ELSEVIER Clinica Chimica Acta 249 (1996) 149—165

Alterations of erythrocyte morphology and lipidcomposition by hyperbilirubinemia

Maria A. Britoa, Rui M. Silvaa, Deolinda C. Matosb,António T. da Silvab, Dora T. Brites*a

Cenlro de Metabolismos e Geneflca da Faculdade de Farmáeia da Universidade de Lisboa,A v. das Forças Armadas, 1600 Lisboa, Portugal

Serviço de Pediatria. Hospital Garcia de Orla. Almada, Portugal

Rcceived IS August 1995; revised 16 January 1996; accepted 19 January 1996

Abs(ract

Morphology and membrane lipid composition of erythrovytes from neonates (jaundicedand healthy) and adults (before and after incubation with bilirubin) were sludied. Themorphological index, expressing the reiative proportions of Lhe differcnt stagcs of cclidistortion, and lhe membrane cholesterol, phospholipids and cholcstcrol/phosphoiipidsmolar ratio, were determined. In jaundiced neonates a significant increase in lhe morphoIogicai index (P < 0.01) was found. Afler incubation with bilirubin, aduil erythrocytesalso showed an increase iii Lhe morphoiogical index (P < 0.01). Hcmolysis occurred underthese conditions, and lhe red ccii ghosts obtaincd (vesicles) showed a rounded morphology.1-Iighcr choleslerol/phosphoiipid ratio and lower phospholipid content were found injaundiced neonates compared with heaithy babies (P <0.05) and adults (P < 0.01), as wcIIas in the celis (P <0.05) and vesicles (P <0.01) obtained after bilirubin incubation.Bilirubin cytotoxicity may occur in a stepwise manner: deposition of bilirubin in membraneproduces echinocytosis, which is followed by disintegration of lhe lipid bilayer with Ioss oiphospholipids from lhe membrane.

Keywords: Bilirubin toxicily; Neonates; Erythrocytc morphology; Erythrocyte membraneIipids

Corresponding author

0098-898l/96/$15.00 © 1996 Elsevier Science B.V. Ali rights reservedP11 S0009-8981(96)06285-7

150 M.A. Brigo et ai. / Clínica Chimica Acta 249 (1996) 149—165

1. Introduction

Neonatal unconjugated hyperbilirubinemia, an extremely common occurrence among neonates in the first week of life [1—3], can represent abenign physiologic process or be the harbinger of serious illness withassociated severe neurotoxicity [4]. At high bilirubin (Bb) leveis, Bbbinding to ccii membranes is increased and in the particular case of thecentral nervous system can lead to brain damage [5,6]. Bilirubin encephalopathy ranges from moderate, reversible neurological abnormalities tosevere, irreversible deposition of unconjugated Bb in selected brain regions, generating the ciassical syndrome of kernicterus [4,5,7].

Multisite toxicity towards the metabolism of living cells has beendescribed [5,8—11]. However, the mechanisms of neurotoxicity and thepathways through which Bb reaches the inside of the cdl are still unclear[5,7,12].

Like other celi types, erythrocytes are capable of binding unconjugatedBb to tbeir membranes [6], as demonstrated by severa! studies conductedboth in vivo and in vitro [10,13,14]. Since the amount of Bb bound byerythrocytes may refiect the Bb binding to other cells [6,13], red bloodcelis (RBC) may act as markers of Bb cytotoxicity, and have been widelyused for this purpose.

The normal discoid shape of human RBC can be aitered by druginteraction [15], possibly as a result of intercalation of the drug moleculesinto the lipid bilayer. By way of illustration, cationic drugs producestomatocytic red edIs whereas anionic drugs produce crenated red cells[15—18]. Depending on the concentration of the drug and the duration ofinteraction with erythrocytes, the distorted morphoiogy can be restored tothe norma! biconcave disc shape or can evolve towards hemoiysis,indicating irreversible ceil damage and membrane destruction [15—17].

