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Vitamin E-dependent anemia in the premature infant. I. Effects of large doses of medicinal iron
October, 1971 T h e Journal of P E D I A T R I C S
569
Vitamin E-dependent anemia in the premature infant. I Effects of large doses of medicinal iron Description of a hemolytic anemia associated with vitamin E deficiency in premature infants prompted study of the relationships of medicinal iron, vitamin E, and hematologic parameters during the early life of such infants. A total o] 186 patients categorized according to birth weight and gestalional age were placed in study groups: (1) no iron or vitamin E supplement, (2) oral vitamln E supplement, (3) oral iron supplement, and (4) iron and vitamin E supplements. Significantly lower mean hemoglobin and serum vitamin E concentrations, with higher reticulocyte count and hydrogen peroxide #agility, were found during the second month of life in infants with a gestational age less than 36 weeks who were not receiving vitamin E. Hemoglobin values were lowest and reticulocyte counts highest in vitamin E-deficient infants of low gestational age who received supplemental #on. These observations suggest that therapeutic doses of iron increase red cell hemolysis during a period of vitamin E deficiency.
David K. Melhorn, M.D.,* and Samuel Gross, M.A., M.D., with the technical assistance of Geraldine Childers CLEVELAND~ O H I O
I ~ 1967, Oski and Barness 1 reported the occurrence of a mild hemolytic anemia during the second month of life in vitamin E-From the Department of Pediatrics, CaseWestern Reserve University School of Medicine, and University Hospitals of Cleveland. Supported in part by the Mead Johnson Laboratories, Evansville, Indiana, the Health Fund of Greater Cleveland; the Frackelton Memorial Fund, the Wellington, Ohio, United Appeal, and the Natlonal Institutes of Health Grants Nos. RR 05410-09 and FR-87. Presented in part at the Thirteenth annual meeting of the American Society of Hematology, San Juan, Puerto Rico, December 8, 1970. ~Reprlnt address; Babies and CMldrens Hospital, 2103 Adelbert Rd., Cleveland, Ohio 44106.
deficient premature infants. Concurrent studies showed associated abnormal in vitro sensitivity of erythrocytes when exposed to hydrogen peroxide (H202). These observations have also been noted by others. 2, 3 Since one certain biological action of vitamin E See related article, p. 581. (tocopherol) is that of an antiperoxidant at the cellular level,4 the lack of this vitamin may accelerate red cell lysis as a result of an increased rate of peroxidation of the lipid component of the red blood cell (RBC) Vol. 79, No. 4, pp. 569-580
570
Melhorn and Gross
membrane, s The combination of vitamin E deficiency and abnormal in vitro, red cell H202 has also been described in cystic fibrosis and other malabsorptive disorders without associated anemia. 6 It is reasonable to postulate that other factors coexist with deficiency of tocopherol as determinants of the vitamin E-dependent anemia of prematurity. Exogenous iron is often given in relatively large amounts to premature infants to prevent the "late anemia" of prematurity, which develops as a result of rapid growth and outstripping of body iron stores during the latter part of the first year of life. 7 Valent iron has been shown to act as a catalyst in the autooxidation of unsaturated fatty acids, s Since such lipids are essential components of the RBC membrane, excess iron may increase RBC lipid peroxidation when the usual antiperoxidant mechanisms of the red cell are defieientY Because iron is often administered to premature infants during the period of vitamin E deficiency, the present study was designed to investigate these relationships during the first 4 months of life of the premature infant. MATERIALS
AND
METHODS
Included in the initial enrollment were 234 infants admitted to the premature nursery unit of Babies and Childrens Hospital, University Hospitals of Cleveland, during the first day of life in the period between January, 1969, and July, 1970. The gestational age of each infant was ascertained by a combination of the following criteria: (1) reliable maternal estimation of length of gestation, (2) neurologic evaluation of maturity according to the method of Amiel-Tison, 1~ and (3) evaluation of external physical characteristics as described by Usher and associates.1~ When correlation between gestational age and birth weight placed infants below the tenth or above the ninetieth percentile on the graph derived by Lubehenko and associates, a2 patients were excluded from the study. Thus "small-for-date" infants and those infants whose birth weights were excessive for estimated gestatlonal age were not included.
The Journal of Pediatrics October 1971
Infants with hemoglobin concentrations less than 14.0 Gm. per cent in the first 24 hours of life and those with blood group incompatibilities, RBC enzyme deficiencies, hemoglobinopathies, infections, or defects requiring surgical intervention were also excluded. Infants who developed the respiratory distress syndrome were included in the study; those with severe and moderate respiratory distress were assigned to classes I and II, respectively, as suggested by Rudolph and associates? 3 Estimation of gestational age was carried out within the first 48 hours of life, and the infants were assigned to 1 of 3 birth weightgestational age (BWG) categories: Category A = birth weight < 1,500 Gin., < 32 weeks' gestation; Category 13 = birth weight 1,501 to 2,000 Gin., 32 to 36 weeks' gestation; Category C ----- birth weight 2,001 to 2,400 Gm., 36 to 40 weeks' gestation. Infants in each of the 3 BWG categories were then placed at random into one of 4 groups receiving the following therapeutic regimens. Therapy group
Type o[ supplement
1 2 3 4
None Vitamin E alone Iron alone Iron and vitamin E
The form, dosage, and timing of administration of iron and vitamin E supplements are shown below. Form Vitamin E atoeopherol acetate ~ Iron ferrous sulfatet
Dosage 25 units per day orally !0 rag. elemental iron per day orally while weighing < 1 , 5 0 0 Gm.; 15 rag.: 1,501 to 2,000 Gin.; 20 rag.: )>2,001 Gin.
Duration Eighth to forty-second day of life Fifteenth to forty-second day- of llfe
~Aquasol E, United States Vitamin and Pharmaceutical Corporation, New York, N. Y. tFer-ln-sol, Mead Johnson Laboratories, Evansville, Ind.
