LIPIDS lab

Chemical methods for TAG

a. Colorimetric method (Van Handel & Zilversmith)

b. Fluorometric method (Hantzsch Condensation)

Colorimetric method (blue complex)

Van Handel and Zilversmith

glycerol + fatty acids

Van Handel and Zilversmith method:

TAG w/ enzyme:

Alcoholic KOH =

Formaldehyde (HCHO)

Van Handel and Zilversmith method:

Glycerol w/enzyme:

Periodic Acid =

Blue colored compound

Van Handel and Zilversmith method:

HCHO + Chromotropic Acid =

Flurometric method (Diacetyl Lutidine compound)

Hantzsch Condensation

glycerol + FA

Hantzsch Condensation Method:

TAG w/ enzyme:

Alcoholic KOH =

HCHO

Hantzsch Condensation Method:

Glycerol w/enzyme:

Periodic Acid =

Diacetyl Lutidine compound

Hantzsch Condensation Method:

HCHO + diacetyl Acetone + NH3 =

Enzymatic Method for TAG

Relatively specific, rapid and easy

- The analyses are performed directly in plasma or serum = not subject to interference by phospholipids or glucose.

a. Glycerol Kinase Method

b. Modified Van Handel and Zilversmith

Glycerol

Major interference of Enzymatic method for TAG

- It is normally present in plasma in concentrations below 0.163mmol/L

- it is ≈ TAG concentration of 14 mg/dl

Modified Van Handel and Zilversmith (pink end-color)

cdc reference method

- time-consuming manual method which cannot be automated

Saponification

Modified Van Handel and Zilversmith:

Alkaline hydrolysis with aKOH, where there is a removal of water until only the solvent is left

Chloroform

Modified Van Handel and Zilversmith:

solvent extraction using?

Silicic Acid (chomatography)

Modified Van Handel and Zilversmith:

Extracted solvent is then treated with?

- to remove phospholipids from the chloroform extract.

- consists of Silicon, O4, H4 (Si(OH)4)

formaldehyde + formic acid

Modified Van Handel and Zilversmith:

Glycerol + Sodium periodate =

colored end product (Pink)

Modified Van Handel and Zilversmith:

Formaldehyde + Chromotropic acid =

Factors for increased TAG

Hyperlipoproteinemia

Alcoholism

Nephrotic syndrome

Hypothyroidism

pancreatitis

Factors for decreased TAG

Malabsorption syndrome

Hyperthyroidism

Malnutrition

Brain infarction

Methods for Quantitative analysis of Phospholipids

- analysis of phospholipids is rare in laboratory medicine

1. Estimation of serum lipid phosphorus

2. Status of fetal lung maturation

Estimation of serum lipid phosphorus

4%

Each mole of phosphorus contributes about ____ to the total phospholipid mass.

Phospholipid phosphorus conc. (mg/dl) x 25

Estimation of serum lipid phosphorus:

Phospholipid mass determined by: PPc x 25

Status of fetal lung maturation

status of fetal lung maturation is estimated from the evaluation of pulmonary surfactant in amniotic fluid

- through Lecithin/Spingomyelin (L/S) ratio, Phosphatidyl glycerol (PG) and microviscosity

Chromatorgraphy, Densinometric quantitation

L/S ratio and PG is determined by?

what quantitation?

fluorencence polarization

microviscosity is determined by?

Total Cholesterol (TC) concentration

______________ is measured rather than its forms.

- may be assayed from non-fasting blood samples

- this increases with age

- either plasma or serum can be used for measurements

2 mg/dl/year

serum total cholesterol increases at ________________ between 45-65 yrs old.

- decrease estrogen also contributes to this

chemical methods for Cholesterol

- Dehydration and oxidation of cholesterol to form a colored compound.

a. Libermann Burchardt Reaction

b. Salkowski Reaction

Libermann Burchardt Reaction (green color)

Based on the reaction of:

- Cholesterol + acetic acid anhydride + conc. sulfuric acid.

*Mac Gavack method

Cholestadienyl monosulfonic acid

Libermann Burchardt Reaction (green color) end product

Liebermann Burchardt Reagent

color development mixture:

a. Glacial acetic acid

b. Acetic anhydride

c. Concentrated H2SO4

Glacial acetic acid

Acetic anhydride

Concentrated H2SO4

Liebermann Burchardt Reagent

Salkowski Reaction (red color)

Earliest colorimetric chemical raction for qualitative and quantitative determination of cholesterol.

