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The Journals of Gerontology Series A: Biological Sciences and Medical Sciences 61:1030-1038 (2006)
© 2006 The Gerontological Society of America

Age-Related Influence on Thiol, Disulfide, and Protein-Mixed Disulfide Levels in Human Plasma

Daniela Giustarini, Isabella Dalle-Donne, Sauro Lorenzini, Aldo Milzani and Ranieri Rossi

1 Department of Neuroscience, Pharmacology Unit, University of Siena, Italy.
2 Department of Biology, University of Milan, Italy.
3 Department of Clinical Medicine and Immunological Sciences, Rheumatology Unit, University of Siena, Italy.

Address correspondence to Daniela Giustarini, PhD, Department of Neuroscience, Pharmacology Unit, University of Siena, via A. Moro 4, I-53100, Siena, Italy. E-mail: giustarini{at}unisi.it


   Abstract
Top
 Abstract
Methods
 Results
 Discussion
 References
 
In this study, plasma levels of both low-molecular-mass sulfhydryls/disulfides and mixed disulfides with proteins in 41 healthy humans aged 21–92 years were measured, with the aim of assessing whether there is a shift of the thiol/disulfide balance during aging and verifying some of the possible effects of the thiol imbalance. Our data suggest that aging is strictly correlated to a decrease in plasma glutathione and cysteinylglycine with the concomitant increase of most oxidized forms of thiols and a parallel increase in total cysteine and total homocysteine, probably due to an augmented efflux of these amino acids from various organs. The occurrence of two distinct regulatory systems for plasmatic pools of glutathione/cysteinylglycine on the one hand and cysteine/homocysteine on the other hand is hypothesized.

THE mechanisms responsible for the onset of senescence are still unknown. Many theories have been advanced to account for the aging process, among which the "free radical theory," which proposes that the increase in oxidative stress with aging is the primary cause of age-related declines in cellular function, has received the most attention [reviewed in (1)]. However, relevant indications able to clarify whether oxidative stress has an important role in the aging process or is simply a consequence of it are still lacking.

Much support has been obtained for the presence of oxidative/nitrosative stress in aging (2–5). Aging tissue shows a progressive accumulation of oxidatively modified biomolecules such as carbonylated proteins (6), advanced glycation end products (7), lipid peroxidation products (8), and lipofuscin (9). As a consequence, the progressive accumulation of damaged biomolecules could be a factor in the adverse physiological modifications occurring with aging. Additionally, some clinical studies on humans have revealed an age-related shift of plasma thiol/disulfide redox couples (10,11), which could be a contributing factor to the pathogenesis of some age-related diseases including cardiovascular diseases, diabetes, rheumatoid arthritis, and neurological disorders (10,12–15). The cause of the age-related shift of the thiol/disulfide balance is poorly understood; it could be simply due to a higher reactive oxygen and/or nitrogen species burden in the hematic compartment. Alternatively, a decreased supply of the reduced forms through diet may be hypothesized.

Cysteine (Cys), homocysteine (Hcys), glutathione (GSH), and cysteinylglycine (CysGly) are the most abundant low-molecular-mass sulfhydryls (LMM-SH) occurring in the extracellular milieu and, together with albumin, represent almost all the thiols in plasma. All these molecules are metabolically interrelated and are important in determining the redox environment and free radical interactions (16,17). The turnover of plasma thiols is complex in that multiple organs may contribute to various extents to their delivery and uptake. In particular, GSH seems to be released mainly by the liver and, to a minor extent, by muscle, where dietary Cys (or Cys derived from methionine, via transsulfuration pathway [TSP]) is packaged into the tripeptide (18,19). Plasmatic GSH is hydrolyzed at tissue sites rich in the ectoenzyme {gamma}–glutamyltranspeptidase ({gamma}GT) and dipeptidases (mainly kidney and lung) to CysGly and Cys, respectively, which may be taken up locally and/or mixed in the plasma pool (20). Usually, extracellular GSH is cleaved by {gamma}GT into CysGly, which is immediately taken up locally after its scission into the two amino acids by the action of membrane dipeptidases. However, it is not well established if and to what extent some amount of CysGly may escape the action of dipeptidases and thereby remain in the plasma (20,21). Cys is unstable and toxic at high concentration; however, cells and tissues have an absolute requirement for it, and plasma GSH seems to be a critical source for maintaining steady Cys availability. Cys may also derive from intracellular stores of various tissues (e.g., skeletal muscle during starvation) that can deliver it to the plasma; the other main source is diet (19). Methionine can also serve as a source of Cys because it can be converted to this amino acid through the TSP. Hcys does not derive directly from GSH but is an intermediate in the transformation of methionine into Cys, and is probably delivered by all those cells capable of performing the TSP reactions (22). (For a summary, see Figure 1).


