317
Clinica Chimica Acta, 89 (1978) 317-329 0 Elsevier/North-Holland Biomedical Press
CCA 9763
HUMAN ERYTHROCYTE PHENOL 0-METHYLTRANSFERASE: RADIOCHEMICAL MICROASSAY AND BIOCHEMICAL PROPERTIES P.A. PAZMIfiO
and R.M. WEINSHILBOUM
*
Clinical Pharmacology Unit, Departments of Pharmacology and Internal Medicine, Mayo Foundation, Rochester, MN 55901 (U.S.A.) (Received
May 5th, 1978)
Summary A radiochemical microassay for the determination of phenol O-methyltransferase (PMT) activity in human red blood cell membranes has been developed. Acetaminophen was used as the substrate. The apparent Michaelis-Menten (K,) value for acetaminophen was 21.2 X 10m3 M. The apparent KM value for S-adenosyl-I,-methionine, a co-substrate for the reaction, was 4.8 X 10e6 M, and the pH optimum of the reaction was approximately 9.0 with four different buffer systems. Phenol was also tested as a substrate and had an apparent KM value of 2.0 X 10e3 M. Human erythrocyte (RBC) membrane PMT activity did not have the biochemical characteristics of catechol 0-methyltransferase, another RBC membrane methyltransferase enzyme activity. Blood samples obtained from 212 randomly selected adult white subjects had a mean activity of 134.5 + 41.5 pmol of p-acetanisidide formed per mg protein per hour (mean + S.D.). Activities varied from 44 to 282 units. There were no differences in the mean activities of samples from men and women. Experiments in which preparations were mixtures of “low” and “high” activity RBC membrane assayed for PMT provided no evidence that the variations in enzyme activity were due to the presence of endogenous PMT activators or inhibitors. RBC membrane PMT activity in blood from 9 patients with renal failure, a pathological state in which there are elevated circulating levels of phenols, was found to be significantly decreased with average activity of 76.2 + 9.7 (mean + S.E.M.,
P < 0.001). Introduction Phenol and phenolic compounds are widely used in industry and in medicine. The two major metabolic pathways for these compounds are conjugation * Address reprint tion, Rochester,
requests to: MN 55901.
Dr. Richard U.S.A.
Weinehilboum.
Department
of Pharmacology,
Mayo
Founda-
318
with glucuronic acid or with sulphate [ 1,2]. Minor metabolic pathways include methylation and glutathionine conjugation [3,4]. It has recently been shown that quantitatively minor metabolic pathways may play important roles in the toxicity of some drugs (e.g., acetaminophen overdosage) [4]. Because of the possibility that “minor” metabolic pathways might play a role in the variation of responses of individuals exposed to phenols either medically or occupationally, it is important that methods for the assessment of individual variations in these metabolic reactions be developed. As a first step towards increasing our understanding of methylation of phenols by the enzyme phenol O-methyltransferase (EC 2.1.1.25, PMT), we have developed a procedure for the measurement of PMT activity in human erythrocytes. The measurement of this enzyme activity in an easily obtained human tissue may help make it possible to determine whether individual variations in PMT activity play a role in variations in the therapeutic response to or occurrence of toxicity with the use of phenolic compounds in medicine and industry. Materials and methods 1. Blood samples Blood was collected in 5 ml heparinized vacutainer tubes by venipuncture. Plasma was separated from the formed elements of blood after centrifugation at 800 X g for 10 min in a refrigerated centrifuge. Three ml of 0.9% sodium chloride was added to the pellet and the cells were gently resuspended. After repeat centrifugation at 800 X g for 10 min, the supematant and buffy coat were discarded. The cells were washed a second time with saline, and after centrifugation the supematant was again discarded. Two ml of normal saline was added to the pellet, and, after mixing, 2 ml of the resuspended “washed” erythrocytes was then added to 8 ml of ice-cold water to lyse the cells. The erythrocyte (RBC) lysates were centrifuged at 13 000 X g for 10 min, and 1 volume of the packed pellet was resuspended in approximately 10 volumes of Tris-HCl buffer, 5 mM, pH 7.8. The resuspended erythrocyte membranes were centrifuged again at 13 000 X g for 5 min. The supematant was discarded and the packed RBC membranes were resuspended in Tris buffer, 5 mM, pH 7.8 for 2 additional washes and centrifugation steps. At the end of this procedure the packed membranes had a pearly white appearance. The RBC membrane
NHCOCHJ
lQ OH ACETAMINOPHEN (4’-HYDROXYACETANILIDE)
NHCOCH3 I
Q
.7-y SAM-*CH,
SAH
0fCH3 p-ACETANISIDIDE (p-METHOXYACETANILIDE)
Fig. 1. PMT assay reaction sequence for acetaminophen. SAH represents S-adenosyl-L-homocysteine.
SAM represents S-adenosyl-L-methionine
and
319
preparations were then transferred -85°C prior to enzyme assay.
to 6 ml plastic
tubes
and were stored
at
2. PMT assay The PMT reaction sequence is shown in Fig. 1. One hundred ~1 aliquots of RBC membrane suspensions were placed in 15-ml stoppered glass conical centrifuge tubes. Forty ~1 of Tris-HCl buffer, 0.25 M, pH 9.5, was added to “blank” samples, and 40 ~1 of the same buffer which contained acetaminoof phen, 187.5 mM was added to “active” samples. The final concentration acetaminophen was 50 mM. The enzyme reaction was initiated by the addition of 10 ~1 of a mixture of the following reagents (final concentration in 150 r_ll indicated): S-adenosyl-L-[ 14C]methionine (specific activity 55 mCi/mmol) 6.0 X 10e6 M and reduced glutathione 5 X lo-’ M. The reaction tubes were incubated for 45 min at 37°C in a shaker water bath, and the reaction was stopped by the addition of 0.5 ml of 1 M hydrochloric acid. Five ml of 3% isoamyl alcohol in toluene were added, and the tubes were stoppered and were mixed vigorously for 10 s on a vortex mixer. After centrifugation at 700 X g for 10 min in an International Model K centrifuge, 3.5 ml of the organic phase was removed and placed in a liquid scintillation counting vial that contained 10 ml of toluene fluor (5 g 2,5-diphenyloxazole and 0.1 gram of 1,4-bis[ 2-( 5-phenyloxazole)] benzene per liter). Radioactivity was determined in a Packard 3385 liquid scintillation counter. One unit of enzyme activity represented one pmol of p-acetanisidide formed per mg RBC membrane protein per hour of incubation at 37°C. All results were corrected for the extraction of p-acetanisidide into the organic phase (90%) and for quench and counting efficiency. Exactly the same procedure was used when phenol was the substrate for the enzyme reaction except that the final concentration of phenol in the 150 ~1 volume was 10 mM. 3. Catechol 0-methxltransferase (COMT) assay In some experiments COMT activity was measured in RBC membranes. COMT activity was measured by a modification of the assay of Raymond and Weinshilboum [ 51. The modifications included the use of a final S-adenosyl-L[‘4C]methionine concentration of 12 PM with no addition of non-radioactive S-adenosyl-L-methionine and the substitution of reduced glutathionine, 1 mM, for dithiothreitol. In addition, the membrane preparations were not exposed to the solid chelating resin Chelex-100. 3,4-Dihydroxybenzoic acid was used as a substrate for COMT. The 4-methoxy-3-hydroxybenzoic acid (vanillic acid) formed in the presence of S-adenosyl-L-methionine was extracted by organic solvent extraction into toluene and its radioactivity was determined in a liquid scintillation counter. 4. Ery throcyte membrane protein determinations The Biuret method [6] was used to determine protein concentrations in erythrocyte membrane preparations. The membrane preparations were exposed to Triton X-100 at a final concentration of 1% to eliminate turbidity. Bovine serum albumin was used as a standard for the protein assay.
