Validation of an automated assay for the measurement of cupric reducing antioxidant capacity in serum of dogs
Chemicals
Trolox (6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid), potassium chloride
(KCl), sodium chloride (NaCl), potassium phosphate (KH
2
PO
4
), bathocuproinedisulfonic acid disodium salt, and copper(II) sulphate (CuSO
4
) were obtained from Sigma-Aldrich. Di-sodium hydrogen phosphate anhydrous (Na
2
HPO
4
) was obtained from Panreac.
Apparatus
The analyses were performed in the Cobas Mira Plus Biochemical Auto Analyzer (ABX
Diagnostic) and in the Olympus AU400 Automatic Chemistry Analyzer (Olympus Europe
GmbH).
Principle of the assay
The CUPRAC assay is based on the reduction of Cu
2+
into Cu
1+
by the action of the non-enzymatic antioxidants presented in the sample. The oxidant
complex, consisted of Cu
2+
-bathocuproinedisulfonic acid (Cu
2+
-BCS) reacts with the antioxidants of the sample and is reduced to a Cu
1+
-bathocuproinedisulfonic acid (Cu
1+
-BCS), a stable complex which has a maximum absorbance at 490 nm 19]. The antioxidant capacity of the sample is assumed to be equal to the extent of the
complex Cu
1+
-BCS formation 19]. The assay used for CUPRAC in the present study was based on the method described
by Campos et al. 19] with some modifications.
Measurement procedure for the Cobas Mira Plus biochemical auto analyzer
In brief, a 5 ?L volume of sample was pipetted. Then, 195 ?L of the reagent 1 were
added and a first read at 500 nm was taken. Subsequently, 50 ?L of the reagent 2 were
added to the reaction and incubated at 37 °C during 200 seconds. After incubation,
a second read at 500 nm was taken and the difference between the first and the second
read was used to calculate the antioxidant capacity of the sample. Distilled water
was used for blanks.
Measurement procedure for the Olympus AU400 automatic chemistry analyzer
An amount of 195 ?L of reagent 1 and 5 ?L of sample were pipetted. A first read at
480 nm was taken before the addition of the second reagent. Then, 50 ?L of reagent
2 were added to the mixture and incubated at 37 °C during 280 seconds. A second read
at 480 nm was taken and the difference between the first and the second read was used
for calculation of the antioxidant capacity of the sample. Distilled water was used
instead of sample or standard for blanks.
Preparation of standards
Trolox solution at a concentration of 2.0, 1.0, 0.5, 0.25, 0.125, 0.0625 and 0.0 mmol/L
were used. The results obtained for test samples were compared with a standard curve
obtained with Trolox and were expressed as millimoles of Trolox equivalents per liter.
Optimization of reagents concentrations
To adjust the assay for measurements in canine serum, different concentrations of
reagent 1 and reagent 2 were tested with the standards at different concentrations
and also with different samples in the Cobas Mira Plus biochemical analyzer.
Reagent 1 was prepared at 0.1, 0.25, 1.0 and 1.6 mmol/L of bathocuproinedisulfonic
acid disodium salt in 10 mmol/L phosphate buffer (pH 7.4) while reagent 2 was prepared
at 0.1, 0.5, and 0.8 mmol/L of CuSO
4
in ultrapure water.
The optimal concentrations were selected based on the production of a higher signal
with lower background and a lower intra-assay imprecision calculated after analysis
of one sample five times in one assay run.
Analytical validation of the assay
For the analytical validation of the CUPRAC assay, imprecision, accuracy, and sensitivity were
evaluated following previously reported protocols 19]–22].
Imprecision
Imprecision was expressed as coefficient of variation (CV) and was calculated as inter-
and intra-assay variations. The CV was calculated as the standard deviation (SD) divided
by the mean value (X
mean
) of analyzed replicates x 100 % in the formula CV?=?(SD x 100 %)/ X
mean
. To determine inter-assay variation, four serum samples were used. Inter-assay CV
was determined by analyzing the same samples in separate runs performed on five different
days. Five aliquots of each serum sample were stored in plastic vials at ?20 °C until
analysis. On the day of analysis, the samples were brought to room temperature prior
to TAC
c
measurement. The intra-assay CV was calculated after analysis of four samples five
times in one assay run. Intra-assay CV tests were performed for all the different
combination of reagents tested, although in the results only appear the values for
the final concentration selected for the assay.
