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Determination of Glucose Concentration Using Trinder Method

The Trinder method is used to determine glucose concentration only, (Lott et al, 1975). This method was first described by Trinder in 1969 thus named after him, (Lott et al, 1975). It uses an enzyme glucose oxidase for the first reaction and peroxidase for the second reaction thus the name of the enzyme Glucose oxidase/peroxidase (GODPOD), (Meiattin, 1973).
Enzymes are biological or any chemical catalysts that speed up a reaction without it being used up, (Jan, 2010). It functions to catalyse a reaction by lowering the activation energy of the reaction. Activation energy is the energy needed to initiate the reaction. It is a point of high energy and requires more energy than the substrates. An enzyme also contains an active site for the substrate to catalyse the reaction. Its efficiency depends on the concentration of the substrate and conditions like temperature or pH, (Hames et al, 2005).
The Trinder method, ‘is based on two sequential enzymatic reactions, the first one involves the oxidation of glucose to gluconic acid and H2O2,’ (Casabnon et al, 2005). This reaction is catalysed by the enzyme Glucose oxidase. ‘Then, the H2O2produced is quantified by a chromogenic reaction with peroxidase (POD),’ as the enzyme that catalyses the reaction with the reduced dye, (Casabnon et al, 2005). The oxidised dye changes colour to pink or red according to the glucose concentration. ‘The colour formed is stable at room temperature for at least two hours after development,’ (Anon, 2010).
The main advantage of this method is that it is very specific. It doesn’t target other sugars except glucose. It is also simple straight forward and easy to manipulate. Its results are very reliable and specific, (Bauninger, 1974). Its final products are stable as they are not reactive at room temperature.
Other colorimetric methods to identify glucose include, ‘oxidation of glucose in the presence of Cu 2 to give Cu2O,’ and different types of Chromatography, (Casabnon et al, 2005). Glucose can be detected with o-toledine or other amines, (Casabnon et al, 2005).
The experiment had to check for the specificity of the assay thus other carbohydrates were assayed. These were galactose, fructose, maltose and ribose. Maltose is a disaccharide which is made up of two glucose molecules joined together by a glycosidic bond.
Galactose is a major sugar found in milk, (Hames et al, 2005). Its structure consists of six carbons with a glycosidic bond to join the next glucose molecule, (Berg et al, 2007). Fructose is abundant in fruits. It is a monosaccharide with six carbons as its structure. Ribose is a pentose sugar molecule with 5 carbons. It is mostly abundant in the nucleotides.
The aim of the experiment was to determine glucose concentration in different concentrated solutions and unknown solutions. The specificity of the assay was to be determined by application of the assay on different sugar molecules. A standard curve was to be drawn from the absorbencies acquired from the spectrophotometer at 515nm
Materials 12 – Test tubes 2 – Long pipette tips
1ml of 0.5mM Fructose 1ml of 0.5mM Maltose
1ml of 0.5mM Galactose 1ml of Unknown Glucose
1ml of 0.5mM Ribose
5ml of 0, 5mM Glucose 5ml of Distilled water
7ml of 0.1% Phenol 20ml of GODPOD Reagent
9 – pipette tips 8 – Disposable Cuvettes
Black Marker Stop watch
P100 Pipette Automatic pipette
Recoding paper and pen
Spectrophotometer at 515nm Water bath at 37oC
Test tubes rack Blotting paper
Method The test tubes were marked T1 to T6, for those that had to be inoculated with glucose and S1 to S6, for those that had to be inoculated with different types of sugars. They were arranged in order of concentration on the rack. One row was left out for agitating the test tubes.
Inoculation commenced by transferring glucose into different test tubes T1being the least concentrated. 0.5mM of Glucose was transferred using a P100 pipette and not changing the tip. 0.2cm3 was inoculated into T2, 0.4 cm3 into T3, 0.6 cm3 into T4, 0.8cm3 into T5 and 1.0 cm3into T6.
