Anal. &em. '1983, 55, 1385-1390
1385
Determination of Human Serum Lactate Dehydrogenase Isoenzymes by Anion Exchange Chromatography M. P. Menon," Geraldine Anderson, and 0. K. Nambiar School of Sc:lences and Technology, Savannah State Collegt?, Savannah, Georgia 3 1404
A pH-coupled salt gradient elution technique has been developed for the complete fractionation of lactate dehydrogenase (LDH) isoenzymes In human sera by using a miniature polystyrene column packed wlth preequllibrated DEAE-cellulose l o a bed of 8 cm X 0.7 cm. Flve dlfferent buffers containing 0.02 M Tris (tris(hydroxymethyi)amlnomethane hydrochloride) wlth increaslng salt concentratlon and decreaslng pH were used to separate the isoenzymes from 0.3 to 0.5 mL of sera absorbed on the column. Two commonly employed methods for the assay of LDH Involving LDH catalyzed lactate-to-pyruvate (L-P) reactlon and the pyruvate-to-lactate conversion (P-L) were Investigated. Although the LDH Catalyzed P-L reaction was found to be more senskive than the L-P reactlon, the latter was more preclse and quantltatlve than the former. Although the LDH-l/LDH-2 ratios for several samples measured by chromatographlc and electrophoretic methods agree with each other, there were significant dlfferences In the percentages of Isoenzymes In serum samples obtalned by both methods.
It has been widely accepted that the measurement of lactate dehydrogenase (LDH) isoenzymes in serum samples of patients will provide a diagnostic tool for the detection and progress evaluation of a number of diseases including myocardial infarction (1-5). This is based on the belief that the serum elevation of the LDH isoenzymes that are specific cellular constituents of an organ will give an indication of the injury created to the organ. For instance, Roe (3) and Wagner et al. (6) have reported that LDH-1 and LDH-2 isoenzymes that are predominant in heart muscles become elevated in serum samples of patients after the incidence of myocardial infarction (MI) with a greater increase in LDH-1. Since LDH pattern can be monitored for a longer duration than creatine kinase (CK) IMB isoenzyme, which is also used as an indicator of heart ailment, the former will be more useful in detecting the heart condition of a patient suspected of MI but admitted to the hospital 24-48 h after the episode. The LDH-1ILDH-2 ratio is usually greater than normal in the serum samples of cardiac patients (3,8).The LDH-l/LDH-2 ratio in normal serum samples appears to range from 0.45 to 0.76 (7). The isoenzymes, LDH-3 and LDH-4, have been reported to be present in larger amounts in sera of patients with malignant neoplasms arid various types of malignant tumors (5,9). An increase in LDH-5 has been observed in patients with liver diseases (10) and prostatic carcinoma (2). Oliver et a1 (11) and Elhilali et al. (12)have reported that an LDH-5/LDH-1 ratio greater than 1is an indicator of maligancy of prostate tissue. The clinical diagnostic interpretation of LDH isoenzyme distribution in serum samples is being made mostly on the basis of the elledrophoretic data. Mercer (13)reported that the electrophoretic values for LDH isoenzymes, especially 3, 4, and 5 are 20% lower than the chromatographic values. The electrophoretic values are known to depend on the support medium (14), buffer (15),and quantitation procedures (16). 0003-2700/83/0355-1385$01,50/0
Vasudevan et al. (7) and Hsu et al. (17) have employed miniature ion-exchange columns for the separation of LDH isoenzymes in serum samples by using a discontinuous salt gradient elution technique but achieved only partial success. We report a p H coupled salt gradient elution technique for the chromatographic separation of all five LDH isoenzymeri in human sera by using a miniature DEAE-cellulose anionexchange column. We have also improved the quantitationi of LDH isoenzyme activity in fractions collected for eachi isoenzyme.
