International Journal of Biological & Medical Research

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1 Int J Biol Med Res. 2011; 2(1): Int J Biol Med Res Volume 2, Issue 1, Jan ISSN: 0976:6685 BioMedSiDiret Puliations Contents lists availale at BioMedSiDiret Puliations International Journal of Biologial & Medial Researh Journal homepage: International Journal of BIOLOGICAL AND MEDICAL RESEARCH Original Artile Charaterization of antioxidant profile in human saliva during menstrual yle a a a a a Suarayalu Alagendran,, Govindaraju Arhunan,*, R.L. Rengarajan, S. Muthu Kumar,R. Ilayaraja, a a A. Amarnat, Balamuthu Kadalmani, Gariella Fernandez, Rosalinda Guevara Guzman a,*centre for Pheromone Tehnology, Bharathidasan University, Department of Animal Siene, Tiruhirappalli , Tamil nadu, India. Neurosensorial Physiology and Neurotoxiology Researh Laoratory, Department of Physiology and Pharmaology, Faulty of Mediine, Unam, Av. Universidad, C.P 04510,.Mexio D.F A R T I C L E I N F O Keywords: Saliva, Menstrual yle, LH, Ovulation, Antioxidants, Human. A B S T R A C T The aim of this study was to determine the relationship of antioxidants profile with endogenous reprodutive hormone level aross the menstrual yle. Prediting ovulation is the asis on whih the fertile period is determined. Currently there are many methods aessile to identify the ovulatory period. Unfortunately, these methods are not always effiient for aurate detetion of ovulation. Hene, an attempt was made to detet ovulation through antioxidant profile with the help of BBT, saliva ferning and ultrasound. Salivary antioxidants were investigated serially during preovulatory, ovulatory and postovulatory periods of normal menstrual yle in twenty healthy volunteers. The sample was olleted in three onseutive menstrual yles. Salivary antioxidants was estimated and analysis y UVspetrophotometer. The results revealed higher signifiant variations in the antioxidant onentration and physiologial hanges are also essential owing to ovarian hormones during menstrual yle. Alkaline phopshatase, peroxidase, asori aid and latate dehydrogenase were maximum during ovulation and minimum during postovulatory phase. Superoxide dismutase, atalase and glutathione peroxidase were derease in prepuert and menopau yle. Among these antioxidants alkaline phopshatase, peroxidase and asori aid were predominantly exhiited during ovulatory phase than pre and post-ovulatory phase. It is onluded that of 17 â-oestradiol and progesterone infulene the antioxidative status and the vulneraility to oxidative stress exhiited during normal menstrual yle would serve early diagnosis of ovulation detetion. Copyright 2011 BioMedSiDiret Puliations IJBMR - ISSN: 0976:6685. All rights reserved. 1. Introdution At present time, there is no single, ompletely reliale parameter for ovulation predition. Variations from the mean among hormonal and linial parameters during a speifi menstrual yle are troulesome, and there is onsiderale variation from yle to yle, even in the same patient [1, 2]. The primary ojetive of this study is to develop a noninvasive and reliale method for ovulation detetion. Saliva would e a suitale medium for this purpose. The sample olletion proess is noninvasive, painless and onvenient. Colleting saliva samples is possile several times a day and an e onveniently aomplished under irumstanes where it is diffiult to get lood samples. [2]. * Corresponding Author : Dr.G.Arhunan, Centre for Pheromone Tehnology, Department of Animal Siene, Bharathidasan University, Tiruhirappalli , Tamil nadu, India. E.mail: garhu56@yahoo.o.in Copyright 2011 BioMedSiDiret Puliations. All rights reserved. Cyli hanges in various physial properties and iohemial onstituents of saliva are known to reflet aurately the hormonal hanges assoiated with a menstrual yle and may e utilized linially to determine the time of ovulation. For example, the iohemial sustanes like salivary enzymes as well as hormones like estrogen and progesterone have een found to flutuate during the ovulatory period of the menstrual yle [2]. Reative oxygen speies (ROS) play a numer of signifiant, diverse roles in female reprodutive iology inluding uterine environment, ooyte maturation and ovulation, orpus luteum funtion and regression [3]. Estrogens have een shown to have in vitro antioxidant effets on memrane phospholipid peroxidation [4] and experimental evidene indiates an antioxidant and ateroprotetive role [5, 6-9]. The relationship etween sex steroid hormones and the ellular antioxidant enzyme system has also een investigated.

