J. Biomater. Sci. Polymer Edn, Vol. 18, No. 6, pp. 785–797 (2007) VSP 2007.Also available online - www.brill.nl/jbs

Haemocompatibility of vitamin-E-enriched poly(D,L-lacticacid)

FILIPPO RENÒ, VINCENZINA TRAINA and MARIO CANNAS ∗Human Anatomy Laboratory, Research Center for Biocompatibility Tissue Engineering,Experimental and Clinical Medicine Department, University of Eastern Piedmont “A. Avogadro”,Via Solaroli 17, 28100 Novara, Italy

Received 29 August 2005; accepted 21 February 2007

Abstract—Poly(D,L-lactic acid) (P(D,L)LA) is a biocompatible and biodegradable polymer whoseuse is limited to orthopaedic applications. In fact, the mechanical properties of P(D,L)LA are notusually utilized for cardiovascular applications, as the polymer has been proven to activate bothgranulocyte- and platelet-causing inflammation. In order to improve P(D,L)LA haemocompatibilityvitamin E (α-tocoferol, 10–30% (w/w)), a natural biological anti-oxidant and anti-inflammatoryagent, was added during the solvent casting of P(D,L)LA film. The P(D,L)LA films obtainedwere then analysed using FT-IR analysis to assess vitamin E presence; polymer surface wettabilityand human plasma protein adsorption were measured by sessile drop test, spectrophotometricprotein quantification and Western blot, respectively, and polymer haemocompatibility was assessedmeasuring platelet and granulocyte adhesion and whole blood coagulation. Vitamin E presence causedan increase in polymer surface wettability and human plasma protein adsorption. The combination ofboth effects may account for the decrease in platelet and granulocyte adhesion and for the doublingof whole blood clotting time measured onto vitamin-E-enriched P(D,L)LA compared to controlP(D,L)LA. Our results indicate that vitamin E addition improves P(D,L)LA haemocompatibility,making this polymer suitable for cardiovascular application.

Key words: Poly(D,L-lactic acid); vitamin E; haemocompatibility; platelet; granulocyte.

INTRODUCTION

Biodegradable polymers are used in a plethora of clinical applications, but mainlyas drug-delivery systems. One of the most investigated applications of these poly-mers is the metallic stent coating, where polymers deliver drugs or other agents(e.g., nucleic acids) to reduce the thrombogenic tendency of stents and to contrastneointima hyperplasia and consequent vascular stenosis [1]. In-stent restenosis is

∗To whom correspondence should be addressed. Tel./Fax: (39-321) 660-632; e-mail:cannas@med.unipmn.it

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a pathobiological process that still occurs in 10–50% of cases currently treated [2];therefore, a more effective effort to seriously reduce the incidence of this importantside effect of stent implantation is necessary. One of the main challenges for this re-search area is the interaction occurring between the polymer used in the productionof drug-eluting stents and the complex and fast reactive blood environment. In fact,biodegradable polymers, such as polyglycolic acid/polylactic acid (PGLA) or poly-caprolactone (PCL), which are considered as good candidates for this kind of appli-cation on the basis of in vitro tests, after implantation have been demonstrated to in-duce a marked inflammation with subsequent neointimal thickening [3]. Later someother polymers used for drug elution (sirolimus) were found to be biologically inertand stable for at least 6 months [4, 5], and now the research is focused on the use ofbiomimetic substances such as phosphorylcoline [6] that does not interfere with there-endothelization and the degree of neointimal formation. Among biodegradablepolymers poly(lactic acid) (PLA) (both poly(L-lactic acid) (P(L)LA) and poly(D,L-lactic acid) (P(D,L)LA)) is an interesting candidate for stent coating as it is bio-compatible and undergoes scission in the body to lactic acid with L-lactic acid as anatural intermediate in carbohydrate metabolism [7, 8]. In particular, P(D,L)LA isused as a drug-eluting polymer as it is degraded faster than PLLA, but unluckily italso activates both granulocyte [9] and platelet [10]. Recently, P(D,L)LA has beenused as a paclitaxel-eluting coronary stent with good results in inhibiting restenosisin an animal model, but the unloaded polymer induced a long-lasting local inflam-matory response that probably caused an underestimation of the paclitaxel effect onthe restenosis [11]. In our laboratory we produced P(D,L)LA films enriched withvitamin E (α-tocoferol, 1–10% (w/w)), showing an increased wettability [12]. Vita-min E is a natural biological anti-oxidant [13] and anti-inflammatory agent [14, 15],whose properties have been extensively utilized to improve the biocompatibility ofdifferent biomaterials [16, 17] and the haemocompatibility of cellulose membranefor haemodialysis [18, 19]. In this paper, we investigated the effects of additionof vitamin E (10–30% (w/w)) to P(D,L)LA on polymer wettability, human plasmaprotein adsorption and haemocompatibility, characterized in the term of thrombore-sistant properties (clotting time), platelet and granulocyte adhesion.

