ANNALS OF CLINICAL AND LABORATORY SCIENCE, Vol. 20, No. 3 Copyright © 1990, Institute for Clinical Science, Inc. Mechanisms of Hemolysis in Liver Disease EDWARD. E. MORSE, M.D. Department of Laboratory Medicine, University of Connecticut School of Medicine, Farmington, CT 06032 ABSTRACT Liver disease, particularly alcoholic cirrhosis, is associated with a number of interesting chemical changes which result in structural and metabolic abnormalities of the erythrocyte membrane leading to micro- scopically observable cell shape changes and hemolytic anemia varying from very mild to potentially lethal. Increase in unesterified serum cholesterol owing to lecithin cholesterol acyl transferase (LCAT) deficiency in cirrhosis leads to expansion of the lipid bilayer and macrocytosis without megaloblastic changes in precur- sors. Substitutions of phosphotidyl choline (PC) moieties in the erythro- cyte lipid bilayer lead to echinocytes (disaturated PC) or to stomatocytes (diunsaturated PC). In some patients, high density lipoprotein (HDL) abnormalities lead to erythrocyte surface changes causing rapid formation of echinocytes. The rapidity and reversibility of these changes suggest blockade of metabolic transport channels critical to the maintenance of erythrocyte mem- brane shape. Metabolic changes involving vitamin E deficiency leading to lipid per- oxidation and pyruvate kinase instability leading to adenosine triphos- phate (ATP) reduction have also been invoked to explain hemolysis asso- ciated with acute liver damage. The most severe hemolysis in liver disease is associated with acanthocytes (spur cells) and a marked imbalance in cholesterol-phospholipid ratio. These patients usually have hypersplen- ism, as well as rigid erythrocyte membrane transformations which are irreversible. Any of the other erythrocyte membrane shape changes described appear to be reversible if the liver disease abates, but they too may become irreversible if bits of projecting membrane are repeatedly removed by the macrophages of an enlarged spleen. Introduction erythrocyte. There is free exchange of unesterified cholesterol and some phos- The liver is in large part responsible pholipids between the plasma and the for the plasmatic environment of the erythrocyte membrane. The erythro- 169 0091-7370/90/0600-0169 $00.90 © Institute for Clinical Science, Inc.
Mechanisms of Hemolysis in Liver Disease
Mar 25, 2023
Liver disease, particularly alcoholic cirrhosis, is associated with a
number of interesting chemical changes which result in structural and
metabolic abnormalities of the erythrocyte m embrane leading to microscopically observable cell shape changes and hemolytic anemia varying
from very mild to potentially lethal.
Welcome message from author
Increase in unesterified serum cholesterol owing to lecithin cholesterol acyl transferase (LCAT) deficiency in cirrhosis leads to expansion of the lipid bilayer and macrocytosis without megaloblastic changes in precursors. Substitutions of phosphotidyl choline (PC) moieties in the erythrocyte lipid bilayer lead to echinocytes (disaturated PC) or to stomatocytes (diunsaturated PC)
Transcript
ANNA LS O F C L IN IC A L A N D LABORATORY S C IE N C E , Vol. 20, No. 3 C opy rig h t © 1990, In s titu te for Clinical S cience, Inc.
Mechanisms of Hemolysis in Liver Disease
EDWARD. E. MORSE, M .D.
D epartm ent o f Laboratory Medicine, University o f Connecticut School o f Medicine,
Farmington, C T 06032
ABSTRACT
Liver disease, particularly alcoholic cirrhosis, is associated w ith a num ber of interesting chemical changes which result in structural and metabolic abnormalities of the erythrocyte m em brane leading to micro scopically observable cell shape changes and hemolytic anemia varying from very mild to potentially lethal.
Increase in unesterified serum cholesterol owing to lecithin cholesterol acyl transferase (LCAT) deficiency in cirrhosis leads to expansion of the lipid bilayer and macrocytosis without megaloblastic changes in precur sors. Substitutions of phosphotidyl choline (PC) moieties in the erythro cyte lipid bilayer lead to echinocytes (disaturated PC) or to stomatocytes (diunsaturated PC).
In some patients, high density lipoprotein (HDL) abnormalities lead to erythrocyte surface changes causing rapid formation of echinocytes. The rapidity and reversibility of these changes suggest blockade of metabolic transport channels critical to the m aintenance of ery throcyte m em brane shape.
Metabolic changes involving vitamin E deficiency leading to lipid p e r oxidation and pyruvate kinase instability leading to adenosine triphos phate (ATP) reduction have also been invoked to explain hemolysis asso ciated with acute liver damage. The most severe hemolysis in liver disease is associated with acanthocytes (spur cells) and a m arked imbalance in cholesterol-phospholipid ratio. These patients usually have hypersplen- ism , as w ell as rig id e ry th rocy te m em brane transform ations w hich are irreversible.
