erythrocyte metabolism alpha beta thalassemia · sium flux (4). these changes seem to be unrelated...

9
Influence of Hemoglobin Precipitation on Erythrocyte Metabolism in Alpha and Beta Thalassemia DAVID G. NATHAN, THOMAS B. STOSSEL, ROBERT B. GUNN, HAROLD S. ZAmOwsKY, and Mrrsuico T. LAFORET From the Hematology Research Laboratory of the Department of Medicine of the Children's Hospital Medical Center, Boston, Massachusetts 02115, and the Department of Pediatrics, Harvard Medical School, Boston, Massachusetts 02115 A B S T R A C T Certain aspects of the metabolism of centrifuged young and old erythrocytes in hemoglobin H disease have been examined and compared with similar studies of beta thalassemia and normal cells. Glycolysis, hexose monophosphate shunt activity (HMPS), potas- sium flux, and glutathione (GSH) content were mea- sured. The distributions of hemoglobins H and F, as well as the activities of erythrocyte glucose-6-phosphate dehydrogenase (G6PD) and glutamic oxalacetic trans- aminase (GOT), were utilized for estimations of the relative ages of the cell samples. The young erythrocytes in hemoglobin H disease differed in several respects from older hemoglobin H cells. They contained more soluble hemoglobin H and GSH and, after splenectomy, fewer inclusions. HMPS activity was subnormal in hemoglobin H young cells and rose to normal activity in old cells. Potassium flux tended to increase in old cells when in- clusions were present. Beta thalassemia young cells contained less hemoglobin F and, after splenectomy, more inclusions than old cells. In addition, they had markedly increased glycolysis and HMPS activity. GSH was randomly distributed. Potas- sium flux was increased in younger cells and particularly increased when inclusions appeared in younger cells after splenectomy. The results are interpreted to indicate that inclusion formation is associated with increased erythrocyte cation permeability in the thalassemia syndromes. This is not related to the level of intracellular GSH. The decreased HMPS activity in young hemoglobin H cells may be due to the presence of the extra thiols of soluble hemoglobin H which can act as a reducing agent. Received for publication 9 February 1968 and in revised form 13 August 1968. The substitution of hemoglobin H for glutathione in this capacity would then spare the NADPH-requiring glu- tathione reductase system. As a consequence, HMPS activity would decline. However, in older cells the oxidized hemoglobin H precipitates; these must rely upon GSH and glutathione reductase activity for thiol reduction capacity. Accordingly, HMPS activity in- creases to normal in the old cell population. INTRODUCTION The erythrocytes of patients with beta thalassemia and hemoglobin H disease can be separated by centrifugation into young and old cohorts (1-4). In beta thalassemia the young cells contain less hemoglobin F (2, 3) and, if the patient is splenectomized, more insoluble mem- brane-bound inclusions of precipitated alpha chains (4- 7). In hemoglobin H disease, the young cells contain more soluble hemoglobin H, whereas inclusions of pre- cipitated beta chains are detected in the older cells (1, 4, 8, 9). To examine some of the metabolic consequences of these hemoglobin inclusions, we evaluated the char- acteristics of the centrifuged cell cohorts with respect to glycolysis, HMPS activity, lactate production, GSH concentration, G6PD activity and potassium flux. These data were compared to similar studies of centrifuged normal cells. Since the inclusions were localized in co- horts of different ages in the two forms of thalassemia, a unique opportunity was provided for study of their age-dependent influences. The data confirmed that the development of hemoglobin precipitates in thalassemic erythrocytes is associated with alterations of membrane cation permeability characterized by enhanced potas- The Journal of Clinical Investigation Volume 48 1969 33

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Page 1: Erythrocyte Metabolism Alpha Beta Thalassemia · sium flux (4). These changes seem to be unrelated to cellular GSH (10). A second purpose of this study was to evaluate the influence

Influence of Hemoglobin Precipitation

on Erythrocyte Metabolism

in Alpha and Beta Thalassemia

DAVID G. NATHAN, THOMASB. STOSSEL, ROBERTB. GUNN,

HAROLDS. ZAmOwsKY,and Mrrsuico T. LAFORET

From the Hematology Research Laboratory of the Department of Medicineof the Children's Hospital Medical Center, Boston, Massachusetts 02115,and the Department of Pediatrics, Harvard Medical School,Boston, Massachusetts 02115

