Clinical Biochemistry of Domestic Animals (Sixth Edition) - UMK ...

V. Cholesterol
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V. CHOLESTEROL
A. Structure, Properties, and Assay of
Cholesterol
Structurally, cholesterol is composed of a core of phenanthrene
to which a cyclopentane ring is attached, and there
is an eight-carbon side chain attached to the cyclopentane
ring ( Fig. 4-4 ). Cholesterol is found only in animals and
is not present in plants or microorganisms. Cholesterol
is the precursor of steroid hormones, vitamin D, and the
bile acids, and is a constituent of cell membranes and
bile micelles. Cholesterol can be obtained from the diet
if it contains animal products, or it can be synthesized.
The chief synthetic and catabolic organ for cholesterol
is the liver. Steroidogenic endocrine organs (adrenal cortex,
testis, ovary, placenta) can synthesize small amounts
of cholesterol; however, these organs utilize hepatically
synthesized cholesterol for most of their steroid synthesis
( Pedersen, 1988 ). Pure cholesterol and cholesterol esters
are insoluble waxy white solids and must be transported
through plasma as part of lipoproteins.
Enzymatic methods are used almost universally for
assay of cholesterol ( Stein and Meyers, 1994 ). Older nonenzymatic
methods used harsh reagents and lack specificity.
The key enzymes in the assay are cholesterol esterase,
which hydrolyses cholesterol esters, and cholesterol oxidase.
The latter enzyme is of microbial origin and has an
action analogous to that of glucose oxidase (i.e., it uses
dissolved oxygen to oxidize cholesterol to produce cholest-
4-ene-3-one and hydrogen peroxide). In the presence of
added peroxidase, hydrogen peroxide will oxidize an added
organic dye (e.g., dianisidine, ABTS, 4-aminoantipyrine
plus phenol) to generate a colored product that can be
quantified spectrophotometrically. If cholesterol esterase is
included in the reagent, then total cholesterol will be determined.
If cholesterol esterase is omitted from the reagent,
then only nonesterified (i.e., free) cholesterol will be determined.
If the assay is done with and without cholesterol
esterase, then cholesterol ester concentration can be determined
by subtraction.
Because virtually all of the cholesterol and cholesterol
esters in plasma are part of lipoproteins, they must be liberated
before they can be acted on by the enzymes of the
reagent. This liberation can be accomplished by extracting
cholesterol and its esters with an organic solvent before the
assay or, more conveniently, by including small amounts of
detergents (bile acids or artificial detergents) in the reagent
( Stein and Meyers, 1994 ).
B. Metabolism of Cholesterol
As is the case for LCFA and ketones, the substrate for cholesterol
synthesis is acetyl-CoA. The beginning site of cholesterol
synthesis is in the cytosol, so acetyl-CoA, which is
generated primarily in the mitochondria, must be transferred
to the cytosol via the citrate shuttle mechanism discussed earlier.
The process of cholesterol synthesis is shown diagrammatically
in Figure 4-4 . In the cytosol, the first two steps
of cholesterol synthesis are identical to the first two steps of
ketone synthesis except that the process occurs in the cytosol
rather than in the mitochondria. The enzymes that catalyze
the first two steps are acetyl-CoA: acetoacetyl-CoA thiolase
and hydroxymethylglutaryl-CoA (HMG-CoA) synthase:
2 acetyl-CoA ←⎯⎯→acetoacetyl-CoA CoA
FIGURE 4-4 Synthesis of cholesterol. The first two reactions occur in
the cytosol and the remainder in the smooth endoplasmic reticulum.
acetoacetyl-CoA acetyl-CoA
⎯⎯⎯→
HMG-CoA CoA