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Adenosine Triphosphate Release by Osmotic Shock and Hemoglobin Ale in Diabetic Subjects’ Erythrocytes Enrico Petruzzi, Claudia Orlando, Pamela Pinzani, Roberta Sestini, Attilio Del Rosso, Germana Dini, Elisabetta Tanganelfi, Antonio Buggiani, and Mario Pazzagli We investigated the significance of adenosine triphosphate (ATP) release from diabetic subjects’ red blood cells (RBCs) following osmotic shock (OS) and its possible relationship with hemoglobin Al, (HbAr,) and with the RBC membrane protein skeleton. RBCs from type I (insulin-dependent [IDDM]) and type II (non-insulin-dependent [NIDDM]) diabetic subjects and age- and sex-matched control subjects were submitted to OS using NaCl solutions (from 0.9% to 0.045% final concentration). ATP release values were determined by the bioluminescent method. For pattern study, they were expressed both as absolute values and as percentages (%) of ATP maximum release (at 0.045% NaCl solution). Twenty-seven IDDM and 25 NIDDM subjects and two control groups were investigated. ATP content in RBCs was 2.08 2 0.19 pmol/ lo4 RBC in IDDM and 1.23 f. 0.20 pmol/ lo4 RBC in NIDDM subjects. The ATP content of IDDM subjects’ RBCs was significantly higher than that of the corresponding control group. ATP release at 0.49% NaCl OS, both as absolute value and as percentage value, was significantly lower in both diabetic groups, and ATP% was inversely correlated with HbAr, (IDDM: r = -.489, P c .Ol; NIDDM: r = -854, P < .Ol), suggesting a possible relationship between Hb glycation. RBC membrane protein skeleton g&cation, and its influence on ATP release by OS. In conclusion, the proposed method seems useful for measuring RBC ATP content and, at the same time, for monitoring the leak effect of the RBC membrane before it bursts. Copyright 0 1994 by W.B. Saunders Company S EVElRAL RED BLOOD CELL (RBC) membrane alterations have been described in diabetes mellitus, including physicochemical and functional changes.‘~” Wide- spread vascular complications such as microangiopathy, rctinopathy, and neuropathy (at least as far as IYISU nen’o- mm disease is concerned) have been found to be related to these alterations.’ Many parameters, such as deformabil- ity,‘-’ filterability,“~” fluidity,‘L’~” adhesivity,” and viscosity.” have been investigated to understand the nature and the cause of RBC functional alterations in diabetes mellitus; changes in the RBC membrane have been observed in relation to the duration of diabetes mellitus.14 Several methods have been used for studying these RBC dysfunc- tions. Whole-blood or RBC suspension filterability is one of the most. widely used methods in clinical hemorrheology,x.‘5 but shear-rate measurement is also widely used for these studies.“’ sex-matched healthy control subjects (Cl and C?) were asked to participate in this study. Clinical and biochemical data of the investigated subject groups are shown in Table 1. All subjects had a normal RBC count, hemoglobin content. and renal function, as evaluated by blood creatinine level determinations. Moreover, to evaluate whether subpopulations of RBCs could play any role in the RBC ATP release values in OS, we further determined the blood reticulocyte count by Citofluorimeter (Sysmex R IOOO-TOA. Medical Electronics. Kobe, Japan). No difference was observed in the reticulocyte count between normal control and diabetic subjects. Assay Procedure The presence of functional alterations in the RBC membrane may alter the release of intracellular chemical compounds when the RBC undergoes variable osmotic shock (OS). We report a method based on adenosine triphosphate (ATP) rjeleasc from RBCs submitted to increasing OS. ATP assay was performed using the bioluminescent method based on the luciferin-luciferase system, which allows the use of a simple, sensitive, and fast technique of measure- ment.“-‘” In addition to the ATP release pattern during OS, this method allows the measurement of intracellular ATP content. This parameter seems to play an important role in modulating intraerythrocyte sugar transport.“’ RBCs from type I (insulin-dependent [IDDM]). type II (non-insulin- dependent [NIDDM]), and healthy control subjects were investigated using the proposed method, and results were compared with hemoglobin A,, percentage (HbA,,%) levels. RBCs from diabetic and control subjects were obtained from EDTA-anticoagulated blood collected after overnight tasting. Following whole-blood light centrifugation (1.500 rpm x 5 min- utes), plasma with huffy coat was discarded by aspiration, and RBCs were washed once and then diluted I:10 using ice-cold NaCl 0.9% solution. Fifty microliters of the I:10 RBC sample was transferred to an Eppendorf tube containing 950 uL of decreasing ice-cold NaCIcb OS solutions at 1).9c/i,. t).63Si,. 