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

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Chapter | 4 Lipids and Ketones
β-hydroxybutyric acid), and acetone, are important compounds
in the metabolism of birds and mammals. Ketosis
simply means that ketones are present in body fluids in
elevated concentrations. Ketones are important clinically
and have a rather sinister reputation because of the ketoacidosis
that is often present when their plasma levels are
high. In recent years though, the survival value of ketogenesis
has become clearer, and although increased levels of
ketones in biological fluids will continue to be regarded as
a pathological sign in many situations, perhaps the beneficial
aspects of ketogenesis will be more widely appreciated.
B. Chemistry of Ketones
1 . Structure and Properties
The ketones, acetone, 3-hydroxybutyric acid, and acetoacetic
acid, are relatively simple chemical structures. Of the three,
only 3-hydroxybutyric acid can exist as stereoisomers, having
L-( ) and D-( ) forms. Only the D-( ) form is produced
in a free state in intermediary metabolism. The L-( )
form exists only as its CoA thioester produced and destroyed
in β-oxidation ( Newsholme and Leach, 1983 ). Acetone is
relatively volatile, whereas the other two ketones are not.
Acetone has a characteristic organic solvent odor that may
be detectable in the exhaled breath of animals with elevated
blood ketone levels. Anecdotal evidence indicates that people
vary greatly in their olfactory sensitivity for acetone.
Acetone does not ionize appreciably, whereas 3-
hydroxybutyric acid and acetoacetic acid do readily ionize.
Acetoacetic acid has a pKa of 3.58, and 3-hydroxybutyric
acid has a pKa of 4.41 ( Dean, 1985 ). Consequently, at normal
plasma pH of 7.40, 99.9% of either compound exists in
its ionized form. Therefore, the compounds will usually be
referenced by the names of their ions whenever their metabolism
is discussed. Acetoacetic and 3-hydroxybutyric acids
are more powerful acids than the volatile fatty acids (VFA;
acetic, propionic, and butyric acids), which have pKa’s of
4.76 to 4.87 ( Dean, 1985 ). Acetoacetic acid is more powerful,
and 3-hydroxybutyric acid is less powerful as an acid
than lactic acid, which has a pKa of 3.86 ( Dean, 1985 ).
Acetone and acetoacetic acid are miscible in water in all
proportions, and 3-hydroxybutyric acid is exceedingly soluble,
but not in all proportions ( Dean, 1985 ). The common
metallic salts of acetoacetic acid and 3-hydroxybutyric acid
are soluble in water. Acetone and 3-hydroxybutyric acid
and its salts are relatively stable compounds. Acetoacetic
acid spontaneously decomposes to acetone and carbon
dioxide. This reaction occurs readily without catalysis,
and its rate is accelerated by increased temperature and
hydrogen ion concentration. Apparently, there can be some
nonspecific catalysis of acetoacetate decarboxylation by
cellular proteins ( Williamson, 1978 ). The lithium, sodium,
and potassium salts of acetoacetic acid are relatively stable
if stored in dry form below 0°C.
2 . Detection and Assay
a. Qualitative
The most common qualitative test for ketones is the alkaline
nitroprusside test, which is also known as the Rothera
test ( Rothera, 1908 ). This test has been used for decades in
clinical practice and is still exceedingly useful today. The
test relies on the reaction of nitroprusside with acetone or
acetoacetate to produce a purple chromogen. The nitroprusside
test has been used for virtually every body fluid imaginable
including whole blood, serum, plasma, urine, and
milk. The test is most sensitive for acetoacetate (0.5 mmol/l
can be detected), gives only a slight response to acetone,
and is completely insensitive to 3-hydroxybutyrate.
The nitroprusside test is available commercially in the
form of strips, tablets, and powders. The maximum sensitivity
of all three forms is approximately 0.5 mmol/l,
although specific formulations may have a sensitivity less
than this value. The strip form is commonly used for urine.
The powder form and strips are both commonly used for
milk. The tablet form is used for serum, plasma, and whole
blood and can be used for milk and urine as well. The test
is often used in a semiquantitative manner with the result
expressed in adjectival form (negative, weak, strong) or as
a series of pluses ( , , , etc.).
A number of drugs or other substances may appear in
urine and give a false positive with the nitroprusside test.
Some compounds react with nitroprusside to yield a purple
or near purple color. Included in this group are phenylketones,
levodopa, methyldopa, acetaldehyde, paraldehyde
( Caraway and Kammeyer, 1972 ), cysteine, cysteamine, penicillamine,
and mesna ( Csako, 1987 ). In general, substances
with keto, aldehyde, or sulfhydryl groups have the potential
for reacting with nitroprusside. Because the nitroprusside
test is performed in an alkaline medium, some substances,
like sulfobromophthalein and phenolsulfonphthalein, which
may exist in urine and are otherwise colorless, may yield a
purple or near purple color simply because of the alkaline
pH ( Caraway and Kammeyer, 1972 ).
More recently a semiquantitative color test for 3-
hydroxybutyrate in milk has been used for diagnosis of
clinical and subclinical ketosis in dairy cows ( Gutzwiller,
1998 ; Jorritsma et al ., 1998 ). This test, which is quite
specific, is based on the same enzymatic method used for
quantitative determination of 3-hydroxybutyrate (see the
next section) except that a color visible to the human eye
is produced.
b. Quantitative
Commonly used means of quantitative assay for ketone
concentrations in biological fluids include microdiffusion
methods, used primarily for assay of acetone, and enzymatic
methods, used primarily for assay of acetoacetate
and 3-hydroxybutyrate. Regardless of the method to be
used for analysis, proper handling of the samples before