curve for calculation of results. Therefore, reliable results
can be obtained with a simple recorder, and more expensive
integrators are not required. Paraxanthine,
a metabolite of
caffeine, is co-eluted with theophylline. Therefore, precise
measurement of theophylline in plasma cannot be obtained
in subjects exposed to caffeine. This is a known limitation
of
most reversed-phase chromatographic
methods for methylxanthines. Alternative methods have been used to solve this
problem. Adequate resolution can be obtained with normalphase chromatography or, preferably, by adding an ion-pair
reagent to reversed-phase
chromatography
(5, 8, 9). We
have not addressed this issue because it was not pertinent to
our specific application.
We have applied this method successfully to measure
methylxanthines
in only 10 L of plasma. This may have
particular application in neonates and small animals. Moreover, we can reliably measure
concentrations of caffeine in
tissue after its oral administration. The rat is a widely used
model to study the behavioral effects of caffeine, as well as
its potential toxicology.
In addition, methylxanthines
have
been used as potential pharmacological
probes to study the
role of adenosine in physiological and pathological processes
(10, 11). For each of these applications it is important
to
determine the concentrations of caffeine achieved at relevant tissue sites, because concentrations
in tissue may
correlate better with caffeine actions than do caffeine intake
or concentrations in plasma. The tissue concentrations of
caffeine
obtained in this study (slightly <100 ano1fL)
approximate
the Kd found in in-vitro studies for adenosine
receptor antagonism
(12).
We thank Mrs. Dorothea Boemer and Mr. Bolton Smith for their
help in preparing the manuscript and Mr. Bahrat Patel and Mrs.
Suzanna Lonce for excellent technical assistance. Tha work was
supported by grants HL-34021, HL37961, and RR00095 from the
National Institutes of Health. I. B. was a clinical associate physician of the Elliot V. Newman Clinical Research Center. D. R. is a
Burroughs Welicome Scholar in Clinical Pharmacology.
CLIN.CHEM. 34/11, 2348-2351
References
1. Curatolo P, Robertson D. The health consequences of caffeine.
Ann Intern Med 1983;98:641-53.
2. Pickard CE, Stewart AD, Hartley R, Lucock MD. A rapid HPLC
method for monitoring plasma levels of caffeine and theophylline
using solid phase extraction
columns.
Ann Clin Biochem
198623:440-6.
3. Hartley R, Smith LI, Cookman JR. Improved high-performance
liquid chromatographic
method for the simultaneous
determinations of caffeine and its N-demethylated
metabolites
in plasma
using solid-phase extraction. J Chromatogr 1985;342:105-17.
4. Robertson D, Frolich JC, Carr RK, et al. Effects of caffeine and
plasma renin activity, catecholamines,
and blood pressure. N EngI J
Med 1978;298:181-6.
5. Christensen HD, Whitsett TL. Measurements
of xanthines and
metabolites by means of high pressure liquid chromatography.
In:
Hawk GL, ed. Biological/biomedical
applications of liquid chromatography, vol 1. New York: Dekker, 1979:507-37.
6. Kabra PN, Nelson MA, Marten LI. Simultaneous
very fast
liquid-chromatographic
analysis of ethosuximide, primidone, phenobarbital, and carbamazepine in serum. Chin Chem 198329:473-6.
7. Ou C, Frawley VL. Concurrent measurement of theophylline
and caffeine in neonates by an interference-free
liquid chromatographic method. Clin Chem 1983;29:1934-6.
8. Scott NH, Chakrabokty J, Marks V. Determination of caffeine,
theophylline and theobromine
in serum and saliva using high
performance
liquid chromatography.
Ann Clin Biochem
1984;21:120-4.
9. Kester MB, Saccar CL, Rocci ML, Mansmann HC. New simplified microassay for the quantitation of theophylline and its major
metabolites in serum by high-performance
liquid chromatography.
J Chromatogr 1976;380:99-108.
10. Evoniuk A, Von Borstel RW, Wurtman RI. Antagonism of the
cardiovascular
effects of adenosine by caffeine and 8-(p-sulphophyl)theophylline. J Pharmacol Exp Ther 1987;240:428-32.
11. Ohnishi A, Branch RA, Jackson K, Biaggiom I, Deray G,
Jackson EK. Chronic caffeine administration exacerbates renovascular, but not genetic hypertension,
in rats. J Clin Invest
1986;78:1045-50.