Figure 01
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  		Figure 1. Schematic representation of the main biochemical pathways that regulate the production and the intra-/extracellular transfer of low-molecular-mass sulfhydryls (LMM-SHs). THF, tetrahydrofolate; SAM, S-adenosylmethionine; SAH, S-adenosyl-homocysteine

 
Human plasma contains GSH, Cys, CysGly, and Hcys in the 0.1- to 20-µM range (16). These thiols can also be found in disulfide forms, both as low-molecular-mass disulfides (LMM-SS) and as protein/LMM-SH mixed disulfides (Table 1). Usually, disulfide forms (with the exception of glutathione disulfide [GSSG]) are more concentrated than the respective thiol. Considering the total concentration for each thiol species (i.e., reduced + disulfide forms), Cys is usually present at the highest concentration, with Cys > CysGly > Hcys > GSH (13,16). Additionally, the ratio Cys/cystine is significantly shifted to the oxidized species with respect to the ratio GSH/GSSG, suggesting that these may represent two pools with distinct regulation and significance (17).


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  		Table 1. Nomenclature of Different Redox Forms (Reduced, Disulfide, and Mixed Disulfide With Proteins) of Main Low-Molecular-Mass Thiols Found in Plasma.

 
The attempt to increase LMM-SH availability in plasma by treatment with thiol compounds such as N-acetylcysteine (NAC), to hamper age-related damages and increase life span, has been carried out in various experimental models (10,23). Nevertheless, apart from a few promising results, most of these studies have been inconclusive (24). A number of studies have looked at GSH and GSSG levels and Cys/cystine ratio modulation during aging [for a recent review, see (11)]; however, a complete study on all plasma thiols and their disulfides including low-molecular-mass protein-mixed disulfides in human plasma during aging is lacking.

In this study we measured plasma levels of both low-molecular-mass sulfhydryls/disulfides and protein/low-molecular-mass thiol mixed disulfides in healthy persons at different ages, with the aim of exploiting plasma thiols as biomarkers of oxidative stress and evaluating whether an increase in disulfides and a decrease in thiols take place during the aging process. The possibility that the regulation of the delivery of the different LMM-SH by tissues to plasma is impaired with age is also discussed.


   METHODS
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 Methods
Results
 Discussion
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Study Participants
The study group comprised 41 consenting individuals (17 men, 24 women; age range: 21–92 years). All of 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 (serum glutamic oxaloacetic transaminase [SGOT], serum glutamic pyruvate transaminase [SGPT], {gamma}GT, uremia, creatininemia, erythrocyte sedimentation rate (ESR), blood cell number, platelet number, mean cellular volume (MCV), hematocrit, hemoglobin concentration, plasma protein concentration, albumin concentration). In particular, none of the participants had any alterations in renal function (evaluated by the serum levels of creatinine and urea levels) or in hepatic function (evaluated by activity of {gamma}GT and transaminases). All participants declared not to have taken any medicine in the last 15 days, and gave their informed consent.

