Sadržaj lipida u posteljicama trudnica s tipom 1 šećerne bolesti [The content of lipids in the placenta of the pregnant women with type 1 of the diabetes mellitus]

Starčević, Vito (2008) Sadržaj lipida u posteljicama trudnica s tipom 1 šećerne bolesti [The content of lipids in the placenta of the pregnant women with type 1 of the diabetes mellitus]. PhD thesis, Sveučilište u Zagrebu.

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Abstract

The research was created on the basis of the knowledge gained about the influence of the diabetes mellitus type 1 on the local metabolism of the lipids in the placenta of the pregnant women with diabetes mellitus, which is still not studied thoroughly. In general, the data acquired on the content of the lipids in the placenta of the pregnant women are insufficient and not studied deeply enough. The main aim of this investigation is to determine quantitative and qualitative content of lipids in micrograms on 1 gram of the humid tissue of the placenta and register their percentage (%) in the individual groups of lipids; as well as the ratio of the fatty acid in each lipid group. Special interest is devoted to the study of the influence of the type 1 diabetes on the pregnant women themselves, their unborn children and the placenta tissue. Baring in mind that in the studies done so far there are no real papers studying the influence of the well regulated glycemia at pregnant women suffering type 1 diabetes mellitus on the lipid content in their placenta, we decided to enroll two groups of pregnant women. The first group consisting of 38 pregnant women suffering type 1 diabetes mellitus were the study group; and the other group - called control group - was represented by 34 healthy pregnant women. We took the general data from all the women: the first day of the last menstrual period, and the pregnancy was proved by determining the value of -HCG (human chorionic gonadothropin, beta-subunit) within 14 days after the missing period. The pregnant women were received in RCZDT (Referral center for diabetes in pregnancy) several times during their pregnancy in order to perform clinical surveillance, ultrasound checkup and laboratory analyses. We determined the pregnancy to be normal or disturbed according to the determination of the -HCG. During the embryonic development the CRL – crown rump length was measured as well as thereafter the fetal biometry. The first morning glucose level average was measured; the average of all the mean values of the postprandial level of the large glucose profiles was also measured and compared to the values of HbA1c. The quality of the glycemia regulation was controlled by determining the glycosylated (or glycated) hemoglobin A1c / HbA1c. The pregnant women were invited to regular stays in RCZDT. The exact evaluation of the gestation is done by comparing the first day of the last menstrual period and clinical evaluation of the pregnancy and then compared to the ultrasound findings. All the pregnancies from the studied and control group were finished by the caesarean at the term, after the fetal maturity was proved and the indications for the caesarean section done. The samples of the placenta tissue were collected from the central part of placenta of both groups of women. On those samples the content of the individual groups of lipids in the placenta tissue is studied. The placenta tissue without decidua and blood vessels was used to determine the content of the fatty acids. After taking the samples from the fridge unit and the procedure of defrosting them on the room temperature, the so prepared placenta tissue was used in the lipid extraction process in order to determine the concentration of the fatty acids done by the modified Folch method (287). The extraction of the lipids is done by the use of chloroform mixture : methanol in different ratios. The extraction was performed by the means of different solvents' mixtures in order to gain polar and non-polar lipids into solvent. The following day the sample was filtrated into the test tube, already balanced and soon after that the solvent was steamed in the Büchi steamer. The content of the test tube was weighted and mass of the total lipids was seen. In order to determine the precise ratio of the total lipids the heptadecanoic (margaric) acid was used as internal standard. Heptadecanoic (margaric) acid C 17:0 is added as internal standard in order to determine the ration of the individual fatty acids in the phospholipids (PL), free fatty acids (FFA), triacylglycerols/triglycerides (TAG/TG) and ester of the cholesterol (CE) fractions. The rest was build up on the preparative glass plate for thin-film chromatography. The procedure of dividing the lipid fractions was done by the thin-film chromatography method TLC – Thin Layer Chromatography. The fractions were identified by the comparison with the standard (the traveling speed of the unknown fraction equals to the traveling speed of the known standard). After TLC was performed the fractions CE, TAG/TG, FFA i CH were taken from the glass plate (picture 3.) into the test tubes for centrifuging. Samples are after that prepared for the transesterification with methanol (methanolysis). Transesterification is the procedure by which the fatty acids turn into methyl ester FAME – Fatty Acid Methyl Ester (292,293). In this form we can determine the content of the fatty acids; the procedure of determination of the lipid content is done by the method of gas chromatography GC - Gas Chromatography. Gas chromatography GC was performed on chromatograph SRI 8610 GAS CHROMATOGRAPH (picture number 4), which is product of SRI Instruments Chromatography Systems, Torrance, CA, USA equipped with software Peak Simple for