12. Daly JW. Adenosine receptors: targets for future drugs. J Med
Chem 1982;25:197-207.
(1988)
ResultsCompared for TricyclicAntidepressantsas Assayed by LiquidChromatographyand
Enzyme Immunoassay
R. Cameron Dorey,
Sheldon H. Preskom,
and Pamela
K. Widener2
The tncyclicantidepressantsamitnptyline,nortriptyline,imipramine, and desipramine in serum of patientstaking one of
the drugs were quantified in two laboratories by highperformance liquid chromatography (HPLC) and enzymemultiplied immunoassay (EMIT’”; Syva). Results for split
sampleswere highlycorrelated,but EMIT gave higherresults
in mostcases, and the slopesof the correlationlinesfor each
‘Department
KS 67208.
of Chemistry,
Wichita
State University,
Wichita,
2Psychiatry Service, Wichita Veterans Administration
Center, Wichita, KS 67218.
3Department
of Psychiatry,
University
of Kansas
Medicine at Wichita, Wichita, KS 67214.
Received May 20, 1988; accepted August 8, 1988.
Medical
2348 CLINICALCHEMISTRY,Vol.34, No.11, 1988
School of
analyte were >1. Detection limits for the two procedures
were such that 18% of the EMIT resultsfor the drug(s) were
considered negative, as compared with 4% of the HPLC
results.Additionalassay of desmethylor hydroxyantidepressant metabolites by HPLC did not explain the higher EMIT
results. The relatively high detection limit for EMIT greatly
limits its use in therapeutic drug monitoring, where low
concentrationsof tricyclicantidepressantsare as important
as highones for dose adjustmentor determinationof compliance. Other problems with EMIT measurement of tricyclic
antidepressantsare discussed.
Assay of tricyclic
antidepressant
(TCA) drugs amitriptyline (AM!), nortriptyline
(NOR), imipramine
(IMI), and
desipramine
(DM1) in pharmacotherapy
is becoming
an
accepted practice among physicians who prescribe these
drugs (1). This is in large part attributable
to the recent
rapid development of analytical
methods for quantifying
these drugs, whose therapeutic concentrations
in plasma are
generally <300 tg/L, whereas toxicity develops at concentrations <500 pg/L (2).
TCAs are generally quantified by gas chromatography
(CC) or high-performance
liquid chromatography
(HPLC).
In these methods, the parent drug and one or more metabolites can be quantified independently. HPLC is generally
more commonly used than CC for TCA analysis, because
sample preparation is less time consuming:
single-step
liquid or solid extractions often suffice for sample cleanup,
and derivatization
of a secondary amine TCA is not generally needed (3). HPLC procedures, however, cannot separate
as many compounds as GC (particularly
capillary CC), and
most published procedures
display interferences,
either
from some phenothiazine-type
antipsychotic
drugs (4) or
benzodiazepines
(5), although some schemes avoid problems
with both classes of drugs (6).
Interferences depend primarily
on the type of HPLC
column used; use of an alternative
type of column often can
eliminate a specific interference
(3).
In any event, the procedures involved in HPLC or CC are
labor-intensive
for the toxicology laboratory, because samples must be analyzed serially, and each standard
and
patient’s sample may require 10 mm for the chromatographic step alone. Moreover, skill is needed in extraction
of
samples and interpretation of the resulting chromatogram,
so the assay is not a technically trivial one.
Methods for quantifying TCAs must be able to distinguish
between the tertiary amines and their demethylated metabolites-AMI
and NOR, IMI and DM1-because
both will be
present in plasma of patients taking the parent drug (2).
Also, all four compounds
undergo hydroxylation
of the
backbone structure, after which the hydroxy and desmethyl
metabolites are conjugated to the glucuronide for excretion.
At present, the hydroxy metabolites are not often assayed,
there being no clear correlation between their concentrations and therapeutic benefit or side effects. This may
change, however, given a recent report (7) that hydroxy
metabolites of NOR and DM1 are correlated with cardiotoxic
symptomatology in some patients.
