Anal. Chem. 1980, 52,729-733
729
Determination of Serum Isoenzyme Activity Profiles by High Performance Liquid Chromatography Timothy D. Schlabach,' Joe A. Fulton,2 Peter
B. Mockridge,
and E. Clifford Toren, Jr."
Department of Pathology, University of South Alabama, Mobile, Alabama 36688
The determination of serum isoenzyme activities can be accomplished by coupling a high performance liquid chromatographic column to a postcolumn reaction system. Since serum isoenzyme activities vary widely, the postcolumn system must have a comparable dynamic range. This can be achieved by using both absorbance and fluorescence detectors in the system. The response of this system to applied lactate dehydrogenase activity was linear from 1.5 to 1500 U/L, and the response to applied creatine kinase activity was linear from 0.5 to 500 U/L. Profiles of creatine kinase and lactate dehydrogenase Isoenzymes can be obtained in less than 25 mln, and they exhibit base-line resolution with better than 90% recovery of activity applied to the column. Changes in serum isoenzyme profiles detected with thls system are dlagnostlcally useful and In agreement with cllnlcal findings.
Isoenzymes, first observed by staining an electropherogram for enzyme activity ( I ) , are multiple forms of an enzyme differing in primary structure but catalyzing the same reaction (2). Five isoenzymes of lactate dehydrogenase (LD) result from the combination of two different polypeptides, H and M, to form the tetrameric isoenzymic (H4,H3M. . . M4 or LD-1 t o LD-5, respectively) ( 3 ) . These isoenzymes have a similar molecular mass but different net charges a t physiological pH (4). Three isoenzymes of creatine kinase (CK) result from the combination of two different polypeptides, M and B, to form the dimeric isoenzymes, CK-MM, CK-MB, and CK-BB ( 5 ) . These isoenzymes also have a similar molecular mass but different net charges (6). T h e separation of LD and CK isoenzymes by high performance liquid chromatography (HPLC) was first reported several years ago (7,8). These rapid separations were made possible by the development of new chromatographic materials by Chang and Regnier (9). That group devised a post-column reaction system for the continuous detection of enzyme activity eluting from an HPLC column (10) and demonstrated that five LD isoenzymes could be separated in 6 min and that the CK isoenzymes could be separated in 4 min (11). A similar post-column system was developed by Schroeder et al. (12). A disadvantage of those systems was the use of a single detector to monitor the activity of separated isoenzymes. We found that a second detector permitted the direct measurement of enzyme activity ( 1 3 ) ,because a two-point determination of the enzymatic rate could be made. We also developed a method to display just the profile of enzyme activity in an isoenzyme separation by subtracting the transformed response of one detector from the other (14). Changes in serum LD and CK isoenzyme profiles can be used in the diagnosis and evaluation of myocardial infarction (15, 16). Such changes are particularly sensitive and specific 'Present address: Varian, Instrument Division, 2700 Mitchell Dr., Walnut Creek, Calif. 94598. *Present address: Western Electric ERC, P.O. Box 900, Princeton, N.J. 08540. 0003-2700/80/0352-0729$01 .OO/O
markers for acute myocardial infarction and have greater diagnostic specificity than just the electrocardiogram (17). These changes become apparent when the profile of serum isoenzymes from a patient with suspected myocardial infarction is compared with a profile obtained from the serum of a normal individual. Previous experience revealed that our system did not have sufficient sensitivity to obtain isoenzyme profiles from normal sera. We have attempted to correct this deficiency by adding two fluorescence detectors to our post-column reaction system. We use this system to obtain LD and CK isoenzyme profiles from control materials, normal sera, and sera of patients with organ-specific disorders. We present these data and show how they might be used in clinical diagnosis.
