Estimation of Blood Glucose .ps



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:

| |Blank |Standard |Sample |

|WR (mL) |1.0 |1.0 |1.0 |

|Standard (µL) |-- |10 |-- |

|Sample (µL) |-- |-- |10 |

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) ;

[pic] Activity--Don't smoke or exercise strenuously for 8 hours before the test or during the

test.

[pic] 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.

[pic] 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).

[pic] The test must be performed at daytime (morning).

* General description of test

[pic]Test usually takes 3 hours but can last as long as 6 hours (extended OGTT).

[pic] Drink water frequently during the test (the only allowed fluid to drink).

[pic] 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.

[pic] Operator gives a test load of glucose, usually 75 – 100 gram dextrose / 300 ml water,

lemon flavored . Drink the entire solution in 5 minutes.

[pic] 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 |- |*If Σ of results |

| | | |(F + 60min. + 90 min. + 120 min.) > |

| | | |600 mg/dl = Diabetic |

| | | | |

| | | |*If Σ of results < 600 = non diabetic|

|30 min. |- |- | |

|60 min. |>190 ( ½ point |>190 ( +1 | |

|90 min. |- |> 165 ( +1 | |

|120 min. |>140 ( ½ point |> 140 ( +1 | |

|2 ½ hour |>130 ( 1 point |- | |

|Calculation of Results |2 – 3 point (Diabetic |3 ( Diabetic | |

| |½ - 1 ½ point ( Suspect |1 – 2 (Suspect | |

| |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 | 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 |0.9 mmol/L |

|LDL cholesterol | ................
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