Plasma Thiol Measurements
Blood samples were drawn from the antecubital vein in the morning (7:30–8:30 AM) after 12 ± 2 hours of fasting, collected in evacuated plastic tubes containing K3EDTA, acivicin (50 µg/mL; Sigma-Aldrich Chemie GmbH, Steinheim, Germany) and either monobromobimane (mBrB; Calbiochem, La Jolla, CA, for LMM-SH measurement) or N-ethylmaleimide (NEM; Sigma-Aldrich Chemie GmbH, for disulfide measurement) as previously described (16). Samples were then immediately centrifuged for 15 seconds at 12,000 g at 4°C for plasma separation. Time of centrifugation was shortened as much as possible to avoid the output of mBrB–GSH conjugate from erythrocytes that could artefactually increase the measured levels of plasmatic GSH (25). After plasma separation, thiols and disulfides were measured by high-performance liquid chromatography (HPLC) with fluorometric determination as previously described (13).

All HPLC analyses were performed using a Spherisorb C18 column (Varian Inc, Palo Alto, CA) with an Agilent series 1100 HPLC system equipped with a fluorometric detector. For determination of protein SH groups, aliquots of blood samples were collected in evacuated plastic tubes containing only K3EDTA and acivicin (50 µg/mL) and then immediately centrifuged for 15 seconds at 12,000 g at 4°C. When separated, the plasma was kept at 0°C. Protein SH determination (carried out within 1 hour) was performed by spectrophotometry with 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) as titrating agent (13,26). Spectrophotometric analyses were performed using a Jasco V/530 instrument (Jasco Europe s.r.l. Cremella, Como, Italy).

Statistical Analysis
The degree of correlation between the biochemical parameters and aging was measured by calculating the Spearman's correlation coefficient.


   RESULTS
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 Results
Discussion
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HPLC Analysis of Plasma Thiols
Fluorometric thiol derivatization with mBrB combined with the HPLC separation allows the simultaneous analysis of all SH-containing molecules occurring in plasma within a single run. We applied this method to measure the levels of the reduced, disulfide, and protein-mixed disulfide forms of GSH, Cys, CysGly, and Hcys in plasma from 41 participants aged 21–92 years. In Figure 2A, the typical HPLC tracings for plasma LMM-SH in participants of different ages (22, 62, and 83 years) are shown. Additional runs performed in the presence of standard solutions for each tested thiol allowed the peak identification (Figure 2B). Finally, in some blood samples, thiols were alkylated by NEM before the addition of mBrB. The chromatogram obtained by HPLC analysis of these samples was devoid of the fluorescent peaks corresponding to different plasma thiols (Figure 2B). This finding confirmed both that the peaks obtained by HPLC analysis of plasma samples from our study group were due to the conjugation of mBrB with thiols, and that no additional peaks deriving from mBrB hydrolysis interfered with our measurements.


Figure 02
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  		Figure 2. Typical chromatograms obtained from human plasma analysis after derivatization of low-molecular-mass sulfhydryls (LMM-SHs) with monobromobimane (mBrB) for detection. A, Chromatograms from three participants at different ages (22, 62, and 83 years). B, Same chromatogram shown in A for the 62-year-old participant in comparison with both the same sample to which a standard amount of thiols were added and the same sample pretreated with the thiol alkylating agent N-ethylmaleimide (NEM). Standard (Std) solutions of cysteine (Cys), cysteinylglycine (CysGly), homocysteine (Hcys), and glutathione (GSH; at final concentrations of 10, 2, 0.4, and 3 µM, respectively) were added to the plastic tubes together with K3EDTA, acivicin, and mBrB, immediately after blood collection. Alternatively, NEM (50 mM final concentration) was added to the plastic tubes together with K3EDTA, acivicin, and mBrB, immediately after blood collection. Samples were then treated as indicated in the Methods section

 
Thiol/Disulfide Balance Is Influenced by Aging
In Table 1, the list of the plasmatic thiols and their redox forms measured in our study group is shown. The primary data, including age and gender, are shown in Table 2. Correlation coefficients and rank of significance with aging for every measured parameter are reported in Table 3. Some of the significant correlations found are graphically shown in Figures 3 and 4.


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  		Table 2. Plasma Values (µM) for Each Measured Thiol, Disulfide, and Protein-Mixed Disulfide in the Study Participant.

 

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  		Table 3. List of Measured Parameters and Statistical Analysis of Their Correlation With Aging.

