1+A2+A3) / 3 Enzyme activity (U/L) = Δ A X Factor

∆A/min x 1190 = U/L of γ-GT

Units: One international unit (IU) is the amount of enzyme that transforms 1 µmol of substrate per minute, in standard conditions. The concentration is expressed in units per litre of sample (U/L).

REFERENCE VALUES

30ºC 25ºC 5-25 4-18 Wom U/L U/L en 8-38 6-28 Men U/L U/L 2- Alanine Transaminase (ALT)

PRINCIPLE OF THE METHOD Alanine aminotranferase (ALT) o Glutamate pyruvate transaminase (GPT) catalyses the reversible transfer of an amino group from alanine to α-ketoglutarate forming glutamate and piruvate. The piruvate produced is reduced to lactate by lactate dehydrogenase (LDH) and NADH: α-ketoglutarate + L-Alanine ALT (GPT) L-Glutamate + Pyruvate

Pyruvate + NADH+H+ Lactate dehydrogenase (LDH) L-Lactate + NAD

The rate of decrease in concentration of NADH, measured photometrically, is proportional to the catalytic concentration of ALT present in the sample

CLINICAL SIGNIFICANCE The ALT is a cellular enzyme, found in highest concentration in liver and kidney. High levels are observed in hepatic disease like hepatitis, diseases of muscles and traumatisms, its better application is in the diagnosis of the diseases of the liver. When they are used in conjunction with AST aid in the diagnosis of infarcts in the myocardium, since the value of the ALT stays within the normal limits in the presence of elevated levels of AST Clinical diagnosis should not be made on a single test result; it should integrate clinical and other laboratory data.

PREPARATION Working reagent (WR): Dissolve one tablet of R2 Substrate in one vial of R1. Cap and mix gently to dissolve contents. Stability: 21 days at 2-8ºC or 72 hours at room temperature (15-25ºC).

Signs of reagent deterioration: 1- Presence of particles and turbidity. 2- Blank absorbance (A) at 340 nm < 1.00.

SAMPLES Serum or plasma: Stability 7 days at 2-8ºC.. PROCEDURE 1. Assay conditions: Wavelength: ...... 340 nm Cuvette: ...... 1 cm light path Constant temperature ...... 25ºC / 30ºC / 37ºC 2. Adjust the instrument to zero with distilled water or air. 3. Pipette into a cuvette:

14. Mix, incubate for 1 minute. 25. Read initial absorbance (A) of the sample, start the stopwatch and read absorbances at 1-minute intervals thereafter for 3 minutes. 36. Calculate the difference between absorbances and the average absorbance differences per minute (∆A/min).

CALCULATIONS ΔA (mean difference of readings) = (A1 +A2+A3) / 3 ALT enzyme activity (U/L) = ΔA X Factor (F1) ∆A/min x 1750 = U/L of ALT Units: One international unit (IU) is the amount of enzyme that transforms 1 µmol of substrate per minute, in standard conditions. The concentration is expressed in units per litre of sample (U/L).

REFERENCE VALUES 25ºC 30ºC 37ºC Men up to 22 U/L 29 U/L 40 U/L Women up to 18 U/L 22 U/L 32 U/L Normal newborns have been reported to show a reference range of up to double the adult, attributed to the neonate’s hepatocytes. These values decline to adult levels by approximately 3 months of age.

3- Aspartate Transaminase (AST)

Introduction:

Aspartate Transaminase (AST) is also known by its older name, SGOT, this enzyme is needed in the utilization of energy sources. It is found in high concentrations in muscle (cardiac and others), liver, and other organs. This test usually is ordered to follow cardiac and muscle disease .

This test can be performed on specimens from patients who are either in a fasting or non fasting. Adult reference ranges vary widely with different instruments.

PRINCIPLE OF THE METHOD Aspartate aminotransferase (AST) formerly called glutamate oxaloacetate (GOT) catalyses the reversible transfer of an amino group from aspartate to α- ketoglutarate forming glutamate and oxalacetate. The oxalacetate produced is reduced to malate by malate dehydrogenase (MDH) and NADH: α-ketoglutarate + L-Aspartate AST (GOT) L-Glutamate + Oxaloacetate

Oxaloacetate + NADH+H+ Malate dehydrogenase (MDH) Malate+ NAD The rate of decrease in concentration of NADH, measured photometrically, is proportional to the catalytic concentration of AST present in the sample.

CLINICAL SIGNIFICANCE The AST is a cellular enzyme, is found in highest concentration in heart muscle, the cells of the liver, the cells of the skeletal muscle and in smaller amounts in other weaves. Although an elevated level of AST in the serum is not specific of the hepatic disease, is used mainly to diagnostic and to verify the course of this disease with other enzymes like ALT and ALP. Also it is used to control the patients after myocardial infarction, in skeletal muscle disease and other Clinical diagnosis should not be made on a single test result; it should integrate clinical and other laboratory data.

PREPARATION Working reagent (WR): Dissolve one tablet of R.2 Substrate with one vial of R1 Buffer. Cap and mix gently to dissolve contents. Stability: 21 days at 2-8ºC or 72 hours at room temperature (15-25ºC).

Signs of reagent deterioration: 1- Presence of particles and turbidity. 2- Blank absorbance (A) at 340 nm < 1.00.

SAMPLES Serum or plasma: Stability 7 days at 2-8ºC..

PROCEDURE 1. Assay conditions: Wavelength: ...... 340 nm Cuvette: ...... 1 cm. light path Constant temperature ...... 25ºC /30ºC / 37ºC 2. Adjust the instrument to zero with distilled water or air. 3. Pipette into a cuvette:

14. Mix, incubate for 1 minute. 25. Read initial absorbance (A) of the sample, start the stopwatch and read absorbances at 1 minute intervals thereafter for 3 minutes. 36. Calculate the difference between absorbances and the average absorbance differences per minute (∆A/min).