A B S T R A C T Certain aspects of the metabolism ofcentrifuged young and old erythrocytes in hemoglobin Hdisease have been examined and compared with similarstudies of beta thalassemia and normal cells. Glycolysis,hexose monophosphate shunt activity (HMPS), potas-sium flux, and glutathione (GSH) content were mea-sured. The distributions of hemoglobins H and F, aswell as the activities of erythrocyte glucose-6-phosphatedehydrogenase (G6PD) and glutamic oxalacetic trans-aminase (GOT), were utilized for estimations of therelative ages of the cell samples. The young erythrocytesin hemoglobin H disease differed in several respects fromolder hemoglobin H cells. They contained more solublehemoglobin H and GSHand, after splenectomy, fewerinclusions. HMPSactivity was subnormal in hemoglobinH young cells and rose to normal activity in old cells.Potassium flux tended to increase in old cells when in-clusions were present.

Beta thalassemia young cells contained less hemoglobinF and, after splenectomy, more inclusions than old cells.In addition, they had markedly increased glycolysis andHMPSactivity. GSHwas randomly distributed. Potas-sium flux was increased in younger cells and particularlyincreased when inclusions appeared in younger cells aftersplenectomy.

The results are interpreted to indicate that inclusionformation is associated with increased erythrocyte cationpermeability in the thalassemia syndromes. This is notrelated to the level of intracellular GSH.

The decreased HMPSactivity in young hemoglobin Hcells may be due to the presence of the extra thiols ofsoluble hemoglobin H which can act as a reducing agent.

Received for publication 9 February 1968 and in revisedform 13 August 1968.

The substitution of hemoglobin H for glutathione in thiscapacity would then spare the NADPH-requiring glu-tathione reductase system. As a consequence, HMPSactivity would decline. However, in older cells theoxidized hemoglobin H precipitates; these must relyupon GSHand glutathione reductase activity for thiolreduction capacity. Accordingly, HMPS activity in-creases to normal in the old cell population.

INTRODUCTIONThe erythrocytes of patients with beta thalassemia andhemoglobin H disease can be separated by centrifugationinto young and old cohorts (1-4). In beta thalassemiathe young cells contain less hemoglobin F (2, 3) and,if the patient is splenectomized, more insoluble mem-

brane-bound inclusions of precipitated alpha chains (4-7). In hemoglobin H disease, the young cells containmore soluble hemoglobin H, whereas inclusions of pre-cipitated beta chains are detected in the older cells (1, 4,8, 9). To examine some of the metabolic consequencesof these hemoglobin inclusions, we evaluated the char-acteristics of the centrifuged cell cohorts with respectto glycolysis, HMPSactivity, lactate production, GSHconcentration, G6PD activity and potassium flux. Thesedata were compared to similar studies of centrifugednormal cells. Since the inclusions were localized in co-

horts of different ages in the two forms of thalassemia,a unique opportunity was provided for study of theirage-dependent influences. The data confirmed that thedevelopment of hemoglobin precipitates in thalassemicerythrocytes is associated with alterations of membranecation permeability characterized by enhanced potas-

The Journal of Clinical Investigation Volume 48 1969 33

Page 2: Erythrocyte Metabolism Alpha Beta Thalassemia · sium flux (4). These changes seem to be unrelated to cellular GSH (10). A second purpose of this study was to evaluate the influence

sium flux (4). These changes seem to be unrelated tocellular GSH (10).

A second purpose of this study was to evaluate theinfluence of soluble hemoglobin H on the HMPS inerythrocytes. A major role of the HMPSin red cells isto provide NADPHfor glutathione reductase, an enzymeactivity which maintains a renewable pool of reducedglutathione. The latter serves as an oxidant buffer forthe thiol groups of red cell hemoglobin and membraneprotein (11-13). When hemoglobin A is exposed to anoxidant strong enough to cause its denaturation and pre-cipitation in vitro, glutathione is bound, presumably asa disulfide, to the precipitating protein (11). GSHmayalso be bound to certain abnormal hemoglobins whichprecipitate in vivo during the life span of the red cell(1, 10, 14). Among these abnormal hemoglobins is he-moglobin H (1, 8, 9), a tetramer of beta chains witheight rather than two exposed thiols per molecule (15).Recent studies have indicated that hemoglobin H mayhave a functional redox potential very close to that ofglutathione (14). Therefore, it might substitute for glu-tathione and serve as an oxidant buffer for the cell un-til oxidative precipitation of this hemoglobin occurred.Oxidative precipitation of hemoglobin H is irreversibleand the precipitates cannot be reduced by an NADPH-linked enzyme system. If hemoglobin H can act as areducing agent, one might expect HMPSactivity tobe decreased in cells containing soluble hemoglobin Hand to increase to normal when the hemoglobin H pre-cipitates, because the NADPH-dependent glutathionereductase system would then be left to perform this es-sential buffering function. In these studies, it was foundthat in hemoglobin H disease, HMPSactivity was indeedlower in the young cells, where soluble hemoglobin Hpredominated, than in the old cells, where inclusions pre-dominated.

