C. Wateïa, M. Br ytsieniska, W. Leyko
DECREASED ERYTHROCYTE GHOST MEMBRANE FLUIDITY AS A RESULT OF LIPIO COMPOSITION CHANGES IN PLASMA
OF ADULT INSULIN-DEPENDENT DIABETICS
Increased mlcrovlscoelty of erythrocytes membrane ghosts was found in diabetic adult subjects. It seoas vary likely that Membrane cholesterol: phospholipid ratio enhancement is the main reason of decreaeed membrane fluidity in diabetes. Moreover, plain correlation between membrane cholesterol: phospholipid ratio, plasma choleoterol and membrane fluidity was estimated. The quantitative and structural chonges of e- rythrocyte membranes and plasma lipids quantitative altera-tions art like to be a reason of diabetic complications aris-ing In poorly controlled diabetes. Significant increase of plasma protein-bound alallc acid content Indicates that anor-mal high concentrations of acute-phaso proteins may occur In diabetes. It Is likely that mentioned serum alallc acid flu-ctuations may cause the enhancement of membrane sialic acid content in diabetic red cells.
Introduction
As it is generally accepted presently eupramolecular dynamic membrane structure is mostly determined by biochemical membrane composition and the interaction of membrane components Cl?., 1.5]* On the other hand, membrane fluidity may regulate structural and reological properties essential for erythrocyte viability. Hence any rigidization of membrane seems to be an important
fac-tor reducing red blood cell deformabillty end the functions of erythrocytes as oxygen carriers [10, 12]. As the cholesterol/ /phospholipid ratio (C/PL) markedly affects membrane microvisco-sity [4, 14],any quantitative changes of membrane lipids may
disturb physical properties of membrane noticeably [7]. In ery-throcytes and platelets with very restricted lipid metebollem, where the phenomena of cholesterol transfer between membranes and plasma serve as a control of oell membrane fluidity, any
compo-sitional fluctuations of plasma lipids should be reflected by changed physical parameters of membranes.
The purpose of this study was to find out whether any dif-ferences in pyrene lateral mobility in th* erythrocyte membranea of adult normal end diabetic petlents exist, and to estimate the possible reasons of any discrepances found.
Material and methods
We examined control healthy patients (16-52 year* old) and diabetics (21-58 years old) of both sexes. Almost »11 the dia-betics exhibited rather far advanced diabetic complications (aa retinopathy and polyneuropathy), all of them were treated with insulin, and dl****« duration was la range of 0.5-41 years.
Freshly drawn blood collected on 38» sodium citrate was washed three times with 10 mM phosphate buffer eallne (pH 7.4). The ery- throcyte membranes were obtained by the method of hypotonic he-molysis according to 0 o d g e et al. [5], and than labelled with pyrene (in methanol solution) to final pyrene concentration
mfl
of 6.6 x 10 M and final lipid phosphorus concentration of 3
pg/nl in each sample.
The fluorescence measurements were performed with Oobln-Yvon spectrofluorlmeter et the temperature of 310 K. The fluoreecence intensities of pyrene dimer end monomer were measured at 470 nm end 396 nm respectively, and than dimer/monomer ratios were ca-lculated.
Total membrane and plasma cholesterol were determined accord-ing to 8 a b a o n et al. [3], similarly membrane end plasma phospholipids according to R a h e J a et al. Llll. Additio-nally the levels of total membrsne sialic acids as well aa total and "free" (it means non-protein bound) plasma simile acids were estimated according to the method described by W a r r e n [17].
Results and discussion
It turned out that mlcrovisooelty of diabetic erythrocyte membranes is signlficantly enhanced« as well as the content of diabetic erythrocyte membrane cholesterol and C/PL ratio. On the contrary, the amount of erythrocyte membrane phoepholipid content diminishes in diabetes, and it is undoubtedly the real reason of the increese in membrane C/PL ratio of diabetic arythrocyte ghosts. Next, the membrane sialic acids is a little, but appa-rently, elevated. Moreover, plasma cholesterol and phospholi-pids as well as plasma C/LP ratio are significantly increased (Tab. 1). The level of total plasma sialic acids is highly ele-vated in diabetic patients and the content of plasma free sia-lic acids la lowered. The per cent content of fraction of plas-ma free sialic acids hence might be expected to be diminished in diabetes, and it is really the case.
It seems that quantitative changes of membrane lipids found are one of the main reasons of pyrene motional restriction in hy-drophobic bllayer of erythrocyte ghost membranes. This enhance-ment of erythrocyte membrane mlcroviscosity In diabetes correla-tes with membrane C/PL ratio (r - 0.562) and plasma cholesterol level (r - 0.553) and the restrained values of correlation co-efficients Indicate, that besides of C/PL ratio also other fac-tors as sphingomyelin/lecithin ratio, lipid/protein ratio or uneaturation degree of acyl chains may markedly influence membra-ne fluidity L H , 151. The data presented in the table i seem to confirm an idea about broadly developed metabolic complications in diabetes. It is not striking that membrane microviscosity en-hancement in diabetes correlates positively with the estimated changes in plasma lipids, since diabetic hyperllpoproteineemla happen to be often accompanied by an accumulation of some plas-ma lipoprotein fractions Ll]. It can «Iso not be excluded that increased membrane sialic acids diminish the membrane- fluidity in diabetes.
