A simple automated procedure for thiol measurement in human serum samples

unitermos abstract Thiol groups have been described as the main responsible for antioxidative effects of plasmatic proteins. Also, thiol serum levels have shown a positive correlation with total antioxidant capacity (TAC) in many studies. Measurement of TAC by substract oxidation-based methods have been widely used as a reference to measure antioxidant status; however, in many cases these methods are inexact or imprecise, usually when performed by manual procedures. In this paper we describe a simple automated procedure for the determination of total thiols in serum, which was based on Ellman’s method. It was correlated with several markers of oxidative/antioxidative status, such as TAC and thiobarbituric acid reactive substance test (TBARs). Serum thiol levels were correlated positively with TAC (r = 0.298, p < 0.001) and negatively with TBARs levels (r = -0.330, p < 0.001). The novel automated procedure for thiol groups measurement can be a great tool in estimation of antioxidant status together with TAC assay. This procedure makes the determination of total thiol groups in large scale possible in clinical chemistry or research laboratories where this approach is necessary. Thiol groups


key words y y ey o ds y y key words y y y y y y y y y y y unitermos unitermos unitermos u te os unitermos unitermos abstract
Thiol groups have been described as the main responsible for antioxidative effects of plasmatic proteins.Also, thiol serum levels have shown a positive correlation with total antioxidant capacity (TAC) in many studies.Measurement of TAC by substract oxidation-based methods have been widely used as a reference to measure antioxidant status; however, in many cases these methods are inexact or imprecise, usually when performed by manual procedures.In this paper we describe a simple automated procedure for the determination of total thiols in serum, which was based on Ellman's method.It was correlated with several markers of oxidative/antioxidative status, such as TAC and thiobarbituric acid reactive substance test (TBARs).Serum thiol levels were correlated positively with TAC (r = 0.298, r p < 0.001) and negatively p with TBARs levels (r = -0.330,r p < 0.001).The novel automated procedure for thiol groups measurement p can be a great tool in estimation of antioxidant status together with TAC assay.This procedure makes the determination of total thiol groups in large scale possible in clinical chemistry or research laboratories where this approach is necessary.

Introduction
Oxidative stress is a commonly used term to denote the imbalance between the concentrations of reactive oxygen and nitrogen species and the antioxidative defense mechanisms of the body (11,13,14,21) .An antioxidant is a substance that is able to protect a substrate susceptible to oxidation from peroxidative injury, being itself present at fairly low concentrations in relation to the substrate (12,29) .Oxidative stress has been associated with a number of human diseases, such as cancer, coronary heart disease, arthritis, diabetes, cataract and degenerative processes (3,5,9,19) .Over time, humans are continuously exposed to oxidants of both exogenous and endogenous origins (9) .The determination of the oxidative stress status of a person not only allows comparison with the average of a population, but can also indicate the risk of suffering disorders and diseases that are product of oxidative stress (2) .Thiols are those compounds which contain the sulfhydryl group (-SH) attached to a carbon atom.They are endogenous molecules that assist aerobic cells maintaining a reducing state, despite an oxidizing environment (9,25) .Thiols are extraordinarily efficient antioxidants protecting cells against consequences of damage induced by free radicals, due to their ability to react with the latter (3,27) .Both intracellular and extracellular redox states of thiols play a critical role in the determination of protein structure and function, regulation of enzymatic activity of transcription factors and antioxidant protection (29) .
Thiol groups are found in all body cells and are indispensable for life (3,24) .Some sulfur-containing antioxidant compounds are cysteine, methionine, taurine, glutathione, lipoic acid, mercaptopropionylglycine, N-acetylcysteine, and the three major organosulfur compounds of Gallic oil (diallyilsulfide, diallyldisulfide and diallyltrisulfide) (3,10) .Proteins constitute the main antioxidant component of serum and their sulfhydryl groups are mainly responsible for their antioxidative effects.In a recent study it was calculated that SH protein groups contribute 52,9% to the measured serum total antioxidant capacity in healthy subjects (17) .
The standard method for measuring human serum thiols was originally described by Ellman (15) and further modified by Hu (22) .However, the measurement of thiols and disulfides in this sample has proven to be difficult.Most thiols are present in low concentration (0.1-10nmol/mL) and are very unstable in the isolated serum (16) .Determining a new method for thiol measurement has been the focus of many researches.High-performance liquid chromatography (HPLC) is the most popular method for measurement of isolated thiol contents.This method, however, has some disadvantages, including being time-consuming and having complicated derivatizations.Also, HPLC-based method usually consumes more reagents and is, therefore, high costly (4) .
In the present paper we describe a novel automated method for determination of thiols in human serum samples, which was based on Ellman's method, and was adapted to a chemical analyzer COBAS MIRA ® Plus (Roche Diagnostic Systems, Welwyn, UK).The levels of total thiol groups were directly proportional to total antioxidant capacity and inversely proportional to serum levels of malondialdehyde (MDA).That new methodology makes the determination of serum thiols in large scale possible in clinical chemistry or research laboratories.

