Laposata's Laboratory Medicine: Diagnosis of Disease in the Clinical Laboratory, 3e

CHAPTER 8: Blood Vessels

ATHEROSCLEROSIS
Dietary Fat

Most of the lipid in the diet is in the form of triglycerides. Humans ingest grams of triglycerides daily. Nearly
all (97%) of the mass of the triglyceride molecule is made up of the three fatty acids bound to the glycerol
backbone. The triglyceride molecules are degraded in the gastrointestinal tract by lipases which liberate the
fatty acids from the glycerol backbone of the triglyceride. Fatty acids are long chain carbon molecules with a
carboxyl (COOH) end and a methyl (CH3) end. Commonly encountered fatty acids in the diet are as long as 22
carbons. If there are no double bonds in the carbon chain, the fatty acid is saturated. If there is a single
double bond, the fatty acid is monounsaturated. Two or more double bonds make the fatty acid
polyunsaturated. For those fatty acids with a double bond, the number of carbons between the double bond
and the methyl end identifies the family to which the fatty acid belongs. When the double bond nearest the
methyl end of the fatty acid is three carbons inward, the fatty acid is known as an omega-3 or n-3 fatty acid.
Similarly, if it is six carbons from the methyl end, it is known as an omega-6 or n-6 fatty acid. Other families of
fatty acids include omega-9 and omega-7. Oils are composed of mostly omega-6 fatty acids (e.g., vegetable
oil), omega-9 (e.g., olive oil), or omega-3 (e.g., fish oil). Humans are unable to synthesize omega-3 or omega-6
fatty acids, and for this reason they are known as essential fatty acids because they are essential in the diet.
Saturated fatty acids and fatty acids in the omega-9 and omega-7 family are essential for life, but because
these fatty acids can be made from smaller molecules in humans, they are known as nonessential fatty acids.
There is strong evidence that saturated fatty acids are not healthy; polyunsaturated fatty acids can be
healthy, but it depends to some extent upon the family. Notably, omega-3 fatty acids have beneficial e ects
for reducing cardiovascular risk and, as an adjunct therapy, for patients with depression. Omega-3 fatty acids
are present in higher amounts in certain fish, especially those that swim in cold water. The two major omega-
3 fatty acids, which are highly unsaturated, are EPA (eicosapentaenoic acid) which has 20 carbons and 5
double bonds and DHA (docosahexaenoic acid) which has 22 carbons and 6 double bonds. Omega-6 fatty
acids, even though they are polyunsaturated, can lead to the generation of compounds which promote
inflammation. Another classification is made for only those fatty acids with a double bond. If the hydrogen
atoms are on the same side of the double bond in an unsaturated fatty acid, the fatty acid is known as a cis
fatty acid. This is the natural form of the double bond. Alternatively, when the hydrogens are on the opposite
sides of the double bond, the fatty acid is known as a trans fatty acid. Trans fatty acids are not healthy, and
although small amounts are generated endogenously, much of the trans fatty acid in humans is from the
diet, despite the growing restriction of trans fatty acids in commercially prepared food.
Unlike triglycerides which are eaten in grams daily, only milligrams of cholesterol are ingested. When
cholesterol is bound to a fatty acid, it becomes a cholesterol ester. Cholesterol ester molecules are highly
insoluble in water. Therefore, when cholesterol is delivered into the blood vessel wall, it is much more easily
removed than a cholesterol ester molecule, which is created a er the cholesterol is delivered into a growing
plaque. Atherosclerotic plaques contain significant amounts of cholesterol esters.

Lipoproteins carry triglycerides and cholesterol esters because they are insoluble in the blood. The
lipoprotein has a shell of phospholipid which has enough polarity to interact with water. Free cholesterol,
that is cholesterol not bound to a fatty acid, is also polar and resides in the shell of the lipoprotein. Inside the
shell, in the core of the lipoprotein, interacting with the nonpolar portion of the phospholipid molecule, are
cholesterol ester and triglyceride. Lipoproteins also have certain apolipoproteins which a ect their
metabolism and distribution.

