Medical and Diagnostic Biochemistry

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Medical and Diagnostic Biochemistry

Introduction

Glucose also called a dextrose is a natural sugar present in honey and fruits and it belongs to a

group of carbohydrates called monosaccharide’s and has a formula C6H12O6 .Glucose makes

most of the sugar circulating in the blood of animals hence its other name blood sugar. Cells in

the body get their energy from glucose; therefore it is important to regulate its metabolism in the

human body. In the human body glucose is derived from the breakdown of carbohydrates

ingested in the food we eat or the one stored in form of glycogen. It is also gotten from the

synthesis of proteins. Excess glucose in the human body is converted into fats and glycogen and

stored in the liver, muscles and adipose tissue. When the levels of glucose intake are not

adequate to provide the energy needs breakdown of carbohydrates stores occurs in order to form

glucose (Encyclopedia).

Blood sugar measurements are done to determine glucose levels in human body and are carried

out in hospitals and chemistry heath care laboratories. Diabetes mellitus is the most common

disorder for carbohydrate metabolism and it is as a result of high levels of blood sugar in the

body (Encyclopedia).

A metabolic disorder characterized by high levels of blood is called diabetes mellitus. The high

level of blood sugar is as a result of production of insufficient insulin produced by the pancreas

since insulin reduces glucose levels in the blood (hyperglycemia) (Buse JB,2011). The two types

of diabetes are: type 1 and type 2.

Type 1 diabetes mellitus is as a result of lack of insulin in the blood which leads to

hyperglycemia and is insulin-dependant for management, whereas type 2 diabetes mellitus is not

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dependent on the levels of insulin in the blood for management and and is not linked to HLA

markers (Encyclopedia).

Diabetes mellitus type 2 is further grouped as non-obese type, obese type and gestation diabetes

mellitus which is a glucose intolerance recognized during pregnancy. The symptoms of type1are:

glycosuria, hyperglycemia, rapid weight loss, fatigue, hunger and thurst; Type 2 diabetes

mellitus is common in old and obese persons and its symptoms are: polyuria (increased urine

urine output) and polydipsia (Mccance DR,1997).

The diagnosis of diabetes mellitus is based on two fasting plasma glucose levels of 126mg per

dL (7.0 mmol per L) or higher. The other methods used for diagnosis of diabetes mellitus are two

two-hour postprandial plasma glucose (2hr PPG) which represents readings of 200mg per dL

(11.1 mol per ) or after a glucose load of 75g (Mccance DR,1997).The preferred diabetes

mellitus diagnostic test is the measurement of plasma glucose, this is because it predicts adverse

outcomes, it is more reproducible than the other two and its is easier to perform in a hospital or

chemical laboratory setting. The cut-off point for plasma glucose is as a result of strong evidence

of various complications due to the glycemic status of the patient. The risk of developing type 2

diabetes mellitus is associated with impaired fasting glucose and glucose tolerance (susman,

1997)

The glycemic control of persons with diabetes mellitus is done by measurement of glycated

haemoglobin also called hemoglobin A1 cor haemoglobin A1 or glycohemoglobin which aid in

evaluation of stable linkage of glucose to minor haemoglobin components and comprises 4-6%

of total hemoglobin (Encyclopaedia, 2012). Measurement of glycohemoglobin can also be used

in diagnosis of diabetes mellitus since its levels are highly correlated to clinical outcomes and the

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specimen can be collected without regard to the time the patient ate last. Chronic hyperglycemia

is characterised by glycohemoglobin levels exceeding 6% (susman, 1997).

Enzyme assay techniques are used in measurement of blood glucose in which hexokinase

orglucose oxidase are widely used. This method is based on a coupled enzyme assay that uses

HK and glucose-6-phosphate dehydrogenase (G-6-PD). Phosphorylation of glucose is done by

the HK using ATP in presence of Mg2+ forming glucose-6-phosphate.This product is then

oxidised by G-6-PD to -phosphogluconate in the presence of NADP+ .The amount of NADP+ is

directly propotional to the amount of glucose in the sample and is measured by increase in

absorbance at 340nm.

