LAB # 9: SERUM GLUCOSE ASSAY;GLUCOSE TOLERANCE TEST -THE DIAGNOSIS OF DIABETESObjectives: By the end of this lab session, you should: 1. Understand the role of insulin in the regulation of carbohydrate metabolism. 2. Understand what happens in diabetes mellitus (Type I), where there is an insulin deficiency. 3. Understand the principle of the glucose tolerance test, and how it is used in the diagnosis and treatment of diabetes mellitus. 4. Understand the general idea of end-point enzymatic assays and their relevance to determining serum glucose concentrations. 5. Appreciate a specific real-world example of how spectrophotometry can be used to determine the concentration of a physiologically relevant metabolite in blood. BACKGROUND Diabetes in Greek means "to pass through a siphon.” The disease is so named for the excessive urination that accompanies it. Aretaeus, a Cappadocian physician of the second century A.D., wrote: "The epithet diabetes has been assigned to the disorder, being something like passing of water by a siphon." He perceptively characterized diabetes as "being a melting-down of the flesh and limbs into urine." In fact, in severe, untreated diabetes, the victim's body wastes away despite repeated efforts to satisfy a powerful hunger and thirst.Mellitus is from Latin and means "sweetened with honey". This term refers to the presence of sugar (glucose) in the urine of patients having the disease.This lab is our first involving an analytical procedure routinely performed in hospital clinical chemistry labs. We will analyze data from a glucose tolerance test, which involves determining glucose concentrations in blood samples at specific intervals following administration of a large amount of oral glucose. However, before we begin, we need to consider some background information on carbohydrate metabolism and its regulation. The digestion of starch and disaccharides from the diet yields glucose, fructose, and galactose, but these catabolic pathways all converge very quickly to that of glucose itself. Glucose thus lies at the center of carbohydrate metabolism in humans. Glucose derived from digested foodstuffs enters the bloodstream from the intestinal tract, and most is soon removed from the circulation by various tissues. Muscle cells take up glucose and use it either immediately to make ATP (glycolysis and oxidation through the TCA cycle) or to replenish their glycogen reserves. Liver cells similarly store glucose as glycogen, and the liver is able to release this glucose back into the bloodstream by breaking down glycogen when the blood sugar level falls and needs to be maintained (fasting state - glucagon).Virtually all of the monosaccharides in blood consist of glucose. After several hours of fasting, the normalrange for the blood glucose level, called the normal fasting level, is 80-100 mg/dL of serum. Recall that mg/dL = mg/100 mL. This unit is the standard used by clinical chemistry labs for expressing concentrations in body fluids. If you will be working in a medical setting, you will encounter it daily, so you need to become familiar with it.Wide variations from the normal fasting level of blood glucose generally signify that something is wrong, so a special vocabulary exists to define such variations. Hypoglycemia (hypo = under; glyc = sugar; emia =in blood) is the condition of a blood sugar level below normal, while hyperglycemia (hyper = above) is thecondition when this level is above the normal fasting level. If the blood sugar level becomes high enough,the kidneys are unable to return to the circulation all of the glucose that leaves the blood during renal filtration. Glucose then appears in the urine and the term for this condition is glucosuria (gluco = glucose;uria = in urine). The blood glucose level at which glucosuria happens is called the renal threshold for glucose, and is in the range of about 140-160 mg/dL.When hypoglycemia develops rapidly (this is unusual in normal individuals), dizziness and sometimes fainting may ensue. The usual antidote for this condition, which sometimes occurs when students have been studying for prolonged periods of time without nourishment, is to eat a sugar-laden candy bar whilegulping down an equally sugar-laden cola. Dizziness vanishes abruptly, allowing conscientious students tocontinue their work with renewed vigor and intensity. People with diabetes who unknowingly take too much insulin experience this hypoglycemia in a severe form known as insulin shock. Behind such responses by the central nervous system to hypoglycemia is the brain's almost total reliance on glucose from the circulation for its minute-by-minute energy needs. The brain consumes about 120 g/day of glucose, and a quick onset of hypoglycemia actually starves the brain cells. Although they have the ability to eventually switch over to other nutrients, brain cells cannot do so rapidly enough to prevent dangerous side-effects. Whenever hyperglycemia develops, something is probably wrong with the mechanisms whereby tissues withdraw glucose from the circulation. Diabetes is a common cause of hyperglycemia.Insulin, a polypeptide hormone synthesized in the -cells of the pancreas, has a powerful lowering effect on the blood glucose level. The release of insulin is stimulated by an increase in the blood glucose level, such as normally occurs after eating a meal rich in carbohydrates. Insulin binds to receptors on the cell membranes of muscle and adipose tissue. These insulin-receptor complexes make it possible for glucose molecules to be taken up more easily into the cells, which in turn lowers the blood glucose level. Insulin promotes uptake of glucose by muscle and adipose tissues.Insulin deficiency or insulin resistance cause diabetes mellitus. Diabetes mellitus is a complex disease that affects several hundred million people worldwide. In the United States, there are nearly 15 million known cases of diabetes, and almost as many others are also believed to have this disease. Diabetes is characterized by impaired glucose uptake by peripheral tissues, which results in elevated levels of glucosein the blood and in the urine. As noted above, glucose is excreted in the urine when the blood glucose level exceeds the resorptive capacity of the renal tubules. Water accompanies the excreted glucose, and so an untreated diabetic in the acute phase of the disease is both hungry and thirsty. The lack of