Archives of Medical Research 44 (2013) 136e141

ORIGINAL ARTICLE

Plasma Protein Hydroperoxides During Aging in Humans: Correlationwith Paraoxonase 1 (PON1) Arylesterase Activity and Plasma

Total Thiols

Mohammad Murtaza Mehdi and Syed Ibrahim Rizvi

Department of Biochemistry, University of Allahabad, Allahabad, India

Received for publication November 7, 2012; accepted January 18, 2013 (ARCMED-D-12-00641).

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Background and Aims. Oxidative stress is thought to play a major role in the develop-ment of several age-dependent diseases. Proteins are major targets for oxidative attack.Protein hydroperoxides are formed by hydroxyl and singlet oxygen attack on protein,forming relatively stable hydroperoxides on histidine, tyrosine and tryptophan residues.This study investigated the levels of plasma protein hydroperoxides and antioxidantpotential of plasma during aging in humans. We correlated the protein hydroperoxideformation with plasma antioxidant potential, paraoxonase 1 (PON1) arylesterase activityand plasma total thiols.

Methods. The protein hydroperoxides and antioxidant potential were measured in plasmaof human subjects aged between 20 and 81 years of both genders.

Results. Increase in plasma protein hydroperoxides and decrease in plasma antioxidantpotential were observed as function of human age.

Conclusion. This study provides strong correlation between plasma protein hydroper-oxides formation and decrease in plasma antioxidant potential during aging. PON1 ar-ylesterase activity and plasma total thiols levels were also found to show significantcorrelation with increasing levels of plasma protein hydroperoxides during aging.The plasma protein hydroperoxides provide a reliable marker of long-term redoxbalance and degree of oxidative stress during aging process. � 2013 IMSS. Publishedby Elsevier Inc.

Key Words: Aging, Protein oxidation, Protein hydroperoxides, Antioxidant potential, Oxidative

stress.

Introduction

The free radical theory of aging implied that the targetsof reactive oxygen species are cumulative, random andindiscriminate (1). In living systems an intricate antioxidantdefense system counteracts the burden of ROS productionand the balance between antioxidant defense and ROSproduction determines the degree of oxidative stress (2).Within certain limitations, the generation of reactive oxygenspecies is necessary to maintain homeostasis; they partici-pate in essential defense mechanism to combat infections

requests to: Dr. Syed Ibrahim Rizvi, Associate

t of Biochemistry, Faculty of Science, University

habad, Uttar Pradesh, 211002 India; Phone:

ail: sirizvi@gmail.com

nt matter. Copyright � 2013 IMSS. Published by Elsevier.1016/j.arcmed.2013.01.003

in the process of phagocytes, and mediate the proliferativeresponse of growth factors (3). A rise in intracellular oxidantlevel has important effects such as damage to biomole-cules and altered activation of specific signaling pathways;numerous cellular processes linked to aging and age relateddiseases can be influenced by these effects (4).

Being the most abundant component of biological fluids,tissues and cells, proteins are not necessarily but likelyto be the major targets for oxidative attack (5). Althoughoxidative stress can modify all proteins, certain tissues andspecific protein targets may be especially sensitive; thesulfur-containing, aromatic and basic amino acids are moreprone to oxidation. Loss of sulfhydryl groups, protein frag-mentation, formation of disulfide and dityrosine crosslinks,carbonyls, methionine sulfoxide, nitrotyrosine, glyoxida-tion, and peroxidation products can be induced by chloro-,

Inc.

137Humans Plasma Protein Hydroperoxides During Aging

sulfo-, nitro-, oxo- and hydroxyl-derivatives produced byamino acid-oxidation (6). A progressive loss of a particularbiochemical function can be induced by oxidative damageto a specific protein, especially at the active site (7). Expo-sure of proteins to radicals in the presence of oxygen givesrise to side-chain oxidation and backbone fragmentation (8).Cellular functions like native protein turnover and modifiedmaterial clearance may be modulated by the formation ofoxidized proteins (9).

Hydroperoxides are major product of hydroxyl and singletoxygen attack on protein, forming relatively stable hydro-peroxides on histidine, tyrosine and tryptophan residues(10e12). These hydroperoxides can give rise to secondaryoxidative damage which can happen by two ways, first viaone electron (free radical) reaction to get secondary radicals,and second via two electron (molecular) reactions with suit-able nucleophiles (13). The second pathway can result inhydroperoxides-mediated inactivation of critical thiol-dependent cellular enzymes such as protein tyrosine phos-phatases, glyceraldehydes 3-phosphatase dehydrogenase,sarcoplasmic-endoplsamic reticulum calcium transporterand caspases (14e17). Thiol-dependent cysteine proteases,efficiently inactivated by protein hydroperoxides, maycontribute to the damaged protein accumulation in cellsunder oxidative stress (18). Active-site cysteine residue ofprotein tyrosine phosphatases affected by protein hydroper-oxides may contribute to altered redox signaling (16).