MATERIALS AND METHODS

Preparation of P(D,L)LA films

P(D,L)LA (100% D,L, average molecular mass 75–120 kDa) and vitamin E (α-tocopherol) were purchased from Sigma-Aldrich (Milwaukee, WI, USA). P(D,L)LAfilms were prepared by the casting of a 0.05 g/ml P(D,L)LA solution in chloroformin 150-mm glass dishes. 10, 20 and 30% (w/w) vitamin E was added to theP(D,L)LA/chloroform solution. After 5 min shaking the solution was added toglass dishes and the solvent was evaporated at room temperature for 24 h and under

Haemocompatibility of vitamin-E-enriched poly(D,L-lactic acid) 787

vacuum for 3 h in the dark. Film sheets (approx. 1 mm thick) were then cut understerile conditions into square samples (approx. 1 cm2) and stored at 4◦C for no morethan 1 week.

IR spectroscopy

Fourier transformed infrared spectroscopy (FT-IR) spectra for polymers surfaceswere obtained at 4 cm−1 resolution using a Bruker IFS 113v spectrophotometerequipped with MCT cryodetector. The spectra for IR analysis were executed onthin transparent films of control and vitamin E P(D,L)LA (area = 1 cm2 surface).IR spectra were executed in transmission and recorded in the mid infrared region ata nominal temperature of approx. 300 K.

Wettability tests

Contact-angle measurements were carried out in order to evaluate the wettabilityof the vitamin-E-enriched P(D,L)LA films. An equal volume of distilled water(100 µl) was placed on every sample by means of a micropipette, forming a drop orspreading on the surface. Photos were taken through lenses (Leitz IIA optical stagemicroscope equipped with a Leica DFC320 video-camera) to record drop images.Measure of the contact angle was performed by analyzing drop images (3 for eachsamples) using Scion Image software.

Protein adsorption

Protein adsorption assay was performed in triplicate using human plasma poolobtained from 10 healthy donors. Blood (10 ml) was centrifuged at 200 × g for10 min to obtain platelet-rich plasma (PRP). PRP was then centrifuged at 1600 × g

for 10 min to separate platelets [20]. Plasma was then stored at −20◦C prior to use.PS disks (Nalge Nunc. Roskilde, Denmark), P(D,L)LA and P(D,L)LA/vitamin Efilms (1 cm2) were covered with 200 µl of the undiluted human plasma pool andincubated for 1 h at 37◦C. At the end of incubation, plasma was removed and filmswere washed three times with PBS. Adsorbed proteins were collected by incubatingsamples with 1 ml 2% sodium dodecyl sulfate (SDS) solution in PBS for 4 h at roomtemperature under vigorous shaking. the amount of adsorbed proteins was measuredin triplicate using a commercial protein quantification kit (BCA, Pierce, Rockford,IL, USA). The sample optical density was read at 562 nm against a calibrationcurve created using bovine serum albumin (BSA, 25–2000 µg/ml). The results wereexpressed as µg total protein adsorbed/cm2 ± standard deviation (SD).

