1. Glucose test

2. Introduction
Glucose is monosaccharide found in fruits and also derived from the breakdown of carbohydrates in the diet & the conversion of glycogen by liver.
Glucose in the bodies main source for cellular energy, brain.
The glucose level in the body is maintained by insulin(cause lowering the glucose level) and glucagon, adrenalin and cortisol (cause elevation the glucose level).
The various disorders in carbohydrate metabolism may be grouped in to several categories dependent primarily upon laboratory finding:
those associated with a raised plasma glucose concentration (Hyperglycemia).
those associated with a decreased plasma glucose concentration (Hypoglycemia).

3. The most frequently encountered disorder of carbohydrates metabolism is a high blood glucose due to DM.
Estimates of the glucose concentration in blood are required to:
Help in the diagnosis of diabetes mellitus.
Management of DM patients.
And monitoring of treatment in DM patients.

4. Methods of analysis
In the past, the majority of the quantitative test for glucose determination depended upon the oxidation of glucose by hot, alkaline copper solutions or solutions of potassium ferricyanide.
These were replaced by the ortho-toluidine test and later by enzyme methods.
Enzymatic methods give “true” glucose determination because of the high specificity of an enzyme for a particular substrate and don’t consume the time, other methods contain substances dangerous for the body such as o-toludine.

5. Methods of analysis
Reduction methods: These methods depend on the reductive property of glucose(aldose)
Ferriccyanide( Hoffman’s) method.
Copper sulfate method.
Smogi-Nelson method.
N.B. The reduction methods need alkaline medium &heat
These methods are qualitative & semi-quantitative.
2. Aromatic amines methods:
O-toludine +glucose (aldhyde) heat &acidity glucosamine (colored )

6. Trinder’s (Enz.-Dye Colorimetric ) method
Methods of analysis
Enzymatic methods:
Hexokinase methods (The reference method).
Glucose +ATP +HKADP+G6P
G6P +NAD +G6PD 6 P-gluconolactone +NADH+H
(measured at 340)
Glucose Dehydrogenase Method:
Glucose oxidase methods
Glucose +NAD GDH Gluconolactone +NADH+H (measured at 340)
Kinetic method
Trinder’s (Enz.-Dye Colorimetric ) method

7. Specimen collection Serum, plasma is suitable for samples.
Whole blood and hemolysis are not recommended for use as a sample.
Freshly drawn serum is the preferred specimen.
Stability: serum , heparin or EDTA plasma (with addition of glycolytic inhibitor):
2 days at C
7 days at 2-8 C

8. Cont:
The addition of 2mg sodium fluoride per ml blood to be collected will prevent glycolysis.
The glucose loss, upon standing in a warm room, may be as high as 10 mg/dl per hour.
The decrease in serum glucose concentration is negligible if the blood sample is kept cool and the serum separated from the clot within 0.5 hours of drawing.

9. 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.
It is determined by measuring the increase in absorbance at nm.

10. Procedure:
1. Pipette into a cuvette: 2. Mix and incubate for 10 min at 37ºC or min at room temperature (15-25ºC). 3. Read the absorbance (A) of the samples and standard, against the Blank.
Blank
Standard
control
Sample
WR (mL)
1.0
Standard (µL) -- 10
Control (µL)
Sample (µL)

11. 4. Use beers equation to receive to the concentration of glucose in the patient sample.
Glucose concentration (mg/dL)=
Absorbance of Sample x Standard concentration
Absorbance of Standard
Conversion factor: mg/dL x = mmol/L.

12. Normal Range:
Glucose level
Indication
From 70 – 110 mg/dl
Normal fasting glucose
Up to 140 mg/dl
Normal 2 hrs postprandial
in diabetes mellitus, the level of fasting blood glucose is 126 mg/dl.
The patient considered critically hypoglycemic if, Serum/Plasma glucose level < 45mg/dl
Renal threshold for glucose is 160 – 180 mg/dl.
When the blood glucose exceed the renal threshold for glucose and presences in urine reffered to glucoseuria.
In pregnancy, renal threshold is reduced.

13. ORAL GLUCOSE TOLERANCE TEST (OGTT)

14. 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.

15. Patient preparation
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).

16. Cont:
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.

17. Sample
Blood samples: fasting(basal) sample, 30 minute after oral glucose load, 60min, 90min, 120min.
Urine samples: first fasting urine and the hourly collected urine samples.

18. Calculation Glucose sample Wilkerson Criteria Fajan-conn criteria
Revised Summation
Fasting
> 130 1 point -
*If Σ of results
(F + 60min min 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
½ - 1 ½ point  Suspect
Zero  Non diabetic
3  Diabetic
1 – 2 Suspect

20. 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.
Impaired Fasting Glucose: When a person has a fasting glucose equal to or greater than 110 and less than 126 mg/dl. This is considered a risk factor for future diabetes
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.

21. 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.
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.