 

Figure 03
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  		Figure 3. Scatter diagrams relating measured plasma values for cysteinylglycine (CysGly), glutathione (GSH), cystine (CySS), homocystine (HcySS), protein/cysteine mixed disulfides (CySSP), and protein/homocysteine mixed disulfides (HcySSP) with aging. Regression line, calculated by applying the Spearman's correlation rank, is shown. Thiols and disulfides were measured in plasma samples by using high-performance liquid chromatography fluorescence detection immediately after blood derivation with monobromobimane (for thiol measurements) or N-ethylmaleimide (for both disulfide and protein-mixed disulfide analyses)

 

Figure 04
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  		Figure 4. Scatter diagrams relating calculated plasma values for cysteine/total cysteine (Cys/tCys), cysteinylglycine/total cysteinylglycine (CysGly/tCysGly), homocysteine/total homocysteine (Hcys/tHcys), glutathione/total glutathione (GSH/tGSH), (CysGly+GSH)/low-molecular-mass sulfhydryls (LMM-SH), and (tCysGly/tGSH)/(tCys+tHcys) with aging. Regression line, calculated by applying the Spearman's correlation rank, is shown. Thiols and disulfides were measured in plasma samples by using high-performance liquid chromatography fluorescence detection immediately after the blood derivation with monobromobimane (for thiol measurements) or N-ethylmaleimide (for both disulfide and protein-mixed disulfide analyses)

 
Among thiols, the concentration of GSH and CysGly is strongly inversely correlated with age, whereas Cys and Hcys did not show any significant correlation. Conversely, the oxidized forms of Hcys (i.e., HcySS and HcySSP) and Cys (i.e., CySS and CySSP) showed a significant direct correlation with aging, evidencing an increase in the elderly participants (Figure 3). Interestingly, residuals on the linear regressions of HcySS (and, particularly, HcySSP) do not appear to have a random distribution with respect to age; in fact, the data seem to possess a biphasic distribution, with an evident increase in their concentration only after the sixth decade of life. Upon analyzing the ratios between thiols and their disulfide forms (e.g., GSH/GSSG), some highly significant correlations were found, with a decrease in all thiol/disulfide ratios analyzed being a general feature (Figure 4). Total Cys (tCys) and total Hcys (tHcys), but not total CysGly (tCysGly) or total GSH (tGSH) concentrations increased significantly with aging. Protein thiols were found to significantly decrease in elderly participants, probably due to their increased transformation into mixed disulfides with LMM-SH.

Taking into consideration data from Figures 3 and 4 and Table 3, it is evident that: (a) the oxidative burden increases with aging; (b) GSH, CysGly, and their disulfides behave differently with aging with respect to Cys and Hcys; and (c) an increased delivery of Cys and Hcys, probably by TSP, but not of GSH efflux, by liver and/or other organs occurs with increasing age. This latter phenomenon is evidenced by the fact that neither tGSH nor tCysGly (which can derive exclusively from GSH) was found to increase. Moreover, as indicated in Figure 4, a highly significant inverse correlation with age was found for the ratios (CysGly+GSH)/LMM-SH and (tGSH+tCysGly)/(tCys+tHcys). Consequently, the percentage (to total thiols) of plasmatic thiols deriving exclusively from GSH load in plasma tends to decrease with aging, both as free thiols and as total forms. In summary, our data suggest that the oxidative burden increases with aging, as indicated by the decrease in thiol/disulfide ratios in parallel with (or as a consequence of) an increased delivery to the plasma of Hcys and Cys.


   DISCUSSION
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 Abstract
 Methods
 Results
 Discussion
References
 
In the present study we have focused our attention on plasma thiols with the aim of: (a) assessing whether during aging there is a shift in the thiol/disulfide balance, and (b) finding evidence for the age-related variation in the relative percentages of all thiol forms (i.e., reduced, disulfide, and protein-mixed disulfides).