Description

Atherosclerotic vascular disease is a major cause of mortality and morbidity in the Western world. It is the
consequence of an accumulation of lipid in large arteries including the aorta and, thereby, a narrowing of the
lumen of the arteries, which results in decreased blood flow. When an atherosclerotic plaque ruptures, a
thrombus can form over the ruptured plaque and totally occlude blood flow. Atherosclerotic disease is
vascular in origin, in that lipid deposition and cell proliferation occur within the blood vessel wall. The end-
organ damage depends on the anatomic location of the occluded artery. It is common to have generalized
atherosclerosis with multiple vascular beds a ected.

The causes of atherosclerotic vascular disease include:

Ingestion of excess or atherogenic dietary fat, which is primarily saturated fatty acids and cholesterol. This is
the most common cause of atherosclerotic vascular disease.

Primary lipid disorders, also known as primary hyperlipidemias, which result in an increase in cholesterol,
triglyceride, or both in the plasma. Many of these disorders are a result of genetic mutations that perturb the
metabolism of cholesterol. They are not uncommon.

Nonlipid disorders causing elevations in the concentration of plasma lipids, usually cholesterol and/or
triglyceride. These are called secondary hyperlipidemias. Disorders or conditions that adversely a ect lipid
metabolism include hypothyroidism, nephrotic syndrome, liver disease, diabetes, obesity, and alcohol
abuse. In addition, many medications can alter plasma lipid levels.

Though not common, elevated levels of lipoprotein(a) (Lp(a) and pronounced “L-P-little a”) with or without
other lipid or lipoprotein abnormalities.

Also not common, disorders that are associated with direct damage to the blood vessel wall, independent of
lipid levels, such as high circulating concentrations of homocysteine.
Diagnosis

The initial approach to the patient for routine evaluation or for monitoring the status of atherosclerotic
vascular disease is to determine if the patient has an elevation in serum or plasma total cholesterol and LDL
cholesterol concentrations, and a low HDL cholesterol concentration, and if so, to first determine if the likely
cause is excess intake of dietary fat.

The initial approach to the patient for routine evaluation or for monitoring the status of atherosclerotic
vascular disease is to determine if the patient has an elevation in serum or plasma cholesterol, and if so, to
first determine if the cause is excess intake of dietary fat.

An elevated total cholesterol level (>200 mg/dL) prompts the need to determine the basis for the elevation.
For plasma or serum concentrations of low-density lipoprotein (LDL) cholesterol, a high level is bad, and for
high-density lipoprotein (HDL) cholesterol, a high level is good. Table 8–1 describes the lipoproteins that
transport lipids in the plasma.
 TABLE 8–1
The Major Plasma Lipoproteins

Lipoprotein Lipid Synonyms Predominant Apolipoproteinsa

Class Components for
of Core Lipoprotein
Classes

Chylomicrons Triglycerides None B48 (also in chylomicron remnant), A-I, A-IV, C-I, C-II, C-III, E
>>
cholesterol
esters

Very-low- Triglycerides Pre-beta B-100, C-I (especially in hyperlipidemic patients), C-II

density > cholesterol lipoproteins (especially in hyperlipidemic patients), C-III, E (especially in
lipoproteins esters hyperlipidemic patients)
(VLDL)

Low-density Cholesterol Beta B-100

lipoproteins esters > lipoproteins
(LDL) triglycerides

High-density Cholesterol Alpha A-I, A-II, A-IV, C-I (especially in normolipidemic patients), C-
lipoproteins esters > lipoproteins II (especially in normolipidemic patients), C-III, E (especially
(HDL) triglycerides in normolipidemic patients)

aMajor apolipoprotein in bold.