EXPERIMENTAL Apparatus. The apparatus is shown in Figure 1. Chromatography was with the gradient, high-pressure liquid chromatograph (A), Model 8500 (Varian Instrument Division, Palo Alto, Calif.). The pre-column injection valve (C) used a 102.8-pL loop for LD isoenzyme separations and a 216.7-pL loop for CK isoenzyme. The post-column injection valve (E) used a 21.27-pL loop for total LD activity and a 102.8-pL loop for total CK activity. These valves were Rheodyne Model 7120. The chromatographic column (D) was described earlier (14). The reagent pump (B), a Model 3500 (Spectra-Physics, Santa Clara, Calif.), introduced the enzyme reagent through the 1.6-mm tee fitting (F). The reagent was pre-heated in a 3.05-m coil (G). The enzyme reaction began in the 15.25-m lag-phase coil (H) and linearly developed after the first absorbance and fluorescence detectors (J1and K,, respectively). Coils were made from 0.508-mm i.d. stainless steel tubing wrapped around 4-cm 0.d. plastic tubes. The reaction developed in the 30.5-m reaction coil (I) and was measured in the second absorbance and fluorescence detectors (Jzand Kz, respectively). The coils were maintained at 37 "C unless otherwise specified. Absorbance was measured with a Model 440 dualchannel detector with 340-nm filters (Waters Associates, Milford, Mass.); fluorescence, with Aminco FluoroMonitors (American Instrument Company, Silver Spring, Md.), with Corning 7-60 primary and Wratten 2A secondary filters. The reponse from each detector was acquired with an analog-to-digital converter (L),of the computer, a DEC LAB ll/V03 (Digital Equipment Corp., Maynard, Mass). These data were reduced and plotted with the DEC GT-46 system described earlier (14). Reagents. The weak buffer (buffer A) used in all isoenzyme separations was 0.02 M Tris.HC1 adjusted to pH 7.8. The strong buffer (buffer B) for the elution of LD isoenzymes was 0.15 M NaCl in buffer A; the strong buffer for separating CK isoenzymes was 0.30 M NaCl in buffer A. The post-column reagent for assaying LD activity has been previously described (13). The post-column assay reagent for CK activity was made from the CPK-10 reagent kit (Biodynamics, Indianapolis, Ind.). Each vial was reconstituted with 50 mL of 0.40 M triethylamine buffer (pH 7.0) containing 0.02 M magnesium acetate and 0.04 M D-glucose. Four such preparations were combined and to this solution was added a vial of hexokinase and glucose-6-phosphatedehydrogenase coupling enzymes (catalog no. H8629, Sigma Chemical Co., St. Louis, Mo.) to produce the CK assay reagent. LD and CK assay reagents were kept in an ice bath for the duration of each experiment. Vials of the CK/LD Isoenzyme Control (Helena Laboratories, Beaumont, Texas) were reconstituted with buffer A. Solutions containing bovine LD-1, human LD-1or human LD-2 0 1980 American Chemical Society
730
ANALYTICAL CHEMISTRY, VOL.
52, NO. 4, APRIL 1980
relation coefficient of 0.999 and a standard error of the estimate of 14.5 U/L. The limit of sensitivity was about 1 U/L, a tenfold improvement over our previous method (13). CK activity was determined by the Rosalki method (19) that employs the following reactions:
El---
ADP ATP
were prepared by dilution of the commercial preparations (catalog nos. L0377, L3632, L3757, Sigma) with buffer A that also contained 0.5 mg/mL of human serum albumin (catalog no. A9511, Sigma). Rabbit CK-MM or rabbit CK-BB (catalog nos. C3755, C6638, Sigma) were reconstituted with the same buffer. Frozen serum samples were obtained from the University of South Alabama Medical Center. Serum samples were also obtained from a healthy, 30-year-old male and used on the day of collection. Methods. Gradient elution of LD isoenzymes was begun 2 min after injection. The gradient started at 5% strong buffer and was advanced linearly at a rate of 4% per min. Gradient elution of CK isoenzymes started immediately after injection, beginning at 15% buffer B and was advanced at a rate of 5% per min. Three aliquots, 2.0 mL each, of fresh serum were used in each recovery experiment. To each of two aliquots was added 200 pL of a solution containing the desired isoenzyme, and to a third was added 200 pL of buffer A. The applied LD activity in the dynamic range study was determined at 30 "C with LDH-L reagent (Calbiochem. La Jolla, Calif.); the applied CK activity was also determined at 30 OC with the CPK-10 reagent (Biodynamics). Solutions containing LD and CK activity in this study were injected below the column: sample activity was determined from the resulting peak areas. Samples injected below the column were treated the same as activity eluting from the column. Absorbance peaks were directly converted into activity as previously described ( 1 3 ) ;fluorescence peak areas required the use of a calibration factor. This factor was determined by calibrating the fluorescence detectors against the absorbance detectors with solutions of reduced form of nicotinamide adenine dinucleotide,NADH (catalog no. 340-375, Sigma) in buffer A. In all separations, the total LD or CK activity applied to the column was determined by direct injection below the column, and is referred to as the post-column method. All samples were pre-filtered with 0.22-pm Millex filters (Millipore, Bedford, Mass.). Electrophoresis was performed by the Clinical Laboratory at the University of South Alabama Medical Center.