Granulocyte and platelet separation

Human peripheral venous blood (20 ml) was obtained from 10 healthy donors(age range 20–36 years) using EDTA as anticoagulant. All the blood sampleswere used within 3 h from sampling. Granulocytes were separated from whole

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blood using a modification of the method of Boyum [21]. Blood (10 ml) waslayered onto a Ficoll-Hypaque density gradient and centrifuged for 20 min at2000 rpm to separate mononuclear cells from erythrocytes and granulocytes. Themononuclear fraction was discharged and erythrocytes were then lysed using anammonium chloride lysing solution (150 mM NH4Cl, 10 mM NaHCO3, 1 mMEDTA, pH 7.4) for 20 min at 4◦C. Pellet containing granulocytes was thencentrifuged twice in sterile phosphate buffer (PBS), then cells were counted inoptical microscopy using the Trypan blue exclusion test (viability > 98%) andsuspended at a concentration of 1 × 107 cells/ml in RPMI 1640 (Euroclone, Milan,Italy) medium supplemented with 10% heat-inactivated fetal calf serum (Euroclone)containing penicillin (100 U/ml), streptomycin (100 mg/ml) and L-glutamine(2 mM) (Euroclone) in polypropylene tubes. Granulocyte suspension (200 µl)was seeded onto cell-culture-grade polystyrene disks (area approx. 1 cm2) andP(D,L)LA and P(D,L)LA/vitamin E films (area = 1 cm2), and incubated for 1 hin a humidified atmosphere containing 5% CO2 at 37◦C.

Platelets were obtained as previously indicated and re-suspended in 10 ml RPMI1640 medium supplemented with 10% heat-inactivated fetal calf serum (Euroclone).Aliquots of platelet suspension (200 µl) were seeded onto PS disks, P(D,L)LA andP(D,L)LA/vitamin E films for 0.5 h in a humidified atmosphere containing 5% CO2

at 37◦C.

Analysis of adherent platelets and granulocytes

Cell counting and morphological analysis of adherent platelet and granulocyte wereperformed using an Aristoplan Leitz fluorescence microscope equipped with a LeicaDFC320 digital camera. At the end of the incubation time adhered platelets andgranulocytes were washed three times with cold PBS (pH 7.4) and fixed for 15 minat room temperature using a solution of formaldehyde (3.7%) and sucrose (3%) inPBS (pH 7.4). Platelets were treated for 5 min with Triton X-100 solution (2%,v/v) in PBS and stained with 0.1 µM phalloidin-TRIC (Sigma-Aldrich) for 1 h at37◦C. Platelet adhesion was measured as surface coverage with phalloidin-stainedplatelets (% area coverage ± SD) by measuring the fluorescence presence in 10different fields for sample observed at 10× magnification. Fluorescence presencewas measured using Leica QWin software.

Adherent granulocytes were stained for 10 min at room temperature in thedark with 0.2% of Acridine Orange (AO) solution and counted in 10 differentfields per sample at 10× magnification. Scoring was performed by three separateobservers, blind to the sample treatment using Leica QWin software and expressedas granulocytes adhered/cm2 ± SD. Platelet morphology was observed at 40×magnification while granulocytes were observed at 25× magnification.

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Clotting time

The tromboresistance properties of the P(D,L)LA and vitamin-E-enriched P(D,L)LAfilms were evaluated using fresh human blood by the kinetic clotting method[22]. For this test, 100 µl fresh blood was taken directly from the plastic syringeused for the blood collection and immediately dropped onto the film specimensand onto polystyrene (PS) disks. After a predetermined contact time (10, 20,30 or 50 min), specimens were transferred to plastic tubes, each containing 20ml distilled water, and incubated for 5 min. The surface ability to induce bloodclotting was deduced by the quantity of free haemoglobin measurable at everytime point. In fact, the red blood cells that had not been trapped in a thrombuswere haemolysed and the concentration of free haemoglobin dispersed in water wascolorimetrically measured by monitoring the absorbance at 540 nm. The absorbancevalues were plotted versus the blood contacting time and the clotting times werederived using optical density curves. Each absorbance value represents the averageof 10 measurements ± SD.

Statistical analysis

The statistical analysis of data was performed using Graph Pad Prism 2.01 softwarefor Windows and using the ANOVA test followed by Dunnett’s post-hoc test, takingp < 0.05 as the minimum level of significance.

RESULTS

FT-IR analysis

In Fig. 1 the FT-IR absorbance spectra of P(D,L)LA and P(D,L)LA enriched withvitamin E at various concentrations recorded in the region 4000–1500 cm−1 areshown.