To clarify these aspects, the age-derived changes of the main thiols present in plasma have been analyzed, i.e., protein thiols and LMM-SH (Cys, CysGly, Hcys, and GSH). Additionally, redox forms of LMM-SH have been measured, i.e., low-molecular-mass disulfides and mixed disulfides with proteins. This is, to our knowledge, the first study in which all redox forms of plasma thiols have been investigated in relation to aging in the age range of 21–92 years. Our data (Tables 2 and 3, Figures 3 and 4) show that a strong inverse correlation exists between aging and levels of CysGly and GSH, whereas neither their disulfides nor tGSH and tCysGly changed. Conversely, direct correlations were found between the disulfide forms of Cys and Hcys and age, whereas Cys and Hcys did not show any correlation with age. Interestingly, tCys and tHcys were positively correlated with age. In addition, the ratios between reduced and oxidized forms decreased for all LMM-SHs considered. As expected, the concentration of protein SH groups decreased with age, as a consequence of the increased levels of mixed disulfides with Cys and Hcys, and the decline of the plasma concentration of albumin that takes place in elderly persons (10). All these facts, as a whole, suggest that a higher oxidative burden in elderly persons shifts the redox state of plasma thiols and induces an increased delivery of some LMM-SHs to the hematic compartment.

Some of the data we obtained in our study agree with some previous results. Most of the data available in the literature on this issue derive from analyses of plasma total thiols. In particular, tCys increase has previously been well evidenced (10,27,28). Additionally, the increase in tHcys plasma levels with aging has also been recently described (27,29), and this observation may be of particular interest, because hyperhomocysteinemia is widely considered an independent risk for vascular disease (30,31). In contrast to our results, some existing data also indicate a possible decrease in tGSH levels with aging (29); it is possible that differences in sample storage before analyses (our measurements were taken within a few hours from blood collection) may have influenced the results. Finally, tCysGly levels were not found to change with aging (27).

Fewer data from the literature describing the possible modulation in elderly persons of the different redox forms of thiols normally occurring in plasma are available. Among reduced thiols, the age-related decline in GSH levels that we found has been previously observed in a number of senescent organisms, including humans, in plasma and/or various organs (14,17,23,32,33). Additionally, in contrast to our data, Cys was also shown to decrease with aging (but this phenomenon was less evident) and seems to significantly decrease only after the fifth decade of life (17). To our knowledge, no data on Hcys and CysGly levels in relation to aging are available from the literature.

It has also been observed in previous work that an oxidative shift of Cys and GSH towards disulfide forms occurs in human plasma in elderly persons (10,19,34). Specifically, a linear oxidation for Cys/CySS was found to increase throughout the entire life span, whereas the GSH/GSSG ratio was found to increase only after the fourth decade of life. These data have suggested a possible occurrence of two different pools regulated separately in vivo (19).

It should be emphasized that plasma thiol redox state cannot be simply considered as an indicator of oxidative status, as it has been previously observed that even minimal oxidation shifts of the Cys/CySS or GSH/GSSG redox couple (e.g., 21 and 9 mV changes, respectively, observed in aging) are sufficient to cause a large increase in the oxidized forms of intracellular proteins bearing vicinal thiols (17), and thus are able to influence specific signaling pathways. Such proteins include the N-methyl-D-aspartate (NMDA) receptor, protein disulfide isomerase, the epidermal growth factor receptor, the extracellular signal-regulated kinase, and many others (3,35–37). Moreover, the Cys/CySS ratio seems to strictly regulate many adhesion factors of endothelial cells and to have a role in the atherogenetic process (12).

The complete analysis of LMM-SH performed on all redox forms allows us to infer some hypotheses about the age-derived impact on the levels of thiols and disulfides. From data reported in Tables 2 and 3 and Figures 3 and 4, it can be presumed that: (a) there is a constant decrease in GSH and CysGly levels, and an increase in tCys and tHcys over the life span; (b) the increase in tCys and tHcys is not due to an enhanced concentration of reduced forms, but to the increase in their disulfides; and (c) tGSH and tCysGly remained unchanged. These findings and the turnover of plasma thiols suggest that an increased production of reactive oxygen/nitrogen species in aging tends to decrease all plasma thiols, and that this can be balanced by an increased delivery of thiols from other tissues. In all probability, GSH delivery cannot be increased (nor, consequently, can CysGly) and, thereby, there is a decrease in GSH and CysGly levels, whereas an increased output of Cys takes place (Figure 5). Thus, the increased oxidative stress and Cys load are mirrored by a stable level of Cys (the oxidative stress is balanced by its increased output) and a huge increase in its disulfide forms (CySS and CySSP). The increase in tHcys could be a simple consequence of the activation of the TSP, due to the need to increase the delivery of Cys to the plasma.


