Serum Cholesterol

Sample

1. The best sample is after 12 hours of fasting, and that is the morning sample.
2. The best sample is serum and separated within 2 hours of the collection.
3. This test may be done on Serum or Plasma (EDTA) but not oxalate, citrate, or fluoride.
4. The serum is stable at 4 °C for 5 to 7 days and 3 months at -20 °C.
5. Avoid freezing and thawing.
6. The result with EDTA plasma is 3% lower than serum.
7. Sample with Cholesterol above 500 mg/dL should be diluted.

Precautions for serum cholesterol

1. Try to separate serum or plasma from the cells within 2 hours.
2. The best sample is after 12 to 14 hours of fasting.
3. Pregnancy may give a high level.
4. Drugs that may increase the level are anabolic steroids, corticosteroids, oral contraceptives, vitamin D, phenytoin, thiazide diuretics, and cyclosporin.
5. Drugs that may decrease the level are captopril, colchicine, erythromycin, isoniazid (INH), lovastatin, neomycin, bile salt binding agents, androgens, niacin, and nitrates.

Purpose of the test (Indications) for serum cholesterol

1. Cholesterol level is estimated to determine the coronary artery disease risk.
2. This test may be needed to rule out hyperlipidemia.

Pathophysiology of cholesterol

1. Total serum cholesterol comprises all of the Cholesterol found in the various lipoprotein.
2. Cholesterol is an organic molecule, is sterol or modified steroid, a type of lipid.
3. This is an essential part of all cell membranes.
4. Cholesterol is the major component of LDL and a minority part of VLDL and HDL.
5. Cholesterol is the best-known steroid because of its association with atherosclerosis.
   1. Cholesterol has biochemical significance because this is the precursor for important steroids like bile acids, adrenocortical hormones, sex hormones, Vitamin D, cardiac glycosides, sitosterol of the plant kingdom, and some alkaloids.
6. Cholesterol is steroid alcohol with 27 carbon atoms.
   1. 1. Cholesterol is present virtually in all cells and body fluids.
   2. Cholesterol is absorbed in the diet and synthesized in the liver and gut.
      1. It is a precursor of steroid hormones, bile acids, sex hormones, and cellular membranes.

Sources of Cholesterol:

1. Cholesterol sources:
   1. Exogenous sources are present in the diet, mainly from animal sources, and absorbed by the gastrointestinal tract.
   2. Endogenous sources are like the liver and other tissues.
      1. All the cells have the ability to synthesize Cholesterol, and 90% occur in the liver and intestines.
   3. Malnutrition will give low cholesterol formation.

Cholesterol in the body and their calculation from LDL-cholesterol formula:

1. Three different lipoproteins carry Cholesterol in the fasting serum. So the total cholesterol sum equals the sum of these three components.
2. Total cholesterol = HDL-cholesterol + VLDL-cholesterol + LDL-cholesterol
   1. The amount of Cholesterol in the HDL fraction can be found by precipitating from the serum.
   2. The level of Cholesterol in VLDL = Triglycerides/5
      1. This formula works in the fasting sample and when:
         1. The triglycerides are <400 mg/dL.
         2. No chylomicrons are seen.
         3. Does not work in type III dyslipidemia (β-VLDL).
3. LDL – Cholesterol = The separation of LDL from the lipoproteins is very difficult, so the Friedewald equation is used, which is:
   1. 1. 1. 1.  LDL-cholesterol = (Total cholesterol mg/dL) – (HDL-cholesterol mg/dL) – (triglycerides mg/dl)/5.

Cholesterol level changes in the body and its distribution along with other lipoproteins:

1. There is day-to-day variation in the cholesterol level.
2. This day-to-day variation may be seen in persons around 15%.
3. An 8% difference can be found on the same day.
4. There is a positional change in the level.
   1. The level may be decreased by as much as 15% in the recumbent position.
5. There is a low level in the hospitalized patient.
6. Because of these variations, two samples must be tested for heart disease risk.
7. There is a decreased level of Cholesterol in acute MI, as much as 50% for 6 to 8 weeks.