In earlier reports we have shown the formation ofadvanced oxidation protein products (AOPPs) and proteincarbonyls as markers of oxidative stress during human aging(19). The increased oxidative stress leading to altered proteinfunction has also been highlighted in our recent study wherewe have reported a significantly reduced activity of serumparaoxonase 1 (PON1) arylesterase activity during agingwhich correlates with plasma redox status (20). In thisstudy we correlate plasma protein hydroperoxides andplasma antioxidant capacity as a function of human age.We have also correlated protein hydroperoxides values withdifferent parameters such as paraoxonase 1 (PON1) aryles-terase activity (20) and plasma thiol contents (19), in aneffort to determine the involvement of protein oxidativedamage in age-dependent pathologies.

Materials and Methods

Sample Collection and Processing

The study was carried out on 80 normal healthy subjects ofboth sexes (53 males and 27 females) between the agesof 20 and 81 years. The criteria for selecting subjects werethe same as reported recently (20). All volunteers werescreened for asthma, tuberculosis, diabetes mellitus orany other major illness. None of the subjects were smokersor were taking any medication. Care was also taken toexclude volunteers taking any nutritional supplements

(previous 3 months). Elderly subjects were living at homebut functionally independent without any cognitive impair-ment. All persons gave their informed consent for the useof their blood samples for the study. Venous blood was ob-tained at fasting condition in the morning by venipuncturein sterile polystyrene tubes containing heparin. Plasma wasobtained by centrifuging the blood at 800 � g for 10 min at4�C. Antioxidant capacity measurements were performedimmediately, for further analyses the plasma was frozenand stored at �80�C for not more than 2e3 days. Theprotocols of study were in conformity with the guidelinesof the Allahabad University Ethics Committee.

Plasma Protein Hydroperoxides

Plasma protein hydroperoxides were measured by themethod described by Gay et al. (21). Proteins were precip-itated from 100 mL of plasma by adding 500 mL of 0.2 Mperchloric acid. After a 5-min ice bath, samples were centri-fuged at 6500 � g for 10 min. The pellet was then dissolvedin 1.1 mL of 6 M guanidine hydrochloride. The protein solu-tion was further washed twice with chloroform containing4 mM butylated hydroxytoluene. The washed protein solu-tion was mixed well with 0.5 M perchloric acid (40 mL),5 mM xylenol orange (25 mL) and 5 mM ferrous sulfate(10 mL) and incubated at room temperature for 60 min.Absorbance was taken at 560 nm against an appropriateblank (1.1 mL of 6 M guanidine hydrochloride, 40 mL of0.5 M perchloric acid, 25 mL of 5 mM xylenol orange and10 mL of 5 mM ferrous sulfate). The molar concentrationsof protein hydroperoxides were calculated with the molarabsorption coefficient value of 37,000 M�1 cm�1.

Plasma Antioxidant Potential

Plasma antioxidant potentials were measured by themethod of Miller et al. (22) as follows: 5.0 mL (7 mM)aqueous solution of 2,2’-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) was prepared and added to88 mL (140 mM) potassium persulfate. This solution waskept in the dark for 12 h to make an activated solution ofABTS�þ. This solution was further diluted to obtain anoptical density at 734 nm with about 0.800 unit of absor-bance. Subsequently, 1.9 mL of ABTS�þ solution wasmixed with 100 mL of diluted plasma (ten times). The solu-tion was mixed and remained for 5 min before reading theabsorbance at 734 nm, taking water as a blank. Percentplasma antioxidant potential was measured by using theformula % AOP 5 100 (1-As/A0).

Plasma PON1 Activity

This assay was performed by the method developed byAyub et al. (23). Enzyme activity toward phenyl acetate (ar-ylesterase activity) was determined by measuring the initialrate of substrate hydrolysis in the assay mixture (3 mL)

Figure 1. Plasma protein hydroperoxides as a function of human age.

138 Mehdi and Rizvi/ Archives of Medical Research 44 (2013) 136e141

containing 2 mM substrate (phenyl acetate), 2 mM CaCl2and 10 mL of plasma in 100 mM Tris-HCl (pH 8.0). Theabsorbance was monitored for 3 min at 270 nm and theactivity was calculated from E270 5 1310 per M/cm bytaking the substrate solution as blank. The results are ex-pressed in U/mL, 1 U of arylesterase hydrolyzes 1 mmolof phenyl acetate per minute.

