hemoglobin H disease, six patients with Cooley's anemia, .... to one adult patient with Cooley's anemia. 5 days ..... a grant from the John A. Hartford Foundation.
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), potassium flux, and glutathione (GSH) content were measured. The distributions of hemoglobins H and F, as well as the activities of erythrocyte glucose-6-phosphate dehydrogenase (G6PD) and glutamic oxalacetic transaminase (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 inclusions 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. Potassium 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 glutathione 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 increases 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 membrane-bound inclusions of precipitated alpha chains (47). In hemoglobin H disease, the young cells contain more soluble hemoglobin H, whereas inclusions of precipitated 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 characteristics 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 cohorts 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
sium flux (4). These changes seem to be unrelated to top and bottom fractions each contained approximately 20% of the initial volume of cells. cellular GSH (10). White blood cell counts, reticulocyte counts, inclusion body A second purpose of this study was to evaluate the counts and measurement of red cell indices were performed influence of soluble hemoglobin H on the HMPS in on all fractions by standard methods (17). Inspection of erythrocytes. A major role of the HMPS in red cells is reticulocyte smears revealed virtually absent platelets. The to provide NADPH for glutathione reductase, an enzyme ratio of leukocytes to red cells was less than 2 X 10' in top cell suspensions and less than 10-' in bottom layer activity which maintains a renewable pool of reduced layer suspensions. glutathione. The latter serves as an oxidant buffer for Minimum HMPS activity by evolution of 14CO2 from the thiol groups of red cell hemoglobin and membrane glucose-1-`C was studied in duplicate by modifications of the protein (11-13). When hemoglobin A is exposed to an method of Murphy (18). After incubation in the presence of oxidant strong enough to cause its denaturation and pre- 1 ,uc of glucose-1-"4C, a small piece of filter paper, which had suspended from the inner edge of the serum cap of the cipitation in vitro, glutathione is bound, presumably as been incubation flasks before incubation, was soaked with KOH. a disulfide, to the precipitating protein (11). GSH may The blood was acidified with 0.1 ml of 0.5 N H2SO4. The also be bound to certain abnormal hemoglobins which acid and alkali additions were made with syringes and precipitate in vivo during the life span of the red cell hypodermic needles. The filter papers and appropriate stan(1, 10, 14). Among these abnormal hemoglobins is he- dards applied to filter paper were dried and counted for 14C in a liquid scintillation spectrometer (19). The moglobin H (1, 8, 9), a tetramer of beta chains with activity diffusion stystem for 14CO2 was evaluated with Na2,4COs eight rather than two exposed thiols per molecule (15). standards and found to be quantitative. Blanks were perRecent studies have indicated that hemoglobin H may formed by acidification and diffusion immediately after the have a functional redox potential very close to that of addition of glucose-1-14C to the incubate. Murphy's method of calculation was utilized to derive a value for shunt activity glutathione (14). Therefore, it might substitute for glu- in terms of mm glucose/liter cells per hr. tathione and serve as an oxidant buffer for the cell unThe effects of stimulation of HMPS activity by glucose til oxidative precipitation of this hemoglobin occurred. and methylene blue were measured by the methemoglobin Oxidative precipitation of hemoglobin H is irreversible reduction technique of Beutler and Baluda (20). Methemoand the precipitates cannot be reduced by an NADPH- globin and hemoglobin were assessed by the technique of Evelyn and Malloy (21). The total hemoglobin in the linked enzyme system. If hemoglobin H can act as a hemolyzates remained constant even in samples containing reducing agent, one might expect HMPS activity to hemoglobin H. Failure to recognize possible precipitation of be decreased in cells containing soluble hemoglobin H hemoglobin H by methylene blue might have been because and to increase to normal when the hemoglobin H pre- the molar hemoglobin to dye ratio was approximately 300 cipitates, because the NADPH-dependent glutathione rather than the ratios approaching unity that have been in other studies of hemoglobin H precipitation by reductase system would