INTRODUCTION
Over the years, several procedures have been reported to determine copper
in biological fluids. Most of the available methods are based on spectrophotometric
(Landers and Zak, 1958; Williams et al., 1977; Kossman, 1983),
Atomic Absorption Spectrophotometry (AAS) (Meret and Henkin, 1971; Makino
et al., 1981) or neutron activation analysis (Smeyers-Verbeke and
Massart, 1973). Although methods based on AAS are most accurate, specific,
reproducible and reliable (Meret and Henkin, 1971) but it is costlier
and the instrument is not available in all the clinical laboratories.
Spectrophotometrically copper in biological fluids has been determined
using various chromogens like diethyl dithiocarbamate, biquinoline, dimethyl
diphenyl phenanthroline (Mikac-Devic, 1969) and BCDS (Landers and Zak,
1958; Zak, 1958; Kossman, 1983). All spectrophotometric methods that are
in use either contain several steps or require large amount of sample
(Landers and Zak, 1958; Mikac-Devic, 1969). Various micromethods are also
available to determine copper using small sample volume but involve several
steps comprising plasma protein precipitation, centrifugation, extraction
into organic solvent, or incubation at low and high temperatures (Kossman,
1983; Smeyers-Verbeke and Massart, 1973; Mikac-Devic, 1969). Measurement
of serum copper is required in number of clinical situation like Wilsons
disease, chronic renal failure and in neonatal neurological disorders.
Due to lack of availability of simple and cost effective methods serum
copper estimation is not routinely done in all small laboratory set ups.
In this study, we have modified literature known method into few simple
steps to determine serum copper, which can be adopted in any clinical
laboratory set-ups.
MATERIALS AND METHODS
Pooled serum samples were obtained from Department of Biochemistry, Clinical
Laboratory Division, which was sent for routine analysis. BCDS and guanidine
hydrochloride were obtained from Sigma Chemicals, St. Louis, MO, USA.
All others reagents were of analytical grade. Metal free double distilled
deionized water was used throughout the study. Glass wares were acid washed
and all necessary precautions were taken to avoid trace element contamination.
Reagents
Stock Copper Standard
Copper sulphate (CuSO4.5H2O) (100 μg mL-1),
working copper standard (2 μg mL-1), 0.09 M hydroquinone
in 1 N HCl, 0.89 mM BCDS in sodium acetate and 0.89 mM BCDS in 2 M tris
solution, 0.1 M sodium acetate/acetic acid buffer pH 4.8, 6 M guanidine
hydrochloride and 0.1 M ascorbic acid in 0.1 M acetate buffer pH 4.8.
Procedure
Calibration Curve
Serial copper working standards were prepared with concentration ranging
from 40 to 200 μg dL-1. One hundred microliter of each
copper working standard was taken in different tubes into which 200 μL
of guanidine hydrochloride containing ascorbate was added. After incubating
at room temperature for 10 min, 200 μL of BCDS in tris was added
and the volume is made up to 500 μL by using tris solution, vortex
mixed and incubated at room temperature for 5 min and absorbance was read
at 480 nm using Genesys 10 UV spectrophotometer. Calibration curve was
prepared after subtracting absorbance of each standard by absorbance of
reagent blank.
Sample
Three tubes were labeled as Sample Test (ST), Sample Blank (SB) and
Reagent Blank (RB). One hundred microliter of serum was added to each
ST and SB and 100 μL of tris solution to RB. Two hundred microliter
guanidine hydrochloride containing ascorbate was added to each tubes,
vortex mixed and incubated at room temperature for 10 min. 200 μL
BCDS in tris was added into each tubes except SB, into which 200 μL
of tri sol added. Absorbance was read at 480 nm after 5 min. The corrected
absorbance of the sample (ST-{SB + RB}) was used to determine amount of
copper by using extinction co-efficient derived from the calibration curve.
We have compared our modified micromethod with that of literature known
method (Landers and Zak, 1958) using 22 serum samples. Accuracy of the
assay was checked by recovery experiments where serum was enriched with
known concentrations of copper and analyzed by the proposed micro method.
The reproducibility of the proposed micro method was checked by precision
assay where same serum sample was analyzed for within-run, within-day
and day-to-day variations.
