Robert Rej, Jean-Pierre Bretaudlere, and Barbara Graffunder. Six procedures ..... The DEAE-Sephadex Ion-exchange procedure of Sampson et al. (9) and the.
CLIN.
CHEM.
27/4,
535-542
(1981)
Measurement of Aspartate AminotransferaseIsoenzymes: Six Procedures Compared Robert Rej, Jean-Pierre
Bretaudlere,
and Barbara Graffunder
Six procedures were evaluated for aspartate aminotransferase (EC 2.6.1.1) isoenzyme assay in human serum and tissue homogenates. Results of procedures based on immunochemical precipitation by use of antibodies directed against either the mitochondrial or (with greater precision) soluble isoenzyme correlated well with those by a differential kinetic assay involving both different pH conditions and adipate inhibition. Results with a DEAESephadex ion-exchange chromatographic procedure correlated well with these techniques for specimens containing purified isoenzymes, but showed substantial positive bias for determination of the mitochondrial isoenzyme in human serum. An assay based on the differential effects of pH alone discriminated between the isoenzymes with less bias than did the chromatographic assay. Precision of the two differential pH assays was limited by significant reagent blank activity resulting from destruction of NADH at pH 6.0 or 6.2. An electrophoretic procedure in which diazonium salt is used to make oxalacetate visible was least accurate for measuring samples for which the isoenzyme composition was known. Mammalian aspartate aminotransferases (AspAT, EC 2.6.1.1) exist in two predominant forms (1-3), one mitochondrial (m-AspAT), the other of soluble or cytosolic origin (s-AspAT). Although diagnostic utility of AspAT isoenzyme measurements has been suggested (4-7), these measurements are not yet routine, perhaps because (8) of the unavailability of separation techniques that are sufficiently accurate and (or) convenient. However, improved techniques have been described for measuring AspAT isoenzymes: chromatographic (9), immunochemical (10-12), differential kinetic (13, 14), and electrophoretic (8). Here we compare the relative merits of these techniques.
Materials and Methods Total AspAT activity was measured in the presence of exogenous pyridoxal phosphate by coupling oxalacetate production with NADH in reaction with malate dehydrogenase (EC 1.1.1.37). Absorbance change was monitored at 339 nm; all units (U) are tmol/min at 30#{176}C. Assay concentrations, in mmolfL, were: L-aspartate, 180; 2-oxoglutarate, 15; NADH, 0.18; pyridoxal phosphate, 0.11; and tris(hydroxymethyl) aminomethane (Tris), 89. The pH was 7.8, and malate dehydrogenase activity was 1.0 kU/L at 25 #{176}C. Unless otherwise specified, the volume fraction of specimen was 0.083 (1:12). Reactions were initiated by adding 2-oxoglutarate after the specimen had incubated for 10 mm in an otherwise complete reaction mixture. Aspartate-free and reagent blank measurements were performed as specified further on. Activities
were calculated by using a molar absorptivity 6.22#{149} j3 L mol cm’ for NADH at 339 nm.
value
of
Rates of absorbance change were monitored and recorded with a Cary 219 spectrophotometer (Varian Associates, Palo Alto, CA 94303). Reaction mixtures were maintained at 30 ± 0.1 #{176}C with a Model FK fluid circulator bath (Haake Instruments, Rockville, MD 20852); temperatures were verified with a Model 45 CU cuvette thermometer and gallium cell (Yellow
Springs Instruments,
Yellow Springs, OH 45387).
