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Estimation of Blood Glucose

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					                                      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 Biochemistry – Part II:

                                  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
          -Polarigraphic method:
           using O2 electrode to detect O2 utilization.

          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
        -D-glucose +H2O+O2          Glucose oxidase          D-gluconic acid+H2O2
        H2O2+ 4-aminophenazone+phenol                  Peroxidase    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):
 Dissolve the contents of one vial R 2 Enzymes in one bottle of R 1 Buffer.
Cap and mix gently to dissolve contents.
The reagent is stable 1 month after reconstitution in the refrigerator (2-8ºC) or 7 days at room
temperature (15-25ºC).

Signs of reagent deterioration:
- Presence of particles and turbidity.
- 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. 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:

           Sample        Standard         Blank
           1.0           1.0              1.0   WR (mL)
           --            10               --    Standard (µL)
           10            --               --    Sample (µL)

4. Mix and incubate for 10 min at 37ºC or 15-20 min at room temperature (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
(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. + 120
        90 min.                          -                       > 165  +1
                                                                                         min.) > 600 mg/dl =
       120 min.                  >140  ½ point                  > 140  +1
                                                                                         Diabetic
       2 ½ hour                   >130  1 point                      -
                           2 – 3 point Diabetic           3  Diabetic                  *If Σ of results < 600 = non
                           ½ - 1 ½ point  Suspect         1 – 2 Suspect                diabetic
Calculation of Results
                           Zero  Non diabetic             Zero  Non 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 minutes       <200                        <200                       <200
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:

Calculation: % HbA1c =      A1 X 100 /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) therapeutic goal                         Less than 7%
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:

          Triglycerides + H2O      lipase       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:

                  Sample            Standard           Blank
                  1.0               1.0                1.0           WR (mL)
                  --                10                 --            Standard (µL)
                  10                --                 --            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 esters + H2O                          Cholesterol esterase   Cholesterol + FA

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

    H2O2 + 4-AAP + Phenol                           Peroxidase       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.
   3. Pipette into a cuvette:

           Sample      Standard    Blank
           1.0         1.0         1.0         WR (mL)
           --          10          --          Standard (µL)
           10          --          --          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 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 200-239 mg/dL
      High       240 mg/dL and above
                                                      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

     Cholesterol esters + H2O                          Cholesterol esterase    Cholesterol + FA

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

     H2O2                    catalase            2 H2O + O2

      2º Measurement of LDLc

      Cholesterol esters + H2O                                    Cholesterol esterase   Cholesterol + FA

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

     H2O2 + 4-AAP + Phenol                                     Peroxidase       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:
                                Blank Standard      Sample
             R1(µL)             300     300         300
             Standard (µL)      -------    4        ------
             Sample (µL)        ------- --------    4

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

               R2 (µL) 100          100      100

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
                                            1. Pipette into a centrifuge tube:

       100 R (µL)
       1.0 Sample (mL)
    2. Mix well; allow to stand for 10 min at room temperature.
    3. Centrifuge at 4000 r.p.m. for 20 min or 2 min at 12000 r.p.m..
    4. 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:

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



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

           Urea + H2O                 Urease    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
      - Serum or heparinized plasma: Do not use ammonium salts or fluoride as anticoagulants.
      - 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
    1. Assay conditions:
 Wavelength: . . . . . . . . . . . . . .. . . . .. . . . . . 580 nm
 Cuvette: . . . . . . . . . . . . . . . . . . . . .. 1 cm light path
 Temperature. . . . . . . . . . . . . . . . . .. . . 37ºC / 15-25ºC
    2. Adjust the instrument to zero with distilled water.
    3. Pipette into a cuvette:

             Sample      Standard        Blank
             1.0         1.0             1.0   WR (mL)
             --          10              --    Standard (µL)
             10          --              --    Sample (µL)
    4. Mix and incubate 5 min at 37ºC or 10 min at room temperature (15-25ºC).
    5. Pipette:

           Sample Standard Blank
           1.0    1.0      1.0   R 2 (mL)

        6. Mix and incubate 5 min at 37ºC or 10 min at room temperature (15-25ºC).
        7. 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:
         - Presence of particles and turbidity.
         - Blank absorbance (A) at 492 nm 1.80.