Since the toxicity of Bb appears to depend on its passage across theplasma membranes [19], the interactions between Bb and biologicalmembranes have been extensiveiy studied [8,13,14,20,21]. The toxic effectof Bb was demonstrated to result from its association with membranelipids [22,23]. In fact, there is evidence that lipids, and not proteins,constitute the Bb binding sites on human erythrocyte membranes[6,23,24] and that those Bb binding sites may be distributed on both outerand inner membrane surfaces [12]. Recent resuits obtained by Zucker etai. [25] point to the choiesteroi/phosphoiipid ratio as the principaldeterminant of Bb transfer rates across membranes, overriding thephysicochemicai effects of the various phosphoiipids species.

Although the in vitro Bb—RBC interaction has been widely studied,reports concerning the in vivo interaction are few and not very recent

M. A. Brita eta!. / Clínica Chimica Acta 249 (1996) 149—165 151

[10,13]. On the other hand, despite the broad information regarding theparticular biochemical characteristics and biophysical properties of normal newborn RBC [26—30], little is known about the erythrocytes inneonatal hyperbilirubinemia.

Our present study was therefore undertaken to evaluate the alterationsof RBC morphology and membrane lipid composition in unconjugatedneonatal hyperbilirubinemia and to establish their relationships with thecorresponding changes observed in adult RBC after in vitro Bb interaction.

2. Materiais and methods

2.1. ChemicaisBilirubin (Bb) and phosphorus standard solution were purchased from

Sigma Chemical Co. (St. Louis, MO); osmium tetroxide was obtainedfrom BDH Chemicais (Poole, UK); Triton X-100 and cholesterol oxidaseenzymatic assay kit were from Boehringer Mannheim (Germany);glutaraldehyde and ali other chemicals were of analytical grade and wereobtained from Merck (Darmstadt, Germany).

2.2. SubjecisVenous blood from 13 (9 male and 4 female) jaundiced neonates (JN)

was collected between the 2nd and the 4th days of life; umbilical cordblood from 16 (7 male and 9 female) apparently healthy neonates (HN)was obtained immediately after delivery. Ali the children were full-term,with a good weight for the gestational age and with no malformations(Table 1). Babies of the JN group were breast fed and no child from theHN group has returned to the hospital due to hyperbilirubinemia. Venousblood from 18 (6 male and 12 female) healthy young adult donors wasalso collected in order to compare the results with those obtained in thetwo newborn groups.

Ethical permission was obtained from the Ethics Committee of theGarcia de Orta Hospital, Almada.

2.3. Sample coliection and preparationBlood samples were collected into 50 U/ml heparin, stored at 4°C,

protected from Iight and used within 3 h. For the morphological analysis,a blood aliquot was fixed with 2.5% glutaraldehyde immediately aftercollection, as described below.

Erythrocytes were separated from plasma and buffy coats by centrifugation at 600 x g for io mm at 4°C and washed three times with ice cold5 mml/I sodium phosphates~ 145 mmol/l NaC1, pH 7.4 (buifer A).

152 M.A. Brigo ei ai. / Clinica Chi,nica Acta 249 (1996) 149 /65

Table 1Characteristics of healthy and jaundiced neonates

Healthy neonates Jaundiced neonates(cord blood) (venous blood)

Range Mean ± S.D. Range Mean ± S.D.

Birth weight (g) 2780 3850 3233 ± 289 2490—4205 3436 ± 485Gestational age (weeks) 37 41 39.1 ± 1.1 37—42 39.2 ± 1.4Apgar score 1 miii 7—9 8.7 ± 0.7 6—9 8.5 ± 0.9

5 mm 10—10 10 ± O 9—10 9.9 ± 0.3Jaundiçe appearance 30.0—84.1 52.7 ± 17.5

(hours of lhe)Blood coliection 39.0—81.7 60.3 ± 11.6

(hours of life)

2.4. Incubation of red blood celLv with bilirubinWashed adult RBC were re-suspended (i0~ celis/mi) in 7.5 mmol/1

sodium phosphates, 145 mmol/l NaC1, 5 mmol/1 glucose, 1 mmol/lMgSO4, pH 7.4 (buifer B). A stock solution ofBb was prepared,just beforeuse, by dissolving 5 mg of Bb in 1 ml of 100 mmol/l NaOH and addingto the RBC suspension ata final concentration of 171 pmol/l. The additionof Bb solution was immediateiy foliowed by an equivalent volume of 100mmol/l HC1 in order to restore the pH to 7.4, and an incubation of 3 h,at 37°C ia buifer B, ia the dark, was performed. Controis were preparedia the absence of Bb in a celi suspension containing 100 mmol/1 NaOHand 100 mmol/l HCI ia equivalent amounts, as in the samples.

