- Email: [email protected]
Determination of protein thiolation index (PTI) as a biomarker of oxidative stress in human serum
Analytical Biochemistry 538 (2017) 38e41
Contents lists available at ScienceDirect
Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio
Determination of protein thiolation index (PTI) as a biomarker of oxidative stress in human serum Daniela Giustarini a, Federico Galvagni b, Graziano Colombo c, Isabella Dalle-Donne c, Aldo Milzani c, Anna Maria Aloisi a, Ranieri Rossi d, * a
Department of Medicine, Surgery and Neurosciences, University of Siena, Via A. Moro 2, I-53100 Siena, Italy Department of Biotechnology, Chemistry and Pharmacy, University of Siena, Via A. Moro 2, I-53100 Siena, Italy degli Studi di Milano, Via Celoria 26, I-20133 Milan, Italy Department of Biosciences, Universita d Department of Life Sciences, Laboratory of Pharmacology and Toxicology, University of Siena, Via A. Moro 2, I-53100 Siena, Italy b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 22 June 2017 Received in revised form 7 September 2017 Accepted 18 September 2017 Available online 20 September 2017
We have introduced protein thiolation index (PTI), i.e. the molar ratio of the sum of all low molecular mass thiols bound to plasma proteins to protein free cysteinyl residues, as a sensitive biomarker of oxidative stress. According to the original procedure its determination requires a rapid separation of plasma and a specific treatment of samples to stabilize thiols. Here we demonstrate that samples can be collected without use of any anticoagulant to prevent blood clotting and without any stabilization of thiols too. This simplification of the determination of PTI makes its analysis more feasible also in routine clinical laboratories. © 2017 Elsevier Inc. All rights reserved.
Keywords: Protein sulfhydril S-thiolation Serum Oxidative stress Biomarkers
We have recently proposed that plasma protein thiol redox state may be a sensitive yet convenient parameter for assessing wholebody oxidative stress. In particular, we introduced the molar ratio of the sum of all low molecular mass thiols (LMM-SHs) bound to plasma proteins (forming, as a whole, S-thiolated proteins) to protein free cysteinyl residues (P-SH). We called this new parameter the protein thiolation index (PTI) and demonstrated that it can be used as a suitable biomarker of oxidative stress [1]. In fact, in proteins, eSH groups are the most reactive ones towards oxidants. Additionally, proteins bearing eSH groups or mixed disulfides with LMM-SH circulate in plasma for several days before being removed; therefore, they can be considered a good sensor of cumulative oxidative perturbations. Finally, since PTI is calculated as molar ratio between two oxidative states, it is not influenced by the total content of plasma proteins, which can vary in some pathological conditions. We also demonstrated that PTI can increase with ageing and in patients suffering from alkaptonuria [1]. More recently, PTI was demonstrated to increase in patients with end stage renal disease on maintenance haemodialysis [2] and to be inversely
* Corresponding author. E-mail address: [email protected] (R. Rossi). https://doi.org/10.1016/j.ab.2017.09.010 0003-2697/© 2017 Elsevier Inc. All rights reserved.
associated with spontaneous intake of calories and proteins in the same patients [3]. For PTI analysis, we developed and validated a spectrophotometric method to detect quickly total S-thiolated proteins (PSSX) in plasma samples [1]. The procedure is easy to perform but it requires a pre-treatment of sample to stabilize thiols in blood and the rapid separation of plasma (i.e., blood specimen centrifugation at 12,000g for 15 s to be carried out within 1 min from blood sampling), which makes it of difficult application in blood specimens coming from routine clinical laboratories. Samples stabilized by this procedure are stable for months at 20 C [4]. In this study, we report on a deeper investigation on the preanalytical procedure required to assess PTI, which demonstrates that whole blood samples can be collected without use of any anticoagulant to prevent blood clotting. Even if PTI can increase with time due to oxidation of thiols to form disulfides, probably the only reactive cysteine (Cys) residue at position 34 in albumin cannot form dimers because of steric hindrance of the protein itself. In addition, we demonstrate that the reaction between low molecular weight disulfides (LMM-SS) and the albumin Cys34 -SH group by transsulfuration is very slow. Conditions of storage and possible drawbacks are also investigated.