RESULTS AND DISCUSSION
We found 200 μL of 0.89 mM BCDS was sufficient for chromogen formation
(Fig. 1) when compared to other methods requiring 500
μL of BCDS (Landers and Zak, 1958). We have rechecked the absorbance
maxima of copper-BCDS chromogen and it was found to be at 480 nm. We have
utilized the fact that a guanidine hydrochloride at its concentration
of 4 M or greater was sufficient to completely relax and alter the tertiary
structure releasing protein bound metal ions (Tanford et al., 1966).
The copper released from ceruloplasmin will form complex with copper specific
chromogen BCDS, which can be estimated spectrophotometrically. The extinction
co-efficient calculated from the Cu+-BCDS standard graph was
2.388x103 M-1 cm-1 (Fig.
2).
The minimum detection limit of our proposed micromethod is 0.4 μg
mL-1 as that of literature known method (Landers and Zak, 1958).
Thus our micromethod is as sensitive as the currently followed spectrophotometric
method. Serum copper values of our modified micromethods compared well
with that of literature known method.
|
| Fig. 1: |
Optimum volume of BCDS color reagent required for chromogen
formation, keeping concentration of copper standard constant (600
μg dL-1) |
|
| Fig. 2: |
Calibration curve of copper-BCDS complex using guanidine
hydrochloride
|
|
| Fig. 3: |
Comparison between proposed micromethod
with literature known method |
The resulting correlation coefficient was r2 = 0.9941 (Fig.
3). Recoveries were related linearly to serum copper concentration
over wide range with mean value of 92.3% of copper being recovered (Table
1). The precision of the proposed method is statistically significant
with coefficient of variance (CV) of about 2% in all the three assays;
within-run, within-day and day-to-day variations assay (Table
2). We measured serum copper levels (in μg dL-1) using
both our modified micromethod and literature known method in ten serum
samples each of healthy individuals (112.40±2.2, 110.60±1.2),
chronic renal failure (210.45±4.8, 212.42±2.4) and hyperlipidemia
(54.6±4.2, 56.1±3.2), respectively.
The rise in copper level has been reported in various diseases like chronic
renal failure (Navarro-Alarcon et al., 2006; Panichi et al.,
2004), Hodgkin`s lymphoma (Cunzhi et al., 2001), brain tumors (Floriañczyk
et al., 2003). In these conditions, the amount of sample that is
available for determination of copper along with other battery of tests
that are done for the patient management will be less specially in neonatal
disorders. So it becomes important to minimize the sample volume. To our
knowledge the modified micromethod needs less sample (100 μL) when
compared to other spectrophotometric methods which requires 1000 μL
of sample (Landers and Zak, 1958).
Use of guanidine hydrochloride in our proposed micromethod avoids protein
precipation and denaturation steps used in literature known methods and
also it avoids the additional steps like centrifugation. This simple modification
makes our proposed micromethod simple and avoids possible trace element
contamination present in steps like precipitation and centrifugation.
The proposed micromethod is simple without plasma protein precipitation,
centrifugation, or extraction steps. Our proposed method is as sensitive
as the existing spectrophotometric method. Although AAS is more accurate
method to estimate copper in biological samples (Meret and Henkin, 1971;
Smeyers-Verbeke and Massart, 1973), but the cost factor restricts its
use in small laboratories. The difference in copper estimation between
AAS and spectrophotometry was studied by Smeyers-Verbeke and Massart (1973)
and observed only small difference (2%) and such a small difference can
be compromised in situations where the AAS is not available because of
its cost and expertise to handle it.
In conclusion, our modified micromethod is simple, sensitive, rapid, cost
effective, requires less amount of sample and color reagent.The method
can be adopted in any clinical laboratories using simple spectrophotometer.
| Table 1: |
Analytical recovery of the modified micromethod to determine
serum copper (μg dL-1) |
|
| Table 2: |
Precision of the modified micromethod to determine serum
copper (μg dL-1) |
|
ACKNOWLEDGMENTS
We are thankful to our professor and head of the Department Dr. Sudhakar
Nayak for his kind support and encouragement and Dr. Nalini K., Associate
Professor, for her kindness in providing the necessary reagents for the
study.
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