I. Chemical Methods
1. Alkaline Copper Reduction ACT + Glucose & Heat = Cuprous Ioins
a. Folin Wu Phosphomolyb-dic Acid (-denum BLUE)
A. Oxidation-Reduction b. Nelson Somogyi Arsenomolyb-dic Acid (-denum BLUE)
c. Neocuproine (2,9 Dimethyl 1,10 Phenantroline Cuprous Neocuproine Complex (Y/Y-O)
Hydrochloride
d. Benedict's (Modified Folin Wu)
2. Alkaline Feric Reduction (Hagedorn Jensen) Yellow Ferricyanide to Colorless Ferrocyanide (INVERSE)
Glucose
B. Condensation 1. Ortho-Toluidine (Dubowski Method) Glycosamine + Schiff's Base
1. Glucose Oxidase Method Measures Beta-D Glucose
a. Colorimetric GOM (Saifer Gernstenfield) Glucose Oxidase & Peroxidase
b. Polarographic GOM Glucose Oxidase, Catalase, Molybdate
II. Enzymatic Methods 2. Hexokinase Hexokinase & G6PD = NADPH
3. Glucose Dehydrogenase GD, Diaphorase = MTTH + NAD
4. Dextrostics (Cellular Strip) Before: Glucose Oxidase, Peroxidase, Chromogen (Now: Hexokinase)
5. Interstitial Glucose Monitoring Device Glucose "trend" analysis
I. Estimation of Serum Phosphorus Phospholipid Mass = Phospholipid Phosphorus X 25
Phospholipid
II. Fetal Lung Maturation (L/S ratio) TLC + Densitometry or Phosphatidyl Glycerol by Fluorescence Polarization
1. Liebermann Burchardt Reaction Cholestadienyl Monosulfuric Acid
Cholesterol (3- I. Chemical Methods
2. Salkowski Reaction Cholestadienyl Disulfuric Acid
hydroxy-5,6-
1. Cholesterol Oxidase Raection Quinoneimine Dye
cholestene) II. Enzymatic Methods
2. Abell, Levy and Brodie (CDC Reference Method)
1. Colorimetric Method (Van Handel & Zilversmith) HCHO + Chromotrophic Acid = Blue
I. Chemical Methods
2. Fluorometric Method (Hantzsh Condensation) Diacetyl Lutidine Compound
Triglycerides
II. Enzymatic Methods
(Triacylglycerol,
1. Reaction A Lipase, Glycerol Kinase, Pyruvate kinase, LDH
Neutral Fat) A. Glycerol Kinase Method
2. Reaction B Lipase, Glycerol Kinase, Glycerol PO4 DH, Diaphorase
B. Modified Van Handel and Zilversmith CDC Reference Method HCHO + Chromotrophic Acid = Blue
1. Ultracentrifugation (Density Gradient) Reference Method Reagent: Potassium Bromide with 1.063 density
2. Electrophoresis HDL > VLDV > LDL > Chylomicrons Preferred Medium: Agarose Gel (Sensitive)
3. Chemical Precipitation Polyanions: HeparinSO4, DextranSO4, Phosphotungstate / Divalent cations: Mg2+, Ca2+, Mn2+
a. HDL CDC Reference/3-step: (1)Ultracentrifugation (2)Heparin Mn2+ Precipitation (3)Abell-Kendall Assay
Lipoproteins
b. LDL Ultracentrifugation at least 18hrs at 105 Kxg + Chemical Precipitation
4. Chromatographic Methods Gel or Affinity Chromatography
5. Immunochemical Methods
6. Immunoassay/Immunonephelometry Apoliporotein Assay - Immunoturbidimetric: Lp(a)
1. Exogenous
a. Inulin
b. Iothalamate125
c. 99mTc-DTPA (metastable technitium99-labeled diethylene triamine pentaacetic acid)
d. Iohexol
e. Chromium51-labeled ethylenediaminetetraacetic acid (51Cr-EDTA)
Glomerular Filtration Rate
f. Nonradiolabeled iothalamate
2. Endogenous
a. Creatinine Clearance
b. Urea Clearance
c. Cystatin C
d. Beta-trace Protein
Renal Blood Flow
I. Blood Urea Nitrogen