RESULTS AND DISCUSSION Samples injected a t the bottom of the column or peaks eluting from the column are mixed with the assay reagent a t the tee connection (F in Figure 1). LD catalyzes the conversion of L-lactate to pyruvate with NAD (nicotinamide adenine dinucleotide) as the cofactor. LD activity is determined from the rate of NADH production according to reaction 1.
LD
pyruvate
+ NADH
-
CK
creatine
glucose-6-phosphate
+
Figure 1. Schematic diagram of the apparatus. See text for identification of parts
+ NAD
+ glucose
HK
-
(1)
The reaction rate increases rapidly with increases in temperature (It?), but since the reaction coils are good heat exchangers, the post-column reaction temperature can be maintained a t the water bath temperature (13). The sensitivity and dynamic range of the post-column method (PCM) to applied LD activity were determined at a water bath temperature of 30 "C by direct sample injection below the column. Duplicate injections (21.3 pL) were made of samples containing bovine LD-1. The resulting regression equation for applied LD-1 activity ( x ) , ranging from 1.45 to 1480 International Units per liter (U/L), and recovered ac- 0.535 ( n = 22) with a cortivity (y), PCM, was y = 0 . 8 5 1 ~
+ ATP + ADP
-+
(2) (3)
GGPDH
glucose-6-phosphate NADP 6-phosphogluconolactone
I
L-lactate
+ creatine phosphate
NADP (4)
where ATP, ADP, HK, GGPDH, NADP, and NADPH are adenosine triphosphate, adenosine diphosphate, hexokinase, glucose-6-phosphate dehydrogenase, nicotinamide adenine dinucleotide phosphate, and its reduced form (the measured product), respectively. The rate of NADPH production in reaction 4 is equivalent to the rate of ATP production in reaction 2 only when the intermediates reach steady-state levels. The time required for this is termed the lag phase, which under our conditions has been reported to be about 1.5 min (20). This is slightly less than the 1.6 min required for CK activity to reach the first detector. The substrate concentrations for reactions 2, 3, and 4 in the post-column reaction coil were about four times less than those in the commercial CK reagent kit to reduce reagent cost. Samples assayed with diluted reagent yielded only 45% of the CK activity found with the full-strength reagent but did not affect the dynamic range of detection because samples are greatly diluted by the separation process. The sensitivity and dynamic range of the PCM to applied CK activity were determined by direct injection as before. Duplicate injections (102.8 pL) were made of samples containing rabbit CK-MM activity. The resulting regression equation for applied CK-MM activity ( x ) ranging from 0.45 to 470 U/L, and recovered activity (y) for PCM was y = 0.494~ - 0.047 ( n = 22) with a correlation coefficient of 0.999 and a standard error of the estimate of 1.27 U/L. Commercial control sera containing complete sets of LD and CK isoenzymes were used to establish peak identity, resolution, and recovery. The isoenzyme activity profiles obtained resulted from the transformation and subtraction of the response of the first detector from the second (14). This technique was evaluated and substantidy similar LD profiles of control materials were subsequently reported (21). The total LD activity applied to the column was 298.4 U/L (PCM). The sum of the isoenzyme activities after separation was 276.5 U/L; the recovery of activity applied to the column was 92.7% by fluorescence detection. The isoenzyme activities obtained by absorbance detection were similar to those by fluorescence and the recovery was 91.2%. The profile of CK isoenzymes in the same control material was obtained as above and is shown in Figure 2 to illustrate a complete chromatogram and corresponding retention times. The total CK activity (PCM) applied to the column was 117.8 U/L. The recovery of activity was 97.9% by fluorescence detection and 97.3% by absorbance detection. The profile of