effects of lead on electrophoretic mobility,

Industrial Health, 1993, 31, 113-126113

Effects of Lead on Electrophoretic Mobility,

Membrane Sialic Acid. Deformabilitv

and Survival of Rat Erythrocytes

Kazuyuki TERAYAMA

Department of Hygiene, Asahikawa Medical College, 3-11, 4-5 Nishikagura, Asahikawa, Hokkaido 078, Japan

(Received May 10, 1993 and in revised form June 28, 1993)

Abstract : The anemia frequently observed in lead poisoning is thought to result from the

shortening of erythrocyte life span in combination with inhibition of hemoglobin synthesis.

However, the exact mechanism by which lead shortens the life span of red blood cells

(RBCs) remains unclear. In the present study, the effects of injected lead on electrophoretic

mobility, membrane sialic acid content, deformability and survival of rat RBCs were

investigated in order to clarify the relationships between them. As indices of lead exposure,

RBC counts, hemoglobin (Hb) levels, hematocrits (Ht), mean corpuscular volume (MCV),

mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC)

and blood lead (blood Pb) levels in the rats were also examined. Exposure to lead

significantly decreased RBC counts, Hb levels, Ht, MCV and MCH. Similarly, exposure to

lead significantly decreased the mobility, sialic acid content and deformability of rat RBCs. A shortening of erythrocyte survival time was also observed in the rats exposed to lead. It

is speculated that decreases in membrane sialic acid content and deformability of RBCs

induce a shortening of erythrocyte survival time in anemia caused by lead.

Key words: Lead-Anemia-Erythrocyte-Erythrocyte membrane-Erythrocyte survival-

Electrophoretic mobility-—Sialic acid-Erythrocyte deformability

INTRODUCTION

Lead poisoning has been recognized as a clinical entity since ancient times° The hematological effects of lead consist of two types : firstly, interference with heme and hemoglobin synthesis, and secondly, effects on erythrocyte morphology and survival2). Lead exposure may give rise to microcytic anemia, shown by biochemical methods to be caused by a combination of inhibition of hemoglobin synthesis and shortened life span of circulating red blood cells (RBCs)3). It has been reported that lead exposure often induces a shortening of erythrocyte survival time4-9). According to numerous studies, lead affects several steps in the

pathway of hemoglobin synthesis1-4). On the other hand, the exact mechanism by which lead shortens the erythrocyte survival time remains unclear.

114 K. TERAYAMA, et aL

The electrophoretic mobility of RBCs is determined by the negative surface charge density of the cells10), and the carboxyl groups of sialic acid in the membrane are mainly responsible for the surface charge11). Some investigators have reported that old red cells showed decreases in mobility 12-14) and in mem-brane sialic acid content14-18) compared with young cells. Furthermore, erythro-cyte deformability is important in the removal and subsequent destruction of abnormal or senescent RBCs from the circulation19,20). However, the effects of lead on mobility, membrane sialic acid content and deformability of RBCs in relation to the erythrocyte survival are not yet clearly understood.

Previously, the author reported that exposure to lead significantly decreased the mobility21) and the membrane sialic acid content22) of rat RBCs. In the present

study, the effects of lead, injected intraperitoneally, on electrophoretic mobility, membrane sialic acid content, deformability and survival of rat RBCs were investigated in order to clarify the relationships between them. Futhermore, the

possible mechanism by which lead shortens erythrocyte survival time was dis-cussed.

MATERIALS AND METHODS

Animal treatment and sample collection

One hundred and thirty-eight rats were used for three series of experiments to

accomplish this study. Sixty rats were used to measure red blood cell (RBC)

count, hemoglobin (Hb) level, hematocrit (Ht), mean corpuscular volume

(MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin

concentration (MCHC), erythrocyte deformability and blood lead (Pb) in Expt.

1. Another 60 rats were used to measure electrophoretic mobility, membrane

sialic acid content of RBCs and blood Pb in Expt. 2. The remaining 18 rats

were used for erythrocyte survival studies in Expt. 3.

