Brtish Journal of Industrial Medicine 1982;39:295-299

Effect of lead in vitro on water metabolism andosmotic fragility of human erythrocytesI KARAI,' K FUKUMOTO,2 K KAGEYAMA,3 AND S HORIGUCHI'

From the Department of Preventive Medicine and Public Health, Osaka City University Medical School,Abeno-ku, Osaka Prefectural Institute ofOccupational Health,2 Higashi-ku, and Department ofPhysiology, 3Osaka City University Medical School, Abeno-ku, Osaka, 545 Japan

ABSTRACr The addition of lead to normal human blood was previously found to cause a decreasein erythrocyte osmotic fragility in vitro. The mechanism of the decreased osmotic fragility causedby lead has not been completely clarified, but the following hypothesis has been proposed. Leadcauses a leakage of water from erythrocytes, thus more water can enter the cell before haemolysisoccurs. There has been no report, however, of the direct measurement of the intracellular watercontent of erythrocytes treated with lead. This study has tried to clarify the relation betweenintracellular water and the osmotic fragility of lead-treated erythrocytes in vitro. The resultsshowed that 0.05 ,uM/ml of lead decreased the osmotic fragility, the intracellular water content,and intracellular potassium and mean corpuscular volume, increased the plasma water contentand trapped water content, and contracted the erythrocyte shape. These changes correspondedwell with each other, and close coincidence of the osmotic fragility and the intracellular watercontent was also observed.

In previous papers'2 we observed a decreased eryth-rocyte osmotic fragility in workers occupationallyexposed to lead compared with controls and found adose-response relationship between the blood leadconcentrations and the osmotic fragility in leadworkers using the coil planet centrifuge (CPC)method.3 We also observed, by an in vitro experi-ment,4 that the addition of lead to normal humanblood caused a decrease in the osmotic fragilityusing the CPC method.The exact mechanism of the decreased osmotic

fragility is not completely understood, but the fol-lowing hypothesis has been generally accepted. Theaddition of lead to blood causes a leakage of potas-sium accompanying water from the erythrocytes,which means that more water can enter the cellbefore haemolysis occurs.5 There has been noreport, however, of the direct measurement of theintracellular water content of erythrocytes treatedwith lead. We have tried to clarify the relation bet-ween the intracellular water content and the osmoticfragility of erythrocytes treated with lead in vitro

Received 27 April 1981Accepted 16 October 1981

and have studied the effects of lead on intracellularpotassium and sodium, mean corpuscular volume(MCV), plasma water, trapped water, intracellularwater contents, osmotic fragility, and erythrocyteappearance.

Methods

The experiment was performed in quadruplicate inthis study. Water content was measured byKageyama's method.6 One millilitre of heparinisednormal human blood was incubated for two hours at37°C in an atmosphere of 5% CO2 and 95% air with10 ,ul of lead acetate solution (lead concentrations inblood; 0, 10, 50, 100, 500, 1000 nMJml) and 10 Alof physiological saline containing 2 ,Ci 3H-sucrose(Radiochemical Centre, Amsterdam) and 0.1 mgsucrose was added; this mixture was centrifuged for30 minutes at 3000 rpm. The haematocrit value wasread, and then 50 ,ul of the supernatant transferredwith a micro syringe into a sample bottle coveredwith a rubber cap containing 1-95 ml of methanoland 4.1% of n-buthanol as an internal standard withtwo glass beads to enhance the stirring efficiency.The sample bottle was shaken well and centrifugedfor 10 minutes at 3000 rpm.

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The supernatant and buffy coat were completelyremoved from the haematocrit. The remainingpacked erythrocytes were mixed well with a capil-lary pipette, and 50 ,ul of the erythrocytes wasinjected quickly into the sample bottle with a mic-rolitre syringe (Precision Sampling Co, USA). Thesample in the bottle was shaken vigorously and cen-trifuged at 3000 rpm for 10 minutes. Next, 2 ,ul ofthe supernatant was taken from each bottle contain-ing plasma or packed erythrocytes and analysed forwater content by gas chromatography (Shimadzu,thermal conductivity detector Model GC-4BPTE,Japan). The water content of the plasma and packederythrocytes was estimated by the method describedabove and the calibration curve for water wasdetermined using standard samples with knownwater contents.