Late in 2013, the American College of Cardiology and the American Heart Association, in conjunction with the
National Heart, Lung, and Blood Institute, developed new guidelines. These contained substantial changes
from the previous guidelines that were established more than a decade earlier by the Adult Treatment Panel
III(ATP III). Previous targets for LDL cholesterol of 100 mg/dL, with the optional goal of less than 70 mg/dL,
were removed as indicators of treatment success. Instead, four treatment groups for statin therapy have been
identified. They are individuals with clinical atherosclerotic vascular disease; those with LDL cholesterol
levels greater than 190 mg/dL; those with diabetes between 40 and 75 years old with LDL cholesterol levels
between 70 and 189 mg/dL without evidence of atherosclerotic vascular disease; and individuals without
evidence of cardiovascular disease or diabetes who have LDL cholesterol levels between 70 and 180 mg/dL
and a 10-year risk of atherosclerotic vascular disease greater than 7.5%. The risk for stroke has been added to
the coronary events traditionally covered by cardiovascular risk assessments. Specific recommendations on
lifestyle for cardiovascular disease prevention include eating a diet rich in fruits, vegetables, whole grains,
fish, low-fat dairy, lean poultry, nuts, legumes, nontropical vegetable oils with restriction of saturated fats,
trans fats, sweets, sugar sweetened beverages, and sodium; and engaging in aerobic physical activity of
moderate to vigorous intensity lasting 40 minutes per session three to four times per week. With regard to
weight loss, the new recommendation is that patients with a BMI of 25, not 30 as in the past, who have even
one comorbidity, such as an elevated waist circumference, should begin treatment for weight loss.

LDL Cholesterol

The most common method for determining LDL cholesterol is a calculation that requires the use of a fasting
sample with a triglyceride level less than 400 mg/dL. The LDL is calculated according to the Friedewald
formula, which is: calculated LDL cholesterol = total cholesterol − HDL cholesterol − (triglycerides/5). The
very-low-density lipoprotein (VLDL) fraction is represented by (triglycerides/5) with the assumption that
there is very little triglyceride in LDL and HDL. Because the plasma and serum triglyceride concentration
increases with ingestion of dietary fat, a fasting sample is required for an accurate calculation of LDL
cholesterol using the Friedewald formula. Although it is acceptable to drink water, the patient may not ingest
any calories 8 to 12 hours before the blood sample is collected. If the patient does not fast, and the
triglyceride is elevated above baseline, the calculated LDL cholesterol will be falsely low. Another challenge
to an accurate determination of LDL cholesterol is that there is substantial biological variability, independent
of any change in dietary habits, in the total cholesterol level. This would also have a significant impact on the
calculated LDL value. For that reason, testing for total cholesterol and LDL cholesterol should be repeated on
a second sample drawn 1 to 8 weeks later. The mean value from these two samples is used, as long as the
di erences between them are less than 30 mg/dL in total cholesterol. If the di erence is greater than 30, a
third sample should be obtained and the mean of the three samples calculated. The day-to-day variability in
total cholesterol within a single individual is typically at least 10% and can be as high as 30%. This level of
variability, in a patient whose LDL cholesterol is calculated using the Friedewald formula, could span a range
of values from 125 to 165 mg/dL if the patient has a true value of 145 mg/dL. The Friedewald calculation fails
if the triglyceride concentration in the fasting sample is higher than 400 mg/dL. At such high concentrations,
the (triglyceride concentration/5) is no longer a reasonable estimate of the VLDL cholesterol concentration.

Assays that directly measure LDL are also available. These are not dependent on the Friedewald formula, and
are therefore independent of the triglyceride concentration. For that reason, fasting is not required if a direct
LDL assay is performed. The direct LDL cholesterol assay circumvents the issues associated with the
calculated LDL cholesterol when it is greater than 400 mg/dL. However, even though these assays are
routinely used, some studies have shown substantial imprecision with the assay, although this conclusion
has been challenged by other studies. Direct LDL cholesterol measurement is not useful in patients with a
dyslipidemia.

HDL Cholesterol

Low levels of HDL cholesterol (<40 mg/dL) represent a cardiac risk factor. However, an elevated HDL
cholesterol concentration greater than or equal to 60 mg/dL reduces cardiovascular risk. The patient does
not need to fast prior to sample collection for performance of the HDL cholesterol.

Total Cholesterol

The total cholesterol concentration is the sum of HDL cholesterol, LDL cholesterol, VLDL cholesterol,
intermediate-density lipoprotein cholesterol (IDL cholesterol), and cholesterol associated with Lp(a). In the
vast majority of patients, the cholesterol in IDL and in Lp(a) is very small relative to the other lipoproteins.
The patient does not need to fast prior to sample collection for performance of the total cholesterol. Like HDL
cholesterol, total cholesterol is not a ected by recent dietary intake.