then be left to perform this es- utilized such dyes (6). Because it is difficult to remove all nitrite sential buffering function. In these studies, it was found from the cells (22), this technique indicates the net rate that in hemoglobin H disease, HMPS activity was indeed of methemoglobin reduction rather than absolute reduction lower in the young cells, where soluble hemoglobin H rates. Therefore, only the periods of linear methemoglobin predominated, than in the old cells, where inclusions pre- reduction were considered in the calculations of comparative reduction rates. dominated. Glucose consumption and lactate production during incubation were measured on perchloric acid filtrates by the METHODS glucose oxidase (23) and the LDH: NADH (24) methods Studies were performed on blood from five patients with respectively. GSH concentrations in top and bottom cell suspensions hemoglobin H disease, six patients with Cooley's anemia, two patients with S-thalassemia and a group of eight normal were measured by the method of Beutler, Duron, and Kelly controls. Percentages of hemoglobins A and H were deter- (25). G6PD and GOT activities in hemolyzates were mined by starch granule electrophoresis (16). None of the respectively measured by suitable modifications of the technique of Zinkham and Lenhard (26) and by the method of patients had received blood transfusions in recent years. Venous blood was collected with preservative-free heparin 1 Karmen, Wroblewski, and LaDue (27). Potassium fluxes were measured by micromodifications of (0.1 mg/ml blood) and studied immediately. The blood was separated into layers of young and old cells by centrifuga- the methods of Solomon (28). The top and bottom layer cells were of different size tion of 15 ml of whole blood in celluloid tubes at 20,000 g for 1 hr. After this procedure, the relatively younger large distribution, the top cells having a larger mean volume than cells are found at the top of the tube, and the older smaller the bottom. To develop meaningful comparisons between the cells segregate at the bottom of the tube. The buffy coat layers the measurements described above (with the excepand unavoidably the top 1% or 2% of erythrocytes were tion of G6PD and GOT activities which were expressed as removed by aspiration. The remaining top (reticulocyte-rich changes of optical density at 340 ,u/110" erythrocytes), were or young) and bottom (reticulocyte-poor or old) 1.5-ml modified in terms of a standard cell volume of 82 ,A. fractions were then collected and resuspended at hematocrits For example, the measured value for glucose consumption of approximately 40% in the autologous plasma. Thus, the per liter of bottom layer cells was multiplied by the ratio of (MCV bottom layer cells)/(MCV standard cells) in which 1 Connaught Medical Research Laboratories, Toronto, MCV stands for mean corpuscular volume. The corrections were not entirely suitable for statistical compariOntario, Canada.
34
Nathanw Stossel Gunn, Zarkowsky, and Laforet
sons of normal cells to the cells of beta thalassemia. Size distribution studies showed that the cells of splenectomized beta thalassemia patients were not merely smaller than normal cells. The variances of their mean volumes were considerably greater than those of normal cells so that simple statistical comparisons on the basis of mean volume were of limited significance.2 On the other hand, the variances of the mean volume of beta thalassemia and hemoglobin H disease top and bottom layer cells were not as widely different. Nor were the variances of normal and hemoglobin H cells sufficiently dissimilar to limit statistical comparisons of their metabolic functions on the basis of a correction for cell volume. With these limitations considered, the corrected data were recorded in the tables and figures.
RESULTS The adequacy with which centrifugation separated cells into young and old populations is illustrated in Table I where the substantial differences in reticulocyte counts and GOT and G6PD activities between top and bottom fractions are tabulated. As expected (1, 2, 4, 29), the young cells were in the top layers and the older cells in the bottom fractions.! Glucose metabolism in young and old cells. The rates of total glucose consumption, lactate production, glucose oxidation by the HMPS, and reduction of methemoglobin with methylene blue and glucose are shown 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 the validity of the separation method by administration of '9Fe to one adult patient with Cooley's anemia. 5 days after isotope administration nearly all of the radioactivity was localized in the large hypochromic cells of the upper layer which were rich in reticulocytes and poor in hemoglobin F.
