Erythrocyte lipids and vitamin E in Type II congenital dyserythropoietic anemia

March 1974 The Journal o f P E D I A T R I C S

355

Erythrocyte lipids and vitamin E in Type H congenital dyserythropoietic anemia An 8-year-old girl with Type H congenital dyserythropoietie anemia, characterized by morphologic abnormalities of erythroid precursors, immunologic alterations, hyperbilirubinemia, and chronic anemia, was found to be vitamin E deficient. Nutritional history and vitamin E absorption studies indicated that neither dietary lack nor intestinal malabsorption was the cause of the deficiency. Striking changes in the patient's hematologic status following administration o f vitamin E included rise in hemoglobin, decrease in bilirubin and reticulocyte count, and a marked increase in red blood cell survival Erythrocyte phospholipids, altered while the patient was vitamin E deficient, returned to normal levels during therapy. However, hematologic improvement was not complete, immunologic abnormalities persisted, and morphologic aberrations in erythrocyte precursors actually increased during vitamin E therapy. It is therefore concluded that vitamin E deficiency played a secondary role in the production o f the child's hematologic disorder and may have been a result o f the increased utilization of the vitamin in stabilization o f defective cellular membranes.

Sean O'Regan, M.B., B.Ch., David K. Melhorn, M.D.,* Arthur J. Newman, M.S., M.D., and Richard C. Graham, M.D., Cleveland, Ohio

CONGENITAL dyserythropoietic anemias comprise a group of hematologic disorders characterized by chronic a n e m i a of varying severity, i n a p p r o p r i a t e l y low reticulocyte response, hyperbilirubinemia, and a spectrum of morphologic abnormalities of erythroid precursors in the bone marrow. Criteria f o r the diagnosis of Type II congenital dyserythropoietic anemia (CDA) include erythroblastic multinuclearity, a positive acidified serum hemolysis test, 1and increased sensitivity of erythrocytes to lysis by anti-I and anti-i antibodies? Red cell survival may be shortened, and the presence of storage cells in the bone marrow has recently been noted. 3 Several features of Type II CDA suggest that hemolysis plays a significant role in the production of the anemia, although the causes of premature red blood From the Departments of Pediatrics and Medicine, Case Western Reserve University School of Medicine, and University Hospitals o f Cleveland. Supported in part by the National Institutes o f Health Grant No. R R 05410-09 and by the Hastings Fund. *Reprintaddress."Rainbow Babies and ChildrensHospital 2103 Adelbert Rd., Cleveland, Ohio44106.

cell (RBC) destruction have not been clearly elucidated. This report describes a patient with Type II CDA in whose erythrocytes membrane abnormalities were observed in association with unexplained vitamin E (tocopherol) deficiency. The effects of administration of

Abbreviations used CDA: congenital dyserythropoieticanemia RBC: red blood cell BSI: bound serum iron TIBC: total iron-binding capacity ETI: vitamin E tolerance index MDA: malonyldialdehyde PE: phosphatidyl ethanolamine SM: sphingornyelin

vitamin E on the patient's clinical course, hematologic values, and erythrocyte and serum lipids are also presented. CASE REPORT

T. S., an 8-year-old white girl, was born at term to parents of German-Italian ancestry. Unexplained hyperbilirubinemia ne-

Vol. 84, No. 3, pp. 355-361

356

O'Regan et al.

The Journal of Pediatrics March 1974

4.0--

3.5-

:3.0-

.,,,,.,.

t~ 2 . 5 2.0-

1.51.0~ PATIENT

0.5--

0.O O

I

I

I

4

8

24 HOURS

Fig. 1. Vitamin E tolerance curves in the patient and control subjects (mean values).

