BIOCHEMICAL

MEDICINE

AND

METABOLIC

BIOLOGY

33-44

46,

(1991)

Membrane-Bound Hemoglobin as a Marker of Oxidative Injury in Adult and Neonatal Red Blood Cells RENU SHARMA AND B. R. PREMACHANDRA” Department of Pediatrics, University and *Department of Biochemistry,

of Florida, University

Vniversiry Hospital at Jacksonville, Florida 32209; of the Pacific, San Francisco, California 94115

Received October 19, 1990 A comparative study of the effect of hydrogen peroxide on adult and neonatal red blood cell (RBC) membrane protein composition has been carried out. The results indicate that (a) the native neonatal RBC membranes contain higher levels of membrane-bound hemoglobin (MBHb) than the adult RBC membranes. (b) The content of MBHb increases when RBCs are incubated with increasing concentrations of hydrogen peroxide (HZOZ), more so in neonatal than in adult RBCs; however, neonatal RBC membrane proteins are less susceptible to HZOz oxidation than adult ones. This could be attributed to the fact that Hb F, which is more susceptible to oxidation than Hb A, adds to the reduction potential of neonatal RBC (in which it is present in large amounts) and partially protects neonatal membrane proteins against oxidant stress compared to Hb A in adult RBC. (c) In both neonatal and adult RBCs, Spectrin 1 is relatively more susceptible to oxidant stress than spectrin 2, and spectrins in adult RBC are more labile for peroxidation than the spectrins in neonatal RBC. (d) Based on electrophoretic studies with and without reduction of membranes with mercaptoethanol, we have classified two types of MBHb: Type I is adsorbed to membrane by noncovalent interactions and Type II MBHb is chemically crosslinked to membrane components by disulfide bridges; the content of both these types increases when RBCs are incubated with increasing concentrations of H,O,. (e) Band 6 protein is present in higher amounts in neonatal than in adult RBC membranes. (I) Since the total content of MBHb increases linearly with the level of oxidant stress, we suggest that it could be used as a marker for oxygen radical-induced injury to tissues. o 19% Academic PKSS, IX.

It has been thought that during fetal development, antioxidant defense mechanisms are less active than those of the mature newborn infant or adult (l-3). Oxygen radical-induced injury has been postulated to be a prime factor in pathological states such as bronchopulmonary dysplasia (4)) retinopathy of prematurity (5), and enhanced susceptibility to hemolysis in both preterm and term infants (6). Studies on antioxidant defense potential in human erythrocytes have revealed reduced levels of glutathione peroxidase and vitamin E in preterm and term groups compared with adults (7); such reduced antioxidant potential may well lead to the generation of oxygen free radical species with the concomitant oxidant injury to the cell. It has also been reported that hemoglobin (Hb) itself can catalyze the formation of hydroxyl-radical by Fenton type reaction (8). 33 0885-4505191

$3.00

Copyright Q 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.