Six-weeks old rats were housed in groups of 3 or 4 in a room maintained at

a temperature of 24 -•}1 •Ž and a relative humidity of 55 •} 5% before use in the

experiment. They were fed a diet of pellets (CE-2, Clea Japan, Japan) and tap

water ad libitum throughout the experiment. The administration of lead was

started when the rats were 11 weeks old. Twenty mM lead acetate solution was

injected intraperitoneally once a week for 5 weeks, with a volume of 1 m1/100 g

of body weight ; specifically, the rats were administered with 200 iumol/kg of lead

at day zero, and the 7th, 14th, 21st and 28th days. The solution was freshly

prepared with distilled, deionized boiled water for each injection stage. The

control rats were injected with 1 m1/100 g of 145 mM NaCl solution. All rats

were injected between 10 and 11 am. About 24 h after each injecion, the rats

were anesthetized by i.p. injections of 50 mg/kg of sodium pentobarbital. Blood

samples were obtained by cardiac puncture using disposable syringes containing 1

ml of acid-citrate-dextrose solution as anticoagulant. Five ml of each blood

EFFECTS OF LEAD ON RBC MEMBRANE AND SURVIVAL 115

sample was collected in a lead-free glass tube and placed in an iced water bath.

Erythrocyte deformability Six exposed and 6 control rats were used for the measurement of erythrocyte

deformability at each injection stage. One ml of blood from each rat was used

for the determinations of RBC count, Hb level, Ht, MCV, MCH and MCHC

using an automatic analyser (Model S plus 4, Coulter Electronics, USA). Fifty

,a1 of blood from each individual were frozen at - 20•Ž for subsequent blood Pb

measurement.

The remaining blood was centrifuged at 1500 g for 10 min to remove the

plasma and buffy coat. The RBCs were washed 3 times in a 12 mM Tris-Ringer

buffer (pH 7.4) containing 0.25% bovine serum albumin (Sigma, USA), and then

were diluted to a 2% suspension for the determination of erythrocyte deforma-

bility. The deformabiliby was measured according to Durocher et al.23) by the

filtration properties of the RBC suspension as determined by the time (filtration

time) required for 2 ml of the 2% RBC suspension to filter through a 3-ƒÊm

polycarbonate filter (Nuclepore, USA) under 10 cm water negative pressure at

room temperature. All samples were coded and filtered in triplicate.

Electrophoretic mobility and sialic acid content

Another 12 rats (Exposed group, n = 6 ; Control group, n = 6) were used for

the determination of electrophoretic mobility and membrane sialic acid content of

RBCs at each injection stage. Immediately after cardiac puncture blood collec-

tion, 50 ƒÊl of each blood sample was frozen at - 20•Ž for blood Pb measurement

and the remaining blood was centrifuged at 1500 g for 10 min. The plasma and

buffy coat were removed and 50,ƒÊlo of the RBCs were washed 3 times in 10 ml of

cold isotonic buffer, made up of 145 mM NaCl and 0.3 mM NaHCO3 (approx.

pH 7.2)24). The washed cells were suspended in 100 ,u1 of the buffer, kept at 4 •Ž

and used for electrophoretic measurement within 24 h after collection. The

electrophoretic mobility was measured according to the method of a previous

paper21). The remaining RBCs were washed 3 times in 40 ml of phosphate-

buffered saline (PBS) (pH 7.2), made up of 144 mM NaCl and 10 mM phosphate

buffer18). The washed RBCs obtained as packed cells were immediately used for

the determination of the membrane sialic acid content of rat RBCs according to

the method described previously22).

Erythrocyte survival

Eighteen rats (3rd-injection group, n = 6 ; 5th-injection group, n = 6 ; Control

group, n = 6) were used in erythrocyte survival studies. About 24 h after the 3rd

or 5th lead-injection, 150 ,ƒÊl of blood was collected from the rat tail vein by tip

incision without anesthetic21), and 50 ,ƒÊl of each was frozen at - 20 •Ž for

measurement of blood Pb. The remaining 100 ,ƒÊl of blood was immediately

116 K. TERAYAMA, et al.

incubated with 100 iil of 370 kBq/ml of Na251Cr04 (New England Nuclear, USA)

in 0.15 M NaCl solution for 30 min at room temperature with frequent mixing.