The band at approx. 3500 cm−1 indicates the O–H stretching and it is present inevery sample, as OH groups are present in both P(D,L)LA and vitamin E structures.Two bands at approx. 3000 cm−1 represent the –CH3 stretching. The –CH3

functions are also P(D,L)LA and vitamin E structures, but in the vitamin E there areboth aromatic and aliphatic –CH3, the former emitting at slightly higher frequenciesthan the latter. The intensity of bands at 3500 and 3000 cm−1 increased withthe vitamin E concentration added to the P(D,L)LA, indicating the dose-dependentvitamin E presence.

Wettability

In the wettability test performed using the control P(D,L)LA surface, a drop ofdistilled water on the polymer surface formed an angle of almost 90◦ (89.6 ±1.5◦), while the vitamin E addition surprisingly decreased the water contact angle

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Figure 1. FT-IR spectra of control P(D,L)LA and P(D,L)LA enriched with 10%, 20% and 30% (w/w)vitamin E.

Table 1.Water contact angle of control and vitamin-E-enriched P(D,L)LA films

Sample Water contact angle ± SD (◦)

Control P(D,L)LA 89.6 ± 3.5P(D,L)LA/10% vitamin E 62.3 ± 1.5*

P(D,L)LA/20% vitamin E 58.2 ± 1.9*

P(D,L)LA/30% vitamin E 49.4 ± 2.3*

* Significantly different from control, P < 0.001.

starting from 10% concentration (water contact angle 62.3 ± 1.5◦, P < 0.001)(Table 1). The 20% vitamin E P(D,L)LA wettability (water contact angle 58.2 ±1.9◦) was not significantly higher than the one measured for 10% vitamin E films,while wettability increased significantly for 30% vitamin E samples (water contactangle 49.4 ± 2.3◦). The increased wettability of vitamin-E-enriched P(D,L)LA wasunexpected, as vitamin E is hydrophobic. The only explanation we could give tothis phenomenon was that it could be due to the increased presence of OH groupson the film surface in vitamin E-P(D,L)LA films, as shown by the FT-IR analysis(Fig. 1).

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Figure 2. Quantification of protein adsorbed onto polystyrene (PS), control P(D,L)LA (PLA) andP(D,L)LA enriched with 10% (PLA10), 20% (PLA20) and 30% (PLA30) vitamin E. *P < 0.05,**P < 0.001, compared to control P(D,L)LA.

Human plasma protein adsorption

As expected from the results of the wettability test, the protein adsorption assayevidenced that addition of vitamin E to P(D,L)LA induced a higher total proteinadsorption compared to control P(D,L)LA (70 ± 32.9 µg/cm2) and cell-culture-grade PS (66.3 ± 34.1 µg/cm2) (Fig. 2). The adsorbed protein quantity measuredfor 10%, 20% and 30% vitamin E P(D,L)LA was 162 ± 6.9, 226 ± 22.5 and 400.7± 52.5 µg/cm2, respectively.

Platelet and granulocyte adhesion

In vitro platelet adhesion testing was performed to study the quantity and themorphology of adherent platelets onto control P(D,L)LA and vitamin-E-enrichedP(D,L)LA. As shown in Fig. 3A the percentage of area covered by platelets adheredonto PS and P(D,L)LA was 45.5 ± 0.6% and 42.3 ± 2.9%, respectively. Plateletadhesion on 10% and 20% vitamin E P(D,L)LA films decreased slightly (36.1 ±2.0% and 34.4 ± 1.4%, respectively; P < 0.05 compared to control P(D,L)LA),even if no statistically significant difference was observed as regards the percentagesof covered area measured for the two vitamin-E-enriched polymers. Plateletadhesion on 30% vitamin E P(D,L)LA films dropped dramatically; only 4.4 ± 1.7%of the polymer area was covered by platelets (P < 0.001). As shown in Fig. 3Bgranulocytes adhered both to PS (619 200 ± 104 840 cells/cm2) and P(D,L)LA(806 300 ± 17 900 cells/cm2) after 1 h incubation. The presence of 10% and 20%vitamin E in P(D,L)LA films strongly decreased granulocyte adhesion (360 300 ±4500 cells/cm2, P < 0.001, and 317 000 ± 37 200 cells/cm2, P < 0.001,

