CLINICAL CHEMISTRY LAB 1 FINALS ● UREMIA: Azotemia + Acidosis + Electrolyte

BLOOD UREA NITROGEN, CREATININE imbalance (hyperkalemia)

*elevated potassium levels

CLINICALLY SIGNIFICANT NPNs

● Urea 45%
● Uric Acid 20%
● Creatinine 20%
● Ammonia 0.2%
● Amino acid 20%
● Creatinine 1-2%
*urea is elevated first in renal disorders

BLOOD UREA NITROGEN (BUN)

UREA
● Major end-product of protein and amino acid
metabolism (from ammonia and amino acid)
*catabolism: breaking down of protein into simple
substances
● 45% most abundant of NPN; 80% total
nitrogen excreted
● Ammonia to Urea: Takes place in the liver
Roles of the Kidney: ● Sensitive but not specific method for renal
● Excretion disorder
● Homeostasis *we have to correlate it to creatinine
● Osmoregulation (maintaining overall fluid ● Filtered and reabsorbed at proximal tubules
balance) *we do not directly measure urea, we measure the
● Regulation of salts in the body nitrogen content of urea
● Regulation of pH
● Production of a hormone (EPO) SPECIMEN CONSIDERATIONS
*if there is a kidney disease/disorder, BUN and ● Non-fasting
creatinine is elevated ● Serum, Plasma, Whole blood, Urine
*kidney filters waste materials: food, medications, *whole blood is the least option
other toxic substances ● Fluoride and citrate will inhibit urease
*kidney produces Erythropoietin which is used for *do not use gray and blue top tubes
RBC production ● Avoid bacterial contamination in urine
*kidney regulates blood pressure *prolonged standing of urine will lead to bacterial
contamination
NON-PROTEIN NITROGEN ● Should be refrigerated when not analyzed
● Non-Protein Nitrogen: The major substance immediately
excreted by the kidneys
*waste materials PRINCIPLE OF THE TEST
● AZOTEMIA: Elevated NPNs in the blood 1. Chemical Method
(urea) ● Direct method
*urea is the most abundant NPN ● Diacetyl monoxime (DAM)
● Urea + DAM= Yellow diazine
derivative
*chemical method is not used in the lab anymore after reading (A1), read and record
2. Enzymatic Method absorbance (A2).
● Urease enzyme ● Calculate change in absorbance (△A= A2 -
*more specific and accurate A1) per minute.
*coupled enzymatic method
PROCEDURES (for Chem Machine)
● Prepare BUN working reagent according to
instructions.
● Pipette 1000 uL working reagent into tubes
labeled “standard”, “control”, “patient”
● Add 10 uL of sample and mix.
*Enzymes: Urease and GLDH (Glutamic ● After 2 minutes, feed onto the machine.
Dehydrogenase) ● Analyze and record your results.
*Urease converts Urea to Ammonium carbonate
*GLDH catalyzes the conversion of NADH to NAD+ REFERENCE RANGE: 8-23 mg/dL
*Final product is NAD+ and measured in the
spectrophotometer at 340 nm INCREASED BUN
● Chronic renal disease
REAGENT PREPARATION ● Stress
● Reconstitute BUN Working Reagent (12 mL ● Burns
distilled water) ● High protein diet
*usually powdered form ● Dehydration
*reconstitution: dissolve in distilled water using
serologic/automatic pipettes DECREASED BUN
● Urea Nitrogen Standard is ready to use. ● Poor nutrition
● Hepatic disease
MATERIALS ● Impaired absorption
● Spectrophotometer ● Pregnancy
● Distilled water *protein anabolism
● Semi-automated machine
● Constant temperature block or bath 37°C, or CREATININE
temperature-controlled cuvette ● Final product of creatine phosphate in
● Accurate pipetting devices muscles. (muscle metabolism)
*same pipettes used with glucose and cholesterol ● Produced from 3 AA- Methionine, Arginine,
test Lysine
● Test tubes and rack ● Partially secreted by PCT
● Timer ● Specific but not sensitive to renal disorders.
● Not affected by protein diet
PROCEDURES (for Spectro) ● Index of overall renal function
● Prepare BUN working reagent according to ● Solely a waste product
instructions. *creatinine is more specific than BUN
● Pipette 1000 uL working reagent into tubes
labeled “standard”, “control”, “patient” SPECIMEN CONSIDERATIONS
*standard, blank, patient ● Non-fasting
● Add 10 uL of sample and mix. ● Hemolyzed samples should be avoided
● After exactly 30 seconds, read and record ● Cephalosphorin (antibiotic) - false
absorbance (A1). 7. At exactly 60 seconds increase/elevation

PRINCIPLE
1. Chemical Method (Jaffe Method)
● A yellow-orange tautomer of creatinine
picrate is formed when creatinine is
mixed with alkaline picrate
● Female: 0.7 - 1.37 mg/dL
INCREASED CREATININE
● Impaired renal function
● Chronic nephritis
● Congestive heart failure
*weak pumping of blood can lead to decreased blood
flow and therefore decreased removal of waste

*measure the colored complex on a

DECREASED CREATININE
spectrophotometer at 510 nm
● Decreased muscle mass
*the rate of formation of color change is directly
● Advance severe liver disease
proportional to the creatinine concentration
● Pregnancy
● Inadequate protein diet
WORKING REAGENT
● 1000 uL sodium Picrate
● Combine equal volumes of creatinine picric
acid reagent (500 uL, reaget A) and
creatinine sodium hydroxide (500 uL,
reagent B), mix well.
● Combined (working) reagent is stable for up
to one (1) month.

PROCEDURES (for Spectro)

● Prepare creatinine working reagent according
to instructions.
● Pipette 1000 uL working reagent into tubes
labeled “standard”, “control”, “patient”.
● Add 50 uL of sample and mix.
● After exactly 30 seconds, read and record
absorbance (A1). 7. At exactly 60 seconds
after reading (A1), read and record
absorbance (A2).
● Calculate change in absorbance (△A = A2 -
A1) per minute.

PROCEDURES (for Machine)

● Prepare creatinine working reagent according
to instructions.
● Pipette 1000 uL working reagent into tubes
labeled “standard”, “control”, “patient”.
● Add 50 uL of sample and mix.
● Incubate for 2 minutes.
● Feed onto your machine.

REFERENCE RANGE
● Male: 0.9 -1.50 mg/dL