METHODSStudies were performed on blood from five patients withhemoglobin H disease, six patients with Cooley's anemia,two patients with S-thalassemia and a group of eight normalcontrols. Percentages of hemoglobins A and H were deter-mined by starch granule electrophoresis (16). None of thepatients had received blood transfusions in recent years.

Venous blood was collected with preservative-free heparin 1(0.1 mg/ml blood) and studied immediately. The blood wasseparated into layers of young and old cells by centrifuga-tion of 15 ml of whole blood in celluloid tubes at 20,000 gfor 1 hr. After this procedure, the relatively younger largecells are found at the top of the tube, and the older smallercells segregate at the bottom of the tube. The buffy coatand unavoidably the top 1% or 2% of erythrocytes wereremoved by aspiration. The remaining top (reticulocyte-richor young) and bottom (reticulocyte-poor or old) 1.5-mlfractions were then collected and resuspended at hematocritsof approximately 40% in the autologous plasma. Thus, the

1 Connaught Medical Research Laboratories, Toronto,Ontario, Canada.

top and bottom fractions each contained approximately 20%of the initial volume of cells.

White blood cell counts, reticulocyte counts, inclusion bodycounts and measurement of red cell indices were performedon all fractions by standard methods (17). Inspection ofreticulocyte smears revealed virtually absent platelets. Theratio of leukocytes to red cells was less than 2 X 10' in toplayer cell suspensions and less than 10-' in bottom layersuspensions.

Minimum HMPSactivity by evolution of 14CO2 fromglucose-1-`C was studied in duplicate by modifications of themethod of Murphy (18). After incubation in the presence of1 ,uc of glucose-1-"4C, a small piece of filter paper, which hadbeen suspended from the inner edge of the serum cap of theincubation flasks before incubation, was soaked with KOH.The blood was acidified with 0.1 ml of 0.5 N H2SO4. Theacid and alkali additions were made with syringes andhypodermic needles. The filter papers and appropriate stan-dards applied to filter paper were dried and counted for 14Cactivity in a liquid scintillation spectrometer (19). Thediffusion stystem for 14CO2 was evaluated with Na2,4COsstandards and found to be quantitative. Blanks were per-formed by acidification and diffusion immediately after theaddition of glucose-1-14C to the incubate. Murphy's methodof calculation was utilized to derive a value for shunt activityin terms of mmglucose/liter cells per hr.

The effects of stimulation of HMPSactivity by glucoseand methylene blue were measured by the methemoglobinreduction technique of Beutler and Baluda (20). Methemo-globin and hemoglobin were assessed by the technique ofEvelyn and Malloy (21). The total hemoglobin in thehemolyzates remained constant even in samples containinghemoglobin H. Failure to recognize possible precipitation ofhemoglobin H by methylene blue might have been becausethe molar hemoglobin to dye ratio was approximately 300rather than the ratios approaching unity that have beenutilized in other studies of hemoglobin H precipitation bysuch dyes (6). Because it is difficult to remove all nitritefrom the cells (22), this technique indicates the net rateof methemoglobin reduction rather than absolute reductionrates. Therefore, only the periods of linear methemoglobinreduction were considered in the calculations of comparativereduction rates.

Glucose consumption and lactate production during incu-bation were measured on perchloric acid filtrates by theglucose oxidase (23) and the LDH: NADH (24) methodsrespectively.

GSH concentrations in top and bottom cell suspensionswere measured by the method of Beutler, Duron, and Kelly(25). G6PD and GOT activities in hemolyzates wererespectively measured by suitable modifications of the tech-nique of Zinkham and Lenhard (26) and by the method ofKarmen, Wroblewski, and LaDue (27).

Potassium fluxes were measured by micromodifications ofthe methods of Solomon (28).