Figure 05
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  		Figure 5. Schematic representation of age-derived oxidative stress and its supposed effects on plasma low-molecular-mass sulfhydryls. Cys, cysteine; CysGly, cysteinylglycine; Hcys, homocysteine; GSH, glutathione

 
The relative percentage of each LMM-SH measured in our study was shown to change over the entire life span. In particular, GSH and CysGly percentages decreased linearly at about 2% per year (Figure 3). Due to its intrinsic chemical feature, each individual thiol may have a different behavior and role in the redox homeostasis of the plasma. For example, it has been recently shown that NAC treatment is able to increase the plasmatic levels of Cys, but not of GSH, in patients with age-related macular degeneration (38). Additionally, the reactivity of the thiol group of CysGly is known to be largely greater than that of other LMM-SHs (39). Probably because of its high reactivity, CysGly can be considered a "Janus" molecule because, on one hand, it is particularly efficient in capturing electrophiles and potentially dangerous thiol-reactive species and, on the other hand, in the presence of metal ions and other particular conditions, it is able to generate free radicals by acting as a prooxidant (40). Consequently, it is not known if a decrease in CysGly levels during aging could be a useful adaptive condition or a potentially dangerous situation. For instance, it is widely accepted that LMM-SH may have a pivotal role in activating some nitric oxide donors such as S-nitrosoproteins (41–47) via transnitrosation reactions.

Several studies are now available showing that the plasma thiol/disulfide redox state becomes oxidized with aging [for a review, see (19)]. Among the well-documented changes, there is a decrease in GSH and an increase in both CySS and tHcys (34). In particular, a linear decrease of 0.2 mV/year for the Cys/CySS redox couple and a linear decrease of 0.7 mV/year for the GSH/GSSG couple were depicted (17). More generally, humans show, on the average, a significant oxidative shift between the 3rd and 10th decade of life (48). It is possible that, because a group of redox-regulated biological signaling pathways responds to changes in the thiol/disulfide redox state (12,49), this variation in the plasmatic redox state can be expected to contribute to aging-related diseases. One interesting example of this is represented by the findings of Go and Jones (12), which showed that the oxidation of the thiol/disulfide redox state over the range found in vivo in human plasma is a key determinant of early events in vascular disease development. Moreover, the authors suggested that the plasma thiol/disulfide state may provide a useful and early marker of atherosclerosis development, and that nutritional and therapeutic means to normalize the oxidized redox state could provide a strategy for protecting against disease development. However, available evidence for the specific contribution of each extracellular thiol/disulfide redox couple has not yet been established.

Here we have provided a view of the levels of the different main thiols in all redox forms in human plasma and the effect of aging on their levels. Our data suggest that aging is correlated to a decrease in plasma GSH and CysGly, with a concomitant rise in most oxidized forms of LMM-SH and a parallel increase in tCys and tHcys, probably due to an augmented afflux of these amino acids from various organs. The diminished levels of CysGly may have both useful and detrimental effects. Therapeutic interventions designed to increase one single plasmatic LMM-SH rather than all of them could be an interesting approach to evaluate the contribution of each LMM-SH in many possible pathological events.


   Acknowledgments
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This work was supported by grants from Fondazione Monte dei Paschi di Siena and by FIRST 2005 (Fondo Interno Ricerca Scientifica e Tecnologica), University of Milan.

We are grateful to Dr. Laura Keith for her assistance with the English corrections.


   Footnotes
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Decision Editor: Huber R. Warner, PhD

Received January 9, 2006

Accepted March 22, 2006


   References
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