CC - Specimen Considerations and Methodologies for Lipid Determination

Lipids

Fatty acids, phospholipids, triglycerides, cholesterol, fat-soluble vitamins.

Lipoproteins

Chylomicrons, VLDL, LDL, HDL.

Lipids and lipoproteins

Important indicators of coronary heart disease (CHD) risk — the major reason for their measurement in research and clinical practice.

Lipid profile

• Recommended for initial screening in adults age 20 or older.
• Includes TC, LDL, HDL and TAG.
• Repeat at least once every 5 years.

Biological variation

• Usual diet for 2 weeks, neither gaining nor losing weight, before cholesterol measurement.
• Oral contraceptives, postmenopausal estrogens and some antihypertensive drugs significantly alter lipid levels.

Fasting before lipid testing

At least 12 hours before venipuncture.

TC and HDL-C

Can be measured in NONFASTING individuals.

TAG and LDL-C

REQUIRE FASTING.

Posture

Postural change from upright to supine decreases TAG by almost 50%.

Posture (NCEP)

Patient seated for at least 5 minutes prior to venipuncture.

Plasma or serum

Either can be used when only cholesterol, TG and HDL-C are measured and LDL-C is calculated from these three measurements.

Plasma

Preferred when lipoproteins are measured by ultracentrifugal or electrophoretic methods.

EDTA

Preferred anticoagulant for lipid and lipoprotein testing.

Long-term storage

−70 °C or lower.

Short-term storage

−20 °C (up to a month or two).

Increased TAG

Hyperlipoproteinemia (types I, IIb, III, IV, V), nephrotic syndrome, pancreatitis, hypothyroidism, alcoholism.

Decreased TAG

Malabsorption syndrome, hyperthyroidism, brain infarction, malnutrition.

TAG fasting requirement

12–14 hours.

TAG and age

Like total cholesterol, increases at a rate of 2 mg/dL/year between 50–60 years old.

TAG interferences

Phospholipids and glucose may cause interference in TAG measurement.

Alcohol before TAG test

Avoid alcohol consumption at least 2 days before the test.

TAG principle

Hydrolysis of triglycerides and measurement of the glycerol released in the reaction.

TAG chemical methods

1. Colorimetric (Van Handel and Zilversmit)
   2. Fluorometric (Hantzsch condensation)

TAG enzymatic method

Glycerol kinase method.

Van Handel and Zilversmit

Colorimetric TAG method:
• Triglycerides + alcoholic KOH → glycerol + free FA
• Glycerol + periodic acid → formaldehyde
• Formaldehyde + chromotropic acid → BLUE colored product (the same reaction is called a PINK chromophore under the former CDC method; the product is pink-to-violet)

Hantzsch condensation

Fluorometric TAG method:
• Triglycerides + alcoholic KOH → glycerol + free FA
• Glycerol + periodic acid → formaldehyde
• Formaldehyde + diacetyl acetone + ammonia → diacetyl lutidine compound (YELLOW fluorescence)

Alcoholic KOH

Triglycerides + alcoholic KOH → glycerol + free FA (chemical TAG methods).

Periodic acid

Oxidizes glycerol to formaldehyde in the chemical TAG methods.

Chromotropic acid

Reacts with formaldehyde — Van Handel and Zilversmit colorimetric TAG method.

Diacetyl lutidine compound

YELLOW fluorescence — end product of the Hantzsch condensation.

Enzymatic TAG methods

• Now universally used for TAG analysis.
• Specific, rapid and easy to use.
• Performed directly in plasma or serum.
• Not subject to interference by phospholipids and glucose.

Endogenous glycerol

Major interference in enzymatic TAG methods. Remedy: blanking technique.

Glycerol kinase method

• Triglycerides →(lipase) glycerol + fatty acids
• Glycerol + ATP →(glycerokinase) glycerophosphate + ADP
• Glycerophosphate + NAD →(glycerophosphate dehydrogenase) dihydroxyacetone phosphate + NADH + H⁺

Glycerol kinase method

Measured at 340 nm. The lecture says "Disappearance of NADH"; in the glycerophosphate dehydrogenase step NADH is formed, so absorbance rises.