Plasma Total Thiols

Plasma total thiol (TSH) concentration was estimated (24)by the ability of the -SH group to reduce 5,50 dithiobis, 2 ni-trobenzoic acid (DTNB). Two hundred mL of plasma mixedwith 500 mL of sodium dodecyl sulfate (10%) and 2.50 mLof EDTA-phosphate buffer (0.01 M, pH 8.2), kept foran additional for 10 min and centrifuged for 5 min. The2.50 mL supernatant was further taken and mixed with10 mL freshly prepared DTNB solution (10 mM) and incu-bated for 15 min to form a yellow-colored anionic productwhose absorbance is measured at 412 nm against a reagentblank. The concentration of TSH is expressed as mmol/L ofplasma.

Statistical Analysis

Statistical analysis was done using the software GraphPadPrism v.5.01. Skewness was calculated as a test for normaldistribution and was found within the normal limits forall parameters. To assess relationships between parame-ters, Pearson’s correlation coefficient (r) was derived at95% confidence interval by taking p value !0.05 assignificant.

Figure 2. Plasma antioxidant potential as a function of human age.

Results

Our study shows an increase in plasma protein hydroper-oxides and a decrease in plasma antioxidant potentialsas a function of age. Plasma protein hydroperoxidesincrease with age (Figure 1, Pearson’s r 5 0.80, p !0.001). Percent plasma antioxidant potentials decreasewith age (Figure 2, Pearson’s r 5 0.79, p !0.001). Wehave already reported a decrease in PON1 activity andplasma total thiols during human aging (19,20). In thepresent study we use these values to emphasize the rela-tionship of these parameters with age-dependent decreasein plasma protein hydroperoxide. Figure 3A shows thecorrelation between the quotients of percent plasma anti-oxidant potentials and plasma protein hydroperoxides withage (Pearson’s r 5 �0.84, p !0.001). Figure 3B showsthe correlation of quotients of paraoxonase 1 (PON1) ary-lesterase activity and plasma protein hydroperoxideswith age (Pearson’s r 5 �0.86, p !0.001). Figure 3Cshows the correlation of quotients of plasma total thiolsand plasma protein hydroperoxides with age (Pearson’sr 5 �0.88, p ! 0.001).

Discussion

The role of protein oxidation and its consequences in agingand age-related diseases have long been a major researchtopic (25). The implications of elevated levels of proteinoxidation, extracellular and intracellular removal or decom-position, the stability and role of plasma protein hydroper-oxides and its correlation with other markers of oxidativestress during aging are matters of intense scientific interest.Our results show that the levels of plasma protein hydroper-oxides correlate positively with aging. This result is also inagreement with similar studies on different populationgroups (26) in which the authors have shown a decreasein plasma protein hydroperoxide in middle-age and older-age individuals compared to young subjects. In earlierreports we showed an increased carbonyl formation andAOPP as a function of human age (19); however, the corre-lation between plasma protein hydroperoxides and aging isquite high in comparison with protein carbonyls andadvanced oxidation protein products. The higher peroxida-tion index could be explained on the basis of higherstability of protein hydroperoxides compared to proteincarbonyls and AOPP.

Figure 3. (A) Correlation of quotient of plasma antioxidant potential and

plasma protein hydroperoxides with human age. (B) Correlation of

quotient of total plasma thiols and plasma protein hydroperoxides with

human age. (C) Correlation of quotient of paraoxonase 1 (PON1) arylester-

ase activity and plasma protein hydroperoxides with human age.

139Humans Plasma Protein Hydroperoxides During Aging

The decomposition of some amino acid and peptidehydroperoxides is found to occur extracellularly via theinvolvement of cell-surface reducing system, such as trans-plasma membrane electron transport system or via redox

cycling of trace transition metal ions (27). Amino acid,peptide and protein hydroperoxides also react with numberof enzymes and low-molecular mass species, resulting ineither detoxification or further damage. However, theremoval of protein hydroperoxides, either by enzymaticor by low-molecular-mass species, is less rapid due to elec-tronic and steric factors, consistent with the slow decay ofthese species within intact cell (28).