CALCULATIONS ΔA (mean difference of readings)/min. = (A1 +A2+A3) / 3 AST enzyme activity (U/L) = ΔA X Factor (F1) ∆A/min x 1750 = U/L of AST Units: One international unit (IU) is the amount of enzyme that transforms 1 µmol of substrate per minute, in standard conditions. The concentration is expressed in units per litre of sample (U/L).

REFERENCE VALUES 25ºC 30ºC 37ºC Men up to 19 U/L 26 U/L 38 U/L Women up to 16 U/L 22 U/L 31 U/L

Bone Profile Testes

Calcium Determination

Introduction:

Calcium is required for cell function overall and for bone metabolism. Too little calcium gets you either a loss of tissue function or soft bones (osteoporosis) while too much gives you tetni ( cardiac arrest and/or lock jaw is from over clenching of muscles) or over brittle bones. Changes in calcium are used to assess bone function. Higher blood levels usually mean lower bone levels. Usually performed in conjunction with Phosphorous determinations.

OBJECTIVES:

-Ionized calcium constitutes 48 to 52 % of the total calcium, the un-ionized diffusible form constitutes 5 % approx. and about 43 – 47 % of the total plasma calcium is protein bound, primarily to albumin, but also to some extant to the α-, β- and γ-globulins.

-To know the status body calcium (Tetany) METHODS:

i. Chelation with o-Cresolphthalein Complexone(Colorimetric) ii. Atomic absorption Spectrophotometry (AAS) iii. Flame photometer iv. ISE PRINCIPLE OF THE METHOD The measurement of calcium in the sample is based on formation of color complex between calcium and o-cresolphtalein in alkaline medium: Ca++ + o-Cresolphtalein OH Colored complex O-Cresolphthalein Complex one gives violet color in alkaline medium. The intensity of the colour formed is proportional to the calcium concentration in the sample

CLINICAL SIGNIFICANCE Calcium is the most abundant and one of the most important minerals in the human body. Approximately 99% of body calcium is found in bones. A decrease in albumin level causes a decrease in serum calcium. Low levels of calcium are found in hypoparathyroidism, pseudohypoparathyroidism, vitamin D deficiency, malnutrition and intestinal malabsortion. Among causes of hypercalcemia are cancers, large intake of vitamin D, enhaced renal retention, osteoporosis, sarcosidosis, thyrotoxicosis, hyperparathyroidism. Clinical diagnosis should not be made on a single test result; it should integrate clinical and other laboratory data.

PREPARATION All the reagents are ready to use. To prepare monoreagent, mix according to this proportion: 50 vol. of R1 and 1 vol. of R2.

SAMPLES - Serum or plasma: Separated from cells as rapidly as possible. Blood anticoagulants with oxalate or EDTA are not acceptable since these chemicals will strongly chelate calcium. - Urine: Collect 24 hour urine specimen in calcium free containers. The collecting bottles should contain 10 ml of diluted Nitric acid (50% v/v). Record the volume. Dilute a sample 1/2 in distilled water. Mix. Multiply results by 2 (dilution factor). Stability of the samples: Calcium is stable 10 days at 2-8ºC.

PROCEDURE 1. Assay conditions: Wavelength: ...... 570 nm (550-590) Cuvette: ...... 1 cm. light path Temperature ...... 37ºC / 15-25ºC 2. Adjust the instrument to zero with distilled water. 3. Pipette into a cuvette:

Blank Standar Sampl d e R1 (mL) 2.0 2.0 2.0 R2 (2 drop) 1 1 1 Standard ----- 20 ----- (µL) Sample (µL) ------20

4. Mix and incubate for 5 min at 37ºC / 15-25ºC. 5. Read the absorbance (A) of the samples and calibrator, against the Blank. The color is stable for at least 40 minutes.

CALCULATIONS Serum and plasma (A) Sample x 10 (Standard conc.) = mg/dL calcium (A) Standard Conversion factor: mg/dL x 0.25= mmol/L.

REFERENCE VALUES Serum or plasma: Adults 8.5-10.5 mg /dL = 2.1-2.6 mmol/L Children 10 -12 mg/dL = 2.5 - 3 mmol/L Newborns 8 -13 mg/dL = 2 - 3.25 mmol/L

RESULTS:

CLINICAL SIGNIFICANCE:

* HYPOCALCEMIA 1. Vitamin D deficiency 2. Hypoparathyroidism 3. Alkalosis (Alkalemia)

* HYPERCALCEMIA 1. Hyperparathyroidism 2. Malignancy of bone 3. Thyrotoxicosis 4. Vitamin D intoxication 5. Idiopathic

DISCUSSION:

TETANY

PRECAUTIONS:

1. Avoid venous stasis (Increase protein & calcium) 2. Do not use contaminated glass ware (Increase calcium) 3. Lipemic Samples (Prepare blank 0.05 ml sample + 2.5 D.W)

QUESTIONS:

1. What is HYPOCALCEMIA? Write a short note on Tetany. 2. Give the principle for the determination of serum calcium by colorimetric method. 3. Enumerate different methods for the determination of serum calcium.

Quantitative determination of phosphorus

PRINCIPLE OF THE METHOD Inorganic phosphorus reacts with molybdic acid forming a phosphomolybdic complex. Its subsequent reduction in alkaline medium originates a blue molybdenum colour. The intensity of the color formed is proportional to the inorganic phosphorus concentration in the sample

CLINICAL SIGNIFICANCE Phosphorus is an essential mineral for tissue bone formation and is required by every cell in the body for normal function. Approximately 85% of the body phosphorus is found in bone and in teeth. Low levels of phosphorus, can be caused by hypervitaminosis 0, primary hyperparathyroidism, renal tubular disorders, antacids or malabsortion. High levels of phosphorus can be caused by diet, bone metastases, liver disease, alcohol ingestion, diarrhea and vomiting Clinical diagnosis should not be made on a single test result; it should integrate clinical and other laboratory data.

