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    upc s?: 81 > CODEN POBILD. ‘sswoosisase, he Original scientific paper Post-traumatic differences in the titer of autoantibodies against oxidised low density lipoproteins (oLDL) in the sera of patients with bone fractures and traumatic brain injury SUZANA BOROVIC:, NEVEN 2ARKOVIC"*, RENATE WILDBURGER®, FRANZ ‘TATZBER? and MISLAV JURIN' INTRODUCTION Posttraumatic damage of tissue is often caused by ‘ischemia Tepertusion jury’ that (gen iree-radicals (19, 32). Consequently, lipid onda. ton occurs and ondatively modified low density lino: proteins (OLDL) are produced (14), leading ta the ap- pearance of modified epitopes on protein comoo: nents of LDL, They are ammunogenic inducing the production of specific anbodies which are released. im the blood (17). Anubodies against oLDL are also found im the sera of healthy people. One of the rea- ‘sons for the appearance of these autoantizodies, ‘could be association with the development of athero. sclerosis, related to the LDL metabolism (25). “ent 2 possibility to follow progression of atheroscisrosis ‘by measuring the titer of eutcantbodies against OLD, seems credible (21). On the other band, “tere are the evidence indicatng that ter of autoanabo¢- 3s against oLDL differs between healtny peovie: ents with acute or chronic diseases / ‘The aims of our study were: |) to test wh matic injury is accompanied by the change of autoantibodies against oLDL and , if so, ut possible differences in time course of the ‘autoantibodies against the oLDL in patents ferent imines, Atparticular times, Le, once ig the first month following trauma, muned the tter of ant-oLDL antibodies in ‘nan long done iracrures, “mth taumaue drain e"mth both bone fractures and traumate bra: OLDL values in the normal, control human MATERIALS AND METHODS Patients Blood samples have been obtained from forty sub- Jects who were assigned to one of four groups Group J ncluced $ patients (age M.31.5 years), 7 male and 2 female, with ‘ractures of either long bones eriod biol, Vol 97, No 4, 1995 or large join's. Three of the patients had a femur frec- ture, one had a femur fracture and an acetabulum fracture, as well as a rupture of the symphysis, one ‘ad acetabulum fractures on both sides and one had ‘a dislocation fracture of the elbow. The consolidation ‘of the femur fractures lasted on average 4 months, ‘while the acetabulum fractures healed in about 4 ‘months, There were no periarticular ossifications noz hypertrophic callus formations observed. Group If included 11 patients (age M26.5 years), 9 male and 2 female, with severe head injures. All of them were in coma lasting on average 14 days. Three ‘of them underwent neurosurgical treatment. Six of the patients had fracture of the skull or facial fac: tures. Three of the ‘acial fractures had to be plated. ‘Three of the patients aad a post-traumatic hem:- plegia, but all of them recovered. Group fT included 19 panents (age M-18 years) male and 2 female, wih severe head injury and 3 ure of the femus, one pation: had femur fractures on both sides. Two patients had an additional frac of the palms and one had a fracture of the humerus days. Five patents had Sacture tures, One gatient underwent neurosurgery, 2 fo patents the facial acturas have been plated. ‘The first callus formatoa occurred benvean ond and he fourth meek after injury In every was very extended nearty along the complete lenge. of the femur, Heterotopic osstications around the ‘bow and on the lateral femur condivie were noticed == one patient after a supraconcyiar rail extension. Group IV compnsed 10 healthy subjects (age Mt 27.8 years), $ male and 3 female. All the penents wer Preparation of the sera Blood was taken for the first ume dunng the frst five days after injury, and afterwards once a week unng the frst four weaks. Slood samples were cen- ‘fuged within one hour at ~4°C, for 10 min at times gravity. The sera were collected, immediately 289 Suzana Boron eta bodies Measurement of the titer of anti-oLDL antibodies Novel ELISA.immunoassay “oLAb’ (elfTec, Vienna Austria) was used ‘or the determination of autoant bodies against oxidised LDL, as descnbed before (22, 27), According to the repeated measurements of the standard samples of the known antbocy tier ‘more than 60% inter- as well as intra-precision of the assay was determined. We were using 1 : 5 diluted standards (of 37, 75, 180, 300, 600 and 1200 maU of the antibody titer) and the sampies of the patients’ sera prepared as described. The assay butfer (200 l) was mixed ynth 20 ul of the respective standards or sera in OLDE, coated wells of the 26 micro-well plate and