EXPERIMENTAL SECTION Materials: The unewollen DEAE-cellulose anion exchange (Sigma Chemical Co.) was equilibrated with tris(hydroxymethy1)aminomethanehiydrochloride (Tris-HC1)buffer according to the manufactor's instructions. The final equilibration was carried out with the starting buffer (0.02 M Tris-HC1, pH 8.0) before packing a bed of (3.0 cm X 0.7 cm in a polystyrene column. Before the samples are alpplied, the column is washed with at least 8 mL of the starting buffer. A mixture of standard LDH isoenzymes 1, 2, and 5 (Sigma Chemical Co., Saint Louis, MO), myocardial extract from heart muscles prepared from autopsy material (Memorial Medical Center, Savannah, GA), and the extract from platelets of healthy persons (VA Medical Center, Charleston, SC) were used to study the elution pattern of the isoenzymes from the miniature column. The following five buffer systems containing O.Of! M Tris-HC1 with increasing salt concentration and decreasing pH were finally used to achieve complete separation of LDH isoenzymes: buffer A (0.02 M Tris-HC1, pH 8.0) 6 mL; buffer B (0.02 M Tris-HC1+ 0.06 M NaCl, pH 7.8) 8 mL; buffer C (0.02 M Tris-HC1 + 0.10 M NaCl, pH 7.6) 8 mL; buffer D (0.02 M Tris-H[Cl+ 0.15 M NaC1, pH 7.4) 10 mL; and buffer E (0.02 M Tris-HC1 0.25 M NaC1, pH 7.2) 10 mL. All the chemicals used for this study including @-nicotinamideadenine dinucleotide (NAD') and NADH (reduced form) were obtained from Sigma Chemical CID. Human sera used for the study were obtained from two local hospitals (Savannah, GA) and also from the VA Medical Center [Charleston, SC) and stored at 5 "C for no more than 1 week before analysis. Column Separation. The LDH-1, -2, and -5 standards were mixed in such proportions as to give a totalLDH activity of about 600 units/mL. Myocardial and platelet extracts were diluted to such an extent as to give approximately the same total LDH activity. Half a milliliter of the standard mixture or the extract was diluted to 2.5 mL with water and absorbed into the column (8 cm X 0.7 cm). After calmplete absorption, the LDH was eluted with 6,8,8,10, and 10 mL,of buffers A, B, C, D, and E successively with no mixing and 0.7-mL fractions were collected, starting from the time when the sample was introduced. A 0.5 mL of alternate fractions was mixed with 2.5 mL of the substrate for the measurement of the enzyme activity in these fractions. It was found, from repeated measurements that fractions 3-9 (4.9 mL), 10-14 (3.5 mL), 15-21 (4.9 mL), 22-37 (11.2 mL), and 38-55 (12.5 mL) contain LDH-5, -4, -3, -2, and -1,respectively. LDH-1 was found at times to leave a tail in the remaining fraction, and therefore, the activity in the remalining eluate was also measured to determine the total LDH-1 activity. Chromatographic Procedure for the Separation of Serum LDH Isoenzymes. Depending on the total LDH content of the serum sample, a 0.2-0.5 mL sample was diluted to 2.5 mL with water to reduce the chloride concentration below 0.02 M and applied to the miniature column. Volumes of 0.2 mL samples containing 600 units/mL or more of total LDH, 0.3 mL for those
+
0 1983 American Chemical Socllety
1386
ANALYTICAL CHEMISTRY, VOL. 55, NO. 8, JULY 1983
containing 400-600 units/mL, and 0.5 mL for samples having 400 units/mL or less were employed. The first two 0.7-mL fractions after absorption of the sample were discarded. By following the same elution scheme used for the standards with the five different buffer systems as discussed before, fractions containing 4.9 mL, 3.5 mL, 4.9 mL, 11.2 mL, and 12.5 mL were colleded and labeled as LDH-5, LDH-4, LDH-3, LDH-2, and LDH-1 fractions. The remaining eluent in the column was also collected as the last fraction. The elution curve for fractionating the LDH isoenzymes from serum samples were also constructed, occasionally, by collecting 0.7-mL fractions using the same elution scheme. Miniature columns, when once made, were not used for more than three or four separations. Measurement of LDH A c t i v i t y . Two commonly employed methods, the method of Wacker et al. involving LDH-catalyzed conversion of lactate to pyruvate (L-P) in the presence of NAD' (18) and the method of Wroblewski and LaDue (19) which makes use of the conversion of pyruvate to lactate (P-L) in the presence of NADH, were investigated for better precision and sensitivity. In the former method the final concentrationsin mmol L-l of each reagent of the assay mixture were the following: sodium phosphate