A. Chemical Method (Direct) 1. Diacetyl Monoxime Method Yellow Diazine Derivative
B. Enzymatic Method (Indirect) 2. Hydrolysis of Urea by Urease Measure Ammonia by Berthelot and CO2
3. Coupled Urease/Glutamate Dehydrogenase UV Enzymatic Method
3. Isotope Dilution Mass Spectrometry Reference Method
II. Creatinine
A. Chemical Method 1. Direct Jaffe Method Creatinine Picrate
a. Folin Wu Sensitive but not specific
Kidney Function Test
b. Lloyd's (Sodium Aluminum Silicate) Sensitive and Specific
c. Fuller's Earth (Aluminum Magnesium Silicate) Time-cosuming; Not routinely used
B. Kinetic Jaffe Method
C. Enzymatic Method 1. Creatinine Aminohydrolase-CK Method
2. Creatinase-Hydrogen Peroxide Method
D. IDMS
III. Blood Uric Acid
A. Chemical Method 1. Reduction-Oxidation
a. Sodium Cyanine Folin Newton Brown Benedict
b. Sodium Carbonate Archibald Henry Caraway
B. Enzymatic Method 1. Uricase Method
C. IDMS Reference Method
Tubular Function
I. Excretion Tests A. Para-amino Hippurate Test (Diodrast) RV: 600-700 mL/minute
B. Phenolsulonthalein Dye Test (6 mg PSP IV) RV: 1200 blood flow per minute
II. Concentration Tests A. Specific Gravity RV: 1.005-1.030
B. Osmolality RV: Serum (275-295 mOsm/kg); 24hr-Urine (300-900 mOsm/kg)
1. Direct Method
a. Freezing point Osmometry - Popular method
b. Vapor Pressure Osmometry (Seebeck Effect)
2. Indirect Method 1.86 x Sodium + Glu/18 + BUN/2.8
I. Synthetic Function
A. Total Protein 1. Kjeldahl Method (Reference Method) R:H2SO4 / EP: Ammonia
2. Biuret Method (IFCC Recommended) @545nm R: Alkaline Copper SO4, Rochelle Salt (NaK Tartrate, NaOH, KI
3. Folin-Ciocalteu (Lowry) Method
4. Ultraviolet Absorption Method @210nm 280nm: Tryptophan, Tyrosine, Phenylalanine
5. Serum Protein Elecrophoresis Most important: Monoclonal spike identification
6. Refractometry Alternative test
7. Turbidimetric/Nephelometric Sulfosalicylic and Trichloroacetic acid
8. Salt Fractionation Sodim Sulfate Salt
9. Coomasie Brilliant Blue Protein as little as 1ug
10. Ninhydrin Widely used for amino acids and peptides after chromatography
B. Prothrombin Tme 1. Vitamin K Response Test (10mg daily for 1-3 days) Prolonged PT in Intrahepatic VS Normal PT in Extrahepatic
C. Albumin a. Bromcresol Green Most common
Liver Function Test b. Bromcresol Purple Most specific
c. Methy Orange
d. Hydroxyazobenzene Benzoic Acid (HABA)
II. Conjugation and Excretion
A. Bilirubin Assay 1. Evelyn and Malloy (Methanol) Pink to Purple Azobilirubin
2. Jendrassik and Grof (Caffeine Benzoic Acid) Pink to Blue Azobilirubin
B. Bromsulfonthalein (BSP) Dye Excretion 1. Rosenthal White (Double Collection Method) 2mg/kg BW after 5 mins (50%) and after 30mins (0%)
2. Mac Donald Method (Single Collection Method) 5mg/kg BW after 45mins (+/-5% retention)
III. Detoxification
A. Enzyme Tests
B. Ammonia 1. Digestion (Kjeldahl Method) Catalyst: Copper SO4, Merury, Selenium
2. Nesslerization Low to Moderate (Yellow) / Hign (Orange-brown)
3. Berthelot Reaction Indophenol Blue
4. Glutamate Dehydrogenase Glutamate + NADP
Reference/s: Blue book, Ciulla