LD isoenzymes in a human serum sample is shown in Figure 3A: the total LD activity (PCM) applied to the column was 99.3 U/L and is within the normal range for our method, 75 to 165 U / L (13). The addition of human LD-2 to this serum produced the isoenzyme profile shown in Figure 3B. The total LD activity (PCM) of the spiked sample was 172.6 U/L, and the activity recovered from the column was 155.6 U/L (90.1%). The difference in applied activity between the two samples was 73.3 U/L. Since the difference between the LD-2 activities in A and B is 67.2 U/L, the recovery of added LD-2 activity was 91.7%, assuming that only LD-2 was added. The addition of human LD-1 to the
ANALYTICAL CHEMISTRY, VOL. 52, NO. 4, APRIL 1980 40.00
-
MM
70' 00
0.00i - - t - - - t - - - - c ' i
e
0.00
16
T I r E WIN,
Figure 2. Profile of CK isoenzyme activity in a CK/LD isoenzyme control
with fluorescence detection. The isoenzyme activities and percentages of total activity for CK-BB, CK-ME, and CK-MM were 16.4 (13.9%), 14.4 (12.2%), and 87.0 U/L (73.9%), respectively
"
"
"
TIllE
(WIN)
16
I
85.0 U/L
L D1
h
0,aea
A
Figure 4. Changes in the CK isoenzyme profile after the addition of CK-MM (C) or CK-BB (B) to a human serum sample (A). The activity of CK-MM in (A) was 22.5 U/L. The CK-MM and CK-BB activities in (B) were 20.3 and 46.7 U/L, respectively. The CK-MM activity in (C)
was
0.040
MM
T
0
731
i
0
~
~
~
T1r-E
~
(-1%:
~
0.125
T
LO5
~
24
Flgure 3. Changes in the LE isoenzyme profile after the addition of LD-1 (C) or LD-2 (B) to a human serum sample (A). The isoenzyme activities in (A) were 17.8, 6.6, 1.7, 7.1, 34.0 and 22.3 U/L for LD-5,
0. 0 0 0
d ' " ' , ' ' TIME (flIN)
24'
and LD-1, respectively. The isoenzyme activities in (B) were similarly 16.1, 5.2, 3.0, 7.8, and 101.2, and 22.3 U/L, respectively. The isoenzyme activities in (C) were similarly 17.0, 4.1, 1.5, 6.2, 32.6, and 117.8, respectively
Figure 5. Comparison of LD isoenzyme profiles from the sera of a normal (A) and an abnormal (B) individual. The isoenzyme activities in (A) were 19.3, 0, 6.1, 2.6, 7.2, 37.0, and 26.2 U/L for LD-5, LD-4, LD-3", LD-3', LD-3, LD-2, and LD-1, respectively. The isoenzyme activities in (B) were similarly 131.9, 15.3, 22.3, 17.9, 61.7, 121.8, and
same serum produced the isoenzyme profile shown in Figure 3C. The total LD activity (PCM) of this spiked sample was 193.1 U/L, and the activity recovered from the column was 179.5 U / L (93.0%). The difference in applied activities between this sample and the control was 93.8 U/L, and the recovery of added LD-1 activity was 102%. Spiking human serum with either LD-1 or LD-2 appeared to affect only the corresponding activities in the control serum, and better than 90% of the activity was recovered. This agrees with previous studies using bovine LD isoenzymes ( 1 4 ) . The difference in retention times and the shape of the LD-3 peaks is attributed to insufficient washing of the column with weak buffer between runs. It is apparent that all LD isoenzymes are resolved to base line and that multiple LD-3 activities are present. Multiple LD-3 peaks have been observed previously ( 1 3 , 2 2 )and may represent the complexation of LD-3 with other biological molecules (23). The CK isoenzyme profile from the serum of the normal donor is shown in Figure 4A. Observe that the MB and BB isoenzymes are absent. The total CK activity (PCM) applied to the column was 22.5 U/L,and the recovery of applied activity was 100%. The addition of rabbit CK-BB to this