Table I. Basic hematologic findings Hemoglobin Hematocrit Reticulocytes Leukocytes Platelets Bilirubin Direct Coombs

6.5 (Gm. %) 18 (%) 5.0 (%) 7,200/mm.3 321,0001mm. 3 0.8/3.0 (D/T, mg./100 ml.) Negative

cessitated two exchange blood transfusions in the first three days of life. No blood group incompatibility was identified. At 7 weeks of age she was found to have a hernatocrit of 20 per cent and during the next three years she maintained a hematocrit between 24 and 26 per cent. A variety of hematinic agents, including iron, vitamin B12 and B6, and folic acid failed to produce a favorable response. At 4 years of age she was first seen at University Hospitals for evaluation of her anemia. Physical examination revealed a small girl with a sallow complexion, slight icterus, "and mild splenomegaly. Her height and weight were in the tenth percentile (consistent with her previous growth pattern). Laboratory data included: hematocrit, 25 per cent; hemoglobin, 8.6 Gin. per cent; reticulocyte count, 4.4 per cent; white blood cell count of 5,000 per cubic millimeter with a normal differential and adequate platelets on smear. Red blood cell morphology was characterized by moderate poikylocytosis and some fragmented forms. Screening tests for glucose-6-phosphate dehydrogenase, pyruvate kinase, and glutathione reductase were normal, as

were RBC osmotic fragilities and hemoglobin electrophoreses. Other studies included: bound serum iron (BSI), 225 ~zg per cent; total iron-binding capacity (TIBC), 320/zg per cent; saturation, 70 per cent. Serum vitamin BI2 and folate concentrations were normal. Bone marrow examination revealed erythroid hyperplasia with large numbers of multinucleated RBC precursors. During the subsequent four years, the patient's hematocrit gradually fell and splenomegaly increased, although no blood transfusions were required. At 8 years of age she was readmitted for further evaluation. Routine laboratory studies. Results of base-line hematologic findings are shown in Table I. Serum concentrations of vitamin B12 and folate, RBC osmotic fragilities, autohemolysis, hemoglobin electropboreses, A2, and fetal hemoglobin concentrations were within normal limits. Special studies. The following determinations were obtained prior to and during the administration of alpha-tocopherol acetate (as Aquasol-E, U, S. Vitamin and Pharmaceutical Corp., New York, N. Y.). Maintenance dosage was 200 I.U. per day given orally. Some studies were conducted during a period between the first and second sequences of tocopherol administration. Serum concentrations of vitamin E were determined according to the method of Quaife and associates,4 using 0.6 ml. of serum with appropriate readjustment of the reagents. Vitamin E tolerance studies were carried out according to a modification of the method of Filer and associates.5 Alpha-tocopherol acetate was given in an oral dose of 25 I.U. per kilogram and venous blood was obtained for determination of free serum tocopherol at base line, 4, 8, and 24 hours following the test dose. The vitamin E tolerance index (ETI) was calculated on the basis of the tocopherol level using the following formula:

ETI =

8 hr. tocopherol level--base-line tocopherol level

x 100

Mg. alpha-tocopheroi acetate administered per Kg. The initial serum vitamin E level was 0.32 mg. per 100 ml., abnormally low. (The normal range in this laboratory is 0.70 to 1.40 mg. per 100 ml. in children of similar age.) As shown in Table II, serum tocopherol increased markedly following the initial four-week trial of tocopheral. When therapy was stopped, serum level of vitamin E fell to 0.48 rng. per 100 ml., rising again to within the normal range when tocopherol therapy was resumed. The results of the patient's tocopherol test are compared with mean tolerance values in five normal children, 4 to 10 years old (Fig 1). The vitamin E tolerance curves of the patient and controls, as well as the ETI's, are similar (patient's ETI, 10.8; mean control ETI, 11.3). The results of in vitro hydrogen peroxide (H202) erythrocyte fragilities and RBC malonyldialdehyde (MDA) determinations are also recorded in Table I1. H202 fragilities were performed according to the method of Gordon and colleagues,6 and evaluation of MDA, a measure of the degradation products of erythrocyte membrane lipids, by the technique of Stocks and Dormandy. 7 RBC hydrogen peroxide fragility was increased prior to institution of alpha-tocopherol therapy. Following four weeks of

Volume 84 Number 3

Type 11 dyserythropoietic anemia

E sufficient

E deficient .,-..rI ~

357

ethonolarnine

~

~'

~, I--,~--phosphatidyl

~

~

~

[ ~'~

choline

- origin -

l

w

"

----

L _ ce iis ..-J~,-- plas ma-.-F

~,----c e IIs ....-~~-.-p lasm a ---j

Fig. 2. Thin-layer chromatography of RBC and plasma phospholipids before and during vitamin E therapy. A striking increase in erythrocyte PE was observed during vitamin E administration. T a b l e I1. Vitamin E, e r y t h r o c y t e H202 fragility, a n d M D A prior to and during vitamin E t h e r a p y

Patient, E deficient Patient, E sufficient* Control patients--mean value Range of con t rols

Vitamin E (mg. %)

HeOJragility (% hemolysis)

MDA (nm./Gm. Hgb.)