Trapped water content-The radioactivity of 50 ,ulof the methanol supernatant was measured with aliquid scintillation spectrometer (Packerd, Model3330, USA), and the trapped water content wasestimated as: plasma radioactivity . packed eryth-rocyte radioactivity x plasma water content.

Intracellular water content was calculated by sub-tracting the trapped water content from the packederythrocyte water content.Osmotic fragility of erythrocytes was determined

by CPC with a coil planet centrifuge, a coiled sampletube with a range of 150-30 mOsmol, a coilincubator, and a scanning photodensitometer (SankiEngineering, Japan). Three haemolytic points of thehaemoglobin distribution pattern in the coiled tubewere determined (HSP, haemolysis of the startingpoint: HMP, haemolysis of the maximum point:HEP, haemolysis of the end point (fig 1).

Erythrocyte count-An automatic blood cellcounter (Coulter Electronics, Coulter CounterModel ZF, USA) was used.Mean corpuscular volume was calculated by divid-

ing the haematocrit value by the erythrocyte count.Intracellular potassium and sodium-A 20- ,pl

sample of blood was washed well with 0*1 M MgCl2and centrifuged. After decantation the blood washaemolysed with 4 ml of deionised water and cen-trifuged. Sodium and potassium in the supematantwere determined by flame photometry (Hitachi,Model 170-50A, Japan), and these values weredivided by the haematocrit value.

Erythrocyte shape was observed by scanning elec-tron microscopy. The erythrocytes were fixed with0 1M phosphate buffer solution containing 1%glutaldehyde and with 0-5% osmic acid solution.The sample was then dehydrated with alcohol, driedat critical temperature, placed on the sample stage,ion-coated, and observed with a scanning electronmicroscope (JEOL, JSM-50A, Japan).

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Fig 1 Haemolysis ofnormal human blood samples incoiled tube determined by CPC after treatment with lead invitro at 37°C for two hours. Three haemolytic points ofhaemoglobin distribution pattern were recorded by scanningphotodensitometer (haemolysis ofstarting point, HSP;haemolysis ofmaximum point, HMP; haemolysis ofendpoint, HEP). Greatly decreased osmotc fragility in500 nM/ml are clear.

Results

Figure 1 shows the haemolysis of normal humanblood samples in the coiled tube determined by CPCafter treatment with lead at 37°C for two hours: fig 2shows the change of three points of haemolysis. Theosmotic fragility at all three points of haemolysisdecreased with 50 nM/ml of lead; it was greatlydecreased with 500 nM/ml of lead.The intracellular water content decreased consid-

erably with 50 nM/ml of lead, then graduallydecreased with increased blood lead concentration(fig 3). Plasma water content, however, graduallyincreased with increasing blood lead concentration(fig 4). The trapped water content increased with50 nM/ml of lead and increased greatly with500 nM/ml of lead (fig 5).The changes in intracellular sodium and potas-

sium with lead treatment are shown in fig 6. Intracel-lular potassium decreased with 50 nM/ml of leadand decreased greatly with 500 nM/ml of lead.

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Effect of lead in vitro on water metabolism and osmotic fragility ofhuman erythrocytes

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Fig 2 Change in osmotic fragility oferythrocytes. Normalhuman blood was incubated for two hours at 370C withlead.

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Fig 3 Change in intracellular water content. Normalhuman blood was incubated for two hours at 370C withlead.

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Fig 5 Change in trapped water content. Normal humanbiood was incubated for two hours at 37°C with lead.

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Fig 6 Change in intracellular sodium and postassium.Normal human blood was incubated for two hours at 37°Cwith lead.

more with 1000 nM/ml of lead, and most of theerythrocytes were contracted and looked like flatdiscs without concaveness. The presence of echino-cytes was observed.