Windows. We served 30M Quadrex column Cat. No. 007-23-30-0.25F, FSCC/Fused Silica Capillary Column, product of Quadrex Corporation, Woodbridge, USA. Column diameter was 0,25 milimeter and impregnated with thin layer of 78% cyanopropyl-methyl-polysiloxan and 0,25 µm thickness. The results of the research done were as follows: - The total mass of lipids in the placenta samples were less represented in the placenta of the pregnant women suffering diabetes mellitus type 1 compared to the group of healthy pregnant women. The difference was not statistically significant. In the total number of lipids the most frequent were phospholipids, followed by total free fatty acids, then total cholesterolesters and in the smallest amount were triacylglycerols/triglycerides. The statistically significant difference is found in the group of total free fatty acids and in the group of total cholesterol esters: less number of total fatty acids and cholesterol esters at the pregnant women suffering from diabetes–type 1, than at women belonging to the control group. The total phospholipids was found and the group of triacylglycerols/triglycerides were less represented in the studied group than in the control group, but with no statistical significant difference. - The presence of each individual fatty acid was determined in the studied and control group of pregnant women. - In the group of total phospholipids there was statistically significant difference in the presence of myristic C 14:0 and lignoceric C 24:0 fatty acid. The myristic C 14:0 fatty acid was much more present in the pregnant women suffering from diabetes – type 1 than in the control group. Lignoceric fatty acid C 24:0 was statistically less present in the group of pregnant women suffering diabetes than at the women belonging to the control group. - In the group of triacylglycerols/triglycerides there was statistically more lauric C 12:0 fatty acids at the pregnant women with type 1 diabetes mellitus compared to the control group women. Statistically significant less number of docosahexaenoic C 22:6 n-3 acid and lignoceric C 24:0 fatty acids at the pregnant women with type 1 diabetes mellitus compared to the results received in the control group. - The total free fatty acids were statistically less present in the placenta of the pregnant women suffering diabetes mellitus type 1 compared again to the control group. In this group of total free fatty acids there were more important statistical differences in the presence of the individual fatty acids compared to the control group. Statistical significant higher quantity was established in lauric C 12:0 fatty acid, whereas lower quantity of the palmitic C 16:0; stearic C 18:0; oleic C 18:1 n-9; miristoleic C 14:1 n-5, linoleic C 18:2 n-6 and docosahexaenoic fatty acid C 22:6 n-3 have been found within the placenta of the pregnant women suffering from diabetes mellitus type 1 compared to the quantities obtained in the placenta of the women in the control group. - In the group of total cholesterol-esters there was significantly more lauric C 12:0 fatty acid and significantly less palmitic C 16:0; linoleic C 18:2 n-6; lignoceric C 24:0; docosahexaenoic fatty acid C 22:6 n-3; stearic 18:0 and miristoleic C 14:1 n-5 fatty acids in the placenta samples taken from the pregnant women suffering from the diabetes mellitus type 1 compared to the placenta samples of the women in the control group. Strict regulation of glycemia probably had influence on the changes in the content of the lipids in the placenta of the pregnant women suffering diabetes mellitus type 1. Regulation of glycemia was done by repeated insulin injection, diabetes diets and recommended special diet for the pregnant women suffering diabetes mellitus. The diet for diabetes patients consisting of 1800 kcal followed by the strict glycemia regulation with insulin and recommended food habits changements where a certain quantities of carbohydrates are interchanged with other nutrients, could have a specific influence on the content of placenta lipids. Due to the above mentioned actions certain changes happen in the composition of the fatty acids in the individual fractions of the placenta lipids. Some changes are of statistical importance and some do not undergo any statistically important changes. Statistically important changes are noticed in the presence of fatty acids with smaller number of carbon atoms in the carboxyl acid chain. At the carboxyl acids with the larger number of carbon atoms (longer chain fatty acids) had smaller presence in the placenta samples in both groups of the pregnant women. On the basis of the given experiments we can presume that the composition of the fatty acids is determined with more than one factor. We have to bare in mind that the interactive substitution can take place within the presence of the individual fatty acids. The diabetes diet in the permanent metabolic system, like the one found with the pregnant women suffering diabetes mellitus type 1 possibly maintains the stability of the cellular membrane. This membrane is in charge of fluidity and transport of the individual lipid fractions keeping their ration which is at the same time the basic parameter for penetration and function of the each cell within placenta. The stabile metabolism of the carbohydrates and good glycemia regulation at the pregnant women suffering diabetes mellitus type 1 can in this manner lower the lesion of the cell organelle, lesion of the cellular membrane, the damage of the incorporative lipids within the cellular membrane and at the same time lower the higher level of lipids sedimentation in the placenta tissue. This regulation also lower the frequency of the secondary complication of the main disease.

