Determination At different wavelengths molecules will absorb different amounts

Determination of Glucose Concentration in Patient Samples Utilising a
Calibration Curve

 

Charlotte Wooler

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Introduction

Spectrophotometers are used
in biochemistry to measure the concentration of solutes in solution by
measuring the amount of the light that is absorbed by the solution in a cuvette
that is placed in the spectrophotometer.  At different
wavelengths molecules will absorb different amounts of energy. The technique
is to measure the intensity of as a function of wavelength. This gives a result which is on the colour
wavelength spectrum. ‘Spectrophotometry works by shining light of a specific
wavelength through a sample of the solution being tested and detecting the
amount of light absorbed during its passage through the sample. If the solution
is of high concentration, then a lot of light is absorbed, and only a little
light passes through the sample to be detected. By contrast, if the solution is
of low concentration, only a little light is absorbed by the sample, and much
of the light passes straight through it, and is then detected.’ (Crowe,
Bradshaw and Monk 2006: 319).

 

Absorbance values from
spectrophotometers, by themselves, do not describe the concentration of a substance.
However, the concentration of a substance in a solution can be determined by
using a standard curve. A standard curve translates absorbance values into
concentration, which is created by making solutions with a known concentration
of the substance being measuring and then measuring the absorbance. Plotting
the concentration on the x-axis and the absorbance on the y-axis, a linear
relationship can be seen between concentration and absorbance. A standard curve
is not really a curve, but a straight line. Beer Lambert Law describes this
linear relationship:

 

 

A = absorbance

? = molar absorption coefficient (the amount absorbed by a 1M
solution)

C = concentration (in moles/L)

L = path length (usually 1cm)

 

A is therefore proportional
to c. The absorption of a substance is directly proportional to the
concentration.

 

Glucose is a monosaccharide
that is used by cells as an energy source, although when it is not available,
most cells in the body use fatty acids as their fuel. After a meal, glucose is
absorbed into the bloodstream, elevating the concentration of glucose found in
the blood. Insulin is released as a result, and all cells switch to burning
glucose. Diabetes is diagnosed by measuring the amount of glucose in blood
after an 8-hour fast from food. A serum glucose blood test measures the amount of glucose in a blood
sample taken from a patient. The test is usually performed to check for
elevated blood glucose levels which may be an indication of diabetes or insulin
inhibition. Normal blood glucose levels are between 4.0 to 6.0 mmol/l when
fasting and up to 7.8 mmol/l two hours after eating (www.diabetes.co.uk)

 

Aims & Objectives

1.    To learn how to use a spectrophotometer.

2.    To gain experience of producing a standard
curve.

3.    To perform a glucose assay.

4.    To determine the concentration of glucose in
two patient serum samples.

5.    To gain additional experience in data
interpretation and presentation.

 

Materials & Methods

Refer to practical handout. For the serial dilutions used in the
practical see table below.

Tube

mM Glucose

ml 20mM Glucose

ml H20

1

0.0

0

1

2

5

0.25

0.75

3

10

0.50

0.50

4

15

0.75

0.35

5

20

1

0

 

Results

Tube

Absorbance at 340nnm

1

0.000

2

0.265

3

0.514

4

0.759

5

1.006

P1

0.374

P2

0.197

QC

0.314

 

 

Tube

Glucose concentration (mM)

P1

7.8

P2

3.7

Quality Control (QC)

6.3

 

Discussion

The concentration of glucose is measured by a Hexokinase assay linked to
the reduction of NAD+. Glucose + ATP is converted to glucose-6-phosphate and
ADP, catalysed by hexokinase. In the presence of NAD+, glucose-6-phosphate is
converted to 6-Phosphogluconate and NADH, catalysed by glucose-6-phosphate
dehydrogenase.

 

First reaction:

 

Glucose + ATP                                                 Glucose-6-Phosphate
+ ADP

 

Second reaction:

 

G6P + NAD+                                                    6-Phosphoglutonate + NADH

 

The amount of NADH present at the end of the second reaction is the same
as the amount of glucose present at the start of the first reaction, because
the two reactions are equimolar and linked. The presence of NADH can be
measured spectrophotometrically at 340 nm and is proportional to the original
glucose concentration. Glucose levels in patient samples can then be quantified
by using a standard curve derived from known glucose concentrations.

The first reaction is the rate limiting step as it is linked to the
amount of glucose in the sample. The rate determining step is the slowest step
of a chemical reaction that determines the speed (rate) at which the overall
reaction proceeds.

 

If the temperature of the reaction mixture fell significantly whilst
monitoring the rate of the reaction, the results would decrease as heat is a
catalyst. Providing all the sample are within the standards and the QC then the
results will be acceptable. If the results were not within the standards, then the
results would be invalid, and the samples would have to be repeated.

 

The appropriate concentration of glucose-6-phosphate dehydrogenase for
the coupled reaction is chosen

 

The concentration of the quality control sample falls within the
allowable QC range of between 5 and 7mM. The calibration curve shown in figure
1 is accurate and therefore the assay is valid. ‘Quality control is designed to
detect, reduce, and correct deficiencies in a laboratory’s internal analytical
process prior to the release of patient results. Quality control samples are
special specimens inserted into the testing process and treated as if they were
patient samples by being exposed to the same operating conditions.’ (http://www.clinlabnavigator.com/quality-control.html)

If the QC fails it can because an error has been made, such as inaccurate
pipetting by the user or the sample not being heated up to the correct temperature.
 

 

Errors in results can also be caused by how samples are collected and
handled, such as a delay in separation via centrifugation. ‘In the absence of
appropriate preservatives, uncentrifuged blood shows a rapid decrease in
glucose levels that can be as much as 75% per hour at room temperature. When
clotting is permitted and followed by centrifugation, glucose levels in the
serum remain stable for two days if the specimen is kept refrigerated at 4– 8 ?
C.’ (Dods 2013: 224).

 

Patient 1 has a glucose level of 7.8 which is within the normal range
for a non-fasting sample, but a fasting sample would be required for
confirmation because the result is borderline as the normal range before meals
is 4-7mM and the normal range 2 hours after meals is <9mM.   Patient 2 has a glucose level of 3.7 which suggest that the patient has low blood sugars (hypoglycaemia). Hypoglycaemia occurs when blood glucose level fall below 4 mmol/l (diabetes.co.uk). Diabetes is a lifelong condition in which the body's ability to produce or respond to the hormone insulin is impaired, resulting in abnormal metabolism of carbohydrates and elevated levels of glucose in the blood. There are two types of diabetes; type 1 and type 2. Type 1 is a chronic condition in which the pancreas produces little or no insulin. Insulin is a hormone needed to allow sugar (glucose) to enter cells to produce energy. Type 2 is when the pancreas does not produce enough insulin or the body's cells do no react to insulin. References J. Crowe, T. Bradshaw, P. Monk (2006) Chemistry for the Biosciences. First edition. Oxford: University Press. R. F. Dods (2013) Understanding Diabetes: A Biochemical Perspective. First edition. New Jersey: John Wiley & Sons, Inc. http://www.clinlabnavigator.com/quality-control.html https://www.diabetes.co.uk/diabetes_care/blood-sugar-level-ranges.html