Any of the o ther ery throcyte m em brane shape changes described appear to be reversible if the liver disease abates, bu t they too may becom e irreversib le if bits of projecting m em brane are repeated ly removed by the macrophages of an enlarged spleen.
Introduction erythrocyte. T here is free exchange of unesterified cholesterol and some phos-
The liver is in large part responsible pholipids betw een the plasma and the for the plasm atic env ironm ent of the ery th rocy te m em brane. The ery thro-
169 0091-7370/90/0600-0169 $00.90 © Institute for Clinical Science, Inc.
1 7 0 MORSE
cy te , hav ing no glycogen sto res , is dep en d en t upon plasm a glucose, the level of which is maintained by the liver. In liver disease, alterations in lecithin cholesterol acyl transferase (LCAT) and in lipoproteins may produce changes in the erythrocyte cholesterol/phosopholi- pid ratio resulting in reduced membrane flexibility and, with processing, by the spleen, irreversible changes in cell shape and hemolysis.
Metabolic functions of the erythrocyte may also be affected by components pro duced by an abnormal liver. Interference with glucose transport across the m em b rane or w ith glycolytic m etabolism w ithin the ery th rocy te may decrease adenosine triphosphate (ATP) which may re su lt in c o n tra c tile p ro te in tetany , osm otic im b a la n c e , shape changes, abnormal m em brane rigidity and early removal of the erythrocyte in the spleen. Portal hypertension as a result of fibrosis (cirrhosis) in the liver may cause conges tive splenomegaly and a form of hyper- splenism with sequestration of erythro cytes in the spleen. In addition, recent evidence exists to suggest that the avail ability of vitamin E may be decreased, particularly in ch ildren , w ith chronic liver disease, possibly accounting for an increased susceptibility of the erythro cytes to intravascular oxidative hem o lysis through lipid peroxidation.
This paper will review the existing data that support the various proposed mechanisms of erythrocyte damage in association with liver disease and will attem pt to provide a unifying hypothesis for the prem ature destruction of erythro cytes in patients with abnorm alities of liver function.
Review of D ata
Lipids account for about 50 percent of th e w eigh t of re d cell m em b ran es .4 Mammalian red cell membranes have a molar ratio of cholesterol to phospholipid of about one.19 Four phospholipids pre
dom inate in hum an erythrocytes: leci th in (phosphatidyl choline), sphingo m y e lin , p h o s p h a tid y l s e r in e , an d phosphatidyl ethanolamine. The interac tions betw een cholesterol and phospho lipids have important consequences for m em brane structure and function.12
C holesterol increases the packing of phospholipids in both layers of the lipid bilayer of the erythrocyte m em brane.8 The close interpositioning of sterols with p h o sp h o lip id s in the lip id b ilayers , which acquire excess cholesterol and phospho lip ids from plasm a, causes a d eg ree of im m obility to be im posed upon the acylcarbon atoms near the lipid bilayer’s inner or outer surface, while increasing the freedom of motion deep w ith in th e hyd rophob ic core of the bilayer creating an in term ediate fluid state .3 The num ber of saturated double bonds w ith in th e ph o sp h o lip id acyl chains in the bilayer is an im portan t determ inant of their fluid qualities.20
Cholesterol itself also has an important effect on m em brane fluidity. By in te r posing itself betw een adjacent phospho lipid acyl fatty acid chains, it increases the degree of o rder w ithin the m em brane bilayer.7 Unesterified cholesterol and phospholipids of plasma lipoproteins readily exchange with their counterparts in the lipid bilayers of the erythrocyte m em brane.5 In patients whose erythro cytes acqu ire excess cho lestero l and phospholipids from plasma lipoproteins in a one to one ra tio , th e red cells become target cells and the m em brane expands in a uniform fashion.6'21 This phenom enon is seen in cirrhosis, hepati tis, and obstructive jaundice. In obstruc tive jaundice, the increase in cholesterol may be as great as 75 percent and that of phospholipid up to 60 percent.4 A simi lar abnorm ality has been described in patients with congenital absence of leci- th in -c h o le s te ro l acyl tran sfe ra se (L- CAT).10 This L-CAT deficiency has com monly been found in liver disease,6 but
MECHANISMS OF HEMOLYSIS IN LIVER DISEASE 1 71
correlation betw een serum L-CAT activ ity and abnorm alities of red cell lipids has been difficult to demonstrate.