0 10 50 100 500 1000Lead concentration (nM/ml)

Fig 4 Change in plasma water content. Normal humanblood was incubated for two hours at 37°C with lead.

Intracellular sodium did not change even with1000 nM/ml of lead.Figure 7 shows that MCV was decreased greatly

with 50 nMIml of lead, then decreased graduallywith increasing blood lead concentration.

Figure 8 shows the morphological changes inerythrocytes observed with scanning electron micro-graphs. Erythrocytes were slightly flattened withlead compared with the controls but retained a dis-cocyte appearance. Erythrocytes were flattened

Discussion

Changes in osmotic and mechanical fragilities oferythrocytes have been observed in vitro and invivo, and these changes showed alterations in themorphology and permeability of the erythrocytemembrane."' The mechanism for the decrease inosmotic fragility has not been completely clarified,however. Griggs5 proposed that lead disturbs theactive transport of potassium leading to its leakageand an accompanying water loss from the erythro-cytes and then a decrease in MCV. These changesindicate that more water can enter the erythrocytebefore the critical spherical form and lysis occur.Measurement of the intracellular water content

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and the water metabolism of lead-treated erythro-cytes have not previously been reported. In thisstudy we found that the addition of lead to normalhuman blood causes a disturbance of the watermetabolism, and we also found close relationshipsbetween the change in osmotic fragility and those inintracellular water content, intracellular potassium,MCV, trapped water content, and erythrocyteshape. Therefore, our results seem to agree wellwith Grigg's hypothesis.5The contracted shape of erythrocytes observed by

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Karai, Fukumoto, Kageyama, and Horiguchi

scanning electron microscope indicates a decrease inerythrocyte flexibility, which leads to increases inintercellular space and trapped water content ofpacked erythrocytes. We assume that a pronouncedincrease in trapped water content with 500 and1000 nM/ml of lead is related to the formation ofechinocytes with crenation of cell membrane surfaceand generation of spicules caused by a pronounceddecrease in intracellular potassium accompanyingthe water loss from the erythrocytes.The MCV value increased a little with 1000 nM/

ml of lead, and was not correlated with the results ofintracellular water and potassium, trapped watercontent, and osmotic fragility. The MCV value con-sists of cell volume and intracellular space. There-fore, we assume that the increased MCV with1000 nMIml of lead is due to the considerablyincreased intercellular space and trapped water con-tent.

Lcso 100

Lead concentration(nM/ml)

We thank Dr S Uni for his kind advice in operatingthe electron microscope.

This work was supported in part with Grant-in-Aid for Encouragement of Young Scientist (1981)from the Ministry of Education, Science, andCulture of Japan.

Fig 8 Scanning electron micrographsoferythrocytes. Normal human bloodwas incubated for two hours at 37°Cwith lead. (a) control, (b) 50 nM/ml oflead, (c) 100 nM/ml oflead,(d) 500 nM/ml oflead,(e) 000 nM/ml oflead.

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0 10

Fig 7 Change in mean corpuscular volume oferythrocytes.Normal human blood was incubated for two hours at 37°Cwith lead.

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Effect oflead in vitro on water metabolism and osmotic fragility ofhuman erythrocytes

References

Horiguchi S, Teramato K, Nakano H, Shinagawa K, Endo G,Kiyota I. Osmotic fragility test of red blood cells of leadworkers by coil planet centrifuge. Osaki City Med J1 974;20: 51-3.

2 Karai I, Fukumoto K, Horiguchi S. Studies on osmotic fragility ofred blood cells determined with a coil planet centrifuge forworkers occupationally exposed to lead. Int Arch Occup Envi-ron Health 1981;48:273-81.

Ito Y, Weinstein MA, Aoki I, Harada R, Kimura E, Nunogaki K.The coil planet centrifuge. Nature 1966;212:985-7.

