Clinical Chemistry: Lipids and Lipoproteins Overview

Biological Variation

Physiologic variations in cholesterol, triglyceride, and lipoproteins can be several times greater than analytical error.

Fasting

Fasting for the 12 hours before venipuncture is ideal.

Posture

Significant pre-analytic variables may affect measured lipid and lipoprotein levels.

Blood Source

Lipoprotein cholesterol is used to evaluate lipoprotein concentration.

Storage

Significant pre-analytic variables may affect measured lipid and lipoprotein levels.

Cholesterol

The coefficient of physiologic variation of an individual averages about 6.5%.

Triglycerides

Chylomicrons (CM) are usually present in postprandial plasma and can increase plasma triglyceride concentration.

Phospholipids

Part of the estimation of plasma lipids.

Lipoproteins

Lipoprotein concentrations have been measured and described in several ways.

Apolipoproteins

Lipoproteins, and Apolipoproteins.

Ultracentrifugation Methods

One of the methods for estimating lipoproteins and lipoprotein cholesterol.

Electrophoretic Methods

One of the methods for estimating lipoproteins and lipoprotein cholesterol.

Polyanion Precipitation Methods

One of the methods for estimating lipoproteins and lipoprotein cholesterol.

HDL-C Measurement

One of the methods for estimating lipoproteins and lipoprotein cholesterol.

LDL-C Measurement

One of the methods for estimating lipoproteins and lipoprotein cholesterol.

NCEP Recommendation

The cholesterol screening should be repeated every 5 years.

Dietary Intake

Dietary intake of saturated fat and cholesterol significantly influences plasma lipid levels.

Saturated Fats

Examples of saturated fats to limit includes: fat on meat, processed meats, pastries & pies, fried foods, butter & ghee, coconut oil, crisps, ice cream, and chocolate.

Oral Contraceptives

Several common medications significantly alter lipid levels.

Postmenopausal Estrogens

Several common medications significantly alter lipid levels.

Antihypertensive Drugs

Several common medications significantly alter lipid levels.

Friedewald Formula

[LDL Cholesterol] = [Total Cholesterol] - [HDL Cholesterol] - [Plasma TG]/2.175.

Beta Quantification Formula

LDL Cholesterol= [Bottom Fraction Cholesterol] - HDL Cholesterol.

Chylomicrons (CM)

CMs are almost completely cleared within 6-9 hrs.

Anticoagulants

EDTA is preferred but cholesterol and triglyceride in plasma are 3% lower than in serum.

LDL-C and HDL-C concentrations

Concentrations of LDL-C and HDL-C decline transiently after eating due to CETP-mediated compositional changes.

Total cholesterol and HDL-C levels

Can be measured in non-fasting individuals, facilitating screening and monitoring.

Non-fasting HDL-C levels

Can be a few milligrams lower than fasting levels but will not lead to misclassification of patients.

Fasting effect on plasma total cholesterol

Fasting has little effect on plasma total cholesterol levels.

Fasting requirement for triglyceride and LDL-C measurement

Fasting is required as postprandial appearance of CMs can lead to underestimation of LDL-C.

NCEP ATPIII fasting recommendation

Recommends fasting for at least 9 hours before blood specimens are taken for lipids and lipoprotein measurements.

Shorter fasting periods

Should produce only minor or clinically insignificant errors in estimation of triglycerides, LDL-C and HDL-C.

12-hour fasting

Considered appropriate when lipoprotein measurements are made for clinical and epidemiological studies.

Analysis of frozen samples

Total cholesterol, triglycerides, and HDL-C can be analyzed in frozen samples; LDL-C can be estimated using Friedewald equation.

Frozen samples for ultracentrifugal analysis

Frozen samples are NOT appropriate for ultracentrifugal analysis as triglyceride-rich lipoproteins do not withstand freezing.

Storage temperature for serum or plasma

Must be maintained at -70°C or lower for longer periods.

Short-term sample storage

For short-term (1-2 months), samples can be kept at -20°C.

Self-defrosting freezers

Do not store samples in self-defrosting freezers.

Freeze-thaw cycles

Should be avoided as they could lead to deterioration of blood samples and variability in measurements.

Estimation of plasma lipids

Of greatest interest in the diagnosis and management of lipoprotein disorders.

Patient position during venipuncture

The position should be standardized, preferably to the sitting position, to prevent hemoconcentration.

NCEP ATPIII seating recommendation

Recommends that patients be seated for 5 minutes before sampling.

Cholesterol in plasma

Accounts for almost all of sterol in plasma, consisting of unesterified (30-40%) and esterified (60-70%) forms.

Total cholesterol and lipoprotein cholesterol concentrations

Usually expressed in terms of the sterol nucleus without distinguishing esterified and unesterified fractions.

Distinguishing cholesterol forms

It is not necessary to distinguish the two forms except in cases where the contribution of the fatty acid moiety must be accounted for.

Venous Blood

Blood obtained from veins, generally assumed to be equivalent to capillary samples.

Capillary Blood

Blood obtained from capillaries, which tends to be a little lower than venous blood samples.

Fingerstick Samples

Capillary blood samples that tend to be more variable than venous samples obtained at the same time.

Pre-analytic Sources of Error

Factors that can contribute to variability in measurements, particularly in fingerstick samples.

Epidemiological Data

Data from which risk levels for lipoproteins and lipids are derived, based on measurements in venous samples.