The top and bottom layer cells were of different sizedistribution, the top cells having a larger mean volume thanthe bottom. To develop meaningful comparisons between thelayers the measurements described above (with the excep-tion of G6PD and GOT activities which were expressed aschanges of optical density at 340 ,u/110" erythrocytes), weremodified in terms of a standard cell volume of 82 ,A.For example, the measured value for glucose consumptionper liter of bottom layer cells was multiplied by the ratio of(MCV bottom layer cells)/(MCV standard cells) in whichMCV stands for mean corpuscular volume. The correc-tions were not entirely suitable for statistical compari-

34 Nathanw Stossel Gunn, Zarkowsky, and Laforet

Page 3: Erythrocyte Metabolism Alpha Beta Thalassemia · sium flux (4). These changes seem to be unrelated to cellular GSH (10). A second purpose of this study was to evaluate the influence

sons of normal cells to the cells of beta thalassemia. Sizedistribution studies showed that the cells of splenectomizedbeta thalassemia patients were not merely smaller thannormal cells. The variances of their mean volumes were con-siderably greater than those of normal cells so that simplestatistical comparisons on the basis of mean volume were oflimited significance.2 On the other hand, the variances ofthe mean volume of beta thalassemia and hemoglobin H dis-ease top and bottom layer cells were not as widely different.Nor were the variances of normal and hemoglobin H cellssufficiently dissimilar to limit statistical comparisons oftheir metabolic functions on the basis of a correction forcell volume. With these limitations considered, the correcteddata were recorded in the tables and figures.

RESULTS

The adequacy with which centrifugation separated cellsinto young and old populations is illustrated in TableI where the substantial differences in reticulocytecounts and GOTand G6PD activities between top andbottom fractions are tabulated. As expected (1, 2, 4,29), the young cells were in the top layers and theolder cells in the bottom fractions.!

Glucose metabolism in young and old cells. Therates of total glucose consumption, lactate production,glucose oxidation by the HMPS, and reduction ofmethemoglobin with methylene blue and glucose areshown in Table I.

2 The authors are grateful to Dr. Frederick Stohlman, Jr.for performing some of these size distribution studies.

3 As previously noted (4), we have further evaluated thevalidity of the separation method by administration of '9Feto one adult patient with Cooley's anemia. 5 days after iso-tope administration nearly all of the radioactivity waslocalized in the large hypochromic cells of the upper layerwhich were rich in reticulocytes and poor in hemoglobin F.

4.4

4.0

TOw~P 3.6-BOTTOM

RATIO 3.2

2.8'

2.4-

2.0O

1.6-

I.2~

0.8

0.4-

H

Lif

rA

As anticipated (30-33), total glucose consumptionand lactate production in normal young cells wasgreater than in normal old cells. In beta thalassemiaerythrocytes the same pattern was detected, an expectedfinding in view of the large number of reticulocytesand nucleated red blood cells in the top layer cells ofthese patients. However, in four of the five studies ofhemoglobin H, glucose consumption and lactate pro-duction were greater in old cells than in young cells,despite relatively large numbers of reticulocytes in thetop cell layers. HMPSactivity in young normal cells,measured either by the '4CO2 or by the methemoglobinreduction methods, was greater than HMPSactivity inold normal cells (31-33). The same was true to an evengreater extent of the cells of patients with beta thalas-semia. On the contrary, the HMPSactivity of old cellsfrom patients with hemoglobin H disease was equal toor greater than that of young cells from these patients.These differences in the cellular localization of shuntactivity are demonstrated in Fig. 1 in which the top andbottom cell ratios for HMPSactivity are graphicallyshown. This figure shows that the differences in shuntactivity between young and old cell fractions were easilydemonstrable by the glucose-1-"C technique whereasthey were barely if at all discernible by the the methemo-globin reduction technique since the latter only mea-sures the potential maximal activity of the shunt basedlargely upon available glucose, NADP, G6PD, andNADPH:methylene blue reductase activity in the bot-tom and top layer cells.

G6PD activity and GSHconcentrations in young andold cells. The relative youthfulness of all cells from

EI MEASUREDWITH 4 C-I -GLUCOSEmMGLUCOSE/liter CELLS/hr

[ MEASUREDWITH METHEMOGLOBINREDUCTION

%METHEMOGLOBINREDUCED/min

N(8)w

FIGURE 1 Relative predominance of total HMPSactivity in top and bottom cell layers measured inpatients with hemoglobin H disease (H), beta thal-assemia (pt), and normals (N).