Accuracy
The accuracy was evaluated through assessment of linearity and spiking recovery. The
linearity was evaluated by linearity under dilution, then duplicate determinations
of TAC
c
were made of a canine serum diluted at 1/2, 1/4, 1/8, 1/16 and 1/32 using ultrapure
water. Dilution of a Trolox solution (2.0, 1.0, 0.5, 0.25, 0.125, and 0.0625 mmol/L)
was also analyzed. For the spiking recovery, two canine serum samples with a known
TAC
c
concentration were mixed in different percentages (12.5, 25, 50, 75 and 87.5 %). The
percentages of the measured TAC
c
concentrations to the expected TAC
c
concentrations were then calculated.
Sensitivity
The detection limit was calculated on the basis of data from 20 replicate TAC
c
determinations of ultrapure water as mean value plus 3 SDs. The lower limit of quantification
(LLOQ) was calculated based on the lowest TAC
c
concentration that could be measured within a CV less than 15 % 20].
Effects of hemolysis and lipemia
In order to examine the effect of hemolysis and lipemia, serum samples from three
dogs were mixed with various concentrations of hemoglobin and lipids solution, respectively,
and TAC
c
was measured 23]. To study the effects of hemolysis, fresh hemolysate was prepared by the addition
of distilled water to packed, washed canine red blood cells from one dog, followed
by centrifugation to remove cell debris. The hemoglobin concentration was adjusted
to 80, 40, 20, 10, 5, and 0.0 g/L. Ten ?L of each concentration were added to three
90 ?L samples of canine serum to produce test samples with final hemoglobin concentration
of 8, 4, 2, 1, 0.5, and 0.0 g/L, respectively. The 0.0 g/L concentration was reached
by adding 10 ?L of distilled water to 90 ?L of the serum sample. Prepared samples
were used to determine the TAC
c
concentrations.
To investigate the effects of lipemia, a commercial fat emulsion (Lipofundina 20 %;
Braun Medical S.S.) with triglycerides concentration of 200 g/L was serially diluted
to 50, 25, 12.5, 6.25,3.125 and 0.0 g/L. Ten ?L of each dilution were added to 90 ?L
of the serum samples and were used to determine the TAC
c
concentration. The final concentrations of triglycerides in the samples were 5, 2.5,
1.25, 0.625, 0.3125, and 0.0 g/L (10 ?L of distilled water were added to 90 ?L of
the serum samples).
The TAC
c
measurements to evaluate the effect of hemolysis and lipemia in the assay were made
in the Olympus AU400.
Clinical validation
TAC
c
levels were determined in healthy (control) dogs and dogs with inflammatory bowel
disease (IBD). The control samples were from ten (seven males and three females) clinically
healthy dogs of several different breeds aged between 3 and 8 years old, that were
presented for routine checkups and had normal physical examination. Twelve dogs with
IBD were included in this study. They were four female and eight male dogs aged between
3 and 8 years old also of different several breeds. A diagnosis of IBD was made on
the basis of clinical signs (vomiting, diarrhea, and weight loss) of at least 3 weeks’
duration, and detection of lymphoplasmacytic inflammation during histologic examination
of duodenal biopsy samples following the criteria of Ohta et al. 24]. Exclusion of other causes of chronic gastrointestinal tract signs including urinalysis,
abdominal ultrasound, fecal exam, complete blood count and serum biochemistry were
made 25], 26].
Blood samples of the healthy and diseased dogs were collected from each via jugular
or lateral saphenous venipuncture into tubes without anticoagulant. Samples were centrifuged
at 3,500 x g for 5 min at 20 °C. The serum samples were stored in plastic vials at
– 20 °C until analysis.
Statistical analysis
Arithmetic means, medians, intra- and inter-assay CVs were calculated by use of routine
descriptive statistical procedures and computer software (Excel 2013, Microsoft; GraphPad
Statistics Guide). Linearity under dilution was investigated by linear regression.
To compare the TAC
c
results from both analyzers a Spearman correlation coefficient was used. The influence
of hemolysis or lipemia on TAC
c
concentration was investigated by use of 1-way ANOVA and Dunnett posttests. Interferograms
were prepared to show the differences in TAC
c
concentrations when hemoglobin or lipids were added. Kolmogorov-Smirnov’s test was
performed to assess normality of data. Comparison of the TAC
c
concentrations between healthy dogs and dogs with IBD were made by use of Student’s
t test once a parametric distribution was given. For all tests, P??0.05 was considered as statistically significant.