2 Suarayalu Alagendran / et.al Int J Biol Med Res. 2011; 2(1): The expression of various iomarkers of oxidative stress has een demonstrated in normal yling human ovaries [1, 2, 10, 11]. All folliular stages were examined for the expression of the SOD inluding primordial, primary, preantral, nondominant antral folliles in preovulation phase, dominant folliles and atreti folliles [12]. There is a deliate alane reative oxygen speies and the antioxidant enzymes in the ovarian tissues. The antioxidant enzymes neutralize reative oxygen speies prodution and protet ooyte and the emryo from oxidative stress. Oxidative stress is involved in the modulation of ylial hanges in the endometrium. Changes in the expression of SOD in the endometrium have een studied. SOD is an enzyme involved in savenging the superoxide radial and proteting the ells from oxidative radial's toxiity. Inrease in the SOD and ROS levels in the endometrium in the late seretory phase just efore menstruation indiates that these hanges in the level of expression indiated involvement in menstruation [13, 14]. Estrogen and progesterone withdrawal led to inreased expression of ylooxygenase-2, mrna and inreased prostaglandin F2 - á synthesis in endometrial ells ultured in vitro. Gluthatione and gluthatione perioxidase are oth antioxidants that neutralize the free radials and lipid peroxides to maintain the intraellular homeostasis and redox alane. The role of oxidative stress in modulating a gamete of physiologial funtions and its role in pathologial proesses affets the female reprodution. Oxidative stress influenes a host of reprodutive proesses in a woman life. There is onsiderale evidene that estrogen dereases ROS and free radials prodution oth in vitro and in vivo, and depressed estrogen synthesis in postmenopausal woman is onsidered to e responsile for enhaned oxidative stress [1, 12, 16, 17]. On the other hand, reprodutive system dysfuntion ranges from post ovulation phase defiieny, hypoestrogenism to an ovulation, and even amenorrhea is often assoiated with vigorous physial ativity [18]. This evokes onern regarding adverse effets of physial effort on antioxidant defense in premenopausal females [19]. However, the data onerning effets of female hormonal status on antioxidant protetion in physially ative sujets are itty. Diner et al [20] revealed that in eumenorrhei female athletes, erythroyte glutathione peroxidase ativity in the early folliular phase (day 3 5) was lower than in their amenorrhei ounterparts. The aove data might suggest that hormonal disturanes due to amenorrhea affeted at least erythroyte glutathione system. The data otained in this study indiate nearly that the derease in G-Px levels in muus samples otained either from the ervix or from the vaginal anal provides three or more day's predition of ovulation in the human female and the proedures provides a women with a simple, noninvasive means for natural fertility ontrol. The proedure desried here for prediting ovulation an also e applied to advantage to female animals where ervial muus exhiits hormonal ontent and hanges during estrus similar to that oserved in the human female. For ex; it is known that in female ovine animals the preovulation and post ovulation hanges in reprodutive hormones suh as progesterone, estrogen, FSH and LH follow very losely the hanges that our in the human female [10, 11, 19]. Uri aid appears to e the dominant antioxidant present in saliva and displays a onentration similar to that of serum. Other salivary antioxidants inlude asori aid and alumin, ut onentrations of these are lower than that of serum [21]. Asori aid has long een assoiated with fertility, ut no onsistent study of its mehanism of ation in reprodutive tissues has een made. This artile onsiders how three of asori aid's prinipal funtions, namely its promotion of ollagen synthesis, its role in hormone prodution, and its aility to protet ells from free radials, may explain its reprodutive ations [22]. Data relating to oth ovary and testis are reviewed sine asorate aumulates in oth tissues. Both gonads exhiit yles of tissue remodeling and of peptide and steroid seretion that an e assumed to e asorate-dependent. Asori aid may also prevent gametes from damage y free radials during prodution and