D. Giustarini et al. / Analytical Biochemistry 538 (2017) 38e41
Material and methods Materials All the other reagents were purchased by Sigma Aldrich (Milan, Italy), with the exception of mBrB that was from Calbiochem (Milan, Italy). The HPLC solvents were from Carlo Erba (Milan, Italy). Blood collection Blood samples were taken from the antecubital vein of healthy people, after oral consent, in the morning after about 12 h of fasting. The study group comprised 55 consenting volunteers (32 females and 23 males, age range: 19e72 years). All the participants reported that they were in good health and none of them had any abnormality on physical examination or in routine laboratory blood tests. Active smokers: 8 of whom 5 smoked <5 cigarettes/day, 3 smoked >5 < 20 cigarettes/day. None of the participants was under antioxidant treatment at the time of the study and all were on free diet. Two aliquots of whole blood (3 ml each) were collected, one in evacuated plastic tubes containing K3EDTA and the other one in evacuated plastic tubes containing separating gel only. Plasma was obtained from K3EDTA-treated aliquot by centrifugation at 12,000g for 15 s carried out within 1 min from blood collection and then immediately stored at 20 C as described in the original method [1]. Serum was separated by centrifugation (10 min at 800g) of blood specimens left at room temperature for 2 h after collection. Both plasma and serum samples were analyzed using the same method for PTI detection [1]. Briefly, one aliquot (0.05 ml) of either plasma or serum was used for spectrophotometric analysis of P-SH groups by endpoint colorimetric reaction with 5,50 -dithiobis(2nitrobenzoic acid) (DTNB) [5]. Another aliquot (0.15 ml) of plasma/serum was analyzed for PSSX content by colorimetric reaction with ninhydrin. PTI was calculated as the molar ratio of PSSX to the concentration of free, DTNB-titratable P-SH groups [1].
39
include, as main constraint, the rapid separation of plasma and its immediate freezing [1]. Here, PTI was assessed both in plasma and in serum from the same donors (Fig. 1). The obtained values measured in the two matrices were not significantly different, as highlighted by the strong correlation (r2 ¼ 0.995, Fig. 1A) and the 95% limits of agreement calculated by the Bland-Altman plot (Fig. 1B). This suggests that the blood coagulation process and the consequent deprivation of some proteins do not influence the results. Additionally, a 2-h time lapse, needed to obtain serum, does not lead to protein eSH group oxidation and consequent increase in PTI. We further considered the possibility that samples can be stored for times longer than 3 h and/or at varying temperatures. To study the effect of these variables on PTI levels, serum samples were divided into three aliquots and processed after standing 3 h at 20 C (~room temperature), 24 h at 20 C, 24 h at 30 C or three
Method validation and statistics Data are expressed as mean ± SD. Differences between means were evaluated using Student's t-test. A value of p < 0.05 was considered statistically significant. Correlation analysis was performed by calculating the Pearson's product-moment correlation coefficient. The agreement between plasma and serum values was evaluated by the Bland-Altman plot [6]. Results and discussion We have proposed PTI as a new biomarker of oxidative stress a few years ago. Mounting evidence has shown that perturbations in the thiol/disulfide homeostasis and, in particular, an oxidative shift in the thiol/disulfide redox potential in plasma are associated with aging and with several different diseases, such as atherosclerosis, chronic renal failure and neurodegenerative diseases [2,3,7e11]. Cys, homocysteine, glutathione (GSH) and cysteinylglycin (CysGly) exist in plasma in reduced, disulfide, and protein-bound forms, interacting with each other through thiol-disulfide exchange reactions [11]. Cys prevails over the other LMM-SH but the largely more abundant free thiols in plasma are represented by P-SH, with the relative molar ratio of LMM-SH, low molecular mass disulfides (LMM-SS), PSSX and P-SH being about 1:20:20:40 [11]. Albumin provides almost all free thiols in plasma since it is the most abundant protein, with typical concentrations ranging from 0.5 to 0.75 mM [12]. Previously, we validated the method for PTI determination in human plasma, and we supposed that the procedure should
Fig. 1. Correlation between PTI measured in plasma and serum. PTI was measured in plasma and serum of healthy people. Panel A: data set and correlation. Equation regression: y ¼ 0.995x þ 0.00175. Panel B: Bland-Altman plot, n ¼ 55. The reported values for each individual are the mean of 3 analyses.
