Clin Bi~,chem, Vol. 25, pp. 41-46, 1992

0009-9120/92 $5.00 + .00 [~pyright © 1992 The Canadian Society of Clinical Chemists.

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Diabetes Mellitus Induces Red Blood Cell Plasma Membrane Alterations Possibly Affecting the Aging Process LAURA MAZZANTI, AHMAD KANTAR,

Istituto di Biochimica

EMANUELA FALOIA, 1 ROSA ANNA RABINI, ROBERTO STAFFOLANI, ROSAMARIA FIORINI, BEN SWOBODA, ROBERTO DE PIRRO, 1 and ENRICO BERTOLI a n d 1 C a t t e d r a di E n d o c r i n o l o g i a ,

Various alterations of red blood cell (RBC) plasma membrane appear both in diabetes mellitus and during the physiological aging process. Diabetes mellitus decreases RBC life-span; therefore, it may change the plasma membrane by acting through its effect on the aging process. In order to clarify the issue, RBCs from normal subjects and insulin-dependent diabetic patients were fractionated in five subpopulations of different mean age (fraction 1 : early young RBC, fraction 5: mature RBC). Thereafter, plasma membranes were prepared and enzymatic activities, membrane fluidity and lipid peroxidation were evaluated. NA +, K +-ATPase activity decreased during aging and it was higher in all RBC subpopulations from normal subjects in comparison to diabetic patients. Next, lipid peroxidation and fluidity increased during aging in both the study groups; in this case, however, in all subpopulations, except for that from fraction 1, RBCs from diabetic patients showed higher membrane fluidity and lipid peroxidation in comparison to normal subjects. Data herein reported suggest that diabetes mellitus affects the plasma membrane independently of (lipid peroxidation and fluidity) or dependently on (Na +, K+-ATPase) its effect on aging. In the case of lipid peroxidation and fluidity diabetes mellitus seems to affect the membrane by decreasing RBC life span, whereas in the case of Na+K+-ATPase it seems to alter this

enzymatic activity which in turn might affect RBC aging. Acetylcholinesterase activity decreased during aging in RBCs from normal subjects, but it increased in RBCs from diabetic patients; RBC subpopulation from fraction 1, on the other hand, showed similar values in normal subjects and diabetic patients. In this case the effect of diabetes mellitus appears only during aging.

KEY WORDS: erythrocyte aging; cell membrane; membrane fluidity; Na+/K+-ATPase activity; Acetylcholinesterase activity; lipid peroxidation; diabetes mellitus, insulin-dependent. Introduction

uring aging several variations appear in red D blood cell (RBC) plasma membrane such as peroxidative damage of membrane lipids (1) and reduced insulin receptor number (2), and in RBC cytosol such as reduced activity of glycolytic enzymes Correspondence: Prof. Laura Mazzanti, Istituto di Biochimica, FacoltA di Medicina e Chirurgia, Via Ranieri, 60131 Ancona, Italy. Manuscript received July 16, 1990; revised March 26, 1991 and August 13, 1991; accepted September 11, 1991. CLINICAL BIOCHEMISTRY,VOLUME 25, FEBRUARY 1992

U n i v e r s i t & di A n c o n a ,

Ancona,

Italy

(3) and decreased intracellular K ÷ concentration (4). In comparison to normal subjects, RBCs from patients with diabetes mellitus show decreased lifespan (5) as well as various alterations in t h e i r plasma membrane such as: i) increased fluidity (6); ii) variations in total cholesterol content, phospholipid content, cholesterol/phospholipid ratio (7,8), and insulin receptor number (9); and iii) variations in both the activity and enzymatic properties of membrane-bound enzymes (6,10). To our knowledge no study has evaluated the possibility that some variations may have a role in the reduced RBC life-span, whereas others may be the effect of diabetes mellitus on cell aging. In order to address this question, enzymatic activities, membrane fluidity and lipid peroxidation were investigated in RBCs from both normal and insulindependent diabetic subjects during the RBC aging process. Materials and methods PATIENTS