fertilization [11, 18, 19, 22]. This may indiate an ative seretion system for salivary antioxidants rather than passive diffusion from the irulation. This eomes further evident when antioxidant onentrations are ompared in unstimulated and stimulated saliva. Stimulated saliva ontains a lower onentration of antioxidants ut when flow rates are taken into aount, antioxidant apaity is higher than in unstimulated saliva [21, 23]. Reently, the importane of another salivary defense system has eome ovious. The antioxidant system appears to e less effiient with age [6, 24]. Similar to other iologial systems, the salivary antioxidant system inludes various moleules and enzymes of whih the most important are the uri aid moleule and the peroxidase enzyme, oth of whih are water-solule. The lipid-solule antioxidants arried y lipoproteins, whose onentration in saliva is very low, ontriute not more than 10% of the saliva antioxidant apaity [13, 25, 26]. The purpose of this study was to determine the timing and magnitude of hanges in parameters of antioxidant quantity status in relation to the flutuation of steroid hormone derivatives during menstrual yle support as iomarker for ovulation detetion. 2. Materials and Methods 2.1. Sujets The studies were performed in 20 different healthy female adults. The volunteers were instruted to astain from smoking; eating and drinking anything ut water for 10 hr prior to testing. In addition, all volunteers were asked to astain from tooth rushing to prevent minimal gingival leeding. Anthrophotometri index shows the alulation of ovulation day using BBT and salivary ferning [2]. Antioxidant profiles like Alkaline phosphase, latate dehydrogenase, superoxide dismutase, atalase, peroxidase, gluthatione peroxidase, asori aid, uri aid and salivary hormone like LH, FSH, Estradiol and progesterone are the indiator for identify the exat day of ovulation Colletion of samples The human saliva was olleted from female volunteers during various periods viz, Preovulatory, ovulatory, postovulatory phases and also from prepuertal and menopause stages. Saliva samples were olleted after patients had reeived their routine hek-up. With the patients seated, the saliva was olleted over a 5-min

3 384 Suarayalu Alagendran / et.al Int J Biol Med Res. 2011; 2(1): period with instrutions to allow saliva to pool in the ottom of the mouth and drain to a olletion tue when neessary. Sujets were asked not to swallow any saliva for the duration of the olletion to allow the alulation of salivary flow rates. At the end of the olletion period, saliva volume was measured, the tue sealed and then frozen in dry ie until taken ak to the laoratory for proessing. Prior to analysis, the saliva was plaed into Salivette (Sarstedt, Leiester, U.K.) tues using a natural otton swa insert, and entrifuged at 4000 g for 10 min at 4?C. The supernatant fration was then aliquotted into storage vials and kept at - 80?C until required for analysis (Step I). The use of the Salivette tues was neessary to redue the high visosity of the saliva samples that would otherwise prevent aurate pipetting. Preliminary work to ompare fresh saliva entrifuged in standard entrifuge tues with saliva prepared y entrifugation in three types of Salivette (natural otton insert, itri aid impregnated insert or plain polyester insert) demonstrated that the antioxidant profile from samples prepared using the natural otton inserts was idential to the native sample. It was inferred from this that the analytes of interest in this study were not asored into the inserts [27] Extration of samples The saliva sample was extrated with 80% ethanol (if neessary, heat the mixture to 80ºC for 30 mins in a water ath). The extrat was entrifuged at 12,000xg for 15 mins. Conentrate the lear supernatant and used for assay (Step. II). Salivary sample was used to evaluate the antioxidant and hormone ontent during reprodutive phases of normal menstrual yle Bioanalytial methods Salivary Alkaline phosphatase ativity (IU/L) Alkaline phosphatase ativity was determined using ommerial test kits omprising 1 mol/l diethanolamine HCl uffer ph 9.8, 0.5 mmol/l magnesium hloride and 10 mmol/l of the sustrate p-nitrophenylphosphate. The improved method utilizes p-nitrophenyl phosphate that is hydrolyzed y