Hemoglobin Precipitation and Red Cell Metabolism in Thalassemia 35

:

I

t N(8)

Page 4: Erythrocyte Metabolism Alpha Beta Thalassemia · sium flux (4). These changes seem to be unrelated to cellular GSH (10). A second purpose of this study was to evaluate the influence

TABLE I

Metabolic

Hemo-Sple- globin Hemo-nec- Cell inclu- globin Reticu- Lactate Glucose

Patient Diagnosis* tomy fraction MCV sions H or F locytes production consumption

u3 % %

H (9%) Yes Top 75 1.6 14 HBottom 53 6.6 6.2 H

L. D. H (8%) No TopBottom

M. L. H (10%) Yes TopBottom

A. R. H (15%) Yes TopBottom

W.D. H(8%) No TopBottom

S. D. CA Yes TopBottom

J. C. CA No TopBottom

C. Z. CA Yes TopBottom

G. P. CA No TopBottom

P. M. CA Yes TopBottom

P. F. CA Yes TopBottom

S. G. S-T

A.G.R. S-T

Yes TopBottom

No TopBottom

75 0 18 H50 0.1 5 H

84 3.854 12.8

75 3.453 11.8

12.21.0

10.02.3

15 H 22.64 H 8.0

30 H 16.28 H 2.3

81 0 12 H63 6.2 2 H

69 50 7 F47 5 32 F

65 0 8 F51 0 34 F

113 45 22 F71 7 56 F

75 0 20 F64 0 40 F

89.5 38 22 F74 3 37 F

102 47 30 F68 5 44 F

95 15 13 F75 2 25 F

75 0 7 F63 0 13 F

10.81.8

18.61.0

7.20.3

30.05.0

15.04.0

28.01.4

25.01.2

14.40.9

10.02.0

mM/lifer cells per hr:

10.5 4.612.3 5.3

8.58.6

10.012.0

7.010.0

11.88.8

10.64.6

12.1

3.44.3

4.25.2

3.74.4

4.93.6

5.03.8

3.52.0

5.32.7

4.51.3

6.13.4

5.03.0

Normal controls (8) No Top 91 ±3.0§ 0Bottom 82 43.9 0

3.1 40.8§ 5.8 ±0.7§ 3.1 +0.5§0.1 0.1 2.8 40.4 1.5 40.3

* H, hemoglobin H; CA, Cooley's anemia; S-T, S-thalassemiat Corrected to a standard cell volume of 82 ;pB.§ Mean ASDof eight separate controls.11 Range of three determinations.

patients with hemoglobin H disease and beta thalassemiais shown by their high G6PDand GOTactivities com-

pared with those of normal cells (Table I and II).The G6PD and GOTactivites of young cells from allsubjects were greater than those of old cells.

As described previously (14), the GSH content ofnormal old cells was only slightly less than that of

young cells; but the mean GSH concentration of oldhemoglobin H erythrocytes was only 47.8 +7.5 mg/100ml cells, significantly less GSH than was measured innormal old cells (P <0.01) and 32% less GSH thanwas found in young hemoglobin H cells (P < 0.05).

The distribution of glutathione in young and old cellsin the beta thalassemia patients was quite widely scat-

36 Nathan, Stossel, Gunn, Zarkowsky, and Laforet

V. J.

Page 5: Erythrocyte Metabolism Alpha Beta Thalassemia · sium flux (4). These changes seem to be unrelated to cellular GSH (10). A second purpose of this study was to evaluate the influence

Studies

Hexose monophosphateshunt activity

Methemoglobin reduc-Consumption tion by nitrited ery- Enzyme activity Potassium flux

of 14C-.. throcytes withglucose methylene blue G6PD GOT Glutathione in out net

mM/liter %methemoglobin units/lO10 cells mg/1OO mEq/liter cells per h4rml cellst

65.345.9

- 61.936.0

- 71.2- 47.0

- 82.4- 58.9

70.451.4

52.460.7

54.465.9

3.43.6

2.32.1

3.23.6

2.53.5

3.52.2

7.52.9

5.52.7

- - 12.7- - 5.4

8.35.1

4.1 -0.74.4 -0.8

2.9 -0.62.3 -0.2

3.4 -0.23.8 -0.2

2.7 -0.23.2 +0.3

3.4 +0.12.1 +0.1

8.1 -0.63.4 -0.5

5.8 -0.32.5 +0.2

13.94.4

9.05.5

-1.2+1.0-0.7-0.4

8.3 90.03.1 60.0

12.04.0

85.060.0

65.056.6

66.3- 58.9

8.53.2

2.91.8

8.7 -0.22.6 +0.6

3.0 -0.12.0 -0.2

0.27 4-0.14§ 0.90 -+0.18§ 2.32 --0.50§ 2.1-3.111 87 4-5.0§ 1.8 ±t0.2§ 1.7 4-0.2§ 0.1 -±0.01§0.17 -40.07 0.75 -10.25 1.47 -0.50 0.6-0.9 77 ±-7.0 1.9 --0.2 1.7 -±0.2 0.2 ±-0.1