The incubation was stopped by centrifugation at 600 x g for 10 mm at4°C. The celi pellet obtained showed two layers of celis: the top layerconsisted of yellow coloured celis (designated as top edis ia the text) andthe remaining peiiet had the usual coloration (designated as bottom celisia the text). Both top and bottom celis were washed three times with buiferA. As hemolysis occurred in the assay tubes during the incubation period,the supernatants from these tubes were re-centrifuged at 6000 x g for 20mm at 4°C to sediment vesicular materiais, which were presumed to beerythrocyte membranes or ghosts of the Iysed celis (designated as vesiciesin the text). The extent of hemolysis was determined by hemoglobinmeasurement at 542 nrn in the supernatants. Results were expressed aspercentage of total hemolysis, which was complete lysis of the sarnevolume of ceil sarnple by addition of water. A set of five experiments wasperformed.

AIA. Brito ei a?. / C?inica Chimica Acta 249 (1996) 149—165 153

Aithough in this condition some Bb aggregation may occur, the absenceof albumin in the incubation medium aliows one to achieve high free Bbleveis capable of exerting cytotoxyc effects in short incubation periods. Infact, most of the bilirubin studies using albumin oniy achieve significantresuits at high biiirubin/albumin molar ratios [7,8,31], exceeding thebinding stoichiometry, which can aiso lead to some co-aggregation of Bbwith albumin [32].

2.5. Morpho!ogica! analysisCelis were prepared for scanning eiectron microscopy (SEM) observa

tion accordingiy to Kaui et ai. [10]. Briefly, to 50 pi of packed ceiis wasadded 150 pi of 2.5% glutaraidehyde in 100 mmoi/i phosphates, pH 7.4(buifer C). After 30 mm, at room temperature, the edis were pelleted bycentrifugation, washed with buifer C and post-fixed by the addition of 150gi of 1% osmium tetroxide in buifer C for 1 h. Finally the celis werewashed three times with deionized water and dehydrated by suceessivewashings with 50%, 70%, 80%, 90%, 95% and 100% ethanol. Forobservation, the sampies were diiuted in a solution of ethanoi andpropyiene oxide (1:1 v/v); a drop was deposited directiy onto the microseope support, air dried, coated with goid by sputtering and examined inan 151 (Internationai Scientific Instruments) DS 130 microscope.

In order to express the extent of shape changes quantitativeiy, stages ofmorphoiogicai aiterations were evaiuated (as percentages) on eiectronmicrographs, in conformity with the ciassification of Hsu et ai. [33], asechinocytes 1, echinocytes 2, echinocytes 3 and spheroechinocytes. Approximateiy 200 edis were counted, distributed in six fieids randomiyseleeted. The morphoiogicai index was caicuiated, as defined by Fujii et ai.[17] according to the formula: morphologicai index = E (morphologicalseore) x [(number of distorted ceils)/(totai ccii number)], where morphoiogicai scores of + 1, + 2, + 3 and + 4 correspond to each shape stagereferred to above, respectiveiy; the discocytes score was considered as 0.

2.6. Exiraction and quantjfication of membrane cholesterol andphospho!ipids

RBC erythrocyte membranes were obtained by the method of Dodge etai. [34], ali the steps being performed at 4°C.

Lipids were extracted and the phosphoiipids (P1) were analyzed in termsof inorganic phosphorus (Pi) and choiesterol (Ch) content determined byadaptation of previousiy described methods [35,38]. In detail, 200 pi ofRBC membranes was mixed with 200 p1 of water and after 15 mm at roomtemperature 2.5 mi of isopropanoi was added with vortexing. After 1 hincubation, 1.5 ml of chloroform was added with vortexing and incubated

