Cell Science Oral Glucose Tolerance Test Biology Essay

Published:

This essay has been submitted by a student. This is not an example of the work written by our professional essay writers.

The body has the extraordinary ability to maintain the blood glucose levels in a specific range, using a feedback system. The failure for physiological systems to maintain blood glucose level could results in homeostatic diseases such as diabetes mellitus.

The Oral glucose tolerance test is a method of checking if one has diabetes. A solution consisting of 250ml of water and 75g glucose is given to patients and their blood glucose levels are monitored (Lee, 2009). The quick rise in blood glucose levels after the consumption of the solution is quickly maintained by normal individuals through the production of insulin by the islet of langerhans. However, diabetics cannot maintain the increase blood glucose levels as they are incapable of producing insulin. This in turn results in improper functionality of the kidney, resulting in excess glucose in the urine.

This experiment aimed to categorise patients (A-C) who have had an OGTT, according to their blood glucose levels before fasting and after 2 hours. Moreover, to categorise patient D who was given a random plasma glucose test (this patient did not fast and did not receive oral glucose tolerance test).

Glucose standards were prepared using the 1.5ml of stock solution of 300 mg/mL glucose that was provided, Gilson 200µL pipette was used to prepare 500 µL each of a sequence of glucose standards containing 240 mg/mL, 180 mg/mL, 120 mg/mL, 60 mg/mL and 0 mg/mL, respectively. The sets of solutions were duplicated and 200 µL of each solution where place in LP4 tubes.

Samples A, B and C were given OGTT and the blood was collected in fluoride/EDTA anticoagulant and blood cells were separated using a centrifuge.

Both Fasting which was designated as (0) and 2 hour samples (2) where provided, labelling them as A0 and A2, respectively. Samples B, C, D and A0 were used directly without further dilution. Sample A2 was diluted, using a glass pipette, 1ml of sample A2 was added into a LP4 tube along with 2ml of distilled water. Duplicate of sample A-D was pipetted into LP4 tubes using Gilson.

GOD/HRP enzyme reagent where prepared. 2ml of GOD/HRP was added using a glass pipette to the 24 LP4 tubes each containing 200µL of sample or standard solution. Once added the GOD/HRP was allowed to settle in the LP4 tubes for 20 minutes allowing the catalyse to oxidise the glucose producing a blue colour. All the LP4 tubes where measured for the absorbance at 575 nm using a spectrophotometer, after using water as a blank.

Results

Table .1- Table to show the absorptions (575nm) of glucose standards.

Table .1 represent the absorption of the glucose standard solutions at 575nm; two reading where taken to increase reliability. Mean absorbance rounded to 2 decimal points.

Glucose standard (mg/dL)

Absorbance (575nm)

Trial 1 Trial 2

0

0

0

60

0.023

0.019

120

0.027

0.036

180

0.034

0.039

240

0.047

0.042

300

0.052

0.048

Table .2 - Absorbance (575nm) of the sample solution, showing both fasting (0) and 2 hour sample (2), Mean absorbance and the glucose concentration (Deduced, first from using the graphical formula and inputting sample absorbance in place of y to get glucose standards, then using the method from table.4).

Sample Solution

Absorbance (575nm)

Trial 1 Trial 2

Mean Absorbance (575nm)

Glucose concentration (mmoles/L)

A0

0.041

0.043

0.042

9.5

A2

0.031

0.029

0.030

18.7

B0

0.029

0.027

0.028

5.7

B2

0.038

0.036

0.037

8.2

C0

0.030

0.033

0.032

6.8

C2

0.027

0.031

0.029

6.0

D

0.032

0.036

0.034

7.3

Table .3 - Preparation of Glucose standard solution using glucose and water. Sample calculation on how the volumes were obtained are below.

Glucose standard (mg/dL)

Glucose (µL)

Water (µL)

0

0

500

60

100

400

120

200

300

180

300

200

240

400

100

300

500

0

Sample calculation for glucose standard (mg/dL):

C­­1V1x C2V2 = ((Glucose standard÷ 300) Ã- total volume) = Volume of glucose.

e.g ((240÷300) Ã- 500) = 400 µL of glucose.

Therefore, 400 µL of glucose must be added to 100 µL of water, resulting in 240 mg/dL of glucose stock solution.

Table .4 - Conversions of glucose standard (mg/dL) to glucose concentration (mmoles/L).

Samples

A0

A2

B0

B2

C0

C2

D

Glucose concentration (mg/dL)

173.5

113.5

103.5

148.5

123.5

108.5

133.5

Glucose concentration (mmoles/L)

9.5

18.7

5.7

8.2

6.8

6.0

7.3

Sample calculation: Mean absorbance's for each sample where inputted into the graphical equation, replacing Y, which gave the glucose concentration in (mg/dL) e.g

0.0002x + 0.0073, replacing mean absorbance for A0 (0.042) with y. which gives 0.042 = 0.0002x + 0.0073 then solve to find x, giving the glucose concentration in (mg/dL).

The conversion from mg/dL to mmoles/L was deduced by multiplying each glucose concentration (mg/dL) by 0.055. E.g 267 Ã- 0.055 = 14.7 mmoles/L.

This factor is specific to glucose only because 1 mmoles/L = 18 mg/dL (Martin & Blumer 2004).

A2 concentration was multiplied by 3, as the sample was diluted by a factor of 3.

Graph

Graph.1 - Absorption of glucose standards at 575nm showing both trial 1 and 2.