The 51Cr-labeled RBCs were separated from the unreacted isotope by 4 washes

with PBS and were then resuspended in 100 ,ƒÊl of PBS. After the rats were

anesthetized, each 51Cr-labeled RBC suspension was autologously transfused thr-

ough the rat femoral vein. One hundred kil of blood was withdrawn 30 min after

the transfusion as the sample for 0 time ; 20 ƒÊl was used for Hb measurement

and the remaining 80 ƒÊ1 was frozen at - 20 •Ž for the determination of radioac-

tivity. In order to monitor the survival of labeled RBCs, animals were bled

every other day until the radioactivity of the samples dropped to below 50 % of

the counts per minute (c.p.m. ) at 0 time. The Hb level of each blood sample

was measured, and specific activity was obtained as c.p.m./gHb after correction

for decay of the radioisotope. Gamma counting was done in an automatic

gamma counter (1280 Ultro Gamma, LKB-Wallac, Sweden).

The blood Pb was measured by using the method of a previous paper21).

Statistics

Student's t-test or linear regression analysis were used for most studies. The data of erythrocyte survival studies were subjected to analysis of variance

(ANOVA) and multiple range test (Tukey's method)25).

RESULTS

Fig. 1 shows the changes in mean RBC counts, Hb levels and Ht of lead-

exposed rats. Exposure to lead significantly decreased the RBC counts, Hb and

Ht after the 2nd injection (p < 0.05 or p < 0.01), and decreases were clearly

observed after the 3rd-5th injections.

The MCV and MCH in the lead-exposed rats significantly decreased after the

3rd injection (p < 0.01), whereas injected lead had no effect on the mean MCHC

(Fig. 2).

Fig. 3 shows the effects of lead on erythrocyte deformability and blood Pb.

Freshly collected normal RBCs had a filtration time of 8.2 •} 1.8 sec (Mean ± SD)

in our experimental system. The RBCs of lead-exposed rats showed a signific-

antly prolonged filtration time after the 2nd-4th injections (p < 0.05 or p < 0.01),

although the filtration time was restored to almost the original level after the 5th

injection. The mean blood Pb level of lead-exposed rats showed a marked

increase in proportion to the amount of injected lead.

Fig. 4 shows the changes in the electrophoretic mobility, the membrane sialic

acid content of RBCs and the blood Pb in the lead-exposed rats. Lead exposure

significantly decreased the RBC mobility after all the injection stages compared

with the control (p < 0.05 or p < 0.01). The change in the sialic acid content of

lead-exposed group was very similar to that in mobility, although the significant


Fig. 1. Changes in mean RBC count, Hb level and Ht of lead-exposed

rats (•œ).

The rats were injected intraperitoneally with 200 gmol/kg of lead at day

zero, and at 7th, 14th, 21st and 28th days. The control rats (•›) were

injected with 1 ml/100 g of 145 mM NaC1 solution in the same manner.

Each point is the mean of 6 animals. Vertical bars represent SD. *

and * * indicate significant differences from control at p< 0.05 and p<

0.01, respectively.


Fig. 2. Changes in MCV, MCH and MCHC of lead-exposed rats (•œ).•

See legends for Fig. 1.


decreases were observed after the 2nd injection (p< 0.01). The change of blood

Pb level was almost the same as the result indicated in Fig. 3.

Fig. 5 shows the relationship between the electrophoretic mobility of rat RBCs

and the blood Pb level. The RBC mobilities in control rats (•›) showed an

almost constant value (2.51•}0.02 gm/sec/V/cm) corresponding to blood Pb

levels of 1-9 ƒÊg/100 ml. In the lead-exposed rats (•œ), decreases in mobility were

evident to some extent below a blood Pb level of 100 ƒÊg/100 ml and generally

present at a level of 100 ,ag/100 ml and higher. A significant negative correlation

between the mobilities in the exposed rats and the logarithms of blood Pb level

was also found (p < 0.01).