4Karai I, Fukumoto K, Horiguchi S. The effects of heavy metal

ions on osmotic resistance of human red blood cells. OsakaCity Med J 1979;25:153-7.

Griggs RC. Lead poisoning: hematologic aspects. Prog Hematol1964;4:1 17-37.

6 Kageyama K. Effect of incubation on red cell shape and watercontent as measured by gas-liquid chromatography. Nouv RevFr Hftmatol 1979;21161-9.

Joyce CRB, Moore H, Weatherall M. The effect of lead, mercuryand gold on the potassium turnover of rabbit blood cells. BrJ Pharmacol 1954;9:463-70.

'Vincent PC, Blackburn CRB. Effect of heavy metal ions on thehuman erythrocyte. 1 Comparison of the action of severalheavy metals. Aust J Exp Biol Med Sci 1958;36:471-80.

The May 1982 issueTHE MAY 1982 ISSUE CONTAINS THE FOLLOWING PAPERS

Health implications of exposure toradiofrequency/microwave energies S M MICHAEL-SON

Coalworkers' simple pneumoconiosis and exposureto dust at 10 British coalmines J F HURLEY, JBURNS, LIZ COPLAND, J DODGSON, AND M JACOBSEN

Tetrachlorodibenzodioxin: a survey of subjects tenyears after exposure G MAYElemental mercury exposure: peripheral neurotox-icity S P LEVINE, G D CAVENDER, G D LANGOLF, ANDJ W ALBERS

Actual hazard of methyl bromide fumigation in soildisinfection R VAN DEN OEVER, D ROOSELS, AND DLAHAYE

Epilepsy in the British Steel Corporation: an evalua-tion of sickness, accident, and work recordsA K DASGUPTA, M SAUNDERS, AND D J DICK

Absences attributed to respiratory diseases in wel-ders R F FAWER, A WARD GARDNER, AND D OAKES

Mechanism of increased osmotic resistance of redcells in workers exposed to lead I KARAI, KFUKUMOTO, AND S HORIGUCHI

Assessment of the body burden of chelatable lead: amodel and its application to lead workers S ARAKIAND K USHIO

Value of the simultaneous determination of Pco2 inmonitoring exposure to 1,1,1 -trichloroethane bybreath analysis M GUILLEMIN AND E GUBERANAngiographic study of digital arteries in workersexposed to vinyl chloride P FALAPPA, N MAG-NAVITA, A BERGAMASCHI, AND N COLAVITA

Urinary excretion of hydroxyproline in workersoccupationally exposed to vibration T KASAMATSU,K MIYASHITA, S SHIOMI, AND H IWATA

Adrenocortical suppression in workers employed inmanufacturing synthetic glucocorticosteroids: solu-tions to a problem R W NEWTON, MARGARET C KBROWNING, P C NICHOLSON, AND D A E MOWAT

Lack of allergic reactions in workers exposed to Pru-teen (bacterial single-cell protein) R W MAYESTLC separation of hippuric, mandelic, and phenyl-glyoxylic acids from urine after mixed exposure totoluene and styrene GRAZYNA BIENIEK, EWAPALYS, AND T WILCZOK

Analysis of fibres in human lung tissue B GYLSETH,R H BAUNAN, AND L OVERAAE

Cancer morbidity among polishers B JARVHOLM, GTHIRINGER, AND 0 AXELSON

Notes and miscellaneaInteraction between drugs and solvents as a cause offatty change in the liver? C EDLINGHanford radiation study R H MOLEBook reviewsInformation sectionCorrections:Healthy worker effect in the total Finnish popula-tion (May 1980, p. 180-184)A study of the mortality of Cornish tin miners(November 1981, p. 378-380)Relationship between type of simple coalworkers'pneumoconiosis and lung function. (November1981, p. 313-320)

Copies are still available and may be obtained from the PUBLISHING MANAGER, BRITISHMEDICAL ASSOCIATION, TAVISTOCK SQUARE, LONDON WC1H 9JR, price £425(USA $9-20), including postage

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