320
5. Thin-layer chromatography The radioactive product of the enzyme reaction was identified by thin-layer chromato~phy on Eastman Chroma~am sheets of silica gel, 100 micron in thickness which contained a fluorescent indicator. The two solvent systems Iused were benzene/methanol/acetic acid (90 : 8 : 4, v/v) and chloroform/ methanol (9 : 1, v/v). Chromatographic sheets were activated by drying for 15 min at 80°C immediately prior to use. After development the p-acetanisidide spots were identified by the use of ultraviolet light. The chromatograms were marked and cut into 1 cm strips that were placed in counting vials that contained 1.0 ml of Soluene-100 (Packard Instrument Company). After 1 h, 1 ml of absolute ethanol and 10 ml of toluene liquid scintillation counting fluid were added to each vial and the samples were placed in a liquid scintillation counter for the measurement of radioactivity. All results were corrected for quench after the addition of an internal standard of [ 14C]toluene. 6. Source of biood samples Blood samples were obtained from 212 randomly selected adult white blood donors at the Blood Bank of the Mayo Clinic in Rochester, Minnesota. The donors were not related and were not taking medications at the time that the blood samples were obtained. 7. Kinetic analysis Michaelis-Menten (KM) values were determined by the method of Wilkinson [ 71 with a Fortran program written by Cleland [S]. A Control Data Corporation 3500 Computer was used for these calculations. 8. Materials S-Adenosyl-L-[ 14C] methionine (specific activity 55 mCi/mmol) was purchased from New England Nuclear Corporation, Boston, Massachusetts. Tris(hydroxymethyl) aminomethane base, reduced glutathione, S-adenosyl-L-homocysteine, 3,4dihydroxybenzoic acid and cyclohexyl~inopropane sulfonic acid (CAPS) were purchased from Sigma Chemical Company, St. Louis, MO. 4’-Hydroxy-acetanilide, p-acetanisidide, and N-ethylmaleamide were purchased from Eastman Kodak Company, Rochester, N.Y. Phenol was obtained from Fisher was purchased from Regis Scientific Company, Fairlawn, N.J. Tropolone Chemical Company, Morton Grove, IL., and dithiothreitol (Cleland’s reagent) was obtained from ~~biochem, San Diego, CA. SKF 525A was kindly provided by Dr. R. Van Dyke of the Mayo Clinic. Results f. Choice of blank Lysates of human erythrocytes contain an enzyme activity that results in the formation of radioactively labelled methanol in the presence of S-adenosyl-L[14C]methionine [9]. This activity was originally thought to be a “methanol forming activity”, but it is now known that it results from the action of a protein carboxymethylase which methylates endogenous protein substrate(s) [ 101. During organic solvent extraction these radioactively labelled protein esters are
321
cleaved nonenzymatically, and the radioactively labelled methanol is extracted into the organic solvent [ 111. Our initial experiments with human RBC membrane preparations demonstrated that heated blanks consistently gave significantly lower counts per min (cpm) than did samples in which the assay was performed in the absence of substrate. For example, in one series of experiments it was found that a “no substrate” blank yielded average counts per min of 217 f 7 (mean + S.E.M., N = 5) and heated enzyme blanks yielded average counts per min of 127 f. 2. Whether or not a protein carboxymethylase similar to that found in erythrocyte lysates is present in RBC membranes is not clear, but it is obvious that a heated enzyme sample is not an appropriate blank for this assay. 2. Linearity of reaction with increasing concentration of lysate PMT activity increased with increasing quantities of RBC membrane in a linear fashion over a range of 25 to 100 ~1 of RBC membrane preparation (approximately 0.1 to 0.4 mg membrane protein, Fig. 2). All assays were performed with concentrations of RBC membranes that fell within this linear range. 3. Time course The time course of the enzyme reaction was linear to 60 min (Fig. 3). All assayed were performed at an incubation time of 45 min. 4. pH optimum The effect of pH on PMT activity in RBC membrane preparations is shown in Fig. 4. All pH determinations were made at 20°C in the presence of the membrane preparation and all components of the reaction mixture. A pH optimum of approximately 9.0 was found with four different buffer systems. All buffers were present in a final concentration of 0.07 M. Tris buffer was chosen for use in the assay since the pH optimum of 9 is within the range that Tris is an adequate buffer_ and since, unlike some of the other buffer systems, TrisHCl did not appear to inhibit the enzyme reaction. HUMAN
25 RBC
RBC
PMT
100
50 MEMBRANES,
vi
Fig. 2. Effect of increaehg quantities of RBC membrane on PMT activity. Each Point represents the mean of three determinations.
322
HUMAN
x
-
I $
1.6 -
RBC
PMT
lz
0 0
I I5
I 30
1 45
TIME,
MINUTES
Fig. 3. Effect of increasing three dbferminations.
1 60
I 75
time of incubation
I 90
on RBC
PMT activity.
Each
point
represents
the mean of
5. Relation of enzyme activity to substrate concentrations PMT activity in human RBC membranes was measured in the presence of varying concentrations of acetaminophen and S-adenosyl-L-[14C]methionine (14C-SAM), the two co-substrates for the reaction (Fig. 5). Because of the limited solubility of acetaminophen in water, several different solvents were used in an attempt to dissolve the compound more easily. These solvents included methanol, ethanol, dimethyl sulfoxide (DMSO), dimethylformamide (DMF), and acetonitrile. Although all of these solvents dissolved the acetaminophen they also apparently inhibited the enzyme reaction (see Fig. 5 for the effect of 25% DMF). DMSO did not inhibit the PMT reaction, but the use of DMSO resulted in a significant increase in the values of blanks, and thus, a
HUMAN
RBC
PMT
1.6 -
Polaarium
Phosphate
TRIS D Aspwlic
Acid
CAPS
o’,
, 7.0
I
,
,
(
9.0
9.0
,
10.0
PH Fig. 4. Effect of pH on RBC membrane PMT activity. CAPS represents acid. Each point represents the mean of three determinations.
cyclohexylaminopropane
sulfonic
323 HUMAN RBC PMT (Al BI_~
0
1.6 n ‘0
50
25 A~ETAMINOPHEN,
mM re
1.2
e-
x 0.6 I "0 0.4 0
Fig. 5. Effect of substrate concentration on PMT activity. (A) RBC membrane PMT activity was determined in the presence of increasing concentrations of acet~nophen dissolved fn water (open circles) and acetaminophen dissolved in 26% dimethylformamide DMF fsquares). Each point represents the mean of three determinations. (B) Effect of varying concentrations of SAM (S-adenosyl-L-methionine) on PMT activity, on “blank” values, and on the ratio of counts per min (CPM) in active samples divided by CPM in blanks. Each point represents the mean of three determinations.