1. Alkaline Copper Reduction ACT + Glucose & Heat = Cuprous Ioins
a. Folin Wu Phosphomolyb-dic Acid (-denum BLUE)
A. Oxidation-Reduction b. Nelson Somogyi Arsenomolyb-dic Acid (-denum BLUE)
c. Neocuproine (2,9 Dimethyl 1,10 Phenantroline Cuprous Neocuproine Complex (Y/Y-O)
Hydrochloride
d. Benedict's (Modified Folin Wu)
2. Alkaline Feric Reduction (Hagedorn Jensen) Yellow Ferricyanide to Colorless Ferrocyanide (INVERSE)
Glucose
B. Condensation 1. Ortho-Toluidine (Dubowski Method) Glycosamine + Schiff's Base
1. Glucose Oxidase Method Measures Beta-D Glucose
a. Colorimetric GOM (Saifer Gernstenfield) Glucose Oxidase & Peroxidase
b. Polarographic GOM Glucose Oxidase, Catalase, Molybdate
II. Enzymatic Methods 2. Hexokinase Hexokinase & G6PD = NADPH
3. Glucose Dehydrogenase GD, Diaphorase = MTTH + NAD
4. Dextrostics (Cellular Strip) Before: Glucose Oxidase, Peroxidase, Chromogen (Now: Hexokinase)
5. Interstitial Glucose Monitoring Device Glucose "trend" analysis
I. Estimation of Serum Phosphorus Phospholipid Mass = Phospholipid Phosphorus X 25
Phospholipid
II. Fetal Lung Maturation (L/S ratio) TLC + Densitometry or Phosphatidyl Glycerol by Fluorescence Polarization
1. Liebermann Burchardt Reaction Cholestadienyl Monosulfuric Acid
Cholesterol (3- I. Chemical Methods
2. Salkowski Reaction Cholestadienyl Disulfuric Acid
hydroxy-5,6-
1. Cholesterol Oxidase Raection Quinoneimine Dye
cholestene) II. Enzymatic Methods
2. Abell, Levy and Brodie (CDC Reference Method)
1. Colorimetric Method (Van Handel & Zilversmith) HCHO + Chromotrophic Acid = Blue
I. Chemical Methods
2. Fluorometric Method (Hantzsh Condensation) Diacetyl Lutidine Compound
Triglycerides
II. Enzymatic Methods
(Triacylglycerol,
1. Reaction A Lipase, Glycerol Kinase, Pyruvate kinase, LDH
Neutral Fat) A. Glycerol Kinase Method
2. Reaction B Lipase, Glycerol Kinase, Glycerol PO4 DH, Diaphorase
B. Modified Van Handel and Zilversmith CDC Reference Method HCHO + Chromotrophic Acid = Blue
1. Ultracentrifugation (Density Gradient) Reference Method Reagent: Potassium Bromide with 1.063 density
2. Electrophoresis HDL > VLDV > LDL > Chylomicrons Preferred Medium: Agarose Gel (Sensitive)
3. Chemical Precipitation Polyanions: HeparinSO4, DextranSO4, Phosphotungstate / Divalent cations: Mg2+, Ca2+, Mn2+
a. HDL CDC Reference/3-step: (1)Ultracentrifugation (2)Heparin Mn2+ Precipitation (3)Abell-Kendall Assay
Lipoproteins
b. LDL Ultracentrifugation at least 18hrs at 105 Kxg + Chemical Precipitation
4. Chromatographic Methods Gel or Affinity Chromatography
5. Immunochemical Methods
6. Immunoassay/Immunonephelometry Apoliporotein Assay - Immunoturbidimetric: Lp(a)
1. Exogenous
a. Inulin
b. Iothalamate125
c. 99mTc-DTPA (metastable technitium99-labeled diethylene triamine pentaacetic acid)
d. Iohexol
e. Chromium51-labeled ethylenediaminetetraacetic acid (51Cr-EDTA)
Glomerular Filtration Rate
f. Nonradiolabeled iothalamate
2. Endogenous
a. Creatinine Clearance
b. Urea Clearance
c. Cystatin C
d. Beta-trace Protein
Renal Blood Flow
I. Blood Urea Nitrogen