Target cells do not leave the circula tio n p re m a tu re ly in th e absence of hypersplenism ; however, larger imbal ances in the cholesterol/phospholip id ratio often lead to formation of echino- cytes (with sea urchin-like projections) and eventually to acanthocytes (spur or thorn cells) which are more rigid and are rapidly removed from the circulation by the spleen.
R ecent studies have suggested that echinocytes are m ore readily found in the blood of patients with liver disease if the cells are examined in wet films or by scanning e lec tron m icroscope ra th e r than in fixed smears by light m icros copy. 12 Owen et al18 showed that echino- c y tes can b e p ro d u c e d from nom al w ashed erythrocytes w hen incubated with the high density lipoprotein (HDL) fraction from some jaundiced patients. O ther plasma fractions (LDL, VLDL, Alb) are not apparently echinocytogenic. This type of echinocyte change is rapidly reversible if the abnormal cells are incu bated in normal HDL. The echinocyte change occurs in m inu tes and is not accom panied by change in m em brane cho lestero l con ten t no r by up take of lysolecithin nor bile acids.
T he H D L ’s from th ese jau n d ic ed patien ts show binding characteristics that were saturable at an estimated 5000 sites per cell. Normal H D L or nonechin- ocytogenic p a tie n t H D L ’s show only n onsatu rab le b ind ing characteristics. Pretreatm ent of erythrocytes with pro- nase or trypsin reduces or elim inates b ind ing by abnorm al H D L . Thus, it seems likely that echinocytosis in associ ation w ith liver d isease may, in some cases, be associated w ith m em brane changes induced by a rapid shift (in glu cose or electrolytes) induced by H D L related binding to specific receptor sites in the erythrocyte surface membrane.
G ra h n ,14 as early as 1968, d em on strated that serum from echinocyte-con- taining blood could induce echinocyte form ation in norm al cells. Similarly, serum from blood containing acantho- cytes also induces formation of echino cytes, but not acanthocytes. Thus, it is argued, an additional factor acting in vivo may be necessary for the production of acan thocy tes, p robab ly by excess m em brane removal in the spleen.
S ubsequen t stud ies by H ui e t a l15 show that some low density lipoprotein (LDL) fractions of norm al serum p ro duce echinocytes from discocytes. The active constituent appears to be apolipo- protein B (apo B). The H D L of patients with liver disease is enriched with apo E, which competes with apo B of normal LD L for apo B, E receptors on the sur face of a num ber of nucleated cells.18 In the N etherlands,16 phosphatidyl choline specific transfer protein from beef liver has been used to replace native phos phatidyl choline (PC) molecules with a variety of PC species differing in fatty acid composition w ithout changing the total phospholipid content of the m em brane. No morphological changes were noted when PC was replaced by mono sa tu ra ted species like 1-palm itoyl, 2 oleoyl PC; 1 palmitoyl, 2 linoleoyl PC, egg PC or PC from rat liver microsomes; however, replacem ent with disaturated species, 1,2 dimyristoyl PC, 1,2 dipalmi- toyl PC, and 1,2-distearoyl PC resulted in formation of echinocytes. I t should be n o ted th a t PC rep lacem en t using C 14 labe lled com pounds is m easured in hours (10 to 20 p e rc e n t in one to two hours, 40 to 60 percent in four to six hours).
In te re s tin g add itional observations include the form ation of stom atocytes upon replacem ent with PC species con taining two unsaturated fatty acids, e.g., 1,2 dioleoyl PC and 1,2 dilinoleoyl PC. These studies suggest that the discoid shape of the hum an erythrocyte is opti
1 7 2 MORSE
mally stabilized by PC species that con tain one saturated and one mono- or di unsaturated fatty acid, and that the cell tolerates only lim ited variations in the species composition of its PC. Saturated PC seem s to concentrate in the outer monolayer causing outward projection, while unsaturated PC inserts deeper in the lipid bilayer causing inward projec tion and stomatocytosis.
D ra m a tic changes in e ry th ro c y te shape are observed when the unsatura tion index of PC, defined as the total num ber of double bonds relative to that of the fatty acids, becomes lower than 0.5 or higher than 1.0. If the erythrocyte PC is replaced by dimyristoyl PC, dipal- mitoyl PC or distearoyl PC, the index decreases to 0.5. Echinocytes are formed when about 25 percent of native PC is rep laced by dipalm toyl PC. Stomato- cytes form if diunsaturated PC is substi tuted at 20 to 30 percent and hemolysis occurs at about 40 percent replacement.