Distilled water was then inoculated into the test tubes using a different tip to avoid cross contamination. One centimetre cubed was inoculated into T1, 0.8 cm3 into T2, 0.6 cm3 into T3, 0.4 cm3 into T4 and 0.2 cm3 into T5. There was no water inoculated into the last tube T6.
Phenol was then inoculated into all the twelve test tubes. It was transferred using a different tip to avoid cross contamination.
One centimetre cubed of different sugars were inoculated in specific S tubes. 0.5mM of Galactose was inoculated into S1, 0.5mM of Glucose was inoculated into S2, Glucose unknown was inoculated into S3, 0.5mM of Fructose was inoculated into S4, 0.5mM of Maltose was inoculated into S5 and then 0.5mM of Ribose was inoculated into S6. These transfers were done with different tips for different sugars.
One and half millilitres of GODPOD reagent was then inoculated into all the test tubes using an automatic pipette and a long pipette tip. The test tubes were then agitated on the rack and incubated in the water bath for forty minutes. The temperature was constantly checked during incubation.
After forty minutes, the solutions changed colour from colourless to light pink according to the concentration. These different solutions were then read on a spectrophotometer in a cuvette. The spectrophotometer was zeroed at first then absorbencies of Glucose and other sugars were read and recorded.
A cuvette was wiped on the soft side to minimize absorbencies caused by contamination. These different absorbencies were recorded on a table.

Discussion The reactions of glucose with the GODPOD were slow due to the fact that the enzyme was stored in ice thus it took long for the reaction to take place. The enzyme’s structure was disrupted because it was kept in cold thus it took time to equilibrate with the conditions.
The reason why GODPOD was slow to react was because it is sensitive to its environment, (Teal et al, 1985). Enzymes are sensitive to pressure, temperature and pH. This added to the fact that the colour produced was not very dark because the enzyme was adjusting to the conditions. The enzyme also didn’t denature because it was kept in ice at 4oC not in the heat above 40oC.
On the graph, the points that are not on the line of best fit might have appeared because there might have been a competitive inhibitor thus the reaction didn’t go on well owing to reduced absorbance. The inhibitor might have been so because of cross contamination. The same pipette might have been used to transfer the solutions thus cross contamination.
The specificity of the enzymes might have caused the other solutions not to produce reliable results. Enzymes are sensitive to pH, (Jan, 2010). The pH of the test tube might have been so low or higher than the required thus some of the points are not in the line of best fit. This might have been avoided by carrying out the experiment repeatedly and then getting average values.
When the enzyme was applied to the other sugars, there was no absorbance at all because the enzyme is specific to one substrate thus it didn’t catalyse the reaction of other sugars and GODPOD. There might have no absorbance because these sugars might have their own wavelengths they absorb the light. This might have been avoided by scanning the various wavelengths and determine the exact wavelength.
The unknown glucoses were determined by the use of the graph. The line of best fit was used to determine the glucose concentration. An equation was used also to determine the concentrations.
The other points not on the line of the best fit might be there because the transitional state might have been great, thus when thirty minutes had passed, the enzyme had not gone past the transitional stage. This might have been characteristic of the colours produced according to the concentration of the different solutions.
The unknown glucose solutions showed to have the same concentration as the stock solution. It might have the same compounds and properties as the stock solution.
The results of the experiment were according to the literature values. This was highlighted by the absorbencies of stock solutions and different sugars. The specificity of the reaction was achieved.
In conclusion, the aims of the experiment were achieved by obtain reliable data and results. The standard curve showed the absorbencies of the different solutions and unknowns.

