Download and anglographic data of the patients and

Erythrocyte CuZn Superoxide
Dlsmutase and the Extent of
Coronary Atherosclerosis
from coronary angiography
(5). Erythrocyte CuZn SOD activity was determined
by a previously
described
method (6). The statistics were performed by t-test and correlation regression analysis.
Compared
with the controls, the
patients tended to have higher total
cholesterol and triglyceride concentrations and lower high-density
lipoprotein
and
lower ejection
fractions
(Table 1). The patients also tended to
have lower CuZn SOD values, but the
difference between the two groups was
not significant
statistically (P >0.05).
The correlation
between CuZn SOD
activity
and CAD was negative and
weak (r = 0.2), as was the correlation
between CuZn SOD and the number of
vessels affected (r = 0.16). The correlation between the concentrations
of
CuZn SOD and cholesterol was weak
but significant
(r = 0.3, P <0.05).
There was no correlation
between
CuZn SOD and other lipid variables.
The negative findings in our study,
the lack of a significant
trend towards
lower CuZn SOD values in patients
with atherosclerosis,
and no correlation between CuZn SOD activity and
the extent of atherosclerosis as judged
by the coronary score or ejection fraction may be due to the small number
of patients studied. Other factors affecting erythrocyte
SOD may be operative, despite our attempt to exclude
clinical situations
that affect SOD activities. Also, erythrocyte
CuZn SOD
may not adequately indicate the tissue CuZn SOD activity in the body.
Other indices of free radical activity,
e.g., glutathione,
catalase,
plasma
thiols, or plasma malondialdehyde,
which were not measured
in this
To the Editor:
Superoxide
dismutases
(SODs) are
considered
important
for protecting
living cells against toxic oxygen derivatives, e.g., lipid peroxides,
which
have been implicated in the initiation
of atherogenesis
and coronary artery
disease (CAD) (1). Recent trials, showing that several antioxidants
delay or
prevent the progression of atherosclerosis, support
this implication
(2).
Moreover,
preliminary
experiments
have shown that SOD, catalase, and
glutathione
peroxidase might be biological indicators
of chronic diseases
such as diabetes, alcoholism, and cancer (3). Erythrocytes
have some of the
highest CuZn SOD content of any tissue in the human body (4). Possibly,
therefore, CuZn SOD activity in a patient may be an important
marker for
the development of atherosclerosis.
To determine
whether erythrocyte
CuZn SOD activities
are a biological
marker of atherosclerosis,
we determined these values in patients with
angiographically
proven CAD and
compared
them with those in healthy
controls.
Patients with other disease
states that alter SOD activity, such as
diabetes, renal disease, anemia,
altered thyroid states, obesity, alcoholism, and smoking, were excluded.
We studied 43 patients with angiegraphically
proven CAD (36 men, 7
women, mean ages 51± 10 years) and
31 healthy controls matched for Body
Mass Index with the patients (12 men,
19 women, mean ages 47 ± ii years).
The coronary
scores were determined
study, may be more important markers. We conclude that we could not
demonstrate
that erythrocyte
CuZn
SOD was an important marker of coronary atherosclerosis.
Further studies
in a larger group of patients are
needed to clariir this matter.
References
1. Stringer MD, Gorog PG, Freeman A,
Kjikksor VV. Lipid peroxides and atherosclerosis. Br Med J 1989;298:281-4.
2. Stampfer MJ, Hennekens
CH, Manson
JE, Colditz AG, Rosner B, Wfflett C. Vitamin E consumption and the risk of coronary disease in women. N Engi J Med
1993;328:1444-8.
3. Guemouri L, Artur Y, Herbeth B, Jeandel C, Cuny G, Siest G. Biological variability of superoxide dismutase, glutathione
peroxidase, and catalase in blood. Clin
Chem 1991;37:1932-7.
4. Marklund S. Distribution of CuZn superoxide dismutase and Mn superoxide diamutase in human tissues and extracellular
fluids. Acta Physiol Scand 1980;492:19-23.
5. Gensini GO. Coronary arteriography.
Mount Kis, CO: Futura Publishing,
1973:
269-74.
6. Winterbourn
CC, Hawkins ER, Brian
M, Carrell WR. The estimation of red cell
superoxide dismutase activity. J Lab Clin
Med 1972;85:337-41.
Fatih Sinken
S Lale Tokgozoiu’
Nurten Renda
Sel#{231}uk
Adabag
Dept. of Cardiol. and Biochem.