ALP into a yellow olored produt (maximal asorane at 405nm). Salivary alkaline phosphatase ativity was quantified as IU/L Salivary Latate dehydrogenase LDH assay is ased on the redution of the tetrazolium salt MTT in a NADH-oupled enzymati reation to a redued form of MTT whih exhiits an asorption maximum at 565 nm. The intensity of the purple olor formed is diretly proportional to the enzyme ativity [28]. Samples were assayed using UV-spetrophotometer and the Latate dehydrogenase ontent was measured y IU/L Salivary peroxidase Salivary peroxidase ativity was measured in saliva aording to the nitroenzoi aid (NBS) assay were determined aording to Putter and Beker [29]. The reagents used for salivary analysis were 20 mm 2.2 azino-di-(3-etil-enzotiazolin-(6)-sulphoni aid) diammonium aid (ABTS) in 67 mm phosphate uffer ph value 6.0 and 10 mm hydrogen peroxide and peroxidase in quantity of 250 J/kg. The assay was ased on 0.5 ml of saliva sample whih was diluted with 1.5 ml of phosphate uffer ph value 6.0. Reative mixture onsisted of 2 ml of diluted saliva sample, 0.2 ml of ABTS solution and 0.2 ml of hydrogen peroxide whih were mixed in the reative uvette and put into spetrophotometer was read at a wavelength of 570 nm for enzyme ativity. Peroxidase assay uses H2O2 and an eletron donor dye that forms a pink olor during the peroxidase reation. Salivary peroxidase was measured as U/L Salivary Gluthione peroxidase Glutathione peroxidase (GPx) ativity was measured y the method desried y Rotruk et al [30]. Briefly, reation mixture ontained 0.2 ml of 0.4 M Tris-HCl uffer ph 7.0, 0.1 ml of 10 mm sodium azide, 0.2 ml of homogenate (homogenized in 0.4 M, Tris- HCl uffer, ph 7.0), 0.2 ml glutathione, 0.1 ml of 0.2 mm H O. The ontents were inuated at 37 C for 10 min. The reation was arrested y 0.4 ml of 10% TCA, and entrifuged. Supernatant was assayed for glutathione ontent y using Ellmans reagent (19.8 mg of 5, 5'-dithioisnitro enzoi aid (DTNB) in 100 ml of 0.1% sodium nitrate). Redued glutathione (GSH) was determined y the method of Ellman 1.0 ml of supernatant was treated with 0.5 ml of Ellmans reagent and 3.0 ml of phosphate uffer (0.2 M, ph -8.0). The measured derease in optial density at 340 nm is diretly proportional to the enzyme ativity in the sample. The GPX ativity is expressed as U/L Salivary atalase The determination of Catalase (CAT) was assayed olorimetrially at 620 nm and expressed as ìmoles of H2O2 onsumed/min/mg protein as desried y Sinha [31]. The reation mixture (2.0 ml, vol) ontained 1.0 ml of 0.01 M phosphate uffer (ph 7.0), 0.1 ml of erythroyte lysate and 0.4 ml of 2 M H2O2. The reation was stopped y the addition of 2.0 ml of dihromate-aeti aid reagent (5% potassium dihromate and glaial aeti aid were mixed in 1:3). An improved assay diretly measures atalase degradation of H2O2 using a redox dye. The hange in olor intensity at 570 nm is diretly proportional to the atalase ativity in the sample. The atalase ativity is expressed as U/L Colletion of samples The superoxide dismutase assessment was ased on the generation of superoxide radials produed y xanthine and xanthine oxidase, whih reats with 2-(4-iodophenyl)-3-(4- nitrophenol)-5-phenyl tetrazolium hloride (INT) to form a red formazan dye. The SOD ativity was measured y the degree of inhiition of this reation. The method is a modifiation of the NBT assay. XTT is redued y the superoxide anion oxygen generated y xanthine oxidase. Formazan is read at 470 nm [15]. SOD inhiits this reation y savenging the anion oxygen. One unit of the enzyme is defined as the amount of enzyme needed for 50% inhiition of asorption in the asene of the enzyme [32]. The superoxide dismutase ativity was expressed as U/L Salivary Asori aid Two milliliters of paraffin-stimulated whole saliva were olleted and entrifuged, and the supernatant was mixed with 10% trihloraeti aid (TCA). The TCA-saliva mixture was entrifuged and the TCA-solule fration analyzed for asorate as