Abstract in Croatian

Provedeno istraživanje je nastalo, na osnovi dosadašnjih spoznaja o utjecaju tipa 1 šećerne bolesti, na još uvijek slabo proučen, lokalni metabolizam lipida u posteljicama dijabetičkih trudnica. Uopće, dosadašnji podaci o sadržaju lipida u posteljicama trudnica su nedostatni i slabo proučeni. Zadatak ove disertacije je bio utvrditi: a) kvalitativni i b) kvantitativni sadržaj lipida u mikrogramima na 1 gram vlažnog tkiva posteljice, te izraziti njihov udio u postotku (%) zastupljenosti u pojedinim skupinama lipida; te c) zastupljenost pojedinih masnih kiselina u svakoj skupini lipida. Poseban je interes posvećen proučavanju utjecaja tipa 1 šećerne bolesti na same trudnice, njihovu nerođenu djecu i posteljično tkivo. S obzirom, da u do sada dostupnoj literaturi ne postoje radovi koji su proučavali utjecaj dobro reguliranih glikemija kod trudnica s tipom 1 šećerne bolesti na sadržaj lipida u posteljicama takvih trudnica u istraživanje su bile uključene dvije skupine trudnica. Prva skupina je bila skupina od 38 trudnica s tipom 1 šećerne bolesti, koja je predstavljala istraživanu skupinu trudnica; druga je bila kontrolna skupina, koju su predstavljale 34 zdrave trudnice. U svih trudnica su uzeti opći podaci, podaci o prvom danu posljednje menstruacije, a trudnoća je dokazana određivanjem vrijednosti -HCG-a unutar 14 dana od izostale menstruacije. Trudnice su zaprimljene u Referentni centar za dijabetes u trudnoći (RCZDT) više puta tijekom trudnoće, radi kliničkog nadzora, UZV pregleda i laboratorijske obrade. Serijskim je određivanjem -HCG-a utvrđeno da li se radilo o normalnoj ili poremećenoj trudnoći. Tijekom embrionalnog razvoja praćen je rast udaljenosti tjeme-trtica (CRL - crown rump length), a kasnije je praćena fetalna biometrija. Određivana je srednja vrijednost prve jutarnje vrijednosti GUK-a; srednja vrijednost prve postprandijalne razine GUK-a; srednja vrijednost, svih srednjih vrijednosti velikih profila GUK-a; vrijednosti HbA1c. Kvaliteta regulacije glikemije kontrolirala se određivanjem glikoziliranog hemoglobina A1c / HbA1c). Trudnice su dolazile na redovite kontrolne boravke u RCZDT. Točna procjena gestacije određena je usporedbom prvog dana posljednje menstruacije i kliničke procjene trudnoće, koja je uspoređena s UZV nalazom. Sve trudnoće, istraživane i kontrolne skupine trudnica dovršene su carskim rezom u terminu, nakon procjene fetalne zrelosti i postavljene indikacije za carski rez (disproporcija, stav zatkom, itd.). Prikupljeni su uzorci posteljičnog tkiva iz centralnog dijela posteljica istraživane i kontrolne skupine trudnica, te je analiziran sadržaj pojedinih frakcija lipida u posteljičnom tkivu. Posteljično tkivo, bez decidue i krvnih žila koristilo se za određivanje sadržaja masnih kiselina. Nakon uzimanja uzoraka iz hladnjaka i postupka odleđivanja na sobnoj temperaturi pripremljeno posteljično tkivo je korišteno za postupak ekstrakcije lipida, za određivanje koncentracije masnih kiselina, koja je provedena modificiranom metodom prema Folch-u (287). Ekstrakcija lipida je učinjena primjenom smjese kloroforma : metanola u različitim omjerima. Ekstrakcija se provodila s različitim smjesama otapala kako bi polarni i nepolarni lipidi prešli u otapalo. Sljedeći dan uzorak je filtriran u prethodno izvaganu epruvetu, te je slijedilo uparavanje otapala u Büchijevom uparivaču. Sadržaj epruvete se vagao i tako se odredila masa ukupnih lipida. Za određivanje točnog udjela ukupnih lipida korištena je heptadekanska kiselina, kao interni standard. Heptadekanska kiselina C 17:0 dodaje se kao interni standard, zbog određivanja udjela pojedinih masnih kiselina u frakcijama fosfolipida (PL), slobodnih masnih kiselina (SMK/FFA), triacilglicerola/triglicerida (TAG/TG) i estera