Non-high-density Lipoprotein Cholesterol

Non-HDL cholesterol is the di erence between the total cholesterol concentration and the HDL cholesterol
concentration. The remaining lipoprotein particles—LDL, VLDL, IDL, and Lp(a)—are all atherogenic. In some
clinical trials, non-HDL cholesterol was shown to be superior over LDL cholesterol as a measurement of
cardiovascular risk. Because neither the total cholesterol nor the HDL cholesterol is a ected by the
triglyceride level or ingestion of dietary fat, non-HDL cholesterol can be calculated on nonfasting specimens.
Use of non-HDL cholesterol measurements instead of calculated LDL cholesterol measurements avoids the
problem of calculating LDL cholesterol in patients who have triglyceride concentrations greater than 400
mg/dL.

High-sensitivity C-reactive Protein

The level of persistent inflammatory processes relates to the risk of cardiovascular events. Patients who have
an existing inflammatory process typically have CRP levels greater than 3.0 mg/L. Transient elevations in CRP
associated with benign processes occur frequently, and for that reason repeat testing within 2 weeks of an
elevated value is recommended to determine if the CRP level exceeds 3.0 mg/L. The American Heart
Association and the National Cholesterol Education Panel concur that CRP levels should be measured only
a er traditional lipid parameters have been assessed completely, with the CRP levels used to classify only
those patients who are considered to have borderline cardiovascular risk. Values for CRP of less than 1.0
mg/L represent low risk; 1.0 to 3.0 mg/L is intermediate cardiovascular risk; in patients with greater than 3.0
mg/L CRP is considered to be at high cardiovascular risk. The CRP assay was not originally designed for a
high-sensitivity analysis, and previously was only used to measure much higher values than the levels used
in cardiovascular risk assessment.

Metabolic Syndrome

Metabolic syndrome is present when a series of risk factors for developing heart attack stroke and diabetes
are present. In the United States, metabolic syndrome is extremely common, a ecting more than 40% of
people above the age of 60. Patients with the metabolic syndrome do not have physical symptoms. However,
patients who have the metabolic syndrome, over time, commonly develop atherosclerotic vascular disease
(myocardial infarction and stroke), kidney dysfunction, and type 2 diabetes with its associated risks and
complications. Di erent expert groups have established the risk factors and their levels that are part of the
metabolic syndrome from the American Heart Association and the National Cholesterol Educational
Program.

Waistline: For men, greater than 40 in; for women, greater than 35 in

Elevated triglycerides: Equal to or greater than 150 mg/dL

Reduced HDL cholesterol: For men, less than 40 mg/dL; for women, less than 50 mg/dL

Elevated blood pressure: Equal to or greater than 130/85 mm Hg or use of a medication for hypertension

Elevated fasting glucose: Equal to or greater than 100 mg/dL or use of a medication for hyperglycemia

Assessment of Cardiovascular Risk

Patients for whom testing for lipid status and inflammation is most useful include those with factors that
increase cardiovascular risk, such as cigarette smoking, hypertension, diabetes mellitus, obesity, physical
inactivity, or a family history of coronary heart disease, who are currently asymptomatic and have no history
of coronary heart disease. As noted below in the scoring calculations for cardiovascular risk, decisions about
patient management involve an assessment for both non-laboratory-test- and laboratory-test-associated risk
factors. There are a number of risk calculations based on a combination of data from clinical history, signs
and symptoms, and laboratory test results. The following are risk scores and their associated parameters.
Collectively, they represent a risk for developing atherosclerotic plaques and to a much lesser extent, with
inflammatory markers included, the risk of plaque rupture.

The Framingham score: total cholesterol, HDL cholesterol, age, gender, smoking status, and blood pressure.

The Reynolds score: high-sensitivity C-reactive protein (hs-CRP), total cholesterol, HDL cholesterol, age,
gender, parental history, smoking status, and blood pressure. In women with diabetes, the hemoglobin A1C
result is also included.

The PROCAM score: LDL cholesterol, HDL cholesterol, triglycerides, family history of coronary artery disease,
smoking status, age, and presence of diabetes. This score is validated only in men.