34

SHARMA

AND PREMACHANDRA

Red cells can be separated by age, based on their relative densities. RBC membranes isolated from old cells have been observed to contain significant amounts of MBHb, and this has been attributed to a gradual oxidative crosslinking process during red cell aging (9,lO). Such a supposition is supported by the observation that in vitro incubation of erythrocytes with hydrogen peroxide results in crosslinking of Hb to membrane components resulting in alteration of its properties (llJ2); of particular interest is the complex between hemoglobin and spectrin, a major component of the erythrocyte membrane skeletal system. Spectrin performs a variety of membrane functions including regulation of membrane deformability (a vital property of erythrocyte for its survival in vivo) and stability. In addition, spectrin may also regulate cell shape and surface characteristics by its ability to attach to the lipid bilayer and the integral membrane protein, Band 3 (the anion channel (13)). Crosslinking of Hb to spectrin has been correlated with increased red cell membrane rigidity (12). The membranes of neonate and adult red cells differ in a number of poorly defined ways; for example, neonatal membranes contain more phospholipid and cholesterol per cell (and as a consequence have a large surface to volume ratio), are slightly more osmotically resistant, more rigid, and mechanically fragile, and have decreased life span (average 45-70 days) than adult red cells (14). Although protein composition of neonatal and adult cells is similar, little is known about their organization in the membrane milieu in neonatal cells, and no information is available on peroxide-induced damage in neonatal cells either in vivo or in vitro, and on Hb interactions with the neonatal RBC membrane. Hence, we studied the interaction of Hb with neonatal and adult cell membranes in vivo and the effect of hydrogen peroxide on the composition of neonatal and adult cell membrane proteins in vitro on a comparative basis, In an effort to understand the organization of proteins in the membranes, electrophoretic analysis was carried out with and without reduction by mercaptoethanol after peroxide crosslinking. We have also investigated the possibility of using MBHb as a marker for oxidative injury to RBC membranes. MATERIALS AND METHODS Blood. Adult blood was collected by venipuncture from normal, healthy volunteers (30-40 years of age) into sterile heparinized tubes containing citrate phosphate dextrose (CPD). Venous cord blood samples were obtained from the separated placenta within 10 min of the birth of the babies of Caucasian origin. Hemolysis was prevented by gentle aspiration using a sterile syringe and a 19French-gauge needle into sterile, heparizined tubes containing CPD as in the case of adults. The mothers were healthy and their babies weighed between the 10th and 90th percentile and showed no evidence of birth asphyxia. RBC preparation. The heparinized blood samples were immediately centrifuged (75Og, 10 min). After separation of plasma and leukocytes, the erythrocytes were washed three times in a phosphate-buffered saline (PBSG; 5 mM sodium phosphate buffer containing 150 mM sodium chloride and 5 mM glucose, pH 7.4). The washed erythrocytes were adjusted to a hematocrit of 50% with PBSG containing 1 mM sodium azide. Hydrogen peroxide was added to the cell suspension to achieve

MEMBRANE-BOUND

Hb AS MARKER

35

final concentrations of 0.1, 0.5, and 1.0 mM and the mixture was incubated at 37°C in a shaking water bath for 1 hr. After the incubation, the cells were washed three times with cold (5°C) PBSG, and the supernatant washings were pooled to determine the extent of hemolysis. Electrophoretic analysis of membrane proteins. The erythrocyte membranes were prepared from the RBCs by hypotonic hemolysis using 10 mM Tris-HCl; buffer, pH 7.4, at 4°C by the method of Dodge et al. (15). Equal amounts of sodium dodecyl sulfate (SDS)-solubilized membrane proteins obtained from a defined number of ghosts were electrophoresed on 10, 12.5, and 15%, polyacrylamide gels as described by Laemmli et al. (16). The electrophoresis was carried out with and without mercaptoethanol reduction separately, for each set of samples. Authentic sample of purified Hb was used to identify the Hb band on the gels during electrophoresis. The gels were stained by Coomassie blue and scanned using EC910 densitometer with a built-in integrator. The individual peaks were quantitated three different ways: (a) by integrating the area under the peak; (b) by carefully cutting and weighing the peaks; and (c) by measuring the peak intensity, i.e., percentage transmittance (for MBHb only). Protein Estimation. The protein content of membrane preparations was estimated by the method of Lowry et al. (17). RESULTS The SDS-PAGE analysis patterns of adult and neonatal RBCs incubated with H202 (0.1 to 1 mM) are shown in Figs. la (without mercaptoethanol reduction) and lb (with mercaptoethanol reduction). Without reduction by mercaptoethanol, considerable amounts of gel polymerization products were evident on the stacking gel as well as at the top of the running gel. Adult cells seem to be more sensitive to increasing concentrations of H202, as the observed polymerized bands at the top of the gel were more intense in adults than in neonatal RBCs. The spectrin band, in particular a-spectrin, was found to polymerize relatively to a higher extent in adult RBCs compared to neonatal RBCs, and this enhanced polymerization was found to increase with increasing levels of H202. A significant amount of MBHb was found in both adult and neonatal cells without mercaptoethanol reduction and the intensity of this band was higher in the case of neonatal cells than in adult RBC; its amount was also found to increase with increase in H202 concentration. Upon reduction with mercaptoethanol (Fig. lb), except for a small fraction of the observed high-molecular-weight polymers (HMWP), most of the bands at the top of the gel disappeared with the concommitant increase in intensity of all the protein bands that entered the gel; the MBHb was also found to increase following the same pattern, i.e., (a ) more in neonatal than in adult control, and (b) at increasing levels with an increase in H202 concentration used during incubation. The small fraction of HMWP that could not be further reduced by increasing the concentration of mercaptoethanol in sample preparations resembled those reported in previous studies (18). The gels were scanned using a densitometer and the results are shown for adult and neonatal RBC, with and without treatment with 1.0 mM H,Oz (Figs. 2a-2d).