incubated for $0 minutes at 27°C. Alter washing the vwells with butfer, 100 ul of antihuman-IgG peroxdase conjugates were added to each well and the lates ‘were incubated for 30 minutes at room temperature. Afterwards, plates were washed again mih ouiler and 100 yl of the prepared (included in the ki) tetramethyl-benzidine solution was added in avery well and incubated for 15 minutes in dark at room temperature. The reaction was stopped by addition of 80 yl of the prepared stop reagent (2 mol/L sulphuric acid included in the iat) and the absorpuon (of 450 nm wavelength light (with the reference of $70 ‘am wavelength) was measured by microplate reader (model 450, Bio-Rad Laboratories, Richmond. USA). Statistics Results were calculated as mean values anda per- centage of normal values obtained from healthy vol- unteers, Significance was calculated acon e Student's test and the values of P<(0.05, considered as significant. RESULTS he second week ><0.02), Afterwards, dunng the third and the fourta week after injury an increase (to 76% of normal values) in iter of autoantibodies was observed, thus. the values ze- turned to the normal (both for the thira and for the fourth week in comparison to the controls, P>0.08). Patients with traumatic brain injury only igure 2) showed a decrease in tter of autoantibodies cunng the first and the second week after :njury (the frst ‘week 84% and the second week 73% of normal val: ues, in both cases P<0.06). However, there was 3 (great increase in tter of autoantibodies opservec even above ihe normal values during the third weak (18186 P<0.02) andi the fourth week (135% P-<0.06), 290 roanibedies agamst LDL in the sea of injured patents Weeks after inary FIGURE 1. Post-traumatic change of the autoanubodies tar against LDL in he sera of pationts with bone frac ‘ures. Mean values obtained for the triplicates of the pooled sera (N=9) samples prepared during the four onsecutive weeks aiter injury are presented (SD < 26% of the respective mean). Normal values were ob- tained for the pool of 10 denors. * The difference con: sidered as significant o the control (P < 0.08 accord ing tothe Student'sttest) was observed for the first and ‘or tte second week, IRE 2 Post-traumatic change ofthe titer bodies against oLDi in tie sera of patients matic brain injury, Mean vaiues abtained for 2 of the pooled sera {N = 11) samples unng the four consecutive weeks after injury are pre- nied (SD < 15% of the respective mean). Norm: ues were obtained for the pool of 1Odanors. * The ci ence considered as significant to the consol (P < 0008 according tothe Student's west) was observed {=r she all four veks. Period biol. Yo! 97, No 4. 1995 Weeks after inurr FIGURE 3, Post-traumatic change ofthe titer of autoan- bedies against oLDL in the sera of patients with com: bined injury (both bone fractures and treumatic brain ‘injury). Mean values obtained for the tnplicates of the pooled sera ‘N = 10) samples arenared during the four consecutive weeks alter inury are presented (SD ‘< 10% of the respective mean). Normal values were abained for the pool of 10 donors. * The difference considered as significant to the control (P < 0.05 ac: cording to the Student's test) was abserved for the 1@ second and the fourth week. ‘The sera of the patients with combined traumatic rain injury and bone fractures (Figure 3), showed in ne vst and in the second week after inury decrease 1 the tier of aulcanbbodies (in the first week 45%, 001, and in the second week 87% of normal val es, 2<0.08), During the thicd week after injury there vas an snerease .n the titer of autoantibodies ob- served, thus reacting the values of S0% of the conzoi ‘nm comparison to the control, ?>0.1). In the fourth "eek further increase of the iter of autoantibodies "ras Gatacted reaching the values of 135% of normal flues (?<0.0 srmore, there were ditferences cbservad é jured patients, oo. Thus, during he vere signiicanty (P<0.08) higher both in the sera. ‘panents wth brain injury alone and in the sera of sa sents vnth combined injury if compared to the values of the patienis with the bone fractures only. Finally fo values of autoantibodies were different (higher, ?