Twenty-three insulin-dependent diabetic patients (24-43 years), and 26 h e a l t h y subjects ( 2 6 - 4 7 years) were studied. No patient had nephropathy, neuropathy or any other disease state. All diabetic patients were treated with insulin and diet, without any other drug. At the time of the study they were in good metabolic control (HbAlc: 7.3 -+ 1.2%). Stable HbAlc was determined by high-performance liquid chromatography (HPLC; Diamat-Biorad). Both diabetic patients and healthy subjects had a similar diet (carbohydrates 50-60%, proteins 30-35%, lipids 15-20%) and Body Mass Index; furthermore, healthy subjects were drug-free and with a negative family history of diabetes mellitus. REAGENTS

Percoll was p u r c h a s e d from P h a r m a c i a LKB (Uppsala, Sweden). 1,6 diphenyl-l,3,5 hexatriene (DPH) was purchased from Molecular Probes (Eu41

M A Z Z A N T I , FAL01A, RABINI, zT AL.

gene, OR, USA). Bovine Serum Albumin (BSA), ouabain, Fiske and Subbarow Reducer as well as other items were purchased from Sigma (St. Louis, MO, USA). The pyruvate kinase activity was measured using the Boehringer Mannheim GmbH test combination. PREPARATION OF RED BLOOD CELL (RBC) SUBPOPULATIONS

Blood samples were collected, at 8 a.m. after overnight fasting, in lithium-heparin and RBCs were isolated by elution on microcrystalline cellulose: alpha-cellulose (1:1) columns as described by Beutler et al. (11). Isolated RBCs were washed three times in Hepes Buffered Stock Solution (HBSS) consisting of NaC1 2.66 mol/L, KC1 0.09 mol/L, Hepes 0.2 mol/L, pH 7.4 diluted 1:20, and finally resuspended in a total volume of 5 mL of this solution. RBCs were separated into 5 subpopulations on Percoll/BSA density gradients according to the method of Salvo et al. (12), with minor modifications. Briefly: RBC preparation (0.5 mL) was placed at the top of a discontinuous gradient of 60, 63, 66 and 69% Percoll/5.263% BSA in HBSS at room temperature and was centrifuged at 1000 x g for 8 min. Five fractions of RBCs were collected, washed 3 times with HBSS (1:20) and resuspended in a final volume of 1.5 mL. MEMBRANE PREPARATION

RBCs from each fraction were lysed hypotonically in 5 mmol/L phosphate buffer (pH 8) and centrifuged at 20,000 x g for 20 min at 4 °C. Membranes were then washed with phosphate buffers of decreasing molarity in order to completely remove hemoglobin as described by Burton et al. (13). NA *, K +-ATPASE ASSAY The Na +, K ÷-activated Mg 2 +-dependent ATPase activity was determined on RBC plasma membranes by the method of Kitao and Hattori (14). RBC ghosts were incubated at 37 °C in i mL of medium containing MgC12 5 mmol/L, NaC1 140 mmol/L, KC1 14 mmol/L in Tris HC1 40 mmol/L (pH 7.4). The ATPase reaction was started by the addition of 3 mmoUL Na2ATP; the reaction was stopped after 20 rain by the addition of 1 mL of 15% trichloracetic acid. Inorganic phosphate (Pi) released in the reaction was measured according to Fiske and Subbarow (15). Enzyme activity was expressed as the difference in the inorganic phosphate released in the presence and absence of ouabain 10 mM. The ATPase activity assayed in the presence of ouabain was subtracted from the total Mg 2 ÷ dependent ATPase act i v i t y to calculate the a c t i v i t y of the ouabainsensitive Na +, K+-ATPase. Results are expressed as ~mol Pi/mg membrane protein/60 min.

cording to Ellman et al. (16). The enzymatic activity is expressed as ~mol acetylthiocholine hydrolysed/ mirdmg membrane protein. Protein concentration was determined by the method of Lowry et al. (17), using albumin as a standard. FLUORESCENCE MEASUREMENTS

