| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Technical Briefs |
1
Department of Legal Medicine, Hamamatsu University School of Medicine, 1-20-1 Handayama, Hamamatsu 431-3192, Japan
2
Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa-ku, Nagoya 464-8601, Japan
aauthor for correspondence: fax 81-53-435-2239, e-mail kminakat{at}hama-med.ac.jp
Plasma copper is reportedly increased in pregnancy,
infections, and inflammation and decreased in corticosteroid and
corticotropin treatment (1). The usual and convenient
methods for measurement of metal ions are colorimetry and atomic
absorption spectrophotometry (AAS). These methods, however, are not
suitable for copper. For example, the molar absorptivity at 480 nm of
copper complex with bathocuproine disulfonic acid, the most suitable
reagent, is not strong (
= 1.3 x
104) (2)(3)(4), and the atomic absorption
at 324.7 nm specific to copper is marginal. As a consequence, both
methods require 2 mL of serum, although the copper concentration in
plasma is fairly high, e.g., 15.7 µmol/L (1 µg/mL). A more
sensitive but selective and convenient method is needed for
quantification of small amounts of copper in blood as well as in
polluted water, food, and tissues.
Electron spin resonance (ESR) is a very sensitive method that requires only 109 spins for detection. We examined the use of ESR for measurement of copper because Cu2+ is paramagnetic, and thus can be measured by ESR, although Cu+ is diamagnetic.
We confirmed the results of a previous report that
diethyldithiocarbamate (DDC) reacts with Cu2+ at
pH 0–10 (5). The Cu2+-DDC complex
could be extracted with most organic solvents at these pHs
(5)(6). We found that the addition of
Na2SO4 facilitated the
extraction of the complex with 1-butanol. As shown in Fig. 1A
(spectrum a), Cu2+-DDC in
1-butanol shows a characteristic four-line ESR spectrum with hyperfine
splitting of 80 gauss and a splitting factor (g) of 2.047. The
height of the peak indicated by the arrow at the highest magnetic field
of 3350 gauss was used for the quantification of copper.
|
To liberate copper from bound protein in plasma, the pH of the solution should be adjusted to <5 (7). To determine suitable acids, we added 0.15 mL of 1 mol/L solutions of several acids to 1 mL of plasma.
The addition of HNO3,
HClO4, or
H3PO4 gave the same amounts
of copper obtained from wet-ashed plasma, but the addition of HCl,
HCOOH, CH3COOH, or
H2SO4 gave only 
80%. In
the present work, HNO3 was used to acidify
plasma. The final pH was 3.3 or 1.5 after the addition of 0.1 or 0.15
mL of 1 mol/L HNO3, respectively, to 1 mL of
plasma. DDC can react with all Cu2+ when the
concentration of DDC is high enough (8). For the
determination of copper concentration in tissue, tissue was wet-ashed
with concentrated HNO3 and the solution was
diluted until its pH was >0.
Among dozens of chelators, DDC is the one that binds Cu2+ in the presence of ceruloplasmin (7). We confirmed that the present method could be used for plasma containing bilirubin, ascorbic acid, or anticoagulants such as EDTA, oxalic acid, succinic acid, and heparin, although these substances made colorimetric assay of copper difficult (4)(9).
ESR measurements were performed on a JEOL JES-FE2XG ESR spectrometer. For comparisons with ESR, copper was also measured by AAS using a Shimadzu AA-6200 flame atomic absorption instrument. A centrifuge with maximum 6000g and a vortex-type mixer were also used. Polypropylene tubes (0.5 mL) were obtained from Eppendorf, pipettes and 0.1–10 µL pipette tips were from Gilson, and 20-µL quartz hematocrit capillaries [60 mm long x 0.8 mm (o.d.)] were from Drummond Scientific. We could not use glass or plastic hematocrit capillaries because of the strong ESR signals produced by impurities. To seal the capillaries, we used a vinyl plastic putty (CRITOSEAL; Oxford Labware). All chemicals were of atomic absorption grade or biochemical grade from Wako Pure Chemical Ltd. The ultra-pure water (specific resistance, 18 megaohms · cm) was used. All glassware and plastics were soaked in concentrated HNO3 or 0.3 mol/L HNO3, respectively, overnight and rinsed >10 times with ultra-pure water.
On the basis of these findings and using materials and instruments
mentioned above, we used the following procedure to prepare plasma and
tissue samples for quantification of copper: (a) 5 µL of
plasma (or 5 µL of copper calibration solution) was placed in a small
tube that could tolerate acid at pH 0 as well as centrifugation at
6000g; (b) 0.75 µL of 1 mol/L
HNO3 was added to the tube and mixed for 10
s in a vortex-type mixer; (c) 0.75 µL of an aqueous
solution containing 100 g/L DDC was added and mixed for 10 s (DDC
solution stored at 4 °C in the dark is stable for 4 weeks);
(d) 6 µl of 1-butanol was added and mixed for 10 s;
(e) 3 mg of
Na2SO4 was added and mixed
for 30 s; (f) the tube was centrifuged at
6000g for 2 min; (g) 5 µL of the top
(1-butanol) layer was removed and placed in a hematocrit capillary, and
the top and bottom of the capillary were sealed with putty;
(h) the capillary was inserted in a quartz ESR cell and
placed in the ESR cavity.