decrease in the sensitivity of the assay. Therefore, it was elected to use acetaminophen dissolved in a relatively large volume of Tris buffer. The data obtained from the acetaminophen substrate curve were used to calculate an apparent Michaelis-Menten (KM) constant, 21.1 X 1O’3 M. The apparent KM value for S-adenosyl-L-methionine was 4.8 X 10e6 M. With increasing concentrations of radioac~vely labelled ~-adenosyl-L-methionine, blank values rose, and the “signal to “noise ratio” (cpm in active samples divided by cpm in blanks) decreased. This fact, plus the expense involved in the use of high concentrations of S-adenosyl-L-[14C]methionine resulted in the choice of 6.0 PM as the concentration of SAM used in the assay. This choice was made with the full knowledge that the assay was performed with less than saturating conditions for S-adenosyl-L-me~ionine. Therefore, the results reported here can only be compared with results obtained from assays in which similar concentrations of S-adenosyl-L-methionine are utilized. It was not possible to add non-radioactive S-adenosyl-L-methionine to the reaction mixture because the decrease in specific activity which occurred after this maneuver resulted in an appreciable loss in the sensitivity of the assay. Tritiated S-adenosyl-L-methionine of higher specific activity was not used in the assay because of lack of stability of this reagent and a difficulty with day-to-day reproducibility of results. 6. Thin-layer chromatogmphy of reaction product The organic solvent extract from an assay in which RBC membrane PMT had
324
been determined was dried under a stream of nitrogen and chromatography was performed with two solvent systems (see Methods above). The Rf values of the radioactive product formed were identical with those of authentic p-acetanisidide in both solvent systems (Fig. 6). 7. Coefficient of variation of the assay procedure The coefficient of variation for the assay of RBC membrane PMT with acetaminophen as substrate was determined with membrane preparations obtained from two separate subjects. The coefficients of variation for 10 assays for each of these preparations were 5.8% and 5.7%. 8. Phenol as a PMT substrate Phenol was also tested as a substrate for PMT (Fig. 7). Data from the substrate curve were used to calculate an apparent K, value of 2 X 10-j M. Although this apparent KILl was much lower than that for acetaminophen, the enzyme activity was noted to decrease at concentrations of phenol above 10 mM. Whether this phenomenon was due to substrate inhibition or to destruction of the enzyme by high concentrations of phenol is not clear. 9. Characteristics of RBC PMT activity PMT in the guinea pig liver is a membrane-associated enzyme [3]. To determine whether human RBC PMT is a membrane-associated enzyme, it was measured in erythrocyte membranes and in the supematant from lysates of 10 subjects. There was no detectable PMT activity in the supematants. Therefore, human RBC PMT is also a “membrane bound” enzyme. Erythrocyte membranes contain a variety of enzyme systems. Included among these is catechol 0-methyltransferase (EC 2.1.1.6, COMT) [12]. Axelrod and Daly have clearly differentiated PMT from COMT activity in guinea pig liver membrane preparations [ 31. To determine whether human RBC PMT is
SOCVENT FRONT
ORIGIN
STRIP Fig. 6. Thin-layer chromatography dark bars represents the migration
ORlGlN
SOLVENT FRONT
STRIP of the product of PMT activity of authentic p-acetanisidide.
using acetaminophen
as substrate.
The
325 HUMAN
RBC
PMT
I 25
0
I 50
PHENOL, mM Fig. 7. Effect of varying substrate mean of three determinations.
concentration
of phenol
on PMT activity.
Each
point
represents
the
separate from RBC membrane COMT activity, a series of experiments was performed in which the characteristics of the PMT activity were compared with those of RBC membrane COMT. COMT is a magnesium dependent enzyme and is inhibited by a variety of compounds including tropolone, S-adenosyl-Lhomocysteine and calcium [13,14]. As can be seen in Table I, RBC membrane PMT activity was not magnesium dependent. In addition although RBC membrane COMT activity was dramatically inhibited by both calcium and tropolone, calcium had no inhibitory effect on PMT and tropolone showed only a quantitatively small inhibition (Table I). The S-adenosyl-L-homocysteine inhibition of both enzymes was not surprising since this compound inhibits a wide variety of methyltransferase enzymes [ 151. Axelrod and Daly also showed that guinea pig liver membrane PMT is inhibited by SKF 525A @-diethylaminoethyldiphenyl propyl acetate) and by N-ethylmaleamide [3]. The human RBC membrane PMT activity that remained after the addition of N-ethylmaleimide (1 X lop4
TABLE
I
CHARACTERISTICS
OF RBC MEMBRANE
PMT AND COMT
Results for COMT are expressed as percentage of assay with [Mg2+l = 1 mM and for PMT as percentage All COMT determinations of assay in the absence of Mg 2+. SAH represents S-adenosyl-L-homocysteine. and ail with the exception of the assay with no Mg2+ were performed in the presence of 1 mM M&l?, assays for PMT with the exception of that performed with 1 mM MgCl2 had no magnesium present. Values are mean f S.E.M. for three determinations. Enzyme
Assay conditions
Relative
COMT
Mg2+, 1 mM No Mg2+ Tropolone. 1 mM Ca2+, 1 mM SAH, 0.1 mM
100 0 14 * 4 10 f 1 5r1
PMT
No Mg2+ Mg2+, 1 mM Tropolone. 1 mM Ca2+, 1 mM SAH, 0.1 mM
100 116 f 1 72 f 4 116 f 1 2*1
activity
(W)
326
M), SKF 525A (5 X 10W4M) and Triton X-100 (0.1%) to reaction mixtures was 52%, 35%, and 6% of control values respectively. Taken together these results demonstrate that the PMT activity in the human RBC membrane is not the result of COMT activity and that the characteristics of human RBC membrane PMT are similar to those of the PMT activity in the guinea pig liver. 10. Effect of sample handling on RBC PMT uctivity Blood samples obtained from human subjects under clinical conditions might be handled in very different fashions. Therefore, PMT activity was measured in blood obtained from 20 randomly selected adult subjects (16 male and 4 female) and the effects of various methods of h~dling the samples were compared. As can be seen in Table II, there was no significant difference between the enzyme activities measured immediately after the blood samples were obtamed as compared with enzyme activity measured after storage of erythrocyte membranes at -85°C for up to 2 weeks or in membranes prepared from blood samples which had remained at room temperature for 24 h prior to the preparation of the membranes. These observations have obvious practical importance in a clinical setting if PMT activity is to be measured in blood samples obtained from patients. Because the use of washed e~throcytes makes sample prep~ation a lengthy, time-consuming procedure, an experiment was performed in which RBC membranes from 3 subjects were prepared by direct lysis of 2 ml heparinized whole blood in 8 ml of ice-cold water. The membranes were then washed twice with 5 mM Tris-HCl, pH 7.8. The mean PMT activity of these 3 samples was 131.3 + 0.9 units/mg protein. This value was very similar to the average value of 128.5 & 3.2 (mean ?r S.E.M.) in RBC membranes prepared in the usual fashion (i.e., with washed erythrocytes from the same 3 samples). These results indicate that it is probably not necessary to perform the time consuming process of “washing” the erythrocytes before tysis. The washing procedure was used for the studies described here because simultaneous experiments were being performed in which other enzyme activities were measured in lysates. Plasma contamination altered some of these enzyme activities.
11.PMT activity in blood of randomly selected population PMT activity was measured in erythrocyte membranes of blood samples obtained from 212 randomly selected adult subjects. The mean PMT activity was
TABLE 11 EFFECT OF METHOD OF HANDLING AND STORAGE ON RBC PMT ACTIVITY Blood sampfes from 20 subjects were treated in a variety of fashions prior to the determination of enzyme activity (see text for de&&s). All values are mean + S.E.M. Treatment
PMT activity/units
Immediate preparation and assay Preparation and assay after 24 h at room temperature Storage of membranes at 45% for 48 h Storage of membranes at -85’C for 2 weeks
139.6 140.1 141.6 138.1
f. 5.9 t 5.6 f 5.8 -?z6.3
327
134.8 -t 41.5 units (mean + SD.) with a range from 44 to 282 units. There was a four-fold variation of enzyme activity within the range of t2 standard deviations. The activity for female subjects was 135.0 + 42.9 units {N = 83) and that in membrane preparations from male subjects was 133.8 + 40.7 units (N = 129). The frequency distribution histograms for RBC membrane PMT activities for this population are shown in Fig. 8. To determine whether or not the relative PMT activity was similar when phenol was used as a substrate, RBC membranes from 14 randomly selected subjects were assayed with both phenol and ace~minophe~ as substrates. There was an excellent correlation between the relative PMT activities with the two substrates (r = 0.92, P < 0.001, Fig. 9). Finally, an experiment was performed to help determine whether the variation in PMT activity in RBC membranes from randomly selected subjects might reflect differences in the levels of endogenous PMT activators, inhibitors or un~ticipa~d competing enzyme systems rather than differences of PMT activity itself. Membrane preparations from 7 subjects with relatively low enzyme activities were mixed with samples from 7 individuals with relatively high PMT activities and the enzyme activity was measured. As is shown in Table III, the PMT activities in the mixtures were not significantly different than the expected arithmetic mean values-a result which makes it ‘less likely that endogenous enzyme activators or inhibitors can account for the variations in RBC PMT activity observed in a population of randomly selected subjects.