buffer (pH 8.8) 50.0, sodium lactate 50.0, NAD+ 5.0. The final concentrations (in mmol L-I) of reagents in the latter method were as follows: sodium pyruvate 0.333, NADH 0.085, and sodium phosphate buffer (pH 7.5) 96.6. Although the final volumes of the reaction mixture, in each case, were kept constant at 3 mL at a temperature of 37 A 1"C, the reagent concentrations in the substrate and the substrate volumes used for analysis were different depending on the size of the sample used for the assay. For the analysis of LDH activity in 0.7-mL fractions of the eluate, 0.5 mL of the sample and 2.5 mL of the substrate were used. For the measurement of total LDH activity in serum samples, 0.2 mL of the original or diluted serum, depending on activity level, and 2.8 mL of the substrate were employed. To determine a relatively smaller amount of activity, especially that of LDH-3, LDH-4, and LDH-5, in separated isoenzyme fractions, 2 mL of the sample and 1mL of a more concentrated substrate were used. The absorbance of the vortexed mixture at 340 nm was measured every 30 s for 3 min for total LDH determination and for 2 min for the relative activity measurement of the isolated isoenzyme fractions with a Perkin-Elmer Model Lambda 3 UV/Visible spectrophotometer. The initial absorbance was always measured 20 s after mixing the substrate and sample to avoid possible initial interference in serum samples following the customary practice (20). Although the LDH catalyzed P-L reaction was found to be more sensitive than the L-P reaction, the latter was more precise than the former (see section on Results and Discussion), and therefore, all the assays were done by using the L-P reaction. In an attempt to compare the precision and sensitivity of the assays of total LDH in serum samples and of the isoenzymes in separated fractions, we have employed the L-P (18) and P-L (19) reaction methods for the measurement of activities of a few selected serum samples. We used 0.2 mL of the original or diluted serum sample and 2.8 mL of the substrate for the determination of total LDH in four randomly chosen serum samples. In both methods the absorbance was measured at 340 nm every 30 s for a period of 3 min starting from 20 s after mixing the substrate and sample. Although fluctuations in absorbance with time were observed in the case of the P-L method, the average U / m i n was computed from the absorbance change for a 3-min period in both cases. To study how absorbance changes with time for the measurement of low-level enzymatic activity of separated LDH isoenzymes using both L-P and P-L methods, 2 mL of the isoenzyme fractions of two serum samples were used, each mixed with 1 mL of concentrated substrate. Electrophoretic I d e n t i f i c a t i o n and Measurement of LDH Isoenzymes. Electrophoresis on the peak fractions of the eluted isoenzymes was performed on polyacrylamide gel (5.5%) by using a vertical temperature-regulated electrophoretic apparatus, Polyanalyst (Buchler Instruments, Inc., Fortlee, NJ). A 30-pL sample was applied to the top of the polymerized gel (5 mm X 42 mm) followed by the addition of 30 pL of 40% sucrose solution and electrophoresed at 2 mA per gel with a Tris-glycine buffer system at pH 8.3 (20). The isoenzyme bands were visualized by use of a staining solution as proposed by Dietz and Lubrano (21) and identified by comparing the bands of the unknown with those
FRACTION NUMBER
Flgure 1. Fractionation of LDH isoenzymes from a synthetic mixture
of LDH-5, LDH-2, and LDH-1 by minlature ion-exchange column chromatography (polystyrene column, 8 cm X 0.7 cm, 6 sldrop). (UIL = unitslml.)
4
8
12
16 20 24 28 32 FRACTION NUMBER
36
40
44
Flgure 2. Chromatographic separation of LDH isoenzymes from human myocardial extract by use of a miniature column. (U/L = units/mL.)
obtained in an LDH isoenzyme standard, isotrol (Sigma Chemical Co.), processed in the same manner as the sample. Similar procedures were employed for the electrophoretic separation of LDH isoenzymes from several serum samples. A Helena Model Quick Scan densitometer was used to scan the colored band, using a 570-nm filter, and to integrate the areas under the peaks. From the relative areas under the peaks corresponding to the five isoenzymes, the percent composition of the serum LDH isoenzymes was computed. Some of the samples were also scanned with Beckman Model CSD-200 densitometer to compare the electrophoretograms measured by the instruments.