4 Suarayalu Alagendran / et.al Int J Biol Med Res. 2011; 2(1): desried y Omaye et al. [33]. Asori aid is oxidized y asorate oxidase resulting in the prodution of H2O2 whih reats with a speifi dye to form a pink olored produt. The olor intensity at 570 nm is diretly proportional to the asori aid onentration in the sample measured as mg/dl Salivary uri aid onentration Uri aid onentration was measured with a kit supplied y Sentinel CH (Milano, Italy) as previously desried [32]. In the assay, uri aid is transformed y uriase into allantoin and hydrogen peroxide, whih, under the atalyti influene of peroxidase, oxidizes the hromogen (4-aminophenazone/ N- ethylmethylanilin propan-sulphonate sodi). This reation forms a red ompound whose intensity of olor is proportional to the amount of uri aid present in the sample and is read at a wavelength of 546 nm. The measurement of salivary uri aid was expressed as mg/dl Salivary Hormone Analysis Salivary estradiol and progesterone were analyzed using solid phase radio-immunoassay tehnique RIA tehnique (Diagnosti Produts, Los Angeles, CA, USA) with an interaassay and interassay oeffiient of variation of 4.7 % and 6.4 %, respetively for estradiol and 4.7% and 7.9%, respetively for progesterone. Serum onentrations of FSH and LH were measured using Immulite hemiluminesent assay kits (Diagnosti Produts Cooperation, Los Angeles, CA, USA).The detetion ranges of FSH and LH were and 0.7 miu /ml, respetively [34, 11] Values are expressed as Mean± SEM and differenes were onsidered those means in the same vertial olumn that are not marked with the same supersript symols are signifiantly different at p<0.05. The data were ompared y one-way ANOVA followed y the Dunan multiple range (DMRT) omparison test. As shown in Tale II. ALP, POD, LDH and Asorate showed statistially signifiant differene among the five groups y one way ANOVA. However, the levels differed only slightly etween ovulatory phase and preovulatory phases. To onfirm the differene desries aove p values were adjusted y the Dunan multiple omparison post ho test. After adjustment, the differenes remained signifiant for (prepuertal vs preovulatory phase; NS, preovulatory vs ovulatory; p 0.05). When the salivary levels of these enzymes and antioxidant ontent were ompared aording to prepuertal and menopause, no signifiant differene was oserved. The patterns of 17-â estradiol onentration, salivary antioxidant derivatives was oserved over the tripliates during the normal menstrual yle are illustrated in Fig.1 and 2 Tale -3. respetively. Figure 1. Salivary enzymes and antioxidant profiles during menstrual yle.? p<0.05 signifiantly higher during ovulatory phase of menstrual yle 3. Statistial methods Results are reported as means ± SEM and differenes were onsidered those means in the same vertial olumn that are not marked with the same supersript letters are signifiantly different at p<0.05. The data were ompared y one-way ANOVA followed y the Dunan multiple range (DMRT) omparison test. 4. Results Among 20 volunteers enrolled, 17 women ompleted the study suessfully and three were found to have an irregular menstrual yle eause of onsumption of oral ontraeptives and were exluded. Age, anthropometri data inluding weight, height, ody mass index and the length of the menstrual yle are shown in Tale 1. Tale 1. Age, anthropometri data and estimated menstrual yle length for sujets at enrollment of the studies Menstrual yle Charateristis Age (years) Mean± SEM 28.5±2.52** NS p 0.05 The pattern of all the parameters exept ALP and LDH were more onsistent in range as ompared to other parameters. These flutuations were signifiant over the menstrual yle whih revealed that 17-â estradiol was dereased during the preovulatory phase. Likewise, Asori aid and peroxidase onentration was high during the ovulatory phase whih was statistially signifiant at 0.05 levels (F=2.561; 17.54±2.46 mg/dl) and (F=3.652; 48.20±6.46 U/L) as ompared to prepuertal, preovulation, post ovulation and menopause stages whih were drastially dereased. In ontrast to the hanges in glutathione peroxidase, atalase, superoxide dismutase sequentially varied from stages to stages in normal menstrual yle. Figure 2. Salivary hormone flutuate in antioxidant profile during menstrual yle Height (m) 128.0±3.64* p 0.05 Weight (Kg) 54.5±3.93** NS BMI (kg/m2) 23.6±1.60* pp Cyle length (d) (Salivary Ferning and BBT) 28.2±1.49* (85%; 0.72 F spike during Ovulation) pp