RBC PMT 212
UNRELATED
SUBJECTS
>
HUMAN RBC
-j 129 UNRELATED
MALE
SUBJECTS
l
83
1 0
UNRELATED
FEMALE
SUBJECTS 550
80
RBC PMT
160 ACTIVITY,
PMT
240
UNITS
.
3.
l
r = 0.918 P < ,001
.
N.
I4
PMT ACTIVITY. CPWMG PROTEIN (ACETAMINOPHEN SUBSTRATE)
Fig. 8. Frequency distribution histo8ram of RBC! PMT activity; top, 2x2 unrelated subjects; middle, ,129 unrelated male subjects; and bottom, 83 unrelated female subjects. Fig. 9. Correlation of RBC memkme PMT activity with two different substrates. PMT activity in RBC membranes of 14 individuals were measured with both acetaminophen and Phenol as substrates.
328 TABLE III HUMAN RBC MEMBRANE
PMT
Equal volumes of RBC membranes from 7 subjects with relatively low and 7 subjects with relatively high PMT activity were mixed and the PMT activity was measured. All values are mean i S.E.M. Low activity (units)
Mixture Actual
Mean
r S.E.M.
Range
58.5 +_3.8 46 to 70
139.1
High activity (units)
_--__. Expected + 8.2
134.7
._
+ 7.6
207.0 f 11.5 163 to 258
12. RBC PMT in blood of patients with renal failure It has been reported that phenol levels are elevated in the blood of patients with renal failure, and it is thought that phenols may play a role in the symptoms suffered by these patients [ 16-181. Therefore it was elected to examine RBC membrane PMT in patients with chronic renal failure on maintenance hemodialysis as the first clinical application of this new test. Blood was obtained from 9 patients randomly selected prior to hemodialysis, and their PMT activity was measured. The average activity was 76.2 2 9.7 (mean + S.E.M.). This value is significantly lower than that present in blood from a randomly selected population (P < 0.001). Whether this observation reflects an effect of elevated phenol levels, is related to the altered hematopoiesis present in azotemic patients, or is due to some other factor remains to be determined. However this limited and preliminary observation does demonstrate that the application of the RBC PMT assay procedure may begin to open new areas for further clinical investigation. Discussion Phenol ~-methyltransfer~e (PMT) is a membr~e-bound enzyme which catalyzes the O-methylation of many phenols 131. We have demonstrated that there is a PMT activity in human erythrocyte membranes and that the biochemical properties of this enzyme activity appear to be similar to those of guinea pig liver PMT [ 31. An assay procedure for the measurement of human RBC PMT activity has been developed, and a four-fold variation in RBC PMT activity in blood from a large population of randomly selected subjects has been demonstrated. Finally, preliminary studies have shown that there is a significant decrease in RBC PMT activity in blood of patients with renal failure on chronic hemodialysis as compared with values in a randomly selected population. Although the role of PMT in the metabolism of drugs and other xenobiotic substances is not clear, it is known that qu~ti~tively minor pathways of drug metabolism can play important roles in the toxicity of some compounds [ 191, For example, although acetarninophen is metabolized primarily by glucuronate and sulphate conjugation, the quantitatively minor pathway of glutathione conjugation is crucial with regard to the determination of whether or not hepatocellular damage will occur after ingestion of a toxic dose of acetaminophen
329
[4,19]. It is important that we begin to characterize the biochemical basis of individual variations in the metabolism of drugs and other xenobiotic agents if we are to begin to understand individual differences in drug response and toxicity. The measurement of PMT activity in an easily obtained human tissue, blood, represents a first step in the expansion of our knowledge of this drug metabolizing enzyme activity in humans. Whether or not the PMT activity measured in the human erythrocyte membrane is biochemically similar to that found in other human tissues, and whether its regulation in the erythrocyte is similar to that in other organs must be determined in future experiments. However, the availability of a sensitive assay procedure for the measurement of PMT activity in human blood may make it possible for us to begin to attempt to answer these questions and to determine whether individual variations in this drug metabolizing enzyme activity may play a role in individual response to drugs and other xenobiotics. Acknowledgements This study was supported in part by NIH grants NS 11014 and HL 17487. Dr. Weinshilboum is an Established Investigator of the American Heart Association. We thank Fredrick Raymond, Joel Dunnette and Luanne Wussow for their assistance with these studies. We also thank Drs. J.C. Mitchell, P. Frohnert and V. Torres for providing blood samples from patients. References 1 Mandel, H.G. (1971) in Fundamentals of Drug Metabolism and Drug Disposition (La Du, B.N.. Mandel. H.G. and Way, E.L., eds.). pp. 149-181. Williams and Wilkins, Baltimore 2 Goldstein, A., Aronow. L. and Kabnan. S.M. (1974) in Principles of Drug Action, 2nd edn.. PP. 227-289. Wiley, New York 3 Axelrod. J. and Daly, J. (1968) Biochim. Biophys. Acta 159.472478 4 Mitchell, J.R.. Jollow. D.J., Gillette, J.R. and Brodie, B.B. (1973) Drug Metab. Dispos. 1, 418423 5 Raymond, F.A. and Weinshilboum. R.M. (1975) Clin. Chim. Acta 58.185-194 6 Gomall. A.G.. Bardawill. C.J. and David, M.M. (1949) J. Biol. Chem. 177.751-766 7 Wilkinson, G.N. (1961) Biochem. J. 80.324-332 8 Cleland, W.W. (1963) Nature 198.463465 9 Axelrod, J. and Cohn, C.K. (1971) J. Pharmacol. Exp. Tber. 176.650-654 10 Diliberto, E.J. and Axelrod. J. (1974) Proc. Natl. Acad. Sci. U.S.A. 71,1701-1704 11 Kim,S. and Paik, W.K. (1970) J. Biol. Chem. 245.1806-1813 12 Poitou, P., Assicot. M. and Bohuon. C. (1974) Biomedicine 21.91-93 13 Guldberg. H.C. and Marsden. C.A. (1975) Pharmacol. Rev. 27.135-206 14 Weinshilboum, R.M. and Raymond, F.A. (1976) Biochem. Pharmacol. 25.573-579 15 Coward, J.K., DWrsoScott, M. and Sweet, W.D. (1972) Biochem. Pharmacol. 21,1200-1203 16 Wardle. E.N. and Wilkinson. K. (1976) Clin. Nephrol. 2.361-364 17 Rabiner, S.F. and Molinas, F. (1970) Am. J. Med. 49.346-364 18 Wengle, B. and Hellstrom. K. (1972) Clin. Sci. 43.493-398 19 Mitchell, J.R. and Jollows. D.J. (1975) Gastroenterology 68, 392410
Clinica Chimica Acta, 89 (1978) 317-329 0 Elsevier/North-Holland Biomedical Press
CCA 9763
HUMAN ERYTHROCYTE PHENOL 0-METHYLTRANSFERASE: RADIOCHEMICAL MICROASSAY AND BIOCHEMICAL PROPERTIES P.A. PAZMIfiO
and R.M. WEINSHILBOUM
*
Clinical Pharmacology Unit, Departments of Pharmacology and Internal Medicine, Mayo Foundation, Rochester, MN 55901 (U.S.A.) (Received
May 5th, 1978)
Summary A radiochemical microassay for the determination of phenol O-methyltransferase (PMT) activity in human red blood cell membranes has been developed. Acetaminophen was used as the substrate. The apparent Michaelis-Menten (K,) value for acetaminophen was 21.2 X 10m3 M. The apparent KM value for S-adenosyl-I,-methionine, a co-substrate for the reaction, was 4.8 X 10e6 M, and the pH optimum of the reaction was approximately 9.0 with four different buffer systems. Phenol was also tested as a substrate and had an apparent KM value of 2.0 X 10e3 M. Human erythrocyte (RBC) membrane PMT activity did not have the biochemical characteristics of catechol 0-methyltransferase, another RBC membrane methyltransferase enzyme activity. Blood samples obtained from 212 randomly selected adult white subjects had a mean activity of 134.5 + 41.5 pmol of p-acetanisidide formed per mg protein per hour (mean + S.D.). Activities varied from 44 to 282 units. There were no differences in the mean activities of samples from men and women. Experiments in which preparations were mixtures of “low” and “high” activity RBC membrane assayed for PMT provided no evidence that the variations in enzyme activity were due to the presence of endogenous PMT activators or inhibitors. RBC membrane PMT activity in blood from 9 patients with renal failure, a pathological state in which there are elevated circulating levels of phenols, was found to be significantly decreased with average activity of 76.2 + 9.7 (mean + S.E.M.,
P < 0.001). Introduction Phenol and phenolic compounds are widely used in industry and in medicine. The two major metabolic pathways for these compounds are conjugation * Address reprint tion, Rochester,
requests to: MN 55901.
