The Effect of Vitamins C and E on Lipid Peroxidation in Stored Erythrocytes*

Transcription

1 ANNALS O F CLINICAL AND LABORATORY SCIENCE, Vol. 23, No. 1 Copyright 1993, Institute for Clinica! Science, Inc. The Effect of Vitamins C and E on Lipid Peroxidation in Stored Erythrocytes* JOSEPH A. KNIGHT, M.D.,tt ROBERT C. BLAYLOCK, M.D.,t and DOUGLAS A. SEARLES, B.S.Î Laboratory Service, Salt Lake VA Medical C enterf and Department o f Pathology, University o f Utah School o f Medicine, t Salt Lake City, UT ABSTRACT Vitamins C and E are both naturally occurring free radical scavengers; as such, their presence assists various other m echanism s in decreasing numerous disruptive free radical processes from taking place, including lipid peroxidation (LP). Vitamin C, being water soluble, is an excellent antioxidant in plasma, while lipid soluble vitamin E is effective in cell membranes. In this communication, our study is reported of the effects of oral vitamin C and E supplem entation in human volunteers on stored red cell LP. It was found that supplem entation with these vitamins for 10 days prior to blood donation significantly decreased LP in stored red cells in both irradiated and non-irradiated samples in comparison to pre-vitamin red cell LP. Plasma potassium levels were also decreased on days 7 and 14 after storage at 4 C, but not after four additional days (day 18) at 37 C. In agreem ent with prior studies, in which various metal chelators and other antioxidants were shown to decrease LP, this study further supports the suggestion that, in addition to adding various im portant nutrient solutions to donor blood, as well as the possible in vitro addition of various metal chelators and other antioxidants, the oral supplem entation w ith vitamin free radical scavengers may further improve the longevity and viability of stored red cells. Introduction Since the introduction of acid-citratedextrose (ACD) in to anticoagulate and preserve w hole blood for transfu sio n s, v ario u s im p ro v e d n u trie n t fo rm u las h av e b e e n in tro d u c e d to * Address reprint requests to: Joseph A. Knight, M.D., Department of Pathology, University of Utah School of Medicine, Salt Lake City, UT increase storage tim e and red cell viability. Thus, Orlina et al16 noted in 1957 that blood stored in citrate-phosphatedextrose (CPD) was more stable than blood stored in ACD. Later, adeninesupplem ented solutions, used in G ermany and Sweden since the late 1960s, and a citrate-phosphate-dextrose-adenine solution supplem ented with additional glucose (CPDA-1) used in the U nited States since 1978,5 extended the allow /93/ $00.90 Institute for Clinical Science, Inc.

2 52 KNIGHT, BLAYLOCK, AND SEARLES able storage time from 21 days for CPD to 35 days for CPDA-1. In this regard, M oroff an d a s so c ia te s 15 n o te d th a t CPDA-1 improved the m aintainance of red cell adenosine triphosphate levels over CPD. Additional additives and/or substrate concentration changes have m ore recently b een suggested to improve further the erythrocyte viability and longevity.1,18 In addition to these im portant contrib u tio n s, o th ers have ex a m in ed the antioxidant properties of red cells and compared them to erythrocyte survival. For example, Lachant and co-workers13 noted the increasing formation of Heinz bodies and decreased glutathione levels in stored red cells. Furthermore, L ee14 reported that the addition of reduced glutathione (GSH) and ethylenediam inetetraacetic acid (EDTA) decreased lipid peroxidation (LP) in plasma stored at 4 to 5 C. W hen both w ere present, their effect was additive. More recently, reduced LP in stored blood following the addition of various m etal-b in d in g chelators was reported by us.10,11 These metal ligands bind the released iron from degenerating red cells and thereby reduce the metalcatalyzed production of oxygen