5 386 Suarayalu Alagendran / et.al Int J Biol Med Res. 2011; 2(1): Tale II. Salivary Antioxidants parameters during menstrual yle. Parameter Prepuertal (6-9 yrs) Preovp (6-12 days) Ovp (13-14 days) Postovp (15-26 days) Menopause (Aove 45 yrs) ALP (IU/L) 54.29±2.79 a 80.39± ± ± ±2.68 e LDH (IU/L) 14.61±4.07 a 12.06± ± ±2.02 d 50.12±4.67 e Peroxidase(U/L) 17.12±3.18 a 28.19± ± ±3.87 d 13.77±2.79 ea GPx(U/L) 1.71± ± ± ± ±0.36 ea Catalase(U/L) 2.03± ± ± ± ±0.37 SOD(U/L) 2.04± ± ± ± ±0.37 Asori Aid (mg/dl) 4.94±0.70 a 9.87± ± ±1.29 d 3.45±0.43 e Uri Aid (mg/dl) 8.98± ± ± ±1.69 d 10.18±2.84 ea Note: Values are mean ± SEM. The data were ompared y one-way ANOVA followed y the Dunan multiple range (DMRT) omparison test. a P<0.05 (Prepu vs. Preovul) P<0.05 (Preovul vs. Ovul) P<0.05 (Ovul vs.post Ovul) d P<0.05 (Post Ovul vs.menopause). e P<0.05 (Menopause vs.prepu) Tale II: Salivary hormone quantified various reprodutive phases during menstrual yle. Parameter Preovp (6-12 days) Ovp (13-14 days) Postovp (15-26 days) Menopause (Aove 45 yrs) LH (miu/l) a 45.6± ± ±8.28 d 56.66±2.68 FSH (miu/l) a 62.06± ±11.10 d 67.43±6.02 d 50.12± eta Estradiol (pg/ml) a 48.19± ±5.46 d 22.33±3.87 da 13.77±2.34 Progesterone (pmol/l) a 7.35± ± ±3.58 ea 1.95±0.36 Values are expressed as Mean± SEM and differenes were onsidered those means in the same vertial olumn that are not marked with the same supersript letter are signifiantly different at p<0.05. The data were ompared y one-way ANOVA followed y the Dunan multiple range (DMRT) omparison test. a P<0.05 (Preovul vs. Ovul) P<0.05 (Ovul vs.post Ovul) P<0.05 (Post Ovul vs.menopause). d P<0.05 (Menopause vs. Preovu)

6 Suarayalu Alagendran / et.al Int J Biol Med Res. 2011; 2(1): Human salivary alkaline phosphatase ativity inreased in all sujets during the ovulatory phase while ompare to pre and postovulatory phases during menstrual yle (Fig. 2). The mean inrease expressed as IU per litre units was (F= 4.352; ± 15.76; p 0.05) in other phases of the yle the onentration remarkaly dereased from Preovulatory phase, prepuertal, menopause and postovulatory stages. In prepuertal saliva the antioxidant markers like G-px, atalase, superoxide dismutase and uri aid oviously dereased. It was also interesting to note that salivary antioxidant levels of preovulatory sample are similar to that of postovulatory samples. But, ontraditory, the antioxidant ativity are found to e dereased after ovulatory phase i.e.; in prepuertal and menopause. The onentration of antioxidant enzyme is very muh redued partiularly Gluthatione peroxidase, atalase and superoxide dismutase during prepuertal and menopause. Tale 2 shows the levels of hormones ompared with antioxidants oserved the atalase, SOD and GPx in the saliva were signifiantly lower in onentration while in preovulatory and postovulatory. Further, salivary hormone onentrations indiate predominantly high during ovulatory phase as LH (F=2.423; ±15.76 miu/l; p 0.05). During prevoualtory phase FSH and estradiol show the variation from the day 7. It indiates that the surge of ovulation was realized during the day of 13. Progesterone was analyzed in all phases of menstrual yle onsistently elevated during postovulatory phase (F=2.134; 19.33±3.58 pmol/l; p 0.05) and ovulatory phase The hormone assay was not deteted in prepuertal and a mild hange was in menopause stages whih do not differ with respet to the days of menstrual yle. Neither in reprodutive status nor in prepuertal and menopause women saliva ALP, LDH, POD, G-Px, SOD, Asorate and urate levels were orrelated with irulating ovarian hormone onentrations. However, it has een shown that the antioxidant ailities of oth estrogen and progesterone an up-regulate the ativities of peroxidase, asori aid and GPx. These results provide further evidene that estrogen and progesterone at as antioxidants and are free radial savengers. 