Cholesterol distribution in the lipoproteins:

Type of lipoprotein	Cholesterol	Triglycerides	Phospholipids	Proteins
Chylomicron	3% to 5%	85% to 95%	5% to 10%	1% to 2%
HDL (High-density lipoprotein)	20%	Very small amount	30%	50%
LDL (Low-density lipoprotein)	45%	5% to 10%	20% to 30%	15% to 25%
VLDL (Very-low-density lipoprotein)	10% to 15%	60% to 70%	10% to 15%	10%

Cholesterol absorption:

1. The primary source of Cholesterol is diet. Another source is endogenous.
   1. Cholesterol is an important constituent in the assembly of the cell membrane.
   2. Cholesterol is the basic part of synthesizing bile acids and steroid hormones (cortisol, estrogen, androgen).
   3. In the normal biologic process, Cholesterol undergoes changes like synthesis, recycling, and degradation.
2. Cholesterol enters the intestine from the following sources:
   1. For food like egg yolk, meat, fat-diary products, and seafood, the average amount is 400 to 700 mg/day.
      1. Plant sterols (200 to 300 mg) are ingested daily. These are poorly absorbed.
   2. Bile provides another fraction of Cholesterol.
   3. Intestinal secretions and turnover of the intestinal mucosal cells are other sources.
3. Bile acids are detergents and are the most critical factor affecting micelles formation.
   1. In the absence of bile acids, digestion and absorption of Cholesterol and triglyceride are severely affected.
4. Most absorption of Cholesterol takes place in the jejunum and ileum.
   1. When Cholesterol enters the intestinal cells, it is packaged with triglycerides, phospholipids, and apolipoprotein into large lipoproteins called chylomicrons.
   2. An increased amount of fat in the diet, particularly triglycerides 98%, will result in more micelles formation, allowing more Cholesterol to be solubilized and absorbed.
   3. Approximately 60% of the total Cholesterol in the male plasma is carried by LDL 22%, HDL 13%, and Chylomicrons 5%.

Cholesterol esterification: 

1. Cholesterol, once formed, enters the blood circulation.
2. Cholesterol is esterified and combined with apoprotein and apolipoprotein and ultimately carried as a lipoprotein.
   1. Cholesterol esterification helps in the packing of Cholesterol into lipoproteins.
   2. Both dietary and synthesized Cholesterol are converted to cholesteryl esters, easily packed into lipoproteins.
   3. Esterification increases the lipid carrying capacity of the lipoproteins in plasma and prevents the toxicity effect of Cholesterol.
3. 75% bound to LDL.
4. 25% bound to HDL.

Biosynthesis of Cholesterol 

1. 90% of cholesterol synthesis takes place in the liver and gut.
2. Cholesterol synthesis takes place in three stages.
3. The starting molecule is acetate.
4. All body cells have the ability to synthesize Cholesterol from Acetyl-CoA.

5. Knowing the endogenous synthesis of Cholesterol, drugs are developed that lower the cholesterol level like mevastatin, lovastatin, simvastatin, pravastatin, and atorvastatin, which will suppress the HMG-CoA reductase.

Cholesterol exists in two forms:

1. Free Cholesterol.
2. Esterified Cholesterol.
   1. 75% to 85% of the total Cholesterol is in the form of esterified Cholesterol.
3. Cholesterol can be derived from dietary sources like meat, eggs, butter, and plants.
   1. 30 to 60% of dietary Cholesterol is absorbed.
4. Once Cholesterol is absorbed into the mucosal cells, it becomes part of the large micelle called Chylomicron.

Cholesterol complications:

• Cholesterol’s main complication is atherosclerosis which leads to heart disease and stroke.

Cholesterol  distribution in other lipids of the body:

Cholesterol found in:	Cholesterol % in other lipids
Chylomicron	2%
VLDL	5% to 8%
LDL	13%
HDL	6%

Cholesterol functions:

1. Cholesterol is a blood lipid (fat).
2. Cholesterol is a normal part of RBC.
3. Cholesterol, when increased, leads to atherosclerosis and is the main lipid.
4. A total raised level of Cholesterol is associated with an increased risk of cardiovascular disease. Its subtypes LDL-C and HDL-C are more important than the total Cholesterol.

5. Cholesterol is the basic unit of hormone produced by adrenal glands, ovaries, and testes.
6. Cholesterol is needed for bile acids and cellular membranes.
7. Cholesterol is transported in circulation by carrier proteins.
   1. Cholesterol bound to high-density lipoprotein (HDL- cholesterol) is known as good Cholesterol. HDL  will prevent atherosclerosis.
   2. Cholesterol bound to low-density protein (LDL-cholesterol) is known as bad Cholesterol and increases atherosclerosis chances.
      1. Also, the same is Cholesterol bound to very low-density lipoprotein (VLDL) is bad Cholesterol.
   3. Increased Cholesterol level is associated with atherosclerosis and coronary vessel disease.
      1. It is needed to form steroids, sex hormones, and bile acids.
      2. This is the part of cellular membranes.
      3. It gives the risk of heart disease.