Spheroechinocytes can also be found if the original PC is replaced by 1-palmi- toyl, 2 arachidonoyl PC. At 20 percent replacem ent, shape change begins. At 60 percent replacem ent, when the double bond index becomes greater than 1.0, spheroech inocy tes are obvious along with many cells showing typical dimples, which may be due to the loss of internal osmotic pressure, since the cells begin leaking ions at this stage.
Stomatocytes, produced by a bending or folding of the m em brane inwards, form after replacem ent of more than 40 percent of the PC with 1,2 dioleoyl PC. Typical stomatocytes are observed when 20 to 30 percent of the PC is replaced by 1,2 dilinoleoyl PC after a six hour incu b a tio n . F u r th e r increase causes cell lysis.
Stomatocytosis has been reported in association w ith alcoholic c irrhosis .9 Four patien ts developed hem olysis of varying severity, and one appeared to have an intrinsic intracorpuscular defect
because norm al cells incubated in his serum did not acquire the shape change, nor did they become stomatocytes after infusion in to his b lood in vivo . The defect appeared to be rela ted to acute alcoholic binges in these four patients and were not accompanied by lipidemia.
Stomatocytosis has also been reported in association with a hereditary red cell anomaly in which there is a remarkable reversal of sodium and potassium con centrations in the erythrocyte.17 It seems possible that lipid changes in the eryth rocyte m em brane during acute alcoholic bou ts m ay affect th e p e rm eab ility of e ry th ro cy te s to g lucose o r calcium , thereby decreasing ATP, causing tetany of contractile proteins and d irectly or indirectly interferring with the sodium, potassium pump.
Hem olytic anemia and susceptibility to H20 2 hemolysis have been described in ch ildren w ith vitam in E deficiency and chronic liver disease.10 Thirty-four children (one to 14 years) with severe chronic liver diseases were studied, 16 of whom w ere also v itam in E defic ien t (vitamin E < 5 mg per ml). Vitamin E may be deficient in liver cirrhosis as a consequence of intestinal malabsorption related to cholestasis. Tocopherol (vita min E) provides a protective effect on lip id perox idation w ithou t w hich the cells may be susceptible to oxidative lysis and a shortened survival. W ith respect to controls, cirrhotic ch ild ren in both vitamin E and non vitamin E deficient g ro u p s show ed s ig n ifican tly h ig h e r values of serum unesterified cholesterol, triglycerides, and total serum phospholi pid concentration. There was an intense increase in phosphatidyl choline content in the erythrocyte membrane, as well as in the serum, that was statistically signif icant in the vitamin E deficient group. The fundamental biochemical lesion in the erythrocyte m em brane may be due to accelerated lipid peroxidation induc ing fo rm ation of m alo ny ld ia ldehyde
MECHANISMS OF HEMOLYSIS IN LIVER DISEASE 1 7 3
which promotes polymerization of cyto- s k e le ta l p ro te in s . T h e s u b s e q u e n t decrease in cell flexibility leads to p re m ature removal of the cell from the cir culation.
In adult patients, a variety of erythro cyte metabolic defects may be induced including pyruvate kinase instability.1213 There is an interesting acute hemolytic anem ia seen in patien ts w ith alcohol- in d u c e d fa tty liv e r a sso c ia te d w ith hypertriglyceridem ia.22 There is little in vitro or in vivo evidence that increased triglycerides per se are associated with h em o ly tic anem ia in th is syndrom e (Zieve’s syndrom e). C u rren t be lief is that hypersplenism accom panying the a lc o h o l-in d u c ed fa tty liv e r m ay be responsib le ,4 or tha t the high density o lder cells in Z ieve’s syndrom e have d e c re a s e d s ta b ili ty o f th e p y ru v a te kinase enzyme, thus, potentially in ter fering with ATP production and support of the sodium, potassium pum p and the contractile elem ents of the cell m em brane.
The m ost severe type of hem olytic anemia associated with liver disease is sp u r cell anem ia, an ab n o rm ality of erythrocyte m em brane cholesterol and phospholipid content found in patients with severe alcoholic cirrhosis, neonatal hepatitis, and Wilson’s disease. Jaundice is prom inent, splenomegaly is constant. Ascites and encephalopathy are often seen. In many patients, spur cell anemia precedes death by only a few weeks or months. Only a few patients have been o b se rv ed to reco v e r from sp u r cell h em o ly tic anem ia if th e ir liv e r d is ease abates.4
In vitro studies show that phospholi p id s are p re se n t in th e e ry th ro cy te m em brane in normal amounts, bu t there is a disproportionate increase in lecithin (PC) and cholesterol is increased 25 to 65 percent. This change results in a C/PL ratio of as m uch as 1.60 com pared to normal of 0.95. The constitution of the
erythrocyte m em branes in this situation seem s to be m ore d e p e n d en t on the C /PL ratio of low density lipopro tein (LDL) than on total serum cholesterol.4
There appears to be a dynamic process of exchange betw een the plasm a LD L and the ery th rocy te m em brane since tra n sfu sed no rm al e ry th ro cy te s w ill develop the same shape changes as the patients own cells. In vitro , these lipid shifts result in cell m em branes with less fluidity. Folding and scalloping of the cell margins are observed suggesting dis tribution of excess cholesterol within the m em brane is uneven.