154 M.A. Brito et ai 1 Clinica Chimica Acta 249 (1996) 149—165

for a further 1 h. The mixture was centrifuged at 600 x g for 10 mm, theisolated organic layer was dried under a nitrogen stream at 37°C and theIipid extract was reconstituted in 150 pi chloroform/methanol (1:1, v/v).For lipid phosphorus determination, 10 p1 of this extract was mineralizedby the addition of 500 p1 of 70% perchloric acid and heating at 180°—200°C for 30 mm. Pi was quantified by adding 7.5 ml of 0.22% ammoniummolybdate in 2% H2S04 and 300 pi of Fiske and Subbarow reagent [37],boiling the solution during 15 mm, after which it was cooled and theabsorbance read at 830 nm, against a reagent blank. Cholesterol wasdetermined by the cholesterol oxidase method (Boehringer Mannheim).Briefly, 10 p1 of the lipid extract was incubated with 1 ml of the enzymaticsolution, containing 0.5% Triton X-100, and the absorbance was read at500 nm against the enzymatic solution as blank. A 5.17 nmol/l cholesterolsolution was used as a standard.

2.7. Other methodsTotal plasma bilirubin was determined in the HN by an adaptation of

the Jendrassik and Grof method [39] (Boehringer Mannheim) and in theJN by the method of Pays and Beijean [40]. Membrane protein determination was performed by Lhe method of Lowry et ai. [41] and membraneresidual hemoglobin was quantified [42] in order to correct the proteincontent for Lhe remaining hemoglobin present in the ghosts.

2.8. Siatistical analysisA two-tailed Student’s t-test was used for the statistical analysis of data.

Values are expressed as means ± standard deviations. Differences with aP value less than 0.05 were considered statistically significant and astatistical trend was considered to be present when P < 0.1.

3. Results

3.1. Bilirubin conteMThe total Bb concentrations in the plasma of Lhe newborn infants were

194.9 ± 41.0 pmol/l (135.1—277.1 pmol/l) in the JN and 27.4 ± 10.3 pmol/1(10.3—42.8 pmol/I) in the HN. These results showed a statistically significant difference (P < 0. 01).

3.2. Red ceil morphologyThe morphology of RBC in Lhe HN group showed a majority of normal

biconcave disc appearance celis (Fig. lA). In the JN group, besides Lhepresence of some intact discocytes, a wide variety of abnormal celis,comprising celis with minor membrane protuberances (echinocytes 1),

M. A. Brito ei ai. 1 Clinica Chhnica Acta 249 (1996) 149—165

Fig. 1. Scanning electron micrographs of erythrocytes obtained from cord blood of heakhyneonates (A) and venous blood ofjaundiced neonates (B). Bar = 10 pm.

156 M.A. Brigo ei aí! Clínica Chimka Acta 249 (1996) 149—165

Table 2Morphological shapes (%) and index values observed in jaundiced and healthy neonateserythrocytes

Morphology Jaundiced neonates Healtby neonates P

Discocytes 45.7 ± 17.2 73.6 ± 5.88 <0.01Echinocytes 1 45.7 ± 12.9 23.0 ± 6.86 <0.05Echinocytes 2 7.79 ± 6.29 2.63 ± 0.88 <0.05Echinocytes 3 0.41 ± 0.51 0.61 ± 0.09 N.S.Spheroechinocytes 0.47 ± 1.08 0.14 ± 0.19 N.S.Morphological index 0.64 ± 0.24 0.31 ± 0.05 <0.01

Stages of morphological alterations were evaluated and the morphological index wascalculated as described in Materiais and methods. Values are mean ± S.D.; N.S., notsignificant.

with two or more spiny protuberances and losing the central concavity(echinocytes 2 and 3) and spheroechinocytes were observed (Fig. 18).While in cord blood of HN 73.6% of lhe celis were discocytes, in lhe JNgroup only 45.7% discocytes were present (P < 0.01) (Table 2). Moreover,in lhe latter group, besides the lower percentage of intact discocytes, awide variety of altered celis, from echinocyles 1 to spheroechinocyles, werepresent. The quantificalion of lhe morphological shape changes alsorevealed thal JN had a significant increase (P < 0.05) in echinocytes 1and 2, as well as in lhe morphological index (P < 0.01), when comparedwith the data from HN~

The hemolysis observed afler in vilro Bb inleraction with aduil RBC(11.4 ± 1.3%) is in agreemenl wilh lhe observations of olher authors[11,43]. The scanning electron micrographs of the control, bottom and topcelis, as weIl as of lhe vesicles, are shown in Fig. 2. Higher proportions ofaltered cells were observõd either in lhe boltõm (Fig. 2B~ or in the top(Fig. 2C) celis when compared with the conlrol edis (Fig. 2A). Regardingthe vesicles (Fig. 2D), they present an erythrocyle similar morphologywith a semitransparent appearance, probably due lo lhe withdrawal ofhemoglobin.