L-Aspartic acid, 2-oxoglutaric and /3-NADH were obtained
acid, pyridoxal 5’-phosphate, from Boehringer Mannheim
Biochemicals, Indianapolis, IN 46250, and Sigma Chemical Co., St. Louis, MO 63178. Porcine-heart malate dehydrogenase,
supplied
in glycerol,
and porcine
heart
s-AspAT
were
purchased from Boehringer Mannheim; Tris and adipic acid from Sigma; polyethylene glycol 6000 (M 6000-7500) and hydroxydimethylarsine
oxide (cacodylic
acid) from J. T. Baker
Chemical Co., Phillipsburg, NJ 08865. Sephadex ion-exchangers were from Pharmacia, Piscataway, NJ 08854, and were prepared for use as directed. All other chemicals were of reagent grade or higher purity. Distilled, de-ionized water with a resistivity of 15 M - cm at 25 #{176}C was used throughout. Wherever possible, reagents were passed through filters of 0.22-tm av. pore diameter (Millipore Corp., Bedford, MA 01730).
We prepared human s-AspAT from erythrocytes and mAspAT from human liver, as described previously (10, 11, 15). Both preparations were homogeneous by the criteria of polyacrylamide gel electrophoresis and isoelectric focusing, and their specific activities exceeded 150 kU/g at 30 #{176}C. Patients’ sera with a wide range of AspAT activities were selected from a hospitalized population. The specimens were stored at < -60 #{176}C within 24 h of collection and used within eight weeks. Results by various methods were compared by the multivariate technique of correspondence analysis (16) and robust correlation techniques (17).
Techniques lsoenzymes
for Measurement
We used six procedures
of AspAT
to estimate
AspAT isoenzyme
ac-
tivities. Column chromatography on DEAE-Sephadex. In the chromatographic procedure of Sampson et al. (9) we followed the authors’ optima] conditions. The specimen (1.0 mL) was applied to a 6 X 0.6-cm column of DEAE-Sephadex A-50
equilibrated
with a buffer containing
Tris and NaCI (each 50
mmol/L), pH 8.5, and washed with 5.0 mL of this buffer mL aliquots. The eluate (6.0 mL) contains m-AspAT.
in 1.0 The
column was then washed with 6.0 mL of a buffer containing Tris (50 mmol/L) and NaCI (200 mmo]/L), and 6.0 mL of eluate containing s-AspAT at low AspAT activities,
was collected. To improve since the serum specimen
precision is diluted
12-fold in this method, we adjusted the reagents to allow a larger volume fraction (0.50) of eluate; final assay conditions Division of Laboratories and Research, New York State Department of Health, Albany, NY 12201. Received Dec. 10, 1980; accepted Jan. 13, 1981.
were identical to those described above for measurement of total AspAT activity. However, due to the presence of chro-
matographic
buffers in the eluates, higher concentrations CLINICAL CHEMISTRY,
Vol. 27, No. 4, 1981
of 535
160
w U
U I-J 140
I-
D I-
>
Ia
Phosphote #{149},5 Tris O,#{149} s-AspAT A,& m-AspAT O,A
DISTANCE
FIg. 1. Electrophoretlc separation cellulose acetate
of AspAT lsoenzymes
on
IOC
Specimen was pertly puifled (55#{176}C for 20 mln and .butanol treatment) human liver ASpATcontainIng 60% m-AspAT and 40% s-AspAT. Ai’eas under cwves ‘e 32% m-AspAT and 68% s-AspAT. inset: Reaction prowess cwves for (0)
60
s-ASpAT and () m-ASpAT. Isoenzymes were adjusted to IdentIcal concentrations. and oxalacetate production was messwed dect1y using lO-nim pelhlenglh cuvettes at 260 nm. Assay reagents were 125 nwno1of 1-aspartate per liter, 6.7 mmol of 2-oxoglutarate per liter, and 90 mmol of phosphate, pH 7.5, per lIter
pH
FIg. 2. pH dependence of activity of human A5pAT isoenzymes Trlsngl.s show m-A5pAT, cArclesshow s-ASPAT.Buffers were phosphate (cen symbols) and Tris (closed symbols), each at 90 mmol/L. L-Aspartato and 2oxoglutarate were as In FIgure 1 Inset; 0.2 ml of enzyme was used In a total volume of 3.0 mL. Mean values and 1 SD error bars are shown
Tris and variable amounts of NaCl are also introduced to this assay. Immunochemical precipitation of s-AspAT. The immunochemical precipitation procedure of Rej (12) was used to quantitate m-AspAT. Antibodies to the human soluble isoenzyme were incubated with the specimen in the presence of
= (1.0
m-AspAT
pH 6.2 and adlpate (50 mmol/L)8
s-A5pAT m-AspAT
92 7
84 15
-
s-AspAT
to inhibit s-AspAT with only a slight effect upon m-AspAT. The procedure of Martinez-Carrion et al. (13) was selected, in which specimens were measured (a) at pH 8.0 without adipate and (b) at pH 6.2 with 50 mmol/L adipate. Other reagents, identical in both systems, were, in mmol/L: cacodylate,
Table 1. Inhibition (%) of A5pAT Isoenzymes pH 80a Ref..