Dr. Richard U.S.A.
Weinehilboum.
Department
of Pharmacology,
Mayo
Founda-
318
with glucuronic acid or with sulphate [ 1,2]. Minor metabolic pathways include methylation and glutathionine conjugation [3,4]. It has recently been shown that quantitatively minor metabolic pathways may play important roles in the toxicity of some drugs (e.g., acetaminophen overdosage) [4]. Because of the possibility that “minor” metabolic pathways might play a role in the variation of responses of individuals exposed to phenols either medically or occupationally, it is important that methods for the assessment of individual variations in these metabolic reactions be developed. As a first step towards increasing our understanding of methylation of phenols by the enzyme phenol O-methyltransferase (EC 2.1.1.25, PMT), we have developed a procedure for the measurement of PMT activity in human erythrocytes. The measurement of this enzyme activity in an easily obtained human tissue may help make it possible to determine whether individual variations in PMT activity play a role in variations in the therapeutic response to or occurrence of toxicity with the use of phenolic compounds in medicine and industry. Materials and methods 1. Blood samples Blood was collected in 5 ml heparinized vacutainer tubes by venipuncture. Plasma was separated from the formed elements of blood after centrifugation at 800 X g for 10 min in a refrigerated centrifuge. Three ml of 0.9% sodium chloride was added to the pellet and the cells were gently resuspended. After repeat centrifugation at 800 X g for 10 min, the supematant and buffy coat were discarded. The cells were washed a second time with saline, and after centrifugation the supematant was again discarded. Two ml of normal saline was added to the pellet, and, after mixing, 2 ml of the resuspended “washed” erythrocytes was then added to 8 ml of ice-cold water to lyse the cells. The erythrocyte (RBC) lysates were centrifuged at 13 000 X g for 10 min, and 1 volume of the packed pellet was resuspended in approximately 10 volumes of Tris-HCl buffer, 5 mM, pH 7.8. The resuspended erythrocyte membranes were centrifuged again at 13 000 X g for 5 min. The supematant was discarded and the packed RBC membranes were resuspended in Tris buffer, 5 mM, pH 7.8 for 2 additional washes and centrifugation steps. At the end of this procedure the packed membranes had a pearly white appearance. The RBC membrane
NHCOCHJ
lQ OH ACETAMINOPHEN (4’-HYDROXYACETANILIDE)
NHCOCH3 I
Q
.7-y SAM-*CH,
SAH
0fCH3 p-ACETANISIDIDE (p-METHOXYACETANILIDE)
Fig. 1. PMT assay reaction sequence for acetaminophen. SAH represents S-adenosyl-L-homocysteine.
SAM represents S-adenosyl-L-methionine
and
319
preparations were then transferred -85°C prior to enzyme assay.
to 6 ml plastic
tubes
and were stored
at
2. PMT assay The PMT reaction sequence is shown in Fig. 1. One hundred ~1 aliquots of RBC membrane suspensions were placed in 15-ml stoppered glass conical centrifuge tubes. Forty ~1 of Tris-HCl buffer, 0.25 M, pH 9.5, was added to “blank” samples, and 40 ~1 of the same buffer which contained acetaminoof phen, 187.5 mM was added to “active” samples. The final concentration acetaminophen was 50 mM. The enzyme reaction was initiated by the addition of 10 ~1 of a mixture of the following reagents (final concentration in 150 r_ll indicated): S-adenosyl-L-[ 14C]methionine (specific activity 55 mCi/mmol) 6.0 X 10e6 M and reduced glutathione 5 X lo-’ M. The reaction tubes were incubated for 45 min at 37°C in a shaker water bath, and the reaction was stopped by the addition of 0.5 ml of 1 M hydrochloric acid. Five ml of 3% isoamyl alcohol in toluene were added, and the tubes were stoppered and were mixed vigorously for 10 s on a vortex mixer. After centrifugation at 700 X g for 10 min in an International Model K centrifuge, 3.5 ml of the organic phase was removed and placed in a liquid scintillation counting vial that contained 10 ml of toluene fluor (5 g 2,5-diphenyloxazole and 0.1 gram of 1,4-bis[ 2-( 5-phenyloxazole)] benzene per liter). Radioactivity was determined in a Packard 3385 liquid scintillation counter. One unit of enzyme activity represented one pmol of p-acetanisidide formed per mg RBC membrane protein per hour of incubation at 37°C. All results were corrected for the extraction of p-acetanisidide into the organic phase (90%) and for quench and counting efficiency. Exactly the same procedure was used when phenol was the substrate for the enzyme reaction except that the final concentration of phenol in the 150 ~1 volume was 10 mM. 3. Catechol 0-methxltransferase (COMT) assay In some experiments COMT activity was measured in RBC membranes. COMT activity was measured by a modification of the assay of Raymond and Weinshilboum [ 51. The modifications included the use of a final S-adenosyl-L[‘4C]methionine concentration of 12 PM with no addition of non-radioactive S-adenosyl-L-methionine and the substitution of reduced glutathionine, 1 mM, for dithiothreitol. In addition, the membrane preparations were not exposed to the solid chelating resin Chelex-100. 3,4-Dihydroxybenzoic acid was used as a substrate for COMT. The 4-methoxy-3-hydroxybenzoic acid (vanillic acid) formed in the presence of S-adenosyl-L-methionine was extracted by organic solvent extraction into toluene and its radioactivity was determined in a liquid scintillation counter. 4. Ery throcyte membrane protein determinations The Biuret method [6] was used to determine protein concentrations in erythrocyte membrane preparations. The membrane preparations were exposed to Triton X-100 at a final concentration of 1% to eliminate turbidity. Bovine serum albumin was used as a standard for the protein assay.