tered and exceeded the range observed in the cells ofnormal individuals. The mean value was not distinguish-able from that of normal. Certainly the uniform reduc-tion of the GSH concentration of old cells in hemo-globin H disease was not regularly observed in thebeta thalassemia patients. These data are graphicallypresented in Fig. 2.

Relationship between cellular GSH, G6PD activityand HMPSactivity with special reference to hemo-globin H disease. Fig. 3 reveals that the distributionof glutathione among top and bottom (young and old)cells was strongly associated with the relative rate ofshunt activity, the shunt activity being higher as glu-tathione declined. However, Table II shows that the

Hemoglobin Precipitation and Red Cell Metabolism in Thalassemia

cells per hr4 reduced/mint7.322.28

0.210.43

0.050.18

0.190.18

0.150.18

0.190.24

1.090.96

0.250.10

0.550.13

1.461.84

3.152.83

2.062.33

1.701.80

1.251.25

2.902.30

1.501.00

3.322.21

1.741.10

3.662.16

4.092.54

3.801.62

2.301.74

3.561.82

3.970.86

0.350.17

0.420.15

2.162.43

7.124.51

37

Page 6: Erythrocyte Metabolism Alpha Beta Thalassemia · sium flux (4). These changes seem to be unrelated to cellular GSH (10). A second purpose of this study was to evaluate the influence

TABLE I IHemoglobin HDisease, Relationship of HMPSActivity to Cell Age and G6PDActivity

Hemoglobin H disease Normal

Top Bottom Top Bottom

HMPSactivity, mM/liter cells per hr 0.16 ±0.06 0.24 40.096 0.27 ±0.14 0.17 ±0.07P = 0.02-0.01 P = 0.1-0.05

G6PDunits 3.32 ±0.82 1.92 ±0.5 2.32 40.40 1.47 ±0.50P < 0.001 P = 0.02-0.01

HMPS P < 0.001Ratio of G6PD0.049 ±0.03 0.129 40.04 0.116 ±0.037 0.115 ±0.046

P = 0.02-0.01

influence of glutathione content on shunt activity maybe quite indirect at least in hemoglobin H disease. Thistable compares shunt activity and G6PD activity in topand bottom cell layers in hemoglobin H disease and innormals. It is notable that the utilization of glucose viathe hexose monophosphate shunt was absolutely lowerin hemoglobin H disease top layer cells than in normaltop layer cells. When an attempt was made to normalizethe potential shunt activity of normal and hemoglobin Htop layer cells by construction of a ratio of shunt ac-tivity to G6PD activity, the difference between thehemoglobin H and normal top layer cells was evenmore marked. On the other hand, bottom layer (old)hemoglobin H cells had a somewhat higher shunt ac-tivity than bottom layer normal cells. When normalizedto a constant G6PD activity, the shunt rates in bottomlayer hemoglobin H cells and normal bottom layer cells

1.8.

1.6-GSH

mg/IOOml CELLS 1.4,TOP/BOTTOM

RATIO 1.2

1.0

0.8

0.6

0.4

0.2

H

.'N N8

FIGURE 2 Relative predominance of glutathione concentra-tion in top and bottom cell layers.

were the same. Thus, it would appear that the fallingGSH concentration in hemoglobin H cells was associ-ated with an increase of HMPSactivity from belownormal toward normal rather than an increase to levelsabove normal. Table I shows that the decline of glu-tathione in aging hemoglobin H cells was also associatedwith a reduction in soluble hemoglobin H, a fact ex-pected from the previous investigations of Rigas andKoler (1).

Potassium flux in young and old cells. There wereno detectable differences in potassium flux among topand bottom cell layers of normal erythrocytes. In patientswith either beta thalassemia or hemoglobin H diseasewho were not splenectomized and whose red blood cellsdid not contain inclusions, the young red cells had ahigher rate of flux than bottom layer cells (Fig. 4).This result was expected in view of the predominanceof young cells in the upper layers (34). Splenectomizedpatients with the two forms of thalassemia had definitedifferences in red cell potassium fluxes. In splenecto-mized patients with beta thalassemia, potassium fluxwas considerably higher in the young cells in whichinclusions were present. In hemoglobin H disease, thefluxes tended to be equal in top and bottom layer cellswith a slight predominance in bottom layer cells whereinclusions were present.