Volume 79 Number4
Vitamin E was administered in a single dose midway between formula feedings. The iron was also given between feedings in 2 divided doses and was separated from administration of vitamin E (in Group 4) by a minimum of 4 hours. Infants in all therapy groups were placed on a standard low solute formula. ~ After discharge from the hospital, infants in Groups 1 and 2 continued to receive the same formula, while infants in Groups 3 and 4 were provided with a similar formula except that the iron content was 8.0 mg. per quart. The patients remained on the assigned formulas until 4 months of age. Infants also received 0.3 c.c. of an oral multivitamin preparation t containing vitamins A, C, and D, which was usually begun, on the third day of life and cominued throughout the study period. Adequate supplies of the formula were provided throughout the study period. When discharge occurred before the age of 6 weeks (as was usually true for the more mature infants in BWG Category C), vitamin E and/or iron supplementation was continued at home until 6 weeks of life. At any time during the study period that the hemoglobin concentration fell below 7.5 Gm. per cent, the infant was removed from the study and treated appropriately. At the end of the study period, those infants not previously given supplemental iron were treated to ensure adequate iron stores during the first year of life. More than 80 per cent of infants in each BWG category were observed during the entire study period; loss of patient contact was similar for therapy groups within each BWG category. Exclusion of infants originally considered for a variety of conditions enumerated above resulted in a final number of 186 infants studied, distributed in BWG categories as follows: Category A, 48 infants; Category B, 89 infants; and Category C, 49 infants. Of the 38 infants removed from the study, there were 10 whose hemoglobin con*Enfamil, Mead Johnson Laboratories, Evansville, Ind. Elemental iron content 1.4 rag. per quart; tocopherol content approximately 5.0 rag. per quart or 0.15 rag. per 100 Gm. of unsaturated fatty acids. 20 calories per ounce. "~Tri-vi-sol, Mead Johnson Laboratories, Evansville, Ind.
Vitamin E-dependent anemia
571
centration fell below 7.5 Gin. per cent; they are considered in a subsequent study. 14 Capillary blood for laboratory determinations was obtained from the heel after warming. Hemoglobin concentration, microhematocrit, reticulocyte count, platelet count, and peripheral blood smear for red cell morphology were determined by usual methods. Red cell H202 fragility tests were performed according to the method of Gordon and associates. 1~ Serum levels of free tocopherol were determined by the technique of Quaife and associates~6 on 0.6 ml. of serum with proportional readjustment of the reagents. A serum tocopherol level less than 0.50 mg. per cent was held to reflect vitamin E deficiency.6 The method of Schade and associates a~ was used to measure bound serum iron. Patients were tested for sickle hemoglobin and other hemoglobinopathies and for glucose-6-phosphate dehydrogenase deficiency.iS, 19 Blood typing and direct Coombs test were performed by usual methods. Hematologic determinations (other than screening procedures) were carried out during the first 24 hours of life, at weekly intervals until the fourth week, and at 2 to 4 week intervals thereafter until completion of the study period. Initial H~O2 fragility, vitamin E levels, and bound serum iron (BSI) levels were obtained at 7 days of age and subsequently according to the above schedule. Blood required for these determinations totalled less than 2.5 ml. per week. RESULTS
The race, sex, mean birth weight, and gestational age for infants in therapy groups within each BWG category are shown in Table I. Within each BWG category, birth weight and gestational age were similar for the therapy groups, as were mean weights at the chronologic age of 8 weeks. Weight gain during this period was not affected by the differences in iron and vitamin E supplementation. Also shown is the number of infants with significant respiratory distress in all therapy groups during the first week of life. Although the incidence of respiratory distress was increased in infants of lower
572
Melhorn and Gross
The Journal o/ Pediatrics October 1971
Table I. Descri ~tion of therapy groups within birth weight-gestational age (BWG) categories
Sex M/F 6/5 6/6 5/6
Mean gestational age (wk.) 30 31 31
3/11
8/6
30
5
1,210
2,310
4/18 4/18 2/20
11/11 13/9 12/10
34 34 34
10 8 8
1,700 1,775 1,775
2,820 2,750 2,710
5/18
12/11
34
8
1,765
2,860
0/12 1/12 3/8
6/6 7/6 6/5
38 38 37
2 3 1
2,190 2,240 2,243
3,610 3,470 3,510
13 3/10 6/7 37 3 2,168 II, according t o the criteria of Rudolph and associates,is
3,420
BWG Type of Categories supplement A No supplement 1,000-1,500 Gm. Vitamin E 28-32 wk. Iron Iron and vitamin E
No. of patients 11 12 11
14
B No supplement 1,501-2,000 Gin. Vitamin E 32-36 wk. Iron Iron and vitamin E
22 22 22 23 12 13 tI
C No supplement 2,001-2,400 Gin. Vitamin E 36-40 wk. Iron Iron and vitamin E eInfants in respiratory distress classes I and
Race Caucasian/ Negro 1/10 2/10 2/9
gestational age, there was no significant difference in the occurrence of this complication among therapy groups within each BWG category. Mean hematologic determinations are presented in Figs. 1 and 2 and in Table II. As shown in Fig. 1, hemoglobin concentrations throughout the study period were generally lower in infants of lesser gestational age. Within each BWG category no significant differences in hemoglobin concentration appeared among therapy groups during the first 4 weeks of life. In the most gestationally mature BWG Category C infants, there were essentially no differences in hemoglobin concentration among therapy groups throughout the study period. Between 6 and 10 weeks of life, however, those infants in groups within BWG Categories A and B, whose diets were supplemented solely with vitamin E, maintained higher mean hemoglobin concentrations than did those in other groups; progressively lower hemoglobin concentrations were seen in groups receiving iron and vitamin E, those given no supplement, and those receiving iron alone, respectively. Hemoglobin concentrations in infants given vitamin E were significantly higher than those of groups given no supplements and those of groups
Significant respiratory distress~ (No.) 5 4 5
Mean birth weight (Gin.) 1,170 1,220 1,280
Mean weight age 8 wk.
(Gin.)