serum produced the isoenzyme profile shown in Figure 4B. The total CK activity (PCM) of this spiked sample was 71.8 U/L, and the activity recovered from the column was 67.0 U/L (93.3%). The difference in applied activity between the two samples was 49.3 U/L. Because the activity of CK-BB in Figure 4B was 46.7 U/L, the recovery of added CK-BB was 94.7%. Spiking this serum with rabbit CK-MM produced the isoenzyme profile shown in Figure 4C. The total CK activity and added CK-MM recoveries were both 101%. Serum with an elevated level of LD activity exhibited the isoenzyme profile of Figure 5B. This profile can be compared with that obtained from a normal donor (Figure 5A). The isoenzyme activities in B are substantially greater than the corresponding activities in A. The only qualitative difference between the two profiles is the absence of LD-4 in A. When comparing the two profiles, the greatest differences are seen in the LD-5 and LD-3 peaks. The high level of LD-5 suggests liver damage, while the high level of LD-3 indicates pancreatic disease, because the liver and pancreas are rich in LD-5 and LD-3, respectively (16). The clinical diagnosis was "necrotizing pancreatitis" accompanied by systematic infection. Liver damage was manifest by moderate jaundice and elevated
LD-3", LD-3', LD-3, LD-2,
57.1 U/L
732
ANALYTICAL CHEMISTRY, VOL. 52, NO. 70. 00
0.00
T
0
4,
APRIL 1980
MM h
TInE (MIN)
’““T
0.“
16
Figure 6. Comparison of CK isoenzyme profiles from the sera of a normal (A) and an abnormal individual (B). The CK-MM activity in (A) was 23.4 U/L. The actlvities of CK-MM and CK-MB in (A) were 123.2 and 13.5 U/L, respectively
serum transaminases. The clinical findings agree with evidence obtained by comparing the suspect profiie with a normal profile. Elevated serum CK activity is usually associated with cardiac damage (8). The profile of CK isoenzymes obtained from the serum of a patient with an acute myocardial infarction is shown in Figure 6B. The presence of CK-MM is usually associated with damaged heart tissue (24))and the appearance of CK-MF3 strongly indicates myocardial infarction (25). The presence of CK-MB was confirmed by electrophoresis. Comparing Figure 6A (obtained from the serum of a normal individual) with B reveals a dramatic increase of CK-MM activity as well as the appearance of CK-MB. Serum isoenzyme profiling is most commonly used in the clinical laboratory to confirm the occurrence of myocardial infarction. Since post-infarction changes in these profiles do not occur for several hours and do not attain a maximum for one or two days afterwards (26))consecutive profiles are usually obtained to detect these changes and to evaluate their magnitude. An increase in LD-1 activity, especially if LD-1 becomes predominant over the other LD isoenzymes, indicates infarction (15). We obtained serum from a patient admitted to the Medical Center with pericardial chest pain. This serum exhibited the isoenzyme profile shown in Figure 7A. Although the total LD activity of this sample was in the normal range, the slight predominance of LD-1 suggests possible heart damage. The patient suffered a cardiac episode during the night and was diagnosed as having a “nontransmural, myocardial infarction of the inferior wall”. A serum sample obtained 10 h after this episode (18 h after the fmt sample) gave the isoenzyme profile shown in Figure 7B. Comparison of the two profiles reveals a pronounced increase in LD-1 activity, about fourfold, and a substantial but lesser increase in LD-2 activity. The detection of isoenzyme activity in some systems can be impaired by the presence of sample interferences, because serum contains components that interfere with the fluorescence detection of low levels of NADPH (27). Denton et al. (28)reported that serum interferences eluted in the CK-MB region from an anion-exchange column and produced a false CK-MB peak with fluorescence detection. We have also found that serum samples produced background peaks with fluorescence detection. The position of those peaks is apparent in Figure 8, which shows the fluorescence profile recorded at the first detector (A) and the profiie recorded at the second detedor (B). The serum sample, used to obtain these profiles, was that used to produce the
LD1
TIME ( f l I N )
24‘