PREPARATION Working reagent (WR): Mix equal volumes of R 1 (Molybdic) and R 2 (Catalyzer) Stability: 24 h at 2-8C, protected from light.

SAMPLES - Serum: Free of hemolysis. Serum should be removed from the clot as quickly as possible to avoid elevation of serum phosphorus from hydrolysis or leakage of phosphate present in erythrocytes. Stabilitr 7 days at 2-8C. - Urine· (24 h): Collect the specimen into a bottle containing 10 mL of 10% v/v hydrochloric acid (HCI) to avoid phosphate precipitations. Adjust to pH 2. Dilute the sample 1/10 with distilled water. Mix. Multiply the result by 10 (dilution factor). Stability: 10 days at 2-BoC. PROCEDURE 1- Assay conditions: Wavelength: ...710 nm (620-750) Cuvette: ...... 1 cm. light path Temperature ...... 37°C 1 15-25°C 2- Adjust the instrument to zero with distilled water. 3- Pipette into a cuvette:

Sample Standard Blank 1.5 1.5 1.5 (WR (mL -- 50 - (Standard (µL 50 -- -- (Sample (µL 4. Mix and incubate for 10 min at 37°C or 30 min at room temperature (15-30°C). 5- Read the absorbance (A) of the samples and calibrator, against the Blank. The colour is stable for at least 2 hours.

CALCULATIONS Serum

(A) Sample x 5 (Calibrator cone.) = mg/dL of phosphorus in the sample (A) Calibrator Conversion factor: mg/dL x 0.323 = mmol/L. REFERENCE VALUES Serum: Children : 4.0 - 7.0 mg/dL = (1.3 - 2.2 mmol/L) Adults : 2.5 - 5.0 mg/dL = (O.8 - 1.8 mmo/lL)

Quantitative determination of alkaline phosphatase (ALP)

PRINCIPLE OF THE METHOD Alkaline phosphatase (ALP) catalyses the hydrolysis of p-nitrophenyl phosphate at pH 10.4, liberating p-nitrophenol and phosphate, according to the following reaction: p-Nitrophenylphosphate + H20 p-Nitrophenol + Phosphate The rate of p-Nitrophenol formation, measured photometrically, is proportional to the catalytic concentration of alkaline phosphatase present in the sample

CLINICAL SIGNIFICANCE Alkaline phosphatase is an enzyme present in almost all weaves of the organism, being particularly high in bone, liver, placenta, intestine and kidney.

Both increases and decreases of plasma ALP are of importance clinically. Causes of increased plasma ALP: Paget's disease of bone, obstructive liver disease, hepatitis, hepatotoxicity caused by drugs or osteomalacia. Causes of decreased plasma ALP: Cretinism and vitamin C deficiency1,5,6. Clinical diagnosis should not be made on a single test result; it should integrate clinical and other laboratory data. PREPARATION working reagent (WR): Dissolve one tablet of R 2 Substrate in one vial of R 1 Buffer. SAMPLES Serum or heparinzed. plasma _Use unhemolyzed .serum, separated from the clot as soon as possible. Stability: 3 days at 2-8°C.

PROCEDURE 1-Assay conditions: Wavelength: ...... 405 nm Cuvette: ...... 1 cm light path Constant temperature ...... 25°C 30°C 37°C 2-Adjust the instrument to zero with distilled water or air. 3-Pipette into a cuvette:

WR (mL) 1.2

(µL)Sample 20

4. Mix, incubate for 1 minute.

5-Read initial absorbance (A) of the sample, start the stopwatch and read absorbances at 1 minute intervals thereafter for 3 minutes. 6-Calculate the difference between absorbances and the average absorbance differences per minute (ΔA/min).

CALCULATIONS (ΔA/min) x 3300 = U/L de ALP Units: One international unit (IU) is the amount of enzyme that transforms 1 µmol of substrate per minute, in standard conditions. The concentration is expressed in units per litre of sample (U/L).

REFERENCE VALUES1 25°C 30°C 37°C Children (1-14 years) < 400 U/L < 480 U/L < 645 U/L Adults 60 - 170 U/L 73 - 207 U/L 98 - 279 U/L Factors affecting ALP activities in a normal population include exercise, periods of repaid growth in children and pregnancy.

Cardiac profile Testes

Quantitative determination of creatin kinase (CK)

CLINICAL SIGNIFICANCE Creatine kinase is a cellular enzyme with wide tissue distribution in the body. Its physiological role is associated with adenosine triphosphate (ATP) generation for contractile or transport systems. Elevated CK values are observed in diseases of skeletal muscle and after myocardial infarction Clinical diagnosis should not be made on a single test result; it should integrate clinical and other laboratory data.

PRINCIPLE OF THE METHOD Creatine kinase (CK) catalyses the reversible transfer of a phosphate group from phosphocreatine to ADP. This reaction is coupled to those catalysed by hexokinase (HK) and glucose-6-phosphate dehydrogenase (G6P-DH): Phosphocreatine + ADP CK Creatine + ATP ATP + Glucose HK ADP + Glucose-6-phosphate G6P + NADP + G6P-DH 6-Phosphogluconate + NADPH + H + The rate of NADPH formation, measured photometrically, is proportional to the catalytic concentration of CK present in the sample

PREPARATION Working reagent (WR): Dissolve 1 tablet of R 2 Substrate with one vial of R 1. Cap vial and mix gently to dissolve contents. Stability: 5 days at 2-8ºC or 24 hours at room temperature (15-25ºC).