Physiology of Manic Depression

Bre Wilson
Manic Depression, or more commonly known as Bipolar disorder, is a disorder that causes the patient affected to cycle through periods of depression and mania (NIMH n.d.). There are three general types of Bipolar Disorder and then a fourth category for those who do not fall within any of the previous categories. Bipolar I Disorder is defined by manic episodes that last at least 7 days and then a depressive episode lasting at least two weeks. Symptoms may be severe enough to seek immediate medical care. Bipolar II Disorder is defined by recurring depressive, and hypomanic episodes, but not to the extent of Bipolar I. Cyclothymic Disorder is defined by numerous periods of hypomania and depression that lasts longer than two years in adults, (for one year for children), but the symptoms exhibited do not fit the within the categories of a simple hypomanic episode or a simple depressive episode. The final category is Other Specified and Unspecified Bipolar and Related Disorders which is defined as bipolar tendencies, and symptoms, that do not meet the requirements within the previous three categories (NIMH n.d.). The true cause of Bipolar Disorder is unknown but scientists believe that it could be due to many environmental and genetic factors, as well as chemical imbalances within the brain- such as: GABA, serotonin, dopamine and norepinephrine (Bressert 2016). Some people experience “rapid cycling” of episodes while others experience a prolonged period of time between cycles of episodes. The exact cause behind the rapid cycling is unclear but researchers believe that it’s due to the consistent misfiring of receptors causing mayhem within the Central Nervous System. These neurotransmitters play a huge role in a person’s circadian rhythm, their emotional expression and regulation, their behavior and how well they respond to stress.
After years of study, Manic Depression is now thought to be a result of serotonin, norepinephrine and dopamine imbalance, as well as a GABA defect within the hippocampus (Frey 2007). GABA, a relative of Glutamate, is a primary inhibitory neurotransmitter which means that it mediates other neurotransmitters to prevent over exertion. Glutamate is the most abundant neurotransmitter present within the Central Nervous System. It’s hypothesized that erratic misfiring of the Glutamate receptors within the brain could possibly be the cause of GABA fluctuations (Frey 2007). This causes the neurotransmitters that GABA mediates, (norepinephrine, dopamine and serotonin), to fluctuate unpredictably as well causing the mood swings, sleeplessness, depression and self-destructive behavior expressed by patients diagnosed with Manic Depression. Dopamine, which acts on the D receptors in the brain, is the neurotransmitter that regulates a person’s feelings of pleasure and emotional reward. Norepinephrine, which acts on alpha and beta adrenergic receptors, is the terminal neurotransmitter of the Sympathetic Nervous System. Norepinephrine is the “fight or flight” molecule. Serotonin, which acts on the 5-HT receptors within the brain, is the neurotransmitter that fosters feelings of happiness and well-being. Modifying the effectiveness, or quantity, of these three neurotransmitters is what leads mania, hypomania, and depression exhibited in patients with Manic Depression.
People who are experiencing a manic episode often engage in risky behaviors, (binge drinking or promiscuity), exhibit restlessness, and experience disruptions in their sleep patterns. Manic episodes can be triggered chemically within the brain, or they can be triggered by external factors such as: excessive stress, a change in diet or exercise, excessive caffeine intake, antidepressant medication, and certain other medications as well (Bressert 2016). Dopamine plays a role in the behaviors exhibited by patients in manic episodes. Dopamine acts on the D receptors within the brain and cause a person to be emotionally satisfied. When the receptors cannot receive dopamine due to a blockage, or antagonist, emotional satisfaction is dramatically lowered- causing patients to engage is risky behavior to try to fulfill their emotional needs. Rapid firing of norepinephrine receptors cause unpredictable, behavior due to the influence on the sympathetic nervous system. Some studies suggest that mania is due to certain circadian cues and insist that treatment for manic depression be scheduled around a person’s circadian rhythm (Roybal et al 2007). Serotonin plays a role in mania because patients have decreased binding affinity at 5-HT receptors during a manic episode (Yatham 2010). Serotonin and norepinephrine play a major role in mania but has also been linked to the depressive episodes as well.
Depressive episodes almost always follow manic episodes which is most likely the result of neurotransmitter reuptake and are the result of neurotransmitter imbalance within the brain. Many times, manic depression is often misdiagnosed as depression since depressive episodes are easier to diagnose than manic episodes, and therefore an antidepressant is prescribed. However, when a patient takes an antidepressant, such as a serotonin reuptake inhibitor, it doesn’t allow the active reuptake of serotonin which often leads to a manic episode because of the reduced binding (Yatham 2010). In contrast with manic episodes, brain serotonin 5-HT receptor binding is increased during bipolar depressive episodes. Treating manic depression is often very complex and combines a process of finding just the right regime of medication, as well as psychotherapy.