The Syva Co. has recently developed an assay of TCAs
based on their proprietary homogeneous enzyme-multiplied
immunoassay
technology (EMrrTh) (8). This assay, which
incorporates a single solid-phase extraction and nonisotopic
immunoassa3,
is adaptable to different automated analyzers
for TCA analysis
in laboratories
without HPLC or CC
equipment (9-11).
To investigate
local reports that results by EMIT were
generally higher than by HPLC, we compared the results by
EMIT
assays with those by a generally accepted HPLC
technique. Also, because published reports often have not
compared the results between the two techniques for NOR
and DM1 when patients were taking AM! or IMI (11,12), we
have done so. Because the total TCA concentration
is
important therapeutically,
increases in the reported metabolite concentrations,
from cross-reactions
or otherwise, can
affect the course of therapy.
Nonstandard abbreviations: EMIT, enzyme-multiplied immunoassay technique; TCA, tricyclic antidepressant(s); AM!, amitriptyline; NOR, nortriptyline; IMI, imipramine; DM1, desipramine;
CC, gas chromatography.
Materials and Methods
Specimens. We obtained patients’ specimens from two
sources: patients of the Psychiatry Service2 and patients
participating
in a study involving amitriptyline
administration at the University of Kansas Medical School at Wichita.
All specimens were collected into 10-mL red-top (no additive) Vacutainer Tubes (Beeton Dickinson, Rutherford, NJ)
and allowed to clot, then centrifuged.
The serum was then
pipetted into polypropylene tubes for storage at -20 #{176}C
until
assay. Results by either procedure were not made known to
the other assay site until after the sample was analyzed by
both methods.
Statistical analysis. For statistical analysis of the comparison study, we used linear-correlation
methods. The statistical program used, the Stats-2 (StatSoft, Tulsa, OK) program, calculates linear correlations of one variable with one
or more other variables, so that any cross-reactivity of the
EMIT antibodies with metabolites
could be factored into the
total response of the assay.
Samples with results by HPLC that were below the stated
detection limit for the EMIT assay (50 g/L for DM1, 25 pgtL
for AM!, IMI, or NOR) were not included in the regression
analysis.
One patient’s sample contained thioridazine and
metabolites, which interfere with both procedures; thus it
was not included in any analyses of results.
HPLC assay. We performed the HPLC assays at the
Research Psychopharmacology
Laboratory,2 using a modification of the method of Koteel et al. (4), with the extraction
step optimized for recovery of hydroxy metabolites.
It is a
typical HPLC method and has been validated against a GC/
mass spectrometric method (13) at the Medical College of
Virginia, where it has been in use for routine clinical
monitoring for the past five years.
We prepared serum-based standards by adding aqueous
solutions of the tertiary and analogous secondary TCAs
(prepared from the hydrochloride salts) to drug-free serum
to give final concentrations of 50, 100, and 200 gfL. The
internal standard used was protriptyline hydrochloride (no
patients in this study were taking protriptyline), which was
eluted later than either of the TCAs of interest or their
hydroxy metabolites.
We prepared the samples for HPLC analysis as follows:
We first combined 1 mL of serum with 50 L of an aqueous
10 ig/L solution of protriptyline hydrochloride, 1 mL of
sodium carbonate solution (2 mollL, pH not further adjusted), and 4 mL of ethyl acetate/hexane/isobutanol
(50/49/1 by
vol) extraction solvent. We vortex-mixed this mixture for 10
s, centrifuged at 2000 x g for 5 mm, and pipetted the
extraction solvent into a second tube. We then evaporated
the solvent under reduced pressure at 40 #{176}C
in a Model SVC100H vacuum centrifuge (Savant Instruments,
Farmingdale, NY), reconstituted
the residue in 200 .u.Lof the HPLC
mobile phase (see below), and injected 100 L of this into the
HPLC.
We used a Supelco LC-PCN column, 15 cm x 4.6 mm
(i.d.), and a mobile phase consisting of acetonitrile, methanol, and phosphate buffer (10 mmol/L, pH 7.0), 60/15/25 by
vol. Drugs were detected by their absorbance at 254 rim, for
which we used a Model 440 fixed-wavelength
detector
(Waters, Millipore Corp., Milford, MA). Calibration curves
were constructed by correlating the ratio of peak heights of
the analyte and internal standard with the concentrations of
the corresponding TCA in the plasma standards.