STORAGE AND STABILITY All the components of the kit are stable until the expiration date on the label when stored tightly closed at 2-8ºC, protected from light and contaminations prevented during their use. Do not use the tablets if appears broken. Do not use reagents over the expiration date.

Signs of reagent deterioration: 1- Presence of particles and turbidity. 2- Blank absorbance (A) at 340 nm ≥ 1.60.

SAMPLES Serum or plasma: Stability 7 days at 2-8ºC, protected from light. The creatin kinase activity decreases 10% after 1 day at 2-5ºC or after 1 hour at 15-25ºC.

PROCEDURE 1. Assay conditions: Wavelength: ...... 340 nm Cuvette: ...... 1 cm light path Constant temperature ...... 25ºC / 30ºC / 37ºC 2. Adjust the instrument to zero with distilled water or air. 3. Pipette into a cuvette:

25 - 30ºC 1.0 WR (mL) Sample (µL)

14. Mix, incubate for 2 minutes. 25. Read initial absorbance (A) of the sample, start the stopwatch and read absorbances at 1 minute intervals thereafter for 3 minutes. 36. Calculate the difference between absorbances and the average absorbance differences per minute (∆A/min).

CALCULATIONS

∆A / min x 4127 = U/L CK ∆A / min x 8095 = U/L CK

Units: One international unit (IU) is the amount of enzyme that transforms 1 µmol of substrate per minute, in standard conditions. The concentration is expressed in units per litre of sample (U/L).

REFERENCE VALUES 25ºC 30ºC 37ºC Men, up to 80 U/L 130 U/L 195 U/L Women, up to 70 U/L 110 U/L 170 U/L Quantitative determination of creatine kinase MB (CK-MB)

PRINCIPLE OF THE METHOD An antibody to the anti CK-M inhibits completely CK-MM and subunit (M) of the CK- MB. The activity of the non-inhibited CK-B subunit is then assayed by the following series of reactions: Phosphocreatine + ADP CK Creatine + ATP ATP + Glucose HK ADP + Glucose-6-phosphate G6P + NADP + G6P-DH 6-Phosphogluconate + NADPH + H + The rate of NADPH formation, measured photometrically, is proportional to the catalytic concentration of CK-B present in the sample

CLINICAL SIGNIFICANCE CK-MB is an enzyme formed by the association of two subunits from muscle (M) and nerve cells (B). CK-MB is usually present in serum at low concentration; it is increases after an acute infarct of myocardium and later descends at normal levels. Also is increased, rarely, in skeletal muscle damage. Clinical diagnosis should not be made on a single test result; it should integrate clinical and other laboratory data.

PREPARATION - Working reagent (WR) Dissolve one tablet of R 2 in one vial of R 1. Cap vial and mix gently to dissolve contents. Stability: 8 days at 2-8ºC or 24 hours at 15-25ºC.

Signs of reagent deterioration: 1- Presence of particles and turbidity. 2- Blank absorbance (A) at 340 nm≥ 1.60.

SAMPLES Serum or plasma: Stability 7 days at 2-8ºC, protected from light. CK-MB activity decreases a 10% after 24 hours at 4ºC or 1 hour at 25ºC. PROCEDURE 1. Assay conditions: Wavelength: ...... 340 nm Cuvette: ...... 1 cm light path Constant temperature ...... 25ºC / 30ºC / 37ºC 2. Adjust the instrument to zero with distilled water or air. 3. Pipette into a cuvette: 1. (WR (mL 0 40 Sample ((µL

4. Mix. Incubate for 10 minute. 5. Read initial absorbance (A) of the sample, start the stopwatch and read again after 5 minutes (A2). 6. Calculate the difference between absorbances : ΔA= A2 – A1.

CALCULATIONS

ΔA x 1651 = U/L de ΔA x 825 = U/L de CK-MB CK-B

Units: One international unit (IU) is the amount of enzyme that transforms 1 mol of substrate per minute, in standard conditions. The concentration is expressed in units per litre of sample (U/L).

Percentage of CK-MB activity in sample:

CK-MB Activity % CK-MB Activity = x 100 CK total Activity

REFERENCE VALUES

Heart infarct probability is high at the following conditions: 25ºC 30ºC 37ºC CK-MB > 10 U/L > 15 U/L > 24 U/L CK-MB activity is between 6 and 25% of total CK activity.

Amylase - blood

Amylase is an enzyme that helps digest carbohydrates. It is produced in the pancreas and the glands that make saliva. When the pancreas is diseased or inflamed, amylase releases into the blood.

A test can be done to measure the level of this enzyme in your blood.

Amylase may also be measured with a urine test.

No special preparation is needed. However, you should avoid alcohol before the test. The health care provider may ask you to stop taking drugs that may affect the test. NEVER stop taking any medications without first talking to your doctor.

Drugs that can increase amylase measurements include:

 Asparaginase  Aspirin  Birth control pills  Cholinergic medications  Ethacrynic acid  Methyldopa  Opiates (codeine, meperidine, morphine)  Thiazide diuretics What Abnormal Results Mean

Increased blood amylase levels may occur due to:

 Acute pancreatitis  Cancer of the pancreas, ovaries, or lungs  Cholecystitis  Gallbladder attack caused by disease  Gastroenteritis (severe)  Infection of the salivary glands (such as mumps) or a blockage  Intestinal blockage  Macroamylasemia  Pancreatic or bile duct blockage  Perforated ulcer

 Tubal pregnancy (may have burst open)

Decreased amylase levels may occur due to:

 Cancer of the pancreas  Damage to the pancreas  Kidney disease  Toxemia of pregnancy

Appendix (1)

Collective Knowledge of Most Common Lab.Tests

Blood Tests

Glucose: Glucose is the primary blood sugar test and indicates blood sugar level at the time blood was drawn. High values are seen in diabetics. In addition to pancreatic functions, Glucose may be altered by diet and medication. Normal fasting value is 70-110.