Normal Cholesterol level

Source 1
Adult

1. The desired level to prevent coronary disease is < 200 mg/dl
2. Moderate risk is 200-239 mg/dl
3. High risk is > 240 mg/dl
4. CSF <0.5 mg/dl

Source 2

Sample	Male mg /dL	Female mg /dL
Cord blood	44 to 103	50 to 108
0 to 4 years	114 to 203	112 to 200
5 to 9 years	121 to 203	126 to 205
10 to 14 years	119 to 202	124 to 201
20 to 24 years	124 to 218	122 to 216
30 to 34 years	138 to 254	130 to 230
40 to 44 years	151 to 268	147 to 252
50 to 54 years	158 to 277	162 to 285
60 to 64 years	159 to 276	172 to 297
>70 years	144 to 265	173 to 280

•  To convert into SI unit x 0.0259 = mmol/L

Cholesterol and its relation to heart disease:

1. Desirable level is = < 170 mg/dl
2. Moderate risk is = 170-199 mg/dl
3. High risk is = > 200 mg/dl

• Above 240 mg/dL   =  needs treatment.
• Nowadays cardiology association recommends the level to be around 160 mg/dl.

Cholesterol mg/dL	Risk
<160	Very low
160 to 199	low risk
200 to 239	moderate risk
240 to 279	high risk
>280	very high risk

• According to the latest NHS research study, there is no relation between total cholesterol and heart disease risk.
  • https://www.nhs.uk/news/heart-and-lungs/study-says-theres-no-link-between-cholesterol-and-heart-disease/

ATP III (Adult treatment panel) recommendations for blood lipids  are:

Chemical substance (Lipid)	Clinical effects on the people
Cholesterol	Desirable level = <200 mg/dL Borderline level = 200 to 239 mg/dL High level            = >240 mg/dL
HDL	Low level = <40 mg/dL  High level = >60 mg/dL
LDL	Optimal level = <100 mg/dL Near-optimal =  100 to 129 mg/dL Borderline level = 130 to 159 mg/dL High level            = 160 to 189 mg/dL Very high             = >190 mg/dL

ATP  III (Adult treatment panel) LDL target level for coronary vascular disease:

Risk factors	Details of risk factors	The target LDL level required ( This level is mandatory)
Presence of coronary heart disease (CHD) or CHD-equivalent disease	CHD heart disease risk factors are: Diabetes Vascular diseases Noncoronary atherosclerosis Framingham risk of MI within 10 years of >20%	<100 mg/dL
2 or more major risk  factors	Major risk factors including are: Smoking Hypertension >140/90 mmHg HDL <40 mg/dL Family H/O premature CHD Male first degree relatives = <55 years Female first degree relatives = <65 years Age men (>45 years), women (>55 years)	<130 mg/dL
<2 major risk factors		<160 mg/dL

Framingham formula calculations (Risk score counting):

Risk factors	Points for the calculation for women	Points for the calculation for men
Age  20 to 34 years Age 35 to 39 years Age 40 to 44 years Age 45 to 49 years Age 50 to 54 years Age 55 to 59 years Age 60 to 64 years Age 65 to 69 years Age 70 to 74 years Age 75 to 79 years	-7 points -3 points 0 point 3 points 6 points 8 points 10 points 12 points 14 points 16 point	-9 points -4 points 0 points 3 points 6 points 8 points 10 points 11 points 12 points 13 points
Total cholesterol: age 20 to 39 years  cholesterol level is <160 mg/dL 160 to 199 mg/dL  200 to 239 mg/dL  240 to 279 mg/dL  28o or higher mg/dL Total cholesterol Age 40 to 49 years = <160 mg/dL  160 to 199 mg/dL  200 to 239 mg/dL  240 to 279 mg/dL 280 or higher mg/dL Total cholesterol Age 50 to 59 years  cholesterol < 160 mg/dL 160 to 199 mg/dL 200 to 239 mg/dL 240 to 279 mg/dL 280 or higher or mg/dL Total cholesterol Age 60 to 69 years cholesterol < 160 mg/dL 160 to 199 mg/dL 200 to 239 mg/dL 240 to 279 mg/dL 280 or higher mg/dL Total cholesterol Age 70 to 79 years cholesterol < 160mg/dL 160 to 199 mg/dL 200 to 239 mg/dL 240 to 279 mg/dL 280 or higher mg/dL	0 point 4 points 8 points 11 points 13 points 0 point 3 points 6 points 8 points 10 points 0 point 2 points 4 points 5 points 7 points 0 point 1 point 2 point 3 points 4 points 0 point 1 point 1 point 2 points 2 points	0 point 4 points 7 points 9 points 11 points 0 point 3 points 5 points 6 points 8 points 0 point 2 points 3 points 4 points 5 points 0 points 1 point 1 point 2 points 3 points 0 point 0 point 0 point 1 point 1 point
Cigarette smoking  age: 20 to 39 years Age 40 to 49 years Age 50 to 59 years Age 60 to 69 years Age 70 to 79 years	9 points 7 points 4 points 2 points 1 point	8 points 5 points 3 points 1 point 1 point
All non-smokers	0 point	0  point
HDL  cholesterol  = 60 mg/dL or higher level 50 to 59 mg/dL 40 to 49 mg/dL <40 mg/dL	-1 point 0  point 1 point 2 points	-1 point 0 point 1 point 2 points
Systolic blood pressure untreated under 120 mmHg 120 to 129 mmHg 130 to 139 mmHg 140 to 159 mmHg 160 or higher mmHg Treated cases of blood pressure  under 120 mmHg 120 to 139 mmHg 130 to 139 mmHg 140 to 159 mmHg 160 or higher mmHg	0 point 1 point 2 points 3 points 4 points o point 3 points 4 points 5 points 6 points	0 point 0 point 1 point 1 point 2 point 0 point 1 point 2 point 2 point 3 point