320
5. Thin-layer chromatography The radioactive product of the enzyme reaction was identified by thin-layer chromato~phy on Eastman Chroma~am sheets of silica gel, 100 micron in thickness which contained a fluorescent indicator. The two solvent systems Iused were benzene/methanol/acetic acid (90 : 8 : 4, v/v) and chloroform/ methanol (9 : 1, v/v). Chromatographic sheets were activated by drying for 15 min at 80°C immediately prior to use. After development the p-acetanisidide spots were identified by the use of ultraviolet light. The chromatograms were marked and cut into 1 cm strips that were placed in counting vials that contained 1.0 ml of Soluene-100 (Packard Instrument Company). After 1 h, 1 ml of absolute ethanol and 10 ml of toluene liquid scintillation counting fluid were added to each vial and the samples were placed in a liquid scintillation counter for the measurement of radioactivity. All results were corrected for quench after the addition of an internal standard of [ 14C]toluene. 6. Source of biood samples Blood samples were obtained from 212 randomly selected adult white blood donors at the Blood Bank of the Mayo Clinic in Rochester, Minnesota. The donors were not related and were not taking medications at the time that the blood samples were obtained. 7. Kinetic analysis Michaelis-Menten (KM) values were determined by the method of Wilkinson [ 71 with a Fortran program written by Cleland [S]. A Control Data Corporation 3500 Computer was used for these calculations. 8. Materials S-Adenosyl-L-[ 14C] methionine (specific activity 55 mCi/mmol) was purchased from New England Nuclear Corporation, Boston, Massachusetts. Tris(hydroxymethyl) aminomethane base, reduced glutathione, S-adenosyl-L-homocysteine, 3,4dihydroxybenzoic acid and cyclohexyl~inopropane sulfonic acid (CAPS) were purchased from Sigma Chemical Company, St. Louis, MO. 4’-Hydroxy-acetanilide, p-acetanisidide, and N-ethylmaleamide were purchased from Eastman Kodak Company, Rochester, N.Y. Phenol was obtained from Fisher was purchased from Regis Scientific Company, Fairlawn, N.J. Tropolone Chemical Company, Morton Grove, IL., and dithiothreitol (Cleland’s reagent) was obtained from ~~biochem, San Diego, CA. SKF 525A was kindly provided by Dr. R. Van Dyke of the Mayo Clinic. Results f. Choice of blank Lysates of human erythrocytes contain an enzyme activity that results in the formation of radioactively labelled methanol in the presence of S-adenosyl-L[14C]methionine [9]. This activity was originally thought to be a “methanol forming activity”, but it is now known that it results from the action of a protein carboxymethylase which methylates endogenous protein substrate(s) [ 101. During organic solvent extraction these radioactively labelled protein esters are
321
cleaved nonenzymatically, and the radioactively labelled methanol is extracted into the organic solvent [ 111. Our initial experiments with human RBC membrane preparations demonstrated that heated blanks consistently gave significantly lower counts per min (cpm) than did samples in which the assay was performed in the absence of substrate. For example, in one series of experiments it was found that a “no substrate” blank yielded average counts per min of 217 f 7 (mean + S.E.M., N = 5) and heated enzyme blanks yielded average counts per min of 127 f. 2. Whether or not a protein carboxymethylase similar to that found in erythrocyte lysates is present in RBC membranes is not clear, but it is obvious that a heated enzyme sample is not an appropriate blank for this assay. 2. Linearity of reaction with increasing concentration of lysate PMT activity increased with increasing quantities of RBC membrane in a linear fashion over a range of 25 to 100 ~1 of RBC membrane preparation (approximately 0.1 to 0.4 mg membrane protein, Fig. 2). All assays were performed with concentrations of RBC membranes that fell within this linear range. 3. Time course The time course of the enzyme reaction was linear to 60 min (Fig. 3). All assayed were performed at an incubation time of 45 min. 4. pH optimum The effect of pH on PMT activity in RBC membrane preparations is shown in Fig. 4. All pH determinations were made at 20°C in the presence of the membrane preparation and all components of the reaction mixture. A pH optimum of approximately 9.0 was found with four different buffer systems. All buffers were present in a final concentration of 0.07 M. Tris buffer was chosen for use in the assay since the pH optimum of 9 is within the range that Tris is an adequate buffer_ and since, unlike some of the other buffer systems, TrisHCl did not appear to inhibit the enzyme reaction. HUMAN
25 RBC
RBC
PMT
100
50 MEMBRANES,
vi
Fig. 2. Effect of increaehg quantities of RBC membrane on PMT activity. Each Point represents the mean of three determinations.
322
HUMAN
x
-
I $
1.6 -
RBC
PMT
lz
0 0
I I5
I 30
1 45
TIME,
MINUTES
Fig. 3. Effect of increasing three dbferminations.
1 60
I 75
time of incubation
I 90
on RBC
PMT activity.
Each
point
represents
the mean of
5. Relation of enzyme activity to substrate concentrations PMT activity in human RBC membranes was measured in the presence of varying concentrations of acetaminophen and S-adenosyl-L-[14C]methionine (14C-SAM), the two co-substrates for the reaction (Fig. 5). Because of the limited solubility of acetaminophen in water, several different solvents were used in an attempt to dissolve the compound more easily. These solvents included methanol, ethanol, dimethyl sulfoxide (DMSO), dimethylformamide (DMF), and acetonitrile. Although all of these solvents dissolved the acetaminophen they also apparently inhibited the enzyme reaction (see Fig. 5 for the effect of 25% DMF). DMSO did not inhibit the PMT reaction, but the use of DMSO resulted in a significant increase in the values of blanks, and thus, a
HUMAN
RBC
PMT
1.6 -
Polaarium
Phosphate
TRIS D Aspwlic
Acid
CAPS
o’,
, 7.0
I
,
,
(
9.0
9.0
,
10.0
PH Fig. 4. Effect of pH on RBC membrane PMT activity. CAPS represents acid. Each point represents the mean of three determinations.
cyclohexylaminopropane
sulfonic
323 HUMAN RBC PMT (Al BI_~
0
1.6 n ‘0
50
25 A~ETAMINOPHEN,
mM re
1.2
e-
x 0.6 I "0 0.4 0
Fig. 5. Effect of substrate concentration on PMT activity. (A) RBC membrane PMT activity was determined in the presence of increasing concentrations of acet~nophen dissolved fn water (open circles) and acetaminophen dissolved in 26% dimethylformamide DMF fsquares). Each point represents the mean of three determinations. (B) Effect of varying concentrations of SAM (S-adenosyl-L-methionine) on PMT activity, on “blank” values, and on the ratio of counts per min (CPM) in active samples divided by CPM in blanks. Each point represents the mean of three determinations.