Fig. 6 shows the relationship between the membrane sialic acid content of rat

RBCs and the blood Pb level. The sialic acid contents in control rats (0)

showed 134•}5,ƒÊg/ml RBC corresponding to blood Pb levels of 1-9,ƒÊg/100 ml.

Similar to the manner of electrophoretic mobility, decreases in the sialic acid

content of injected rats (•œ) were evident to some extent below a blood Pb level

Fie. 3. Changes in mean filtration time and blood Pb level of lead-

exposed rats (•œ).



Fig. 4. Changes in mean electrophoretic mobility, membrane sialic acid

content of erythrocytes and blood Pb level of lead-exposed rats (•œ)



Fig. 5. Relationship between the electrophoretic mobility of rat eryth-

rocytes and the blood Pb level.

The regression line of mobility on logarithms of blood Pb of lead-

exposed rats (•œ) is shown as a solid line. The mean electrophoretic

mobility of control rats (•›) is also indicated as a dotted line.

Fig. 6. Relationship between the membrane sialic acid content of rat

erythrocytes and the blood Pb level.

The regression line of sialic acid content on logarithms of blood Pb of

lead-exposed rats (•œ) is shown as a solid line.

The mean sialic acid content of control rats (•›) is also indicated as a

dotted line.

122 K. TERAYAMA, et aL

Fig. 7. Relationship between the electrophoretic mobility and mem-

brane sialic acid content of erythrocytes in exposed rats.

The regression line of electrophoretic mobility on sialic acid content of

erythrocytes in lead-exposed rats (•œ) is also indicated as a solid line.

Fig. 8. Disappearance from the circulation of 51Cr-labeled erythrocytes

in rats of control (•›), 3rd-injection (•œ) and 5th-injection (•£) groups .

Each point indicates as mean-•} SD.


of 100 ,ƒÊg/100 ml and generally present at a level of 100 ƒÊg/100 ml and higher.

A significant negative correlation between the sialic acid content in the exposed

rats and ' the logarithms of the blood Pb level was also observed (p < 0.01).

Fig. 7 shows the relationship between the electrophoretic mobility and mem-

brane sialic acid content of RBCs in exposed rats. The RBC mobility positively

correlated to the sialic acid content of erythrocytes (p < 0.01).

Fig. 8 shows the erythrocyte survival curves in the rats. The reduced half-life

times (T1/2) of 51Cr-labeled RBCs in the rats both of 3rd-injection (•œ) and 5th

-injection (•£) groups were observed compared with that of the control (•›).

The values of mean •} SD were calculated from the T112 of each animal as

follows : 3rd-injection group, 8.8•}0.4 days ; 5th-injection group, 10.2•}0.4 days ;

Control group, 14.0 •} 0.7 days. There was a significant difference in the half-life

times of these groups (F = 154>>F 215 (0.01) = 6.36). From the multiple range

test, significant differences were found in the T112 between all the pairs of two

groups (p < 0.01). The blood Pb levels in the control, 3rd- and 5th-injection

groups are 2.4 •} 1.3 ,ƒÊg/100 ml, 276•}97 ,ƒÊg/100 ml and 698 •} 280 ,ƒÊg/100 ml, re-

spectively.

DISCUSSION

In the present study, injected lead significantly decreased RBC counts, Hb level, Ht, MCV and MCH in the rat, whereas MCHC was not changed. This fact shows that microcytic, hypochromic anemia was induced by the dosage condition used in this study.