RESULTS AND DISCUSSION The elution curves for the chromatographic separation of the isoenzymes from a synthetic mixture of LDH-5, LDH-2, and LDH-1 standards and from a myocardial extract are shown in Figures 1 and 2. It can be seen from these figures that all the isoenzymes were separated except the LDH-3, -2,
ANALYTICAL CHEMISTRY, VOL. 55, NO. 8, JULY 1983
-
/ " " " " " " " " I zoo ..
Table I. Results of the Analysis of total LDH in a Few Serum Samples by Lactate-Pyruvate (L-P) and Pyruvate-Lactate (P-L) Methods total LDH, units/L serum samples L P methoda P-L method
LDH-I
180
IS0
t
serum-1 serum-2 serum-3 serum-4
a
Ii IO0
5
80
1151 570 428 206
2443 890 668 3 54
a L-P method by Wagner et al. N . Engl. J. Med. 1956, P-L method by Wroblewski and LaDue Proc. 295,449. S O ~E. X ~Biol. . Med. 11965,70, 216. .-
LDH-2
2
The results of the total LDH assay in a few serum samplerr using both the L-P and P-L methods are presented in Table I. The enzymatic activity was computed by using the following equation: enzymatic activity (units/mL or U/L) = 4
12
8
16
FRACTION NUMBER
r-
'1 LDH-3
I
120
20
[
1
11
1
LDH-2
R
LDH-4 LDH-5 5
10
(AA/min) 0.001
v
20 7.4 28 32 36 40 44 48 52 56 6 0 6 4
Figure 3. Minlature column separation of LDH isoenzymes from a human serum sample contalnlng elevated LDH. (UL = unlts/mL.)
140
1387
Ad
I
I5
20
25
30
35
40
45
FRACTION NUMBER
Flgure 4. Mlniiature column separation of LDH isoenzymes from the extract from a pooled platelet sample. (UL = units/mL.) and -1in the myocardial sample. Lack of the resolution among these peaks in the myocardial sample resulted from the use of a large-sized original sarnple (0.3 mL) which is needed to observe the peaks of LDH-5 and LDH-4 that are present in relatively smaller quantities. The total LDH activity in this sample was more than 6000 units/mL. Figure 3 shows the chromatographic fractionation of LDH isoenzymes in a typical human serurn sample while Figure 4 reveals the separated isoenzyme pattern of a platelet sample pooled from those obtained from normal healthy individuals. The peak fractions of the separated isoenzymes were electrophoresed by using 30 pL volumes and subsequently stained to detect the characteristic band and to identify the isoenzymes. Although LDH-1 fraction contained about 5% of LDH-2 activity, LDH-2 was completely pure. Very faint bands were obtained for other isoenzymes. It is obvious from Figures 1-4 that all five isoenzymes in human sera, platelets, and myocardial samples can be separated by use of miniature column chromatography with DEAE-cellulose as anion-exchanger provided the activity in not large.
where V is the volume of the sample used for the assay. This is based on an arbitrarily defined unit of activity (18)which is equal to an increase (L-P reaction) or a decrease (P-L reaction) of 0.001 Almin. The percent of error (standard deviation X 100/mean value) involved in each assay was determined by measuring the activity of a random sample five times and computing the standard deviation. The value obtained for the percent error was 11.9. It is evident from Table I that the value for the total LDH activity measured by the P-L method is higher than that obtained by using L-P method and hence the foirmer is a more sensitive method than the latter. In both methods AA/min was computed by using the absorbance change for a 3-min measurement (18)in spite of the fact that the absorbance fluctuated in the case of P-L method. Table I1 lists the absorbance data as a function of time for the isoenzyme analysis in two randomly chosen serum samples. It can be seen from this table that there is a steady increase in absorbance in all cases when L-P method was used, but most of the samples did not show a steady decrease in absorbance when P-L method was employed. Table I1 also reveals that in the measurement of low-level activity of the separated LDH isoenzymes, the L-P method always gives a positive value for a4/min while the P-L method fails to give a consistent negative absorbance change per minute. The lack of steady decrease or consistency and the apparent loss of precision in the assay when the P-L method was used made us choose the L-P method for all of our assays. Thiers and Valee (22) and Amador et al. (23) have attributed the erratic behavior of the absorbance change in the P-L method to the enzyme inhibition by pyruvate. The change in absorbance per minute is plotted against time for two serum samples and two isoenzyme fractions in Figure 5. None of the plots gives a constant AA/min over time, although samples with AA/min of 0.10 tend to have a constant absorbance change. Bull et al. (24) have observed similar nonlinearity in absorbance measurements (constant AA/min) even in the assay of purified LDH-1 and LDH-5 and attributed this to several factors including inhibition by the product. Our work shows that the activity computed using AA/min from a 1 min measurement will be quite different from that based on absorbance measured for 3 min, especially when U / m i n is more than 0.05. Interlaboratory comparison of the results of total LDH activity measurements will therefore be valid only if all the conditions of analysis including the sample size and time of absorbance measurements are identical.