kolesterola (CE/EC). Ostatak je nanošen na preparativnu staklenu ploču za tankoslojnu kromatografiju. Postupak razdvajanja frakcija lipida proveden je metodom tankoslojne kromatografije TLC – Thin Layer Chromatography. Frakcije su identificirane uspoređivanjem sa standardom (brzina putovanja nepoznate frakcije jednaka je brzini putovanja poznatog standarda). Nakon TCL-a sa ploče su sastrugane frakcije CE, TAG/TG, SMK/FFA i CH (slika br. 3) u epruvete za centrifugiranje. Uzorci su potom pripremljeni za transesterifikaciju (metanolizu). Transesterifikacija je postupak pri kojem masne kiseline prelaze u oblik metilnih estera FAME – Fatty Acid Methyl Ester (292,293). U tom se obliku određuje sastav masnih kiselina; tj. postupak određivanja sadržaja lipida proveden je metodom plinske kromatografije GC - Gas Chromatography. Pri transesterifikaciji dolazi do kidanja postojećih esterskih veza u frakcijama lipida (CE, TAG/TG, PL) i stvaranja esterskih veza između masnih kiselina iz tih frakcija, kao i SMK/FFA i metanola pri čemu se stvaraju metilni esteri masnih kiselina (FAME) (293). Plinska kromatografija je izvedena na kromatografu SRI 8610C GAS CHROMATOGRAPH (slika br. 4), proizvod: „SRI Instruments Chromatography Systems, Torrance, CA, USA“, opremljen softverom Peak Simple za Windows-e. Za plinsku kromatografiju su korištene 30 M Quadrex kolone Cat. No. 007-23-30-0.25F, FSCC/Fused Silica Capillary Column, proizvođač: „Quadrex Corporation, Woodbridge, USA“. Kolone su promjera 0,25 mm impregnirane tankim slojem 78% cyanopropyl-methyl-polysiloxan-om debljine 0,25 µm. Kolone su specifične i idealne za razdvajanje cis/trans izomera metilnih estera masnih kiselina (FAME). Prilagođeno je vrijeme zadržavanja tzv. retencijsko vrijeme (Rt') i prilagođen je volumen zadržavanja tzv. retencijski volumen (VR). Termostatirani prostor kromatografske kolone održava temperaturu u rasponu od nekoliko desetinki oC. Prolaskom uzorka kroz kromatografsku kolonu određuje se sastav masnih kiselina, koji se pouzdano očitava na nedestruktivnom FID detektoru GC-a, koji ima osjetljivost 10-8-10-15g sastojka/mL plina nositelja. Rezultati su preko pojačala vezani za PC registriraju se odazivom detektora kroz faktor vrijeme, te su bili ispisani na ekranu s različitim amplitudama (pikovima) u obliku kromatograma, koji su predstavljali vrijednosti registriranih masnih kiselina. Identifikacija metilnih estera masnih kiselina određena je usporedbom s poznatim standardom metilnog estera. Rezultati provedenog istraživanja bili su sljedeći: ukupni lipidi u uzorcima posteljica su bili manje odlagani u posteljice trudnica s tipom 1 šećerne bolesti, nego u kontrolnoj skupini zdravih trudnica, razlika nije bila statistički značajna. U ukupnim lipidima najzastupljeniji su bili ukupni fosfolipidi, zatim ukupne slobodne masne kiseline, ukupni kolesterol-esteri, a najmanje zastupljeni su bili ukupni triacilgliceroli/trigliceridi. Statistički značajne razlike bile su prisutne u skupini ukupnih slobodnih masnih kiselina i u skupini ukupnih kolesterol-estera. Bilo je statistički značajno manje ukupnih slobodnih masnih kiselina i statistički značajno manje ukupnih kolesterol-estera u ukupnim lipidima, kod trudnica s tipom 1 šećerne bolesti, nego u kontrolnoj skupini trudnica. Skupina ukupnih fosfolipida i skupina ukupnih triglicerida bila je manje zastupljena u istraživanoj, nego u kontrolnoj skupini trudnica, ali bez statistički značajne razlike. U ukupnim lipidima je određena zastupljenost svake pojedine masne kiseline u istraživanoj i kontrolnoj skupini trudnica, čija je zastupljenost međusobno uspoređena. U skupini ukupnih fosfolipida je bila statistički značajna razlika u zastupljenosti miristinske C 14:0 i lignocerinske C 24:0 masne kiseline. Miristinska C 14:0 