36

SHARMA

A C

P-

2%

NANANA c 0.1 --”

AND PREMACHANDRA

0.1 .--q+0.5

0.5 -r? 1.0

I”,

HAb “!I

FIG. 1. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic (SDS-PAGE) patterns of adult and neonatal red cells treated with varying concentrations of hydrogen peroxide. In (a), samples were not reduced with mercaptoethanol, while in (b), they were reduced with 5% (v/v) mercaptoethanol. The samples loaded into lanes are designated as follows: A, adult; N, neonatal; Hb, hemoglobin standard. The numbers indicate the concentration of H202 (mM) used for incubation of red cells. The arrows indicate the location of membrane-bound hemoglobin band. Each lane has been loaded with identical amounts of protein (40 pg). Increasing amounts of high-molecular-weight polymers are apparent with increasing concentrations of H,O, in (a), which could be reduced by treatment with mercaptoethanol as in (b). The MBHb bands are more intense in neonatal (N lanes) than in adult RBCs (A lanes) and the amount increases further with increasing concentrations of Hz02 (compare N-C, N-0.1, N-0.5, and N-1.0.

In the absence of mercaptoethanol, the intensity of almost all the bands was very much reduced. The band corresponding to MBHb is distinctly identifiable and is more intense in case of 1 mM H,O,-treated samples; neonatal membranes contained more MBHb than adult RBCs (compare Figs. 2b and 2d). Upon reduction of membranes with mercaptoethanol, the intensity of all the bands increased with

MEMBRANE-BOUND

Hb AS MARKER

37

the simultaneous disappearance of high-molecular-weight polymers (found near the top of the gel). It should be noted here that, since the amount of proteins loaded into each lane for both adult and neonatal samples is identical and the gels have been subjected to identical treatment, the peaks in the densitometric scans are both qualitatively and quantitatively comparable. An examination of Figs. 2a and 2b indicates that the protein pattern is qualitatively similar in both adult and neonatal controls; however, from the quantitative point of view, the peak intensities for Band 6 protein and MBHb were markedly higher in the case of neonatal membranes and this observation was consistent in all the samples analyzed. In HzOz-treated (1 mM) samples, after reduction with mercaptoethanol, the individual peak intensities were enhanced (as observed in case of controls); however, comparison of Fig. 2a with 2c reveals the following differences. (i) the intensities of spectrin bands 1 and 2 are significantly reduced in Fig. 2c suggesting a partial and irreversible loss of these bands and this loss is irreversible by prior reduction of membrane proteins with mercaptoethanol; (ii) the intensity of spectrin 1 compared with spectrin 2 is much more reduced in Fig. 2c than in 2a indicating that it is more sensitive to peroxide injury; (iii) we noticed that the spectrin 1 in adult red cells is much more sensitive to oxidative injuries than neonatal spectrin 1 (compare 2c and 2d); (iv) the content of MBHb was considerably increased upon mercaptoethanol treatment and more so in neonatal than in adult RBC membranes; and finally (v) except for these differences, the patterns of other protein bands were qualitatively and quantitatively similar in both adult and neonatal membranes. Figures 3a and 3b reveal the relative amounts of MBHb realized in adult and neonatal membranes, respectively; the values have been shown with and without mercaptoethanol reduction of the membranes. It should be noted here that the value of MBHb without mercaptoethanol reduction represents the fraction of Hb bound to membranes without involving any chemical links (i.e., by adsorption); we designate this MBHb as Type I. The MBHb realized after mercaptoethanol reduction represents total membrane-bound Hb. The difference between these values (Total MBHb-Type I MBHb) represents the fraction of hemoglobin that is chemically crosslinked to the membrane components by S-S bridges (as they could be cleaved by mercaptoethanol); we designate this MBHb as Type II. The values of MBHb (total, chemically crosslinked, and adsorbed) were higher in neonatal membranes than in adult; also these values increased with increasing concentrations of HzOz used in the incubation of red cells. Linear regression analysis of ogives of the bars in Figs. 3a and 3b suggests that this increase for total MBHb is linear with H202 concentration (correlation coefficient, r for Adult = 0.9928 and Neonatal = 0.9952). It is interesting to note that H202 treatment not only increases the content of peroxide-induced crosslinking of Hb to membranes but also markedly enhances the adsorption of Hb to membranes. Also, adsorbed hemoglobin accounts for nearly 4566% of the total MBHb in all the cases. The extent of individual classes of Hb bound to membranes expressed as percentage total is summarized in Table 1. When the data are analyzed this way, it is apparent that the relative proportion of Type I MBHb (adsorbed hemoglobin,