The blood glucose determination method in the practice exercise was carried on using three

serum samples (diluted, adding ascorbic acid and adding uric acid) in order to determine the

possible interferences that these compounds cause in the glucose concentration determination,

and a glucose standard with known concentration, in order to determine the accuracy and

precision of the method. It was expected to find a good precision and accuracy for the method

and no interference caused by the ascorbic acid and uric acid.

The estimation of glycated haemoglobin A1Cby affinity chromatography was performed in a

normal blood sample and in a diabetic blood sample, in order to compare both results with a

reference range established and determine the differences in the values. It was expected for the

normal blood sample to obtain results for the percentage of glycated haemoglobin and

haemoglobin A1C inside the reference range; while for the diabetic blood sample, both values

were expected to be higher than those obtained in the normal blood and out of the intervals set in

the reference range.

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Aim

The main objectives of this practice were to estimate test of G-6-P with an exact time showing an

overlapped of ascorbic acid and uric acid and to estimate a rate sample of haemoglobin A1c in

both normal and diabetic blood.

Method

Four samples with one blank sample of serum diluted 10%, 5.0 m mole/L ascorbic acid and 0.5

m mole/L uric acid were taken and added. The samples were then used in spectrophotometric

device at 340 nm with the blank sample being the first to be put which ensured that the device

was ready to for reading the reaction cuvettes. Some additions were then put with waiting time.

The absorbance for each cuvette was read and recorded.

In experiment B, two samples of normal and diabetic blood were taken and the required

additions such as haemolysate reagent were put in the samples. Affinity chromatography was

done for a period of more than five minutes so as enough separated samples for non-glycated and

glycated haemoglobin was obtained. The absorbance in spectrophotometric device at 415 nm for

both samples was obtained.

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RESULTS Table 1

Calculated glucose concentration in the three samples of sera and the glucose stock standard

using the hexokinase/glucose-6-phosphate dehydrogenase method

Serum Number/Standard

solution

Glucose in serum [mmol/L] Averange

1A 9.2 9.2

1B 9.17

2A 7.31 8.22

2B 9.13

3A 8.25 8.44

3B 8.62

Glucose stock 1 8.87 8.18

Glucose stock 2 7.49

Serum 1 corresponds to the stock serum diluted 10% using 0.9% saline; serum 2 to the stock

serum spiked with 5.0 mmol/L ascorbic acid; and serum 3 to the stock serum spiked with 0.50

mmol/L uric acid. The glucose stock standard concentration theoretical value is 5.0 mmol/L

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Table 1 shows the glucose concentration calculated for the samples of serum utilizing the

glucose-6-phosphate method, which were 9.20 mmol/L for the stock serum diluted 10% using

0.9% saline (serum 1); 8.22 mmol/L for the stock serum spiked with 0.50 mmol/L uric acid

(serum 3); and 8.44 mmol/L for the stock serum spiked with 5.0 mmol/L ascorbic acid

(practically there was no much difference between the three results).

The experimental result obtained for the glucose stock standard concentration was 8.18 mmol/L,

being 5.0 mmol/L of the the theoretical value for this standard

Table 2

Glycated haemoglobin, calculated as %GHb, in the normal blood sample and the diabetic blood

sample provided.

Blood type Glycated haemoglobin (%GHb)

Normal blood 10.55

Table 2 shows the percentage of glycated haemoglobin (%GHb) present in the normal blood

sample, calculated using the absorbance data obtained from the eluted fractions after an affinity

chromatography process. The %GHb for the normal blood was 10.55%.

Table 3. Haemoglobin A1C, calculated as %HbA1C, in the normal blood sample and the

diabetic blood sample provided.

Blood type Haemoglobin A1C (%HbA1C)

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Normal blood 8.2

Table 3 provides the results for the percentage of Haemoglobin A1C present in the normal blood

and diabetic blood samples, calculated from the data obtained for their respective %GHb value.

The %HbA1C obtained was 8.2% for the normal blood.