This study also revealed that both 3 and 5 doses of 200 ,umol/kg of lead injected 7 days apart, clearly shortened the erythrocyte life span of rats. Expo-sure to lead often induces a shortening of the survival time of circulating

erythrocytes4-9). The anemia frequently observed in lead poisoning is thought to result from this shortening of erythrocyte survival time in combination with the inhibition of hemoglobin synthesis1-5). It is known that lead exerts influences on erythrocyte membrane ; e.g. changes in the osmotic and mechanical fragility of

erythrocytes1,4), enhancement of potassium loss from the cells26), reduction of the membrane Na+/K+-ATPase activity27), decreases in the membrane fluidity28), and changes in the composition of erythrocyte membrane lipids29) or proteins30). This author tried to elucidate the relationship between the shortening of erythrocyte survival time and our previous observations ; exposed lead decreased the

mobility21) and the membrane sialic acid content22) of rat erythrocytes. The electrophoretic mobility of RBCs is determined by the negative surface

charge density of the RBCs 10), and the carboxyl groups of sialic acid in the membrane are mainly reponsible for this surface chargen). A significant positive correlation between the electrophoretic mobility and the membrane sialic acid content of the erythrocytes in the exposed rats was found in this study (Fig. 7).


Accordingly, it may be appropriate to concentrate on the effect of lead exposure

on sialic acid content of RBCs in relation to erythrocyte survival.

As shown in Fig. 4, the change in the sialic acid content of RBCs in

lead-exposed rats was very similar to that in cell mobility, although a significant

decrease was not observed after the 1st injection. These decreases were evident

to a degree below a blood Pb level of 100 ,ƒÊg/ ml and generally present at a

level of 100 ƒÊg/100 ml or higher, along with the decreases in cell mobility (Fig.

5 and 6). Some investigators have reported that old RBCs showed decreases in

mobility12-14) and in membrane sialic acid content14-18)compared with young cells. Moreover, desialylated RBCs (treated with sialidase) showed a markedly short-ened life Spa1131-34) as well as decreased mobility34'35). Therefore, it is highly conceivable that a decrease in sialic acid and a resulting decrease of mobility may relate to a shortening of erythrocyte survival time induced by lead exposure.

The changes in the sialic acid content in the present study were relatively small ; i.e. decreases of 10-20 % were the largest changes observed. Baxter and

Beeley16) found that the oldest and most dense cell fractions consistently showed a 15 % lower sialic acid content than that of the cells at the top of the gradient. Aminoff et al.33) indicated that the loss of viability of sialidase-treated dog RBCs could be elicited by the removal of 12 % of the total sialic acid. Furthermore, it has been reported that the desialylated RBCs were sequestered in the liver and

spleen35-37). Therefore, a relatively small decrease in sialic acid content of RBCs observed in lead poisoning may induce a clearance in the liver and spleen. However, it is unlikely that injected lead per se cleave the glycosidic linkage of sialic acid in glycoproteins of erythrocyte membrane. Possibly, the decrease in sialic acid content of erythrocytes in lead-exposed rats may be related to a self-digestion process which can release sialic acid ; e.g. liberation of N-acetylneuraminic acid by the action of sialidase38), release of sialoglycopeptide39'4°) by proteinase linked to the membrane, and so on. As a result of this self-diges-tion process accelerated by injected lead, the decrease in sialic acid content of rat erythrocytes may be observed.

Deformability of RBCs is required for their normal passage through the capillaries of the microcirculation and the splenic sinusoids20). In the present study, the RBCs of lead-exposed rats showed a significantly prolonged filtration time after the 2nd-4th injections. This result shows that lead exposure decreased the deformability of rat RBCs. However, the filtration time was restored to almost the original level after the 5th injection. This restoration of erythrocyte deformability may be a certain adaptation to lead exposure. The erythrocyte survival studies showed the T172 of 51Cr-labeled RBCs in the rats of the 5th -injection group was significantly longer than that of the 3rd-injection group . This result may be concerned with the restoration of erythrocyte deformability after the 5th injection.

The author speculates that a decrease in membrane sialic acid content of RBCs


mainly induces a shortening of erythrocyte survival time in lead poisoning.

Possibly, a decrease in erythrocyte deformability may further promote a clearance

of leaded red cell with sequestration by the reticuloendothelial system in the liver

and spleen.