2,320 2,350 2,390
given iron during the period between 6 and 8 weeks (p < 0.001). Comparison of the differences between groups receiving no supplements and those receiving only iron are given special attention below. Also documented in Fig. 1 are mean reticulocyte responses. Within individual BWG categories, reticulocyte counts among therapy groups were similar during the first month of life. Significant differences appeared at 6 weeks and continued through 10 weeks of age among therapy groups in BWG Categories A and B. The most striking reticulocyte elevations were seen in patients whose diets were supplemented solely with iron. Elevation of reticulocyte count also occurred, but to a lesser degree, in the infants receiving no supplement. In addition, during this period of time reticulocyte counts were significantly more elevated in groups not given vitamin E than in groups receiving vitamin E alone or vitamin E and iron (Table II, p < 0.001). Reticulocytosis in all groups within BWG Categories A and B was maximum (except for the first 24 hours of life) at the time when hemoglobin concentration was falling or at lowest levels, and was most elevated in infants of least gestational maturity. Mean platelet counts are shown in Fig. 2. Differences among therapy groups were not
Volume 79 Number 4
Vitamin E-dependent anemia
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Fig. 1. Mean hemoglobin concentration and reticulocyte count in study groups within each BWG category. Significant differences in the figure represent P values from ~0.01 to ~0.001, and are graphically demonstrated by relating (+) to (O). significant, except during the period between 6 and 8 weeks of age in infants in BWG Categories A and B, when the platelet counts in patients receiving either no supplement or iron alone were significantly more elevated (p < 0.01). Bound serum iron concentrations are shown in Fig. 3. During the first month, mean bound serum iron in all therapy groups remained above 75/zg per cent regardless of the type of supplementation; bound serum iron concentrations were generally similar among therapy groups within BWG categories. After 6 weeks, however, the mean bound serum iron was significantly more elevated in groups receiving iron alone than in those receiving no iron supplement. By the end of the study period, bound serum
iron in groups not supplemented with iron had fallen to mean levels between 50 and 60 /xg per cent. Groups given supplementary iron appeared capable of satisfactory intestinal absorption, and there was no clear indication that absorption of iron was related to gestational age. However, the mean bound serum iron in therapy groups in all BWG categories in which infants received vitamin E in addition to iron was consistently lower between 4 and 8 weeks of age than in groups receiving iron alone. This difference was most striking in BWG Category A, in which the pattern of mean bound serum iron concentrations in the group given both iron and vitamin E more closely approximated those of the groups not given iron than those of the groups receiving iron alone.
5 74
Melhorn and Gross
The Journal of Pediatrics October 1971
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BWG Category Irvpe of supplement I Hemoglobin--mean Gm. per cent (+- S.D.)
wk.
I
8 wk
I
10 wk.
A 1,000-1,500 Gin. 28-32 wk.
No sapplement Vitamin E Iron Iron and vitamin E
8.8 9.7 8.2 9.1
(0.90)t (0.65)* (0.80)t (0.70)
8.7 9.7 8.0 8.9
(1.0)t (0.80)* (0.80)t (0.80)
8.9 9.8 8.3 9.1
(0.60)t (0.70)* (0.90)t (0.80')
B 1,501-2,000 Gm. 32-36 wk.
No supplement Vitamin E Iron Iron a n d v i t a m i n E
9.6 10.6 8.9 10.3
(0.90)t (0.90)* (1.0)t (1.1)
9.4 10.4 9.1 9.9
(0.70)t (0.60)* (1.0)t (0.80)
9.7 10.4 9.7 10.4
(0.90) (0.90) (1.1) (1.0)
Reticulocyte count--mean per cent (+ S.D.) A 1,000=1,500 Gm. 28-32 wk.
No supplement Vitamin E Iron Iron and vitamin E
10.9 4.8 12.1 7.9
(2.6)t (2.1)* (2.8)* (3.0)t
8.7 4.9 9.4 6.2
(2.2)t (1.9)* (2.3)t (2.0)
6.6 4.6 7.4 4.9
(3.0) (1.6) e (2.6)t (2.4)
B 1,501-2,000 Gm. 32-36 wk.
No supplement Vitamin E Iron Iron and vitamin E
6.4 3.8 8.8 5.1
.(2.4)t (2.0) r (3.1)t (2.6)
5.6 3.0 6.4 4.4
(2.4)t (1.4) e (3.6)t (2.1)
5.0 2.6 5.1 3.3
(2.2)t (1.2)* (1.8)t (1.4)
p R a n g e < 0.01 to < 0.001 between groups given vitamin E alone (*) and groups indicated by dagger ( ~ ) .
Serum free tocopherol levels a n d values for erythrocyte H20~ fragility are recorded in Fig. 4. Between 6 a n d 10 weeks, those infants in all groups who received vitamin E h a d higher tocopherol levels; these differences
were significant only in B W G Categories A and B (p < 0.001). Serum tocopherol levels during the first ten weeks of life were related not only to vitamin E s u p p l e m e n t a t i o n b u t to gestational age as well. T h e percentage of
Volume 79 Number 4
Vitamin E-dependent anemia
BWG "C"
BWG "B"
575
BWG "A"
150
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Fig. 3. Mean bound serum iron in study groups within each BWG category. Significant differences shown represent a P value <0.01, and are graphically demonstrated by relating (+) to ( i ) . infants with serum tocopherol levels below 0.50 rag. per cent between 4 and 10 weeks was greatest in the lowest gestational age infants, irrespective of whether vitamin E was given (Table I I I ) . This relationship was even more striking, considering that the dosage of vitamin E was the same for all treated patients; i.e., the dosage on a per kilogram basis was greater for the infants of lower birth weight. Also of interest was the pattern of serum tocopherol levels in those infants not given supplementary vitamin E. As seen in Fig. 4, unsupplemented groups in BWG Category C maintained, with the exception of the second week, mean tocopherol levels above 0.50 mg. per cent throughout the study. By contrast, the unsupplemented groups in Categories A and B consistently had mean serum tocopherol levels less than 0.50 mg. per cent until the tenth and eighth weeks of life, respectively. Although the differences were not significant, the mean serum toeopherol levels in all patients who received both iron and vitamin E supplements were regularly lower than
Table I I I . Per cent of infants with vitamin E deficiency (tocopherol < 0.50 rag. per cent) in each BWG category. BWG Category A 1,000-1,500 Gm. 28-32 wk. B
1,501-2,000 Gm. 32-36 wk. C 2,001-2,400 Gm. 36-40 wk.