Strong relation between plasma and membrane C/PL ratios, and particularly the correlation of plasma cholesterol level and mem-brane fluidity seem to be an unambiguous evidence, that elevated levels of plasma lipids (cholesterol mainly) can seriously
sf-T u b i e 1 Statistical comparison o f the parameters measured
In adult healthy control
and in insulin-dependent diabetic subjects Statystyczne porównanie mierzonych parametrów
w kontroli dorosłych
i przypadkach cukrzycy insullnozaleinej
Pa ram«ter ... .. Group I n ' X SO Significance (p) 0/M ratio normal 25 0.679 0.065 p < 0.001 diabetic 20 0.528 0.038 Mesnbra na normal 20 0.883 0.144 p < 0.001 choleste rol nM/g protein diabetic 28 1.091 0.148 Membrane normal 17 0.988 0.166 p < 0.001 i phospholipid mM/g protein diabetic 24 0.780 0.129 .. i ' Membrane normal 17 0.119 0.025 p < 0.02 sialic ecid mM/g protein diabetic 24 0.132 0.015 Plasma normal 38 6.70 1.34 p < 0.0 01 cholesterol ! bM/1 diabetic 31 8.93 1.65 >Plasma normal 22 2.53 0.35 p < 0.001 phospholipid j nijyi diabetic 23 3.04 0.38 j r , , , 1 -r
' Plastse total normal 12 2.15 0.13
p < 0.001 i sialic acid»
j mM/1
dlqbetic 23 3.08 0.27
Plaaaa free normal 12 280.0 21.3 p < 0.001
sialic acids pM/i dla etlc 23 217.5 34.2 \% free sialic ecid fraction normal 12 12.96 1.12 p < 0.001 diabetic 23 7.17 1.36
feet tiasua metabolism in diabetes and contribute to the de-velopment of diabetic complications.
Though elevated levels of plasma glucose may Influence dsta- rioratively on the development of such complications [2,6,8], we fsei sure that the enhanced plasma cholesterol content could cau-se tha complications in the cau-seme degree. The type of quantitati-ve changes In p l a s m lipid« found by us corresponds with
hyper-betallpoprotelnaemla, which Is responsible for artherlosclero- si; [1]. Indeed, in examined group of diabetics the microan-giopathic changes occur rather as a rule, so then there is much to be said in favour of the hypothesis, that strong posi-tive correlation between plasma levels of sows lipids and ralcro- anglopathy exists.
The observed quantitative fluctuations of plasma sialic acids in diabetes can be an oblique proof for the occurence of meta-bolic disturbances in the anabolism of soms plasma proteins, ma-inly so called acute-phase proteins [26]. As reported previously by M c M i l l a n and othars the fluctuations of above men-tioned proteins may account for plasma vlecoelty incroase and diminished blood flow velocity [9] - the phenomena of particu-lar importance in microcirculation processes.
Zt would be very desirable, we think, to elucidate the pa- thomechanlem of auch dlaturbancea in diabetes, since it is com-monly known, that reduced oxygen transport, enhanced hemostasia and erythrocytes or platelets aggregation as well as changed in-tracellular metabolism of sorbitol and myoinositol together with the effects of enhanced tissue hypoxia in diabetes [8, 9, 13, 18] could by means of interaction with changed blood flow parameters, cause ths diabetic microangiopathies.
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University of Lodz
C. Wstała, M. Bryazawska, W. Layko
OBNIŻONA PŁYNNOŚĆ BŁON ERYTROCYTARNYCH 3AK0 WYNIK ZMIAN ILOŚCIOWYCH LIPIDÓW OSOCZA W CUKRZYCY INSULINOZALEŻNEO U LUDZI DOROSŁYCH
W pracy stwierdzono zmniejszono ruchliwość lat*r*lnę piranu w błonach orytrocytarnych ludzi dorosłych chorych na cukrzycę lnsu- llnozaieZn«. Zmiano« tym towarzyszyły następująca zalany iloś-ci ows lipidów błon i osocza: zwiększanie stosunku cholesterol/ /fosfolipidy w błonie w cukrzycy, podwyższeni* poziomu chol**t*- rolu w osoczu diabetyków. C*«dnoczoónie wykazano zwiększani* po-ziomu całkowitych kwasów ejałowych oraz obniżenia związanych z
białkiem kwasów «Jałowych w osoczu w cukrzycy. Opierając się na korelacji zaian płynności 1 zmian Ilościowych lipidów, au-torzy «ugoru 1*. Za zalany powyższa aog* być letotn« przyczyn« zaian płynności błon w cukrzycy. Obaarwuwans fluktuacjo Ilo-ściowa kwasów «jałowych o«oeza w cukrzycy aog« natomiast świa-dczyć o występowaniu w cukrzycy zsburzsri aetabollcznych w ana- bollzale niektórych blatak osocza.
