Experiments in Clinical Biochemistry

Experiment#11 -A

Handout

Clinical Biochemistry

• Developed from a need to create an alternative method for quantifying the level of biological molecules in an individual

• Due to the advances in clinical chemistry, medical technology has surged over the last several decades

• Clinical chemistry provides medical professionals with an extensive arsenal of tests to aid in the early detection of diseases or conditions

Sample Matrix

• Extracellular body fluids– interstitial fluid (between tissues and cells)

– lymph

– cerebrospinal fluid– blood plasma or serum (the most common source)

• since every cell in the body comes in contact with capillary blood, any unusual types of metabolism due to infection, condition, or toxin exposure, will be revealed by what the cell secretes into the bloodstream.

• Excretory fluids

Clinical Biochemistry

• Purpose: to become familiar with several assays that are currently used in the field of biochemistry– Analyses include:

• serum glucose

• serum urea nitrogen

• serum cholesterol

Reaction type

• All three analyses make use of a coupled enzyme catalyzed reaction– “catalyst” = a substance that increases the rate of a chemical reaction

without being consumed or produced in the reaction. Enzymes are biological catalysts.

• Procedural Note:– The rate of the enzyme catalyzed reaction should be directly

proportional to the concentration of the analyte being measured

– The reaction should always be carried out at optimal pH, temperature, and ionic strength

– The enzyme selected should be substrate specific

Analytes

• Glucose– high blood glucose levels associated with:

• diabetes

• hyperactive thyroid or adrenal gland

– low blood glucose levels associated with:• hypoglycemia

• underactive endocrine system

• some forms of liver disease

– glucose analysis assesses the overall integrity of carbohydrate metabolism

• NAD+ is reduced to NADH during the conversion of glucose-6-phosphate into 6-phosphogluconate

• The production of NADH will be monitored spectrophotometrically at 340 nm

• moles of NADH produced = moles of glucose in the reaction mixture

Glucose Assay

Glucose Assay

Analyte

• Urea Nitrogen– Ammonia , a toxic byproduct of amino acid

metabolism, is converted in the liver to a water-soluble form of nitrogen (urea)

– Urea is then passed to the blood, which carries it to the kidneys to be filtered and excreted as urine

– Increased urea concentrations in the blood are indicative of impaired liver function due to disease or infection (hepatitis)

Urea Nitrogen Assay

– relies on coupling the production of ammonia and carbon dioxide from urea

– then the transamination of alpha-ketoglutarate to glutamate

– to finally the oxidation of NADH

– the consumption of NADH will be monitored spectrophotometrically

Urea Assay

• Urea + H2O       urease         2NH3 + CO2

• NH3 + a-Ketoglutarate + NADH + H+

   glutamate dehydrogenase     L-glutamate + NAD+ + H2O

Analyte

• Cholesterol– Not the “bad guy” that it is made out to be

• Naturally produced in the body (80% made in the body, 20% taken in from diet)

• The building block for male and female sex hormones

• Vitamin D, necessary for calcium metabolism, is synthesized from cholesterol

• Adrenal gland hormones produced from cholesterol

• Essential for the construction of every cell wall in the body

Cholesterol

• HDL (high density lipoprotein)– the type of lipoprotein that transports

cholesterol back to the liver from peripheral cells (composed primarily of protein)

• LDL (low density lipoprotein)– derived from very-low-density lipoproteins

(VLDL) as cells remove triglycerides from them (composed primarily of cholesterol)

Cholesterol / Heart Disease

• As a result of faulty diet, poor digestion and metabolic dysfunction of the liver….– Increase in buildup of calcium, fats, and cholesterol

• when fats adhere to calcium and cholesterol adheres to the fat, plaque builds up and clogs the arteries

– High LDL cholesterol is associated with a high risk of heart attack

– High HDL cholesterol seems to have a protective effect

Cholesterol / Other Disease

• High blood cholesterol (hypercholesterolemia) is associated with the following conditions– diabetes mellitus

– atherosclerosis

– diseases of endocrine system, liver, or kidney

** Determination of cholesterol is used in conjunction with other clinical measurements mainly for confirmation of a diseased condition, not a particular ailment***

Cholesterol Assay

– cholesterol esters are hydrolyzed by cholesterol esterase

– Cholesterol is oxidized to cholest-4-ene-3-one• peroxide is produced as a byproduct

– The production of peroxide is coupled with a reaction to convert peroxide to a colored chromogen (monitored at 510 nm)

Procedure• Blood glucose assay

– Preparation of standard curve• Set up the following test tubes

– #1– 750 uL glucose reagent + 8 uL Standard 1 (5.00 mM)– #2--750 uL glucose reagent + 8 uL Standard 2 (10.00 mM)– #3--750 uL glucose reagent + 8 uL Standard 3 (20.00 mM)

• Mix tubes well and incubate at room temperature for 15minutes

• Add 375 uL of deionized water and record the absorbance at 340nm (zero spectrophotometer with deionized water as reference)

– Analysis of serum• Perform like standards (using 8 uL of bovine serum)• Dilute the serum if necessary to ensure it is in the range of the

standards

Procedure• Blood urea nitrogen assay

• Preparation of standard curveSet up the following test tubes– Standard #1– 1mL urea nitrogen reagent– Standard #2--1mL urea nitrogen reagent– Standard #3--1mL urea nitrogen reagent– Serum-- 1mL urea nitrogen reagent

• Incubate the reagent at 37degrees C for 10min• Zero spectrophotometer at 340nm with deionized water• When ready to measure the absorbance for each sample

– Transfer heated solution from test tube to cuvette– Add 10uL of appropriate standard or serum to the heated reagent,

mix and record absorbance after 30 seconds. (A1)– Record absorbance again after 60 seconds and 90 seconds(A2 &A3 )– Repeat procedure for each sample– Dilute serum if necessary

Procedure

• Cholesterol assay • Measurement of Total cholesterol

– Set up the following 5 test tubes:• #1 10 uL of deionized water (0 mg/dL)

• #2 10 uL of 50 mg/dL cholesterol standard

• #3 10 uL of 100 mg/dL cholesterol standard

• #4 10 uL of 200 mg/dL cholesterol standard

• #5 10 uL bovine serum

• #6 10 uL 2x diluted bovine serum

(5uL deionized water + 5uL bovine serum)

Procedure• Total cholesterol assay ( continued)

– Pipet 0.5 mL of cholesterol reagent. Parafilm and gently invert 3-5 times

– Incubate at 37oC for 10 minutes– Add 1.0 mL of saline. Mix by inversion– Transfer blank to a cuvette and set 0 and 100%T at 510 nm– Record absorbances for all other samples at 510 nm (Be sure

to record all absorbances within 30 minutes of removing from bath)

– Blank correction for turbidity is not necessary– Dilute serum if necessary

Data Analysis– Prepare a standard curve (Beer’s Law Plot) using the concentrations

of the standards and their respective absorbances for each analyte measured

• *** For the urea nitrogen analysis, plot concentration vs. change in absorbance*** (Find the most linear plot type using either absorbances directly or the absorbance changes)

– Using linear regression, calculate the equation of the line• y= mx +b• Absorbance = slope (concentration) + y-intercept

– Using the absorbances from the serum samples, calculate the concentration of the appropriate analyte

– *** Make sure that the absorbances of all serums were in the range of the standard curve** Dilute when necessary