2. Objective
At the end of this chapter the student will be
able to
Define terminologies associated with lipids
and lipoproteins
Describe classes of lipids and lipoproteins
Explain significance of lipids
Discuss about chylomicrones, VLDL, LDL, and
HDL
Discuss about Apolipoproteins
3. Objective, continued…
Describe the laboratory procedures used for
determination of lipids and lipoproteins
Interpret laboratory results
4. Outline of lipid lecture
Definition of terms
Introduction about lipids classification of lipids
Lipoproteins: function,
classification(Chylomicrons,VLDL,LDL,
HDL)
Apolipoproteins: functions and classification
Physiological changes in lipids and lipoproteins level
Abnormlities in Lipids and lipoproteins metabolism
Atherosclerosis
Lipids and lipoproteins: laboratory diagnosis, reference
range and interpretations
5. Outline of lipid lecture,
continued…
Atherosclerosis:
Lipoprotein: protein combined with lipid components
Chylomicrons: parcel of lipids and proteins made from
dietary fats (especially triglycerides) during intestinal
absorption
Micelle: ultramicroscopic particle
Apoprotein: the protein portion of a lipoprotein
6. Introduction
lipids are either compounds that yield fatty acids when
hydrolyzed or complex alcohols that can combine with
fatty acids to form esters
Lipids are carried in the bloodstream by complexes
known as lipoproteins. This is because these lipids are
not soluble in the plasma water.
7. Lipids can serve as:
hormones or hormone precursors
structural and functional elements of biomembranes
sources of biochemical fuel
energy storage depots
insulators to prevent heat loss
8. Classification of lipids
Fatty acids (palmitic, linoleic, etc.)
- Even-numbered fatty acids predominate
- The most common saturated fatty acids are
palmitic (16:0) and stearic (18:0), but
unsaturated fatty acids are more common in
nature, such as Palmitoleic acid (16:19, 9-
hexadecanoic acid) Glycerol esters
(triglycerides)
Other lipids include
- Sterols (cholesterol, hormones, vitamin D)
- Terpenes (vitamins A, E, K)
- Sphingosine derivatives (sphingomyelin
9. Clinical importance of fatty acids
- Fecal fatty acids are sometimes measured to detect
malabsorptive and pancreatic disorders
the test is mostly considered obsolete
- Serum free fatty acids help distinguish between
hyperinsulinemic hypoglycemia (FFA normal) and
disorders of fatty acid oxidation (FFA elevated and
negative ketones)
10. Fatty Acids (FA)
the simpler molecular forms of lipids
they depicted by the chemical formula
RCOOH, R stands for an alkyl chain
Classification based on the number of
carbon atoms
2-4 carbon atoms- short chain fatty acids
6-10 carbon atoms – medium chain FA
12-26 carbon atoms – long chain FA
Important in human nutrition and
metabolism – all monocarboxylic acids
containing even numbers of carbon atoms
in straight chains.
11. Lipoproteins
In order to be transported in blood, lipids must
combine with water-soluble compounds, such as
phospholipids and proteins.