5. Disussion The result of this study showed non-signifiant flutuations in SOD, atalase and G-px levels throughout the menstrual period whih was ontraditory while ompared to prepuertal and menopause stage. In addition to this the level of ALP, LDH, peroxidase, asori aid and uri aid was found to e high in reprodutive periods, whih orrelated with ovarian hormone in relation to detet the ovulation using noninvasive methods. These findings are similar to the findings of others who examined in ervial muus, vaginal muus and plasma. The onstany of enzymes and antioxidant onentration were found to e high in ovulation phase whih was similar to ervial muus and plasma findings [12, 17, 35]. The ovulation phase was onfirmed y monitoring the time of salivary LH surge and serum 17-â estradiol onentration. The LH surge provides a quik ut aurate physiologial marker at the time of estrogen peak and ovulation, sine it ours aout hrs after the estrogen peak and hrs efore ovulation, attaining the peak level aout 18 hrs efore ovum release [36]. The overall pattern of mean values of plasma 17-â estradiol onentration oserved in this studies were in agreement with results previously reported [29], onfirming that the lood samples were otained in an aurate time during eah phase without missing estrogen peak prior to LH surge. The inrease of ativity tended to oinide with the inrease in serum estrogen level. Large yli variation without any lear tendeny ourred in salivary á-amylase and siali aid onentrations; minor yli variations were found in thioyanate, alium, potassium, and protein. None of the measured ompounds proved to e a reliale marker for ovulation. Peroxidase has also een found to e a normal onstituent of human ervial muus and some preliminary studies on the orrelation of peroxidase ativity with different phases of the menstrual yle have een desried [37]. These authors found no ovious orrelations ut their results were otained from single measurements in different women and the time of ovulation had not een determined. In the present study we measured onentrations of serum and urinary gonadotrophins and urinary steroids to permit assignment of the most proale time of ovulation, and levels of peroxidase in samples of ervial muus. In attle the level of peroxidase in the ervial muus an e used for the detetion of ovulation [38]. This test in attle is arried out y means of a suitaly impregnated finger-stall whih an e inserted into the ervix. The intensity of the resulting olor hange is approximately proportional to the amount of enzyme present. The amount of enzyme drops signifiantly at estrus, i.e hr efore ovulation [7, 9]. The present study is the first in whih lood selenium on, G- px ativities and dietary selenium intakes were measured simultaneously to detet phase related hanges during the human menstrual yle. Plasma selenium and G-px ativity have een shown to flutuate similarly during the rat estrous yle [30]. Das and Chowdhary [19] also reported hanges in plasma selenium onentration during menstrual yle, ut their plasma samples were olleted during preovulation and ovulation phase ased on hanges in BBT rather than on hanges in plasma estrogen onentration. Estradiol treatment has inreased erythroyte G-px ativity without hanges in malondialdehyde an indiator of lipid peroxidation [39], suggesting that the inreased G-px ativity did not result from an inrease in lipid peroxide. It has een suggested that estrogen plays a protetive role against ardiovasular disease partly y dereasing the likelihood of LDL peroxidation [20]. Our results suggest that an antioxidant role of estrogen may also e modulation of G-Px ativity. This mehanism would have relevane in the postmenopausal population in whih a derease in estrogen level and the assoiated redution in selenium status may aelerate oxidative damage after menopause, although variations in food intake have een reported during the menstrual yle [23]. Further the study revealed that SOD, G-PX and atalase onentration were affeted y flutuation in ovarian hormones are signifiantly lower in menstrual yle periods, prepuertal and menopause stages. However, the effet of prolonged hormonal disturanes noted in prepuertal and menopause stages on enzyme ativity was more evident than due to physiologi flutuation in hormone seretion. SOD plays a key role in the protetion of various tissues against oxidative damage, and raised SOD is a sensitive marker of inreased ROS prodution [6,36]. Erythroyte SOD is of speial importane to red ells protetion