For tissues, we confirmed that the pH of wet-ashed tissue solution was <0 and added DDC, 1-butanol, and Na2SO4 as in the case of plasma.
The ESR conditions were as follows: spectrometer setting, 3300 gauss;
sweep range, 1000 gauss; sweep time, 8 min; response time, 1 s;
microwave power, 6 mW; modulation width, 20 gauss. Measure the height
of the peak at 3350 gauss. It takes 30 s to measure the
peak.
It took 5 min for the quantification of one sample when we used the
preparation steps listed above. Because some processes, such as mixing
and centrifugation, can be performed simultaneously for multiple
samples, it took only 1 h to process 24 samples (e.g., 4
calibrators and 20 specimens).
Tracing b in Fig. 1A
was obtained from 5 µL of plasma taken from a
Wistar strain rat treated with 1 g Diquat/kg diet for 14 days, and
tracing c was obtained from plasma from control rat without Diquat
treatment. Tracing d in Fig. 1A
represents 0.3 mg of wet kidney taken
from a spontaneous hypertensive od-rat (SHR-od), and tracing e
represents wet kidney from a normotensive od-rat (ODS-rat). Because
od-rats cannot synthesize vitamin C, the rats represented by tracings d
and e received adequate amounts of vitamin C. Human plasma shows a
spectrum similar to those in Fig. 1A
, tracings b and c.
The precision was evaluated by 10 analyses of two plasma samples. The
within-run difference was <5%. The recovery of added copper (7.9,
15.7, 23.6, and 31.4 µmol/L) from plasma with an endogenous copper
concentration of 15.7 µmol/L was >95%. Human plasma from 50 healthy
males 20–60 years of age and 50 healthy females 20–60 years of age
was examined by two methods, ESR and AAS. The mean value (SD) for men
was 14.8 ± 2.2 µmol/L, and that for women was 17.3 ± 2.7
µmol/L, in good agreement with reported values
(4)(9)(10). The regression equation
for the present method (y) and AAS (x) measured
by AAS was: y = 0.94x + 1.0
(r = 0.97; Fig. 1B
).
Most metals can form complexes with DDC, and most of the complexes are
extracted with 1-butanol. Some complexes are diamagnetic and some are
paramagnetic. However, the peaks that interfere with copper are not
observed in plasma, kidney, liver, heart, lung, and spleen, although
Fig. 1A
indicates only the results for plasma and kidney. Because ESR
is a nondestructive method, we might be able to use the extract in the
future for assays of other metals as well as for copper.
Although most organic solvents can extract Cu2+-DDC complex, 1-butanol is used because alcohols such as butanol and cyclohexanol do not dissolve the putty used for sealing the capillary. 1-Butanol and cyclohexanol saturated with putty do not show ESR signals attributable to the putty. When measuring the signal for the Cu2+-DDC complex in a capillary, it is better to adjust the capillary so that the putty remains outside the ESR cavity: although the putty signals do not interfere with the Cu2+-DDC complex signals, they do change the baseline.
Cu2+-DDC complex in 1-butanol is quite stable and is not affected by exposure to room light for a few hours. The peak height of the Cu2+-DDC complex remained unchanged for 3 days when the complex was kept in the dark, and the volume of 1-butanol was maintained.
In the present method, Cu2+-DDC complex is placed
in a hematocrit capillary first and the capillary is placed in the ESR
cell; therefore, when a different sample is to be measured, only the
capillaries are changed. This is very convenient because we do not need
to wash the ESR cell between samples. The wall of the quartz capillary
and the quartz wall of the ESR cell, however, have a weak signal at
3270 guass, as indicated by the arrow in Fig. 1A
, part e, and may
contain some substances that shift the baseline from a lower magnetic
field to a higher magnetic field as observed in Fig. 1A
. Therefore, the
limit of detection is 15.7 nmol of copper (1 ng of copper). The signal
of Fig. 1
, part d, corresponds to 3 ng of copper. The sensitivity may
improve when capillaries and ESR cells that do not have signals near
the signal of copper become available. For the quantification of copper
in plasma, flame AAS and colorimetry require 2 mL of plasma, but
graphite furnace AAS and inductively coupled plasma-optical emission
spectrometry require 5 µL of plasma (11), which is the
same amount as the present method.
In ESR measurements, samples with higher concentrations can be measured without dilution, either by decreasing the gain from 104 to 1 or by decreasing the microwave power from 6 mW to nearly 0 mW. A small bench-top ESR instrument can be obtained at a cost comparable to that of an atomic absorption spectrometer.
Acknowledgments
This work was supported by a Grant-in-Aid for Scientific Research (10670385) from the Ministry of Education, Science and Culture of Japan.
References
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
, 50 females 20–60 years of age.