4.4
rA
4.0
TOw~P 3.6BOTTOM RATIO 3.2 2.8'
As anticipated (30-33), total glucose consumption and lactate production in normal young cells was greater than in normal old cells. In beta thalassemia erythrocytes the same pattern was detected, an expected finding in view of the large number of reticulocytes and nucleated red blood cells in the top layer cells of these patients. However, in four of the five studies of hemoglobin H, glucose consumption and lactate production were greater in old cells than in young cells, despite relatively large numbers of reticulocytes in the top cell layers. HMPS activity in young normal cells, measured either by the '4CO2 or by the methemoglobin reduction methods, was greater than HMPS activity in old normal cells (31-33). The same was true to an even greater extent of the cells of patients with beta thalassemia. On the contrary, the HMPS activity of old cells from patients with hemoglobin H disease was equal to or greater than that of young cells from these patients. These differences in the cellular localization of shunt activity are demonstrated in Fig. 1 in which the top and bottom cell ratios for HMPS activity are graphically shown. This figure shows that the differences in shunt activity between young and old cell fractions were easily demonstrable by the glucose-1-"C technique whereas they were barely if at all discernible by the the methemoglobin reduction technique since the latter only measures the potential maximal activity of the shunt based largely upon available glucose, NADP, G6PD, and NADPH: methylene blue reductase activity in the bottom and top layer cells. G6PD activity and GSH concentrations in young and old cells. The relative youthfulness of all cells from
EI
MEASURED WITH 4 C-I -GLUCOSE mM GLUCOSE/liter CELLS/hr
[
MEASURED WITH METHEMOGLOBIN REDUCTION % METHEMOGLOBIN REDUCED/min
:
2.4-
w N
(8)
2.0O 1.6I.2~ 0.8 0.4-
H
Lif
I
t
N
(8)
FIGURE 1 Relative predominance of total HMPS activity in top and bottom cell layers measured in patients with hemoglobin H disease (H), beta thalassemia (pt), and normals (N).
Hemoglobin Precipitation and Red Cell Metabolism in Thalassemia
35
TABLE I
Metabolic
Patient
Diagnosis*
Splenectomy
V. J.
H (9%)
Yes
Cell fraction
MCV
Hemoglobin inclusions
u3
%
75
14 H 6.2 H
12.2 1.0
10.5 12.3
4.6 5.3
Top Bottom
53
1.6 6.6
Hemoglobin H or F
Reticulocytes
Lactate production
Glucose consumption
mM/lifer cells per hr:
%
L. D.
H (8%)
No
Top Bottom
75 50
0 0.1
18 H 5 H
10.0 2.3
8.5 8.6
3.4 4.3
M. L.
H (10%)
Yes
Top Bottom
84 54
3.8 12.8
15 H 4 H
22.6 8.0
10.0 12.0
4.2 5.2
A. R.
H (15%)
Yes
Top Bottom
75 53
3.4 11.8
30 H 8 H
16.2 2.3
7.0 10.0
3.7 4.4
0 6.2
12 H 2 H
10.8 1.8
11.8 8.8
4.9 3.6
W.D.
H(8%)
No
Top Bottom
81 63
S. D.
CA
Yes
Top Bottom
69 47
50 5
7 F 32 F
18.6 1.0
5.0 3.8
J. C.
CA
No
Top Bottom
65 51
0 0
8 F 34 F
7.2 0.3
3.5 2.0
C. Z.
CA
Yes
Top Bottom
113 71
45 7
22 F 56 F
30.0 5.0
G. P.
CA
No
Top Bottom
75 64
0 0
20 F 40 F
15.0 4.0
P. M.
CA
Yes
Top Bottom
89.5 74
38 3
22 F 37 F
28.0 1.4
P. F.
CA
Yes
Top Bottom
102 68
47 5
30 F 44 F
25.0 1.2
S. G.
S-T
Yes
Top Bottom
95 75
15 2
13 F 25 F
14.4 0.9
A.G.R.
S-T
No
Top Bottom
75 63
0 0
7 F 13 F
10.0
Top Bottom
91 ±3.0§ 82 43.9
0 0
Normal controls (8)
No
10.6 4.6
4.5 1.3
12.1
6.1 3.4 5.0 3.0
2.0 3.1 40.8§ 0.1 0.1
5.3 2.7
5.8 ±0.7§ 2.8 40.4
3.1 +0.5§ 1.5 40.3
* H, hemoglobin H; CA, Cooley's anemia; S-T, S-thalassemia t Corrected to a standard cell volume of 82 ;pB. § Mean ASD of eight separate controls. 1 Range of three determinations.
patients with hemoglobin H disease and beta thalassemia is shown by their high G6PD and GOT activities compared with those of normal cells (Table I and II). The G6PD and GOT activites of young cells from all subjects were greater than those of old cells. As described previously (14), the GSH content of normal old cells was only slightly less than that of
36
Nathan, Stossel, Gunn, Zarkowsky, and Laforet
cells; but the mean GSH concentration of old hemoglobin H erythrocytes was only 47.8 +7.5 mg/100 ml cells, significantly less GSH than was measured in normal old cells (P