Hacettepe University Faculty of Med.
Hacettepe, Ankara, Turkey
‘Author for correspondence.
Erroneous Results
Emit#{174}
Reagents
Table 1. Laboratory
and anglographic data of the patients
Mean
±
and controls.
SD
Patients
Controls
43
31
CuZn SOD, U/g Hb
Cholesterol, mg/L
Triglyceride, mg/L
3475 ± 1018
2070 ± 630
2050 ± 1370a
HDL, mg/L
LDL mg/L
1240 ± 510
3618 ± 1042
1900 ±450
1120 ± 620
570 ± 150
1110 ± 550
62±
Ejection fraction, %
Coronary score
Body Mass Index
450
±
56
7
±
26
±
120
17a
± 4a
3
‘Significantly different from controls: P <0.05.
Hb, hemoglobin; HDL high-densitylipoprotein; LDL, low-density lipoprotein.
with Diluted
0
24±6
To the Editor:
Previous reports
high concentrations
tate
tem
sults
with
CA).
dehydrogenase
samples
with
(1, 2) have linked
of lactate and lac(LD) in postmor-
false-positive
refor ethanol screening performed
Emit#{174}
assays
(Syva, Palo Alto,
Enzyme and substrate
were apparently
present
in sufficient quantities to convert NAD
to NADH and
generate a signal. Here we report
falsely low Emit-measured
drug concentrations
associated
with high LD
activity in serum.
A serum specimen from an adult in-
_________
CUNICAL CHEMISTRY, Vol. 40, No. 8, 1994
1597
tensive-care
patient was analyzed
for
total phenytoin
with Emit homogeneous enzyme assay reagents diluted
20-fold as previously described (3, 4).
Briefly, reagent A, supplemented with
NAD
and glucose 6-phosphate to 4.5
mmol/L each, was added at time 0, and
after 5 mm reagent B was added; the
first absorbance
reading
was taken at
6 mm and the final reading at 10 miii.
A serum ultrafiltrate
was prepared by
centrifugation
through a Centrifree#{174}
device
(30-kDa-cutoff
YMT membrane; Amicon Div., W. R. Grace, Beverly, MA) (4), and free phenytoin was
measured
with undiluted
Emit reagents, according to the manufacturer’s instructions.
The total phenytoin
measured
was 5.6 mgfL and the free
phenytoin
was 4.2 mg/L. Because the
calculated free fraction of 0.75 was not
physiological,
we remeasured
the total
and free phenytoin concentrations
by
HPLC (4). We obtained a nearly identical result for the free phenytoin concentration, but the total phenytoin
concentration
by HPLC was 24.2
mgfL, indicating
an apparent recovery
with the diluted Emit reagents
of only
23%. The free fraction
determined
from the HPLC data was 0.17, a value
consistent
with the patient’s mild hypoalbuminemia
(albumin
30 g/L) and
azotemia
(creatinine
53 mgfL).
Further
analysis of the patient’s serum revealed
an LD activity of 9300
UIL and a lactate concentration
of 21
minolJL.
We hypothesized
that the
high U) activity
could impair
net
NADH
production,
an effect that
would be more pronounced
when using diluted
reagents.
Reagent
depletion by lactate can be ruled out: Even
with a sample lactate concentration
of
20 mmol/L, the NAD/Iactate
ratio in
the reaction mixture was >15. Therefore, other diluted Emit assays should
be similarly affected. Because insufficient serum remained
from the original specimen, in further experiments
we used residual
serum from four additional
samples
collected
from the
same patient over the next 12 h, with
U) concentrations
of 4800-9400
U/L.
Known amounts of theophyllune
(another analyte we assay with diluted
Emit reagents)
were added to the specimens. Analytical
recovery when we
used reagents diluted 20-fold averaged
36% (range 24-47%), values similar to
those observed for phenytoin.
The average recovery of theophylline
when
we used undiluted
Emit reagents was
69% (range 60-78%). Thus, although
reagent
dilution
contributed
to the
problem, undiluted reagents were also
affected.
The Emit method exhibited
good recoveries for free phenytoin.
Because
(molecular
mass 36.5 kDa) would
have been excluded from the ultrafiltrate, we examined the effect of adding
an U) inhibitor
(sodium oxamate;
Sigma Chemical Co., St. Louis, MO;
final concentration
20 mmol/L) andreducing the dilution
to fourfold (the
U)
concentrations
of glucose 6-phosphate
and NAD
in reagent A remained 4.5
mmol/L).