Subjects and blood samples
A total of 244 apparently healthy volunteers (122 female and 122 male) were included in this study.The median age of the entire group was 42 years with a range of 18-91 years.All subjects gave oral informed consent and the study was approved by the local ethical committee of Universidade Federal do Amazonas (UFAM).A 5mL blood sample was collected under 12-hour fasting conditions, in blood collecting test tube (Vacuum II, Villfend Corporation), and the serum was separated by centrifugation at 2.500rpm for ten minutes and stored at -20°C until biochemical determinations.

Biochemical analyses
The serum total concentrations of total protein (TP), albumin (ALB), uric acid (AUC), bilirubin (BIL), magnesium (Mg) and iron (Fe) were measured by using commercial kits (Labtest Diagnóstica, Minas Gerais, Brazil) on the Express Plus biochemical analyzer (Ciba-Corning Diagnostics, Medfield, USA).The intra-assay coefficient of variation was evaluated by performing 15 repetitions in a single analytical run, using serum of healthy subjects.The interassay coefficient of variation was evaluated in triplicate (on three different dispensing cycles) in eight different analytical runs using serum of healthy subjects.The same procedures were used for manual and novel automated assay.

Thiol groups assay
Total serum thiol concentration or sulfhydryl groups (SH) were measured by the methods originally described by Elmman (15) and modified by Hu (22) .Here, thiols interact with 5, 5'-dithiobis-(2-nitrobenzoic acid) (DTNB), forming a highly colored anion with maximum peak at 412 m (e412 = 13,600M -1 cm -1 ).Here, for the first time this method was adapted to an automated biochemistry analyzer (COBAS MIRA ® Plus).Table 1 resumes the main parameters for COBAS MIRA ® Plus programming.In manual protocol, as described by Hu (22) , an aliquot of fresh serum (25µL) was mixed with 1mL Tris-EDTA buffer (0.25mmol/L Tris base, 20mmol/L EDTA, pH 8.2), and absorbance at 412 m was read (A1).Next, a 25L aliquot of DTNB stock solution (10mmol/L in absolute methanol) was added to the solution.After 15 minutes at ambient temperature, the absorbance was read again (A2) together with a DTNB blank (B).The concentration of sulfhydryl groups was calculated by using reduced glutathione as sulfhydryl group standard and the result was expressed in mmol/L.

Total antioxidant capacity assay
The TAC of serum was measured by a method based on 2, 2-azinobis 3-ethylbenzothiazoline-6-sulfonate (ABTS) (17) .In this assay, ABTS is incubated with potassium persulfate to produce oxidation of ABTS.Briefly, an amount of 10mg of ABTS was dissolved in 10mL of an aqueous solution containing 2.5mmol/L potassium persulfate and allowing the mixture to stand in the dark at room temperature for one to four hours before use.For the study of samples, ABTS oxidized stock solution was diluted with deionized water to an absorbance of 0.70 at 734m.After addition of 1mL of diluted ABTS oxidized to 10µL of serum, the absorbance reading was taken ten minutes after initial mixing.The TAC was calculated using Trolox ® as standard and the result was expressed as mEq Trolox ® /L.

Ferric-xylenol orange (FOX 1 ) assay
The total peroxide concentrations were determined using the FOX 1 method (19) .The FOX 1 test system is based on the oxidation of ferrous iron to ferric iron by the various types of peroxides contained in plasma samples in the presence of xylenol orange, which produces a colored ferric-xylenol orange complex whose absorbance can be measured.FOX 1 reagent was prepared with 100µmol/L xylenol orange, 4mmol/L butylated hydroxytoluene, 25mmol/L sulfuric acid and 250µmol/L ammonium ferrous sulfate in a HPLC grade methanol:water (9:1 v/v) solution.An aliquot of 200µL of serum was mixed with 2mL of FOX 1 reagent, vortexed and incubated at room temperature for 30 minutes.The tubes were than centrifuged at 2.500rpm for ten minutes.Absorbance of the supernatant was measured at 560 m.The total peroxide content of the plasma samples was determined by the difference in absorbance between test and blank samples using a solution of H 2 O 2 as standard.The results were expressed in µmol H 2 0 2 /L.