= V7.4
with 2-oxoglutarate at 21.3 mmol/L found by et al. Using purified human isoenzymes, we found different activity ratios by this procedure; these were also used as given in Results. Differential pH plus inhibition. As in the preceding technique, the differential pH sensitivity discriminates between the isoenzymes, and an inhibitor-adipate-is added, further
enzyme and NADH were increased to imIn addition, because this procedure was to assays utilizing pyridoxal phosphate, that
Work
-v/v7.1
isoenzymes Graubaum
tions of coupling prove linearity.
This
et al. (14) were used to
where V is the measured catalytic activity (in U/L) at pH n. These are based upon the differential effects of pH on the two
drogenase was at 1 kUIL. These conditions were as recommended by Graubaum et al. (14), except that the concentra-
H#{149}T. 13
it was not originally
Inhibition
performed for both assay conditions. The algorithms given by Graubaum calculate isoenzyme activities:
and antibodies and assay conditions have been described previously (12). Immunochemical precipitation of m-AspAT. In this assay, antibodies directed against m-AspAT were used to obtain measurements of s-AspAT. The principle corresponds to that above, and m-AspAT was estimated by difference. Assay conditions and preparations have been published (10, 11). Differential p11 assay. The two AspAT isoenzymes have different pH profiles, with m-AspAT retaining significantly greater activity at acidic pH. Following the procedure of Graubaum et al. (14), we assayed each specimen at both pH 6.0 and 7.4. The reagent concentrations were, in mmolfL: Laspartate, 76; 2-oxoglutarate, 21.3; phosphate buffer, 67; NADH, 0.16; and pyridoxal phosphate, 0.11. Malate dehy-
lso.nzym.
here, although
patterns of the holoenzyme were identical in the presence or absence of pyridoxal phosphate; see Results and Discussion for details. Reagent blank measurements were
polyethylene glycol, then centrifuged. Residual m-AspAT activity was measured by the assay for total AspAT activity given above, and s-AspAT was calculated as the difference between total and m-AspAT activities. Preparation of antigen
compared
was included
coenzyme specified.
13, 14
0 0
under Four Assay Conditions or
74b
pH 6,0b ThIs
work
0 0
Ref.
14
66 0
ThIs work
58 0
Other conditions were as specified by Martinez-Carrionet al. (13) and given In Materials and Methods. #{176} Other conditions wereas specified by aubaum aI. (14) and given in Materialsand Methods.
536
CLINICAL CHEMISTRY. Vol. 27, No. 4, 1981
et
Table 2. AnalytIcal Recovery of Purified AspAT Isoenzymes from Human Serum (Apparent m-AspAT Activity, U/L) Addition5
None s-AspAT,
Adipat. Publlehed ratIos
Column (ref. 9)
5.6 9.2(+3.6)”
(ref.
5.9 12.8(+6.9)
Differential PubS.h.d ratIos
13) Antl-s-AspAT
Antl-m-ApAT (rels. 10, 11)
12)
Our ratIos
(rsf.
4.5 4.0(-0.5)
4.2
4.0
3. 1(- 1.1)
4.0(0)
6.7
pH (rsf.
