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

VI. Lipoproteins
91
TABLE 4-1 Composition of Lipoproteins of Domestic Animals a
Triacylglycerol
(% by weight)
Cholesterol
(% by weight)
Free Cholesterol
Esters (% by
weight)
Phospholipid
(% by weight)
Cattle
Chylomicrons 87 4 2 4 3
VLDL 60 5 4 25 6
LDL 1 5 35 36 23
HDL 4 4 30 20 42
Dogs
VLDL 68 6 2 10 14
LDL 27 5 25 22 21
HDL 1 5 23 33 38
Horses
VLDL 57 5 6 18 14
LDL 6 8 36 23 27
HDL 0 2 20 28 50
Protein
(% by weight)
a
Cattle: From Ferreri and Elbein (1982) (chylomicrons) and Stead and Welch (1975) (other lipoproteins). Dogs: From Blomhoff et al . (1978) and Mahley and
Weisgraber (1974). Horses: From Le Goff et al . (1989) and Watson et al . (1993).
highest ratio and, on the other end of the spectrum, the
HDL having the lowest ratio ( Table 4-1 ). More than onehalf
of the lipid in chylomicrons and VLDL is triacylglycerol,
whereas in LDL and HDL the majority of the lipids
are not triacylglycerol ( Table 4-2 ). In domestic species,
HDL is normally the most abundant plasma lipoprotein in
the fasting state.
Chylomicrons and VLDL particles are large enough
to refract light significantly, so they make plasma appear
turbid or creamy if in high enough concentration (lipemic
plasma). The chylomicrons have a low enough density
that they will rise to the top of an undisturbed refrigerated
plasma sample in 6 to 12 hours. This phenomenon is the
basis of the “ chylomicron test, ” in which a milky plasma
sample is placed in the refrigerator overnight. If a “ cream
layer ” has formed at the top, then hyperchylomicronemia
is present, and if the bottom portion of the plasma is turbid,
then elevated levels of VLDL are present.
Because of the expense, time, and complexity involved
with ultracentrifugation, electrophoresis in an alkaline
medium has been used as an alternate method of lipoprotein
classification. A variety of electrophoretic supports,
ranging from paper to acrylamide gels, have been used.
The sample is applied at the cathode end of the support,
voltage is applied for a variable time, and the proteins are
fixed and stained with a lipid stain such as oil red O. A
densitometer is used to quantify the lipoprotein fractions
on the stained electrophoretogram.
Typically, three to five bands of lipoproteins can be discerned;
however, additional bands may be present depending
on the species of animal, electrophoretic technique, and
presence of abnormal lipoproteins. The fastest moving
band is HDL, which is designated as α -lipoprotein. The
next fastest moving band is VLDL, which is designated
pre-β-lipoprotein followed by the LDL band, which is
designated as β-lipoprotein. The slowest moving band,
which is still at the origin and seen primarily in the postprandial
period, is composed of chylomicrons. With some
electrophoresis systems, a separate IDL band, designated
as slow pre-β-lipoproteins, can be discerned between the
VLDL and LDL bands, and sometimes subbands of the
HDL can be discerned. The correlation of electrophoretic
and ultracentrifuge fractions established for humans does
not always apply to animals. For example, bovine LDL
can appear as α - or β-bands on electrophoresis ( Puppione,
1983 ). Usually, two HDL bands can be discerned for dog
plasma ( Rogers, 1977 ). Figure 4-5 illustrates the distribution
of lipoproteins in dog plasma.
Although easier and cheaper to perform than ultracentrifugation,
lipoprotein electrophoresis still requires considerable
time and expense. Consequently, methods have
been developed that involve precipitation of one or more
lipoprotein classes followed by analysis of a particular
lipid, usually cholesterol, in the remaining supernatant. For
example, chylomicrons can be removed by low-speed centrifugation
(they rise to the top), and then precipitation of
VLDL and LDL in human plasma can be accomplished by
treatment with magnesium and dextran sulfate. The main
lipoprotein remaining in the supernatant will be HDL,
and if cholesterol is determined, it will mostly be HDL