Vitamin E supplement None
Per cent deficient 83 89 75 78 28
25 units/day 54 60 55 52 None
65 46 48
5
25 units/day 28 32 20 25
0
None
46
8
40
22
15
10
0
25 units/day 21 Weeks 2
10 4
5 6
6 8
0 12
those recorded in patients given only vitamin E. Marked differences in red cell H202 fragility developed during the 6 to 10 week interval between infants given vitamin E supplement and those given only iron supplement in Categories A and B. H20= fragility values in the groups given iron were significantly
576
M e l h o r n and Gross
I00
The Journal of Pediatrics October 1971
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Fig. 4. Mean serum vitamin E (free tocopherol) leveI and red cell H_~O2fragility in study groups within each BWG category. Vitamin E values below the shaded horizontai line (at the 0.50 mg. per cent level) reflect vitamin E deficiency. more elevated (p < 0.001). During this same period of time, differences were also present between infants who received both iron and vitamin E and those who received no supplement (p < 0.01, age 6 weeks). Mean H202 fragility was usually more elevated, although not significantly, in patients given both iron and vitamin E as compared to those who received vitamin E alone. In infants given no supplement, the values for H202 fragility did not differ significantly from those infants given iron alone. An inverse relationship between mean H202 fragility above 30 per cent and serum tocopherol level below 0.50 mg. per cent was seen in therapy groups only after 4 weeks
of age. Groups in all BWG categories had mean red cell H202 fragility values above 30 per cent in the first 2 weeks of life, during which time there was no clear correlation between H202 fragility and serum tocopherol level. Although the association of H202 fragility greater than 30 per cent and serum tocopherol level less than 0.50 mg. per cent was noted after 4 weeks, no relationship was found between the degrees of each. It is evident from the data presented in Table I I I that a significant minority of patients not given supplementary vitamin E in BWG Categories A and B had serum tocopherol levels above 0.50 mg. per cent between 4 and 8 weeks of age. In order to ascertain
Volume 79 Number 4
the effects of iron administration on the vitamin E-deficient infants in Categories A and B, the hemoglobin concentration and reticulocyte counts in vitamin E-deficient patients treated with iron were compared with those of similar vitamin E-deficient infants who were given no iron supplement (Fig. 5). When considered in this fashion, hemoglobin concentration was significantly lower (8.1 versus 9.3 Gm. per cent, p < 0.001) and reticulocyte count higher (10.2 versus 6.4 per cent, p < 0.001) at 6 weeks of age in those vitamin E-deficient infants who were treated with iron. Similar differences were seen at 8 weeks of age (p = 0.01).
DISCUSSION Relationships between vitamin E deficiency and hematologic changes. Subsequent to the observation that vitamin E deficiency is common early in the life of the premature infant, z~ a search had been carried out to identify pathologic conditions associated with this lack. Our results confirm those of previous studies 1, z, 3 with regard to the relatlonship of vitamin E deficiency to abnormally elevated in vitro red cell H202 fragility, anemia, and increased reticulocytosis in significantly premature infants during the second month of life. The correlation between lack of vitamin E, increased in vitro H.oO2 red cell hemolysis, and lower hemoglobin seen here again suggests that a mild hemolytic process is present and may reflect increased peroxide stress. Red ceil survival studies were not performed in the present study, but shortened RBC lifespan under similar circumstances has been documented previously?, s Hydrogen peroxide fragility during the first 4 weeks of life in infants whose gestatational age was less than 36 weeks, and during the first 2 weeks in infants above 36 weeks' gestation, was not consistently related to serum tocopherol level. During this initial period of life H202 fragility was often elevated ( ~ 30 per cent hemolysis) in patients with vitamin E levels above 0.50 rag. per cent and also occasionally indicated less
Vitamin E-dependent anemia
577
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Fig. 5. Mean hemoglobin concentration and reticulocyte count between 4: and 8 weeks in vitamin E deficient infants in BWG categories A and B who did not receive vitamin E supplement. Study groups receiving only iron had significantly lower hemoglobin concentration and higher reticulocyte counts (P<0.001) than groups receiving no supplements. than 30 per cent hemolysis when tocopherol levels were below 0.50 rag. per cent. It would be tempting to relate abnormal RBC response to peroxidative stress to conditions commonly occurring in the early life of the premature infant, particularly the respiratory distress syndrome associated with prolonged oxygen therapy. In the selection of infants for this study, patients with significant respiratory distress were equally distributed among therapy groups; consequently, the evaluation of these relationships was not feasible. It is also possible that the early increased H202 fragility represents deficiencies in other cellular mechanisms that ordinarily protect against peroxidafion, e.g., decreased levels of glutathione peroxidase31 In noting the relationships between vitamin E deficiency and anemia in premature infants, Ritehie and associatess recorded a coincident thrombocytosls during the period of vitamin E lack and relative anemia. In the present study, platelet counts in groups not
578
Melhorn and Gross
given vitamin E were consistently higher than those occurring in infants receiving vitamin E during the period between 4 and 10 weeks. However, platelet count elevations did not approximate the degrees of thrombocytosis observed by Ritchie and associates. The small number of infants studied by Ritchie and associates had birth weights below 1,500 Gm. and were selected because of the combination of unusually low hemoglobin concentrations and vitamin E deficiency. A similar group of infants, not included in analysis of the present data, is considered in the second part of this study. 14 Relationships between iron and vitamin E in the premature infant. Among the recognized nutritional requirements which must be met for the premature infant during the first year of life is the increased need for utilizable iron. Although the gestationally immature infant is born with iron stores which approximate those of the full-term infant when measured in terms of milligrams per kilogram of body weight, the premature infant's body mass (and consequently blood volume and hemoglobin mass) normally increases at a more rapid rate than does that of the full-term infant during the first year. Since dietary sources of iron are often limited, outstripping of iron stores possessed at birth frequently results in significant iron deficiency in the premature infant during the second 6 months of life. For this reason, supplementation of the premature infant's diet with iron has been recommended. 7 It has been suggested that 1.5 to 3.0 mg. of elemental iron per kilogram per day is adequate to maintain iron sufficiency in the premature infant during the first year of life. Implicit in this calculation is the assumption that the infant will continue to receive such supplementation throughout the first year. Because medical follow-up for the premature infant may be erratic or incomplete, it has become common practice to provide relatively large amounts of iron during the early weeks of life while the infant is in the premature nursery. The effort is made to supply iron requirements for the entire first year of life while the patient is