The morphologic similarity betw een spur cells of liver disease and acantho cytes of patients with congenital abetali- poproteinem ia is striking.11 Although the C/PL ratio of acanthocytes is normal or only slightly increased, the sphingomye lin/lecithin concentrations are reversed resulting in m em brane fluidity changes s im ila r to th e sp u r ce ll. W h ile th e acanthocyte of abetalipoproteinem ia has a markedly shortened life span, anemia is no t severe , p robab ly because the patients do not have portal hypertension or hypersplenism .4
Summary
L iver d isease , e spec ia lly alcohol- induced cirrhosis, has been associated with a variety of erythrocyte membrane lipid changes, (cholesterol, phospholi p ids, phosphatidy l choline su b s titu tions), enzyme inhibition (L-CAT defi ciency, p y ru v a te k inase in stab ility ), electrolyte shifts (echinocytes and sto- matocytes), vitamin E deficiency, and with congestive splenomegaly owing to portal hypertension causing hypersplen ism.
The ab n o rm a litie s in d u ced…
Mechanisms of Hemolysis in Liver Disease
EDWARD. E. MORSE, M .D.
D epartm ent o f Laboratory Medicine, University o f Connecticut School o f Medicine,
Farmington, C T 06032
ABSTRACT
Liver disease, particularly alcoholic cirrhosis, is associated w ith a num ber of interesting chemical changes which result in structural and metabolic abnormalities of the erythrocyte m em brane leading to micro scopically observable cell shape changes and hemolytic anemia varying from very mild to potentially lethal.
Increase in unesterified serum cholesterol owing to lecithin cholesterol acyl transferase (LCAT) deficiency in cirrhosis leads to expansion of the lipid bilayer and macrocytosis without megaloblastic changes in precur sors. Substitutions of phosphotidyl choline (PC) moieties in the erythro cyte lipid bilayer lead to echinocytes (disaturated PC) or to stomatocytes (diunsaturated PC).
In some patients, high density lipoprotein (HDL) abnormalities lead to erythrocyte surface changes causing rapid formation of echinocytes. The rapidity and reversibility of these changes suggest blockade of metabolic transport channels critical to the m aintenance of ery throcyte m em brane shape.
Metabolic changes involving vitamin E deficiency leading to lipid p e r oxidation and pyruvate kinase instability leading to adenosine triphos phate (ATP) reduction have also been invoked to explain hemolysis asso ciated with acute liver damage. The most severe hemolysis in liver disease is associated with acanthocytes (spur cells) and a m arked imbalance in cholesterol-phospholipid ratio. These patients usually have hypersplen- ism , as w ell as rig id e ry th rocy te m em brane transform ations w hich are irreversible.
Any of the o ther ery throcyte m em brane shape changes described appear to be reversible if the liver disease abates, bu t they too may becom e irreversib le if bits of projecting m em brane are repeated ly removed by the macrophages of an enlarged spleen.
Introduction erythrocyte. T here is free exchange of unesterified cholesterol and some phos-
The liver is in large part responsible pholipids betw een the plasma and the for the plasm atic env ironm ent of the ery th rocy te m em brane. The ery thro-
169 0091-7370/90/0600-0169 $00.90 © Institute for Clinical Science, Inc.
1 7 0 MORSE
cy te , hav ing no glycogen sto res , is dep en d en t upon plasm a glucose, the level of which is maintained by the liver. In liver disease, alterations in lecithin cholesterol acyl transferase (LCAT) and in lipoproteins may produce changes in the erythrocyte cholesterol/phosopholi- pid ratio resulting in reduced membrane flexibility and, with processing, by the spleen, irreversible changes in cell shape and hemolysis.