Transport lipid to sites of utilization or storage
Composed of variable amounts of lipid and functional
apoproteins
Plasma lipid levels represent balance between
absorption/synthesis and utilization/clearance
12. Change in blood lipid and
lipoprotein components:
associated with atherosclerosis (thickening of the inner
layer of the arterial wall)
lipid storage disease
14. Chylomicrons
Produced by the intestine
Transport dietary lipid from intestine to fat/muscle
cells
The largest lipoproteins
Mostly lipid, almost 90% triglyceride (TG)
1% protein, mainly Apo-B48, C-II, and E
Lipid poor remnants removed by liver Apo B/E
receptors
15. VLDL
Produced by the liver
Transport liver synthesized fat (mostly TG) to
fat/muscle cells
Mainly lipid, 55% TG
10% protein, Apo-B100, C-II, E
Remnants, termed intermediate density lipoproteins,
cleared by liver Apo-B/E receptors
16. LDL
Produced from IDL with loss of Apo C-II
Transport cholesterol to tissue
Taken up by Apo-B receptors and cholesterol released
In liver, decreases cholesterol synthesis, Apo-B
receptors
Alternate pathway for uptake is scavenger receptors,
mainly on macrophages (also HDL receptors) - leads to
the formation of Foam cells
Does not inhibit cholesterol synthesis
Modifications to LDL increase activity of this pathway
Proatherogenic
Better marker for CHD risk
17. HDL
The smallest and most dense lipoprotein
Synthesized by liver and intestine
Mainly protein (Apo A-I, A-II)
Lipid cholesterol, phospholipid
Involved in "reverse transport" of cholesterol from
tissue to liver
21. Physiological Changes in Lipid
and Lipoprotein Levels
High HDL-C levels are seen in premenopausal women,
persons who exercise regularly,
and those who maintain a low but healthy weight. Insulin,
estrogen, and thyroxine
(T4) have an inverse relationship with total cholesterol
levels. When
estrogen levels are higher, as in women who menstruate,
the total cholesterol level
is lower, preferably 200 mg/dL. The HDL-C level is also
elevated in menstruating
women, while the LDL-C tends to be lower.19 Test
Methodology 8–8 describes the
method of analysis for HDL-C.
24. ATHEROSCLEROSIS
Lipid deposition within wall of arteries
Gradually narrows vessels
Major risk factor for development of AMI and
cerebrovascular accidents (stroke)
25. ATHEROSCLEROSIS RISK FACTORS
Age (> 45 men, > 55 women)
Family history of early MI
Smoking
Hypertension
Diabetes
Lipid levels
Life style
26. LIPIDS AND
ATHEROSCLEROSIS
LDL cholesterol (LDL-C) directly related to risk
HDL-C inversely related to risk
Triglycerides, VLDL associated with lower HDL-C, ?
independent increase in risk
Lowering LDL-C, raising HDL-C lowers risk of
atherosclerosis
Lowering LDL-C can also reverse process of
atherosclerosis
Lipid levels are major modifiable risk factor, target for
therapy
27. LABORATORY DIAGNOSIS METHOD OF TOTAL
CHOLESTEROL
Testing methods for total cholesterol use cholesterol oxidase
reactions along with cholesterol esterase and usually a peroxidase
reaction for the “color” or final determination reaction.
29. Interference
Remove sample from red cells after blood clots or
plasma has been spun down. The peroxidase assay
can be susceptible increases in uric acid, ascorbic
acid, bilirubin, hemoglobin to, or other reducing
substances. Samples should have only the normal
amount of these substances present.
.
30. Specimen
Non hemolyzed serum or plasma. The patient need
not be fasting if this is the only lipid test requested.
However, if total cholesterol is requested as part of a
lipid panel, the patient must be fasting for 10 to 12
hours
32. LABORATORY DIAGNOSIS METHOD OF LDL
CHOLESTEROL
LDL cholesterol (LDL-C) may be calculated or
measured directly.
Friedewald Calculation or Derived Beta-
Quantification
Testing for LDL-C involves a calculation that includes
total cholesterol, HDL cholesterol
(HDL-C), and trigylceride (TG) values using the formula:
33. LDL-C = total cholesterol – [HDL-C _ (TG/5)]
where TG/5 approximates the VLDL cholesterol
concentration in the sample.
34. Interference
This method Cannot be used for TG over 400 mg/dL
For example:
Total cholesterol _ 350 mg/dL;
triglycerides _ 150 mg/dL;
HDL-C _ 30 mg/dL
LDL-C =350 – [30 _ (150/5)] = 350 – (30 _ 30) = 350 –
60 = 290 mg/dL
35. Direct LDL cholesterol
Older direct methods for LDL involved
precipitation with heparin or polyvinyl sulfate
Newer methods involve precipitation of VLDL, IDL,
and HDL with polyvalent antibodies to Apo A and
Apo E
LDL is almost exclusively Apo B-100
37. Direct Measurement of LDL-C
With the advent of homogeneous
reagents, LDL-C is now measured using
the cholesterol reaction along with
reagents that block the contribution of
HDL and VLDL to the resulting
answer.
