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We examined the effect of pyridoxal phosphate sup- cal assay for determining the AST activity of serum.
plementation on the apparent aspartate aminotrans- Reports differ on its effect on serum AST activity.
ferase (EC 2.6.1.1.) activity of human serum. Sup- Karmen et al. (1) were unable to show any measur-
plementation by 25 mol/liter effected an average able effect of pyridoxal phosphate, or of boiled rat
increase of 16% in the results for kinetic assay. The liver extract, on serum aminotransferase activity.
increase was not the result of increased enzymatic Similar conclusions have been reported by others
or nonenzymatic blanks, and, within a small range,
(9-11). Hamfelt (12), however, was able to demon-
sample dilution had no significant effect. Part of the
strate increased serum AST activity after supple-
increase was attributableto the enzyme being pro-
tected against the loss of activity that occurs during mentation with pyridoxal phosphate. The increases
preincubation with L-aspartate. A similar increase he reported varied with health and age of subjects
was not demonstrated in a two-point colorimetric but were not limited to cases of Vitamin B6 deficien-
method, perhaps because of the short reaction time, cy, as has been previously suggested (10).
without preincubation, and the initialpresence of Currently, standardization of optimized reference
both substrates in the assay. We attempted to Corre- enzyme methods is being attempted, so that results
late such stimulation of aminotransferase activity will be repeatable and comparable (13-15). The
and the patient’s diagnosis or treatment. Pyridoxal Deutsche Gesellschaft f#{252}r
Klinische Chemie (14) has
phosphate should be included in the reaction mixture recently recommended measurement of enzyme ac-
when aspartate aminotransferase activity is being tivities under optimal or optimized concentrations of
measured clinically. substrate, cofactors, activators, and buffers. In the
same publication this society proposed optimized
Additional Keyphrases: differences in results by kinetic
and colorimetric methods - serum enzyme activity
methods for determination of the activity of several
enzymes found in serum. The method cited for mea-
The diagnostic use of measurements of serum suring AST activity does not include pyridoxal phos-
AST2 activity is well known. In 1955 Karmen et al. phate supplementation.
(1) demonstrated increased aminotransferase activity Reference methods of lasting importance and use-
in sera from patients with various clinical diagnoses; fulness can be achieved only by thorough investiga-
and AST activity of serum is now known to be in- tion of each factor influencing the activity of the en-
creased in a variety of physiological conditions (2, 3). zyme examined. Toward this end the present study
The requirement of pyridoxal phosphate for amino- was initiated.
transferase activity is also well documented. 0 ‘Kane
Materials and Methods
and Gunsalus (4) in 1947 and Meister et al. (5) in
1954 were able to activate AST in porcine heart Kinetic AST assays were performed by the method
preparations by incubating them with pyridoxal or of Henry et al. (16), with slight modifications. Sam-
pyridoxamine phosphate. Pyridoxal phosphate is ple volume was either 0.2 or 0.5 ml, as specified.
now firmly established as the cofactor for AST and Total assay volume was 3.0 ml in all cases. The final
other aminotransferases (6-8). control substrate mixture contained the following,
Despite this knowledge, pyridoxal phosphate has per liter: 90 mmol of phosphate buffer, pH 7.5; 125
not been incorporated into any commonly used clini- mmol of L-aspartate; 6.7 mmol of 2-oxoglutarate;
0.18 mmol of NADH; 867 U of malate dehydrogenase
From the New York State Health Department, Division of Lab- (2.6 U per test); 6.9 mmol of sodium azide; 225
oratories & Research, Albany, N.Y. 12201.
mmol of glycerol; and 0.2 mmol of (NH4)2S04. Pyri-
1 Present address: City of Kingston Laboratory, Kingston, N.Y.
12401. doxal-phosphate-supplemented substrate was the
2 Nonstandard abbreviations used, and enzyme identification: same except for the additional presence of 25 zmol of
AST, aspartate aminotransferase, EC 2.6.1.1, L-aspartate : 2-oxo-
pyridoxal phosphate per liter. Pyridoxal phosphate
glutarate aminotransferase [formerly known as glutamic-oxalo-
acetic transaminase (GOT)J; malate dehydrogenase, EC 1.1.1.37, and NADH solutions were prepared daily.
L-malate:NAD oxidoreductase;GD, glutamate dehydrogenase, All reagents except malate dehydrogenase, which
EC 1.4.1.3,t-glutamate:NAD (P) oxidoreductase (deaminating); was prepared as a glycerol-water (equal volumes)
and lactatedehydrogenase,EC 1.1.1.27,
L-lactate:NADoxidore-
ductase. suspension, were filtered through filters of 0.22-tm
Received Oct. 18, 1972; accepted Nov. 10, 1972. average pore diameter (Millipore Corp., Bedford,
etal. (19). 2-
a-
Statistical equality was analyzed by use of the t- t 2C
Ui
distribution (20). I-
-J -
.#.- 1
Results 10
Kinetic Assay
Human sera, chosen without conscious bias from
specimens collected for a hospital clinical laboratory, 0 ‘0 20 30 40 50
U/LITER
were kept at 4#{176}C
and assayed within 24 h from the
Fig. 1. Correlation between AST activitydetermined with
time of drawing. Aliquots of 0.2 ml of each specimen and without pyridoxal phosphate supplementation in a
were added to control and to pyridoxal phosphate- kinetic assay
supplemented substrates lacking 2-oxoglutarate, and Each point represents the mean of duplicate analyses by each method.