Metabolic functions of the erythrocyte may also be affected by components pro duced by an abnormal liver. Interference with glucose transport across the m em b rane or w ith glycolytic m etabolism w ithin the ery th rocy te may decrease adenosine triphosphate (ATP) which may re su lt in c o n tra c tile p ro te in tetany , osm otic im b a la n c e , shape changes, abnormal m em brane rigidity and early removal of the erythrocyte in the spleen. Portal hypertension as a result of fibrosis (cirrhosis) in the liver may cause conges tive splenomegaly and a form of hyper- splenism with sequestration of erythro cytes in the spleen. In addition, recent evidence exists to suggest that the avail ability of vitamin E may be decreased, particularly in ch ildren , w ith chronic liver disease, possibly accounting for an increased susceptibility of the erythro cytes to intravascular oxidative hem o lysis through lipid peroxidation.
This paper will review the existing data that support the various proposed mechanisms of erythrocyte damage in association with liver disease and will attem pt to provide a unifying hypothesis for the prem ature destruction of erythro cytes in patients with abnorm alities of liver function.
Review of D ata
Lipids account for about 50 percent of th e w eigh t of re d cell m em b ran es .4 Mammalian red cell membranes have a molar ratio of cholesterol to phospholipid of about one.19 Four phospholipids pre
dom inate in hum an erythrocytes: leci th in (phosphatidyl choline), sphingo m y e lin , p h o s p h a tid y l s e r in e , an d phosphatidyl ethanolamine. The interac tions betw een cholesterol and phospho lipids have important consequences for m em brane structure and function.12
C holesterol increases the packing of phospholipids in both layers of the lipid bilayer of the erythrocyte m em brane.8 The close interpositioning of sterols with p h o sp h o lip id s in the lip id b ilayers , which acquire excess cholesterol and phospho lip ids from plasm a, causes a d eg ree of im m obility to be im posed upon the acylcarbon atoms near the lipid bilayer’s inner or outer surface, while increasing the freedom of motion deep w ith in th e hyd rophob ic core of the bilayer creating an in term ediate fluid state .3 The num ber of saturated double bonds w ith in th e ph o sp h o lip id acyl chains in the bilayer is an im portan t determ inant of their fluid qualities.20
Cholesterol itself also has an important effect on m em brane fluidity. By in te r posing itself betw een adjacent phospho lipid acyl fatty acid chains, it increases the degree of o rder w ithin the m em brane bilayer.7 Unesterified cholesterol and phospholipids of plasma lipoproteins readily exchange with their counterparts in the lipid bilayers of the erythrocyte m em brane.5 In patients whose erythro cytes acqu ire excess cho lestero l and phospholipids from plasma lipoproteins in a one to one ra tio , th e red cells become target cells and the m em brane expands in a uniform fashion.6'21 This phenom enon is seen in cirrhosis, hepati tis, and obstructive jaundice. In obstruc tive jaundice, the increase in cholesterol may be as great as 75 percent and that of phospholipid up to 60 percent.4 A simi lar abnorm ality has been described in patients with congenital absence of leci- th in -c h o le s te ro l acyl tran sfe ra se (L- CAT).10 This L-CAT deficiency has com monly been found in liver disease,6 but
MECHANISMS OF HEMOLYSIS IN LIVER DISEASE 1 71
correlation betw een serum L-CAT activ ity and abnorm alities of red cell lipids has been difficult to demonstrate.
Target cells do not leave the circula tio n p re m a tu re ly in th e absence of hypersplenism ; however, larger imbal ances in the cholesterol/phospholip id ratio often lead to formation of echino- cytes (with sea urchin-like projections) and eventually to acanthocytes (spur or thorn cells) which are more rigid and are rapidly removed from the circulation by the spleen.
R ecent studies have suggested that echinocytes are m ore readily found in the blood of patients with liver disease if the cells are examined in wet films or by scanning e lec tron m icroscope ra th e r than in fixed smears by light m icros copy. 12 Owen et al18 showed that echino- c y tes can b e p ro d u c e d from nom al w ashed erythrocytes w hen incubated with the high density lipoprotein (HDL) fraction from some jaundiced patients. O ther plasma fractions (LDL, VLDL, Alb) are not apparently echinocytogenic. This type of echinocyte change is rapidly reversible if the abnormal cells are incu bated in normal HDL. The echinocyte change occurs in m inu tes and is not accom panied by change in m em brane cho lestero l con ten t no r by up take of lysolecithin nor bile acids.
T he H D L ’s from th ese jau n d ic ed patien ts show binding characteristics that were saturable at an estimated 5000 sites per cell. Normal H D L or nonechin- ocytogenic p a tie n t H D L ’s show only n onsatu rab le b ind ing characteristics. Pretreatm ent of erythrocytes with pro- nase or trypsin reduces or elim inates b ind ing by abnorm al H D L . Thus, it seems likely that echinocytosis in associ ation w ith liver d isease may, in some cases, be associated w ith m em brane changes induced by a rapid shift (in glu cose or electrolytes) induced by H D L related binding to specific receptor sites in the erythrocyte surface membrane.