Former CDC reference method for TAG

Modified Van Handel and Zilversmit:
• Chloroform extraction → silicic acid chromatography → TAG isolated → TAG hydrolysis → glycerol
• Glycerol + sodium periodate → formaldehyde
• Formaldehyde + chromotropic acid → PINK chromophore

New CDC reference method for TAG

Gas chromatography/mass spectrometry (GC-MS).

TAG normal

<150 mg/dL (NCEP).

TAG borderline high

150–199 mg/dL (NCEP).

TAG high

200–499 mg/dL (NCEP).

TAG very high

≥500 mg/dL (NCEP) — acute and recurrent pancreatitis.

TAG conversion factor

0.0113 (mg/dL → mmol/L).

Increased cholesterol

Hyperlipoproteinemia (types IIa, III, V), familial hypercholesterolemia, primary hypothyroidism, poorly controlled DM, nephrotic syndrome, biliary obstruction, biliary cirrhosis, alcoholism.

Decreased cholesterol

Severe hepatocellular disease, malabsorption syndrome, hyperthyroidism, severe burns, malnutrition.

Cholesterol specimen

• Fasting: at least 12 hours; may be assayed from NONFASTING samples.
• If analysis is delayed, refrigerate at 4 °C for several days.
• Precise and accurate timing for color development.
• Avoid hemolyzed and icteric samples; avoid water contamination.

Cholesterol principle

Dehydration and oxidation of cholesterol to form a colored compound.

Liebermann-Burchard reaction

• Reagent: glacial acetic acid, conc. sulfuric acid, acetic anhydride.
• End product: cholestadienyl monosulfonic acid (GREEN), measured at 410 nm.

Glacial acetic acid

Oxidizing agent — Liebermann-Burchard reagent.

Conc. sulfuric acid

Dehydrating agent and color developer — Liebermann-Burchard reagent.

Acetic anhydride

Color stabilizer — Liebermann-Burchard reagent.

Salkowski reaction

End product: cholestadienyl disulfonic acid (RED).

Cholestadienyl monosulfonic acid

GREEN end product of the Liebermann-Burchard reaction (410 nm).

Cholestadienyl disulfonic acid

RED end product of the Salkowski reaction.

Cholesterol chemical methods

Steps: saponification, extraction, precipitation, colorimetry.

One-step method

Direct colorimetric method: sample + color reagent. Example: Pearson, Stern and Mac Gavack methods.

Two-step method

Extraction and colorimetric method: sample → extraction of lipids; lipids + color reagent. Example: Bloor's, Zeolite and Chloroform methods.

Three-step method

Saponification, extraction and colorimetry: sample + saponifying agent → free cholesterol → extraction; extracted lipids + color reagent. Example: Abell-Kendall method.

Four-step method

Saponification, extraction, precipitation and colorimetry. Example: Schoenheimer and Sperry, Parekh and Jung, and Sperry and Webb methods.

Abell-Kendall method

Three-step chemical method for cholesterol determination — the FORMER CDC REFERENCE METHOD.

New CDC reference method for cholesterol

Gas chromatography/mass spectrometry (GC-MS).

Enzymatic cholesterol methods

• Involve measurement of the hydrogen peroxide produced.
• Advantages: rapid, smaller sample (µL), no lipid extraction.
• Interferences: reducing and oxidizing agents.

Cholesterol oxidase method

• Cholesteryl ester + H₂O →(cholesteryl esterase) cholesterol + free fatty acid
• Cholesterol + O₂ →(cholesterol oxidase) cholest-4-en-3-one + H₂O₂
• 2H₂O₂ + phenol + 4-aminoantipyrine →(peroxidase) quinoneimine dye + 4H₂O

Cholesterol oxidase

Reacts only with FREE CHOLESTEROL.