<0.02) between the natients with traumatic bran injury and traumatic brain injury combined with the bone fractures only during the third week alter injury Dunng te third ‘veek after injury differences were not observed for the traumatic brain injury combined smth the bone fractures and bone fractures only (@>0.08), Periad biol. Vol 97. No 4, 19% ioannbocies against LDU n te ser of inured parents DISCUSSION Im the sera of injured patients (those with bone fractures only, twaumatic brain injury only and com- med injury) decreased values of autoantibodies against oLDL were determined dunng the Gist and the second week after injury. While ter of autoant- bodies again reached the normal values in the group of patients with the bone fractures from the thirc ‘week after injury in the sera of patients with traumatic Drain injury and combined injury increased titer of autoantibodies, even above the normal values was ‘measured, However. it should be mentioned that ‘or better evaluation of such differences of the postau- mati changes of the antibody titer individual sera samples of eact of the patients, instead of the pooled sera, should be used (which was not possible due to ‘limited number of oLDi. coated microplates). Nev- ertheless, the obtained results strongly indicate par- ‘cular systomic reaction to injury, ie. involvement of the oxdative stress and consequential modification of LDL in traumatised patients. ‘The mechanism of such post-traumatic events shold be based on sec. ondary post-traumatic “ischemia - reperfusion injury" followed by the generation of oxygen free radicals and lipid peroxidation products (2, §). Ondatve modification of LDL can be further induced by the ‘onic products of lipid perondation, like highly reac: ‘hve aldehydes 4-hydroxynonenal (4-HNE) (3, 12) and malondialdehyde (MDA) (4, 5). Since the modifica- tion of LDL results in the appearance of oLDL epitopes (1/, 18) which are immunogenic, the ant bodies agaist LDLs modified by MDA or 4-HNE can be used for the determination of oxidation specific lipid-protein adducts in the best known pathological disorder based on the appearance of OLDL, i inthe atherosclerotic lesions (20, 31). Moreover, the ¢ of autoantibodies against oLDL was foun: he patients with nypertension, c2 tus, systemic lupus erythematosus, ore- geampaa, neparologial complication, diabess er. observed (26), In acute septicaemia ite 2ars were low at the beginning, but inc ng tie recovery of patents, while in patients "70 died the inci antibodies was not observed iytc therapy in the pauen's arction the decrease 12 ody titer was shown. This 152 sae expected release of the free radicals du sperfusion after prolonged :schemia and con: ai generation of the oLDL (22). Two weeks aite: inury se have also found a decrease autoantibodies mall the mmuzed patents, of the type of injury (bone fracture, Dram injury and combined injury). [tis supposed that the decrease in the tter of autoantibodies in the first evo weeks is, lated to the omdatve sess which appears after 2 jury. Consequently, modification of LDL into iss ‘adised oLDL form should occur and couple the free ‘|n1-OLDL auteantibodies, thus decreasing thetr ter 201 Inthe third and fourth weak after injury the ter of au- coantboaies depended on the kind of injury. ‘Ne me that this could be the consequence of differen’ intensity of oxidative stress induced by different ands of amury. Finally it should be mentioned that post-traumatic oxdatve stress could be related to the humoral echanisias of the enhanced osteogenesis since in the patients wath traumatic brain injury and the bone Enctures hypertophic callus formation was obser ved (28, 2), Namely. ts known that ipid perond- ‘tion reactions (superoxide generation) may develop trough the prostaglancin syntnetc pathwey (2,8 7 24), ile te increase in metabolites of arachidonic acid was observed ater injury (2). Prostaglandins have tmportant, Sut not completely understood role anthe metabolism of bone and influence bone resorp- tionand formation (2). Since subcutaneous adiminis- traton of prostagiandin E, (PGE,) leads to produc tion of a nev bone (8, 9, 16) should be further ana- lyzed ifthe prostaglancin metabolism is involved in the changes of the iter of an-oLDL autoantibodies as woll as of an enhanced osteogenesis in the pa- ‘dens with waumatic Bram inary. Acknowledgements: The authors would like to ox press ther sincere gratnude to Ms, Jasminka Golubié {ocher valuable technical assistance. This stady was supported by the AOJASIF Foundaton, Switzerland, Croatian Mimsty of Science and ElTec GmbH, Aus- ma. REFERENCES BELLOMO G, MAGGI E, POLI M, ACOSTA F G, EOLLAT! F FINARDI G 1995 Automiecis against cndatvely mediied ‘dons Upopectens in NIDDM. Babes 4()6035, UGHLER | M, HALL D 1988 Cencal neous system rauma and soak: |. 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Lancet 123-27 Period biol, Vol 97, No 4, 1995 26 UTM EALWACHS ©, FRURWALD F M4 ZWENER 8 ESTERBAUER: SLEIN W 1998 Transient zeducnon of utarnbodies aganst cx sarcansbacies against oUDL in the sers cfingured eaten. odes a cates om

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