10 years risk for cardiovascular diseases according to Framingham risks in %:

Total points	%  Framingham score (risk)
0 point	<1%
1 to 4 points	1%
2 points	5% to 6%
7 points	3%
8 points	4%
9 points	5%
10 points	6%
11 points	8%
12 points	10%
13 points	12%
14 points	16%
15 points	20%
16 points	25%
17 points	>30%

Total Cholesterol relation to coronary artery disease:

1. Another source:
2. Desirable level = <200 mg/dL
3. Borderline level = 200 to 239 mg/dL
4. High Risk level = >240 mg/dL

Cholesterol and triglycerides risk factors for coronary artery disease (NCEP expert panel and ATP III guideline):

Risk factors grades	Total cholesterol	Total triglycerides	Total cholesterol/HDL-C ratio	LDL/HDL ratio
Normal or low risk	<200 mg/dL	<150 mg/dL	3.3 to 4.4	0.5 to 3.0
Average risk (>optical risk)		150 to 190 mg/dL	4.4 to 7.1
Moderate or borderline risk	200 to 239 mg/dL	200 to 499 mg/dL	7.1 to 11.0	3.0 to 6.0
High-risk group	>240 mg/dL	>500 mg/dL	>11.0	>6.0

Optimal values of  lipids (ATP III guidelines):

Risk level	LDL cholesterol	Total cholesterol	Triglycerides	HDL cholesterol
Optimal fasting values	<1000 mg/L	<2000 mg/L	<150o mg/L	>400 mg/L
Near-optimal fasting level	1000 to 1290 mg/L
Borderline high fasting level	1300 to 1590 mg/L
High fasting level	1600 to 1890 mg/L
Very high fasting level	>1900 mg/L

Increased Cholesterol (Hypercholesterolemia):

1. Familial combined hyperlipidemia.
2. Hyperlipoproteinemia type, 1V, V.
3. Intra- and extrahepatic cholestasis.
4. Glomerulonephritis.
5. Nephrotic syndrome and chronic renal failure.
6. Malignant neoplasm of pancreas and prostate.
7. Hypothyroidism.
8. Gout.
9. Diabetes mellitus.
10. Alcoholism.
11. Glycogen storage disease type 1 (Von Gierke disease).
12. Stress.
13. Biliary cirrhosis.

Decrease Cholesterol (Hypocholesterolemia):

1. alpha-Lipoprotein deficiency.
2. Hepatocellular necrosis.
3. Malignant neoplasm of the liver.
4. Hyperthyroidism.
5. Malabsorption and malnutrition (starvation).
6. Severe acute illness.
7. Extensive burns.
8. Rheumatoid arthritis.
9. Cholesterol-lowering medications.

Prevention, recommendations, and treatment for the cholesterol level:

Total cholesterol <200 mg/dL	Check after 5 years
Total cholesterol 200 to 239 mg/dL	0 to 1 risk factor and no coronary disease. Needs diet control and repeat the test after one year.
Total cholesterol >240 mg/dL	Diet control and drugs intervention

Risk factors  leading to heart diseases are:

1. Cigarette smoking.
2. Cerebrovascular disease.
3. Diabetes mellitus.
4. Hypertension.
5. The family history of coronary heart disease.
6. Sex as in male.
7. Overweight >30%
8. peripheral occlusive vascular disease.

Important:

• • There is variation in the level of Cholesterol so take at least two samples.
  • Critical value = >500 mg/dL.
    

    Chemical substance	Panic value (critical value)
    Cholesterol	>240 mg/dL (>500 mg/dL)
    HDL-cholesterol	<40 mg/dL
    LDL- cholesterol	>160 mg/dL
    Triglycerides	>500 mg/dL

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