DISCUSSIONThe role of unbalanced hemoglobin synthesis and he-moglobin subunit precipitation in the erythrocyte de-struction which characterizes thalassemia has attractedincreasing attention. In hemoglobin H disease, the j4tetramer slowly precipitates from the cell during itslife span (1) although some precipitates may also befound in the bone marrow (35). In homozygous betathalassemia very large precipitates are easily detectedin many marrow cells (5). These either are evulsedfrom the cells by the spleen or the cells containing them

3f Nathan, Stossel4 Gunn, Zarkowsky, and Laforet

Page 7: Erythrocyte Metabolism Alpha Beta Thalassemia · sium flux (4). These changes seem to be unrelated to cellular GSH (10). A second purpose of this study was to evaluate the influence

GSHCONCENTRATIONSTOP/BOTTOMRATIOrng/lOOml CELLS

2.0

1.8

1.6.

1.4 F

I .2

1.0

0.8

0.6

0.4

0.2

0 I Iw _

0 02 0.4

I 0H

FIGuRE 3 Linear relation of GSH con-centration to total HMPSactivity in topand bottom cell layers. The symbol Nrepresents the mean of eight determina-tions in normal subjects.

Q6 0.8 1.0 12 1.4 1:6 1.8 2.0 22 2.4 2.6 2.8

HMPSACTIVITY: TOP/BOTTOMRATIO (mM '4C-I-GWCOSE CONSUJMED/liter CELLS/hr)

are destroyed in the spleen within hours after they arenewly delivered to the circulation(4). Thus inclusionsare found in young cells in beta thalassemia and oldcells in hemoglobin H disease. These differences inprecipitate localization contribute to the differencesin severity of the two syndromes. In hemoglobin H dis-ease, there is less destruction of developing erythro-blasts within the marrow, and the rate of removal ofnewly formed peripheral blood cells, though increasedabove normal, is not extreme (36). In beta thalassemia,however, massive destruction of newly formed cells inthe marrow (38) as well as in the peripheral blood(3) constitutes one of the major clinical problems inthe management of this disorder.

SPLENECTOMIZED

4-

K+ EFFLUXTO TTO

3.

2-

j

At

UO

NONSPLENECTOMIZED

pt

H

FIGURE 4 Relative predominance of K+ efflux in top andbottom cell layers in splenectomized and nonsplenectomizedpatients with hemoglobin H disease and beta thalassemia.

The pathophysiological consequences of unbalancedhemoglobin synthesis in thalassemia also provide amechanism for evaluation of age-dependent separationsof thalassemic erythrocytes. In beta thalassemia hemo-globin F has a longer life span than hemoglobin A (3).Hence, any population of thalassemic erythrocyteswhich contains a higher hemoglobin F concentrationthan that of another sample of cells from the sameindividual must have an older mean age. Advantagewas taken of this fact by Loukopoulos and Fessas (2)in their studies of the nature of hemoglobin distribu-tion in beta thalassemia. It has been further substanti-ated by the present studies of the distributions of thesehemoglobins and of G6PD and GOTactivities in cen-trifuged beta thalassemic blood samples. In hemoglobinH disease, the ,84 tetramer has a higher rate of turnoverthan that of hemoglobin A (37), as was predicted fromthe studies of Rigas and Koler (1). For this reason asample of cells containing more soluble hemoglobin Hthan that of another sample from the same individualmust be a younger cell population. This fact was alsoconfirmed by the present hemoglobin and enzyme dis-tribution studies.

Glutathione may be bound to hemoglobin as it pre-cipitates in red cells (1). Since it has been proposedthat the level of glutathione may influence to some ex-tent the activity of the hexose monophosphate shunt(39), it was of interest to examine the relationshipof HMPSactivity to cell age and glutathione concen-tration in the normal individuals and in the two forms ofthalassemia. In fact, when ratios were established be-