Quinoneimine dye

Colored end product of the enzymatic cholesterol method (peroxidase, phenol, 4-aminoantipyrine).

TC desirable

<200 mg/dL (NCEP).

TC borderline high

200–239 mg/dL (NCEP).

TC high

≥240 mg/dL (NCEP).

TC conversion factor

0.026 (mg/dL → mmol/L).

Lipoproteins by density

HDL, LDL, VLDL, chylomicrons.

HDL

Migration: alpha. Content: cholesterol; alpha-globulin.

VLDL

Migration: pre-beta. Content: triglycerides; pre beta-globulin.

LDL

Migration: beta. Content: cholesterol; beta-globulin.

Chylomicrons

Migration: printed "gamma". Content: triglycerides; printed "gamma-globulin". (Chylomicrons remain at the origin on electrophoresis.)

Lipoprotein buoyant density

Chylomicrons <0.96 · VLDL <1.006 · LDL 1.006–1.063 · HDL 1.063–1.21 (the figure prints 1.006–1.21).

Electrophoretic mobility

Chylomicrons at the origin; then β-lipoproteins, pre-β-lipoproteins and α-lipoproteins, nearest the + pole.

Frozen samples

TC, TAG and HDL-c can be satisfactorily analyzed in frozen samples; LDL-c can be computed from these values.

Serum separator tube

Preferred sample for lipoprotein measurement.

EDTA plasma

Preferred sample for ultracentrifugal or electrophoretic methods and research studies.

EDTA

Why preferred:
1. Samples can be cooled immediately to 4 °C to prevent changes in the lipoproteins at room temp.
2. Heparin alters electrophoretic mobilities.
3. Citrate causes falsely low lipid and lipoprotein concentrations.

Heparin

Can alter the electrophoretic mobilities of lipoproteins.

Citrate

Causes falsely low plasma lipid and lipoprotein concentrations.

Lipoprotein electrophoresis

• Differentiates lipoproteins based on charge and mass in an alkaline medium.
• Preferred supporting medium: agarose gel.

Lipid-staining dyes

Oil red O, Fat Red 7B, Sudan Black B, Scharlach R (Sudan IV).

Ultracentrifugation

• Reference method for quantitation of lipoproteins.
• Employs a density gradient; differentiates by density.
• ↑ protein = ↑ density; ↑ TAG = ↓ density.
• Expressed in Svedberg units.
• Frozen samples are not recommended.

Potassium bromide solution

Ultracentrifugation reagent (density = 1.063).

CDC reference method for HDL

Three-step Abell-Kendall method:
1. Ultracentrifugation to precipitate VLDL (ultracentrifugation floats VLDL off; it does not precipitate it)
2. Heparin-manganese precipitation to remove LDL
3. Abell-Kendall assay of the supernatant cholesterol

Heparin-manganese

Precipitates LDL — step 2 of the CDC reference method for HDL.

Beta quantification

CDC reference method for LDL — quantitates cholesterol based on apo-B containing lipoprotein.

Standing plasma test

• Also overnight standing test / REF test.
• Sample: 2 mL plasma.
• Procedure: store at 4 °C (do not agitate!).

Floating creamy layer, clear sub-layer

Chylomicrons present (standing plasma test).

Turbid, no creamy layer

TAG present (standing plasma test).

Clear plasma on standing

Cholesterol MAY be present (standing plasma test).

Creamy layer with turbidity

TAG and chylomicrons both present (standing plasma test).

HDL-c <40 mg/dL

High risk for CHD (NCEP).

HDL-c ≥60 mg/dL

Low risk for CHD (NCEP).

LDL-c optimal

<100 mg/dL (NCEP).

LDL-c near optimal

100–129 mg/dL (NCEP).

LDL-c borderline high

130–159 mg/dL (NCEP).

LDL-c high

160–189 mg/dL (NCEP).

LDL-c very high

≥190 mg/dL (NCEP).

LDL-c

• MOST IMPORTANT VALUE in assessing cardiac risk and directing therapy.
• Measured, or calculated using the Friedewald equation.