40
D. Giustarini et al. / Analytical Biochemistry 538 (2017) 38e41
months at 20 C. A minimal increase in the mean PTI value was found in both samples left for 24 h either at room temperature or at 30 C (1 and 2% increase, respectively) (Fig. 2B and D). Conversely, both the storage at room temperature for 3 h and that at 20 C for three months did not elicit any observable change (Fig. 2A and C) in the PTI values compared to the basal level (Fig. 2E). These are quite unexpected results, since it is known that thiols are unstable in plasma and disappear rapidly. At the beginning of the eighties, it was demonstrated that GSH added to plasma decreases within minutes because it is converted mainly to glutathione disulfide and, in part, it binds to proteins [13]. More recently, the same problem was faced for other LMM-SH [14,15]. Since plasma is a pro-oxidant milieu and is abundant in disulfides, it is taken for granted that all thiols oxidize fast after blood collection and plasma separation, with typical kinetics depending on the pKa of the eSH group (it varies from ~5 for albumin to ~8 for Cys).This is the reason why some pre-analytical treatments have been proposed to stabilize thiols directly in blood or plasma samples. We have recently suggested to modify the procedure proposed by Williams et al. [15], which basically consists in lowering the pH of sample with sodium citrate and further sample dilution. Our preanalytical procedure was shown to allow measurement of plasma thiols, with good recovery, precision and accuracy for at least 6 months in samples that were stored at 80 C [1]. This procedure was suitable also for P-SH analysis [4]. Instead, the correspondence between the values measured both in plasma and in serum of the people recruited in the study clearly demonstrates that PTI is not influenced by longer storage conditions and deprivation of proteins (e.g. fibrinogen) that are lost during sample coagulation. In particular, samples were shown to be stable for at least three months at 20 C. We identified two different phenomena that may explain this stability (see on line supplementary content). Firstly, our data indicate that plasma proteins are notably protected from formation of disulfides due to steric hindrance. In fact, plasma treatment with the thiol specific oxidant diamide was not able to induce protein aggregates, as evidenced by SDS-page electrophoresis. Secondly, we demonstrated that reduced human serum albumin (rHSA) reacts very slowly with LMM-SS. Exchange reactions among these chemical species are in equilibrium (according to reaction 1) and the content of S-thiolated
Fig. 2. Influence of storage conditions. PTI was measured in serum samples stored under different conditions: A) 3 h at r.t., B) 24 h at r.t., C) 24 h 30 C, D) 3 months at 20 C, E) basal values measured in serum samples just separated, n ¼ 55 for each experimental condition.