free radicals and subsequent LP (Fenton and/or Haber-Weiss reactions); presumably, the viability and longevity o f the erythrocytes w ere increased. Vitamins C and E are both recognized as important naturally occurring free radical scavengers.6,19 T herefore, the current authors w ere in terested in their effects, if any, on LP in stored red cells. This was accom plished by m easuring plasm a m alondialdehyde levels, using high performance liquid chromatography (HPLC), before and after oral vitamin C and E supplem entation to human volunteers. In addition, plasm a potassium levels were m easured and compared with the degree of sample hemolysis during the study time. Materials and Methods Sp e c im e n s a n d P r o c e d u r e s This study was approved by the U niversity of U tah In stitu tio n al R eview B o ard, a n d in fo rm e d c o n s e n t w as obtained from each subject following an explanation of the study. Eleven healthy volunteers, five females and six males, none of whom were taking daily vitamin supplem ents, ingested 250 mg of vitamin C (ascorbic acid) and 200 IU of vitamin E (200 mg dl-alpha tocopheryl acetate)* three times daily with meals for 10 days. All volunteers were in good health and qualified as blood donors. Duplicate 7.0 ml blood samples were obtained before (baseline) and after the 10 day period of oral vitamin supplementation. The blood sa m p les w e re a n tic o a g u la te d w ith CPDA-1 solution in the same ratio as present in the normal blood donor containers (0.8 ml CPDA-1 added to 7.0 ml of whole blood) and stored at 3 C until plasm a analysis for m alondialdehyde (MDA) and potassium (K+), which were com pleted w ithin 12 hours. Paired duplicate blood samples were irradiated just prior to MDA and K+ analysis with 2500 rad.t Duplicate plasma MDA levels were determ ined by high perform ance liq u id chrom atography (HPLC) as previous reported20 with modifications with respect to the equipm ent used.9 Plasma K+ was quantified using a discrete chemistry analyzer according to the m anufacturer s procedure.t For the baseline K+ measurements (day 0), the serum/plasma procedure was followed; for subsequent m easurem ents (days 7, 14, 18) the urine procedure was used. * Interstate Drug Exchange, Amityville, NY t Model IBL 437C, Ma Cis Bio Industries, Bedford, MA t Astra-8, Beckman Instruments, Brea, CA

3 EFFECT OF VITAMINS C AND E ON LIPID PEROXIDATION 53 The samples were then stored at 3 C, mixed several times by gentle inversion every other day, including the day of MDA and K+ analysis, and the plasma analyzed following gentle centrifugation at approximately 75 g for five minutes on days 7 and 14. The samples were then placed in a 37 C incubator to simulate in vivo conditions, again gently m ixed several times by inversion on day 16 and immediately before analysis on day 18. T hey w ere then centrifuged at about 2,500 g for five m inutes and the plasma analyzed for MDA and K+. In addition, visual sample inspection for hemolysis was recorded on each analysis day. R e a g e n t s /S o l u t io n s The phosphoric acid, thiobarbituric acid (TBA), 1,1,3,3-tetraethoxy-propane (TEP), m ethanol-n ao H solution, phosphate buffer, and mobile phase solutions were all purchased and prepared as previously reported.13 Reagents for the K+ determinations were obtained from the chem istry analyzer m anufacturer previously noted. S t a t is t ic a l A n a l y s is Each blood sample was analyzed in duplicate for MDA and the m ean determ ined prior to further calculations; potassium measurem ents were performed singly. Data computations then included the group means, standard deviations, and M ann-w hitney test. Results The results, w ith respect to plasm a MDA changes before and after vitamin C and E intake in both irradiated and nonirradiated blood specimens, are shown in table I and figure 1. As clearly dem