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

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

                     Sample         Standard          Blank
                     1.0            1.0               1.0   WR (mL)
                     --             100               --    Standard (µL)
                     100            --                --    Sample (µL)


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

CALCULATIONS
ΔA Sample –ΔA 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:
0,7 - 1,4 mg/dL = (61.8 – 123.7)µ mol/L                         Male
0,6 - 1,1 mg/dL = (53.0 – 97.2 ) µmol/L                         Female
Urine: 15-25 mg/Kg/24 h
10 - 20 mg/Kg/24 h = 88– 177µ mol/Kg/24 h                       Male
8 – 18 mg/Kg/24 h = 71– 177 µmol/Kg/24 h                        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          Standard       Sample
                       WR (ml)                  1.0              1.0            1.0
                       Standard (µL)            -------          25           --------
                       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 increase in         Kidneys unable to concentrate
                                           fluid intake)                  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:
- Presence of particles and turbidity.
- 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
1. Assay conditions:
Wavelength: . . . . . . . . . . . . . .. . 630 nm (600-650)
Cuvette: . . . . . . . . . . . . . . . . . . . . . . 1 cm light path
Temperature . . . . . . . . . . . . . . . . . . . .. . . . 15-25ºC
2. Adjust the instrument to zero with distilled water.
3. Pipette into a cuvette:

           Sample      Standard      Blank
           1.0         1.0           1.0   R (mL)
           --          5             --    Standard (µL)
           5           --            --    Sample (µL)

4. Mix and incubate for 10 min at room temperature (15-25ºC).
5. 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

                                                NH4+

                                                    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:
- Presence of particles and turbidity.
- Blank absorbance (A) at 540 nm 0.22.

SAMPLES
Serum or heparinized plasma: Stability of the sample: 1 month at refrigerator (2-8ºC).

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

                                                    Blank        Standard             Sample
                            R (mL)                  1.0            1.0                1.0
                            Standard (µL)           ------           25               -------
                            Sample(µL)              ------       ---------              25

4. Mix and incubate 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) 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:
         - Presence of particles and turbidity.
         - 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
1. Assay conditions:
Wavelength: . . . . . . . . . . . . . … . . . . 555 nm (530-580)
Cuvette: . . . . . . . . . . . . . . . ... . . … … . . .1 cm light path
Temperature . . . . . . . . . . . . . . . . . . . . . . . . . . . 15-25ºC
    2. Adjust the instrument to zero with distilled water.
    3. Pipette into a cuvette:

                               Direct B.        Blank      Total B          Blank.
                               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


         4. Mix and incubate exactly for 5 minutes at 15-25ºC.
     5. 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:
         - Presence of particles and turbidity.
         - 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:
                 1.0 WR (mL)
                 100 Sample (µL)

   4. Mix, wait 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 (∆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

               37ºC      30ºC     25ºC
             7-32 U/L 5-25 U/L 4-18 U/L Women
             11-50 U/L 8-38 U/L 6-28 U/L Men
                                           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:
         - Presence of particles and turbidity.
         - 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:

          1.0 WR (mL)
          100 Sample (µL)
    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 (∆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:
         - Presence of particles and turbidity.
         - 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:

                       1.0 WR (mL)
                       100 Sample (µL)

     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 (∆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         Standard        Sample
              R1 (mL)                 2.0            2.0             2.0
              R2 (2 drop)             1              1               1
              Standard (µL)          -----           20            -----
              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:
         - Presence of particles and turbidity.
         - 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:
                       37ºC      25 - 30ºC
                        1.0         1.0        WR (mL)
                        20          40         Sample (µL)

   4. Mix, incubate for 2 minutes.
   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 (∆A/min).

CALCULATIONS

    25º- 30ºC          ∆A / min x 4127 = U/L CK
    37ºC               ∆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:
         - Presence of particles and turbidity.
         - 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:

                1.0 WR (mL)
                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 CK-MB              ΔA x 825 = U/L de 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.
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 hemoglobin            5.5 - 8.5%

( 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 ratio          <5.8                1                         <5.8