In the homogeneous LDL assay,
detergents block the other two lipoprotein
cholesterol products from forming colored
chromogens. Only the LDL-C forms a
colored
chromogen that can be measured
spectrophotometrically by automated
systems or designated analyzers.
38. Direct Measurement of LDL-C,
continued….
The Specimen
Serum, plasma. The patient need not be
fasting if this is the only lipid test
requested.
However, if total cholesterol is requested
as part of a lipid panel, the patient must
be
fasting for 10 to 12 hours.
39. LABORATORY DIAGNOSIS METHOD OF
TRIGLYCERIDE
Triglycerides are composed of three fatty acids and a
glycerol moiety.
Analyzing a serum or plasma sample for triglycerides
typically involves four reactions.
45. Precipitation method
Routine methods (The Precipitation Reaction)
precipitate apolipoprotein B with a
polyanion/divalent cation, includes VLDL, IDL,
Lp(a), LDL, and chylomicrons
This HDL supernatant is then assayed for
cholesterol. The resulting answer (in mg/dL)
represents the amount of HDL in the serum
sample.
The supernatant is tested for cholesterol
concentration.
46. Direct method
Newer automated methods use a modified
form of cholesterol esterase, which
selectively reacts with HDL cholesterol
These methods do not use precipitation, nor
do they require a centrifugation separation
step. This improves the yield of HDL
recovered from the specimen.
47. Principle of direct methods
There are two approaches:
Method one:
uses the first reagent as an antibody
to apolipoprotein B-100 to bind LDL
and VLDL in the sample.
This leaves the HDL-C to react with
the second reagent, which contains
enzymes and substrate for cholesterol
analysis.
48. Principle of direct methods,
continued…
Method two:
The first reagent is a synthetic polyanion
reagent that binds the sites on VLDL and
LDL particles, blocking their products from
forming cholesterol colored products.
The second reagent added has detergent,
enzymes, and substrate that react with
the HDL-C in the sample. Only the HDL
particle cholesterol is allowed to form a
colored product and be measured.
49. Interference
Chylomicrons from non fasting specimens will
interfere in these precipitation methods.
The Specimen
Serum, plasma (depends on the method
used).
50. REFERENCE LIMITS
HDL-CHOLESTEROL
Men (25–29 years old) 31–63 mg/dL
Women (25–29 years old) 37–83 mg/dL
DESIRABLE: > 35 mg/dL (> 0.9 mmol/L)
NEGATIVE RISK: > 60 mg/dL (> 1.6 mmol/L)
HIGH RISK: < 35 mg/dL (> 0.9 mmol/L) Compare
the patient results with the reference range to
assess for hyper- or hypo.
51. Quality Control
A normal & abnormal quality control sample should
be analyzed along with patient samples, using
Westgard or other quality control rules for acceptance
or rejection of the analytical run.
Assayed known samples
Commercially manufactured
Validate patient results
Detects analytical errors.
52. Documentation of all lipide
Results
Record patient results in result logbook
Record QC results in QC logbook
Retain records for recommended time
54. Reference
1. Burtis, Carl A., and Ashwood, Edward R.. Tietz:
Fundamentals of Clinical Chemistry. Philadelphia,
2001.
2. Arneson, W and J Brickell: Clinical Chemistry: A
Laboratory Perspective 1st ed. 2007 FA Davis
3. Burtis, Carl A., and Ashwood, Edward R.. Tietz:
Text book of Clinical Chemistry. Philadelphia, 1999.
lipoproteins are micelle-like complexes composed of phospholipids and protein on the outside with cholesterol, cholesterol esters and triglycerides on the inside. The four main types of lipoproteins are chylomicrons, very low density lipoproteins (VLDL), low-density lipoprotein (LDL), and high-density lipoprotein (HDL)
Just as there is a ying to a yang, so too, are two main forms of lipoproteins. While LDL-cholesterol is considered harmful when in excess, the elevation of HDL-cholesterol is viewed as a positive cardiovascular biomarker for a patient. Elevated HDL-cholesterol has a beneficial effect for the vascular system, due to the role that HDL plays in the body. HDL removes excess cholesterol from tissues and routes it to the liver for re-processing and/or removal.