masna kiselina je bila statistički značajno više zastupljena u trudnica s tipom 1 šećerne bolesti, nego u kontrolnoj skupini trudnica. Lignocerinska C 24:0 masna kiselina je bila statistički značajno manje zastupljena u trudnica s tipom 1 šećerne bolesti, nego u kontrolnoj skupini trudnica. U skupini ukupnih triacilglicerola/triglicerida bilo je statistički značajno više laurinske C 12:0 masne kiseline u trudnica s tipom 1 šećerne bolesti, nego u kontrolnoj skupini trudnica. Statistički značajno manje je bilo dokozaheksaenske C 22:6 n-3 i lignocerinske C 24:0 masne kiseline u trudnica s tipom 1 šećerne bolesti, nego u kontrolnoj skupini trudnica. Ukupne slobodne masne kiseline su bile statistički značajno manje zastupljene u posteljicama trudnica s tipom 1 šećerne bolesti, nego u kontrolnoj skupini trudnica. U skupini ukupnih slobodnih masnih kiselina bilo je više statistički značajnih razlika u zastupljenosti pojedinih masnih kiselina između istraživane i kontrolne skupine trudnica. Statistički značajno više je bilo samo laurinske C 12:0 masne kiseline, a statistički značajno manje palmitinske C 16:0, stearinske C 18:0, oleinske C 18:1 n-9, miristoleinske C 14:1 n-5, linolne C 18:2 n-6 i dokozaheksaenske C 22:6 n-3 masne kiseline u posteljicama trudnica s tipom 1 šećerne bolesti, nego u trudnica kontrolne skupine. U skupini ukupnih kolesterol-estera bilo je statistički značajno više samo laurinske C 12:0 masne kiseline, a statistički značajno manje palmitinske C 16:0, linolne C 18:2 n-6, lignocerinske C 24:0, dokozaheksaenske C 22:6 n-3, stearinske C 18:0 i miristoleinske C 14:1 n-5 masne kiseline u posteljicama trudnica s tipom 1 šećerne bolesti, nego u posteljicama kontrolne skupine trudnica. Na promjene sadržaja lipida u posteljicama trudnica s tipom 1 šećerne bolesti vjerojatno se odrazila i striktna regulacija glikemije s višekratnim davanjem inzulina, dijabetička dijeta, te dijetalne preporuke. Dijabetička dijeta od 1800 kcal, uz striktnu regulaciju glikemije inzulinom, te preporučene modifikacije prehrane u dijabetičkih trudnica sa zamjenom određene količine ugljikohidrata drugim nutritijentima vjerojatno se specifično odrazila na sastav lipida posteljice. Zbog navedenog djelovanja dolazi do promjena u sastavu masnih kiselina u pojedinim frakcijama lipida posteljice, kod nekih na razini statistički značajnih razlika, a kod drugih promjena bez statistički značajnih razlika. Više ili statistički značajno više su bile zastupljene samo masne kiseline sa manjim brojem ugljikovih atoma u lancu karboksilne kiseline, dok su karboksilne kiseline s većim brojem ugljikovih atoma (dugolančane masne kiseline) bile manje zastupljene u uzorcima posteljica, i istraživane i kontrolne skupine trudnica. Na osnovi svega se može pretpostaviti, da je sastav masnih kiselina određen s više čimbenika, pri čemu može doći do međusobne supstitucije u zastupljenosti pojedinih masnih kiselina. Dijabetička dijeta u jednom stalnom dinamičkom metaboličkom sustavu, poput dijabetesa u trudnica s tipom 1 šećerne bolesti održava stabilnost stanične membrane, koja je zadužena za fluidnost i transport pojedinih frakcija lipida, te na taj način održava njihov međusobni omjer, što predstavlja osnovni parametar za propusnost, te funkciju svake stanice u posteljici. Stabilan metabolizam ugljikohidrata i dobra regulacija glikemije u trudnica s tipom 1 šećerne bolesti na taj način vjerojatno može smanjiti oštećenja staničnih organela, oštećenja stanične membrane, oštećenja inkorporiranih lipida u staničnoj membrani, a s tim istovremeno smanjiti i veće odlaganje lipida u posteljično tkivo. S takvom regulacijom se usporedno smanjuje i veća učestalost sekundarnih komplikacija osnovne bolesti.

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