1. Experiments in Clinical Biochemistry
Experiment#11 -A
Handout

2. 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

3. Sample Matrix Extracellular body fluids Excretory 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

4. 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

5. 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

6. 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

7. Glucose Assay
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

8. Glucose Assay

9. 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)

10. 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

11. Urea Assay Urea + H2O urease 2NH3 + CO2
NH3 + a-Ketoglutarate + NADH + H+
   glutamate dehydrogenase     L-glutamate + NAD+ + H2O

12. 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

13. 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)

14. 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

15. 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***

16. 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)

17. 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)
# uL glucose reagent + 8 uL Standard 2 (10.00 mM)
# 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

18. Procedure Blood urea nitrogen assay Preparation of standard curve
Set 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

19. 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)

20. 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

21. 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