0.32 2.2 0.95 0.70 - 1.40

79.0 50.0 7.5 0 - 13

364 165 145 125 - 185

*After four weeks of tocopherol therapy. T a b l e III. RBC a n d plasma lipids prior to a n d during v i t a m i n E t h e r a p y

Total phospholipids

PE

Phosphatidyl choline (%)

SM (%)

Other*

(%)

Cholesterol

6

30

44

16

27

36

22

14

29

31

26

14

1.60 x 10-t~ (mg./cell) 1.75 • 10-l~ (reg./cell) 1.70 X 10-1~ (reg./cell)

(%)

Erythrocytes: Vitamin E deficient Vitamin E? sufficient Controls$ (Mean • 1 S.D.)

3.8 x 10-m (reg./cell) 4.4 x 10-1~ (reg./cell) 4.2 • 10-l~ (reg./cell) (0.5)

(_+ 0.3)

Plasma. Vitamin E deficient Vitamin El" sufficient Controls$ (Mean • 1 S.D.)

275 rag. % 265 nag. % 245 rag. % (38)

0 9 5

68 71 60

26 13 28

6 7 7

150 mg.% 155 rag.% 150 mg.% (• 29)

*Phosphatidyl serine, inositol, and lysolecithin. tArter four weeks of tocopherol therapy. SFive children of age similar to patient. daily vitamin E therapy, a significant decrease was observed, although it did not fall to within the normal range. Results of MDA determinations showed a parallel abnormal elevation of lipid degradation products while the patient was vitamin E deficient, and a subsequent fall as her serum vitamin E level rose. Erythrocyte and plasma lipids are recorded in Table IlL Venous blood was used for RBC and plasma phospholipid, and cholesterol determinations which were performed according to

previously outlined techniques.8 Decreased levels of total red blood cell phospholipids and markedly diminished phosphatidyl ethanolamine (PE) were found in association with the patient's vitamin E deficiency (Table III). Sphingomyelin (SM), on the other hand, was elevated. Following administration of alpha, tocopherol acetate and achievement of significantly higher serum tocopherol levels, the distribution of RBC phospholipids changed dramatically, with total phospholipids, PE, and SM ap-

358

0 'Regan et al.

The Journal of Pediatrics March 1974

realities and storage cells previously described in Type II CDA were present both before and during tocopherol therapy. Multinucleate abnormalities in RBC precursors increased markedly after vitamin E administration was instituted. Electron micrographs of bone marrow specimens obtained before and during tocopherol administration were examined. Although the number of abnormal red cell precursors was greater during therapy, the same aberrations were seen during both periods. Various nuclear abnormalities were observed, particularly in the more differentiated RBC precursors. Proerythroblasts, for the most part, showed no definite structural defects. However, more mature precursors frequently had two or more nuclei. Individual nuclei were often highly irregular

Fig. 3. Characteristic nuclear abnormalities in erythroid precursors in bone marrow. A, Incomplete division of proerythroblast; B, multinuclearity of orthochromatic normoblasts; C, basophilic erythroblast in anaphase; D, incomplete division of a basophilic erythroblast. (Light microscopy, original magnification xl00.) proaching normal levels. During the same period, plasma PE reappeared and SM decreased. The change in PE is illustrated in a graphic comparison of the thin-layer phospholipid chromatograms which were obtained prior to and during tocOpherol therapy (Fig. 2). Microscopic studies. Light microscopic evaluation of the patient's bone marrow is illustrated in Fig. 3. The nuclear abnor-