proteins is probably proportional to the molar ratio of LMM-SS to LMM-SH in plasma [16]. PSH þ LMM-SS 4 PSS-LMM þ LMM-SH (1) It is remarkable that plasma proteins do not form disulfides under our experimental conditions. It is likely that HSA, although it has a free Cys residue, is hindered in forming disulfides with other proteins merely for steric hindrance, whereas a thiol-disulfide exchange occurs with LMM-SS at very slow rate. However, albumin dimers have been found by others research groups in disease processes associated with oxidative stress such as liver cirrhosis [17] or in in vitro experiments by the use of tert-butyl hydroperoxide as oxidizing agent [18]. Thus, this point requires additional investigation to be completely clarified. In conclusion, in this study we show that the pre-analytical procedure required to measure PTI can be greatly simplified, since no specific treatment to stabilize blood is necessary and determinations can be carried out in serum. The possibility to quantify reliably PTI in serum specimens is of greater advance because it makes PTI determination easy to perform and suitable for clinical analyses with a high daily throughput. Therefore, it can be used for routine screening tests and, in particular, when blood samples cannot be immediately processed and/or when only serum samples are available. Acknowledgment All authors have read the journal's authorship agreement and policy on disclosure of potential conflicts of interest. Appendix A. Supplementary data Supplementary data related to this article can be found at https://doi.org/10.1016/j.ab.2017.09.010. References [1] D. Giustarini, I. Dalle-Donne, S. Lorenzini, et al., Protein thiolation index (PTI) as a biomarker of oxidative stress, Free Radic. Biol. Med. 53 (2012) 907e915. [2] G. Colombo, F. Reggiani, M.A. Podest a, et al., Plasma protein thiolation index (PTI) as a biomarker of thiol-specific oxidative stress in haemodialyzed patients, Free Radic. Biol. Med. 89 (2015) 443e451. [3] P. Fanti, D. Giustarini, R. Rossi, et al., Dietary intake of proteins and calories is inversely associated with the oxidation state of plasma thiols in end-stage renal disease patients, J. Ren. Nutr. 25 (2015) 494e503. [4] D. Giustarini, F. Galvagni, M. Orlandini, P. Fanti, R. Rossi, Immediate stabilization of human blood for delayed quantification of endogenous thiols and disulfides, J. Chromatogr. B Anal. Technol. Biomed. Life Sci. 1019 (2016) 51e58. [5] G. Ellman, H. Lysko, A precise method for the determination of whole blood and plasma sulfhydryl groups, Anal. Biochem. 93 (1979) 98e102. [6] J.M. Bland, D.G. Altman, Statistical methods for assessing agreement between two methods of clinical measurement, Lancet 1 (1986) 307e310. [7] S. Ashfaq, J.L. Abramson, D.P. Jones, et al., The relationship between plasma levels of oxidized and reduced thiols and early atherosclerosis in healthy adults, Am. Coll. Cardiol. 47 (2006) 1005e1011. € k, H. Kundi, et al., Plasma thiols and thiol-disulfide ho[8] E. Kızıltunç, M. Go meostasis in patients with isolated coronary artery ectasia, Atherosclerosis 253 (2016) 209e213. [9] M.S. Medeiros, A. Schumacher-Schuh, A.M. Cardoso, et al., Iron and oxidative stress in Parkinson's disease: an observational study of injury biomarkers, PLoS One 11 (2016) e0146129. [10] C. Buhmann, S. Arlt, A. Kontush, et al., Plasma and CSF markers of oxidative stress are increased in Parkinson's disease and influenced by antiparkinsonian medication, Neurobiol. Dis. 15 (2004) 160e170. [11] D. Giustarini, I. Dalle-Donne, S. Lorenzini, A. Milzani, R. Rossi, Age-related influence on thiol, disulfide, and protein-mixed disulfide levels in human plasma, J. Gerontol. A Biol. Sci. Med. Sci. 61 (2006) 1030e1038. [12] S. Curry, H. Mandelkow, P. Brick, N. Franks, Crystal structure of human serum albumin complexed with fatty acid reveals an asymmetric distribution of binding sites, Nat. Struct. Biol. 5 (1998) 827e835. [13] M.E. Anderson, A. Meister, Dynamic state of glutathione in blood plasma,
D. Giustarini et al. / Analytical Biochemistry 538 (2017) 38e41 J. Biol. Chem. 255 (1980) 9530e9533. [14] M. Mansoor, A.M. Svardal, P.M. Ueland, Determination of the in vivo redox status of cysteine, cysteinylglycine, homocysteine, and glutathione in human plasma, Anal. Biochem. 200 (1992) 218e229. [15] R.H. Williams, J.A. Maggiore, R.D. Reynolds, C.M. Helgason, Novel approach for the determination of the redox status of homocysteine and other aminothiols in plasma from healthy subjects and patients with ischemic stroke, Clin. Chem. 47 (2001) 1031e1039. [16] L. Turell, R. Radi, B. Alvarez, The thiol pool in human plasma: the central
41
contribution of albumin to redox porcesses, Free Radic. Biol. Med. 65 (2013) 244e253. [17] M. Naldi, M. Baldassarre, M. Nati, et al., Mass spectrometric characterization of human serum albumin dimer: a new potential biomarker in chronic liver diseases, J. Pharm. Biomed. Anal. 112 (2015) 169e175. [18] Y. Ogasawara, T. Namai, T. Togawa, K. Ishii, Formation of albumin dimers induced by exposure to peroxides in human plasma: a possible biomarker for oxidative stress, Biochem. Biophys. Res. Commun. 40 (2006) 353e358.
