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

VIII. Ketogenesis and Ketosis
99
analysis is crucial for obtaining representative results. In
particular, the volatility of acetone and instability of acetoacetate
must be respected.
Blood samples should be cooled immediately after
collection. Ketones can be determined on whole blood
or plasma. Serum is not recommended because of losses,
particularly of acetoacetate, that may occur during the
time required for clotting. Any of the common anticoagulants
(heparin, fluoride, oxalate, citrate, or EDTA) may be
used. If whole blood is to be used, it should be mixed with
perchloric acid immediately after collection to precipitate
proteins. The tube should be chilled on ice until centrifuged,
which should be performed within a few hours. The
supernatant should be frozen until analyzed. If plasma is
to be used, the red cells should be spun down within a few
hours, and the plasma proteins precipitated with perchloric
acid. The supernatant should be frozen until analyzed.
The microdiffusion method can be used to determine
the concentration of acetone or acetone plus acetoacetate
in any biological fluid. The reagents are relatively simple
and inexpensive although rather corrosive. The diffusion
step requires specialized, but inexpensive, apparatus
and adds to the complexity and time to complete the
assay. The method relies on the reaction of acetone with
vanillin ( Henry et al ., 1974 ) or salicylaldehyde (Nadeau,
1952 ) to produce a colored product that can be quantified
in a spectrophotometer. In the author’s experience, vanillin
provides more sensitivity than salicylaldehyde, but
variability in the purity of vanillin batches from commercial
sources makes salicylaldehyde the reagent of choice.
Salicaldehyde must be stored under nitrogen or argon to
preserve its purity.
The method as described by Henry et al . (1974) was
shown to determine acetone, and there was speculation that
it would also detect acetoacetate simultaneously. In fact,
the method as described by Henry et al . (1974) is specific
for acetone. It has been found in the author’s laboratory
that to use the method for acetone plus acetoacetate, it is
necessary to preincubate the sample with an equal volume
of 10 N sulfuric acid for 4 hours at 50°C in a sealed container
to decarboxylate all of the acetoacetate. The method
can be adapted to measure 3-hydroxybutyrate as well by
introducing a step in which 3-hydroxybutyrate is oxidized
to acetoacetate with potassium dichromate ( Procos, 1961 ).
However, if the primary interest is the determination of
acetoacetate or 3-hydroxybutyrate, rather than acetone,
the enzymatic method described later should be used. For
determination of acetone on large numbers of samples,
flow injection analysis ( Marstorp et al ., 1983 ) or infrared
spectroscopy ( Hansen, 1999 ) has been used.
The enzymatic method for assay of acetoacetate or
3-hydroxybutyrate in biological fluids is accurate and precise
( Williamson et al ., 1962 ) and is probably the most
common method used for quantitative assay of ketone concentrations.
The method has been successfully adapted to a
variety of automated analysis systems ( Harano et al ., 1985 ;
Ozand et al ., 1975 ; Työppönen and Kauppinen, 1980 )
and is a relatively straightforward spectrophotometric or
fluorometric method. For a detailed step-by-step practical
description of the method, see Mellanby and Williamson
(1974) and Williamson and Mellanby (1974) .
The method relies on the reversible reaction catalyzed
by 3-hydroxybutyrate dehydrogenase:
3-hydroxybutyrate NAD
←⎯⎯→acetoacetate
NADH
H
The reaction is run in the forward direction by including
an excess of NAD 1 in the reaction mixture to assay
3-hydroxybutyrate and in the backward direction by
including an excess of NADH in the reaction mixture to
assay acetoacetate. The equilibrium constant of the reaction
is 1.42 10 9 and therefore, is highly favorable
toward the reduction of acetoacetate at pH 7.0 ( Krebs
et al ., 1962 ). To force the reaction to completion in the
direction of oxidizing 3-hydroxybutyrate, hydrazine is used
as a trapping agent to remove acetoacetate as it is formed,
and the reaction mixture is buffered at an alkaline pH.
The change in NADH concentration is measured by the
change in absorbance at 340 nm in either case. Alternately,
a fluorometer can be used to measure the change in NADH
concentration. To avoid interference from lactate or pyruvate
in the sample, the 3-hydroxybutyrate dehydrogenase
should be free of lactate dehydrogenase, or alternately, the
lactate dehydrogenase inhibitor, oxamic acid, can be added
to the reaction mixture ( Harano et al ., 1985 ).
Table 4-3 lists normal blood and plasma ketone concentrations
for several domestic species. The values are for
healthy fed animals. It is assumed that plasma and blood
ketone concentrations should be similar because of the
generally high permeability of cell membranes to ketones
and lack of protein binding of ketones; however, reports of
definitive studies on this problem are not apparent in the
literature. For clinical purposes, there is no lower normal
limit for ketone concentrations.
C. Synthesis of Ketones
Ketones are primarily products of intermediary metabolism.
Only under unusual circumstances would more than
trace amounts be absorbed from the contents of the gastrointestinal
tract. The real source of ketones is fatty acids
including those with short (1 to 4 carbons), medium (5 to
11 carbons), and long ( 11 carbons) chains. Of course, any
compound (glucose, lactate, glycerol, amino acids, etc.)
that can be converted to fatty acids can be considered as a
source of ketones, but for the purposes of this discussion,
the origin of ketones will be considered to be fatty acids,
either esterified or nonesterified.