Activities are reported at 30#{176}C:
sample size was 0.2 ml. Pyridoxal con-
were incubated for 1 h at 30#{176}
C. The composition of the centration was 25 mol/liter; ( ) represents a slope of 1.0; (-)
preincubation mixture was, per liter: 89 mmol of represents the regression fit of y = 1.16 x - 0.26. r = 0.987
0 20 40 60
shown in Figure 2. The best linear model to describe U/LITER
the relationship between control (X) and supple- Fig. 2. Correlation between AST activity determined with
mented .(Y) substrates is Y = X + 0.7. This regres- and without pyridoxal phosphate supplementation in a
sion fit, with a slope of 1.0 and an intercept of 0.7, is colorimetric method
Activities are reported in units described by Babson et al. (17) at 37#{176}c.
consistent with increased blank because of addition Where present, pyridoxal phosphate concentration was 26 tmol/liter.
of pynidoxal phosphate without stimulation of en- Regressionlinedescribesy = x + 0.7:r = 0.985
22. Snell, E. E., Non-enzymatic reactions of pyridoxal and their 34. Henry, R. J., Clinical Chemistry: Principles and Technics.
significance. In Chemical and Biological Aspects of Pyridoxal Ca- Hoeber,New York,N.Y.,1964, p 723.
talysis; E. E. Snell et al., Eds. Macmillan Co., New York, N. Y., 35. Turano, C., Giartosio, A., and Fasella,P., Sulfhydryl groups
1963, pp 1-12. and coenzyme binding in aspartic aminotransferase. Arch. Bio-
23. Hesry, R. J., Clinical Chemistry: Principles and Technics. chem. Biophys. 104,524(1964).
Hoeber, New York,N. Y.,1964, p 509. 36. Banks, B. E. C., Lawrence, A. J., Vernon, C. A., and Woot-
24. Fasce, C. F., Jr.,and Vanderlinde, R. E., Problems in SGOT ton, J. F., Kinetic studies of glutamic-aspartic transaminase (pig
standardization and quality control. Clin. Chem. 13, 679 (1967). heart muscle).In Chemical and Biological Aspects of Pyridoxal
Catalysis; E. E. Snell,et al.,Eds. Macmillan Co., New York,
25. Bergmeyer, H. U., and Bernt, E., Glutamate-oxaloacetate
N.Y.,1963, pp 197-215.
transaminase. In Methods of Enzymatic Analyses; H. U. Berg-
meyer, Ed. Academic Press, New York, N.Y., 1965, pp 837-845. 37. Dempsey, W. B., and Christensen, H. N., The specific bind-
ing of pyridoxal-5-phosphate to bovine plasma albumin. J. Biol.
26. Empfehlungen der Deutschen Gesellschaft f#{252}r Klinische Chem. 237, 1113(1962).
Chemie: Standardisierung von Methoden zur Bestimmung von
Enzymaktivitatenin biologischenFl#{252}ssigkeiten. Experimentelle 38. Scardi,V., Scotto,P.,laccarmno,M., and Scarano, E., The
Begrundung der optimierten Standard-Bedingungen. Z. KIm. binding of pyridoxal 5-phosphate to aspartate aminotransferase of
pig heart. Biochem. J. 88, 172 (1963).
Chem. KIm. Biochem. 10, 182(1972).
39. Holzer, H., and Schreiber,G.,Enzymatisch-OptischeBestim-
27. Schmidt, E., Glutamic dehydrogenase. In Methods of Enzy-
mung von Pyridoxal-5-phosphorsaureester und Pyridoxamin-5-
matic Analyses, H. U. Bergmeyer, Ed. Academic Press, New
phosphorsaureester mit Glutamat/Aspartate-Transaminase aus
York, N.Y., 1965, pp 752-756. Bierhefe. In Chemical and Biological Aspects of Pyridoxal Cataly-
28. Fahien, L. A., Wiggert, B. 0., and Cohen, P. P., Crystalliza- sis; E. E. Snell et a!.,Eds. Macmillan Co., New York, N.Y.,
tionand kineticpropertiesof glutamate dehydrogenasefrom frog 1963, pp 523-532.
liver.J. Biol. Chem. 240, 1083(1965). 40. Wolf, P. L., Williams, D., Coplon, N., and Coulson, A. S.,
29. Test Methodology, Smith Kline Instruments,Palo Alto, Low aspartate transaminase activity in serum of patients under-
Calif., 1969. going chronic hemodialysis. Clin. Chem. 18,567 (1972).