Fructosamine: Indicates blood sugar levels over the past one to three weeks.

HGB A1C (Glycohemoglobin): Indicates blood sugar activity for the past three months.

BUN: BUN stands for Blood Urea Nitrogen and is a waste product which should be removed from the blood by the kidneys. This test measures kidney function. Normal range is 6-20.

Creatinine: Creatinine is a waste product which should be removed from the blood by the kidneys. This test measures kidney function. Normal range is 0.5-1.2.

ASAT/ALT: Material found in the liver cells and muscle (heart) cells. Damage to these cells will increase values. Normal range is 10-60.

LDH: LDH is a material found in blood cells and liver cells. Breakdown of the blood cells as in heart disease or liver damage may increase values. Normal range is 91-180.

Alkaline Phosphorus: A material found in the blood related to liver and bone. Normal range for adult males is 20-125; normal range for adult females is 42-124.

SGOT, SGPT: Two measures of liver function; occasionally affected by muscle injury.

GGTP: The earliest liver function to become abnormal. Total Bilirubin: The level of pigment in the blood. Elevations can be associated with liver disease or breakdown or red blood cells. Slight increases are sometimes seen without significance. Some people normally have isolated elevations of bilirubin called Gilbert's disease. Normal range is 1.0-1.2.

Total Protein: This is a combination of albumin and globulin, which are proteins. Abnormal values occur in liver disease and poor nutrition. Normal range is 6.7-8.0.

Globulin: Globulin helps to combat infection on a normal level. It is the total protein value minus albumin value. Normal range is 2.3-4.0.

A/G Ratio: Albumin value divided by the globulin value. Normal range is 0.8-2.4.

Calcium: The most abundant mineral found in the human body. Abnormalities are found in loss of bone, kidney disease and lack of Vitamin D. Normal range is 8.5-10.5.

Phosphorous: Related to bone activity and usually follows exact opposite of calcium. Normal range is 2.5-4.6.

Uric Acid: A material which, if in excess, can deposit stones in the kidney or in the joints and cause gout. Normal range for males is 4.0-7.0; normal range for females is 2.0-6.0.

Cholesterol: A blood fat related in part to eating animal fats such as eggs, cheese, cream, liver, pork, beef, etc. Increased values may indicate a tendency to have hardening of the arteries. Values of 180 or less are associated with least risk of heart disease; in addition to diet and exercise.

Lipoproteins: Proteins combined with lipids that serve as carriers of cholesterol. LDL ("Bad" Cholesterol); HDL ("Good" Cholesterol). The higher the value, the less likely that cholesterol deposits are in the blood stream and the less likely the chance of coronary heart disease. Cholesterol/HDL ratio measures the coronary risk factors.

Triglycerides: A blood fat related to calories and starch (sweets) in the diet. High levels can impair circulation and lead to hardening of the arteries. Alcohol also will increase the value. Fast overnight test for accurate test results. Normal range for males is 40-160; normal range for feales is 35-135.

Magnesium: An element absorbed in the intestine. Abnormal levels are found in pancreatitis, alcoholism and Addison's disease. Normal range is 1.8-2.4. Socium: A body salt, also termed electrolyte. Kidney disease and some diseases of the adrenal gland and dehydration can cause abnormal results. Normal range is 135-145.

Potassium: A body salt or electrolyte found mostly inside of cells. "Water pills" may lower potassium and increase kidney damage. Normal range is 3.6-5.0.

Chloride: A body salt/electrolyte, it usually follows the same pattern as sodium. Normal range is 101-111.

Co2: Buffer system which assists in the transport of carbon dioxide from the tissue to the lungs. Normal range is 21-31.

HIV antibody: Presence of antibody is associated with having been infected by the virus known to cause AIDS (Acquired Immune Deficiency Syndrome).

PSA: Abnormal levels in the serum are associated with clinical abnormalities of the prostate, including prostate cancer. Because PSA is found in normal, malignant and benign prostatic tissue, clinical discrimination is based upon its serum level.

Complete Blood Count

WBC (White Blood Cells): White blood count is the number of white blood cells. It helps combat infection. Normal range is 4.8-10.8.

RBC (Red Blood Cells): Red blood count is the number of red blood cells. It relates to anemia and oxygen transport. Normal range for males is 4.7- 6.1; normal range for females is 4.2-5.4.

HGB/HCT: Hemoglobin is an iron-bearing protein which is the red coloring matter found in blood. Normal range for males is 14-18; normal range for females is 12-16.

MCH/MCV/MCHC: Mathematical relationship between red blood count size, red blood count number and hemoglobin concentration.

Platelets: Platelets deal with hemostasis and blood coagulation. Normal range is 130-400.

Urine Tests

WBC (White Blood Cells): Indicates possible infection of urinary tract, bladder or kidney. RBC (Red Blood Cells): Possible kidney stone, kidney infection or tumor.

Casts: Possible kidney infection or disease.

Glucose: Sugar in the urine, possibility of glucose intolerance or low renal threshold.