a

-A . .. ... N +c,nssn M B Hb

20

DISTANCE

cm

10

0

b

- CIHSSH

HMP

MBHb

-

6 3

FIG. 2. Densitometric scans of SDS-PAGE patterns. The samples are (a) A (-) and N(...), controls; (c) A and N treated with 1 mM H,O, and reduced with mercaptoethanol; (b) control A and N; (d) A and N treated with 1 mM H,O, without reduction by mercaptoethanol. The gels have been scanned from top (start) to bottom (end) as indicated. The positions of high-molecular-weight polymers and of other protein bands are as indicated. The patterns of gel scans for other concentrations of H,O, treatment (i.e., 0.1 and 0.5 mM) were similar (data not shown). Without mercaptoethanol reduction, very few bands are found in small amounts in both the samples. Upon treatment with mercaptoethanol, all the protein bands are noticeable. The MBHb amounts are more in neonatal than in adult membranes which further increases upon treatment with H,O,. Also note the increased level of band 6 protein in neonatal RBC membranes in all the samples. Patterns have been superimposed to get a direct quantitative idea as they are derived from loading identical amounts of protein in each lane. (40 pg).

38

MEMBRANE-BOUND

MB Hb

39

Hb AS MARKER

C

-Al.OrnW -...... WI.0

mr

+ c,r,sw

21

11 0’

I

START

END I

DISTANCE

20

0

1

cm

1

,D

0

START

END I 20

DISTANCE FIG.

Z-Continued

cm

70

‘1 0

40

SHARMA

AND PREMACHANDRA

a IOOn

W ABSORBANCE

ME +

9% ABSORBANCE

ME.

00-

20

0 0 (CONTROL)

0.1

0.5

1.0

0.5

I.0

HZOZCONCENTRATION WV

b LOO

1

n

9% ABSORBANCE

ME+

80-

; 6o m4 gs m-402 20

-

00 CONTROL

.I

HZOZ CM3;NTRATION

FIG. 3. Content of MBHb as a function of concentration of H202 in (a) adult and (b) neonatal membranes. ME, mercaptoethanol; notations are as indicated in the figure. The values represented by the bars are means f SEM of four separate measurements in each case. The +ME bars (m) represent total MBHb, the -ME bars ( ) represent the adsorbed hemoglobin (Type I MBHb), and the difference between these two values represents covalently crosslinked (by S-S bridges) Type II MBHb. All measurements have been made under identical instrument settings.

MEMBRANE-BOUND

41

Hb AS MARKER

TABLE 1 Binding of Type I and Type II Hemoglobins to Normal and Neonatal Membranes: Effect of H,Oz Treatment of RBCs H@z concn (m4 0

0.1 0.5

1.0

Neonatal MBHb (%)

Adult MBHb (%) Type 1 31.5 41.0 41.4 51.3

"_ k f k

3.9 5.2 4.1 6.6

Type II 62.5 59.0 58.6 48.1

f k f +

3.9 5.2 4.7 6.6

Type 1 36.8 60.8 65.4 65.6

+ k 2 I

5.9 6.3 6.5 8.7

Type II 63.2 39.2 34.6 34.4

k f f 5

5.9 6.3 6.5 8.7

Note. Total MBHb (Type I + Type II) is the amount measured by electrophoresis in the presence of mercaptoethanol and is taken to represent 100% in each case. Type I is adsorbed MBHb measured by electrophoresis in the absence of mercaptoethanol. Type II is MBHb covalently crosslinked by SS bridges to membrane components and its percentage mean value is derived as (lOO-Type I % mean value) in the table; the individual values are means 2 SD of four separate determinations.