The Journal of Pediatrics October 1971
"available." Thus the dose of elemental iron used in the present study exceeded "prophylactic" requirements and was utilized in order to study the effects of the administration of a relatively large amount of iron during the early life of the premature infant. In light of the correlation between vitamin E deficiency and anemia early in the life of the premature infant, the data presented here must raise questions concerning the propriety of administration of large amounts of iron to such infants during the initial weeks of life. The lowest hemoglobin concentrations and highest reticulocyte counts in infants whose gestational age was less than 36 weeks were seen in vitamin E-deficient infants receiving iron. Conversely, infants receiving vitamin E supplement without additional iron maintained the highest hemoglobin concentrations throughout the same period. There is ample experimental evidence that valent iron increases in vitro and in vivo RBC hemolysis when the antiperoxidant protection of the RBC membrane is inadequate. Golberg and associates22 demonstrated an increased accumulation of lipid peroxides in the tissues of vitamin E-deficient rats given overloads of iron. In other studies, vitamin E-deficient mice developed a hemolytic anemia associated with elevated red cell H202 fragility and abnormally increased lipid peroxidation2 The mechanism by which iron increases lipid peroxidation is apparently a function of its ability to act as a catalyst in the nonenzymatic autooxidation of unsaturated fatty acids2 It is clear that such an agent as valent iron, known to enhance the rate of lipid peroxidation, might function adversely in this regard when the normal cellular mechanisms for detoxification of peroxidants are deficient. The function of iron in enhancing peroxidation appears to depend upon its valence. In experiments with in vitro RBC lysis by iron-dextran, Fielding 2~ presented evidence that increased hemolysis was the result of relatively small amounts of valent iron in the preparation. This observation is relevant to the present study, in which oral ferrous
Volume 79 Number 4
sulfate was the form of exogenous iron which is transported in the valent state loosely bound to transferrin. In contrast, exogenous iron given parenterally in the form of irondextran remains almost entirely in the complexed state while in circulation. 24 It may therefore be worthwhile to consider future studies utilizing iron-dextran in investigating such relationships between iron and vitamin E in the premature infant. I t is not the intention of this report to suggest that the practice of administering supplemental iron to the premature infant is unwarranted; the justification for such nutritional supplementation is well established. T h e present study did not evaluate the effects of small amounts of iron administered in iron-supplemented (approximately 10 rag. per quart) low solute formulas, and it is not known whether such supplemented formulas would satisfy the premature infant's need for this mineral without producing the effects of large iron doses noted above. Both conditions would presumably be met, however, only if infants remained on such formulas during the entire first year, and only if vitamin E sufficiency could be maintained during the early weeks of life. Other alternative approaches to administration of iron to the premature infant are suggested. In infants for whom appropriate medical attention during the first year of life seems assured, delay in iron supplementation until the third month of life is reasonable. Since the addition of vitamin E supplement during the period of iron administration early in life appears to negate many of the adverse effects of oral iron, the combination of these supplements may be helpful, but only when the infant is capable of intestinal absorption of tocopherol. Data presented elsewhere ~* clearly indicate that the absorption of vitamin E is, at best, erratic during the first three months in low-gestational-age infants; for these infants it may be necessary to explore alternate routes of forms of vitamin E administration. In any event, the data presented above should caution against overzealous attempts to meet the iron requirements of the prema-
Vitamin E-dependent anemia
5 79
ture infant. At a time when many are urging greater supplementation of proprietary infant formulas and other foodstuffs with iron, 25 the potentially adverse effects of larger doses of iron must be considered. The authors wish to express appreciation for the technical assistance of Miss Carol Luckey and Mrs. Sophie Smith. The cooperation of the nursing service at Babies and Chitdrens Hospital in the premature nursery and premature outpatient clinic divisions requires special thanks, particularly the efforts of Mrs. Mary Herkner, R.N., and Miss Ann Noe, R.N. The counsel of Drs. Howard Gruber, Avroy Fanaroff, and Marshall Klaus of the Neonatology Service was also of benefit. Finally, the study could not have been carried out without the assistance of the pediatric house officers at University Hospitals. REFERENCES
1. Oski, F. A., and Barness, L. A.: Vitamin E deficiency: A previously unrecognized cause of hemolytic anemia in the premature infant, J. P~DIAT. 70: 211, 1967. 2. Hassan, H., Hashim, S. A., Van Itallle, T. B, and Sebrell, W. H.: Syndrome in premature infants associated with low plasma vitamin E levels and high polyunsaturated fatty acid diet, Amer. J. Clin. Nutr. 19: 147, 1966. 3. Ritchie, J. H., Fish, M. B., McMasters, V., and Grossman, M.: Edema and hemolytic anemia in premature infants, New Eng. J. Med. 27: 1185, 1969. 4. Horwitt, M. K.: Vitamin E and lipid metabolism in man, Amer. J. Clin. Nutr. 8: 451, 1960. 5. Jacob, H. S., and Lux, S. E., IV: Degradation of membrane phospholipids in peroxide hemolysis: Studies in vitamin E deficiency, Blood 32: 549, i968. 6. Binder, H. J., Herring, D. C., Hurst, V., Finch, S. C., and Spiro, H. M.: Tocopherol deficiency in man, New Eng. J. Med. 273: 1289, 1965. 7. Hammond, D., and Murphy, A.: The influence of exogenous iron on formation of hemoglobin in the premature infant, Pedlatrias 25: 362, 1960. 8. Smith, G. J., and Dunkley, W. L.: Initiation of lipid peroxidatlon by a reduced metM ion, Arch. Biochem. 98: 46, 1962. 9. Smith, K. A., and Mengel, C. E.: Association of iron-dextran induced hemolysis and lipid peroxldation in mice, J. Lab. Clin. Med. 72: 505, I968. 10. Amiel-Tison, C.: Neurologic evaluation of the maturity of newborn infants, Arch. Dis. Child. 43: 89, 1968, 11. Usher, R., McLean, F., and Scott, K.: Judgment of fetal age. II. Clinical signifi-
580
12.
13.
14.
15.
16.
17.
18.