EMIT assay. EMIT assays were performed
at Roche Biomedical Laboratories, Wichita, KS, with the Syva TCA assay
CLINICALCHEMISTRY, Vol. 34, No. 11, 1988
2349
kits used according to the manufacturers
directions. The
zrr assay involves use of monoclonal antibodies for AM!
and 1MI, and polyclonal antibodies
for NOR and DM1.
Glucose-6-phosphate
dehydrogenase
is linked to drug derivatives for competitive binding with the drugs found in
plasma, and enzyme activity (proportional to drug concentration) is monitored as an absorbance change (NAD to
NADH) with time, at 340 nm. Drugs are extracted from
plasma before assay with a solid-phase extraction technique
(8).
Results
Of the 94 samples analyzed by both methods, results by
for 17 (18%) were below the detection limit for at least
one analyte. In contrast, only four samples (4%) had nondetectable concentrations
by HPLC. None of the samples
negative for TCAs by HPLC gave detectable concentrations
by EMIT. These samples were not included in the regression
analysis comparing the two methods.
Table 1 summarizes
the results for the samples used in
the single-variable
linear regression
analysis.
Curves are
presented separately for patients taking the secondaryamine TCAs (NOR and DM1) and for patients taking the
tertiary amine TCAs (AM! and IM1). The slopes and intercepts for these curves are not statistically
different between
the two groups. This finding shows negligible interference
from tertiary-amine
TCAs in assays of the corresponding
secondary-amine
TCAs.
One sample from a patient taking IMI had DM1 and total
TCA concentrations
four times that of the next highest
patient. Results for this patient so changed the slope of the
calculated regression line that we performed analyses both
with and without this patient’s results. The slopes of the two
regression lines were, however, statistically equal. We cannot conclude from this single sample whether or not there is
any change in the curve at high concentrations.
Multiple correlation studies comparing EMIT results of
NOR plasma concentrations
with HPLC results of NOR and
hydroxy-NOR concentrations
did not indicate any influence
of the hydroxy metabolite on the EMIT results. Similarly, we
compared EMIT results for AM! concentrations
to HPLC
results for AM!, NOR, hydroxy-AMI, and hydroxy-NOR.
Factoring in the metabolite concentrations did not increase
the correlation coefficient found by using only the parent
compound. This suggests no significant interference from
metabolites on parent-compound EMIT values. Analysis of
EMIT
Table 1. Correlations between TCA Plasma
Concentrations as Determined by HPLC and EMIT
Slope,
Drug taken
by patIent
AMI
NOR
IMI
Analyte
AMI
NOR
AMI #{247}
NOR
NOR
IMI
DM1
IMI
+
DM1
n
awr vs HPLC
15
15
15
23
16
(15)
16
(15)
16
1.27
1.19
1.25
1.16
0.97
(15)
DM1
39
(1.09)
0.91
(1.02)
0.90
(1.12)
0.97
pg/L
-5.6
13.1
3.0
9.6
16.8
(8.1)
33.0
(26.5)
57.9
(25.6)
20.0
Because
r
0.96
0.99
0.99
0.94
0.94
(0.95)
0.95
(0.88)
0.95
(0.93)
0.99
the DM1 concentration measured for a patient taking IMI was
fourfold the next-highest concentration measured for the group. the results in
parentheses exclude this point to show its exaggerated effect on the regression line.
2350 CLINICALCHEMISTRY, Vol.34, No. 11, 1988
the DM1 and IM! correlations
between
methods produced
the same results.
Discussion
Results for EMIT assay of TCA concentrations
in plasma
correlate well with HPLC results, but the slopes of the
correlation curves often considerably exceed unity, and the
intercepts of the correlation curves are often positive. These
results are not explained by simple selectivity problems of
the antibodies used in the EMiT assays. We do not see any
significant
direct cross-reactivity
between the tertiary
amines and their secondary metabolites in these assays.
Also, including hydroxy metabolites
in the correlation analyses had very little effect.
However, EMIT evidently will generally produce higher
results than our HPLC method, as happened in 70 of the 77
samples that were reported as positive by both methods.
This is not clinically significant in cases involving TCA
toxicity or long-term
routine
monitoring.
Therapeutic
guidelines for TCA concentrations in plasma cover wide
ranges, and there is no immediate risk of toxicity or rapid
relapse at moderately high concentrations.