Thiobarbituric acid reactive substance (TBARs) assay
Malondialdehyde (MDA) serum levels were determined as described previously by TBARs spectrophotometric test (3) .A portion of serum (200µL) was mixed with 2mL of a solution containing 15% (w/v) trichloroacetic acid, 0.38% (w/v) thiobarbituric acid and 0.25N of hydrochloric acid (HCl).The mixture was heated at 100°C for 30 minutes and, after centrifugation, the absorbance was measured at 535 m.The total MDA content of the serum samples was determined by the difference in absorbance between test and standard samples using a solution of MDA as standard.The results were expressed as µmol TBARs/L.

Statistical analysis
All parameters were given as mean ± standard deviation (SD).A paired sample t test was used to estimate differences between male and female subjects.The Mann-Whitney rank sum test was also used for analysis when the data were not normally distributed.Correlations between different measured parameters were also examined by Pearson correlation test.The tests were performed using the SigmaStat 2.03 software (Jandel Scientific, Chicago).Statistical significance for all tests was accepted at p < 0.05.

Results
Table 2 shows the main parameters as age and oxidative stress index of 244 healthy subjects of this study.
It also shows that when separated by sex no variation was shown for the majority of the analyzed parameters, except for albumin, uric acid and iron, as was already believed (t test, p < 0.05).The average levels were also in accordance with those described in literature.
Table 3 shows Spearman correlation indices between serum markers of oxidative stress.There we can see that there was a statistically significant positive correlation between serum levels of thiol groups and TAC, total peroxides (LOOH) and ALB.The serum levels of thiols were also negatively correlated with the levels of TBARs and Mg.Thiol serum levels were positively correlated with TAC (r = 0.298, p < 0.001) and negatively correlated with malondialdehyde levels, an oxidant status marker (r = -0.33,p < 0.001).The intra-assay and inter-assay coefficients of variation to novel automated thiol measurement were 3.8% and 6.7%, respectively.When manual procedure was used, the intra-assay coefficient of variation was 8.5% and the inter-assay, 10,2%.The detection limit for automated procedure was 0.05mmol/L.

Discussion
In the last years, several methods have been developed to assess the total antioxidant capacity of human serum or plasma because of the difficulty in measuring each antioxidant component separately, and the interactions among different antioxidant components in serum or plasma (1,20,28,30) .The total peroxyl radical trapping parameter assay of Wayner et al. (28)   antioxidant capacity most widely used during the last decade.As Rice-Evans and Miller (26) pointed out, the major problem with the original total peroxyl radical trapping parameter assay lies in the oxygen electrode endpoint: an oxygen electrode will not maintain its stability over the period of time required.More recently, the Trolox ® equivalent antioxidant capacity (TEAC) assay was developed by several authors (6-8, 17, 23) .The TEAC assay is based on the inhibition by antioxidants of the absorbance of the radical cation of 2,29-azinobis (3-ethylbenzothiazoline-6-sulfonate) (ABTS).In case of determination of serum total antioxidant capacity, the main problem in this assay is the instability of oxidized ABTS, turning it difficult to be measured in large scale (17) .
An important piece of data presented in this article was the use of an automated procedure for serum total thiol measurement as one more tool in the determination of antioxidant status marker together with the use of traditional markers of this parameter, such as TAC.The results of this study demonstrate, firstly, that the procedure for dosage of serum thiols can be easily automated in the chemical analyzer COBAS MIRA ® Plus, an analyzer currently used in laboratories of clinical chemistry.On the other hand, this novel procedure significantly reduces the costs of the assay, because the volume of reagent used in the automated assay is about three times smaller than that in the manual procedure.Another advantage of the automated procedure is the reduction of analytical errors when an automated Table 3 analyzer is used.In the present study, there was a reduction of the variation intra-assay coefficient of 8.5% in the manual procedure to 3.5% using the automated method.Here, after analysis of 244 samples, the serum levels of total thiol groups were correlated to TAC.Using the Pearson test, there was a positive correlation between serum levels of thiol groups and the TAC (r = 0.298, p < 0.001).Similar results have been shown in recent publications (13,17) .

Spearman correlation indices between serum markers of oxidative stress
In conclusion, the present study showed that the measure of serum thiols can be easily adapted to the chemical analyzer COBAS MIRA ® Plus, diminishing costs and increasing the efficiency of the method, what makes the use of this procedure in large scale possible for population studies.The measurement of thiol groups also showed a positive correlation to the dosage of TAC, which indicates that this measurement can be used as an auxiliary or substitute to TAC assay in studies or physiopathological conditions in which it is needed to evaluate the total antioxidant status individually.