Contents lists available at ScienceDirect
Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio
Determination of protein thiolation index (PTI) as a biomarker of oxidative stress in human serum Daniela Giustarini a, Federico Galvagni b, Graziano Colombo c, Isabella Dalle-Donne c, Aldo Milzani c, Anna Maria Aloisi a, Ranieri Rossi d, * a
Department of Medicine, Surgery and Neurosciences, University of Siena, Via A. Moro 2, I-53100 Siena, Italy Department of Biotechnology, Chemistry and Pharmacy, University of Siena, Via A. Moro 2, I-53100 Siena, Italy degli Studi di Milano, Via Celoria 26, I-20133 Milan, Italy Department of Biosciences, Universita d Department of Life Sciences, Laboratory of Pharmacology and Toxicology, University of Siena, Via A. Moro 2, I-53100 Siena, Italy b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 22 June 2017 Received in revised form 7 September 2017 Accepted 18 September 2017 Available online 20 September 2017
We have introduced protein thiolation index (PTI), i.e. the molar ratio of the sum of all low molecular mass thiols bound to plasma proteins to protein free cysteinyl residues, as a sensitive biomarker of oxidative stress. According to the original procedure its determination requires a rapid separation of plasma and a specific treatment of samples to stabilize thiols. Here we demonstrate that samples can be collected without use of any anticoagulant to prevent blood clotting and without any stabilization of thiols too. This simplification of the determination of PTI makes its analysis more feasible also in routine clinical laboratories. © 2017 Elsevier Inc. All rights reserved.
Keywords: Protein sulfhydril S-thiolation Serum Oxidative stress Biomarkers
We have recently proposed that plasma protein thiol redox state may be a sensitive yet convenient parameter for assessing wholebody oxidative stress. In particular, we introduced the molar ratio of the sum of all low molecular mass thiols (LMM-SHs) bound to plasma proteins (forming, as a whole, S-thiolated proteins) to protein free cysteinyl residues (P-SH). We called this new parameter the protein thiolation index (PTI) and demonstrated that it can be used as a suitable biomarker of oxidative stress [1]. In fact, in proteins, eSH groups are the most reactive ones towards oxidants. Additionally, proteins bearing eSH groups or mixed disulfides with LMM-SH circulate in plasma for several days before being removed; therefore, they can be considered a good sensor of cumulative oxidative perturbations. Finally, since PTI is calculated as molar ratio between two oxidative states, it is not influenced by the total content of plasma proteins, which can vary in some pathological conditions. We also demonstrated that PTI can increase with ageing and in patients suffering from alkaptonuria [1]. More recently, PTI was demonstrated to increase in patients with end stage renal disease on maintenance haemodialysis [2] and to be inversely
* Corresponding author. E-mail address: [email protected] (R. Rossi). https://doi.org/10.1016/j.ab.2017.09.010 0003-2697/© 2017 Elsevier Inc. All rights reserved.