However, there are two situations in which EMiT is at a
distinct disadvantage.
First, the EMIT assay is inappropriate
for low TCA concentrations in plasma, because of its high detection limits and
the positive intercepts of some of the EMIT vs HPLC correlation curves. This situation is likely in three common settings: when a physician starts a patient at a low TCA dose
and wishes to increase the dosage on the basis of the
concentration in plasma; when there is a question of compliance or rapid metabolism
in a nonimproving
patient; or
where a “test dose” of a TCA is given, with the TCA
concentration in plasma measured after 24 h, from which
daily doses are extrapolated.
Second, the EMIT assay cannot be modified to eliminate
interference from other drugs. In the assay, there is significant interference by chlorpromazine
in therapeutic concentrations (8), and the package
insert
with the assay kit
describes a similar interference with thioridazine.
Two
other phenothiazines
and the benzodiazepines
do not interfere at therapeutic concentrations
(8). Thus, TCA as measured in plasma by EMIT in patients who are taking phenothiazine antipsychotics will often be erroneously high.
The tertiary-amine
TCAs cross-react in each other’s assay, as do the secondary-amine
TCAs (8), although we have
seen minimal cross-reactivity
between the two classes. This
makes the ordering of the correct test (not just “tricyclics”)
imperative for the requesting physician, and will cause
problems when a patient is being switched from one TCA to
another without “washing out” the first drug before checking the concentration of the second drug in the patient’s
plasma.
Laboratories where EMIT is to be the routine method for
quantifying TCAs should have a chromatographic
method
on hand for backup where these cases are suspected.
We gratefully acknowledge partial support of this study from the
Veterans’ Administration Merit Review research program.
References
1. American Psychiatric Association Task Force. Tricycic antidepressanta-blood level measurements
and clinical outcome. Am J
Psychiatry 1985;142:155-62.
2. Preskorn SH, Dorey RC, Jerkovich GS. Therapeutic
monitoring
of tricyclic antidepressants. Cliii Chem 1988;34:822-8.
3. Wong SHY. Measurement of antidepressants
by liquid chromatography: a review of current methodology [Review]. Cm Chem
1988;34:848-55.
4. Koteel P, Mullins RE, Gadsden RH. Sample preparation
and
liquid-chromatographic
analysis for tricycic antidepressants in
serum. Cliii Chem 198228:462-6.
5. Breutzmann DA, Bowers LD. Reversed-phase liquid chromatography and gas chromatography/mass
fragmentography compared
for determination
of tricydic antidepressant drugs. Cm Chem
198127:1907-11.
6. Yang S, Evenson EA. Simultaneous
liquid chromathgraphic
determination of antidepressant drugs in human plasma. Anal
Chem 1983;55:994-8.
7. Pollock BG, Perel JM. Hydroxynietabolites
of tricyclic antidepressants: evaluation of relative cardiotoxicity.
Cliii Pharmacol
Psychiatry.
8. Pankey
In press.
5, Collins C, Jaklitsch
CLIN. CHEM. 34/11,
2351-2354
A, et al. Quantitative
homoge-
neous enzyme inununoassays
for amitriptyline, nortriptyline, imiprainine, and desipramune. Clin Chem 1986;32:768-72.
9. Orsulak PA, Haven MC, Huth JA, Studts DJ. EMIT#{174}
quantitive
tricyclic antidepressant assays applied to the Hitachi 705#{174}
chemistry analyzer [Tech Brief]. Cliii Chem 1987;33:1471.
10. Haven MC, Orsulak PA, Huth JA, Markin
ES.
EMIT#{174}
quanti-
tive tricycic antidepressant assays applied to the Encore” chemistry system [Tech Brief]. Cm Chem 1987;33:1472.
11. Frye R, Mathews S. Evaluation of Syva#{174}
EMIT”
enzyme
immunoassays
for the tricyclic antidepressants
on the Cobas#{174}
centrifugal analyzer [Abstract]. Clin Chem 1986;32;1053.
12. Foglia JP, Perel JM, Stiller
RL, et al. Effect of specimen
collection on immunoassay techniques for selected tricyclic antidepressant drugs [Abstract]. Clin Chem 1986;32:1052.