- cholesterol + conc. sulfuric acid

*pearson + stern method

Cholestadienyl Disulfonic acid (red end color)

Salkowski Reaction (red color) end product

hemolyzed blood

Precautions for cholesterol chem methods:

causes falsely increase levels

icteric specimens

Precautions for cholesterol chem methods:

1mg bilirubin = 5-6mg increase in cholesterol.

- Bilirubin can interfere because of its own spectral properties (500 nm)

General Methods for TC

1. One-step method

2. two-step method

3. Three-step method

4. Four-step method

One-Step Method

Colorimetry (Pearson, Stern and Mac Gavack)

Two-Step Method (C: Bloors)

Extraction + Colorimetry - 620 nm (550-650nm)

Three-Step Method (C: Abell-Kendal)

Saponification + Extraction + Colorimetry

Four-Step Method (C: Schoenheimer, Sperry, Parekh, and Jung)

Saponification + Extraction + Colorimetry + Precipitation

Enzymatic methods for TC

rapid, use microliter quantities, does not require extraction step

- Most common method of quantifying the cholesterol oxidase to measure the amount of hydrogen peroxide produced

a. Cholesterol Oxidase Reaction

b. Abell, Levy, and Brodie Method

high ascorbic acid, hb, and bilirubin

interferences of enzymatic methods for TC

hemoglobin

interference of TC:

has a pseudo-peroxidase activity that can consume the hydrogen peroxide produced in reaction.

- color = falsely increase TC

bilirubin, 5-15%

interference of TC:

this exceeding 5mg/dL decreases cholesterol by______?

Cholesterol Oxidase Reaction

Enzymatic methods for TC:

Quinoniemine dye is the end product for this method

cholesterol + FA

Cholesterol Oxidase Reaction:

Cholesterol ester + H2O w/ enzyme cholesterol esterase =

Cholest-4-en-3-one + H2O2

Cholesterol Oxidase Reaction:

Cholesterol + O2 w/ enzyme cholesterol oxidase =

H2O2 + phenol + 4-aminoantipyrine

Cholesterol Oxidase Reaction:

Quinoniemine dye (end product)

Abell, Levy, and Brodie Method

CDC reference method for cholesterol

- uses Hexane extraction after hydrolysis with aKOH + Liebermann-Burchardt color reagent

Factors for increased Cholesterol

1. Hyperlipoproteinemia types II, III, V

2. Biliary cirrhosis

3. Nephrotic syndrome

4. Poorly controlled diabetes mellitus

5. Alcoholism

6. Primary hypothyroidism

Factors for decreased Cholesterol

1. Severe hepatocellular disease

2. Malnutrition

3. Severe burns

4. Hyperthyroidism

5. Malabsorption syndrome

Non-Enzymatic Methods For Cholesterol

Abell-Kendall method

- cholesteryl esters are hydrolyzed with aKOH

a. type 1

b. type 2a

c. type 2b

d. type 3

e. type 4

f. type 5

Type 1, 2a, 2b

- serum plasma: "cream"/clear or turbid

- deficient LPL

- familial trait: autosomal dominant

- homozygotes: severely affected

Familial lipoprotein lipase (LPL) deficiency

type 1 hyperlipoproteinemia

Familial Hypercholesterolemia

type 2a hyperlipoproteinemia

Mixed defect with hyperlipidemia

type 2b hyperlipoproteinemia

Type 3: Familial Dysbetaliloproteinemia

- serum plasma: +++ turbid

- diet, lipid lowering drugs verry effective

Type 4: Hypertriglycedemia

- serum plasma: ++ turbid

- weight loss: lowers

- VLDL

- high fat diet = may convert to type 5

Type 5

- serum plasma: "cream" ++ turbid

- weight loss: not lowers

- VLDL

Liebermann - Burchard reaction (green)

Abell-Kendall method is measured using this

Methods for measuring Lipoprotein

General methods:

a. ultracentrifugation

b. electrophoresis

HDL Methods:

a. Polyanion Precipitation

VLDL Methods:

a. Friedewald equation

Apolipoprotein Methods:

a. Apo-B

b. Apo A-1

ultracentrifugation and electrophoresis

General methods for measuring Lipoprotein

polyanion Precipitation

HDL Methods for measuring Lipoprotein

Friedewald equation

VLDL Methods for measuring Lipoprotein

Apo-B and Apo A-1

Apolipoprotein Method for measuring Lipoprotein