Protein: Possible kidney infection or disease. Appendix (2)

Common Blood Profiles Reference values for the more commonly employed laboratory tests are given in the following table. The reference values are in the units currently often used and in the International System (SI) of Units. Test Current units Factor SI units

Diabetic Screen Glucose, fasting 65-110 mg/dl 0.055 3.57-6.05 mmol/L Glucose , random 71-180mg/dl 0.055 3.9-10.0 mmol/L Glycosylated 5.5 - 8.5% hemoglobin

( Hba1c )

Heart disease risk factors (fasting lipids ) Total Cholesterol <200 mg/dl 0.0259 <5.2 mmol/L HDL cholesterol >35 mg/dl 0.0259 >0.9 mmol/L LDL cholesterol <150 mg/dl 0.0259 <3.9 mmol/L Triglyceride <205 mg/dl 0.0113 <2.3 mmol/L Total cholesterol/HDL <5.8 1 <5.8 ratio

Liver function tests Total Bilirubin 0.25-1.5 mg/dl 17.1 4.3-25.6  mol/L Direct Bilirubin 0-0.5 mg/dl 17.1 0 - 8  mol/L Indirect Bilirubin 0-0.9 mg/dl 17.1 0-14  mol/L Total Protein 6.5-8.5 gm/dl 10 65-85 gm/L Albumin 3.5-4.8 gm/dl 0.154 0.54 - 0.74mmol/L Globulin 2.0-3.9 g/dl 10 20-39 g/L Albumin/Globulin ratio 1-2.5 1 1-2.5 g -Glutamy 11-50 IU/L 1.67 X 10-8 18-84 X 10-8 Katal/L transpeptidase (GGT) -Male g -Glutamy 7-35 IU/L 12-58 X 10-8 Katal/L transpeptidase (GGT) -Female Alkaline Phosphatase 45-125 IU/L(up 1.67 X 10-8 0.75-2.1 X 10-8 Katal/L to 1000 IU/L in young children) Alanine 5-35 IU/L 1.67 X 10-8 8.4 -58 X 10-8 Katal/L aminotransferase (SGPT / ALT) Aspartate 5-40 IU/L 1.67 X 10-8 8.4 -67 X 10-8 Katal/L aminotransferase (SGOT/ AST)

Renal/Kidney Function Tests Urea (BUN) 8-25 mg/dl 0.357 2.9-8.9 mmol/L Creatinine 0.6-1.7 mg/dl 88.4 53-150  mol/L Uric acid 3.5-8.0 mg/dl 0.059 0.21-0.47 mmol/L Potassium 3.3-4.9 mmol/L 1 3.3-4.9 mmol/L Sodium 135-145 1 135-145 mmol/L mmol/L Total Calcium 8.9-10.3 mg/dl 0.25 2.23-2.57 mmol/L Free Calcium 4.5-5.0 mg/dl 0.25 1.12-1.25 mmol/L Phosphate 2.5-4.5 mg/dl 0.323 0.8-1.5 mmol/L

Other Common Serum Chemistries/Enzymatic Activities Test Current units Factor SI units Ammonia (plasma) 11-35  mol/L 1 11-35  mol/L Blood gases (arterial, 7.35-7.45 1 7.35-7.45 whole blood) - pH Blood gases (arterial, 80-105 mmHg 0.133 10.6-14.0 kPa whole blood) - PO2 Blood gases (arterial, 35 - 45 mmHg 0.133 4.7-6.0 kPa whole blood) - PCO2 Blood gases (arterial, 22-31 mmol/L 1 22-31 mmol/L whole blood) -Carbon dioxide content ß-Carotene 50-300  g/dl 0.0186 0.9-5.6  mol/L Ceruplasmin 0.23-0.58 gm/L 6.7 1.5-3.9  mol/L Chloride 95-105 mEq/L 1 95-105 mmol/L Copper - Male 70-140  g/dl 0.157 11.0-22  mol/L Copper - Female 85-155  g/dl 0.157 13.3-24.3  mol/L Complement (total, 118- hemolytic) 226CH50U/ml C3 81-175 mg/dl 0.01 0.8-1.75gm/L C4 12-34 mg/dl 0.01 0.12-0.34 gm/L Creatinine Clearence 60-120 ml/min Ferritin - Children 13-145 ng/ml 2.25 29-326 pmol/L Ferritin -Male, adult 25-240 ng/ml 56-540 pmol/L Ferritin - Female, adult 12-130 ng/ml 27-292 pmol/L Fibrinogen 150-360 mg/dl 0.01 1.5-3.6 gm/L Folate - Plasma 1.7-12.6 ng/ml 2.27 3.9-29 nmol/L Folate - Red cell 153-602 ng/ml 347-1367 nmol/L Haptoglobin 100-300 mg/dl 0.01 1.10-3.00 gm/L Immunoglobulin - IgA 39-358 mg/dl 0.01 0.39-3.58 gm/L Immunoglobulin - IgM 33-229 mg/dl 0.01 0.33-2.29 gm/L Immunoglobulin - IgG 679-1537 0.01 6.79-15.37 gm/L mg/dl Iron - Male 80-160  g/dl 0.179 14.3-28.6  mol/L Iron - Female 60-135  g/dl 10.7-24.2  mol/L Iron - Binding capacity 250-350  g/dl 44.7-62.6  mol/L Iron - Transferrin 16-57% 1 16-57% saturation Lactate (plasma) 0.3-1.3 mmol/L 1 0.3-1.3 mmol/L Magnesium 1.5-2.1 mEq/L 0.5 0.7-1.1 mmol/L Osmolality 270-290 1 270-290 mOsm/kg mOsm/kg Protein electrophoresis - 0.1-0.5 gm/dl 10 1-5 gm/L Alpha- 1- globulin Protein electrophoresis - 0.3-1.2 gm/dl 10 3-12 gm/L Alpha -2- globulin Protein electrophoresis - 0.7-1.7 gm/dl 10 7-17 gm/L Beta globulin Protein electrophoresis - 0.7-1.7 gm/dl 10 7-17 gm/L Gamma globulin Vitamin A 30-95  g/dl 0.035 1.05-3.32  mol/L Vitamin B12 200-800 pg/ml 0.739 148-591 pmol/L