mean value) increases in adult from 37.5 to 51.3%; this increase is more pronounced in neonatal RBCs (from 36.8 to 65.6%). In other words, the contribution of adsorbed hemoglobin to total MBHb is markedly increased upon oxygen radicalinduced injury. It should also be noted here that although the total MBHb shows a near linear increase with Hz02 concentration, the relative contributions (expressed as percentage total MBHb) of Type I and II do not (and need not) exhibit linear relations with H202 concentrations, and can, therefore, be independent variables. (Values of correlation coefficient, r for data in Table I are: Adult, Type I = 0.942 & Type II = -0.942; Neonatal, Type I = 0.6902 & Type II = - 0.6902). DISCUSSION In the present study, we have attempted to investigate the effect of oxygen radical-induced injury on neonatal and adult RBCs on a comparative basis. Our data suggest that adult RBC membrane proteins are more sensitive to oxygen radical-induced oxidation compared to neonatal RBC membrane proteins. This effect is more pronounced with spectrin band I (which in turn was more susceptible to oxidation than spectrin band II); however, the neonatal Hb was found to bind to its membranes more than adult Hb to adult RBC membranes, and this binding was further enhanced upon Hz02 treatment. We explain this pardox as follows: If fetal Hb is more susceptible to oxidation than adult Hb (as reported in the literature), then fetal Hb adds to the reduction potential of neonatal RBCs which contain this type of Hb in large amounts; therefore, the neonatal RBC membrane proteins are more protected, and hence less susceptible to oxidative damage than adult RBC membrane proteins. If this were so, how could this be related to reduced life span of neonatal RBC? Our speculation is that the enhanced binding affinity of Hb F, further accentuated by peroxidative damage, might contribute more to its reduced life span than oxidation of membrane proteins per se. The enhanced binding of Hb to RBC membranes (total MBHb) was found to

42

SHARMA

AND

PREMACHANDRA

have a linear relationship with the concentration of H202 used in the incubation. By quantitating the amount of Hb with and without mercaptoethanol, we have attempted to classify the MBHb into two types: Type I binds to membranes by adsorption without involving any chemical crosslinks and Type II binds to membrane components with S-S crosslinks. In vitro incubation of red cells with hydrogen peroxide has been shown to induce several cellular changes which include alteration in cell shape, membrane deformability, phospholipid organization, and cell surface characteristics. Formation of spectrin-hemoglobin complex has been implicated as one of the factors that might induce these changes. Hemoglobin has also been found to crosslink to Band 3 protein under oxidative conditions. However, it is not clear in the light of our findings to what extent Type I and Type II membrane-bound hemoglobins contribute to these changes. It may well be that noncovalent interaction of Hb with the membrane might alter the packing pressure of lipids in the inner monolayer lipids and through their condensation, bring cell shape alterations observed in previous studies (11,12). In fact, since the functional consequences of these two types of Hb are not explored separately, it opens up several venues of research; for example, it is reasonable to presume that (despite the available evidence that spectrin-hemoglobin complex has a role in membrane deformability) the Type 1 Hb binding might also contribute to membrane deformability as well as to other altered membrane properties due to oxygen radical injury. A fraction of the high-molecular-weight polymers was not reducible with mercaptoethanol as observed by earlier studies (18). Since peroxide radicals generate malonyl dialdehyde (MDA), a product of lipid peroxidation, this might produce MDA-protein-Hb complexes; this invokes the possibility of a third class of MBHb, bound to membrane components by MDA-induced crosslinks (not susceptible for cleavage by mercaptoethanol). These polymers are excluded in the present study; however, we wish to point out (a) their amounts are relatively small and will not seriously affect our quantitations; (b) more elaborate experiments have to be designed to cleave MDA-protein complexes and procedures are yet to be standardized to achieve their successful resolution. The enhanced binding of neonatal Hb might be due to the presence of fetal Hb F ((y2y2), which is more susceptible to oxidation than adult Hb A ((~4~) present in these cells. This enhanced binding might explain the observed, higher rigidity of neonatal cells. It might play a role in their shortened life span since abnormal hemoglobin-membrane interaction has been implicated in enhanced phagocytosis and increased monocyte adherence (11,12). In this connection, it is pertinent to mention that Low and co-workers (19) have demonstrated that binding of hemichrome to cytoplasmic surface exposes new antigens on the surface of RBCs. It may be relevant to speculate that the higher affinity of fetal Hb to neonatal RBCs might play a role in abnormal immunological surface characteristics that might account for their shortened life span (about 70 days compared to 120 days of adult RBCs); related to this event one interesting aspect (not examined in the present study) is to specifically isolate and characterize the nature and content of hemoglobin (F and/or A) bound to membranes after peroxide treatment. At present this study is pursued in our lab.