onstrated, the oral administration of vitamin C and E resulted in a significant (P < 0.01 TABLE I Plasma Malondialdehyde Levels at Varying Storage Times Before and After Oral Vitamin C and E Intake Before Vitamins After Vitamins Storage Sample \imol/l J.mo//. Day Pretreatment Mean ±S.D. Mean + S.D. 0 Baseline 1.65(0.25) 0.94 (0.19) a 7 Nonirradiated 1.74(0.29) 1.37 (0.19) b 7 Irradiated 1.79(0.23) 1.57 (0.19) c 14 Nonirradiated 1.88(0.24) 1.51 (0.25) b 14 Irradiated 2.08 (0.38) 1.65 (0.28) <= 18 Nonirradiated 7.60(1.27) 5.60 (1.05) b 18 Irradiated 8.76(1.31) 6.34 (1.43) b Statistical significance, levels of MDA (malondialdehyde) after oral vitamins C and E versus no prior vitamin intake with both nonirradiated and irradiated erythrocytes. a P < bp = cp<0.01 and usually equal to or less than 0.001) decrease in MDA production in both irra d ia te d and n o n -irra d iate d sp e c i mens; the m agnitude of the reduction was similar in both groups. As expected, the irradiated specim ens had h ig h er p lasm a M DA le v e ls th an th e n o n irradiated ones, although their overall decrease was considerably less than previously noted in prior experiments with m etal chelators.10,11 The plasma K + levels were also significantly decreased in the samples following vitamin intake at 7 and 14 days (table II), although not in the baseline specimens or in those at 18 days (following four days at 37 C). The significance of these results is not readily apparent. However, it is of some interest that these potassium levels correlate well with the degree of visual hemolysis noted on the days of analysis (table III). Here, there was a distinct decrease in the degree of hemolysis following vitamin supplem entation in all samples at 7 and 14 days in both irradiated and non-irradiated samples. However, the degree of hemolysis

4 54 KNIGHT, BLAYLOCK, AND SEARLES 10- Tfnlm ntf Pré-, Nonirradiated Pré-, Irradiated Post-, Nonirradiated Wk Post-, Irradiated F igure 1. P lasm a malondialdehyde (MDA) levels (umol per L) compared with storage time in v o lu n te e rs b efo re (pre-) and after (post-) oral su pplem entation w ith vitamins C and E in both irradiated and nonirradiated blood specimens. was indistinguishable on day 18 in both pre- and post-vitamin intake groups; as e x p e cte d, th e irra d ia te d specim en s show ed m ore hem olysis than the nonirradiated samples in both pre- and postvitamin intake. Discussion Since the successful introduction of ACD solutions to anticoagulate and preserve whole blood for transfusions,8,17 numerous studies have been carried out to more fully understand the metabolic pathways in erythrocytes. This information has led to the addition of more appropriate nutrients to whole blood in order to prolong storage time and retain red cell viability.1,5,15,16,18 Until recently, however, other possible mechanisms of importance in extending red cell viability and longevity have only infrequently been reported.13,14 In this reg a rd, o u r fin d in g s w e re re c e n tly reported on the extent of LP in stored erythrocytes.10,11 Here, it was noted that the production of hydroxyl free radicals and subsequent LP was significantly reduced w hen selected m etal-binding TABLE II Plasma Potassium Levels at Varying Storage Times Before and After Oral Vitamin C and E Intake Before Vitamins After Vitamins Storage Sample mmol/l mmol/l Day Pretreatment Mean±S.D. Mean ± S.D. 0 Baseline 3.6 (0.2) 3.6 (0.4) ns 7 Nonirradiated 27.6 ( (2.6) a 7 Irradiated 40.9 (3.6) 36.2 (3.1) b 14 Nonirradiated 33.7 (4.4) 28.0 (3.4) b 14 Irradiated 46.2 (3.7) 43.1 (3.7) c 18 Nonirradiated 51.9 (2.9) 53.8 (2.8) ns 18 Irradiated 48.9 (2.9) 50.3 (2.5) ns Statistical significance, levels of potassium (K+) after oral vitamins C and E versus no prior vitamin intake with both nonirradiated and irradiated erythrocytes. a P <0.001 bp <o.01 c P < 0.02 ns Not significant.

