Carbohydrates are the most abundant biomolecules and serve as energy forces, fuel metabolic intermediates and have major structural roles in plants and insects. Carbohydrates are polyhydroxy aldehydes or ketones with the empirical formula (CH2O)n. Monosaccharides or simple sugars consist of a single polyhydroxy aldehyde or ketone unit with at least one asymmetric carbon. Monosaccharides with five or more carbon atoms can exist in closed five-membered ring (furanose) or six-membered ring (pyranose) structures. The hemiacetal or carbonyl carbon is known as the anomeric carbon.

Figure 1. Linear and ring structure of glucose and the effect of oxidization. Glucose is the most abundant monosaccaride in nature (Fig. 1). Relatively mild oxidizing agents (e.g., Fe3+, Cu2+) can oxidize the carbonyl (anomeric) carbon to carboxylic acid of glucose and other sugars with a free anomeric carbon. Sugars capable of reducing these ions are known as reducing sugars.

Under controlled conditions, the amount of Cu2+ reduced to Cu+ is proportional to the amount of reducing sugar. The Cu2O formed from Cu+ can then be measured. In today's practical, we will use the Somogyi-Nelson method to determine the amount of reducing sugar. Following addition of Nelson's arsenomolybdate reagent, which is reduced by Cu+, an intense blue colour is formed which can be measured colourimetrically.

These reducing sugar tests were used routinely to determine glucose content of urine and blood. Serum or plasma glucose normally occurs in the range of 65-115 mg/100 mL or 3.6-6.4 mmol/L. Increased levels of glucose are associated with diabetes mellitus and also hyperactivity of adrenal, pituitary or thyroid glands. Decreased levels of glucose occur in conditions interfering with glucose absorption, hypoadrenalism, insulin secreting tumors and insulin overdose.

Nowadays, blood or urine glucose is usually determined by a test using the enzyme, glucose oxidase, which catalyses: glucose oxidase D - glucose + O2  D - gluconate + H2O2

The peroxide formed is then reacted with various dyes to produce an easily detected colour change. In today's practical you will determine the amount of brown oxidized o-diansidine formed. peroxidase H2O2 + o - diansidine  oxidized o - diansidine (brown)

Both of these assays (Somogyi-Nelson and glucose oxidase) are making use of the principles of the Lambert-Beer Law which states that the absorbance of the solution is directly proportional to the concentration of the solution.

Io A = log __ = lc Ie

where A = absorbance (obtained from spectrophotometer) Io = intensity of incidence light Ie = intensity of transmitted light  = molar absorption coefficient c = amount of absorbing species l = length of light path (usually 1 cm)

The Lambert-Beer Law assumes that the light is monochromatic and parallel. Using this law, the unknown concentration of a solution may be determined.