solution containing lactate and NAD+. NADH generated
at the LDH zones is detected either by its fluorescence
when excited by long-wave ultraviolet light (365 nm)
or by its reduction of a tétrazolium salt (nitroblue tétra-
zolium, NBT) to form an insoluble colored complex via
an intermediate redox carrier (e.g., phenazine methosul-
fate, PMS):
234
PMS
NBT
(colored
and
insoluble)
As long as the total activity of LDH is not very high
so that the substrates are not limiting, the intensity of
the color is approximately proportional to the various
isoenzyme activities. Thus, densitometric scanning of an
electropherogram provides an estimate of the activities of
the individual enzymes as well as quantitation of their rela-
tive intensities. LDH-1 is the most negatively charged and
fastest moving isoenzyme (mobility comparable to that
of the a i-globulin region in serum protein electrophore-
sis). LDH-5 is the least negatively charged and slow-
est migrating isoenzyme (mobility comparable to that of
y-globulin); the other isoenzymes possess intermediate
mobilities. The normal relative intensity of LDH isoen-
zyme fractions in serum is
LDH-1 < LDH-2 > LDH-3 > LDH-4 < = > LDH-5
Since myocardium is rich in LDH-1, injury to that tissue
results in the elevation of LDH-1
and the ratio of LDH-1 to
LDH-2; similarly, since liver is rich in LDH-4 and LDH-5,
elevations of these isoenzymes suggests liver disease.
Because a particular isoenzyme pattern may result from
injury to more than one tissue (Table 13-3), further di-
agnostic tests are required. For example, measurement of
creatine kinase and its isoenzyme determination and tro-
ponin I in the serum is helpful in the evaluation of acute
injury to the myocardium. Because of differences in half-
lives in serum, serial determination of total enzyme activity
and relative isoenzyme composition aids in the diagnosis
and assessment of the magnitude of the injury of the tissue
in question (Chapter
8
). In patients with acute myocardial
infarction, serial analysis every
8 - 1 2
hours during the first
48 hours after the onset of symptoms is useful.
In vitro,
LDH-1(H4) has a low
Km
for pyruvate and is strongly
inhibited by high concentration of pyruvate. This form
may favor the oxidation of lactate to pyruvate in aerobic
tissues such as the heart. LDH-5(M4) exhibits a higher
Km
value for pyruvate and is less susceptible to inhibi-
tion by high concentrations of pyruvate than is LDH-1.
chapter
13
Carbohydrate Metabolism I: Glycolysis and TCA Cycle
TABLE 13-3
Serum LDH Isoenzyme Patterns in Various Disorders*
Isoenzyme Pattern
Disorder
Elevation of LDH-1 and
Myocardial infarction
LDH-2, frequently
Renal cortical infarction
LDH-1 > LDH-2
Pernicious anemia
Elevation of LDH-5
Hemolysis
Muscular dystrophy
(later stages)
Liver disease
Elevation of LDH-3,
Skeletal muscle damage
Some cancers
Some neoplastic diseases
frequently
Lymphoproliférative
LDH-3 > LDH-2
disorders
Elevation of LDH-2
Platelet-related disorders
Pulmonary infarction
and LDH-3
All isoenzymes elevated
Widespread tissue injury
*Normal distribution: LDH-1 < LDH-2 > LDH-3 > LDH-4 < = > LDH-5
This form may occur in anaerobic tissues, in which lac-
tate is the end product of glycolysis. However,
in vitro
differences in kinetic properties of the isoenzyme may be
inappropriate to explain actual physiological actions for
several reasons: differences in kinetic properties between
LDH-1 and LDH-5 are less marked at 37°C (body tem-
perature) than at 25°C, high intracellular concentrations
of the enzyme are present, and differences in the actual
ratio of ketopyruvate to enolpyruvate exist (the enol form
may be the more potent inhibitor). Furthermore, the oc-
currence of similar isoenzyme patterns in widely differ-
ent tissues with divergent metabolic goals (e.g., LDH-5
in liver and muscle; LDH-1 in heart and erythrocytes)
points out our lack of understanding of their precise
role.
Alternative Substrates of Glycolysis
The glycolytic pathway is also utilized by fructose, galac-
tose, mannose, glycogen, and glycerol. The metabolism
of the monosaccharides and glycogen is discussed in
Chapter 15. Glycogenolysis catalyzed by phosphorylase
in a nonequilibrium reaction yields glucose-
1
-phosphate,
which
is
then
converted
to
glucose-
6
-phosphate
(Chapter
15),
bypassing
the
initial
phosphorylation
reaction of glycolysis. Therefore, the conversion of a
glucosyl unit of glycogen to two lactate molecules yields
three ATP, which is 50% more than the yield from a
glucose molecule. Glycerol released by hydrolysis of