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Figure 3. (A) Quantification of platelet adhesion expressed as percentage of area coverage obtainedfrom the fluorescence emitted by platelet stained with phalloidine-TRIC and adherent onto polystyrene(PS), control P(D,L)LA (PLA) and P(D,L)LA enriched with 10% (PLA10), 20% (PLA20) and 30%(PLA30) vitamin E after 0.5 h incubation. (B) Quantification of granulocyte adhesion expressed ascellular count of adherent granulocytes stained with Acridine Orange (AO) and scored on PS, PLAand P(D,L)LA enriched with vitamin E (PLA10, 20, 30) after 1 h incubation. Adherent cells werecounted in 10 different fields per sample at 10× magnification and their number was expressed asgranulocytes adhered/cm2. *P < 0.05, **P < 0.001, compared to control P(D,L)LA.

respectively). Also, for granulocyte adhesion no statistically significant differenceswere observed between 10% and 20% vitamin E polymer films, and the presence of30% vitamin E reduced the cell adhesion (11 100 ± 2890 cells/cm2, P < 0.001).

Platelet and granulocyte morphology

Platelet morphology was altered by the presence of high vitamin E concentration

Haemocompatibility of vitamin-E-enriched poly(D,L-lactic acid) 793

Figure 4. Representative fluorescent images of adherent platelets (A, B) and granulocyte (C, D)observed onto control P(D,L)LA (A, C) and P(D,L)LA enriched with 30% vitamin E (B, D) films.Platelet morphology was observed at 40× magnification, while granulocytes were observed at 25×magnification.

as observed by the actin staining with phalloidin. In fact, as shown in Fig. 4A,adherent platelet observed onto control P(D,L)LA formed aggregates and 50–70%of the platelets showed a spread morphology, while the few platelet adhered on 30%vitamin E-P(D,L)LA films (Fig. 4B) were mostly isolated and their morphology wasmainly roundish.

Granulocytes adhered to P(D,L)LA and stained with AO showed the typicalpolylobate nucleus and a spread morphology observed for activated granulocyte(Fig. 4C), while the few adhered granulocytes observed onto 30% vitamin E-P(D,L)LA films (Fig. 4D) showed a roundish morphology with a lower cellular sizecompared to the granulocyte adhered onto control P(D,L)LA.

Clotting time measurement

In Fig. 5 the blood-clotting profile for PS, P(D,L)LA and vitamin E-P(D,L)LA filmsare shown. The absorbance of the haemolyzed haemoglobin solution varied withtime, and the higher the absorbance, the better the thromboresistance. We definedclotting time as the time at which the absorbance equals 0.02. PS was able to reducehaemoglobin absorbance quickly and PS samples were coagulated completely after

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Figure 5. Absorbance of haemolysed haemoglobin solutions obtained after contact with PS disks,P(D,L)LA and vitamin-E-enriched P(D,L)LA films versus time of contact.

45–47 min. P(D,L)LA samples showed a similar clotting time, but the coagulationprocess seemed to occur more slowly compared to PS. The addition of 30% vitaminE to P(D,L)LA slowed the coagulation process significantly compared to normalP(D,L)LA at every time point, while a statistically significant difference betweenabsorbance values for P(D,L)LA and 10 and 20% vitamin E-P(D,L)LA samples wasobserved only after 50 min (P < 0.001). However, for all vitamin-E-enrichedP(D,L)LA samples coagulation time was 70–75 min (data not shown), indicating anincreased thromboresistance compared to the normal P(D,L)LA.

DISCUSSION

This study demonstrates that vitamin E addition to P(D,L)LA greatly improvespolymer haemocompatibility, as indicated by the decrease in platelet and granu-locyte adhesion and by the increase in blood-clotting time. Furthermore, we ob-served that addition of vitamin E caused an increase in both polymer wettability,probably due to an increase in OH groups on the vitamin-E-enriched PLA surface,