in shape, with reniform and multilobulated forms seen (Fig. 4). Microtubules were frequently observed in red blood cells in all stages of differentiation. These were present as paranuclear bundles in the more immature cells but were more often scattered at the periphery of the more differentiated cells. In addition to occasional discontinuities of the nuclear membrane, numerous cytoplasmic vesicles, often aligned in a linear pattern, were present in the more mature cells. A characteristic abnormality, also identified in Fig. 4, was present in more than half the erythroid precursors. This consisted of a narrow band of cytoplasm separated from the bulk of the cell by a membrane-bound space. Similar structures were not observed in mature, nonnucleated erythrocytes. Nucleated erythrocyte precursors were frequently seen within macrophages among the marrow elements. The cytoplasm of the macrophages also contained polymorphous debris. Iron and erythrokinetie studies. Bound serum iron and TIBC determined by the method of Schade and associates9 were consistently abnormal while the child was vitamin E deficient, with the BSI usually between 180 and 225 tzg per cent, and TIBC between 285 and 320/zg per cent (saturation 55 to 75 per cent). A f t e r eight weeks of continuous maintenance therapy with alpha-tocopherol acetate, the BSI had fallen to 70/xg per cent with a TIBC of 240 ~g per cent and a saturation of 29 per cent. Although the BSI fell to a value within the normal range, TIBC remained relatively low both before and during administration of vitamin E. Ferrokinetic studies conducted under standard conditions with the use of 59Fe while the patient was vitamin E deficient showed that 59Fe plasma clearance was more rapid than normal, plasma iron transport rate was increased, and 59Fe :ncorporation in circulating red blood cells was diminished. There was no unusual accumulation of 59Fe in the spleen or liver during the three-week period. These results and those of autologous erythrocyte survival studies performed by standard 51Cr techniques are shown in Table IV. RBC survival was significantly sn~,rtened while the patient was vitamin E deficient. In contrast, similar studies conducted four weeks after tocopherol therapy was begun showed an increase in RBC survival into the low normal range. Miscellaneous determinations. Patients identified as having Type II CDA have demonstrated an abnormal acidified serum test (Ham test l) in which increased RBC lysis is found under in vitro conditions. Such was the finding in our patient both before and during administration of vitamin E. As in similar patients,2

Volume 84 Number 3

Type H dyserythropoietic anemia

359

Fig. 4. Electron micrograph (magnification • of RBC precursors in bone marrow during vitamin E therapy. The nucleus shows multiple lobulations. A narrow rim of cytoplasm is separated from the interior of the cell by a channel throughout much of the cell circumference. the child's erythrocytes were hemolysed by anti-i anti-I antisera (again, both prior to and following the patient's achievement of tocopherol sufficiency). Neither of these laboratory abnormalities which help to characterize Type I1 CDA was altered by the administration of vitamin E, even though other parameters changed significantly. Hematologic responses to vitamin E. Features of hematologic changes in the patient following administration of vitamin E are shown in Fig. 5. Rise in hemoglobin and hematocrit was accompanied by a fall in both reticulocyte count and serum bilirubin concentration. The decrease in serum bilirubin was clinically complemented by an observed loss of cutaneous and scleral icterus present since infancy. DISCUSSION Vitamin E, erythroeyte HzO z fragility, and M D A determinations. The presence of vitamin E deficiency in

this patient was unexpected, although three patients with Type II presented by Verwilghen and associates 1~ during compilation of this report had decreased serum tocopherol levels. Because it was difficult to correlate what first appeared to be an unrelated vitamin deficiency with the patient's basic erythropoietic defect, efforts were initially directed toward identification of possible abnormalities in intestinal absorption of fats and fat-soluble vitamins. However, careful evaluation of the child's dietary habits made nutritional deprivation unlikely. The normal vitamin E tolerance curve and ETI, together with the prompt achievement o f vitamin E sufficiency following the oral administration of a fat-soluble vitamin E preparation, indicated that the initially low tocopherol level was not the result of intestinal malabsorption. Further evidence that the patient's vitamin E lack had

360

O'Regan et aL

The Journal of Pediatrics March 1974

Table IV. Erythrokinetic data

Vitamin E deficient

Vitam& E sufficient

Normal

20 1.9 25 14

23

65-110 0.5-0.75 75-90 24-29

59Fe plasma clearance (T 89 minutes) Plasma Fe transport rate (mg./Kg./day) Fe utilization, day 7 (%) 51Cr autologous RBC survival (T 89 days)

4"0 t BILIRUBIN 5.O (TOTAL, MG %)2-O- 1 I'O- 1

(./.)

i1 1

............................................... ,........,

~i;r =VITAMIN E ( 200 I.U. / DAY)