A. Chemical Method (Direct) 1. Diacetyl Monoxime Method Yellow Diazine Derivative
B. Enzymatic Method (Indirect) 2. Hydrolysis of Urea by Urease Measure Ammonia by Berthelot and CO2
3. Coupled Urease/Glutamate Dehydrogenase UV Enzymatic Method
3. Isotope Dilution Mass Spectrometry Reference Method
II. Creatinine
A. Chemical Method 1. Direct Jaffe Method Creatinine Picrate
a. Folin Wu Sensitive but not specific
Kidney Function Test
b. Lloyd's (Sodium Aluminum Silicate) Sensitive and Specific
c. Fuller's Earth (Aluminum Magnesium Silicate) Time-cosuming; Not routinely used
B. Kinetic Jaffe Method
C. Enzymatic Method 1. Creatinine Aminohydrolase-CK Method
2. Creatinase-Hydrogen Peroxide Method
D. IDMS
III. Blood Uric Acid
A. Chemical Method 1. Reduction-Oxidation
a. Sodium Cyanine Folin Newton Brown Benedict
b. Sodium Carbonate Archibald Henry Caraway
B. Enzymatic Method 1. Uricase Method
C. IDMS Reference Method
Tubular Function
I. Excretion Tests A. Para-amino Hippurate Test (Diodrast) RV: 600-700 mL/minute
B. Phenolsulonthalein Dye Test (6 mg PSP IV) RV: 1200 blood flow per minute
II. Concentration Tests A. Specific Gravity RV: 1.005-1.030
B. Osmolality RV: Serum (275-295 mOsm/kg); 24hr-Urine (300-900 mOsm/kg)
1. Direct Method
a. Freezing point Osmometry - Popular method
b. Vapor Pressure Osmometry (Seebeck Effect)
2. Indirect Method 1.86 x Sodium + Glu/18 + BUN/2.8
I. Synthetic Function
A. Total Protein 1. Kjeldahl Method (Reference Method) R:H2SO4 / EP: Ammonia
2. Biuret Method (IFCC Recommended) @545nm R: Alkaline Copper SO4, Rochelle Salt (NaK Tartrate, NaOH, KI
3. Folin-Ciocalteu (Lowry) Method
4. Ultraviolet Absorption Method @210nm 280nm: Tryptophan, Tyrosine, Phenylalanine
5. Serum Protein Elecrophoresis Most important: Monoclonal spike identification
6. Refractometry Alternative test
7. Turbidimetric/Nephelometric Sulfosalicylic and Trichloroacetic acid
8. Salt Fractionation Sodim Sulfate Salt
9. Coomasie Brilliant Blue Protein as little as 1ug
10. Ninhydrin Widely used for amino acids and peptides after chromatography
B. Prothrombin Tme 1. Vitamin K Response Test (10mg daily for 1-3 days) Prolonged PT in Intrahepatic VS Normal PT in Extrahepatic
C. Albumin a. Bromcresol Green Most common
Liver Function Test b. Bromcresol Purple Most specific
c. Methy Orange
d. Hydroxyazobenzene Benzoic Acid (HABA)
II. Conjugation and Excretion
A. Bilirubin Assay 1. Evelyn and Malloy (Methanol) Pink to Purple Azobilirubin
2. Jendrassik and Grof (Caffeine Benzoic Acid) Pink to Blue Azobilirubin
B. Bromsulfonthalein (BSP) Dye Excretion 1. Rosenthal White (Double Collection Method) 2mg/kg BW after 5 mins (50%) and after 30mins (0%)
2. Mac Donald Method (Single Collection Method) 5mg/kg BW after 45mins (+/-5% retention)
III. Detoxification
A. Enzyme Tests
B. Ammonia 1. Digestion (Kjeldahl Method) Catalyst: Copper SO4, Merury, Selenium
2. Nesslerization Low to Moderate (Yellow) / Hign (Orange-brown)
3. Berthelot Reaction Indophenol Blue
4. Glutamate Dehydrogenase Glutamate + NADP
Reference/s: Blue book, Ciulla
