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Why do we add a phosphate phenol 4-AAP buffer to each cuvette? (100mM Sodium Phosphate, pH 7.4, containing 1 mM Phenol and 2.5 mM 4-AAP)

The buffer contains two components of the reaction.

- PHENOL: The enzyme Peroxidase (POD) requires an oxidisable substrate to cleave hydrogen peroxide (H202), which is provided as phenol (a source of hydrogen atoms i.e. electrons).

4-AAP (4-aminoantipyrine): This compound reacts spontaneously with oxidised phenol to yield a RED quinoneimine adduct. A coloured product can be measured by spectrophotometry.


How is glucose consumption favoured in the first part of the assay?

Glucose Oxidase (GOD) catalyses the reaction of D-glucose to D-glucono-1,5-lactate, by removing a pair of H atoms from glc. This intermediate product is rapidly hydrolysed to gluconic acid, driving the reaction forward (Glc + FAD-enz --> gluconic acid + FADH2-enz).


How is Hydrogen Peroxide produced in the assay reactions? (H2O2)

After FAD is reduced to FADH2 as a result of GOD catalysing D-glucose to D-glucono-1,5-lactone (because it's the redox cofactor employed by GOD), it is oxidised back to FAD in the presence of O2.

O2 is a natural acceptor of hydrogen atoms, hence bringing about the concomitant production of hydrogen peroxide (H2O2):

FADH2-enz + O2 --> FAD-enz + H2O2


How were the lower and upper limits of the standard curve decided (what is used on the x-axis and in what units)?

The x-axis of the standard curve represents Glucose Concentration, measured in uM.

The concentration of the glucose sample provided to generate the standard curve was 10 mM. For each cuvette used, a different volume of this glucose solution was used: 0-25 uL with 5 uL increments. In calculating the actual number of nmol's in each volume of glucose solution respective to this 10 mM concentration, the [glc] per mL (total volume of assay) could be calculated, hence the final units of uM.

Depending on what concentration was used here, different upper and lower limits may result.


You do not need to do a time course for the unknown samples of glucose measured from your 'Patient' (either A, B or C).
How long should you continue to measure the absorbance of the time course assays?
When should you measure the absorbance of the unknown standard samples?

***check this

Once the absorbances of the standard reactions have plateaued off (~4 min) read the unknown samples also. This should typically be about 40 min.


How do we choose our volumes of unknown solutions based on the standard curve?

In this case, the standard curve runs from 0-250 uM. To obtain a more accurate absorbance, we want a reading in the middle of the curve, at approximately ~125 uM, or 125 nmol/mL.
The BG lies between 4-25 mM. If the sample is 4 mM = 4 umol/mL = 4 nmol/uL then you will need 125/4 = ~30 uL to give an absorbance in the middle of the standard curve. The upper end is done similarly.


After obtaining the standard curve, how would you determine the glucose concentrations in the unknown samples?

The standard curve would be generated from the absorbances (vs. respective [glc], uM) once the readings start to plateau out. After adding a trendline and obtaining its equation, use the relationship between absorbance and [glc] to determine the unknown concentrations in each cuvette (these will be different, corresponding to the various chosen volumes of the unknown). As these concentrations are in nmol/mL, that is, nmol of glucose in the 1mL total volume of the cuvette, determine the original glucose concentration by dividing by the initial volume added to obtain nmol/uL. Average out these calculations between the 4 unknown cuvettes to obtain your approximate unknown glucose concentration of the patient.

e.g: Vol added = 30 uL (corresponding to 4mM lower limit and middle of standard curve). A500 = 0.86. Used Std Curve equation to obtain [glc] in the cuvette (nmol/mL) = 181.7. (If total volume of cuvette wasn't 1mL, divide by the volume to get per mL). Divide again by the volume added (30uL) to get [glc] in nmol/uL. Average with other calculated [glc] in nmol/uL to obtain unknown glc concentration of patient.


An individual's blood analysis revealed high blood insulin but normal glucose responses. What condition might they have?
a) Type II Diabetes
b) Type I Diabetes
c) Insulin Resistance

c) Insulin Resistance

Muscle & fat cells respond inefficiently; more insulin is required to handle blood glucose challenges; exhausted beta cells.


What are the characteristics of T1D vs T2D?

1) Characterised by HIGH blood glucose and NO insulin. (no endogenous insulin production as a result of beta-cells being destroyed by an auto-immune attack).

2) Characterised by HIGH blood glucose and HIGH blood insulin (can be induced).


If we're to measure the glucose concentration in soft drinks, how do we handle the fact some of the sugar in soft drinks exists as sucrose?

Sucrose is a disaccharide composed of one molecule of glucose and one molecule of fructose. Whilst sucrose breaks down over time, to account for remaining levels in soft drink samples, we add SUCRASE (/invertase) to cleave the alpha 1-2 bond between these molecules. This will provide the monosaccharides as individual sugar components, the glucose of which to be measured.


Why did we use Diet soft drinks in this experiment?

We are trying to measure glucose concentrations.

Diet soft drinks contain an artificial sweetener, and will not be effected by the GOD enzyme. Thus it serves as a good NEGATIVE CONTROL.