EXPERIMENTS RELATED TO CARBOHYDRATE METABOLISM

Glucose levels are typically measured in body fluids. Blood and urine samples are most commonly used
for this purpose.
Blood glucose levels can be measured in whole blood, serum, or plasma. When whole blood samples are
kept at room temperature, glucose levels decrease by approximately 7-10% per hour due to glycolytic
enzymes in blood cells metabolizing glucose. Therefore, if whole blood is to be used for measurement, the
procedure should be done immediately. If serum or plasma is to be used, blood cells should be removed by
centrifugation within half an hour and stored in a refrigerator (+4°C).
In routine practice, it is recommended to collect blood samples for glucose measurement in fluoride tubes
(NaFl, gray-capped). The fluoride ion is an inhibitor of the enolase enzyme in glycolysis. Glucose levels in
fluoride plasma samples are stable for 8 hours at room temperature and 72 hours at +4°C.
Glucose measurement is usually performed on venous blood samples taken following 8-12 hours of fasting
(fasting blood glucose test) in the morning. It can also be done on blood samples taken 2 hours after any
meal (postprandial blood glucose) or as a component of the oral glucose tolerance test (OGTT) following
75 g glucose.
Fasting glucose OGTT 2nd hour
Normal glucose tolerance <100 mg/dL <140 mg/dL
Impaired fasting glucose (IFG) 100-125 mg/dL <140 mg/dL
Impaired glucose tolerance (IGT) <100 mg/dL 140-199 mg/dL
Diabetes Mellitus >126 mg/dL >200 mg/dL

Testing for Urine Glucose

Circulating glucose is filtrated into the glomerular filtrate through glomeruli, but under normal conditions,
they are completely reabsorbed by the proximal tubules. So that, glucose or other monosaccharides are not
found in urine.
The presence of glucose in urine usually indicates elevated blood glucose levels. Glucose begins to appear
in urine when blood glucose levels exceed the kidney threshold of 160-180 mg/dL. The presence of glucose
in urine is called “glycosuria”.
Renal glycosuria is defined as the detection of glucose in the urine despite blood sugar being below 160-
180 mg/dL. This could be a congenital condition caused by a reduced number or insufficient function of
Na-Glucose transporters responsible for glucose reabsorption.
Today, urine strips (paper strips) impregnated with glucose oxidase, peroxidase, and a reduced chromogen
are generally used to test for urine glucose because they provide quick results and are easy to use. Special
chemical tests such as polarimetry, fermentation, osazone formation, or Seliwanoff test (fructose), Bial's
orcinol test (pentoses) can be applied to determine the type of sugar(s) present in urine. The presence of
lactose in urine can be seen during postpartum periods physiologically.

Qualitative Testing for Urine Glucose

The presence of glucose or other monosaccharides in urine can be easily demonstrated by tests based on
the reducing properties of carbohydrates. These qualitative tests are methods developed based on glucose's
reducing property in hot and alkaline environments. They are not specific, because other reducing
substances like uric acid, creatinine, and glutathione, may give positive results.
Fehling test
Reagents:
1- Fehling I: 7% CuSO4 solution
2- Fehling II: Solution containing 350g sodium-potassium tartrate and 100g NaOH
Procedure: Equal volumes of Fehling I and Fehling II reagents are placed in a test tube and heated over an
open flame. This process is done to determine if there are any reducing substances in the test environment,
the reagent's color should not change with heating.
After confirming the reagent's color hasn't changed, adding an equal volume of urine to the tube containing
the boiled reagent, tube is heated again. If glucose (or other reducing sugars) is present in the urine, an
orange-red color will form.
Principle:
When Fehling I and Fehling II reagents are mixed, reaction is; CuSO4 + 2NaOH → Cu(OH)2 + Na2SO4.
The blue color copper (II) hydroxide [Cu(OH)2] tends to precipitate in alkaline conditions. The sodium-
potassium tartrate found in Fehling II reagent prevents precipitation. Sugars containing free aldehyde and
keto groups reduce divalent copper (Cu+2) to Cu+1 in hot and alkaline conditions, as a result of reduction
reaction. Cu(OH2) first is converted to yellow copper(I) hydroxide, and with continued heating, to brick-
red color, copper(I) oxide (Cu2O) is generated.
Disaccharides with free aldehyde or keto groups also give (+) results in the Fehling test. If there is no
reducing sugar in the environment, the blue color does not change as a result of heating.

CuSO4 + 2NaOH → Cu(OH)2 + Na2SO4

Cu(OH)2 → Cu2O + H2O
Sucrose (saccharose) is a disaccharide formed by the condensation of glucose and fructose. Since sucrose
doesn't contain free aldehyde or keto groups due the condensation of free aldehyde and keto groups, it gives
negative (−) results with the Fehling test. The Fehling test is not specific for glucose and other sugars.
Many compounds such as uric acid, creatinine, aldehydes, phenols, aminophenols, formic acid, and
resorcinol give positive reactions with Fehling's reagent.
 PROTEIN METABOLISM EXPERIMENTS
Total protein amount in serum: 6.0-8.3 g/dL
Serum albumin level: 3.5-5 g/dL
Globulin level: 2.3-3.5 g/dL
Albumin / Globulin ratio: approximately 2:1
Serum protein levels are measured to evaluate general health and nutritional status, particularly for
diagnosis and monitoring of diseases originating from liver, kidney, and bone marrow. In clinical
biochemistry laboratories, protein measurements are made in serum, urine and also in body fluids including
cerebrospinal fluid, pleural fluid, peritoneal fluid, etc. Serum proteins are evaluated by measuring total
protein and albumin levels. The level of globulins, which are heterogeneous group of proteins, is calculated
by subtracting albumin amount from total protein amount.
Protein electrophoresis is a diagnostic method based on the separation of proteins depending on their net
charge. It enables detection of changes in globulin subgroups (α1-, α2-, β- and γ-globulins). Additionally,
individual levels of globulins can also be measured by immunometric methods.
An increase in serum protein level is called “hyperproteinemia”; a decrease is called “hypoproteinemia”.
Some atypical proteins that are not found in the blood under normal conditions and are structurally similar
to immunoglobulins are called "paraproteins". For example, in multiple myeloma, increased paraprotein
includes immunoglobulin heavy (α, β, γ) and/or light chains (kappa, lambda) are seen.