In lhe morphologicai quantificalion (Table 3), significant deereases(P < 0.01) in lhe percenlages of discocytes were noticed in lhe bottom andtop edIs when compared wilh controis, and significant increases(P < 0.01) were observed in lhe percentages of echinocyles 1, 2 and 3 inthe boltom, and of eehinocytes 2 in lhe top fraction. Both populationsshowed significantly higher (P <0.01) values of lhe morphologicai index.The fact that lhe total number of transformed celis observed in the bottom

M.A. Brito ei tiL 1 Clinica Clziniica Acta 249 (19%) 149—165

o

‘oFig. 2. Scanning electron micrographs of erythrocytes incubatcd in the absence (A) or inthe presence of 171 pmol/1 bilirubin, for 3 h, at 37°C (B, bottom; C, top), and of the vesicles(1)) obtained, by centrifugation of the bilirubin—incubation medium (6000 x g, 20 mm).Bar = lO pm.

Table 3Morphologicai shapes (%) and index values observed after biiirubin—erythrocyteinteraction

Morphology Control Bottom P Top Pfraction fraction

Discocytes 79.8 ± 7.82 14.8 ± 5.12 <0.01 47.7±6.15 <0.01Echinocytes 1 20.1 ± 7.56 65.3 ± 6.33 <0.01 29.4±5.53 N.S.Echinocytes 2 0.17 ± 0.37 17.7 ± 1.72 <0.01 21.0±8.73 <0.01Echinocytes 3 O 2.22±0.88 <0.01 1.26 ± 118 N.S.Spheroechinocytes 0 O N.S. 0.63 ± 1.08 N.S.Morphoiogical index 0.20 ± 0.08 1.07 ± 0.05 <0.01 0.78±0.08 <0.01

Erythrocytes wcre incubated in the absence (control) or presence of bilirubin (bottom andtop fractions) and stages of morphoiogical aiterations and morphological index weredetermineci as described in Materiais and methods. Values are means ± S.D.; P values wereobtained vs. control; N.S., not significant.

158 IdA. Brim et ai 1 Clínica Chimica Acta 249 (1996) 149—165

(85.2%) is considerably higher (P < 0.01) than that found in the topfraction (52.3%) suggests that these two erythrocyte populations maycorrespond to different stages of celi agression induced by Bb. However,when the morphological index of the bottom fraction (Table 3) wascompared with that found in JN (Table 2), a significantly greater value(P <0.01) was observed in the first case. This finding is not surprisingsince the absence of albumin from the incubation medium in the in vitrostudy favors the binding of Bb to the erythrocyte membrane.

3.3. Lipid compositionThe quantification of membrane Ch and P1 content in the neonates and

adults has showed (Fig. 3) that JN presented the highest Ch/P1 molar ratiowhen compared with healthy neonates and adults (P <0.05 and P < 0.01,respectively). This increment seems to be mostly due to a lower content ofthe membrane P1, as indicated by the significant difference observedbetween JN and adults (P < 0.05), and a statistical trend (P < 0.10) wasfound when JN were compared with HN.

Iii the Bb—RBC in vitro interaction studies, significant increases in theCh/P1 molar ratio were found in the top cells (P < 0.05) and in the vesicles(P < 0.01) when compared with the control ones (Fig. 4); these incrementswere also mainly associated with significant reductions (P < 0.05 andP < 0.01, respectively) in the P1 concentration. The fact that no differenceswere observed in the Ch and P1 leveis between the bottom fraction and

0.8~ 2

0.7 1.6

0.6

0.5 1.2

~0A

0.3a 0.2 0.4

0.1

o 0~

OJaundiced neonates • Healttiy neonates üAdults

Fig. 3. Cholesterol (Ch) and phospholipids (P1) content and cholesterol/phospholipids(Ch/PI) molar ratio of erythrocyte membrane observed in jaundiced neonates, bealthyneonates and adults. *~ <0.05; **~ <0.01.