Melhorn and Gross
cance of gestational age and an objective method for its assessment, Pedlar. Clin. N. Amer. 13" 835, 1966. Lubchenko, L. O., Hanssman, C., Dressier, M., and Boyd, E.: Intrauterine growth as estimated from liveborn birthweight data at 24 to 42 weeks of gestation, J. PEDXAT. 71: 159, 1967. Rudolph, A. J., Desmond, M. M., and Pineda, R. G.: Clinical diagnosis of respiratory difficulty in the newborn, Pediat. Clin. N. Amer. 13: 669, 1966. Melhorn, D. K., and Gross, S.: Vitamin E-dependent anemia in the premature infant. II. Relationships between gestational age and absorption of vitamin E, J. P ~ A T . 79; 581, 1971. Gordon, H. H., Nitowsky, H. M., and Cornblath, M.: Studies of tocopherol deficiency in infants and children. I. Hemolysis of erythrocytes in hydrogen peroxide, Amer. J. Dis. Child. 92-" 164, 1956. Quaife, M. L., Scrimshaw, N. S., and Lowry, O. H.: Micromethod for assay of total tocopherols in blood serum, J. Biol. Chem. 188 1229, 1949. Schade, A. L., Oyama, J., Relnhart, 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. M~tulsky, A., and Campbell-Kraut, J. M.: Population genetics of glucose-6-phosphate
The Journal of Pediatrics October 1971
19.
20.
21.
22.
23. 24. 25.
dehydrogenase deficiency of the red ceil, in Blumberg, B., editor: Proceedings of the Conference on genetic polymorphisms and geographic variations in disease, New York, 1962, Grune & Stratton, Inc., pp. 159. Zinkham, W. H., Lenhard, R. E., Jr., and Childs, B.: Deficiency of glucose-6-phosphate dehydrogenase activity in erythrocytes from patients with favlsm, Bull. Hopkins Hosp. 102: 169, 1958. Wright, S. W., Filer, L. J., Jr., and Mason, K. E.: Vitamin E blood levels in premature and full term infants, Pediatrics 7: 386, 1951. Gross, R. T., Bracci, R., Rudolph, N., Schroeder, E., and Kochen, J. A.: Hydrogen peroxide toxicity and detoxification in the erythrocytes of newborn infants, Blood 29: 481, 1967. Golberg, L., Martin, I. E., and Batchelor, Ao: Biochemical changes in the tissues of animals injected with iron. III. Lipid peroxidation, Biochem. J. 83: 291, 1962. Fielding, J.: The hemolytic activity in an iron-carbohydrate complex, J. Clin. Path. 16: 12, 1962. Henderson, P. A., and Hillman, R. S.: Characteristics of iron-dextran utilization in man, Blood 34: 357, 1969. Smith, N. J., editor: Iron nutrition in infancy. Report of the 62nd Ross Conference on Pediatric Research, Columbus, 1970, Ross Laboratories, p. 45.
569
Vitamin E-dependent anemia in the premature infant. I Effects of large doses of medicinal iron Description of a hemolytic anemia associated with vitamin E deficiency in premature infants prompted study of the relationships of medicinal iron, vitamin E, and hematologic parameters during the early life of such infants. A total o] 186 patients categorized according to birth weight and gestalional age were placed in study groups: (1) no iron or vitamin E supplement, (2) oral vitamln E supplement, (3) oral iron supplement, and (4) iron and vitamin E supplements. Significantly lower mean hemoglobin and serum vitamin E concentrations, with higher reticulocyte count and hydrogen peroxide #agility, were found during the second month of life in infants with a gestational age less than 36 weeks who were not receiving vitamin E. Hemoglobin values were lowest and reticulocyte counts highest in vitamin E-deficient infants of low gestational age who received supplemental #on. These observations suggest that therapeutic doses of iron increase red cell hemolysis during a period of vitamin E deficiency.
David K. Melhorn, M.D.,* and Samuel Gross, M.A., M.D., with the technical assistance of Geraldine Childers CLEVELAND~ O H I O
I ~ 1967, Oski and Barness 1 reported the occurrence of a mild hemolytic anemia during the second month of life in vitamin E-From the Department of Pediatrics, CaseWestern Reserve University School of Medicine, and University Hospitals of Cleveland. Supported in part by the Mead Johnson Laboratories, Evansville, Indiana, the Health Fund of Greater Cleveland; the Frackelton Memorial Fund, the Wellington, Ohio, United Appeal, and the Natlonal Institutes of Health Grants Nos. RR 05410-09 and FR-87. Presented in part at the Thirteenth annual meeting of the American Society of Hematology, San Juan, Puerto Rico, December 8, 1970. ~Reprlnt address; Babies and CMldrens Hospital, 2103 Adelbert Rd., Cleveland, Ohio 44106.
deficient premature infants. Concurrent studies showed associated abnormal in vitro sensitivity of erythrocytes when exposed to hydrogen peroxide (H202). These observations have also been noted by others. 2, 3 Since one certain biological action of vitamin E See related article, p. 581. (tocopherol) is that of an antiperoxidant at the cellular level,4 the lack of this vitamin may accelerate red cell lysis as a result of an increased rate of peroxidation of the lipid component of the red blood cell (RBC) Vol. 79, No. 4, pp. 569-580
570
Melhorn and Gross
membrane, s The combination of vitamin E deficiency and abnormal in vitro, red cell H202 has also been described in cystic fibrosis and other malabsorptive disorders without associated anemia. 6 It is reasonable to postulate that other factors coexist with deficiency of tocopherol as determinants of the vitamin E-dependent anemia of prematurity. Exogenous iron is often given in relatively large amounts to premature infants to prevent the "late anemia" of prematurity, which develops as a result of rapid growth and outstripping of body iron stores during the latter part of the first year of life. 7 Valent iron has been shown to act as a catalyst in the autooxidation of unsaturated fatty acids, s Since such lipids are essential components of the RBC membrane, excess iron may increase RBC lipid peroxidation when the usual antiperoxidant mechanisms of the red cell are defieientY Because iron is often administered to premature infants during the period of vitamin E deficiency, the present study was designed to investigate these relationships during the first 4 months of life of the premature infant. MATERIALS
AND
METHODS
Included in the initial enrollment were 234 infants admitted to the premature nursery unit of Babies and Childrens Hospital, University Hospitals of Cleveland, during the first day of life in the period between January, 1969, and July, 1970. The gestational age of each infant was ascertained by a combination of the following criteria: (1) reliable maternal estimation of length of gestation, (2) neurologic evaluation of maturity according to the method of Amiel-Tison, 1~ and (3) evaluation of external physical characteristics as described by Usher and associates.1~ When correlation between gestational age and birth weight placed infants below the tenth or above the ninetieth percentile on the graph derived by Lubehenko and associates, a2 patients were excluded from the study. Thus "small-for-date" infants and those infants whose birth weights were excessive for estimated gestatlonal age were not included.