1388
*
ANALYTICAL CHEMISTRY, VOL. 55, NO. 8, JULY 1983
Table 11. Comparison of Lactate-Pyruvate and Pyruvate-Lactate Reaction Methods for the Measurement of Change in Absorbancea L-P method P-L method A at 20 s A at 50 s A at 80 s A at 140 s AA/min A at 20 s A at 50 s A at 80 s A at 140 s AA/min LDH-1 LDH-2 LDH-3 LDH-4 LDH-5 LDH-1 LDH-2 LDH-3 LDH-4 LDH-5 a
0.244 0.255 0.221 0.196 0.215
0.260 0.293 0.235 0.205 0.222
0.257 0.280 0.231 0.202 0.218
0.228 0.245 0.218 0.237 0.217
0.234 0.262 0.221 0.244 0.222
0.267 0.315 0.241 0.210 0.226
Serum-A 0.012 0.030 0.010 0.007 0.006 Serum-B 0.009 0.027 0.004 0.009 0.006
0.245 0.299 0.226 0.255 0.229
0.238 0.275 0.223 0.249 0.224
1.067 1.067 1.125 1.168 1.094
1.065 1.085 1.237 1.152 1.127
1.048 1.062 1.130 1.145 1.092
1.040 1.072 1.125 1.150 1.090
-0.014 1-0.003 0.0 -0.009 -0.002
1.026 0.952 1.053 1.036 1.026
1.045 0.949 1.066 1.008 1.041
1.037 0.949 1.049 1.010 1.005
1.036 0.947 1.054 1.007
1-0.005 -0.003 1-0.001 -0.015 -0.020
1.001
Measurement of absorbance was started exactly 20 s after mixing the LDH with substrate containing NAD+ or NADH.
I SER-5 (TOTAL LDH) 2 SER-5 (LDH-2) 3.SER-397(TOTALLDH) 4 S E R - 5 (LOH- I )
\
\
\
I
I
30
60
90
120
150
I80
210
TIME IN SECONDS
Figure 5. Rate of change of absorbance as a functlon of tlme for the LDH
catalyzed lactate to pyruvate reaction.
The activities of the isolated isoenzymes and their percentages in ten representative samples out of a total of 36 serum samples assayed are presented in Table 111. The relative percentages of isoenzymes in the samples determined by the electrophoretic method are also included for comparison. The isoenzyme activities were computed by using the equation: isoenzyme activity (units/mL or U/L) =
(AA /min) VF
v,vs
0.00 1
(2) where V , is the total volume of the isoenzyme fraction collected, V , is the volume (2 mL) of each fraction, and V Sis the volume of the serum used for the assay. The sum of the activities of isoenzymes 1to 5 is higher than that of total LDH presented in Table IV. This is because the activities of isoenzymes are based on a 2-min absorbance measurement while the total LDH activity is based on a 3-min measurement. The linearity of the absorbance and the constancy of the rate of absorbance change (AAlmin) with time depends on the level of activity in the sample, extent of interference, and the
duration of the measurement of absorbance. If the activity is high, as is the case for the measurement of total LDH in serum samples, the increase in absorbance reaches a plateau or steady state after a short time. The nonlinearity of absorbance change with time is therefore, more pronounced in the analysis of total LDH than in the assay of isoenzymes which have relatively smaller activities. Bull et al. (24) recommend that the absorbance measurement be limited to 1 min, after the initial 20-8 period. We employed the customary clinical practice following the method of Wacker et al. (18) that recommends a 3-min measurement of absorbance for the assay of total LDH. Table I1 also reveals that there are significant differences in the percentages of the isoenzymes in the serum samples obtained by electrophoretic and chromatographic methods. It appears that in most cases values for LDH-3 are higher while LDH-4 and -5 values are lower in electrophoretic assay than in chromatographic data. However, it is to be pointed out that, from our own experience, the estimated error in the composition of the isoenzymes arising from the poor resolution of the peaks of electrophoretograms and reproducibility of results from multiple scans of the same sample alone can be as high as 7-8%. No estimate of error is available for the data furnished by other laboratories. It