**

5—Ch P1 ChIPI

M.A. Brigo ei tiL / Clinica Chimica Acta 249 (19%) 149—165 159

0.8 2

0.71.6

0.6

105 11.2e’OA

~0.3 ~0.8

0.2 0.4

0.1

o

Ocontrol Eboitom top BvesiclesFig. 4. Membrane cholesterol (Ch) and phosphoiipids (P1) content, and choiesteroi/phospholipids molar ratio (Ch/Pi) of the erythrocytes incubatcd in the absence (contrai)and ia the presence of 171 pmoi/I bilirubin, for 3 li, at 37°C (bottom and top fractions), aswefl as of the ghosts of lhe iysed edis (vesicles) obtained by centrifugation of thebilirubin—incubation medium (6000 x q, 20 mm). *p < 0.05; **~ <0.01.

the corarei celis, but only with the top fraction and vesicles, is inagreement with the existence of different stages of ccli toxicity by Bbinteraction, as pointed out by the anaiysis of the morphological study datapreviously presented.

4. Discussion

The HN inciuded in this study presented cord blood total Bb leveiswithin the range of 10.3—42.8 pmoi/i, and had no ABO or Rh incompatibility or hemolytic disease. Therefore, based on recent literature [44], wemay presume that they were not at risk of developing transient hyperbilirubinemia during the first days of life.

Since the mean Bb levei of the JN group evaiuated in the present paperwas 194.9 pmoi/i, which is considerabiy iower than the 300 425 pmol/iabove which there is an irnportant risk of Bb neurologicai damage infuii-term infants [45—48], we may consider the icterie babies studied as agroup not at risk of Bb neuroiogicai damage and thus corresponding to agroup with a moderate and reversible hyperbiiirubinemia. On the otherhand, the conditions employed in our in vitro study, with a Bb concentration (171 prnoi/i) in the sarne range as that of JN but in the absence of

-ti

Ch P1o

ChIPI

160 M.A. Bailo ei a?. / Clinica Chimica Acta 249 (1996) 149—165

albumin, are favourabie to a much higher free Bb concentration and tendto approach the irreversibie hyperbiiirubinemia that occurs~ in vivo andwhich may iead to the pathoiogicai condition of kernicterus.

The effect of Bb on REC morphology has been studied by other authorsin several different conditions [10,11,13]. Aithough initial reports of invitro Bb—erythrocyte interaction using Bb—aibumin solutions (0.7—2.0molar ratios) [13] did not find any ccli distortion, more recent studiesevidenced red ccii crenation after in vitro interaction with Bb [10,11], asweii as in hyperbiiirubinemic neonates [10], which corroborates ourpresent resuits. However, none of these authors quantified the extent ofshape changes and determined the morphoiogicai index as we did. In fact,Kaui et ai. [10] only mentioned the observation of a moderate to highnumber of crenated edis corresponding to Iow (~ 171 pmol/i) and high(~342 pmoi/i) serum Bb leveis.

The quantification of morphoiogicai shape changes appears to us to bea useful way of reiating ccii aiterations with Bb aggression steps. Based onthis, it is interesting to point out the considerably higher vaiues (P < 0.01)obtained for the total number of transformed ceiis and for the morphological index in the bottom ceiis when compared with those of hyperbilirubinemic babies. As the toxic effects of Bb were proposed to be dueto unbound unconjugated Bb [6,13,49,50], such a difference may resuitfrom the highest leveis of that form present in the in vitro study, asmentioned above; in the serum of JN the binding of Bb to aibumin [51,52]prevents, to a great extent, Bb binding to the circuiating celis.