Haemocompatibility of vitamin-E-enriched poly(D,L-lactic acid) 795

and plasma protein adsorption that could account for the observed cellular effects.In fact an increase in both surface wettability and protein adsorption negativelymodulated platelet adhesion and activation [23], as observed also for hydrophilicmodification of P(D,L)LA surface with 2-hydroxyethyl-methacrylate (HEMA) [9].The rapid adhesion and subsequent activation of platelets causes formation of ag-gregates, thrombi and thromboemboli; therefore, the ability of vitamin-E-enrichedP(D,L)LA to reduce the platelet adhesion could also explain its thromboresistancemonitored using the clotting time for whole blood. Protein adsorption and plateletsadhesion are the first events occurring at the surface of a biomaterial interactingwith blood, followed by the adhesion of polymorphonuclear cells (PMN) or granu-locytes (70–80% neutrophils) and other leukocytes, whose activation is responsiblefor the acute inflammation occurring at the site of biomaterial implantation. In fact,it has been demonstrated that the number of adherent neutrophils correlates to theextent of the inflammatory response after implantation [24] and high levels of gran-ulocyte adhesion and activation have been observed onto P(D,L)LA compared toother biomaterials [10]. Granulocytes interact with the biomaterial surface throughthe layer of adsorbed protein or preferentially through the adherent platelets. In ourin vitro model we observed a significant reduction of granulocyte adhesion in allthe vitamin-E-enriched P(D,L)LA films compared to the control P(D,L)LA in theabsence of platelet and in the presence of 10% heat-inactivated fetal calf serum;therefore, the inhibition of granulocyte adhesion could be addressed to a changein the quantity and/or composition of protein layer. Biomaterials surface physicalparameters such as wettability, solubility and surface roughness strongly influencethe composition of adsorbed proteins. Fibrinogen, albumin and immunoglobulin G(IgG) are the main proteins adsorbed from plasma and they modulate granulocyteadhesion. In particular adsorbed IgG have been shown to enhance neutrophil gran-ulocyte adhesiveness [25] and activation [27], albumin seems to blunt inflammatoryphenomenon and granulocyte activation [26], whereas the role of fibrinogen is stillunclear, as fibrinogen-coated surfaces have been shown to activate platelets [27] andto inhibit granulocyte activation [28].

In this study we did not investigate the composition of the adsorbed plasma proteinlayer, but in a previous paper [12] we observed that the main protein adsorbedfrom serum onto 10% vitamin E P(D,L)LA was albumin, the most abundant plasmaprotein known to be responsible for transport and delivery of a wide variety ofmetabolites, drugs, anionic ligands and cations [29].

Albumin adsorbs preferably to hydrophilic surfaces [30] and it is conceivable thatthe same phenomenon observed for the 10% vitamin E P(D,L)LA might occuralso onto the more hydrophilic surfaces of 20% and 30% vitamin E P(D,L)LA.Therefore, although if this part of the work needs to be clarified further, the presenceof adsorbed albumin could explain the inhibition of granulocyte adhesion ontovitamin-E-enriched polymers.

The utilization of vitamin E as a doping factor for P(D,L)LA was suggested by theattractive anti-oxidative and anti-inflammatory properties of this natural agent and

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by the possibility of improving the biocompatibility of a biomaterial using a naturaland biocompatible agent with no side-effects. However, the physical and biologicaleffects observed seem not to be related to the biological properties of vitamin E asit is not released in the short time used for the adsorption and adhesion experiments(Dr. V. Aina, personal communication) and, in our experimental model, it was notpossible to discriminate between the physical effect (change in wettability) and thepossible biological effects of vitamin E (inhibition of cell adhesion and activation)[14, 15].

CONCLUSIONS

Our data indicate that the addition of vitamin E to P(D,L)LA films alters thepolymer wettability, making the polymer surface more hydrophilic and increasingthe amount of adsorbed protein. The combination of these vitamin-E-enrichedpolymer properties caused the inhibition of platelet and granulocyte adhesionand the increase of P(D,L)LA thromboresistence, making the new polymer morehaemocompatible compared to the native polymer and more suitable as a coatingagent for cardiovascular applications.

Acknowledgements

The authors are grateful to Dr. V. Aina and Prof. C. Morterra, from the ChemistryDepartment, University of Turin, Italy, for the FT-IR analysis and to Dr. E. Grossinifrom the Physiology Laboratory, Department of Medical Sciences, Novara, forblood collection.

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