ACKNOWLEDGMENTS

The author is greatly indebted to Prof. K. Yamamura for his encouragement and continuous guidance throughout this study. I am also grateful to Dr. M. Muratsugu for much discussion. I sincerely wish to thank Mr. M. Kashima, Mr. T. Nagahara and Mr. K. Taguchi (Laboratory for Radio-Active Isotope Re-search), Mr. K. Nakaya (Animal Experiment Center, Asahikawa Medical Col-lege) and Mr. H. Akutsu (Central Laboratory for Research and Educaion, Asahikawa Medical College) for their technical helps. The author is also obliged to Mr. Simon Bayley for correcting the English in the manuscript.

REFERENCES

1) Waldron HA. The anemia of lead poisoning : A review. Br J Ind Med 1966; 23: 83-100.

2) Ratcliffe RC. Lead in man and the environment. Chichester : Ellis Horwood, 1981; 32-50.

3) Task Group on Metal Toxicity. A consensus report from an international meeting organized bythe Subcommittee on the Toxicology of Metals of the Permanent Commission and International

Association on Occupational Health, Tokyo, November 18-23, 1974. In: Nordberg (.114, eds.

Effects and dose-response relationships of toxic metals. Amsterdam : Elsevier Scientific Publishing,

1976; 7-111.

4) Griggs RC. Lead poisoning : Hematologic aspects. Prog Hematol 1964; 4 : 117-37.5) Hernberg S, Nurminen M, Hasan J. Nonrandom shortening of red cell survival times in men

exposed to lead. Environ Res 1967; 1: 247-61.

6) Rubino FG, Prato V, Fiorina L. L'anemia da piombo : sua nature e patogenesi. Folia Med

(Napoli) 1959; 42: 1-20.7) Reikin S, Eng G. Erythrokinetic studies of the anemia of lead poisoning. Pediatrics 1963; 31: 996

—1002 .

8) Dagg JH, Goldberg A, Lochhead A, Smith JA. The relationship of lead poisoning to acuteintermittent porphyria. Quart J Med 1965; 34 : 163-75.

9) Berk PD, Tschudy DP, Shepley LA, Waggoner JG, Berlin NI. Hematologic and biochemical

studies in a case of lead poisoning. Am J Med 1970; 48 : 137-44.

10) Abramson HA, Moyer LS. The electrical charge of mammalian red blood cells. J Gen Physiol1936; 19: 601-7.

11) Eylar EH, Madoff MA, Brody OV, Oncley JL. The contribution of sialic acid to the surfacecharge of the erythrocytes. J Biol Chem 1961; 237: 1992-2000.

12) Danon D, Marikovsky Y. Difference de charge êlectrique de surface entre erythrocytes jeunes et ages.. C R Acad Sci Ser D 1961; 253: 1271-2.

13) Yaari A. Mobility of human red blood cells of different age groups in an electric field. Blood 1969 ; 33 : 159-63.

14) Bartosz G, Grzelinska E, Bartkowiak A. Aging of the erythrocyte. X IX. Decrease in surfacecharge density of bovine erythrocytes. Mech Ageing Dev 1984; 24: 1-7.

15) Greenwalt TJ, Steane EA. Quantitative haemagglutination IV. Effect of neuraminidase treatmenton agglutination by blood group antibodies. Brit J Haematol 1973; 25: 207-15.


16) Baxter A, Beeley JD. Changes in surface carbohydrate of human erythrocytes aged in vivo.Biochem Soc Trans 1975; 3 : 134-6.

17) Cohen NS, Ekholm JE, Luthura MG, Hanahan DJ. Biochemical characterization of density-separated human erythrocytes. Biochim Biophys Acta 1976; 419: 229-42.

18) Seaman GVF, Knox RJ, Nordt FJ, Regan DH. Red cell aging. I. Surface charge density andsialic acid content of density-fractionated human erythrocytes. Blood 1977; 50: 1001-11.

19) Weed RI. The importance of erythrocyte deformability. Am J Med 1970; 49: 147-50.