associated with spontaneous intake of calories and proteins in the same patients [3]. For PTI analysis, we developed and validated a spectrophotometric method to detect quickly total S-thiolated proteins (PSSX) in plasma samples [1]. The procedure is easy to perform but it requires a pre-treatment of sample to stabilize thiols in blood and the rapid separation of plasma (i.e., blood specimen centrifugation at 12,000g for 15 s to be carried out within 1 min from blood sampling), which makes it of difficult application in blood specimens coming from routine clinical laboratories. Samples stabilized by this procedure are stable for months at 20 C [4]. In this study, we report on a deeper investigation on the preanalytical procedure required to assess PTI, which demonstrates that whole blood samples can be collected without use of any anticoagulant to prevent blood clotting. Even if PTI can increase with time due to oxidation of thiols to form disulfides, probably the only reactive cysteine (Cys) residue at position 34 in albumin cannot form dimers because of steric hindrance of the protein itself. In addition, we demonstrate that the reaction between low molecular weight disulfides (LMM-SS) and the albumin Cys34 -SH group by transsulfuration is very slow. Conditions of storage and possible drawbacks are also investigated.
D. Giustarini et al. / Analytical Biochemistry 538 (2017) 38e41
Material and methods Materials All the other reagents were purchased by Sigma Aldrich (Milan, Italy), with the exception of mBrB that was from Calbiochem (Milan, Italy). The HPLC solvents were from Carlo Erba (Milan, Italy). Blood collection Blood samples were taken from the antecubital vein of healthy people, after oral consent, in the morning after about 12 h of fasting. The study group comprised 55 consenting volunteers (32 females and 23 males, age range: 19e72 years). All the participants reported that they were in good health and none of them had any abnormality on physical examination or in routine laboratory blood tests. Active smokers: 8 of whom 5 smoked <5 cigarettes/day, 3 smoked >5 < 20 cigarettes/day. None of the participants was under antioxidant treatment at the time of the study and all were on free diet. Two aliquots of whole blood (3 ml each) were collected, one in evacuated plastic tubes containing K3EDTA and the other one in evacuated plastic tubes containing separating gel only. Plasma was obtained from K3EDTA-treated aliquot by centrifugation at 12,000g for 15 s carried out within 1 min from blood collection and then immediately stored at 20 C as described in the original method [1]. Serum was separated by centrifugation (10 min at 800g) of blood specimens left at room temperature for 2 h after collection. Both plasma and serum samples were analyzed using the same method for PTI detection [1]. Briefly, one aliquot (0.05 ml) of either plasma or serum was used for spectrophotometric analysis of P-SH groups by endpoint colorimetric reaction with 5,50 -dithiobis(2nitrobenzoic acid) (DTNB) [5]. Another aliquot (0.15 ml) of plasma/serum was analyzed for PSSX content by colorimetric reaction with ninhydrin. PTI was calculated as the molar ratio of PSSX to the concentration of free, DTNB-titratable P-SH groups [1].
39
include, as main constraint, the rapid separation of plasma and its immediate freezing [1]. Here, PTI was assessed both in plasma and in serum from the same donors (Fig. 1). The obtained values measured in the two matrices were not significantly different, as highlighted by the strong correlation (r2 ¼ 0.995, Fig. 1A) and the 95% limits of agreement calculated by the Bland-Altman plot (Fig. 1B). This suggests that the blood coagulation process and the consequent deprivation of some proteins do not influence the results. Additionally, a 2-h time lapse, needed to obtain serum, does not lead to protein eSH group oxidation and consequent increase in PTI. We further considered the possibility that samples can be stored for times longer than 3 h and/or at varying temperatures. To study the effect of these variables on PTI levels, serum samples were divided into three aliquots and processed after standing 3 h at 20 C (~room temperature), 24 h at 20 C, 24 h at 30 C or three
Method validation and statistics Data are expressed as mean ± SD. Differences between means were evaluated using Student's t-test. A value of p < 0.05 was considered statistically significant. Correlation analysis was performed by calculating the Pearson's product-moment correlation coefficient. The agreement between plasma and serum values was evaluated by the Bland-Altman plot [6]. Results and discussion We have proposed PTI as a new biomarker of oxidative stress a few years ago. Mounting evidence has shown that perturbations in the thiol/disulfide homeostasis and, in particular, an oxidative shift in the thiol/disulfide redox potential in plasma are associated with aging and with several different diseases, such as atherosclerosis, chronic renal failure and neurodegenerative diseases [2,3,7e11]. Cys, homocysteine, glutathione (GSH) and cysteinylglycin (CysGly) exist in plasma in reduced, disulfide, and protein-bound forms, interacting with each other through thiol-disulfide exchange reactions [11]. Cys prevails over the other LMM-SH but the largely more abundant free thiols in plasma are represented by P-SH, with the relative molar ratio of LMM-SH, low molecular mass disulfides (LMM-SS), PSSX and P-SH being about 1:20:20:40 [11]. Albumin provides almost all free thiols in plasma since it is the most abundant protein, with typical concentrations ranging from 0.5 to 0.75 mM [12]. Previously, we validated the method for PTI determination in human plasma, and we supposed that the procedure should
Fig. 1. Correlation between PTI measured in plasma and serum. PTI was measured in plasma and serum of healthy people. Panel A: data set and correlation. Equation regression: y ¼ 0.995x þ 0.00175. Panel B: Bland-Altman plot, n ¼ 55. The reported values for each individual are the mean of 3 analyses.