Fluorescence polarization was studied with the probe DPH, according to the previously described method (18). The fluorescence polarization measurements were made on a Perkin Elmer spectrofluorimeter MPF 44 equipped with two quartz prism polarizers using excitation at 365 nm. Suspensions of unlabeled membranes, at the same protein concentration as the sample, were used as reference blanks to evaluate the light-scattering contribution. The steady-state fluorescence polarization (P) was calculated using the equation (19) p-

I V - - IH X g IV + IH x g

where I v and IH are respectively the emission intensities polarized vertically and horizontally to the direction of the polarized light and g is an instrumental correction factor. Decreased P levels indicate decreased phospholipid order (i.e., increased membrane fluidity). THIOBARBITURIC ACID

(TBA) REACTIVITY

The product of fatty acid peroxidation, malondialdehyde, reacts with thiobarbituric acid (TBA) to give a product with fluorescence at 553 nm. Membranes were centrifuged at 3,000 x g for 15 min after the addition of 30% trichloroacetic acid, and 0.5 mL of the resulting supernatant was mixed with 1.1 mL of TBA reagent (equal volumes of 0.67% TBA aqueous solution and glacial acetic acid; v/v). The reaction mixture was heated for 60 min at 95 °C in a sand bath. After cooling to room temperature, 5 mL of n-butanol was added and the mixture was shaken vigorously for 2 min. Thereafter, samples were centrifuged at 4,000 x g for 15 min and the n-butanol layer was used for fluorometric measurements at 553 nm with 515 nm excitation, according to the method of Yagi (20). The lipid peroxide level (Lp) is expressed in t e r m s of m a l o n d i a l d e h y d e content (nmol/mg of m e m b r a n e proteins), using 1,1,3,3tetramethoxypropane as standard. STATISTICAL ANALYSIS

Statistical analysis was carried out using paired and non-paired Student's t-test.

ACETYLCHOLINESTERASE ASSAY

Results

Acetylcholinesterase (AchE) activity was measured in triplicate on RBC plasma membranes ac-

RBCs from controls and diabetic subjects were separated into five populations of different mean age

42

CLINICAL BIOCHEMISTRY,VOLUME 25, FEBRUARY 1992

DIABETES AFFECTS RBC MEMBRANES

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Figure 1 - - N a ÷, K ÷ - A T P a s e activities in RBC plasma m e m b r a n e s from 23 insulin-dependent diabetic patients [ ] and 26 normal subjects D- RBCs were fractionated in five subpopulations of different m e a n age and cells from each subpopulation were used to prepare plasma m e m b r a n e s (see Materials and Methods). Results are expressed as M e a n -+ SD; *p < 0.001.

by d e n s i t y - g r a d i e n t centrifugation; thereafter, membranes obtained from each cell population were used to evaluate enzymatic activities and lipid properties. Mean RBC age increased from fraction 1 (early young RBCs) to fraction 5 (mature RBCs); no significant variation was observed in the pyruvate kinase activity between the controls and diabetics (data not shown) (2). Figure 1 shows that aging produces a progressive reduction in membrane Na ÷, K÷-ATPase activity both in normal and diabetic subjects. However, RBC plasma membranes obtained from normal subjects showed higher enzymatic activity in comparison to diabetic subjects in all fractions tested. Figure 2 shows that plasma membranes from normal subjects decreased their AchE activity during aging, while membranes from diabetic patients in-

creased in activity. Comparison of values obtained in normal and diabetic subjects showed that all the RBC subpopulations, except for the subpopulation containing early young cells (fraction 1), had significantly different AchE activity. The possibility that alterations in the Na ÷, K +ATPase and AchE activities may appear in the presence of variations of membrane fluidity and/or lipid peroxidations was evaluated. Fluidity was investigated by means of the polarization P parameter of the fluorescent probe DPH and results are presented in Figure 3. The extent of lipid peroxidation (malondialdehyde content) was also investigated and data are presented in Figure 4. Both membrane fluidity and lipid peroxidation increased on account of RBC aging either in normal subjects or in diabetic patients. It is noteworthy that plasma membranes

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