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

VIII. Methodology
63
peroxidase and a dye. GO catalyzes the conversion of glucose
to gluconic acid:
GO
glucose gluconic acid H O
→ 2 2
The hydrogen peroxide, with peroxidase, oxidizes a dye to
form a colored product. This principle is also used in the
glucose-specific paper strips for urine glucose.
In the HK method, HK catalyzes the phosphorylation
of glucose and the reaction is coupled to a reaction such as
G-6-PD for assay:
HK
glucose ATP → G-6-P ADP
G-6-PD
G-6-P NADP → 6 -PG NADPH
Either NADP or NADPH is measured spectrophotometrically.
In the GD method, GD catalyzes
GD
glucose NAD → gluconolactone NADH
and NAD or NADH is measured spectrophotometrically.
Of these enzymatic methods, the method of Banauch
et al . (1975) was found to be best for the quantitative assay
of urine glucose ( Kaneko et al ., 1978a ).
No matter how accurate the method for blood glucose,
it cannot compensate for loss of glucose in an improperly
handled blood sample. Glucose breakdown (i.e., glycolysis)
by red blood cells takes place very rapidly, about 10% per
hour loss, at room temperature and is even more rapid if
microorganisms contaminate the sample. For these reasons,
the plasma or serum must be separated from the RBCs as
quickly as possible, within the half hour; otherwise, the
glucose in the blood sample must be protected from glycolysis.
This is best accomplished through refrigeration
or by the use of sodium fluoride (NaF) (10 mg/ml blood).
The NaF acts both as an anticoagulant and a glucose
preservative. The NaF can also be added to a blood sample
vial containing an anticoagulant.
2 . Blood Glucose in Animals
The reference ranges for blood glucose are given in Table 3-7
and in Appendices VII, VIII, and IX . A standard sampling
procedure must be used to obtain optimum results and to
minimize variations in blood glucose, especially those
resulting from diet. This is best accomplished in the nonruminant
and in the young ruminant by a standard overnight
(12 to 16 h) fast before sampling. This is not necessary in
the mature ruminant, because feeding elicits no blood glucose
response. Methods for establishing statistically valid
TABLE 3-7 Blood Glucose Levels in Domestic
Animals a
Glucose (Reference Range and Mean SD)
Species mmol/liter mg/d
Horse 4.2–6.4 75–115
(5.3 0.4) (95 8)
Cow 2.5–4.2 45–75
(3.2 0.4) (57 7)
Sheep 2.8–4.4 50–80
(3.8 0.3) (68 6)
Goat 2.8–4.2 50–75
(3.5 0.4) (63 7)
Pig 4.7–8.3 85–150
(6.6 0.9) (119 17)
Dog 3.6–6.5 65–118
(5.0 0.4) (90 8)
Cat 2.8–4.2 50–75
(3.5 0.4) (63 7)
Monkey 4.7–7.3 85–130
( Macaca sp.) (5.9 0.7) (107 13)
Llama 5.7–8.9 103–160
(7.1 0.9) (128 16)
Rabbit 2.8–5.2 50–93
(4.1 0.5) (73 10)
a
Plasma or serum, glucose oxidase method, adult animals.
reference ranges for analytes such as blood glucose, with
examples, are given in Chapter 1 .
B . Indirect Monitoring of Blood Glucose
The phenomenon of glucose molecules irreversibly binding
to proteins is widespread in biological systems, and
the products are known as glycated proteins. The glucose
molecules are covalently bound to free amino groups of
a protein (i.e., lysine) valine by a nonenzymatic glycation
mechanism. The glycated intermediate in the reaction is
unstable and immediately undergoes a classic Amadori
rearrangement to form a stable ketoamine . The carbon
backbone of this ketoamine is identical to fructose. When
the protein of the protein-ketoamine complex is hemoglobin
(Hb), the product is called hemoglobin A1c (HbA1c)
because it was first identified as a fast-moving minor Hb
component by electrophoresis. When the protein of the
complex is albumin or total serum protein, the product is
called fructosamine (FrAm) ( Armbruster, 1987 ). When the
albumin-ketoamine is specifically measured, the product is
sometimes called glycoalbumin (Galb).