CC1_Midterms_Lab: Glucose and TAG determination

Glucose Determination Principle

Glucose + O₂ —(glucose oxidase)→ Gluconic acid + H₂O₂

Second reaction in glucose determination

2 H₂O₂ + Phenol + 4-AAP —(peroxidase)→ Quinoneimine + 4 H₂O

Wavelength used in glucose determination

500 nm

Temperature for glucose assay

37°C

Blank composition (glucose test)

Reagent + distilled water (10 µL)

Standard composition (glucose test)

Reagent + standard (10 µL)

Unknown composition (glucose test)

Reagent + sample (10 µL)

Measurement in glucose assay

Measure ΔA within 300 seconds

Formula for glucose computation

Abs unk / Abs std × 100 mg/dL

Conversion factor for glucose

mg/dL × 0.0555 = mmol/L

Reference value for glucose (mg/dL)

74-106 mg/dL

Reference value for glucose (mmol/L)

3.2-4.6 mmol/L

Stability of glucose reagents

2-8°C, protected from light

Specimen for glucose test

Serum free of hemolysis

Serum stability for glucose

8 hrs at 25°C, 3 days at 2-8°C

Plasma anticoagulants for glucose

Fluoride, heparin, iodoacetate (glycolysis inhibitors)

Plasma stability for glucose

24 hrs at room temperature

TAG Determination Principle

TAG —(lipase)→ Glycerol + Fatty acids

Second step in TAG determination

Glycerol + ATP —(glycerol kinase)→ Glycerol-1-phosphate

Third step in TAG determination

Glycerol-1-phosphate + O₂ —(GPO)→ DAP + H₂O₂

Final reaction in TAG determination

H₂O₂ + 4-AAP + DHBS —(peroxidase)→ Quinoneimine dye + H₂O

Storage of TAG reagents

2-8°C

Specimen for TAG test

Fresh, clear, non-hemolyzed serum (fasting)

Stability of TAG specimen

3 days at 2-8°C

Room temperature storage of TAG sample

Not recommended

Collection precaution for TAG

Avoid glycerol-contaminated devices

Interference in TAG assay

Glycerol contamination

Other interferences in TAG

Lipemic, icteric, hemolyzed samples

Tubes used in TAG procedure

RB, Standard, Unknown

Initial reagent volume in TAG

1 mL reagent in all tubes

Incubation condition before sample addition

37°C for at least 4 minutes

Sample volume added in TAG

10 µL sample

Final incubation in TAG

37°C for 5 minutes

Wavelength for TAG reading

520 nm

Blank used for zeroing spectrophotometer

Reagent blank (RB)

TAG calculation formula

Abs unk / Abs std × 200 mg/dL

Conversion factor for TAG

0.0113

Reference value for TAG (mg/dL)

36-165 mg/dL

Reference value for TAG (mmol/L)

0.4-1.9 mmol/L

G1P aka

Glycerol-3-Phosphate

4-AA aka

4-aminoantpyrene

DAP full name

Dihydroxyacetone phosphate

DHBS full name

3,5-dichloro-2-hydroxybenzene sulfonic acid