40
D. Giustarini et al. / Analytical Biochemistry 538 (2017) 38e41
months at 20 C. A minimal increase in the mean PTI value was found in both samples left for 24 h either at room temperature or at 30 C (1 and 2% increase, respectively) (Fig. 2B and D). Conversely, both the storage at room temperature for 3 h and that at 20 C for three months did not elicit any observable change (Fig. 2A and C) in the PTI values compared to the basal level (Fig. 2E). These are quite unexpected results, since it is known that thiols are unstable in plasma and disappear rapidly. At the beginning of the eighties, it was demonstrated that GSH added to plasma decreases within minutes because it is converted mainly to glutathione disulfide and, in part, it binds to proteins [13]. More recently, the same problem was faced for other LMM-SH [14,15]. Since plasma is a pro-oxidant milieu and is abundant in disulfides, it is taken for granted that all thiols oxidize fast after blood collection and plasma separation, with typical kinetics depending on the pKa of the eSH group (it varies from ~5 for albumin to ~8 for Cys).This is the reason why some pre-analytical treatments have been proposed to stabilize thiols directly in blood or plasma samples. We have recently suggested to modify the procedure proposed by Williams et al. [15], which basically consists in lowering the pH of sample with sodium citrate and further sample dilution. Our preanalytical procedure was shown to allow measurement of plasma thiols, with good recovery, precision and accuracy for at least 6 months in samples that were stored at 80 C [1]. This procedure was suitable also for P-SH analysis [4]. Instead, the correspondence between the values measured both in plasma and in serum of the people recruited in the study clearly demonstrates that PTI is not influenced by longer storage conditions and deprivation of proteins (e.g. fibrinogen) that are lost during sample coagulation. In particular, samples were shown to be stable for at least three months at 20 C. We identified two different phenomena that may explain this stability (see on line supplementary content). Firstly, our data indicate that plasma proteins are notably protected from formation of disulfides due to steric hindrance. In fact, plasma treatment with the thiol specific oxidant diamide was not able to induce protein aggregates, as evidenced by SDS-page electrophoresis. Secondly, we demonstrated that reduced human serum albumin (rHSA) reacts very slowly with LMM-SS. Exchange reactions among these chemical species are in equilibrium (according to reaction 1) and the content of S-thiolated
Fig. 2. Influence of storage conditions. PTI was measured in serum samples stored under different conditions: A) 3 h at r.t., B) 24 h at r.t., C) 24 h 30 C, D) 3 months at 20 C, E) basal values measured in serum samples just separated, n ¼ 55 for each experimental condition.