MEMBRANE-BOUND

Hb AS MARKER

43

It should be noted here that a previous study (20) has reported binding of Hb to senescent neonatal cell membranes; the authors in this study did not observe oxidative protein crosslinking. In this communication, we wish to point out that (a) in contrast to the previous study (20) which has used fractionated neonatal RBCs, we have used whole neonatal RBCs. (b) The authors of the previous study used 5 mM sodium phosphate buffer (pH 8.0) in isolating RBC membranes. This buffer, in our experience, brings about dissociation of Hb-membrane complexes due to its high alkalinity; further, depending on the number of washes, one might lose significant amounts of cytoskeletal proteins due to the low ionic strength of this buffer. For this reason, we selected Dodge’s procedure (15) which uses 10 mM Tris-HCl, pH 7.4 (more physiological), for membrane preparations. (c) Finally, the variations due to blood samples should also be taken into consideration. While reviewing our results in the context of other findings, two more aspects have to be highlighted here: (i) Although abnormal binding of Hb to membranes, probably initiated in viva by peroxide radicals, might be the causative factor for decreased life span of neonatal erythrocytes (as implicated in our studies), the role of other factors like membrane lipid peroxiation may also result in reduced membrane deformability and cell survival (21-23). (ii) Whether the observed differences in susceptibility of neonatal erythrocytes to peroxide can be attributed to differences in the altered membrane lipid asymmetry inherent in them (compared to adult RBC (24)) is yet another question not answered in the present study. In view of the fact that peroxide radicals are readily diffusible across the membrane, it is our speculation that the relative disposition of the lipid may not play a significant role, and as already explained, the higher reductive potential of neonatal Hb may be responsible for the observed differences in susceptibility of neonatal RBC toward oxidative damage. One novel observation from the present study is that the quantitative analysis of SDS-PAGE scans has suggested that Band 6 protein is present in a higher amount in neonatal than in adult cells. Since this band has been associated with the enzyme glyceraldehyde-3-phosphate dehydrogenase it will be of interest to quantitate the activity of this enzyme in these cells (currently in progress). If this enzyme activity is enhanced, then it could be correlated with the higher metabolic activity prevailing in neonatal RBCs compared to adult RBCs. In view of the present finding that the binding of both adult and neonatal Hb to the erythrocyte membrane is sensitive to oxygen radical concentration, we propose that it may be used as a marker of oxidative injury to RBCs in particular and other tissues in general; in other words, it may serve to indicate the level of exposure of other vital tissues to oxygen free radicals as well. This might be particularly helpful to evaluate the extent of injury in neonates with Chronic Lung Disease as well as adults with Adult Respiratory Distress Syndrome who require prolonged oxygen supplementation with mechanical ventilation.

ACKNOWLEDGMENT This work was supported by NIH Grant HL31105-07.