I--

HEMATOCRIT (%)

25

2O '~

IO

~-~-~i~,i:~.~.-~i'~ ~.:-;~'?~5!!~i~;i~i~;"i:~j~i ?~;~:Y

Fig. 5. Changes in hematologic parameters during and between courses of vitamin E therapy are shown.

an etiologic basis other than intestinal malabsorption was the relatively rapid decrease in her serum free tocopherol level when oral vitamin E supplementation was temporarily stopped after the initial trial o f therapy. Such data suggest the possibility of the unusual existence of a situation in which the biologic requirements for tocopherol are heightened, and vitamin E deficiency results even when dietary intake is normal. Although increased red blood cell H20 2 fragility had been identified in a variety of hemolytic disease states in which serum vitamin E levels are normal, 1~ elevated values, have more often been related to coexisting tocopherol deficiency. 12 The same is true for the M D A determination, in which abnormal elevation of the products of peroxidation is consistently found in conjunction with vitamin E lack but also in several anemias in

which deficiency of the vitamin is not present. In this patient, both H20 2 fragility and M D A abnormalities appeared related to her vitamin E-deficiency state, although RBC HjO 2 fragility did not fall within the normal range even after vitamin E sufficiency was achieved. The persistence of H20 2 fragility elevation during vitamin E therapy is unexplained, but may suggest that the RBC in this disease is also susceptible to peroxide stress not directly related to serum tocopherol levels. Lipid studies. The presence of erythrocyte lipid abnormalities in this child while vitamin E deficient is intriguing for several reasons. Although some alterations were seen in other RBC lipid fractions, the almost complete absence of PE was most striking. The depletion of this phospholipid is of particular interest, because it is a m a j o r structural c o m p o n e n t o f the e r y t h r o c y t e membrane. The studies of Jacob and Lux 13 in rats with vitamin E lack indicated that the primary defect in the lipid membrane component was an almost complete depletion of PE, demonstrated following in vitro exposure of cells to H20 2. In both the animals which they described and in the patient considered here, reappearance of membrane PE was quite dramatic when sustained vitamin E sufficiency was achieved. Additional findings showed the inability of mature, circulating red blood cells to repair losses of these lipids from the membrane. Thus, the effect o f antioxidants playing an in vivo role in protecting against membrane lipid degradation is to slow the rate of peroxidation and not to stimulate regeneration of lipids in the mature cell. In situations in which defects in erythropoiesis involve inherent deficiences in construction of an adequate RBC "envelope," oxidant stress may exceed the antioxidant capacities of both tocopherol and the complex group of enzymes which are directly or indirectly involved in the detoxification of free peroxide radicals. In patients with Type II CDA, levels of these enzymes are either moderately or greatly increased. 3 Elevation of these enzymes may represent a response to a heightened requirement for maintenance o f membrane integrity.

Volume 84 Number 3

Type II dyserythropoietic anemia

361

Utilization of tocopherol under such circumstances may also be increased. Clinical response to vitamin E. Vitamin E deficiency has been reported in a number of disease states in both children and adults. With few exceptions, tocopherol lack has been predictably associated with illnesses such as cystic fibrosis 14and other conditions in which the intestinal malabsorption of fats and fat-soluble vitamins constitutes a major pathologic feature. Restoration of serum vitamin E levels to normal in such diseases is

Type II CDA and response to vitamin E therapy is a p h e n o m e n o n unique to this patient or a feature common to all children with this hematologic disorder must await the results of similar studies in other patients with Type II CDA. The authors wish to express their appreciation for the cooperation of T. S. and her parents, the assistance of Dr. F. Miraldi in interpretation of erythrokinetic data, and the technical help of Ms. Arlene Pelavin, Ms. Carol Luckey, and Ms. Shirley Eisenberg.