14)
Our ratios 3.9
12.1(+5.4)
7.9(+4.0)
85 U/L m.-AspAT,
75.3
85.0
92.0
79.0
76.0
105.5
100.1
79.9(+4.6)
94.2(+9.2)
93.5(+1.5)
79.1(+2.1)
78.1(+2.1)
129.6(+24.1)
116.2(+16.1)
75 U/L s-AspAT,
85 U/L and m-AspAT, 75 U/L #{149} PurifIed Isoenzymes were added at activities shown bytheassay for measurement of total A5pAT activity In Materials
and Methods. b Bias Inmeasurement
of m-A8pAT due to presence of s.AspAT (U/L).
30; 2-oxoglutarate, 8; and NADH, 0.14. dehydrogenase was present at :1.0 kU/L. Pyridoxal phosphate, 0.11 mmolfL, was also added for the reason described above. Reagent blanks were measured as described in Results. The following algorithms were used to calculate isoenzyme activities: 50; Tris, 50; aspartate,
tamed
Malate
the published
s-AspAT m-AspAT
= 093. = V8.0
V80-
V6.2
s-AspAT
These relationships were obtained from studies with porcine isoenzymes (13). Using purified human isoenzymes, we found different activity ratios by this procedure; those ratios were also used (see Results). Elect rophoresis on cellulose acetate. The procedure of Rej (8) was followed, with use of the reagents and equipment described. After electrophoretic separation, the membranes were incubated with substrate in agar gel, then with a diazonium salt, to stain for the oxalacetate produced: Fast Violet B salt (6-benzamido-4-ethoxy-m-toluidine diazonium chloride, Grade III from Sigma, rather than the Grade! specified previously).
trophoretic procedure from further study. Initial experiments with the differential kinetic assays and purified human isoenzymes showed inhibition patterns somewhat different from those published (13, 14). The procedure of Martinez-Carrion et al. (13) is based on ratios obtained for porcine isoenzymes. We therefore examined the differential pH sensitivity of the human isoenzymes in Tris and phosphate buffers and found sufficient pH discrimination to form the basis of a differential assay (Figure 2). Because buffer conditions (Figure 2) and substrate concentrations (14) affect the pH-activity proffles, the purified human isoenzymes were assayed by each technique as described; the ratios ob-
were calculated both by ratios (13,14) and by those found in our labo-
ratory.
For the column and both immunochemical procedures, blanks were 1 UIL. For the procedures of Graubaum et al. (14) and Martinez-Carrion et al. (13), reagent blanks were approximately 6 and 3 U/L, respectively, at lower pH. This higher apparent activity is likely due to the instability of NADH at pH I
a.
ANTI-s-AspAT
PRECIPITATION ASSAY
FIg. 3. ComparIson of column chromatographlc and Immunoprocedures for determination of m-AspAT
chemical
The DEAE-Sephadex Ion-exchange procedure of Sampson et al. (9) and the lmmuiochemlcal precipitation assay utIlIzing antibodies dIrected agaInst aA5pAT (12) were used. Results are expressed as (A) catalytic activity of mA5pAT and (B) m-AspAT as a percentageof total AspAT activity. Robust procedures (17) were used to calculate linear correlation equations; correlation coefficients were (A) 0.875 and (B) 0.90. &oken lines represent a slope of 1.0. Specimens were authentic patients sara(#{149}), human liver homogenate (A), and purif led human AspAT Isoenzymes added to albumin (a), human serum (0), or Inactivated bovine serum (0) matrIces
CLINICAL CHEMISTRY,
Vol. 27, No. 4, 1981
537
z
I
0 I-
I
z
FIg. 4. Comparison of two Immunochemical procedures for determInatIon of m-AspAT The procedures used antIbodies dIrected against human m-A8pAT (ref. 10, 11) or s-AspAT (ref. l2 All other details wereas in FIgure3. CorrelatIon coefficients were
(A) 0.998 and (8)0.996
I 0.