1. Qualitative Testing for Urine Protein

Under normal conditions, albumin is filtrated from glomeruli, then completely reabsorbed from proximal
tubulus. Only 20-150 mg of protein is excreted daily in urine, this trace amounts of protein cannot be
detected by routine protein testing methods such as dipsticks, and in practice, it's accepted that there is no
protein in urine. Approximately half of urinary proteins is albumin, and the remainder is uromucoid secreted
from distal tubules. An increase in urine protein level (≥300 mg/day) is called “proteinuria” or formerly
“albuminuria”.
Microalbumin; is defined as albumin excretion in urine above normal values (150 mg/day) but not
measurable with urine strips. Urinary albumin excretion is between 30-300 mg/day and is generally
considered an indicator of kidney disease that may develop in chronic diseases such as hypertension and
Diabetes Mellitus and should be monitored.
Proteinuria is mainly classified into two groups as functional or organic proteinuria.
a. Functional proteinuria occurs without any disease, due to heavy exercise, prolonged standing, high
fever or pregnancy (150 mg/day).
b. Organic proteinuria can be prerenal, renal, or postrenal situations.
Prerenal proteinuria occurs in conditions that impair kidney circulation such as heart failure, fluid
accumulation in abdomen, intra-abdominal tumors, liver diseases, and febrile illnesses.
Renal proteinuria occurs with diseases that cause impairment in kidney filtration capacity, such as nephritis
and nephrosis.
Postrenal proteinuria occurs due to inflammatory diseases, tumors, or trauma of ureters, bladder, prostate,
and urethra.
Qualitative, semi-quantitative, and quantitative methods are used for the determination of urine protein:
Qualitative methods are based on the principle of precipitating urine protein by denaturing it with various
chemicals or physical methods, and the intensity of turbidity is directly proportional to the protein amount.
a. Heat coagulation test
Procedure:
It is the simplest test for detecting protein in urine. Full 2/3 of a test tube with urine and gently heat the
upper half of the fluid. If a cloudy white precipitate (ppt) appears in the heated part, it may be due to the
presence of albumin or phosphate salt. In differential diagnosis of protein and phosphates, few drops of
acetic acid are added to the white ppt. and notice the result. If the ppt. disappeared, that is a phosphate salt.
If not, that is globular protein.
b. Heller's Ring Test
Reagents: Concentrated nitric acid (HNO3)
Procedure: Some urine is placed in a test tube, and the tube is held at about 45° angle. Concentrated HNO3
is slowly dripped using a pipette. Since HNO3 is denser than urine, it moves toward the bottom of the tube.
If protein is present in the urine, a white, cloudy ring forms at the interface between HNO3 and urine due
to protein denaturation by acid effect.

Semi-quantitative methods; use urine strips impregnated with anionic dyes that react with albumin, such
as tetrabromophenol blue, tetrachlorophenol, or tetrabromosulfonphthalein, and buffers. Qualitative and
semi-quantitative methods are generally not suitable for investigating proteins above 300-500 mg/dL. Also,
microalbuminuria or paraproteinuria cannot be detected with using dipstick method.

Sulfosalicylic acid test is another semiquantitative method, based on the protein precipitation by acid effect.
Equal volumes of urine and 3% sulfosalicylic acid solution are mixed, left for 5 minutes, and the turbidity
is evaluated between 1(+) and 4(+).
≤5 mg/dL Negative (-) Clear
≈ 20 mg/dL Trace (±) Opelescent
≈ 50 mg/dL (+) Turbidity with no granulation
≈ 200 mg/dL (++) Turbidity with granulation
≈ 500 mg/dL (+++) Turbidity with granulation and sedimentation
≥1.0 g/dL (++++) Precipitated solid clumps or solid precipitate
2. Quantitative methods for protein measurement: Urine or serum total protein is measured
commonly by biuret method; serum albumin determination by bromcresol green (BCG), bromcresol purple
(BCP) method; and microalbumin measurement in urine by immunometric methods.
Biuret method:
The reaction in the biuret test is a colorimetric reaction where the result is indicated by a color change from
blue to purple or violet.
In an alkaline environment, the cupric (Cu+2) ions in the biuret reagent bind to the nitrogen atoms in the
peptide bonds of proteins forming a violet-colored copper coordination complex. The formation of purple
color indicates the presence of peptide bonds in the sample. The intensity of the developed purple color is
directly proportional to the concentration of peptide bonds present in the solution.

Reagent:
1- Biuret reagent: includes 30 mM potassium iodite, 100 mM Na+-K+ tartarate, 30 mM CuSO4 ve 3.8 M
NaOH
2- Standart: protein solution of 7 g/dL

Three tubes is prepared as follows:

Blank Standart Test
Biuret reagent 2 mL 2 mL 2 mL
Distilled water 40µL - -
Standart solution - 40µL -
Serum - - 40µL

Tubes are mixed and incubated at 37°C'de 10 minutes or at 20-25°C for 20 minutes. And the absorbances
Calculation
Total protein (g/dL) = (Test absorbance/Standart absorbance) x Standart concentration (7g/dL)