Discussion

The aim of the experiment was to determine and categorise sample (A, B, C and D) into groups to see whether they had normal blood glucose concentration or were diabetics. Diabetes is caused by a homeostatic imbalance leading to kidney complications and blindness (Chiras, 2005). Comparing the samples glucose levels in (mmoles/L) against the reference table, it's clear to see that sample A's blood glucose level is above the reference range of a diabetic both at fasting (9.5mmoles/L) and after 2 hours (18.7mmoles/L), showing that sample A in a diabetic. Sample B's blood glucose level indicated that the individual is most likely has a impaired glucose tolerance as they have a blood glucose level of 5.7mmoles/L at fasting and 8.2mmoles after 2 hours, supporting the case for sample B having IGT. Sample C can be classified as normal, their glucose concentration matches with the reference range for a normal individual. Sample D who did not receive the OGTT, but was given a random plasma glucose test shows signs of having IGT, but because the individual was not fasting before the test, they could have had a meal which could of increased their blood glucose concentration, giving the false impression of IGT, it would be advisable to ask the individual to take a OGTT test.

Graph.1 shows a linear relationship. Both trials where plotted to give an indication on how precise the data was. The difference between trial 1 and 2 for the glucose standards where relatively small overall, indicating that the results obtained where precise. However, at glucose standard 120 mg/l there was a significant difference to the rest of the data, showing a wider range between the trials. This could have been caused due to human error, such as placing the LP4 tube incorrectly into the spectrophotometer or the incorrect use of a Gilson pipette. R² value of o.9265 shows that there is a very good correlation between absorbance and glucose standard solution as it is very close to one.

The method used had few errors, the errors which had occurred where due to human error or incorrect use of equipment, to enhance the experiment more repeats have to be prepared for glucose standards and samples to give more reliable result.

Additional Question - An enzyme hexokinase catalyzes the phosphorylation reaction between glucose and ATP producing glucose-6-phosphate glucose-6-phosphate dehydrogenase converts glucose-6-phosphate to 6-phosphogluconate, forming NADPH which can be measured at 340nm. The NADPH has a direct correlation to the glucose intake; therefore the more glucose in one's blood the more NADPH produced which is measured by spectrophotometer (Reginald & Grisham 2008).

Reference

Chiras, D., 2005. Human Biology. 5th ed. Massachusetts: Jones and Bartlett Publishers. Pp. 168- 180.

Lee, M., 2009. Basic Skills in Interpreting Laboratory Data. 4th ed. American Society of Health-System Pharmacists. Pp. 276-278.

Martin, p. Blumer, I., 2004. The everything diabetes book. Cincinnati: Adams Media Corporation. Pp 297.

Reginald, G. Grisham, C., 2008. Biochemistry. Florence: Thomson Brooks/Cole

There were flaws in some aspects of the methodology.

Mention about homeostasis and importance of glucose tests

Mention that sample B had a increase in the absorption between 0 and 2, possible diabetic because individual is unable to 'manage' the glucose. Further testing to confirm ?? in comparison, sample a and b both had a decrease in absorption from 0 to 2 indicating that they where able to control the glucose solution.

Trends in absorption increases as sample glucose increases, likewise for concentration.

In relation to the aims

Linear relationship between the glucose sample and absorption

This is where you discuss your 'findings in light of the material given in the

Introduction. The implications of any practical problems constructively

discussed and theory related to practice. Evidence of further reading

and any inferences drawn from statistical analysis should be included.

You must also show a clear evidence of your own original critical

analysis' (paraphrased; Dr David Perry 2008).

Additional Question - briefly describe one other enzymic method for glucose determination that is used in clinical laboratories.

Hexokinase catalyzes the phosphorylation of glucose in the presence of ATP (Reaction 2.3). The glucose-6-phosphate formed is converted to 6-phosphogluconate by a second enzyme, glucose-6-phosphate dehydrogenase (Reaction 2.4). NADPH is formed in the reaction and can be measured at 340 nm.

Reaction 2.3 glucose + ATP G6P + ADP

Reaction 2.4 G6P + NADP 6-phosphogluconate + NADPH + H+

HBA1C test

Writing Services

Essay Writing
Service

Find out how the very best essay writing service can help you accomplish more and achieve higher marks today.

Assignment Writing Service

From complicated assignments to tricky tasks, our experts can tackle virtually any question thrown at them.

Dissertation Writing Service

A dissertation (also known as a thesis or research project) is probably the most important piece of work for any student! From full dissertations to individual chapters, we’re on hand to support you.

Coursework Writing Service

Our expert qualified writers can help you get your coursework right first time, every time.

Dissertation Proposal Service

The first step to completing a dissertation is to create a proposal that talks about what you wish to do. Our experts can design suitable methodologies - perfect to help you get started with a dissertation.

Report Writing
Service

Reports for any audience. Perfectly structured, professionally written, and tailored to suit your exact requirements.

Essay Skeleton Answer Service

If you’re just looking for some help to get started on an essay, our outline service provides you with a perfect essay plan.

Marking & Proofreading Service

Not sure if your work is hitting the mark? Struggling to get feedback from your lecturer? Our premium marking service was created just for you - get the feedback you deserve now.

Exam Revision
Service

Exams can be one of the most stressful experiences you’ll ever have! Revision is key, and we’re here to help. With custom created revision notes and exam answers, you’ll never feel underprepared again.