and immunochemical assays, using patients’ specimens and purified isoenzymes; m.AspAT is expressed both as amount (U/L, Figure 3A) and percent of total AspAT activity (%, Figure 3B). Figures 4-6 present similar comparisons for the immunochemical assay with use of anti-m..AspAT antibodies, the differential pH, and the differential pH-adipate inhibition assays, respectively. Figures 5 and 6 show data obtained both by using published isoenzyme inhibition data (upper, A and B) and ratios found in our laboratory (lower, C and D). Correlations given in Figures 3-6 are based on robust techniques (17) including all data points. Comparison of the column and immunochemical procedures (Figure 3) showed a nearly one-to-one correlation, but this was influenced by specimens containing purified isoenzymes with m-AspAT 50 UIL. Correlation of the two procedures with patients’ specimens alone gave a regression slope of 1.8 and a correlation coefficient of 0.981 (Figure 3A). This bias appeared not to be related to the level of catalytic activity
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ANTI-s-AspAT
PRECIPITATION
IQO
ASSAY
FIg. 6. Comparison of differential kinetic and immunochemical procedures for determination of m-AspAT The dlfterentlal.pH plus adlpate-inhlbltlon assay ofMartinez-Carrion et al. (13) and the antl-s-AspAT assay (12) were used. The algorIthms were based upon (A, B) published data (13) or (C, 0) data obtained In our laboratory (Table 1). All other detaIls were as In Figure 3. CorrelatIoncoefficients were (A) 0.967, (B)
0.992, (C) 0.962, and (0)0.992
of the serum specimen.
One sample of purified s-AspAT in an
inactivated
bovine serum matrix also showed substantial bias from the column technique, perhaps due in part to the altered
serum
matrix.
Recoveries
of AspAT
activity
from
the
DEAE-Sephadex columns were in the range 90 to 110%. The precision of the column procedure and the agreement between the methods was best for purified materials and for those with
as reflected in the scatter shown in Figure 5. There was a proportional bias of about 25% between the procedures (Figure 5C). Because the assay conditions for the two procedures differ, this was not unexpected and does not indicate an inherent inaccuracy in either procedure. The correlation obtained with patients’ sera alone gave a slightly greater slope (1.34) and a correlation coefficient of 0.930. Due to the nature of calculation of m- and s-AspAT by this procedure it is possible to obtain >100% apparent m-AspAT. This was the case
4 2
100
FIg. 5. Comparison of differential pH and immunochemical procedures for determination of m-AspAT The dIfferential pH assay by Graubaumet al. (14) and the antl-s-AspAT assay (12) were used. The algorithms were based upon (A, B) publisheddata (14) or (C, 0) data obtained inthis iaboratoy (Table 1). All other details as in FIgure 3. Correlation coefficIents were (A) 0.917, (8)0.945, (C) 0.954, and (0)0.951
CLINiCAL CHEMISTRY. Vol. 27, No. 4, 1981
I’
Correlation of the differential pH assay with the immunochemical procedure was best with the calibration ratios found in our laboratory (Figure 5, C and D). Of the five methods examined, the differential pH assay had the poorest precision,
ANTI-s-AspAT PRECIPITATIONASSAY
538
#{149}
higher amounts of m-AspAT. Correlation between the two immunoprecipitation assays was excellent, although the procedure involving anti-rnAspAT was found to have a slightly low bias relative to that involving anti-s-AspAT antibodies (Figure 4). We saw no difference between results for purified human isoenzymes and patients’ sera.
B x
00
IU Li. LI
iso A
I
z
ANTI-s-AspAT PRECIPITATIONASSAY
for two materials containing solely m-AspAT (Figure 5, B and D); each showed about 115% m-AspAT by both published (14) and within-laboratory ratios. The differential-pH plus adipate-inhibition assay correlated well with the immunochemical procedure (Figure 6). As with the differential pH assay, a proportional bias (in this case about 17%; Figure 6C) reflects differing assay conditions and does not necessarily imply an accuracy bias. A nearly oneto-one correlation was found for data expressed as percentage of m-AspAT (Figure 6D). There was no significant difference
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