The formation of protein carbonyls and advanced oxida-tion protein products (AOPPs) occurs differently than proteinhydroperoxides. Common mechanisms for generating proteincarbonyls are oxidation of side-chain functional groupsof lysine, arginine and proline to aldehyde group as well asthreonine to a ketone group (29,30). Increased release ofmyeloperoxidase from activated phagocytes forms mostof the AOPPs (31,32), which comprise several chromophoresincluding pentosidine, carbonyls and protein cross-linked bydityrosine. Although any protein susceptible for oxidativemodification may contribute to the increased level of AOPPs,fibrinogen has been recognized as a key molecule (33). Beingdifferent in formation and degradation as mentioned earlier,the elevated levels of protein hydroperoxides remain in theplasma for a comparatively long time because of their slowremoval, which makes these species more stable than anyother protein oxidation products like AOPPs and proteincarbonyls. Therefore, levels of protein hydroperoxide providea better reflection of the long-term plasma redox state duringhuman aging.

As a consequence of diminished antioxidant defensecapabilities, intrinsic oxidative stress increases during aging,which may cause deteriorative effects in various cells.The involvement of protein hydroperoxides in the etiologyof many neurodegenerative diseases has been well docu-mented (34). Potent antioxidants accessible to brain cellsmay have a role of reduction in progression of neurodegen-erative diseases by reducing oxidation of proteins. Alz-heimer’s disease, Huntington disease, Parkinson’s disease,Creutzfeld-Jakob Disease and frontotemporal dementia havebeen associated with oxidative stress leading to proteinoxidation as a common underlying molecular event (35).Coronary artery disease is also found to be associated withelevated levels of protein hydroperoxides (36). Our observa-tion of a strong correlation between the quotient of plasmaantioxidant potential and levels of plasma protein hydroper-oxides (Figure 3A) with human age may explain the higherincidence of neurodegenerative and other diseases duringold age.

A strong correlation (Figure 3B) between the quotient ofprotein hydroperoxides and plasma total thiols or sulfhydrylgroups with aging can be explained because protein thiolsare readily oxidized by these hydroperoxides during aging.Even exogenous protein and amino acid peroxides makecellular thiols as a major target (37). The reaction rate ofhydroperoxides with free cystine (or cysteine) and otherthiols are comparatively lower than those within the active

140 Mehdi and Rizvi/ Archives of Medical Research 44 (2013) 136e141

site of peroxiredoxins: an antioxidant enzyme family thatalso controls cytokine induced peroxide levels. Proteinthiols are thus readily oxidized by hydroperoxides, poten-tially resulting in enzyme inactivation.

There is considerable evidence that the antioxidantactivity of high-density lipoprotein (HDL) is largely dueto the PON1 located on it (38). The decrease in PON1activity may contribute to the increased susceptibility ofHDL to oxidation during aging (39). We recently reportedthe reduction in human PON1 activity and increased suscep-tibly of LDL oxidation during aging in humans (20). Thepossible role of PON1 in aging and its effect on longevityhas been thoroughly reviewed with a focus on the relation-ship between enzyme activity and genetic polymorphism aswell as its capability to counteract oxidative stress (40).

The correlation between the quotient of PON1 activityand plasma protein hydroperoxides with age (Figure 3C)shows the reduction in PON1 arylesterase activity withincrease in protein hydroperoxides during aging. Theimportance of maintenance of enzyme eSH groups is animportant factor in modulation of PON1 activity. We havealready reported the role of oxidative stress in relation withPON1and aging. The anti-atherogenic properties of PON1 isrelated to reducing characteristics of its cysteine-protectedsulfhydryl group (41) that is significantly decreased duringaging (42), and there is also formation of homocysteineduring age-related macular degeneration (43). The deter-iorative behavior of hydroperoxides towards active siteamino acid residues—especially sulfhydryl groups—maytherefore be a strong factor towards the reduction ofPON1 activity.

To conclude, despite the fact that this study was based on80 subjects, which is a low number to derive firm conclu-sions, we provide evidence of an age-dependent increasein plasma protein hydroperoxides that may predisposetowards many disease and stress conditions. Elevatedprotein hydroperoxide levels may also provide an effectiveand stable marker of oxidative stress in an elderly popula-tion. Although dietary and lifestyle factors may vary indifferent populations and within members of a society, itsstrong correlations with antioxidant potential, plasma totalthiols and paraoxonase1 (PON1) arylesterase activityreflect a better understanding of mechanisms that underliethe development of many age-dependent diseases.

AcknowledgmentsM.M.M. is a recipient of a Senior Research Fellowship from theIndian Council of Medical Research, New Delhi, India. This workwas supported in part by University Grants Commission MajorResearch Project Grant to S.I.R. (F31-392/2009).

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