0.54%, t).Sl%,. 0.49%. 0.35%. 0.42%. 0.40%. 0.36%, O..iOc~, 0.255, 0.18%. and tl.04Sci final concentration. The tubes were gently mixed for 30 seconds and then centrifuged to 13,000 rpm at 4°C for 30 seconds. Ten microliters of the supernatant. obtained from the RBCs after OS, was evaluated for ATP content (l:h.OOO final dilution) by a bioluminescence assay”~“’ using the ERITROLUX Kit (Bouty. Milan, Italy). ATP values were determined by the internal stan- dard procedure. based on the addition of 2.5 pmol ATP standard solution to the previously measured tube, and counted by a BIOLUMAT LB 9500 bioluminometer (Berthold. Wilbad. Ger- From the Depwtments of Gerontology, Clinicul Piqsioputhology, and General Pathology, Ukrrsity of Florence. Laboratoy of l&L IOD, Florence, Itu!y. SUBJECTS AND METHODS Submitted December 2. 1992: accepted June 2. 1993. Supported by a grant from the lJniver.~iq of Florence, Florence. Ita!v. P. P. is a Postdoctoral Research Fellow of Cons&~ Nazionale delie Richer&e. Itu!v. Twenty-seven IDDM and 25 NIDDM patients and. since RBC age-related variations have bern reported.?‘-‘? 10 + IO age- and Address reprint requests to Enrico Petruzzi. MD, Lahoratoy of Endocrinolo,, Kale Pieruccini 6, 50134~Care ratio (Fig 3) WC observed two different cvcnts, as follows: (I) in diabetic subjects, the ATP*/HbC%, ratio maximum value at 0.49!Z NaCl OS, was approximately one-third that of the control subjects: and (2) a shift to the left was observed in diabetic subjects. representing a dccrcasc in ATP release as compared with that of control subjects. The retarded ATP”r release in diabetic subjects could be 18, 0 IDDM 1~27 16. 0 0 NIDDM ix=25 0 0 14. 0 0 (0) y= -.076x + 12.46, r= 512. -.489. p< .Ol 4J *. . . . . . . . . . _ _ 0 10 20 30 40 50 60 70 80 90 ATP 46 Fig 5. Correlation between HbA,,% and ATP% release at 0.49% NaCl OS in IDDM and NIDDM subjects. ATP RELEASE IN DIABETIC ERYTHROCYTES related to RBC’ deformability defects that several investiga- tors’-5 observed in diabetic subjects. In both diabetic subject groups, a significant difference in ATP release at 0.49% NaCl OS in comparison to that of normal control subjects was observed, both when ATP% value (Fig 4A) and absolute ATP value (Fig 4B) were considered. A statistically significant negative correlation was found, both in IDDM and NIDDM subjects, between HbAI,% and ATP% release values at 0.49% NaC1 OS (Fig 5). Since several RBC membrane alterations occur in diabe- tcs mellitus. for both lipid32.33 and protein composition,” such RBC membrane modifications could be implicated in the difference in leak magnitude we observed in diabetic as opposed to control subjects. Schwartz ct al’ reported a positive correlation between glycosylated Hb levels and erythrocyte membrane protein glycation. This finding supports the hypothesis that high HbA,,Ra levels and consequent RBC membrane protein skeleton glycation may have a strong influence on ATP release by OS. 439 Altered ATP release in diabetic subjects cannot be related to morphologic changes of the RBC membrane. With increasing HbA,,% levels, RBC membranes from diabetic subjects investigated by negative-stained electron micrography (1.15 x 10’ magnification). were observed to maintain an intact membrane protein skeleton structure. Our data in normal subjects were in agreement with results from Liu et al.“” In conclusion, the proposed method for ATP release pattern study seems to give different information with respect to the classic Hb osmotic fragility test. In particular, it seems useful for monitoring the leak effect of the RBC membrane before it bursts, and thus for evaluating mem- brane protein skeleton deformability. ACKNOWLEDGMENT The authors gratefully acknowledge Professor Mario Serio, University of Florence. Italy, for his suggestions and criticism during the preparation of the manuscript. The authors also thank Professor Laura Mazzanti. University of Ancona. Italy. for revising the manuscript. REFERENCES I. Schwartz RS, Madsen JW. Rybicki AC, et al: Oxidation of spectrin and deformability defects in diabetic erythrocytes. Diabe- tes 40:701-70X, 1991 2. Mazzanti L, Rabini RA. Testa I, et al: Modifications induced by diabetes on the physicochemical and functional properties of erythrocyte plasma membrane. Eur J Clin Invest 19:X4-89. 19X9 3. Watala C: Hyperglycaemia alters the physico-chemical prop- erties of proteins in erythrocyte membranes of diabetic patients. Int J Biochem 24:1755-1761.1992 3. Kennedy L, Baynes JW: Non-enzymatic glycosylation and the chronic complications of diabetes: An overview. Diahetologia 26:93-9X, I9X4 5. McMillan DE, Utterback NG, La Puma J: Reduced erythro- cyte deformability in diabetes. Diabetes 27:X95-901. 197X 6. 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