Plasma

The liquid component of blood preferred for measuring lipoproteins by ultracentrifugal or electrophoretic methods.

Serum

The liquid that remains after blood has clotted, can be used when only cholesterol, triglyceride, and HDL-C are measured.

Enzymatic Methods

Methods that measure total cholesterol directly in plasma or serum, replacing traditional chemical methods.

Advantages of Enzymatic Methods

Include relative resistance to interfering substances, small sample requirement, no preliminary extraction step, and rapid and precise results.

Enzymatic Values

Values that agree with reference values within 1-2%.

Cholesteryl Esters Hydrolysis

A series of reactions in which cholesteryl esters are hydrolyzed.

3-OH Group of Cholesterol

The functional group that is oxidized in the enzymatic measurement of cholesterol.

Hydrogen Peroxide Quantification

The process of measuring hydrogen peroxide enzymatically in cholesterol analysis.

Sample Turbidity

A result of hypertriglyceridemia that can interfere with cholesterol measurements.

Bilirubin

A substance that interferes with total cholesterol measurements due to its spectral properties and absorbs light at 500 nm.

Significant Interference Level

Bilirubin causes significant interference at levels greater than 5 mg/dL.

Hemoglobin

A substance with pseudo-peroxidase activity that can consume H2O2 and falsely elevate total cholesterol levels.

Products of Hemolysis

Substances such as catalase that compete with peroxidase for H2O2 in cholesterol measurement.

Abell-Kendall Method (Modified)

The reference method for cholesterol used by CDC and secondary reference laboratories.

Cholesteryl Esters Hydrolysis with KOH

A process involving saponification of cholesterol ester by hydroxide in the Abell-Kendall Method.

Unesterified Cholesterol Extraction

The process of extracting unesterified cholesterol with petroleum ether in the Abell-Kendall Method.

Accuracy of Abell-Kendall Method

The accuracy of this method is 0.5% of the true value.

Measurement of hydrogen peroxide (H2O2) produced

Second and third steps/reactions (product side)

Most current method of cholesterol estimation

Oxidatively coupled to two chromogenic substrates (chromogens) by catalysis with a peroxidase, as seen in the third reaction (HRP or horseradish peroxidase)

Chromogens used in cholesterol estimation

Chromogens that are used include phenol and 4-aminoantipyrine

Quinoneimine dye production

When the chromogens are coupled with H2O2 or when it oxidizes, the 4-aminoantipyrine (in the presence of peroxidase) produces a quinoneimine dye, a pink or reddish complex, that can be read photometrically at 500 nm

Disadvantages of HRPs

Catalytic properties of HRPs are very nonspecific

Interference in cholesterol measurement

Third step is subject to interference from other components of the serum or plasma

Measurement of oxygen consumption

Second step/reaction (reactant side)

Advantages of oxygen consumption measurement

Interference by some components with serum/plasma is minimized

Disadvantages of oxygen consumption measurement

Not easily automated and require a lot of cholesterol oxidase

Measurement of cholest-4-en-3-one

Second step/reaction (product side)

Measurement wavelength for cholest-4-en-3-one

After the reaction, it can be measured at 240 nm

Disadvantages of cholest-4-en-3-one measurement

Time-consuming and very difficult to perform

Enzymatic methods of cholesterol measurements

Less subject to interferences by non-sterol substances, however, there are still a few

Sterols in cholesterol measurement

Compete for cholesterol oxidase reaction

Example of sterols interference

Plant sterols present in the plasma (patients with β-sitosterolemia)

Ascorbic acid in cholesterol measurement

A reducing agent that competes with chromogenic substrates for H2O2

Effect of elevated ascorbic acid levels

Elevated levels result in lower measured TC levels

Measurement of triglycerides

Disappearance of NADH is then measured at 340 nm

Measurement wavelength for NADH

Measurement at 340 nm of Disappearance of NADH

Triglyceride blanks

Assays without the addition of lipase which provides a measure of preexisting glycerol.

Measured based on hydrolysis of triglycerides

Measurement of glycerol that is being released afterward.

TG concentrations

Often overestimated due to monoglycerides, diglycerides, and unesterified glycerols not subtracted.

Endogenous glycerol

Not corrected by labs because in most normal individuals, such levels are negligible.

Enzymatic Methods

Methods used for TG measurements in clinical laboratories, replacing traditional methods.

Inter-laboratory coefficient of variations for triglyceride measurement

5-6%.

Accuracy and precision evaluation range

1.299-12.987 mmol/L (50-500 mg/dL).

Hydrolysis of TG

Converts triglycerides to free fatty acids (FFAs) and glycerol.

Chloroform extraction procedure

Used to isolate triglycerides.

Saponification

Alkaline hydrolysis of triglycerides releasing glycerol.

NADH formation

Measured spectrophotometrically at 340 nm.

Absorbance readings in 500 to 600 nm region

Instruments commonly available in clinical laboratories measure absorbance in this region.

Glycerophosphate oxidase

Oxidizes glycerophosphate and measures H2O2 using methods for cholesterol estimation.

Phospholipids in human plasma

Most common types include Phosphatidylcholine (70-75%), Sphingomyelin (18-20%), Phosphatidylserine, Phosphatidylethanolamine (3-6%), and Lysophosphatidylcholine (4-9%).