13. Narasimhachari
N, Sandy J, Friedel RO. Quantitative mapping of metabolites of imipramine and desipramine
in plasma
samples by gas chromatography-mass
spectrometry. Biol Psychiatry 1981;16:937-44.
(1988)
“Flipped”Patterns of Lactate Dehydrogenaselsoenzymesin Serum of Elite College Basketball
Players
hi Rotenberg,’ Richard Seip,2 Larry A. Wolfe,
and DavidE. Bruns4
We kinetically measured total lactate dehydrogenase (LD,
EC 1.1.1.27), total creatine kinase (CK, EC 2.7.3.2), and
aspartate aminotransferase (AST, EC 2.6.1.1.) in 16 elite
college basketball players, before the competition season
and not in close temporal relation to near-maximal exercise,
and in 17 healthy non-athlete controls. LD isoenzymes were
determined by both electrophoretic and immunoprecipitation
methods. CK-MB isoenzyme was measured electrophoretically. We found significantlly higher mean LD-1 values and
LD-1/LD-2 ratios in the players than the controls: 31.6 (SD
3.7)% vs 25.8 (SD 3.2)% (P <0.005) and 1.1 (SD 0.13) vs
0.87 (SD 0.16) (P <0.001), respectively. A “flipped” LD
pattern (LD-1 > LD-2) was found in half the players and in six
of the eight black athletes, but in only two of the control group
and in none of the black controls. Mean CK activity in serum
exceeded normal values in the serum of the athletes and was
higher in comparison with the control group [274 (SD 156) vs
103 (SD 82) U/L]. Mean CK was significantly higher in the
eight athletes with the flipped LD pattern than in those with
LD-1 <LD-2 [322 (SD 163) vs 180 (SD 98) U/L; P
0.05],
and also in comparison with CK in the two controls with
flipped LD pattern. We saw no significant difference in mean
CK between the nine players with normal immunochemical
LD-1/LD ratios and the seven players with above-normal
ratios. CK-MB was not detected in either athletes or controls.
Departments
of Pathology and2 Health and Physical Education,
Schools of Medicine and Education, University of Virginia, Charlottesville, VA 22908.
Present address: ‘Massada Center for Heart Diseases, Beiinson
Medical Center, Petah Tikva 49100, Israel; and3 School of Physical
and Health Education, Queens University,
Kingston, Ontario,
Canada, K7L 3N6.
‘Address correspondence to this authoi- Box 168, Department of
Pathology, University of Virginia Medical School, Charlottesville,
VA 22908.
Received June 29, 1988; accepted August 8, 1988.
None of the players had any clinical or electrocardiographic
evidence for myocardial ischemia or infarction. Evidently the
flipped LD pattern usually found in patients with acute
myocardial infarction and reported in some athletes after
extreme exercise such as ultra-marathon running may also
be found in athletes who are in their “basal fitness shape” but
who are not involved in competitive physical activity.
Numerous reports have been published on the increase in
serum enzyme activities following various types of exercise
(1-10). The most commonly described
increase
is that in
serum creatine kinase (CK, EC 2.7.3.2) (1-4, 6-9, 11).
Increases
in aspartate
aminotransferase
(AST, EC 2.6.1.1)
(1, 7) and lactate dehydrogenase
(LD, EC 1.1.1.27) (2, 6, 7,
11) have also been seen, but the changes were generally
smaller than for CK. Most studies have reported increased
LD only after vigorous exercise, such as marathon running.
Some (4, 9, 12) have even described a “flipped” LD pattern
(LD-1 > LD-2) in certain athletes after long-distance
running, similar
to that described
in patients
with acute
myocardial
infarction (13-16).
The present study was prompted by the unexpected
finding of abnormalities of both CK and LD isoenzymes in
college basketball players. In particular, five of 16 players
demonstrated
a ffipped LD ratio when the athletes underwent routine examinations
on the second day of team
practice. The present prospective study was undertaken
in
the same group a year later, to determine a “basal” proffle of
LD isoenzymes
before the competition season.
Patients and Methods
We studied 16 members of a highly ranked U.S. basket(mean age 20.3, SD 1.3 years), and 17 healthy, tall
ball team
5Nonstandard
abbreviations:
dehydrogenase; AST, aspartate
CK, creatine kinase; LD, lactate
aminotransferase.
CLINICALCHEMISTRY, Vol. 34, No. 11, 1988 2351