decrease in the sensitivity of the assay. Therefore, it was elected to use acetaminophen dissolved in a relatively large volume of Tris buffer. The data obtained from the acetaminophen substrate curve were used to calculate an apparent Michaelis-Menten (KM) constant, 21.1 X 1O’3 M. The apparent KM value for S-adenosyl-L-methionine was 4.8 X 10e6 M. With increasing concentrations of radioac~vely labelled ~-adenosyl-L-methionine, blank values rose, and the “signal to “noise ratio” (cpm in active samples divided by cpm in blanks) decreased. This fact, plus the expense involved in the use of high concentrations of S-adenosyl-L-[14C]methionine resulted in the choice of 6.0 PM as the concentration of SAM used in the assay. This choice was made with the full knowledge that the assay was performed with less than saturating conditions for S-adenosyl-L-me~ionine. Therefore, the results reported here can only be compared with results obtained from assays in which similar concentrations of S-adenosyl-L-methionine are utilized. It was not possible to add non-radioactive S-adenosyl-L-methionine to the reaction mixture because the decrease in specific activity which occurred after this maneuver resulted in an appreciable loss in the sensitivity of the assay. Tritiated S-adenosyl-L-methionine of higher specific activity was not used in the assay because of lack of stability of this reagent and a difficulty with day-to-day reproducibility of results. 6. Thin-layer chromatogmphy of reaction product The organic solvent extract from an assay in which RBC membrane PMT had
324
been determined was dried under a stream of nitrogen and chromatography was performed with two solvent systems (see Methods above). The Rf values of the radioactive product formed were identical with those of authentic p-acetanisidide in both solvent systems (Fig. 6). 7. Coefficient of variation of the assay procedure The coefficient of variation for the assay of RBC membrane PMT with acetaminophen as substrate was determined with membrane preparations obtained from two separate subjects. The coefficients of variation for 10 assays for each of these preparations were 5.8% and 5.7%. 8. Phenol as a PMT substrate Phenol was also tested as a substrate for PMT (Fig. 7). Data from the substrate curve were used to calculate an apparent K, value of 2 X 10-j M. Although this apparent KILl was much lower than that for acetaminophen, the enzyme activity was noted to decrease at concentrations of phenol above 10 mM. Whether this phenomenon was due to substrate inhibition or to destruction of the enzyme by high concentrations of phenol is not clear. 9. Characteristics of RBC PMT activity PMT in the guinea pig liver is a membrane-associated enzyme [3]. To determine whether human RBC PMT is a membrane-associated enzyme, it was measured in erythrocyte membranes and in the supematant from lysates of 10 subjects. There was no detectable PMT activity in the supematants. Therefore, human RBC PMT is also a “membrane bound” enzyme. Erythrocyte membranes contain a variety of enzyme systems. Included among these is catechol 0-methyltransferase (EC 2.1.1.6, COMT) [12]. Axelrod and Daly have clearly differentiated PMT from COMT activity in guinea pig liver membrane preparations [ 31. To determine whether human RBC PMT is
SOCVENT FRONT
ORIGIN
STRIP Fig. 6. Thin-layer chromatography dark bars represents the migration
ORlGlN
SOLVENT FRONT
STRIP of the product of PMT activity of authentic p-acetanisidide.
using acetaminophen
as substrate.
The
325 HUMAN
RBC
PMT
I 25
0
I 50
PHENOL, mM Fig. 7. Effect of varying substrate mean of three determinations.
concentration
of phenol
on PMT activity.
Each
point
represents
the
separate from RBC membrane COMT activity, a series of experiments was performed in which the characteristics of the PMT activity were compared with those of RBC membrane COMT. COMT is a magnesium dependent enzyme and is inhibited by a variety of compounds including tropolone, S-adenosyl-Lhomocysteine and calcium [13,14]. As can be seen in Table I, RBC membrane PMT activity was not magnesium dependent. In addition although RBC membrane COMT activity was dramatically inhibited by both calcium and tropolone, calcium had no inhibitory effect on PMT and tropolone showed only a quantitatively small inhibition (Table I). The S-adenosyl-L-homocysteine inhibition of both enzymes was not surprising since this compound inhibits a wide variety of methyltransferase enzymes [ 151. Axelrod and Daly also showed that guinea pig liver membrane PMT is inhibited by SKF 525A @-diethylaminoethyldiphenyl propyl acetate) and by N-ethylmaleamide [3]. The human RBC membrane PMT activity that remained after the addition of N-ethylmaleimide (1 X lop4
TABLE
I
CHARACTERISTICS
OF RBC MEMBRANE
PMT AND COMT
Results for COMT are expressed as percentage of assay with [Mg2+l = 1 mM and for PMT as percentage All COMT determinations of assay in the absence of Mg 2+. SAH represents S-adenosyl-L-homocysteine. and ail with the exception of the assay with no Mg2+ were performed in the presence of 1 mM M&l?, assays for PMT with the exception of that performed with 1 mM MgCl2 had no magnesium present. Values are mean f S.E.M. for three determinations. Enzyme
Assay conditions
Relative
COMT
Mg2+, 1 mM No Mg2+ Tropolone. 1 mM Ca2+, 1 mM SAH, 0.1 mM
100 0 14 * 4 10 f 1 5r1
PMT
No Mg2+ Mg2+, 1 mM Tropolone. 1 mM Ca2+, 1 mM SAH, 0.1 mM
100 116 f 1 72 f 4 116 f 1 2*1
activity
(W)
326
M), SKF 525A (5 X 10W4M) and Triton X-100 (0.1%) to reaction mixtures was 52%, 35%, and 6% of control values respectively. Taken together these results demonstrate that the PMT activity in the human RBC membrane is not the result of COMT activity and that the characteristics of human RBC membrane PMT are similar to those of the PMT activity in the guinea pig liver. 10. Effect of sample handling on RBC PMT uctivity Blood samples obtained from human subjects under clinical conditions might be handled in very different fashions. Therefore, PMT activity was measured in blood obtained from 20 randomly selected adult subjects (16 male and 4 female) and the effects of various methods of h~dling the samples were compared. As can be seen in Table II, there was no significant difference between the enzyme activities measured immediately after the blood samples were obtamed as compared with enzyme activity measured after storage of erythrocyte membranes at -85°C for up to 2 weeks or in membranes prepared from blood samples which had remained at room temperature for 24 h prior to the preparation of the membranes. These observations have obvious practical importance in a clinical setting if PMT activity is to be measured in blood samples obtained from patients. Because the use of washed e~throcytes makes sample prep~ation a lengthy, time-consuming procedure, an experiment was performed in which RBC membranes from 3 subjects were prepared by direct lysis of 2 ml heparinized whole blood in 8 ml of ice-cold water. The membranes were then washed twice with 5 mM Tris-HCl, pH 7.8. The mean PMT activity of these 3 samples was 131.3 + 0.9 units/mg protein. This value was very similar to the average value of 128.5 & 3.2 (mean ?r S.E.M.) in RBC membranes prepared in the usual fashion (i.e., with washed erythrocytes from the same 3 samples). These results indicate that it is probably not necessary to perform the time consuming process of “washing” the erythrocytes before tysis. The washing procedure was used for the studies described here because simultaneous experiments were being performed in which other enzyme activities were measured in lysates. Plasma contamination altered some of these enzyme activities.
11.PMT activity in blood of randomly selected population PMT activity was measured in erythrocyte membranes of blood samples obtained from 212 randomly selected adult subjects. The mean PMT activity was
TABLE 11 EFFECT OF METHOD OF HANDLING AND STORAGE ON RBC PMT ACTIVITY Blood sampfes from 20 subjects were treated in a variety of fashions prior to the determination of enzyme activity (see text for de&&s). All values are mean + S.E.M. Treatment
PMT activity/units
Immediate preparation and assay Preparation and assay after 24 h at room temperature Storage of membranes at 45% for 48 h Storage of membranes at -85’C for 2 weeks
139.6 140.1 141.6 138.1
f. 5.9 t 5.6 f 5.8 -?z6.3
327
134.8 -t 41.5 units (mean + SD.) with a range from 44 to 282 units. There was a four-fold variation of enzyme activity within the range of t2 standard deviations. The activity for female subjects was 135.0 + 42.9 units {N = 83) and that in membrane preparations from male subjects was 133.8 + 40.7 units (N = 129). The frequency distribution histograms for RBC membrane PMT activities for this population are shown in Fig. 8. To determine whether or not the relative PMT activity was similar when phenol was used as a substrate, RBC membranes from 14 randomly selected subjects were assayed with both phenol and ace~minophe~ as substrates. There was an excellent correlation between the relative PMT activities with the two substrates (r = 0.92, P < 0.001, Fig. 9). Finally, an experiment was performed to help determine whether the variation in PMT activity in RBC membranes from randomly selected subjects might reflect differences in the levels of endogenous PMT activators, inhibitors or un~ticipa~d competing enzyme systems rather than differences of PMT activity itself. Membrane preparations from 7 subjects with relatively low enzyme activities were mixed with samples from 7 individuals with relatively high PMT activities and the enzyme activity was measured. As is shown in Table III, the PMT activities in the mixtures were not significantly different than the expected arithmetic mean values-a result which makes it ‘less likely that endogenous enzyme activators or inhibitors can account for the variations in RBC PMT activity observed in a population of randomly selected subjects.