The Journal of Pediatrics October 1971
Infants with hemoglobin concentrations less than 14.0 Gm. per cent in the first 24 hours of life and those with blood group incompatibilities, RBC enzyme deficiencies, hemoglobinopathies, infections, or defects requiring surgical intervention were also excluded. Infants who developed the respiratory distress syndrome were included in the study; those with severe and moderate respiratory distress were assigned to classes I and II, respectively, as suggested by Rudolph and associates? 3 Estimation of gestational age was carried out within the first 48 hours of life, and the infants were assigned to 1 of 3 birth weightgestational age (BWG) categories: Category A = birth weight < 1,500 Gin., < 32 weeks' gestation; Category 13 = birth weight 1,501 to 2,000 Gin., 32 to 36 weeks' gestation; Category C ----- birth weight 2,001 to 2,400 Gm., 36 to 40 weeks' gestation. Infants in each of the 3 BWG categories were then placed at random into one of 4 groups receiving the following therapeutic regimens. Therapy group
Type o[ supplement
1 2 3 4
None Vitamin E alone Iron alone Iron and vitamin E
The form, dosage, and timing of administration of iron and vitamin E supplements are shown below. Form Vitamin E atoeopherol acetate ~ Iron ferrous sulfatet
Dosage 25 units per day orally !0 rag. elemental iron per day orally while weighing < 1 , 5 0 0 Gm.; 15 rag.: 1,501 to 2,000 Gin.; 20 rag.: )>2,001 Gin.
Duration Eighth to forty-second day of life Fifteenth to forty-second day- of llfe
~Aquasol E, United States Vitamin and Pharmaceutical Corporation, New York, N. Y. tFer-ln-sol, Mead Johnson Laboratories, Evansville, Ind.
Volume 79 Number4
Vitamin E was administered in a single dose midway between formula feedings. The iron was also given between feedings in 2 divided doses and was separated from administration of vitamin E (in Group 4) by a minimum of 4 hours. Infants in all therapy groups were placed on a standard low solute formula. ~ After discharge from the hospital, infants in Groups 1 and 2 continued to receive the same formula, while infants in Groups 3 and 4 were provided with a similar formula except that the iron content was 8.0 mg. per quart. The patients remained on the assigned formulas until 4 months of age. Infants also received 0.3 c.c. of an oral multivitamin preparation t containing vitamins A, C, and D, which was usually begun, on the third day of life and cominued throughout the study period. Adequate supplies of the formula were provided throughout the study period. When discharge occurred before the age of 6 weeks (as was usually true for the more mature infants in BWG Category C), vitamin E and/or iron supplementation was continued at home until 6 weeks of life. At any time during the study period that the hemoglobin concentration fell below 7.5 Gm. per cent, the infant was removed from the study and treated appropriately. At the end of the study period, those infants not previously given supplemental iron were treated to ensure adequate iron stores during the first year of life. More than 80 per cent of infants in each BWG category were observed during the entire study period; loss of patient contact was similar for therapy groups within each BWG category. Exclusion of infants originally considered for a variety of conditions enumerated above resulted in a final number of 186 infants studied, distributed in BWG categories as follows: Category A, 48 infants; Category B, 89 infants; and Category C, 49 infants. Of the 38 infants removed from the study, there were 10 whose hemoglobin con*Enfamil, Mead Johnson Laboratories, Evansville, Ind. Elemental iron content 1.4 rag. per quart; tocopherol content approximately 5.0 rag. per quart or 0.15 rag. per 100 Gm. of unsaturated fatty acids. 20 calories per ounce. "~Tri-vi-sol, Mead Johnson Laboratories, Evansville, Ind.
Vitamin E-dependent anemia
571
centration fell below 7.5 Gin. per cent; they are considered in a subsequent study. 14 Capillary blood for laboratory determinations was obtained from the heel after warming. Hemoglobin concentration, microhematocrit, reticulocyte count, platelet count, and peripheral blood smear for red cell morphology were determined by usual methods. Red cell H202 fragility tests were performed according to the method of Gordon and associates. 1~ Serum levels of free tocopherol were determined by the technique of Quaife and associates~6 on 0.6 ml. of serum with proportional readjustment of the reagents. A serum tocopherol level less than 0.50 mg. per cent was held to reflect vitamin E deficiency.6 The method of Schade and associates a~ was used to measure bound serum iron. Patients were tested for sickle hemoglobin and other hemoglobinopathies and for glucose-6-phosphate dehydrogenase deficiency.iS, 19 Blood typing and direct Coombs test were performed by usual methods. Hematologic determinations (other than screening procedures) were carried out during the first 24 hours of life, at weekly intervals until the fourth week, and at 2 to 4 week intervals thereafter until completion of the study period. Initial H~O2 fragility, vitamin E levels, and bound serum iron (BSI) levels were obtained at 7 days of age and subsequently according to the above schedule. Blood required for these determinations totalled less than 2.5 ml. per week. RESULTS
The race, sex, mean birth weight, and gestational age for infants in therapy groups within each BWG category are shown in Table I. Within each BWG category, birth weight and gestational age were similar for the therapy groups, as were mean weights at the chronologic age of 8 weeks. Weight gain during this period was not affected by the differences in iron and vitamin E supplementation. Also shown is the number of infants with significant respiratory distress in all therapy groups during the first week of life. Although the incidence of respiratory distress was increased in infants of lower
572
Melhorn and Gross
The Journal o/ Pediatrics October 1971
Table I. Descri ~tion of therapy groups within birth weight-gestational age (BWG) categories
Sex M/F 6/5 6/6 5/6
Mean gestational age (wk.) 30 31 31
3/11
8/6
30
5
1,210