is not clear what causes LDH-3 to become higher and LDH-4 and -5 lower in electrophoretic results. Table IV lists the values for LDH-l/LDH-2 ratios in 20 serum samples out of 36 analyzed by us using miniature column technique as well as the electrophoretic results furnished by other laboratories. The total LDH isoenzymes measured by us and also by other laboratories are included in this table. Relative standard deviation, s / X , where s is the standard deviation and 8 is the mean of 18 sets (two in each set) of measurements for total LDH is 5.7%. Standard deviation was calculated on the basis of deviations from the means which were obtained by averaging our values and those reported by other laboratories in each set. The sample, ser6238 which showed a very high LDH level was from a patient who died immediately after admission to the hospital. As the LDH-l/LDH-2 ratio is within the normal range (7) it could be assumed that he did not die due to heart attack. Although the LDH-l/LDH-2 ratios for several samples measured by chromatographic and electrophoretic methods agree with each other, there are significant differences in the ratios for other samples. While chromatographic results are based on the activities of LDH-1 and LDH-2, electrophoretic results are based on the percentages of LDH-1 and LDH-2. An error in the measurement of the peak areas of any one of the isoenzymes will be reflected in the LDH-1/LDH-2 ratios in
ANALYTICAL CHEMISTRY, VOL. 55, NO. 8, JULY 1983
13iB9
Table IV. LDH-l/LDH-2 Ratios in Human Serum Samplt; Measured by Chromatographic and Electrophoretic Methods
serum sample
chromat.ographic electrophoretic method method a -~ total total LDH, LDH, units/ LDH-l/ units/ LDH-l/ mL LDH-2 mL LDH-2
Ser-7 Ser-355 Ser-397 Ser-39 9 Ser-408 Ser-438 Ser-490 Ser-50 8 Ser-4296
783 420 590 515 495 580 480 510 368
0.78 0.67 0.61 0.49
Ser-430 9 Ser-477gC Ser-5086 Ser-53 3 7 Ser-5402
790 258 703 818 320
1.27 0.43 1.80 2.14 1.43
1.00
0.45 0.61 1.24 2.13
810 381 572 409 381 433 335 469 325
0.77 0.62 0.65
810
1.27 0.46 0.58 1.46 0.43
220 700 256
reported condition of patient not known
1.00
1.04 1.31 1.04 1.07 0.78
abdominiil surgery surgery chest pain chest pain angina pregnant alcoholic chest pain chest pain chest pain not known chest pain
Ser-63 14 268 0.44 300 0.64 Ser-6849 250 0.95 263 0.73 Ser-6850 143 0.89 157 0.92 152 S e ~ 6 8 5 7 ~ 183 0.85 Ser-7201 150 137 1.34 a Our results. Results furnished by other laboratories except for those indicated by footnote c. Electrophoresis on these samples was performed by us. J J
electrophoretic measurement. Mercer (13) and Vasudevan et al. (7) have also reported that the electrophoretic method is tedius, time-consuming, and semiquantitative. From OUT own experience considerable skill is required to obtain good electrophoretograms for meaningful analysis. Sample size, staining technique, intubation time, etc. are critical in obtaining bands with colorless background. There are also significant differences in the percentage of isoenzymes obtained by using two different densitometers, Quick Scan andl Beckman 200 CD, for the same electrophoresed samples. In the column chrolmatographic method, when once the' elution curve and the columns of fractionated isoenzymes are established for the miniature column of proper size, good separation among the isoenzymes can be achieved as demonstrated in Figures 1-4. Our new method differs from the previously reported work (11) in two important aspects: (1) we have used a gradient in the range of pH 8-7.2 for elution while previous authors have used a constant pH of 6.3 at which the buffering capacity for Tris-HC1 buffer and ion-exchange properties of anion exchanger are not very