Figure 7. Comparison of sequential, LD isoenzyme profiles from the sera of another patient in the cardiac care unit. (A) Admission profile. The isoenzyme activities in (A) were 6.3, 9.8, 40.8, and 43.2 U/L for LD-5, LD-3, LD-2, and LD-1, respectively. (B) Profile 18 h after admission. The isoenzyme activities in (B) were similarly 7.5, 33.5, 130.0,
and
183.0
U/L, respectively 18800
T
t
mm A
I\
0. e0
TIWE (PlIN)
’ 16‘
Figure 8. Comparison of the first (A) and second (B)fluorescence detector responses that shows the presence of nonreacting peaks in a serum sample. The serum sample was that used in Figure 7 8
isoenzyme profile shown in Figure 7B. The retention time of human CK-MB in Figure 8 is somewhat less than that for the CK-MB peak in the control material (prepared from rat tissue, Figure 2). This effect has been observed previously (22). Background peaks can be readily identified in Figure 8 because they do not increase in area as do peaks with CK activity. One background peak is apparent between CK-MM and CK-MB, and another elutes after CK-MB. The identification of these peaks is the subject of another paper (29). Since we use a two-point method, subtraction of A from B, background peaks are eliminated from the final activity profile, as seen in Figure 9B. The CK isoenzyme profile in the pre-infarction serum sample is shown in Figure 9A. The presence of CK-MB in this sample, confirmed by electrophoresis, indicates that the patient had already suffered some cardiac distress before admission. The large increase in CK-MB activity seen in Figure 9B indicates that the nighttime episode caused substantially more cardiac damage. This system has been shown to detect deviations from the normal in serum isoenzyme profiles that are associated with organ-specific disease, and such a system could be used to search for new correlation between profile changes and organ-specific disorders. Progressive changes in isoenzyme
Anal. Chem.
1980, 52, 733-736
733
zymes. J. Biol. Chem. 1977. 252. 5939. (3) Markert, C. L. Science 1983, 740, 1330. (4) Chamoles, N.; Karcher, D. Clin. Chim. Acta 1970, 3 0 , 337. (5) Dawson, D. M.; Eppenberger. H. M.: Kaplan, N. 0. Biochem. Biophys. Res. Commun. 1985, 21, 346. (6) Hooton, 6.T. Biochemistry 1988, 7, 2063. (7) Kudirka. P. J.; Schroeder, R. R.; Hewitt, T. E.; Toren, E. C. Clin. Chem. 1978, 22, 471. ( 8 ) Chang, S . H.; Noel, R. R.; Regnler, F. E. Anal. Chem. 1978, 46, 1839. (9) Chang, S. H.; Regnler, F. E. US. Patent 4029563, 1977.
(IO) Schlabach, T. D.; Chang, S. H.; Gooding, K. M.; Regnler, F. E.
J.
Chromatogr. 1977, 134, 91. (11) Chang, S.H.; Gooding, K. M.; Regnler. F. E. J. Chromatogr. 1978, 125,
0. 00
' 16'
T I M E (PIIN)
Figure 9. Comparison of sequential, CK isoenzyme profiles from the same serum used in Figure 7. (A) Admission profile. The CK-MM and CKMB activities in (A) were 106.5 and 12.8 U/L, respectively. (B)Profile 18 h after admission. The CK-MM and CK-MB activities in (B) were 168.1 and 30.1 U/L, respectively
102. (12) Schroeder, R. R.; Kudirka, P. J.; Toren, E. C. J. Chmmtogr. 1977, 134. 83. (13) Schlabach, T. D.; Fulton, J. A,; Mockridge, P. B.; Toren, E. C. Clin. Chem. 1979, 25, 1600. (14) Fulton, J. A.; Schbbach, T. D.; Kerl, J. E.; Toren, E. C.; Miller, A. R. J . Chromatog. 1979, 775, 269. (15) Cohen, L.; Djordvitch. J.; Ormiste, Y. J . Lab. Clin. Med. 1964, 64, 355. (16) Roe, C. R.; Limblrd, L. E.; Wagner, G. S.; Nerenberg, S. T. J. Lab. Clln. Med. 1972. 80. 577. (17) Galen, R . S:;Relffel, J. A.; Gambino, S. R. J. Am. Med. Assoc. 1975, 232,145. (18) Williams, E. B.; Lyons, R. 6.Anal. Biochem. 1971, 42, 342. (19) Rosalki, S. B. J . Lab. Clin. Med. 1967, 69, 696. (20) Szasz, G.;Gruber, W.; Bernt, E. Clin. Chem. 1978, 22, 650. (21)FuRon. J. A.: Schlabach. T. D.: Kerl. J. E.: Toren. E. C. J. Chrometwr. I97gS 775, 283. (22)Schlabach, T. D.; Alpert, A. J.; Regnler, F. E. Clin. Chem. 1978, 24,
-
1351
profiies were also detected and correctly predicted myocardial infarction.