Systematic coronary risk evaluation (SCORE): total cholesterol, HDL cholesterol, age, gender, smoking status,
and blood pressure.

Genetic Disorders Classified by Causation of an Excess or Deficiency of a Specific Class of Lipoprotein

Genetic abnormalities are much less common explanations for high blood lipid levels than excess intake of
dietary fat. Described below are selected dyslipidemias and dyslipoproteinemias.

LDL-associated abnormalities:
High LDL-cholesterol

Familial hypercholesterolemia: Defects in the LDL receptor gene cause an accumulation of LDL particles in
the plasma. Patients have an elevated plasma LDL cholesterol and premature coronary artery disease. Gene
with defect: LDLR (LDL receptor).

Familial defective apolipoprotein B: The defective apolipoprotein B has a reduced a inity for the LDL
receptor. A ected individuals usually have an elevated LDL cholesterol, but there is wide variability in the LDL
cholesterol levels. Gene with defect: APOB (apolipoprotein B).

Low LDL-cholesterol

Hypobetalipoproteinemia: Hypobetalipoproteinemia results from a mutation within the apolipoprotein B

gene that results in truncation of the mature apolipoprotein B. This condition is not associated with an
increased risk of cardiovascular disease. Gene with defect: APOB (apolipoprotein B).

Abetalipoproteinemia: This condition is caused by mutation in a gene coding for a protein required for
assembly of apolipoprotein B-containing lipoproteins in the plasma. A ected patients su er from mental and
developmental retardation as children. Gene with defect: MTTP (microsomal triglyceride transfer protein).

Triglyceride-rich lipoprotein (VLDL and chylomicrons [CM]-associated) abnormalities:

High triglyceride

Lipoprotein lipase deficiency: These patients have severe hypertriglyceridemia with associated
complications such as pancreatitis, xerostomia, and xerophthalmia. The hypertriglyceridemia results from
either a markedly reduced or absent level of lipoprotein lipase activity. Gene with defect: LPL (lipoprotein
lipase).

Apolipoprotein C-II deficiency: These patients have familial chylomicronemia due to the absence of the
activator of lipoprotein lipase, which is apolipoprotein C-II. Gene with defect: APOC-II (apolipoprotein C-II).

Dysbetalipoproteinemia: This disorder is characterized by an accumulation in the plasma of remnant

lipoprotein particles, mostly from CM and VLDL. The excess plasma lipoprotein results in pathognomonic
tuberous xanthomas and palmar striated xanthomas. Gene with defect: APOE (apolipoprotein E).

HDL-associated abnormalities:

Low HDL-cholesterol

Apolipoprotein A-1 gene defects: Defects in genes for apolipoprotein A-I, C-III, and A-IV can all decrease the
production of HDL particles. Some of these defects are associated with premature cardiovascular disease.
However, other mutations appear to confer longevity despite very low HDL levels. Gene with defect: APOA1
(apolipoprotein A1).
Lecithin cholesterol acyltransferase (LCAT) deficiency: A deficiency of LCAT results in reduced conversion of
cholesterol to cholesterol esters in the plasma. This results in low HDL cholesterol levels, corneal opacities,
and hemolytic anemia. Gene with defect: LCAT (lecithin-cholesterol acyltransferase).

Tangier disease: Individuals with this disorder have reduced cellular cholesterol e lux. The mutation is in the
gene that codes for the protein known as ATP-binding cassette A1 (ABCA1), which is a transporter protein.
Many di erent mutations within this gene have been associated with Tangier disease, which is associated
with an increased risk for coronary artery disease and extremely low HDL levels. Gene with defect: ABCA1
(ATP binding cassette subfamily A member 1).

High HDL-cholesterol

Cholesterol ester transfer protein (CETP) deficiency: Patients with this deficiency have extremely elevated
HDL cholesterol levels, which are primarily cholesterol esters. A deficiency of this enzyme causes
accumulation of cholesterol esters within the HDL particles, and, therefore, this deficiency is not associated
with premature coronary artery disease. Gene with defect: CETP (cholesterol ester transfer protein).

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