usually not accompanied by significant clinical hematologic change. In this patient, however, improvement in laboratory parameters following administration of vitamin E was accompanied by clinical and hematologic improvement. The administered dose of tocopherol (in addition to her dietary sources) represented an intake considerably greater than that of a normal individual of similar age. A n average dietary intake and (based on her normal ETI) an uncompromised intestinal absorption of vitamin E had obviously been insufficient to maintain a normal serum tocopherol level or to prevent the hematologic effects associated with this deficiency. The fact that hematologic i m p r o v e m e n t d u r i n g vitamin E administration was incomplete, even though erythrocyte survival time increased significantly, suggests a secondary role for vitamin E lack in the production of the patient's anemia. The persistence of the abnormal acidified hemolysis test and continued presence o f " i " and "I" antigens further indicate that the primary dyspoietic abnormalities were not affected by tocopherol therapy in the ,losage administered. The lack of change in the electron micrographic alterations and actual increase in nuclear aberrations seen with light microscopy also emphasize that the basic pathologic process was unaffected. While the biochemical disturbances which produce the extraordiriary morphologic changes in erythroid precursors that characterize Type II CDA are unknown, it is clear that several cellular components and functions are affected. I n particular, the peculiar configurations of the cytoplasmic and nuclear membranes seen in the electron micrographs in the present and previous reports 1~may r e p r e s e n t the morphologic expression o f faulty membrane construction. Since the clinical improvement during vitamin E therapy in the present patient was not accompanied by correction of ultrastructural cellular abnormalities, it is likely that the action of vitamin E is to proyide stabilization of defective membranes, thereby reducing their intramedullary and peripheral rates of destruction. Whether the vitamin E deficiency associates with

REFERENCES

1.

2.

3.

4.

5.

6.

7.

8.

9.

10.

11.

12.

13.

14.

Crookston, J. H., Crookston, M. C., Burnie, K. L., and Francombe, W. H.: Hereditary erythroblastic muttinuclearity associated with a positive acidified-serum test: A type of dyserythropoietic anemia, Br. J. Haematol. 17: 11, 1969. Lewis,S. M., Grammaticos, P., and Dacie, J. V.: Lysis by anti-I in dyserythropoietic anemias: Role of increased uptake of antibody, Br. J. Haematol. 18: 465, ] 970. Murphy, S., and Oski, F.: Congenital dyserythropoietic anemia Type II: Report of two cases and a review of the literature,Pediatrics 50: 858, 1972. Quaife, M. L., Scrimshaw, N. S., and Lowry, O. H.: Micromethod for assay of total tocopherols in blood serum, J. Biol. Chem. 18: 1229, 1949. Filer, L. J., Jr., Wright, S. W., Manning, M. P., and Mason, K. E.: Absorption of alpha tocopherol and tocopherol esters by premature and full term infants and children in health and disease, Pediatrics 8: 328, 1951. Gordon, H. H., Nitowsky, H. M., and Cornblath, M.: Studies of tocopherol deficiency in infants and children. I. Hemolysis of erythrocytes in hydrogen peroxide, Am. J. Dis. Child. 92: 164, 1956. Stocks,J., and Dormandy, T. L.: The auto-oxidation of the human red cell lipids induced by hydrogen peroxide, Br. J. Haematol. 20: 95, 1971. Gross, S., and Melhorn, D. K.: Vitamin E, red cell lipids and red cell stability in prematurity, Ann. N. Y. Acad. Sci. 203: 141, 1972. Schade,A. L., Oyama, J., Reinhart, R. W., and Miller, J. R.: Bound iron and unsaturated binding capacity of serum: Rapid and reliable quantitative determination, Proc. Soc. Exp. Biol. Med. 87: 443, 1954. Verwilghen, R. L., Lewis, S. M., Dacie, J. V., Crookston, J. H., and Crookston, M. C.: Hempas: Congenital dyserythropoietic anemia (Type II), Quart. J. Med. 42: 257, 1973. Melhorn, D. K,, Gross, S~,Lake, G. A., and Leu, J. A.: The hydrogen peroxide fragility test and serum tocopherol level in anemias of various etiologies, Blood 51: 438, 1971. Binder, H. J., Herting, D. C., Hurst, V., Finche, F. C., and Spiro, H. M.: Tocopherol deficiency in man, N. Engl. J. Med. 273: 1289, 1965. Jacob,H. S., and Lux, S. E., IV.: Degradation of membrane lipids in peroxide hemolysis: Studies in vitamin E deficiency, Blood 32: 549, 1968. Underwood, B. A., Denning, C. R., and Navab, M.: Polyunsaturated fatty acids and tocopherol levels in patients with cystic fibrosis, Ann, N. Y. Acad. Sci. 203: 237, 1972.