ACKNOWLEDGMENT We thank William Dean, Corning Glass Works, Corning, N.Y. for supplying us with column support material and Paul Schneider, Biodynamics/bmc, Indianapolis, Ind., for providing some CK assay reagents. We are indebted to Timothy G. Kelly, American Instrument Co., Silver Spring, Md., for writing our file transfer program and assisting in program development and Ruth C. Ray1 and Dorothy N. Vacik for their help in preparing the manuscript. LITERATURE CITED (1) Hunter, R . L.; Markert, C. L. Science 1957, 725, 1294. (2) Report of the Subcommittee on Nomenclature of Interconvertible En-
(23) Tiicha, P. J. Clin. Chem. 1977, 23, 1780. (24)Tsung, S. H. Clin. Chem. 1978, 22, 173. (25)Wagner. G. S.;Roe, C. R.; Limbird, L. E.; Rosatl, R. A.; Wallace, A. G. Circulation 1973, X L V I I , 263. (26) Lederer, W. H.; Gerstbrein, H. L.; McCllntock, W. C. Am. J. Clh. Patho/. 1976, 66, 425. (27) Solni, E.; Hemmila, I. Clin. Chem. 1979, 25, 353. (28) Denton. M. S.:Bostick. W. D.: Dlnsmore. S.R.: Mrochek. J. Cffn. Chem. 1978, 24. 1408. (29) Schlabach, T. D.: Fukon. J. A.; Mockridge, P. B.; Toren, E. C., Clln. Chem., In press.
RECEIVED for review November 1, 1979. Accepted January 16,1980. This work was supported by Grant no. GM 24452 from the National Institutes of Health, and was presented in part at the 31st Annual Meeting of the American Association for Clinical Chemistry in New Orleans, La., July 1979.
Determination of Methanethiol at Parts-per-Million Air Concentrations by Gas Chromatography Rlchard Knarr and Stephen M. Rappaport" School
of Public Health,
Department
of Biomedical and
Environmental Health Sciences, University
A method is described for determining the air concentration of methanethlol. The sampling device, a 37-mm glass fiber filter Impregnated with mercurlc acetate, is suitable for either personal or area monltoring. Methanethiol is regenerated from the mercury mercaptide, formed on the fllter during sampling, by treatment wlth hydrochloric acid and Is dissolved in methylene chlorkle. Chantitation employs gas chromatography wlth flame photometric detection. The detection ilmit of 17 pg/m3 permits use of the method for determlning elther time-welghted average concentrations or 15min ceiling concentrations. The relative error of the method is I 1 0 % , while the relative standard deviation is I 1 %
.
Methanethiol is a commercially important gas which is used extensively in the manufacture of agricultural chemicals, pharmaceutical products, gas odorants, and specialty chemicals. It is also produced in large quantities as an unwanted
of California,
Berkeley, California 94720
by-product of paper manufacture and petroleum refining. The toxic effects of methanethiol have not been extensively investigated. It is known that the LCW (lethal airborne concentration to l/z the animals) for mice was 1664 ppm (I) and that mice and rhesus monkeys exposed to 50 ppm for 90 d experienced mortality rates in excess of 40% (2). One human death has been attributed to exposure to airborne methanethiol ( 3 ) . Given the paucity of information concerning the health effects of methanethiol in man, standards and guides for occupational exposure have been based in part upon the offensive odor of this compound. Although the current OSHA standard for airborne methanethiol is 10 ppm, a Threshold Limit Value (TLV) of 0.5 ppm has been adopted by the American Conference of Governmental Industrial Hygienists (ACGIH) ( 4 ) and proposed by the National Institute for Occupational Safety and Health (NIOSH) (5). Significantly, this 0.5-ppm limit is suggested as an 8-h time-weighted average (TWA) TLV by ACGIH ( 4 ) and as a ceiling concentration
0003-2700/80/0352-0733$01.00/0 0 1980 American Chemical Society