effective. Volumes of fractions of isoenzymes collected are smaller in our work, and the volume of the sample used for activity measurement is larger resulting in an increase in the sensitivity of our measurement. This work was not intended to study the diagnostic use of the LDH isoenzyme pattern of serum sample of patients but rather to improve the chromatographic methodology for the assay of LDH isoenzymes. However, the pathological condition of the patients €rom whom the sera were collected is, in most cases, included in Table IV. Out of 36 samples analyzed over a period of' several months, 11 patients showed LDH-l/LDH-2 ratios > 1,18 patients had a ratio >0.76 which is the cutoff point for MI suggested by Vasudevan et al. (7) and Leung and Henderson (25),and 18 patients showed a range of 0.28 to 0.76 in serum samples. This cutoff has a sensitivity of 100% and a specificity of 91% (25). On the bssis of this interpretation only 4 out of 11,who complained of chest
Anal. Chem. 1983, 55, 1390-1395
1390
pain and found to have LDH-1:2 levels >0.76 might be suspected of stroke or heart attack. Table IV shows also that certain other pathological conditions such as abdominal surgery and angina may also cause a LDH-l:LDH-2 ratio higher than normal. This work indicates that LDH isoenzymes in human serum samples or other bodily fluids can be fractionated completely with a pH-coupled salt gradient elution technique after initial absorption of a diluted sample (0.3 mL). The ratio of any pair of the isoenzymes can be obtained from the activities of the separated isoenzymes rather than from the percentages on the basis of electrophoretic method. Even small quantities of LDH-4 and LDH-5 present in normal serum samples have been measured by this technique. The sensitivity and precision of the assay using the chromatographic technique appear to be higher than that obtained by the electrophoretic method.
ACKNOWLEDGMENT We are grateful to R. M. G. Nair and Jana Johnson of VA Medical Center, Charleston, SC, Jane Jennings and Terry Best of Memorial Medical Center, and Geetha Bala of Candler General Hospital, Savannah, GA, for their assistance in obtaining human serum samples and their results. Laboratory assistance rendered by Jimmy Gregory is also appreciated. Registry No. Lactate dehydrogenase, 9001-60-9.
LITERATURE CITED (1) Cohen, M. D.; Djordjevlch, J.; Jacobsen, S. M e d . Clin. North Am. 1966, 50, 193-209. (2) Heln, R. C.; Grayback, J. T.; Goldberg, E. J. urology 1975. 113, 51 1-516. (3) Roe, C. R. Ann. Clin. Lab. Sci. 1977, 7 , 201-209. (4) Lott. J. A.; Stang, J. M. Clln. Chem. (Winston-Salem, N.C.) 1980, 2 6 , 1241-1250.
Talagerl, V. R.; Nadakarern. S. S.; Gollerken, M. P. Indlan J . Cancer 1977, 1 4 , 43-49. Wagner, 0. S.; Roe, C. R.; Llrnberd, L. E.; Rosali, R. A,; Wallace, A. C. Circulation 1973, 47, 263-269. Vasudevan, G.; Mercer, D. W.; Varat. M. A. Circulation 1978, 57 (E), 1055- 1057. Galen, R. S. Hum. Pathol. 1975, 6, 141-144. Wrlght, E. J.; Cawley, L. P.; Eherhardt, L. J . Clin. Pathol. 1966. 4 5 , 737-741. Wleme, R. J.; Macrche, Y. Ann N . Y . Acad. Sci. 1961, 94, 898-901. Oliver, J. A.; Elhilal, M. M.; Belitsky, P.;Mackinnon, K. J. Cancer 1970, 25. - , 863-867. - .- - .. . Elhilall, M. M.; Oliver, J. A.; Sherwin, A. I.; Mackinnon, K. J. Cancer 1967. 98. 686-689. Mercer, b. W. Clh. Chem. (Wlnston-Salem, NC) 1975, 21, 1 102-1 106. Kreutzer, H. H.;Eggels, P. H. Ciln. Chim. Acta 1965, 12, 80-88. Joseph, R. N.; Schulz, J. J . Lab. Clin. Med. 1962, 6 0 , 349-353. Springell, P. H.; Lynch, T. A. Anal. Blochem. 1976, 74, 251-253. Hsu, Mei-Yung; Kohler, M. M.; Barolla, L.; Bondar, R. J. L. Clin. Chem. (Wlnston-Salem, N . C . ) 1979, 25 (E), 1453-1457. Wacker, W. E. C.; Ulrner, D. D.; Vallle, P. L. N . Engl. J . M e d . 