5 EFFECT OF VITAMINS C AND E ON LIPID PEROXIDATION 55 TABLE III Estimate of Hemolysis at Varying Storage Times Before and After Oral Vitamin C and E Intake Storage Sample Day Pretreatment Before Vitamins After Vitamins 0 Baseline Nonirradiated 1 + Trace 7 Irradiated Nonirradiated Irradiated Nonirradiated Irradiated chelators were added to the blood immediately following its withdrawal. Added glutathione, a natural antioxidant which converts hydrogen peroxide to water in the presence of glutathione peroxidase, was also effective although to a lesser degree than the iron-binding chelators. This positive effect on LP by metal chelatio n was also e ffe c tiv e in irra d i ated erythrocytes.12 Vitamins C and E are both recognized as very important naturally occurring free radical scavengers.6 19 In this regard, supplem ental vitamin E to uremic patients undergoing hemodialysis was shown to be effective in reducing red cell LP and increasing the packed red cell volume w hen com pared to those patients not receiving vitam in E prior to hem odialysis.7 Interestingly, a recent publication4 confirm ed that vitam in C supplem entation leads to increased human longevity, an idea suggested earlier but was less well docum ented.2,3 W ith this inform ation at hand, the effects of LP on red cell storage were studied following oral intake of both vitamin C and E. Table I and figure 1 show that vitam in supplem entation significantly reduces the extent of LP in both irradiated and non-irradiated stored red cells, although at a lesser degree than w hen m etal chelators were added.1011 However, prior vitamin intake was probably more effective than the direct addition of glutathione.10 The combination of pre-donor vitam in su p p le m e n ta tio n, added metal binding chelators, and glutathione w ould probably be additive.14 In table II it is shown that the plasma K+ levels were also significantly reduced following vitam in supplem entation in both irradiated and non-irradiated samples on days 7 and 14, but not at baseline (day 0) or following incubation at 37 C for four days (day 18). These results are not fully understood, although prior studies also showed decreased plasma K+ levels in both irradiated and non-irradiated sam ples w hen m etal chelators w ere added,12 further suggesting that free radical formation and LP have some effect on the cellular release of K+. In this regard, it is in terestin g that the increase in plasma K+ levels corresponded with the degree of hem olysis in irradiated and non-irradiated samples, both before and after vitamin supplem entation. Perhaps the events leading to hemolysis and subsequent increase in K+ levels simply predominate over the process of LP. Since vitamin supplem entation was for only 10 days, and the normal red cell lives for 120 days, the question arises as to whether or not greater decreases in LP may have been noted had the volunteers taken the vitamins for a longer period of time. It should also be noted that, unfortunately, these vitamins cannot be added directly to whole blood since vitamin E is insoluble in aqueous solutions and vitamin C is a prooxidant6 in the presence of iron ions and various other hem e moieties. As such, the in vitro addition of vitam in C to w hole blood lead s to increased L P.10 Summary Prior oral supplem entation of vitamins C and E to blood donors, as well as the addition of various metal binding ligands and glutathione to freshly draw n blood,

