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

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Chapter | 4 Lipids and Ketones
carbon chain lengths of 16 and 18 constitute the greatest
bulk of fatty acids in animal tissues and most animal diets.
The saturated 16-carbon LCFA is palmitic acid, and the
saturated 18-carbon LCFA is stearic acid. Unsaturated 18-
carbon LCFA are common, with double bonds occurring
at C 9 —C 10 (oleic acid); at C 9 —C 10 and C 12 —C 13 (linoleic
acid); and at C 9 —C 10 , C 12 —C 13 , and C 15 —C 16 (linolenic
acid). The double bonds found in fatty acids in nature
are mostly of the cis configuration. Ruminant fat contains
more trans-LCFA than that of nonruminants because rumen
microbes isomerize some plant cis-LCFA to trans isomers.
Unsaturated LCFA have a lower melting point than saturated
LCFA with the same number of carbons and are more
susceptible to spontaneous oxidation ( Gurr et al ., 2002 ).
The 20-carbon polyunsaturated fatty acids, arachidonic
acid (double bonds at C 5 —C 6 , C 8 —C 9 , C 11 —C 12 , C 14 —
C 15 ) and eicosapentaenoic acid (also called timnodonic
acid), which is arachidonic acid with an additional double
bond at C 17 —C 18 , are the precursors of the eicosanoids
(prostaglandins, leukotrienes, thromboxanes).
Long chain fatty acids are relatively insoluble in water
at physiological pH. They dissolve readily in highly alkaline
solutions, forming soaps. LCFA are amphiphilic , being
quite polar (hydrophilic) at their carboxyl end and quite
nonpolar (hydrophobic) at the methyl end. All LCFA must
bind to proteins in order to be transported for any significant
distance, and albumin is the primary transport protein
in plasma ( Gurr et al ., 2002 ).
Plasma LCFA concentrations can be determined spectrophotometrically
with a specific enzymatic reaction,
which involves direct reaction of plasma LCFA to form
LCFA-CoA. Then, LCFA-CoA is oxidized using LCFA-
CoA oxidase, which produces hydrogen peroxide. The
hydrogen peroxide is used to produce a colored product
under the catalysis of peroxidase ( Demacker et al ., 1982 ;
Shimizu et al ., 1980 ). If a sample contains triacylglycerol
and lipase, which is not uncommon, LCFA may be released
if the sample is allowed to stand. Falsely high LCFA may
be avoided by centrifuging blood samples and freezing the
plasma immediately after collection or by adding paraoxon,
a lipase inhibitor (Degen and Van der Vies, 1985).
B. Synthesis of Long Chain Fatty Acids
LCFA may be synthesized in most tissues, but only liver,
adipose, or mammary tissue does it on a large scale.
Synthesis occurs in the cytosol from acetyl-CoA. The precursor
of the acetyl-CoA used for LCFA synthesis is usually
acetate or glucose, with the former being important
in ruminants and the latter being important in nonruminant
mammals. When acetate is the acetyl-CoA precursor,
it is formed from plasma acetate in the cytosol, the same
cellular location as the enzymatic machinery needed to
manufacture the LCFA. However, when glucose is the precursor,
it must go through glycolysis, which has its terminal
enzyme, pyruvate dehydrogenase, located in the mitochondria.
Thus, the acetyl-CoA is produced in the mitochondria,
which is a problem if it is to be used for LCFA synthesis
because the inner mitochondrial membrane is relatively
impermeable to acetyl-CoA ( Rangan and Smith, 2002 ).
This problem has been solved by a mechanism known as
the citrate shuttle, which is shown in Figure 4-1 . Acetyl-CoA
in the mitochondria combines with oxaloacetate under the
catalysis of citrate synthase to form citrate. Citrate is translocated
across the mitochondrial membrane where it is cleaved
into acetyl-CoA and oxaloacetate by ATP-citrate lyase. Thus,
acetyl-CoA has been effectively transported from mitochondrion
to cytosol. What remains is for the oxaloacetate to
reenter the mitochondria to complete the cycle. However,
the inner mitochondrial membrane is also impermeable to
oxaloacetate, so it is first converted to malate-by-malate
dehydrogenase or aspartate-by-aspartate aminotransferase in
the cytosol. The malate or aspartate is translocated into the
mitochondrion where it can be converted back to oxaloacetate
by reversal of the reactions that occurred in the cytosol.
Alternately, malate in the cytosol can be converted to
pyruvate by malic enzyme, and the pyruvate can enter the
mitochondrion and be converted to oxaloacetate by pyruvate
carboxylase ( Rangan and Smith, 2002 ).
Once acetyl-CoA reaches or has been formed in the
cytosol, it must be carboxylated to produce malonyl-CoA
via acetyl-CoA carboxylase if it is to be used for LCFA
synthesis. This biotin-containing enzyme catalyzes the following
reaction:
Acetyl-CoA Carboxylase
CH3CO-CoACO2ATP
⎯⎯⎯→
OOCCH CO-CoAHADP
P
2 i
Acetyl-CoA carboxylase is the main regulatory site in the
synthesis of LCFA, which makes sense because the cell has
little use for malonyl-CoA other than the synthesis of LCFA.
The enzyme is activated by citrate, which is logical because
citrate will be abundant only when there is a plentiful supply
of mitochondrial acetyl-CoA. In addition, acetyl-CoA carboxylase
is directly inhibited by LCFA-CoA, which can be
derived from the synthetic process itself or from uptake and
activation of plasma LCFA. Acetyl-CoA carboxylase is also
regulated by hormones via phosphorylation of the enzyme
itself. Glucagon and LCFA-CoA stimulate phosphorylation,
which inhibits the enzyme. Insulin activates the enzyme
quickly by stimulating dephosphorylation (Rangan and
Smith, 2002) . These controls make sense in that a fasting or
exercising animal will have its capacity for LCFA synthesis
suppressed by increased plasma glucagon and LCFA levels,
decreased plasma insulin, and increased intracellular LCFA-
CoA. Conversely, in a recently fed animal, these controls
will all be reversed to promote LCFA synthesis.
Malonyl-CoA is used as the building block for LCFA
in the cytosol by a large, complex, multiunit enzyme called