7 388 Suarayalu Alagendran / et.al Int J Biol Med Res. 2011; 2(1): against superoxide prodution due to hemogloin autoxidation [17]. On the other hand erythroyte SOD has een reognized as potent antioxidant for other tissues, sine anion hannel allows transport of superoxide and other radials into red ells [12,17]. In onsequene, flutuation in the erythroyte SOD ativity may reflet the hanges in ROS generation not only in red ells ut also in other tissues. However, the lak of differenes in plasma TBARS levels and GSH dependent erythroyte antioxidants system etween prepuertal, menopause and reprodutive phase like preouvlation, ovulation and postovulation phases of the urrent study revealed that there were signs of greater oxidative stress in women with distured ovarian hormones seretion. In addition, an inverse orrelation etween erythroyte SOD ativity and plasma 17-â estradiol levels in ovulating women may imply an inhiitory ation of physiologial hormone onentrations on SOD ativity [36,37]. This assumption is supported y the lak of this effet in prepuertal and menopause women with notiealy lower irulating 17-â estradiol levels. Sine females have enhaned SOD ativity (whih is an important antioxidant enzymati defense) and in view of the nonenzymati defense offered y estrogen, oxidative stress was kept under ontrol, as shown y the LPO data [31]. Although glutathione peroxidase ativity was dereased in females as ompared to male ontrols, the pro-oxidant/antioxidant alane was in the favor of the antioxidants and the oxidative damage was redued in females. Glutathione peroxidase enhanement in male hearts, that do not have estrogen protetion, is not enough to alane free radial prodution. This appears to e an example of oxidative stress-indued up-regulation of an antioxidant enzyme [3]. The antioxidant potential of various steroid hormones (estriol, estradiol, estrone, progesterone, testosterone, androstenedione, ortisol and others) has een evaluated and it was shown that estrogens, espeially estriol and estradiol, are natural antioxidants, while the other steroids do not present signifiant antioxidant ativity [6,7]. The other aim of the present study was to determine whether the removal of sex hormones ould affet the oxidative stress pattern oserved under physiologial onditions. The data pertaining to ovarian hormones effet on erythroyte SOD ativity in human is unreliale. Guemouri et al. [24] showing a 16% derease in erythroyte SOD ativity after menarhe when signifiant elevation in ovarian hormones seretion ourred. The aove data and results of the urrent study indiate that 17-â estradiol may play an important role in the regulation of erythroyte SOD ativity. In onlusion, regularly menstruating women, prepuertal and menopause women with distured progesterone and 17-â estradiol seretion and susequent asene of ovulation are haraterized y markedly derease in SOD ativity in the preovulatory and postovulatory phases of the menstrual yle than their normal ounterparts. This may e due to diminished inhiitory 17-â estradiol on SOD ativity in females with low irulating hormone levels. These findings were similar to the results of Lutoslawska et al. [16] in whih a strong relationship was found among salivary ferning patterns and ovarian hormones in menstrual yle. Results of this study showed that levels of LDH were so high next to ALP. This may e due to the stimulatory effet of oxidative damage. Furthermore, SOD, Catalase and G-px at ovulation and prepuertal and men opause were signifiantly dereased in aordane with previous findings [16]. This finding indiates that the quantifiation of antioxidants in saliva may e onsidered as one of the reliale parameters in detetion of ovulation. 6. Conlusion The levels of several antioxidants were altered in women saliva in the presene of oestradiol and progesterone: the onentrations of glutathione peroxidase and atalase dereased signifiantly while the levels of superoxide dismutase and uri aid did not hange in all reprodutive phases. The alterations in enzyme ativity like alkaline phosphatase and latate dehydrogenase for the period of ell vulneraility in saliva samples were more evident in ovulatory phase with a omination of oestradiol and progesterone. It is onluded that enzymes and ertain antioxidants like peroxidase and asori aid is a key ompound in gonadal physiology on whih further researh is needed and that a reappraisal of its potential linial value in the treatment of various types of female infertility would e timely for the aid of IVF. 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