The normal blood %HbA1C values are inside the reference range.

Discussion

In the first part of the experiment, analysis of blood glucose was done using the enzyme assay of

the glucose-6-posphate.There was no interference in the concentration of glucose since the

values for the samples with ascorbic and uric acid obtained were almost constant. I the second

part of the experiment estimation of haemoglobin A1c in normal blood sample was done by

affinity chromatography, in which it results obtained would be inside the reference range (9%-

17%).The results obtained were inside the reference range. The results for the glucose samples

taken were almost identical hence presence of ascorbic or uric acid in the samples did not affect

the results obtained that are they did not cause interference in the assay. This is because, unlike

the glucose oxidase procedure, in which uric acid or ascorbic acid act as inhibitors of the enzyme

(causing a decreased catalytic activity), this two compounds do not cause an inhibition of the

enzymes hexokinase or G-6-PF and do not affect their catalytic activity (McCance,1997). This

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method also presents the advantages of being simple, requiring small sample and reagent

volumes and it can be used with urine samples as well as blood samples (susman,1997).

Since the experimental glucose concentration for the standard (8.18 mM) was almost the same as

the theoretical value (5.0 mM), it can be established that the method is accurate and precise, and

the formula used in the calculations was adequate. The precision of the glucose measurement in

the assay is due to the complete specificity of the enzyme G-6-PD towards the substrate glucose-

6-phosphate.

In the experimental estimation of haemoglobin A1C by affinity chromatography, the results

obtained in table 2 for the %GHb in normal blood (10.55%) and was almost inside the reference

intervals established by Helena laboratories (between 4.3-7.7%) and the same tendance was

observed for the %HbA1C (table 3). The expected tendency for both results was that, for the

normal blood the percentage should have been inside the normal reference.

Conclusion

The G-6-P method for determining blood glucose is an accurate method since the results

obtained were almost similar to theoretical values and it does not suffer from interference due to

presence of ascorbic and uric acid.

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Appendix

BLANK NORMAL

A0

AFTER 5 MIN

AF

AFTER 1 MIN

AF

AFTER 1 MIN 2

AF

1A 0.206 0.587 0.578 0.583

1B 0.197 0.575 0.577 0.586

2A 0.234 0.535 0.523 0.527

2B 0.197 0.573 0.563 0.566

3A 0.209 0.549 0.545 0.545

3B 0.194 0.549 0.559 0.559

STOCK 1 0.192 0.804 0.801 0.803

STOCK 2 0.195 0.874 0.670 0.671

CALCULATIONS OF GLUCOSE CONCENTRATION

Serum 1

Glucose, mmol/L serum = ΔA x 24.28 x D

Where

ΔA = change in absorbance = (Af – A0)

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24.28 = conversion factor, derived from the ε of NADPH at 340 nm (= 6.22 x 103 L mol-1 cm-1),

and the M.

Wt. of glucose (= 180.16 g/mol)

D = dilution factor = 1.00, since a 20 µL sample was taken

Glucose, mmol/L serum1A=9.2

Serum 1B=9.17

Serum 2A=7.31

Serum 2B=9.13

Serum 3A=8.25

Serum 3B=8.62

Stock 1=14.87

Stock 2=16.49

A. Estimation of glycated haemoglobin A1C by affinity chromatography

B. NGHb = 0.763

C. GHb = 0.450

D. Sample calculation of the percentage of glycated haemoglobin (%GHb) in the normal blood:

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%GHb (percentage of glycated haemoglobin in sample) =

(|.|of GHbTube x100%)(|.|of GHbTube )+5.0(|.|of N−GHbTube )

Abs. of GHb tube = Absorbance of the contents of the GHb collection tube at a wavelength of

415 nm

Abs. of N-GHb tube = Absorbance of the contents of the N-GHb collection tube at a wavelength

of 415 nm

5.0 = dilution factor (15 mL of N-GHb tube/ 3 mL of GHb tube)

100 = percentage conversion factor

%GHb in the normal blood sample =10.55%

%HbA1C in the normal blood sample =8.2%

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