G ra h n ,14 as early as 1968, d em on strated that serum from echinocyte-con- taining blood could induce echinocyte form ation in norm al cells. Similarly, serum from blood containing acantho- cytes also induces formation of echino cytes, but not acanthocytes. Thus, it is argued, an additional factor acting in vivo may be necessary for the production of acan thocy tes, p robab ly by excess m em brane removal in the spleen.
S ubsequen t stud ies by H ui e t a l15 show that some low density lipoprotein (LDL) fractions of norm al serum p ro duce echinocytes from discocytes. The active constituent appears to be apolipo- protein B (apo B). The H D L of patients with liver disease is enriched with apo E, which competes with apo B of normal LD L for apo B, E receptors on the sur face of a num ber of nucleated cells.18 In the N etherlands,16 phosphatidyl choline specific transfer protein from beef liver has been used to replace native phos phatidyl choline (PC) molecules with a variety of PC species differing in fatty acid composition w ithout changing the total phospholipid content of the m em brane. No morphological changes were noted when PC was replaced by mono sa tu ra ted species like 1-palm itoyl, 2 oleoyl PC; 1 palmitoyl, 2 linoleoyl PC, egg PC or PC from rat liver microsomes; however, replacem ent with disaturated species, 1,2 dimyristoyl PC, 1,2 dipalmi- toyl PC, and 1,2-distearoyl PC resulted in formation of echinocytes. I t should be n o ted th a t PC rep lacem en t using C 14 labe lled com pounds is m easured in hours (10 to 20 p e rc e n t in one to two hours, 40 to 60 percent in four to six hours).
In te re s tin g add itional observations include the form ation of stom atocytes upon replacem ent with PC species con taining two unsaturated fatty acids, e.g., 1,2 dioleoyl PC and 1,2 dilinoleoyl PC. These studies suggest that the discoid shape of the hum an erythrocyte is opti
1 7 2 MORSE
mally stabilized by PC species that con tain one saturated and one mono- or di unsaturated fatty acid, and that the cell tolerates only lim ited variations in the species composition of its PC. Saturated PC seem s to concentrate in the outer monolayer causing outward projection, while unsaturated PC inserts deeper in the lipid bilayer causing inward projec tion and stomatocytosis.
D ra m a tic changes in e ry th ro c y te shape are observed when the unsatura tion index of PC, defined as the total num ber of double bonds relative to that of the fatty acids, becomes lower than 0.5 or higher than 1.0. If the erythrocyte PC is replaced by dimyristoyl PC, dipal- mitoyl PC or distearoyl PC, the index decreases to 0.5. Echinocytes are formed when about 25 percent of native PC is rep laced by dipalm toyl PC. Stomato- cytes form if diunsaturated PC is substi tuted at 20 to 30 percent and hemolysis occurs at about 40 percent replacement.
Spheroechinocytes can also be found if the original PC is replaced by 1-palmi- toyl, 2 arachidonoyl PC. At 20 percent replacem ent, shape change begins. At 60 percent replacem ent, when the double bond index becomes greater than 1.0, spheroech inocy tes are obvious along with many cells showing typical dimples, which may be due to the loss of internal osmotic pressure, since the cells begin leaking ions at this stage.
Stomatocytes, produced by a bending or folding of the m em brane inwards, form after replacem ent of more than 40 percent of the PC with 1,2 dioleoyl PC. Typical stomatocytes are observed when 20 to 30 percent of the PC is replaced by 1,2 dilinoleoyl PC after a six hour incu b a tio n . F u r th e r increase causes cell lysis.
Stomatocytosis has been reported in association w ith alcoholic c irrhosis .9 Four patien ts developed hem olysis of varying severity, and one appeared to have an intrinsic intracorpuscular defect
because norm al cells incubated in his serum did not acquire the shape change, nor did they become stomatocytes after infusion in to his b lood in vivo . The defect appeared to be rela ted to acute alcoholic binges in these four patients and were not accompanied by lipidemia.
Stomatocytosis has also been reported in association with a hereditary red cell anomaly in which there is a remarkable reversal of sodium and potassium con centrations in the erythrocyte.17 It seems possible that lipid changes in the eryth rocyte m em brane during acute alcoholic bou ts m ay affect th e p e rm eab ility of e ry th ro cy te s to g lucose o r calcium , thereby decreasing ATP, causing tetany of contractile proteins and d irectly or indirectly interferring with the sodium, potassium pump.