proteins is probably proportional to the molar ratio of LMM-SS to LMM-SH in plasma [16]. PSH þ LMM-SS 4 PSS-LMM þ LMM-SH (1) It is remarkable that plasma proteins do not form disulfides under our experimental conditions. It is likely that HSA, although it has a free Cys residue, is hindered in forming disulfides with other proteins merely for steric hindrance, whereas a thiol-disulfide exchange occurs with LMM-SS at very slow rate. However, albumin dimers have been found by others research groups in disease processes associated with oxidative stress such as liver cirrhosis [17] or in in vitro experiments by the use of tert-butyl hydroperoxide as oxidizing agent [18]. Thus, this point requires additional investigation to be completely clarified. In conclusion, in this study we show that the pre-analytical procedure required to measure PTI can be greatly simplified, since no specific treatment to stabilize blood is necessary and determinations can be carried out in serum. The possibility to quantify reliably PTI in serum specimens is of greater advance because it makes PTI determination easy to perform and suitable for clinical analyses with a high daily throughput. Therefore, it can be used for routine screening tests and, in particular, when blood samples cannot be immediately processed and/or when only serum samples are available. Acknowledgment All authors have read the journal's authorship agreement and policy on disclosure of potential conflicts of interest. Appendix A. Supplementary data Supplementary data related to this article can be found at https://doi.org/10.1016/j.ab.2017.09.010. References [1] D. Giustarini, I. Dalle-Donne, S. Lorenzini, et al., Protein thiolation index (PTI) as a biomarker of oxidative stress, Free Radic. Biol. Med. 53 (2012) 907e915. [2] G. Colombo, F. Reggiani, M.A. Podest a, et al., Plasma protein thiolation index (PTI) as a biomarker of thiol-specific oxidative stress in haemodialyzed patients, Free Radic. Biol. Med. 89 (2015) 443e451. [3] P. Fanti, D. Giustarini, R. Rossi, et al., Dietary intake of proteins and calories is inversely associated with the oxidation state of plasma thiols in end-stage renal disease patients, J. Ren. Nutr. 25 (2015) 494e503. [4] D. Giustarini, F. Galvagni, M. Orlandini, P. Fanti, R. Rossi, Immediate stabilization of human blood for delayed quantification of endogenous thiols and disulfides, J. Chromatogr. B Anal. Technol. Biomed. Life Sci. 1019 (2016) 51e58. [5] G. Ellman, H. Lysko, A precise method for the determination of whole blood and plasma sulfhydryl groups, Anal. Biochem. 93 (1979) 98e102. [6] J.M. Bland, D.G. Altman, Statistical methods for assessing agreement between two methods of clinical measurement, Lancet 1 (1986) 307e310. [7] S. Ashfaq, J.L. Abramson, D.P. Jones, et al., The relationship between plasma levels of oxidized and reduced thiols and early atherosclerosis in healthy adults, Am. Coll. Cardiol. 47 (2006) 1005e1011. € k, H. Kundi, et al., Plasma thiols and thiol-disulfide ho[8] E. Kızıltunç, M. Go meostasis in patients with isolated coronary artery ectasia, Atherosclerosis 253 (2016) 209e213. [9] M.S. Medeiros, A. Schumacher-Schuh, A.M. Cardoso, et al., Iron and oxidative stress in Parkinson's disease: an observational study of injury biomarkers, PLoS One 11 (2016) e0146129. [10] C. Buhmann, S. Arlt, A. Kontush, et al., Plasma and CSF markers of oxidative stress are increased in Parkinson's disease and influenced by antiparkinsonian medication, Neurobiol. Dis. 15 (2004) 160e170. [11] D. Giustarini, I. Dalle-Donne, S. Lorenzini, A. Milzani, R. Rossi, Age-related influence on thiol, disulfide, and protein-mixed disulfide levels in human plasma, J. Gerontol. A Biol. Sci. Med. Sci. 61 (2006) 1030e1038. [12] S. Curry, H. Mandelkow, P. Brick, N. Franks, Crystal structure of human serum albumin complexed with fatty acid reveals an asymmetric distribution of binding sites, Nat. Struct. Biol. 5 (1998) 827e835. [13] M.E. Anderson, A. Meister, Dynamic state of glutathione in blood plasma,
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