RBC PMT 212
UNRELATED
SUBJECTS
>
HUMAN RBC
-j 129 UNRELATED
MALE
SUBJECTS
l
83
1 0
UNRELATED
FEMALE
SUBJECTS 550
80
RBC PMT
160 ACTIVITY,
PMT
240
UNITS
.
3.
l
r = 0.918 P < ,001
.
N.
I4
PMT ACTIVITY. CPWMG PROTEIN (ACETAMINOPHEN SUBSTRATE)
Fig. 8. Frequency distribution histo8ram of RBC! PMT activity; top, 2x2 unrelated subjects; middle, ,129 unrelated male subjects; and bottom, 83 unrelated female subjects. Fig. 9. Correlation of RBC memkme PMT activity with two different substrates. PMT activity in RBC membranes of 14 individuals were measured with both acetaminophen and Phenol as substrates.
328 TABLE III HUMAN RBC MEMBRANE
PMT
Equal volumes of RBC membranes from 7 subjects with relatively low and 7 subjects with relatively high PMT activity were mixed and the PMT activity was measured. All values are mean i S.E.M. Low activity (units)
Mixture Actual
Mean
r S.E.M.
Range
58.5 +_3.8 46 to 70
139.1
High activity (units)
_--__. Expected + 8.2
134.7
._
+ 7.6
207.0 f 11.5 163 to 258
12. RBC PMT in blood of patients with renal failure It has been reported that phenol levels are elevated in the blood of patients with renal failure, and it is thought that phenols may play a role in the symptoms suffered by these patients [ 16-181. Therefore it was elected to examine RBC membrane PMT in patients with chronic renal failure on maintenance hemodialysis as the first clinical application of this new test. Blood was obtained from 9 patients randomly selected prior to hemodialysis, and their PMT activity was measured. The average activity was 76.2 2 9.7 (mean + S.E.M.). This value is significantly lower than that present in blood from a randomly selected population (P < 0.001). Whether this observation reflects an effect of elevated phenol levels, is related to the altered hematopoiesis present in azotemic patients, or is due to some other factor remains to be determined. However this limited and preliminary observation does demonstrate that the application of the RBC PMT assay procedure may begin to open new areas for further clinical investigation. Discussion Phenol ~-methyltransfer~e (PMT) is a membr~e-bound enzyme which catalyzes the O-methylation of many phenols 131. We have demonstrated that there is a PMT activity in human erythrocyte membranes and that the biochemical properties of this enzyme activity appear to be similar to those of guinea pig liver PMT [ 31. An assay procedure for the measurement of human RBC PMT activity has been developed, and a four-fold variation in RBC PMT activity in blood from a large population of randomly selected subjects has been demonstrated. Finally, preliminary studies have shown that there is a significant decrease in RBC PMT activity in blood of patients with renal failure on chronic hemodialysis as compared with values in a randomly selected population. Although the role of PMT in the metabolism of drugs and other xenobiotic substances is not clear, it is known that qu~ti~tively minor pathways of drug metabolism can play important roles in the toxicity of some compounds [ 191, For example, although acetarninophen is metabolized primarily by glucuronate and sulphate conjugation, the quantitatively minor pathway of glutathione conjugation is crucial with regard to the determination of whether or not hepatocellular damage will occur after ingestion of a toxic dose of acetaminophen
329
[4,19]. It is important that we begin to characterize the biochemical basis of individual variations in the metabolism of drugs and other xenobiotic agents if we are to begin to understand individual differences in drug response and toxicity. The measurement of PMT activity in an easily obtained human tissue, blood, represents a first step in the expansion of our knowledge of this drug metabolizing enzyme activity in humans. Whether or not the PMT activity measured in the human erythrocyte membrane is biochemically similar to that found in other human tissues, and whether its regulation in the erythrocyte is similar to that in other organs must be determined in future experiments. However, the availability of a sensitive assay procedure for the measurement of PMT activity in human blood may make it possible for us to begin to attempt to answer these questions and to determine whether individual variations in this drug metabolizing enzyme activity may play a role in individual response to drugs and other xenobiotics. Acknowledgements This study was supported in part by NIH grants NS 11014 and HL 17487. Dr. Weinshilboum is an Established Investigator of the American Heart Association. We thank Fredrick Raymond, Joel Dunnette and Luanne Wussow for their assistance with these studies. We also thank Drs. J.C. Mitchell, P. Frohnert and V. Torres for providing blood samples from patients. References 1 Mandel, H.G. (1971) in Fundamentals of Drug Metabolism and Drug Disposition (La Du, B.N.. Mandel. H.G. and Way, E.L., eds.). pp. 149-181. Williams and Wilkins, Baltimore 2 Goldstein, A., Aronow. L. and Kabnan. S.M. (1974) in Principles of Drug Action, 2nd edn.. PP. 227-289. Wiley, New York 3 Axelrod. J. and Daly, J. (1968) Biochim. Biophys. Acta 159.472478 4 Mitchell, J.R.. Jollow. D.J., Gillette, J.R. and Brodie, B.B. (1973) Drug Metab. Dispos. 1, 418423 5 Raymond, F.A. and Weinshilboum. R.M. (1975) Clin. Chim. Acta 58.185-194 6 Gomall. A.G.. Bardawill. C.J. and David, M.M. (1949) J. Biol. Chem. 177.751-766 7 Wilkinson, G.N. (1961) Biochem. J. 80.324-332 8 Cleland, W.W. (1963) Nature 198.463465 9 Axelrod, J. and Cohn, C.K. (1971) J. Pharmacol. Exp. Tber. 176.650-654 10 Diliberto, E.J. and Axelrod. J. (1974) Proc. Natl. Acad. Sci. U.S.A. 71,1701-1704 11 Kim,S. and Paik, W.K. (1970) J. Biol. Chem. 245.1806-1813 12 Poitou, P., Assicot. M. and Bohuon. C. (1974) Biomedicine 21.91-93 13 Guldberg. H.C. and Marsden. C.A. (1975) Pharmacol. Rev. 27.135-206 14 Weinshilboum, R.M. and Raymond, F.A. (1976) Biochem. Pharmacol. 25.573-579 15 Coward, J.K., DWrsoScott, M. and Sweet, W.D. (1972) Biochem. Pharmacol. 21,1200-1203 16 Wardle. E.N. and Wilkinson. K. (1976) Clin. Nephrol. 2.361-364 17 Rabiner, S.F. and Molinas, F. (1970) Am. J. Med. 49.346-364 18 Wengle, B. and Hellstrom. K. (1972) Clin. Sci. 43.493-398 19 Mitchell, J.R. and Jollows. D.J. (1975) Gastroenterology 68, 392410