Since drug-induced membrane distortion was proposed as a sensitiveindicator of membrane drug toxicity [17,18], the crenation observed inour study indicates that, like other anionic drugs, Bb has a membraneperturbing effect. In fact, the marked increase in the number of crenatedceils observed, both in JN versus HN and in Bb treated erythrocytes(bottom and top) versus controi, may be due to the Bb—membraneinteraction in an anionic form [15], inducing an expansion of the cclimembrane outer ieaflet, resuiting in the membrane evagination [18]. Theappearance of hemoiysis in the in vitro study is in agreement with theobservations of Kirschner-Ziiber et ai. [11], and may result in a subsequent irreversibie stage of Bb interaction with membrane penetration andccli disruption [53,54], ieading to the formation of the vesicies [16]. Thisis also in agreement with previous reports according to which Bb JX~distributes into the iipid apoiar region of model membranes, over a rangeof concentrations from normai to pathological [54], intercalates betweenthe hydrophobic surfaces of integral membrane proteins and the acyichains of iipids [55], dissoives readiiy in the biiayers, diffuses throughthem and rapidiy dissociates into an aqueous medium [54].

M.4. Brito ei ai. / Clínica Chimica Acta 249 (1996) 149—165 161

The iower number of transformed celis (P < 0.01) found in the toppopulation compared with the bottom one, leads us to hypothesize thatthe top fraction may correspond to celis with a Iower Bb deposition at theouter membrane Ieaflet, inducing lesser echinocytosis, or that Bb haspenetrated deeper into the membrane, reaching the inner leaflet. In theIatter case, the expansion of the inner leaflet might have compensated themembrane evagination [17] previously originated by the Bb deposition inthe outer Ieaflet, leading to a decrease in the number of transformed celisand suggesting that top celis may be more affected than the bottom ones.

In fact, the lower P1 content and the higher Ch/P1 molar ratio found inthe top celis, relative to the bottom ones, reinforces the evidence that topcelis are the most affected by the Bb interaction, although presenting lessmorphologicai alterations. This suggests a further stage of ccii toxicity,which may be reiated to a more extensive Bb deposition, where Bbtransiocation to the cytosolic domam of the membrane may have occurred, expiaining the yellowish coloration observed in this fraction.

In the Bb—RBC in vitro study a small decrease of P1 content wasobserved in the bottom fraction (not significant), being more pronouncedin the top fraction (1’ < 0.05) and even more so in the vesicles (P <0.01).These results point to a progressive ceiluiar toxicity, beginning in the celiscorresponding to the bottom fraction (the most crenated ones— stage 1)passing through the top ceils (less crenated but already presenting phospholipid decrease — stage 2) and ending with the vesicies resuiting fromccli iysis (those with the greatest iipid composition modifications — stage3).

It is worth mentioning that even in the moderate conditions of our invivo study, corresponding to a reversible mild hyperbilirubinemia (totalBb mean values of 194.9 MmoI/l), a Iower (P < 0.05) membrane P1 contentwas observed in JN compared with adults (Fig. 3).

The studies conducted with JN, where an increased morphologicaiindex and a decreased P1 content were also observed, together with theconcepts presented above, leads us to infer that an intermediate levei ofBb toxicity has occurred, corresponding to a stage between 1 and 2. Alithis taken together allows the assumption that a ioss of P1 from themembrane may be a resuit of Bb—membrane interaction, in both in vivoand in vitro conditions, depending in its extent on the amount of Bbavailable to interact with the ccli membrane and the duration of theexposure.

Collectively, our results suggest that the Bb cytotoxicity may occur inthree different stages, the first being the deposition in the outer leaflet ofthe ccli membrane, in an anionic form, leading to echinocytosis andconsequently to an increased morphoiogical index (JN and bottom celis).

162 M.A. Brito eta!. / Clínica Chimica Acta 249 (1996) 149 163

In the second ceil interaction step, Bb transiocates through the membrane,reaching the inner leaflet (top); this process is accompanied by the loss ofmembrane P1 causing significant changes in membrane lipid composition.Finalty, the last stage corresponds to an irreversible loss of membraneintegrity, with the Ieakage of hemoglobin and celi destruction (vesicles).

Acknowledgments

We gratefully acknowledge Patrícia Cará and Susana Correia for theirhelp in the experimental work and Dr. May-Jean King for eriticalcomments and suggestions. We also thank the Estação AgronómicaNacional and Mr. Octávio Chaveiro for the use of, and technical assistance with, the scanning electron microscope. This work was supported bya grant of the Junta Nacional de Investigação Científica e Tecnológica(Proj. PBIC/C/SAU/1 570/92).

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