This resulted in an accept-
able theophylline
recovery
of 89%
(range 88-89%). Lack of additional
specimen prevented testing these two
modifications
separately.
Sodium
oxa-
mate did not affect the recoveries in
specimens
with normal LD activity.
Examination
of drug-supplemented
sera from other patients with high U)
activities
by using diluted reagents
without sodium oxaunate showed variable but generally
more modest decreases in drug recovery than that observed in the index case (data not
shown). The addition of sodium pyruvate (0.1 mmoJJL) to the sera further
reduced recovery, but the decrease
in
recovery was stifi less than that seen
in the index case. There was no apparent correlation between serum creatinine and drug recovery.
We hypothesize that, in addition to
increased serum activity of LD in our
index case, increased concentrations
of
pyruvate or other ketoacids were also
present.
These putative interfering
substances
would generate
NAD
when reduced
by a dehydrogenase,
thus depleting
the NADH generated
in the Emit assay and producing
falsely low results. We are not certain
that U) is the only dehydrogenase
involved. It may serve merely as a
marker
for tissue
destruction
and
other enzymes might actually contribute to the problem. However, sodium
oxamate apparently
inhibited the activity of the enzyme(s)
involved and
minimized the problem. We conclude
that sodium oxamate should be added
to Emit reagents to minimize erroneous results in the presence of high concentrations
of LD.
References
1. Badcock NR, O’Reilly DA. False-positive EMIT#{174}-st
ethanol screen with postmortem infant plasma [Tech Brief]. Clin
Chem 1992;38:434.
2. Thede-Reynolds K, Johnson GF. False
positive ethanol results by EMIT#{174}
[Abstract]. Clin Chem 1993;39:1143.
3. Sung E, Neeley WE. A cost-effective
system for performing therapeutic drug assays. I. Optimization
of the theophylline
assay. Clin Chem 1985;31:1210-5.
4. Roberts WL, Rainey PM. Interference in
immunoassay measurements
of total and
1598 CUNICAL CHEMISTRY, Vol. 40, No. 8, 1994
free phenytoin in uremic patients: a reappraisal. Clin Chem 1993;39:1872-7.
William L. Roberts’
Florie S. Santos
Petrie M Rainey
Herbert Malkus
Dept.
Yale
P.O.
New
of Lab. Med.
Univ. School of Med.
Box 208035
Haven, CT 06520-8035
Diagnostic
Chemicals,
Ltd.Jan Holinsky
Oxford, CT 06478
‘Author for correspondence.
Analytical
Performance of
lmmulltem Assay of
ThyroId-StImulatIng Hormone
To the Editor:
In the Immulite”
(Cirrus
Diagnostics, Diagnostics Products Corp., DPL
Division, EURO/DPC, Glyn Rhonwy,
LL55 4EL, UK) immunochemiluminometric assay (ICMA), thyroid-stimulating hormone (TSH; thyrotropin)
is
captured by a murine monoclonal antibody to TSH bound to a polystyrene
bead (solid phase). The detector antibody, polyclonal goat antibody to TSH,
is conjugated to alkaline phosphatase
(ALP). After incubation, unbound conjugate is removed by a centrifugal
wash, and the chemilumunescent
substrate, a phosphate ester of adaniantyl
dioxetane [3-(2’-spiroadamantane)-4methoxy-4-(3’-phosphoryloxy)-phenyl1,2-dioxetane],
is added. In the presence of ALP this ester undergoes
hydrolysis to form an unstable intermediate with the production of light.
The light output, detected by a luminometer, is proportional
to the concentration of TSH in the sample. The original description of the Immulite TSH
method assessed the imprecision of the
assay over the range 0.34 to 32.8
mIU/L (1). In contrast, the assay was
designed for use at TSH concentrations
two orders of magnitude
lower than
this, i.e., as a third-generation
assay
with a functional
sensitivity
(CV of
<20% at 0.01 mIU/L) two orders of
magnitude
below that of typical firstgeneration
TSH radioimmunoassays
(2). In the present study, we have assessed the analytical
performance of
this assay over a lower range, using
the functional
sensitivity
limit suggested by Nicoloff and Spencer (3).
Immulite
TSH assays were performed in singleton according to the
manufacturer’s
instructions.
Results