20) Stuart J, Nash GB. Red cell deformability and haematological disorders. Blood Rev 1990; 4:

141-7.

21) Terayama K, Maehara N, Muratsugu M, Makino M, Yamamura K. Effect of lead on elec-

trophoretic mobility of rat erythrocytes. Toxicology 1986; 40: 259-65.

22) Terayama K, Muratsugu M. Effects of lead on sialic acid content and survival of rat erythro-cytes. Toxicology 1988; 53 : 269-76.

23) Durocher JR, Glader BE, Gaines LT, Conrad ME. Effect of cyanate on erythrocyte deforma-bility. Blood 1974; 43 : 277-80.

24) Heard DH, Seaman GVF. The influence of pH and ionic strength on the electrokinetic stabilityof the human erythrocyte membrane. J Gen Physiol 1960; 43: 635-54.

25) Nakamura E, Kimura M. Bioassay and biostatistics. Tokyo : Hirokawa Publishing, 1974; 63-71.26) Hasan J, Hernberg S, Metsala P, Vihko V. Enhanced potassium loss in blood cells from men

exposed to lead. Arch Environ Health 1967; 14: 309-12.

27) Hasan J, Vihko V, Hernberg S. Deficient red cell membrane (Na K )-ATPase in leadpoisoning. Arch Environ Health 1967 ; 14 : 313-8.

28) Valentino M, Fiorini RM, Curatola G, Governa M. Changes of membrane fluidity in erythro-cytes of lead-exposed workers. Int Arch Occup Environ Health 1982; 51: 105-12.

29) Karai I, Fukumoto K, Horiguchi S. Alterations of lipids of the erythrocyte membranes inworkers exposed to lead. Int Arch Occup Environ Health 1982; 50: 11-6.

30) Fukumoto K, Karai I, Horiguchi S. Effect of lead on erythrocyte membranes. Br J Ind Med 1983; 40 : 220-3.

31) Jancik J, Schauer R. Sialic acid-A determinant of the life-time of rabbit erythrocytes.Hoppe-Seyler's Z Phsiol Chem 1974; 355: 395-400.

32) Gattegno L, Bladier D, Cornillot P. The role of sialic acid in the determination of survival ofrabbit erythrocytes in the circulation. Carbohydr Res 1974; 34: 361-9.

33) Aminoff D, Bell WC, Fulton I, Ingebrigtsen N. Effect of sialidase on the viability of erythrocytes

in circulation. Am J Hematol 1976; 1: 419-32.

34) Durocher JR, Payne RC, Conrad ME. Role of sialic acid in erythrocyte survival. Blood 1975;

45 : 11-20.

35) Landaw SA, Tenforde T, Schooley JC. Decreased surface charge and accelerated senescence ofred blood cells following neuraminidase treatment. J Lab Clin Med 1977; 89: 581-91.

36) Gregoriadis G, Putman D, Louis L, Neerunjun D. Comparative effect and fate of non-entrappedand liposome-entrapped neuraminidase injected into rats. Biochem J 1976; 158: 323-30.

37) Aminoff D, Bruegge WFV, Bell WC, Sarpolis K, Williams R. Role of sialic acid in survival of

erythrocytes in the circulation: Interaction of neuraminidase-treated and untreated erythrocytes

with spleen and liver at the cellular level. Proc Natl Acad Sci USA 1977; 74: 1521-4.

38) Bosmann HB. Red cell hydrolases. III. Neuraminidase activity in isolated human erythrocyte

plasma membranes. Vox Sang 1974; 26: 497-512.

39) Brovelli A, Pallavicini G, Sinigaglia F, Balduini CL, Balduini C. Identification of a sialoglyc-opeptide released by self-degestion from human erythrocyte membranes. Biochem J 1976; 158:

497-500.

40) Brovelli A, Suhail M, Pallavicini G, Sinigaglia F, Balduini C. Self-digestion of human erythrocytemembranes. Role of adenosine triphosphate and glutathione. Biochem J 1977; 164: 469-72.

effects of lead on electrophoretic mobility,

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