1956, 295, 449-453. Wrobleswskl, F.; LaDue, J. S. Proc. SOC.Exp. Biol. M e d . 1955, 9 0 , 216-2 18. Technlon Method No. SG4-0021FH9, Technlcon SMAC System, Aug 1979. Dletz, A. A.; Lubrano, T. Anal. Biochem. 1987, 20, 246-257. Thlers, R. E.; Vallel, B. L. Ann. N . Y . Acad. Scl. 1958, 75. 214-218. Amador, E.; Dorfrnan, L. E.; Wacker, W. E. C. Cftn. Chem. (WlnstonSalem, N . C . ) 1983, 9 , 391-395. Buhl, S. N.; Jackson, K. Y.; Lublnskl, R.; Vanderllnde, R. E. Clln. Chem. (Winston-Salem, N . C . ) 1977, 23, 1289-1295. Leung, F. Y.; Henderson, A. R. Clin. Chem. (Winston-Salem, N.C.) 1979, 25, 209-211.
RECEIVED for review October 22, 1982. Accepted March 7, 1983. The financial support provided by the Division of Research Resources of the National Institutes of Health (MBRS Grant No. 5 SO6 RR08144-08, Project No. 5) is gratefully acknowledged. This paper was presented at the annual meeting of Georgia Academy of Sciences, Columbus, GA, April 24-25,1982.
Characterization of Substitution-Inert Cobalt(I 11) Complex Bonded-Phase Columns for Liquid Chromatography C. Allen Chang,’ Chen-Shl Huang,
and Cheng-Fan Tu
Department of Chemistry, The Universitv of Texas at El Paso, El Paso, Texas 79968
The preparation procedures for several substitution-inert cobalt( I I I ) complex bonded phases, [Co(en),]CI,, [Co(edda)(en)]CI, [Co(dmedda)(en)]CI, and [Co(deedda)(en)]Cl, are described where en = ethylenediamine, edda = ethylenediamlne-N,N’-dlacetate Ion, dmedda = N,N’-dimethylethylenedlamlne-N,N’-dlacetate ion, and deedda = N,N’dlethylethylenedlamlne-N,N’-dlacetateIon. All the complex bonded phases are found to have mlxed surface coverage of both diamine and the complexes. A method Is then deveioped to calculate the surface coverage of the complex. Besides the normal chromatographic characteristics such as the relationshlps of the capacity factor and the number of theoretical plates vs. sample size and flow rate, a simple thermodynamic approach to sort out lndlvldual contribution for the overall mlxed retention is reported. Knowing the AH value of solute transfer from the mobile phase to the stationary phase for the column, the AH value due to complex alone can be calculated provlded the percent surface coverage of the complex Is known. The AH values for all complex bonded phases are then compared and rationalized according to the structural nature of the complexes.
The use of metal ions or complexes to enhance separation has gained momentum only recently in modern high-performance liquid chromatography although the concept is not a new one. For example, it is known that early in 1903-1906, calcium carbonate was used by Tswett to separate plant pigments (I). In general, metal ions and their complexes can be roughly divided i n t ~ two classes, i.e., substitution inert and substitution labile, based on their kinetics of ligand displacement reactions. Many metal complexes are substitution labile and the use of them in liquid chromatography is generally called “ligand exchange chromatography” (2). Depending on whether the metal ion or complex is fixed on the stationary phase or whether it is moved along the column in the mobile phase, one can distinguish two types of ligand exchange chromatography: (1) the chromatography of ligands in which the metal ion is held by the stationary phase via strong complex formation or adsorption; (2) the chromatography of complexes in which the metal ion is bound more strongly toward the ligands in the mobile phase. One novel application of ligand exchange chromatography is the separation of enantiomers, particularly, d- and l-amino
0003-2700/83/0355-1390$01.50/00 1983 Arnerlcan Chemical Soclety