Hemoglobin Precipitation and Red Cell Metabolism in Thalassemia 39

Page 8: Erythrocyte Metabolism Alpha Beta Thalassemia · sium flux (4). These changes seem to be unrelated to cellular GSH (10). A second purpose of this study was to evaluate the influence

tween the top and bottom cell layers, the data suggestedthat HMPSactivity is inversely related to the level ofglutathione in the cells, but the relationship may be co-incidental and not causal. The more interesting fact wasthat the level of HMPSactivity was actually low inyoung cells in hemoglobin H disease. The activity roseto normal as the cells aged and glutathione concentra-tion fell. An explanation for this phenomenon whichappears more reasonable than that of glutathione lossis to be found by consideration of the peculiar sensi-tivity of hemoglobin H to oxidative precipitation. Theadditional reactive thiols in soluble hemoglobin H maywell participate in cellular reduction reactions and sub-stitute in this regard for glutathione. As mentioned inthe introduction to these studies, HMPSactivity wouldbe expected to diminish where the oxidative burden onglutathione is less because the demand for NADPHbyglutathione reductase would be commensurately re-duced. HMPSactivity would be expected to increaseto normal in old hemoglobin H cells containing lesssoluble hemoglobin H, perhaps in part because of lossof glutathione in coprecipitates with hemoglobin H, butmore likely because of loss of the reducing substitutefor glutathione, soluble hemoglobin H.

HMPSactivity was consistently higher in young thanin old red cells in beta thalassemia. The influence of theinclusions, which predominated in the young cells, onHMPSactivity could not be evaluated since the im-mature cells would be expected to maintain a consider-ably higher rate of HMPS activity than old cellswhether inclusions were present or not (32). Sincealpha chains do not contain increased exposed thiolgroups and in addition precipitate extremely rapidlyeven without the influence of oxidative stress, it is un-likely that they serve as a useful substitute for glutathi-one and, hence, it seems as unlikely that their presencewould significantly influence HMPSactivity.

Another result of hemoglobin precipitation in thalas-semia may be an increase in membrane cation perme-ability. Such an increase was associated with cell agingin hemoglobin H disease and was present in the youngcells in the beta thalassemia syndromes. The enhancedpermeability was not related to glutathione content.Although it is tempting to relate the increased potas-sium efflux to the presence of hemoglobin precipitatesin beta thalassemia, we cannot be sure of this inter-pretation. The markedly increased flux observed ininclusion bearing upper layer cells of splenectomizedpatients with beta thalassemia could be related to thelarge number of nucleated erythrocytes and reticulocytesin these fractions as well as to the inclusions them-selves (34). That nucleated erythrocytes do not neces-sarily obscure these studies is suggested by the resultsof one experiment performed in a splenectomized pa-tient with polycythemia vera and iron deficiency (4)

in whose cells inclusions were not present and in whommore nucleated red cells were detected in lower layercells than in upper layer cells. The upper layer potas-sium flux was 20% higher than that of the bottom layercells (4).' More striking was the fact that in splenec-tomized patients with hemoglobin H disease the oldercells contained inclusions and tended to have a higherrate of potassium flux than the young cells. These dataprovide firmer support for the concept that inclusionsof hemoglobin may alter membrane permeability. On theother hand it does not appear likely to us that enhancedcation flux constitutes the most serious threat producedby intra-erythrocyte hemoglobin subunit precipitation.Rifkind (40), Slater, Muir, and Weed (41), and Wenn-berg and Weiss (42) have clearly demonstrated thatlarge intra-erythrocyte inclusions enhance splenic se-questration, erythrocyte pitting and destruction of eryth-rocytes by mechanical interference with red cell pas-sage through tortuous channels. This seems to us toconstitute a much more important pathophysiologicalconsequence of inclusion formation which results fromunbalanced hemoglobin subunit synthesis in the thalas-semia syndrome.

ACKNOWLEDGMENTSWe are grateful to Drs. William Beck 'and BarbaraRosen, Massachusetts General Hospital, and Thomas B.Necheles, Boston Floating Hospital, for encouraging some oftheir patients to participate in these studies. The technicalassistance of Lynne Greer Esler and Jeanne Forgione isdeeply appreciated.

This work was supported by U. S. Public Health ServiceGrants HD-02777-01, T1 HE 5255, and AM 01975, and bya grant from the John A. Hartford Foundation. David G.Nathan is the recipient of U. S. Public Health ServiceCareer Development Award K3 AM35361. Some of theseclinical studies were performed in Clinical Research Centersat the Peter Bent Brigham Hospital and Children's HospitalMedical Center supported by U. S. Public Health ServiceGrants 5MO1-FR3 and FR-00128.

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Hemoglobin Precipitation and Red Cell Metabolism in Thalassemia 41