6 56 KNIGHT, BLAYLOCK, AND SEARLES all decrease LP in stored erythrocytes. The introduction of any or all of these techniques may lead to increased viability and preservation of stored blood. References 1. B r e c h e r, M. E., Z y l s t r a -H o l l i n g, V. W., and PINEDA, A. A.: Rejuvenation of erythrocytes preserved with AS-1 and AS-3. Am. J. Clin. Pathol. 96: , E n s t r o m, J. E. and P a u l in g, L.: Mortality among health-conscious elderly Californians. Proc. Natl. Acad. Sci. USA 79: , E n s t r o m, J. E., Ka n im, L. E., and B r e s l o w, L.: The relationship between vitamin C intake, general health practices, and mortality in Alameda County, California. Am. J. Public Health 76: , E n s t r o m, J. E., Ka n im, L. E., and K l e i n, M. A.: Vitamin C intake and mortality among a sample of the United States population. Epidemiology 3: , Federal Register, 43 (151) 34457, Part 640, August F r e i, B., E n g l a n d, L., and A m e s, B. N.: Ascorbate is an outstanding antioxidant in human blood plasma. Proc. Natl. Acad. Sci. USA 86: , G i a r d i n i, O., T a c c o n e - G a l l a c c i k, M., L u b r a n o, R., R ic c ia r d i-t e n o r e, G., B a n - d in o, D., Sil v i, I., P a r a d is i, C., M a n n a r in o, O., ClTTI, G., ELLI, M., a n d C a s c ia n i, C. U.: E ffe c ts o f alp h a -to c o p h e ro l a d m in istra tio n on re d b lo o d c e ll m e m b ra n e lip id p e ro x id a tio n in h e m o d ia ly sis p a tie n ts. C lin. N e p h ro l. 22: , G ib s o n, J. G., E v a n s, R. D., Au b, ]. C., S a c k, T., and PEACOCK, W. C.: The post-transfusion survival of preserved human erythrocytes stored as whole blood, or in resuspension, after removal of plasma, by means of two isotopes of radioactive iron. J. Clin. Invest. 26: , Kn i g h t, J. A., Sm it h, S. E., Kin d e r, V. E., and An STALL, H. B.: Reference intervals for plasma lipoperoxides: Age-, sex-, and specimen related variations. Clin. Chem. 33: , Kn ig h t, J. A., Vo o r h e e s, R. P., M a r t in, L., and A n STALL, H.: Lipid peroxidation in stored red cells. Transfusion 32: , Kn ig h t, J. A., V o o r h e e s, R. P., and M a r t in, L.: The effect of metal chelators on lipid peroxidation in stored erythrocytes. Ann. Clin. Lab. Sci. 22: , Kn ig h t, J. A., Se a r l e s, D. A., a n d Bl a y l o c k, R. C.: T h e e ffe c t o f m e ta l c h e la to rs o n lip id p e r o x id atio n in irra d ia te d ery th ro c y te s. A nn. C lin. L ab. Sci. 22: , L a c h a n t, N. A., N o b l e, N. A., M y r h e, B. A., and T a n a k a, K. R.: Antioxidant metabolism during blood storage and its relationship to posttransfusion red cell survival. Am. J. Hematol. 17: , L e e, D. M.: Malondialdehyde formation in stored plasma. Biochem. Biophys. Res. Comm. 95: , M o r o f f, G. and D e n d e, D.: Characterization of biochemical changes occurring during storage of red cells: Comparative studies with CPD and CPDA-1 anticoagulant-preservative solutions. Transfusion 23: , O r l in a, A. R. and JOSEPHSON, A. M.: Comparative viability of blood stored in ACD and CPD. Transfusion 9:62-69, Ross, J. F., F i n c h, C. A., P e a c o c k, W. E., and SAMMONS, M. C.: The in vitro preservation and post-transfusion survival of stored blood. J. Clin. Invest. 26: , V a l e r i, C. R., V a l e r i, D. A., G r a y, A., M e l a r a g n o, A., D e n n is, R. C., and E m e r s o n, C. P.: Viability and function of red blood cell concentrates stored at 4 C for 35 days in CPDA-1, CPDA-2, or CPDA-3. Transfusion 22: , W a y n e r, D. D. M., B u r t o n, G. W., I n g o l d, K. U., Ba r c l a y, L. R. C., and L o c k e S. J.: The relative contributions of vitamin E, urate, ascorbate and proteins to the total peroxyl radicaltrapping antioxidant activity of human blood plasma. Biochim. Biophys. Acta 924: , W o n g, S. H. Y., K n i g h t, J. A., H o p f e r, S. M., Z a h a r i a, O., L e a c h, C. N. J r., and S u n d e r - MAN, F. W. J r.: Lipoperoxides in plasma as measured by liquid-chromatographic separation of malondialdehyde-thiobarbituric acid adduct. Clin. Chem. 33: , 1987.