Other common serum enzymatic activities Test Current units Factor SI units Aldolase 1.5-8.1 IU/L 1.67 X 10-8 2.5-13.5 X 10-8 Katal/L Amylase 25-115 IU/L 1.67 X 10-8 42-192 X 10-8 Katal/L Creatine kinase - Male Up to 185 IU/L 1.67 X 10-8 Up to 309X10-8 Katal/L Creatine kinase - Up to 150 IU/L 1.67 X 10-8 Up to 251X10-8 Katal/L Female Lactic dehydrogenase 90-250 IU/L 1.67 X 10-8 150-417 X 10-8 Katal/L Lipase 4-24 IU/dl 10 40-240 IU/L 5' - Nucleotidase 2-16 IU/L 1.67 X 10-8 3-27 X 10-8 Katal/L Phosphatase, acid Up to 0.7 IU/L 1.67 X 10-8 Up to 1.2X10-8 Katal/L

Common Serum Hormone Values Test Current units Factor SI units ACTH, fasting (8 AM) 20-100 pg/ml 0.22 4.4-22 pmol/L Aldosterone 10-160 ng/L 2.77 28-443 mmol/L Cortisol (plasma, 8-25  g/dl 0.027 0.22-0.57  mol/L morning) FSH - Male Up to 20 IU/L 1 Up to 20 IU/L FSH Female - Follicular Up to 20 IU/L 1 Up to 20 IU/L FSH Female - Luteal Up to 15 IU/L 1 Up to 15 IU/L FSH Female - Midcycle 15-30 IU/L 1 15-30 IU/L FSH Female - >40 IU/L 1 >40 IU/L Postmenopausal Gastrin, fasting Up to 130 1 Up to 130 ng/L pg/ml Growth hormone, <5 ng/ml 1 <5  g/L fasting 17- 3-90 ng/dl Hydroxyprogesterone - Prepubertal 17- 27-199 ng/dl Hydroxyprogesterone - Male, adult 17- 15-70 ng/dl Hydroxyprogesterone - Female - Follicular 17- 35-290 ng/dl Hydroxyprogesterone - Female - Luteal Insulin, fasting (72 hr) <10 mU/L 1 <10 mU/L LH - Male Up to 25 IU/L 1 Up to 25 IU/L LH - Female -Follicular Up to 40 IU/L 1 Up to 40 IU/L LH - Female - Luteal Up to 25 IU/L 1 Up to 25 IU/L LH - Female - Midcycle 50-150 IU/L 1 50-150 IU/L LH - Female - >30 IU/L 1 >30 IU/L Postmenopausal Parathyroid hormone 2-10 U/ml 1 2-10 arb units Progesterone - Male Up to 100 ng/dl Progesterone - Female Up to 150 ng/dl -Follicular Progesterone - Female - 250-2800 ng/dl Luteal Progesterone - Female 1300-5000 -1st trimester ng/dl Prolactin - Male 2-12- ng/ml 1 2-12 ug/L Prolactin - Female 2-20 ng/ml 1 2-20 ug/L Renin activity (plasma) 0.9-3.3 0.278 0.2-0.9 ng/L.s ng/ml/hr Testosterone, total - 280-1000 ng/dl 0.0346 10-35 nmol/L Male Testosterone, total - 20 -120 ng/dl 1-4 nmol/L Female Testosterone, free - 52-280 pg/ml 0.00346 0.18-1.0 nmol/L Male Testosterone, free - 1.1-6.3 pg/ml 4-22 X 10-³ nmol/L Female Thyroxine, total ( T4 ) 5.0-11.0 ug/dl 12.9 64-142 nmol/L Thyroxine, free 1.0-2.3 ng/dl 12.9 13-30 pmol/L T3 resin uptake 35-45% 0.01 0.35-0.45 arb units Triiodothyronine (T3) 100-216 ng/dl 0.0154 1.54-3.23 nmol/L T4 index 1.75-4.95 1 1.75-4.95 arb units TSH Up to 10  1 Up to 10 mU/L U/ml Vitamin D, 1,25 20-76 pg/ml dihydroxy Vitamin D, 25 hydroxy 10-55 ng/m

Common Urinary Chemistries Test Current units Factor SI units -aminolevulinic acid 1.3-7.0 mg/day 7.6 9.9-53  mol/day Amylase 0.04-0.30 1 0.04-0.30 IU/min IU/min Calcium < 250mg/day 0.025 < 6.25 nmol/day Catecholamines < 135  g/day 1 Up to 135  g/day Dopamine 90-440  g/day Epinephrine < 13  g/day 5.5 Up to 71 nmol/day Norepinephrine 11-86  g/day 5.9 65-507 nmol/day Copper 15-50  g/day 0.0157 0.2-0.78  mol/day Cortisol, free 20-90  g/day 2.76 55- 248 nmol/day Creatinine - Male 1.0-2.0 gm/day 0.0088 0.009-0.018 mmol/day Creatinine - Female 0.6-1.5 0.005-0.013 mmol/day gm/day 5Hydroxyindoleacetic 1.8-6 mg/day 5.3 9.5-31.8  mol/day acid Hydroxyproline, total 25-77 mg/day 7.63 191-588  mol/day Metanephrine 0.3-1.0 mg/day Oxalate Up to 40 7.93 < 317  mol/day mg/day Porphyrin- 15-125 g/day 1.53 23-191 nmol/day Coproporphyrin Porphyrin-Uroporphyrin < 30  g/day 1.2 Up to 36 nmol/day Protein < 150 mg/day 0.001 Up to 0.150 gm/day Vanillylmandelic acid 0.5-7 mg/day 5.05 2.5-35.3  mol/day (VMA)