Although manic depression is a life-long illness that cannot be totally eradicated, many patients can lead a semi-normal, productive life if properly medicated. Typical treatments for bipolar disorder include: psychotherapy, mood stabilizers, antipsychotics, and some Selective Serotonin Reuptake Inhibitors (NIMH n.d.). In some cases, doctors will prescribe a medication to combat the depression, a medication to combat the mania, and then medication to combat any side effects caused by the other medications. Patients who exhibit a higher rate of serotonin 5-HT binding prior to starting medication had a higher chance of recurring episodes, even while taking medication, than did those who had a lower rate of serotonin 5-HT binding prior to medication (Parsey 2013). Lithium was a drug that works within the nervous system to strengthen the neural connections between cells to increase cell signaling therefore limiting the frequency and intensity of manic-depressive episodes. Lithium was anticipated to have success rates as high as 70%-80% but studies now indicate that Lithium really has a success rate of approximately 40% and comes with a plethora of side effects (Surgeon General Report for Mental Health). Modafinil is an alpha-adrenergic agonist and directly stimulated the receptors within the brain (modafinil.com n.d.). The reuptake of noradrenaline by the noradrenergic is decreased and increase excitatory glutamatergic transmission is increased. This decreases GABAergic transmission, resulting in the elimination of GABA receptor signaling which could combat symptoms of bipolar depression by limiting the abundance of GABA receptor misfiring (modafinil.com n.d.). Studies show a significant improvement in depressive symptoms in the patients who received the experimental drug (Modafinil) by the second week with maintained improvement throughout the remainder of the experiment (Frye 2007). The drug did not seem to affect mania however, because during the experimental period, there was no significant difference between the emergence of mania or in hospitalizations due to mania (one per group) (Frye 2007).
Works Cited
Bressert, S. (2016). Causes of Bipolar Disorder. Psych Central.
Frey B. N., Andreazza A. C., Nery F. G., Martins M. R., Quevedo J., Soares J. C., Kapczinski F. (2007). The role of hippocampus in the pathophysiology of bipolar disorder. Behavioral Pharmacology, 18(5-6):419-30
Frye, M. A., Grunze, H., Suppes, T., Mcelroy, S. L., Keck, P. E., Walden, J., . . . Post, R. M. (2007). A Placebo-Controlled Evaluation of Adjunctive Modafinil in the Treatment of Bipolar Depression. American Journal of Psychiatry, 164(8), 1242-1249.
Modafinil (Provigil). (n.d.). Retrieved March 29, 2017, from https://www.modafinil.com/
National Institute of Mental Health. (n.d.). Retrieved March 28, 2017, from https://www.nimh.nih.gov/health/topics/bipolar-disorder/index.shtml#part_145402
Parsey RV, Olvet DM, Oquendo MA, Huang YY, Ogden RT, Mann JJ. (2013). Higher 5-HT(1A) Receptor Binding Potential During a Major Depressive Episode Predicts Poor Treatment Response: Preliminary Data from a Naturalistic Study. Neuropsychopharmacology.
Roybal, K., Theobold D., Graham A., DiNieri, J. A., Russo, S. J., Krishnan, V., Chakravarty, S., Peevey, J., Oehrlein N., Birnbaum S., Vitaterna, M. H., Orsulak, P., Takahashi J. S., Nestler, E. J., Carlezon Jr., W.A., and McClung C. A. (2007). Mania-like behavior induced by disruption of CLOCK. PNAS.
Surgeon General Report for Mental Health. (n.d.). Accessed March 28, 2017.
Yatham, L. N., Liddle, P F., Erez, J., Kauer-Sant’Anna, M., Lam, R. W., Imperial M., Sossi, V., and Ruth, T. J. (2010). Brain serotonin-2 receptors in acute mania. The British Journal of Psychiatry, 47-51

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