Liver function tests
Total Bilirubin                 0.25-1.5 mg/dl          17.1                4.3-
Direct Bilirubin                  0-0.5 mg/dl           17.1                  0-
Indirect Bilirubin                0-0.9 mg/dl           17.1                  0-
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 transpeptidase         11-50 IU/L        1.67 X 10-8           18-84 X 10-8 Katal/L
(GGT) -Male
g -Glutamy transpeptidase          7-35 IU/L                              12-58 X 10-8 Katal/L
(GGT) -Female
Alkaline Phosphatase           45-125 IU/L(up to    1.67 X 10-8          0.75-2.1 X 10-8 Katal/L
                                 1000 IU/L in
                                young children)
Alanine aminotransferase           5-35 IU/L        1.67 X 10-8           8.4 -58 X 10-8 Katal/L
(SGPT / ALT)
Aspartate aminotransferase         5-40 IU/L     1.67 X 10-8        8.4 -67 X 10-8 Katal/L
(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-
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 mmol/L        1               135-145 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-                  1                 11-
Blood gases (arterial, whole      7.35-7.45           1                   7.35-7.45
blood) - pH
Blood gases (arterial, whole    80-105 mmHg         0.133              10.6-14.0 kPa
blood) - PO2
Blood gases (arterial, whole    35 - 45 mmHg        0.133                4.7-6.0 kPa
blood) - PCO2
Blood gases (arterial, whole     22-31 mmol/L         1                22-31 mmol/L
blood) -Carbon dioxide
content
ß-Carotene                       50-               0.0186              0.9-
Ceruplasmin                     0.23-0.58 gm/L       6.7               1.5-
Chloride                        95-105 mEq/L          1                95-105 mmol/L
Copper - Male                    70-                0.157              11.0-22
Copper - Female                  85-                0.157             13.3-
Complement (total, hemolytic) 118-226CH50U/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 mg/dl      0.01                  6.79-15.37 gm/L
Iron - Male                         80-                0.179                14.3-
Iron - Female                       60-                                     10.7-
Iron - Binding capacity            250-                                     44.7-
Iron - Transferrin saturation         16-57%             1                       16-57%
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 mOsm/kg         1                  270-290 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 - Beta      0.7-1.7 gm/dl       10                      7-17 gm/L
globulin
Protein electrophoresis -           0.7-1.7 gm/dl       10                      7-17 gm/L
Gamma globulin
Vitamin A                           30-                0.035                1.05-
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 - Female           Up to 150 IU/L   1.67 X 10-8           Up to 251X10-8 Katal/L
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, morning)     8-              0.027        0.22-
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 pg/ml     1           Up to 130 ng/L
Growth hormone, fasting         <5 ng/ml         1
17-Hydroxyprogesterone -       3-90 ng/dl
Prepubertal
17-Hydroxyprogesterone -      27-199 ng/dl
Male, adult
17-Hydroxyprogesterone -       15-70 ng/dl
Female - Follicular
17-Hydroxyprogesterone -      35-290 ng/dl
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 -1st   1300-5000 ng/dl
trimester
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 ng/ml/hr    0.278          0.2-0.9 ng/L.s
Testosterone, total - Male     280-1000 ng/dl     0.0346           10-35 nmol/L
Testosterone, total - Female    20 -120 ng/dl                       1-4 nmol/L
Testosterone, free - Male       52-280 pg/ml      0.00346         0.18-1.0 nmol/L
Testosterone, free - Female     1.1-6.3 pg/ml                   4-22 X 10-³ nmol/L
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                                                 1             Up to 10 mU/L
Vitamin D, 1,25 dihydroxy        20-76 pg/ml
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-
Amylase                        0.04-0.30 IU/min     1            0.04-0.30 IU/min
Calcium                         < 250mg/day        0.025         < 6.25 nmol/day
Catecholamines                                      1
Dopamine                        90-
Epinephrine                                         5.5         Up to 71 nmol/day
Norepinephrine                  11-                 5.9          65-507 nmol/day
Copper                          15-               0.0157        0.2-
Cortisol, free                  20-                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 gm/day                 0.005-0.013 mmol/day
5Hydroxyindoleacetic acid       1.8-6 mg/day        5.3         9.5-
Hydroxyproline, total           25-77 mg/day       7.63         191-
Metanephrine                   0.3-1.0 mg/day
Oxalate                        Up to 40 mg/day     7.93
Porphyrin-Coproporphyrin        15-                1.53          23-191 nmol/day
Porphyrin-Uroporphyrin                    /day      1.2         Up to 36 nmol/day
Protein                         < 150 mg/day       0.001        Up to 0.150 gm/day
Vanillylmandelic acid (VMA)     0.5-7 mg/day       5.05         2.5-
                                    Common Hematologic Studies
Test                              Current units      Factor           SI units
Coagulation studies -              2.5-9.5 min        60            150-570 sec
Bleeding time
Coagulation studies -Partial        25-41 sec          1             25-41 sec
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 gm/dl      0.62       8.56-10.7 mmol/L
Hemoglobin - Female              12.1-15.1 gm/dl                 7.50-9.36 mmol/L
Erythrocyte / RBC count -        4.5-5.7 X106         106         4.5-5.7 X 10¹²/L
Male
Erythrocyte / RBC count -        3.9- 5.0X106                     3.9-5.0 X 10¹²/L
Female
Leukocyte count                   3.8-                106          3.8-9.8 X 109/L
Leukocyte profile -              1.2-
Lymphocytes
Leukocyte profile -               0.1-0.7X10
Mononuclear cells
Leukocyte profile -              1.8-
Granulocytes
Platelet count                   190 -                106         190-405 X 109/L
Erythrocyte indices - Mean       26.7-33.7 pg/cell   0.062       1.66-2.09 fmol/cell
corpuscular hemoglobin
Erythrocyte indices - Mean       32.7-35.5 gm/dl      0.62       20.3-22.0 mmol/L
corpuscular hemoglobin
concentration
Erythrocyte indices - Mean        80.0-                             80.0-97.6 fl
corpuscular volume
Erythrocyte indices - Red cell     11.8-14.6%
distribution width
Erythrocyte Sedimentation        Up to 30 mm/hr        1          Up to 30 mm/hr
rate
Reticulocyte count                  0.2-2.0%
                                       Practical Sheet #( )
              Practical Experiment: ...........................................................
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