Hem olytic anemia and susceptibility to H20 2 hemolysis have been described in ch ildren w ith vitam in E deficiency and chronic liver disease.10 Thirty-four children (one to 14 years) with severe chronic liver diseases were studied, 16 of whom w ere also v itam in E defic ien t (vitamin E < 5 mg per ml). Vitamin E may be deficient in liver cirrhosis as a consequence of intestinal malabsorption related to cholestasis. Tocopherol (vita min E) provides a protective effect on lip id perox idation w ithou t w hich the cells may be susceptible to oxidative lysis and a shortened survival. W ith respect to controls, cirrhotic ch ild ren in both vitamin E and non vitamin E deficient g ro u p s show ed s ig n ifican tly h ig h e r values of serum unesterified cholesterol, triglycerides, and total serum phospholi pid concentration. There was an intense increase in phosphatidyl choline content in the erythrocyte membrane, as well as in the serum, that was statistically signif icant in the vitamin E deficient group. The fundamental biochemical lesion in the erythrocyte m em brane may be due to accelerated lipid peroxidation induc ing fo rm ation of m alo ny ld ia ldehyde
MECHANISMS OF HEMOLYSIS IN LIVER DISEASE 1 7 3
which promotes polymerization of cyto- s k e le ta l p ro te in s . T h e s u b s e q u e n t decrease in cell flexibility leads to p re m ature removal of the cell from the cir culation.
In adult patients, a variety of erythro cyte metabolic defects may be induced including pyruvate kinase instability.1213 There is an interesting acute hemolytic anem ia seen in patien ts w ith alcohol- in d u c e d fa tty liv e r a sso c ia te d w ith hypertriglyceridem ia.22 There is little in vitro or in vivo evidence that increased triglycerides per se are associated with h em o ly tic anem ia in th is syndrom e (Zieve’s syndrom e). C u rren t be lief is that hypersplenism accom panying the a lc o h o l-in d u c ed fa tty liv e r m ay be responsib le ,4 or tha t the high density o lder cells in Z ieve’s syndrom e have d e c re a s e d s ta b ili ty o f th e p y ru v a te kinase enzyme, thus, potentially in ter fering with ATP production and support of the sodium, potassium pum p and the contractile elem ents of the cell m em brane.
The m ost severe type of hem olytic anemia associated with liver disease is sp u r cell anem ia, an ab n o rm ality of erythrocyte m em brane cholesterol and phospholipid content found in patients with severe alcoholic cirrhosis, neonatal hepatitis, and Wilson’s disease. Jaundice is prom inent, splenomegaly is constant. Ascites and encephalopathy are often seen. In many patients, spur cell anemia precedes death by only a few weeks or months. Only a few patients have been o b se rv ed to reco v e r from sp u r cell h em o ly tic anem ia if th e ir liv e r d is ease abates.4
In vitro studies show that phospholi p id s are p re se n t in th e e ry th ro cy te m em brane in normal amounts, bu t there is a disproportionate increase in lecithin (PC) and cholesterol is increased 25 to 65 percent. This change results in a C/PL ratio of as m uch as 1.60 com pared to normal of 0.95. The constitution of the
erythrocyte m em branes in this situation seem s to be m ore d e p e n d en t on the C /PL ratio of low density lipopro tein (LDL) than on total serum cholesterol.4
There appears to be a dynamic process of exchange betw een the plasm a LD L and the ery th rocy te m em brane since tra n sfu sed no rm al e ry th ro cy te s w ill develop the same shape changes as the patients own cells. In vitro , these lipid shifts result in cell m em branes with less fluidity. Folding and scalloping of the cell margins are observed suggesting dis tribution of excess cholesterol within the m em brane is uneven.
The morphologic similarity betw een spur cells of liver disease and acantho cytes of patients with congenital abetali- poproteinem ia is striking.11 Although the C/PL ratio of acanthocytes is normal or only slightly increased, the sphingomye lin/lecithin concentrations are reversed resulting in m em brane fluidity changes s im ila r to th e sp u r ce ll. W h ile th e acanthocyte of abetalipoproteinem ia has a markedly shortened life span, anemia is no t severe , p robab ly because the patients do not have portal hypertension or hypersplenism .4
Summary
L iver d isease , e spec ia lly alcohol- induced cirrhosis, has been associated with a variety of erythrocyte membrane lipid changes, (cholesterol, phospholi p ids, phosphatidy l choline su b s titu tions), enzyme inhibition (L-CAT defi ciency, p y ru v a te k inase in stab ility ), electrolyte shifts (echinocytes and sto- matocytes), vitamin E deficiency, and with congestive splenomegaly owing to portal hypertension causing hypersplen ism.
The ab n o rm a litie s in d u ced…
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