This document outlines the key points about lipids and lipoproteins that will be covered in the chapter. It defines important terms, classifies lipids and lipoproteins, and describes the roles and properties of chylomicrons, VLDL, LDL, and HDL. It also discusses apolipoproteins and the clinical significance of abnormalities in lipid and lipoprotein metabolism, including their role in atherosclerosis. Laboratory methods for analyzing lipids and lipoproteins are summarized, including quality control procedures. Reference ranges for lipid results are also provided.

1. Chapter 9.
LIPIDS AND LIPOPROTEINS

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 (16:19, 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

13. Lipoprotein classes

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

18. Apolipoproteins
The protein composition differs from one lipoprotein class to another,
and the protein constituents are called Apolipoproteins

19. Functions of apolipoproteins
• Activate enzymes involved in lipid metabolism
– Lecithin- cholesterol acytransferase (LCAT)
– Lipoprotein lipase (LPL)
• Maintain structural integrity of lipid/protein
complex
• Delivery of lipids to cells via recognition of cell
surface receptors
•

20. Apolipoprotein content of LPs
Lipoprotein electrophoresis
(-) chylomicrons---LDL () —IDL—VLDL, Lp(a) (Pre-) ---HDL().. (+)

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.

22. Clinical Significance
Dyslipoproteinemias
Causes can be primary or secondary
• Primary causes of hyperlipidemia:
– Increased production
– Defective processing
– Defective cellular uptake
– Inadequate removal

23. • Secondary causes of hyperlipidemia
– Starvation
– liver disease
– renal failure
– Diabetes
– Hypothyroidism
– Lipodystrophies and drugs

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.

28. Principle of the test

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

31. REFERENCE LIMITS
TOTAL CHOLESTEROL
• Male (25–29 year old) 130–234 mg/dL
• Female (25–29 years old) 130–231 mg/dL
• DESIRABLE: < 200 mg/dL (< 5.2 mmol/L)
• BORDERLINE HIGH: 200-240 mg/dL (5.2-6.2
mmol/L)
• HIGH: > 240 mg/dL (> 6.2 mmol/L)

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

36. REFERENCE LIMITS
• LDL-CHOLESTEROL
• Male (25–29 years old) 70–165 mg/dL
• Females (25–29 years old) 71–164 mg/dL
• DESIRABLE: < 130 mg/dL (< 3.4 mmol/L)
• BORDERLINE HIGH: 130-160 mg/dL (3.4-4.2
mmol/L)
• HIGH: > 160 mg/dL (> 4.2 mmol/L)

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.

40. Principle of the test

41. The Specimen
• Serum, fasting 10 to 12 hours.
• Reference Ranges (Age-Specific)
• Male (25–29 years old) 45–204 mg/dL
• Females (25–29 years old) 42–159 mg/dL

42. REFERENCE LIMITS
• TRIGLYCERIDE
• Male (25–29 years old) 45–204 mg/dL
• Females (25–29 years old) 42–159 mg/dL
• DESIRABLE: < 200 mg/dL (< 2.3 mmol/L)
• BORDERLINE HIGH: 200-399 mg/dL (2.3-4.5
mmol/L)
• HIGH: > 400-1000 mg/dL (4.5-11.3 mmol/L)
• VERY HIGH: > 1000 mg/dL (> 11.3 mmol/L
• Compare the patient results with the reference range to
assess for hyper- or hypo.

43. Laboratory methods of HDL Diagnosis
• Ultracentrifugation method
• Precipitation method
• Direct method

44. Ultracentrifugation method
• Ultracentrifugation is the most accurate
method
• HDL sediment from the sample based on its
density, 1.063 – 1.21 g/mL

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

53. Summary

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.

55. Next Chapter
Chapter 10
Proteins