Common Hematologic Studies Test Current units Factor SI units Coagulation studies - 2.5-9.5 min 60 150-570 sec Bleeding time Coagulation studies 25-41 sec 1 25-41 sec -Partial thromboplastin time Coagulation studies - 10.8-13.0 sec 1 10.8-13.0 sec Prothrombin time Coagulation studies - 11.3-18.5 sec 1 11.3-18.5 sec Thrombin time Hematocrit - Male 40.7-50.3% 0.01 0.4-0.503 arb units Hematocrit - Female 36.1-44.3% 0.36-0.44 arb units Hemoglobin - Male 13.8-17.2 0.62 8.56-10.7 mmol/L gm/dl Hemoglobin - Female 12.1-15.1 7.50-9.36 mmol/L gm/dl Erythrocyte / RBC count 4.5-5.7 X106/ l 106 4.5-5.7 X 10¹²/L - Male Erythrocyte / RBC count 3.9- 5.0X106/ l 3.9-5.0 X 10¹²/L - Female Leukocyte count 3.8-9.8X 10³/ 106 3.8-9.8 X 109/L l Leukocyte profile - 1.2-3.3X 10³/ l Lymphocytes Leukocyte profile - 0.1-0.7X10³/ l Mononuclear cells Leukocyte profile - 1.8-6.6X 10³/ l Granulocytes Platelet count 190 -405 106 190-405 X 109/L X10³/ l Erythrocyte indices - 26.7-33.7 0.062 1.66-2.09 fmol/cell Mean corpuscular pg/cell hemoglobin Erythrocyte indices - 32.7-35.5 0.62 20.3-22.0 mmol/L Mean corpuscular gm/dl hemoglobin concentration Erythrocyte indices - 80.0-97.6 cu  80.0-97.6 fl Mean corpuscular volume Erythrocyte indices - 11.8-14.6% Red cell distribution width Erythrocyte Up to 30 1 Up to 30 mm/hr Sedimentation rate mm/hr Reticulocyte count 0.2-2.0%

Practical Sheet #( ) Practical Experiment: ...... ………………………………………………………………………………………………… ………………………

Objectives: …………………………………………………………………………………………………. …………………………………………………………………………………………………………… …………………………………………………………………………………………………………… …………………….…………………………………………….….

Principle: …………………………………………………………………………………………………. …………………………………………………………………………………………………………… …………………………………………………………………………………………………………… …………………….……………………. …………………………………………………………………………………………………. …………………………………………………………………………………………………………… …………………………………………………………………………………………………………… …………………….……………………. …………………………………………………………………………………………………. …………………………………………………………………………………………………………… …………………………………………………………………………………………………………… …………………….……………………. …………………………………………………………………………………………………. …………………………………………………………………………………………………………… …………………………………………………………………………………………………………… …………………….……………………. …………………………………………………………………………………………………. …………………………………………………………………………………………………………… …………………………………………………………………………………………………………… …………………….……………………. …………………………………………………………………………………………………. …………………………………………………………………………………………………………… …………………………………………………………………………………………………………… ………………….…………………….………………… Results: …………………………………………………………………………………………………. …………………………………………………………………………………………………………… …………………………………………………………………………………………………………… …………………….……………………. …………………………………………………………………………………………………. …………………………………………………………………………………………………………… …………………………………………………………………………………………………………… …………………….……………………. …………………………………………………………………………………………………. …………………………………………………………………………………………………………… …………………………………………………………………………………………………………… …………………….……………………. …………………………………………………………………………………………………. …………………………………………………………………………………………………………… …………………………………………………………………………………………………………… …………………….……………………. …………………………………………………………………………………………………. …………………………………………………………………………………………………………… …………………………………………………………………………………………………………… …………………….……………………. …………………………………………………………………………………………………. …………………………………………………………………………………………………………… …………………………………………………………………………………………………………… …………………….……………………. …………………………………………………………………………………………………. …………………………………………………………………………………………………………… …………………………………………………………………………………………………………… …………………….……………………. …………………………………………………………………………………………………. …………………………………………………………………………………………………………… …………………………………………………………………………………………………………… …………………….……………………. …………………………………………………………………………………………………. …………………………………………………………………………………………………………… …………………………………………………………………………………………………………… …………………….……………………. …………………………………………………………………………………………………. …………………………………………………………………………………………………………… …………………………………………………………………………………………………………… …………………

COMMENTS & QUESTIONS …………………………………………………………………………………………………. …………………………………………………………………………………………………………… …………………………………………………………………………………………………………… …………………….……………………. …………………………………………………………………………………………………. …………………………………………………………………………………………………………… …………………………………………………………………………………………………………… …………………….……………………. …………………………………………………………………………………………………. …………………………………………………………………………………………………………… …………………………………………………………………………………………………………… …………………….……………………. …………………………………………………………………………………………………. …………………………………………………………………………………………………………… …………………………………………………………………………………………………………… …………………….……………………. …………………………………………………………………………………………………. …………………………………………………………………………………………………………… …………………………………………………………………………………………………………… …………………….……………………. …………………………………………………………………………………………………. …………………………………………………………………………………………………………… …………………………………………………………………………………………………………… …………………….……………………. …………………………………………………………………………………………………. …………………………………………………………………………………………………………… …………………………………………………………………………………………………………… …………………….……………………. …………………………………………………………………………………………………. …………………………………………………………………………………………………………… …………………………………………………………………………………………………………… …………………….……………………. …………………………………………………………………………………………………. …………………………………………………………………………………………………………… …………………………………………………………………………………………………………… …………………….……………………. …………………………………………………………………………………………………. …………………………………………………………………………………………………………… …………………………………………………………………………………………………………… …………………….…………………….