The American National Cholesterol Education Program (ANCEP) provides guidelines for evaluation of lipid panel results in regard to risk factor for cardiovascular problems. HDL (good) cholesterol protects against heart disease, so for HDL, higher numbers are better. HDL-c levels less than 40 mg/dL is considered abnormally low and a major risk factor because it increases the risk of developing heart disease. HDL levels of 60 mg/dL or more help to lower risk for heart disease. High amounts of triglycerides can also raise risk of heart disease. Individuals with triglyceride levels that are borderline high (150-199 mg/dL) or high (200 mg/dL or more) may need treatment. Type 2 diabetics often exhibit elevated cholesterol, triglycerides and LDL-cholesterols with low HDL-cholesterol. Decreasing the LDL-cholesterol is necessary to decrease the risk of cardiovascular disease in the type 2 diabetic.
A summary of the American Heart Association description of factors that can lead to cardio-vascular problems like AMI in type 2 diabetes is listed below: (23)
Being insulin resistant (about 9 out of 10 patients have insulin resistance);
Being obese (about 50 percent of men and 70 percent of women who have diabetes are obese);
Having a lifestyle that does not involve significant physical activity;
Having low HDL ("good") cholesterol levels and high triglyceride levels; and
Having an increased prevalence of high blood pressure
A normal and decreased or increased (depending on the assay) quality control sample should be analyzed along with patient samples, using Westgard or other quality control rules for acceptance or rejection of the analytical run.
If at all possible, use commercially prepared quality control material as it has been preserved to allow for long term storage. If commercially prepared quality control assayed samples are not available, that is, those with established mean, standard deviation and preparation and storage information, laboratory personnel can prepare control materials for their own use.
For the normal control, a large number of samples from similar patients free from most obvious disease, is pooled together in a large container. This is usually salvaged from regular specimens, rejecting those that are haemolysed, lipemic and jaundiced. Specimens are generally collected over a time period and should be frozen at -20 degrees C, tightly covered and held there until ready to determine statistical values. At least 100 mL of serum will be needed to prepare the quality control material.
Upon removing the frozen serum pool from freezer for analysis the container should immediately be placed in a 37 degree C water bath for thawing, if possible. After thawing, the pool should be mixed thoroughly and filtered if needed, prior to analysis. 1.0 mL aliquots of the pool should be placed in test tubes, covered and labelled with a batch number and date. Retain 6-8 of the samples and place the remaining samples back in the freezer.
Mean and standard deviation of the control material is obtained by repeated analysis. A minimum of twenty samples must be analyzed for the particular tests needing quality control information, thawing only what samples will be analyzed immediately. Each sample should be mixed thoroughly prior to analysis. For most accurate representative results, 20 specimens should be run over a three-day period. This will permit more variance to be associated with the results therefore, a more representative of true testing conditions. In order to maximize on specimen availability and eliminate differences the specimen should be run by all methods at the same time, that is automated and manual for which the quality control results will be used.
Once mean and standard deviation is established and recorded, the remaining samples should be labelled with a date of implementation and quality control charts should be prepared to record mean, standard deviation and other identifying information. Aliquots for most analytes will be stable for up to 1 month. So the process is generally repeated monthly in order to have adequate acceptable quality control samples.
For preparation of an abnormal (high or low control), a similar process is used for collection, storage, analysis and aliquotting samples. However, it may be more difficult and take longer to obtain enough abnormal pool. It may be possible to add calibration or standard solution in order to obtain an abnormal high sample or to add appropriate diluent to prepare an abnormally low quality control sample. However, this method of adding to the serum pool or diluting is less desirable.
If unassayed commercially quality control samples are purchased, several vials should be prepared according to the manufacturer’s directions and assayed 20 times over a three day period. Aliquots should be frozen for use during the three day period. Mean, standard deviation can be determined as described above and quality control charts made. This is a desirable alternative, especially for obtaining abnormal quality control samples.