44

SHARMA

AND PREMACHANDRA

REFERENCES 1. Autor AP, Frank L, Roberts RJ. Developmental characteristics of pulmonary superoxide dismutase: Relationship to idiopathic respiratory distress syndrome. Pediatr Res 10:154-158, 1976. 2. Tanswell AK, Freeman BA Pulmonary antioxidant enzyme maturation in the fetal and neonatal rat: Developmental profiles. Pediutr Res l&584-587, 1984. 3. Frank L, Groseclose EE. Late gestational changes in lung antioxidant enzymes in fetal rabbit parallel surfactant system changes. Clin Res 31:512A, 1983. 4. Roberts RJ. Pulmonary oxygen toxicity in the premature and full term infant: Its relationship to the development of RDS and its complications. In Hyaline Membrane Disease, Pathogenesis and Pathophysiology (Stem L, Ed.). New York: Grune and Stratton, 1984, pp 211-232. 5. Slater TF, Riley PA. Free radical damage in retrolental fibroplasia. Lancer 2:467, 1970. 6. Gross RT Bracci R, Rudolph N, Schroeder E, Kochen JA. Hydrogen peroxide toxicity and detoxification in the erythrocytes of newborn infants. Mood 29:481-493, 1967. 7. Ripalda MJ, Rudolph N, Wong SL. Developmental patterns of antioxidant defense mechanisms in human erythrocytes. Pediatr Res X:366-369, 1989. 8. Sayed MH, Graf E, Panter SS, Hallaway PE, Eaton JW. Hemoglobin, a biological fenton reagent. J Biol Chem 259:14354-14356, 1984. 9. Sauberman N, Fortier NL, Joshi W, Piorowski J, Snyder LM. Spectrin-hemoglobin cross-linkages associated with in vitro oxidant hypersensitivity in pathologic and artificially dehydrated red cells. Brit J Hematol54:15-28, 1983. 10. Snyder LM, Liu SC, Garver F, Fortier N, Piotrowski J, Leb L, Fairbanks G. Direct demonstration of Hb associated with purified spectrin from senescent human red cells. Blood 62: 40a (Abstr), 1983. 11. Snyder LM, Fortier NL, Trainor J, Jacobs J, Leb L, Lubin B, Chiu D, Shohet S, Mohandas N. Effect of hydrogen peroxide exposure on normal human erythrocyte deformability, morphology, surface characteristics, and spectrin-hemoglobin cross-linking. J. Clin Invest 761971-1977, 1985. 12. Fortier N, Snyder, LM, Garver F, Kiefer C, McKenny J, Mohandas N. The relationship between in vivo generated hemoglobin skeletal protein complex and increased red cell membrane rigidity. Blood 71:427-1431, 1988. 13. Sheetz MP. Membrane skeletal dynamics: Role in modulation of red cell deformability, mobility of transmembrane proteins, and shape. Semin Hernatol20:175-188, 1983. 14. Bratteyby LE, Garby L, Groth TC, Schneider W, Wadman M. Studies on erythrokinetics in infancy. XIII. The mean life span and the life span frequency of red blood cells formed during fetal life. Actu Pediatr Stand 57:311-320, 1968. 15. Dodge JT, Mitchell C, Hanahan DI. The preparation and chemical characteristics of hemoglobinfree ghosts of erythrocytes. Arch Biochem 100:119-130, 1963. 16. Laemmli UK. Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nafure

117:680-685, 1970.

17. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurements with the Folin phenol reagent. J Biol Chem 193~265-275, 1951. 18. Jain SK, Hochstein P. Polymerisation of membrane components in aging red blood cells. Biochem Biophys Res Commun 92:247-254, 1980. 19. Low PS, Waugh SM, Zinke K, Drenchkhahn D. The role of hemoglobin denaturation and band 3 clustering in red blood cell aging. Science 227:531-533, 1985. 20. Lane PA, Galili U, Iarocci TA, Shew LR, Mentzer WC. Cellular dehydration and immunoglobulin binding in senescent neonatal erythrocytes. Pediutr Res 23(3):288-292, 1988. 21. Jain SK. The neonatal erythrocyte and its oxidative susceptibility. Semin Hematof X(4):286-300, 1989. 22. Jain SK. Membrane lipid peroxidation in erythrocytes of the newborn. Clin Chim Actu 161:301306, 1986. 23. Jain SK. Evidence for the membrane lipid peroxidation during the in vivo aging of human erythrocytes. Biochim Biophys Actu 937~205-210, 1988. 24. Jain SK. Presence of phosphatidylserine in the outer membrane bilayer of newborn human erythrocytes. Biochem Biophys Res Commun w6:914-920, 1986.

Membrane-bound hemoglobin as a marker of oxidative injury in adult and neonatal red blood cells.

A comparative study of the effect of hydrogen peroxide on adult and neonatal red blood cell (RBC) membrane protein composition has been carried out. T...
984KB Sizes 0 Downloads 0 Views