2,310
4/18 4/18 2/20
11/11 13/9 12/10
34 34 34
10 8 8
1,700 1,775 1,775
2,820 2,750 2,710
5/18
12/11
34
8
1,765
2,860
0/12 1/12 3/8
6/6 7/6 6/5
38 38 37
2 3 1
2,190 2,240 2,243
3,610 3,470 3,510
13 3/10 6/7 37 3 2,168 II, according t o the criteria of Rudolph and associates,is
3,420
BWG Type of Categories supplement A No supplement 1,000-1,500 Gm. Vitamin E 28-32 wk. Iron Iron and vitamin E
No. of patients 11 12 11
14
B No supplement 1,501-2,000 Gin. Vitamin E 32-36 wk. Iron Iron and vitamin E
22 22 22 23 12 13 tI
C No supplement 2,001-2,400 Gin. Vitamin E 36-40 wk. Iron Iron and vitamin E eInfants in respiratory distress classes I and
Race Caucasian/ Negro 1/10 2/10 2/9
gestational age, there was no significant difference in the occurrence of this complication among therapy groups within each BWG category. Mean hematologic determinations are presented in Figs. 1 and 2 and in Table II. As shown in Fig. 1, hemoglobin concentrations throughout the study period were generally lower in infants of lesser gestational age. Within each BWG category no significant differences in hemoglobin concentration appeared among therapy groups during the first 4 weeks of life. In the most gestationally mature BWG Category C infants, there were essentially no differences in hemoglobin concentration among therapy groups throughout the study period. Between 6 and 10 weeks of life, however, those infants in groups within BWG Categories A and B, whose diets were supplemented solely with vitamin E, maintained higher mean hemoglobin concentrations than did those in other groups; progressively lower hemoglobin concentrations were seen in groups receiving iron and vitamin E, those given no supplement, and those receiving iron alone, respectively. Hemoglobin concentrations in infants given vitamin E were significantly higher than those of groups given no supplements and those of groups
Significant respiratory distress~ (No.) 5 4 5
Mean birth weight (Gin.) 1,170 1,220 1,280
Mean weight age 8 wk.
(Gin.)
2,320 2,350 2,390
given iron during the period between 6 and 8 weeks (p < 0.001). Comparison of the differences between groups receiving no supplements and those receiving only iron are given special attention below. Also documented in Fig. 1 are mean reticulocyte responses. Within individual BWG categories, reticulocyte counts among therapy groups were similar during the first month of life. Significant differences appeared at 6 weeks and continued through 10 weeks of age among therapy groups in BWG Categories A and B. The most striking reticulocyte elevations were seen in patients whose diets were supplemented solely with iron. Elevation of reticulocyte count also occurred, but to a lesser degree, in the infants receiving no supplement. In addition, during this period of time reticulocyte counts were significantly more elevated in groups not given vitamin E than in groups receiving vitamin E alone or vitamin E and iron (Table II, p < 0.001). Reticulocytosis in all groups within BWG Categories A and B was maximum (except for the first 24 hours of life) at the time when hemoglobin concentration was falling or at lowest levels, and was most elevated in infants of least gestational maturity. Mean platelet counts are shown in Fig. 2. Differences among therapy groups were not
Volume 79 Number 4
Vitamin E-dependent anemia
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Fig. 1. Mean hemoglobin concentration and reticulocyte count in study groups within each BWG category. Significant differences in the figure represent P values from ~0.01 to ~0.001, and are graphically demonstrated by relating (+) to (O). significant, except during the period between 6 and 8 weeks of age in infants in BWG Categories A and B, when the platelet counts in patients receiving either no supplement or iron alone were significantly more elevated (p < 0.01). Bound serum iron concentrations are shown in Fig. 3. During the first month, mean bound serum iron in all therapy groups remained above 75/zg per cent regardless of the type of supplementation; bound serum iron concentrations were generally similar among therapy groups within BWG categories. After 6 weeks, however, the mean bound serum iron was significantly more elevated in groups receiving iron alone than in those receiving no iron supplement. By the end of the study period, bound serum
iron in groups not supplemented with iron had fallen to mean levels between 50 and 60 /xg per cent. Groups given supplementary iron appeared capable of satisfactory intestinal absorption, and there was no clear indication that absorption of iron was related to gestational age. However, the mean bound serum iron in therapy groups in all BWG categories in which infants received vitamin E in addition to iron was consistently lower between 4 and 8 weeks of age than in groups receiving iron alone. This difference was most striking in BWG Category A, in which the pattern of mean bound serum iron concentrations in the group given both iron and vitamin E more closely approximated those of the groups not given iron than those of the groups receiving iron alone.
5 74
Melhorn and Gross
The Journal of Pediatrics October 1971
BWG "A"
BWG " B "
BWG "C"
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400
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BWG Category Irvpe of supplement I Hemoglobin--mean Gm. per cent (+- S.D.)
wk.
I
8 wk
I
10 wk.
A 1,000-1,500 Gin. 28-32 wk.
No sapplement Vitamin E Iron Iron and vitamin E
8.8 9.7 8.2 9.1
(0.90)t (0.65)* (0.80)t (0.70)
8.7 9.7 8.0 8.9
(1.0)t (0.80)* (0.80)t (0.80)
8.9 9.8 8.3 9.1
(0.60)t (0.70)* (0.90)t (0.80')
B 1,501-2,000 Gm. 32-36 wk.
No supplement Vitamin E Iron Iron a n d v i t a m i n E
9.6 10.6 8.9 10.3
(0.90)t (0.90)* (1.0)t (1.1)
9.4 10.4 9.1 9.9
(0.70)t (0.60)* (1.0)t (0.80)
9.7 10.4 9.7 10.4
(0.90) (0.90) (1.1) (1.0)
Reticulocyte count--mean per cent (+ S.D.) A 1,000=1,500 Gm. 28-32 wk.
No supplement Vitamin E Iron Iron and vitamin E
10.9 4.8 12.1 7.9
(2.6)t (2.1)* (2.8)* (3.0)t
8.7 4.9 9.4 6.2
(2.2)t (1.9)* (2.3)t (2.0)
6.6 4.6 7.4 4.9
(3.0) (1.6) e (2.6)t (2.4)
B 1,501-2,000 Gm. 32-36 wk.
No supplement Vitamin E Iron Iron and vitamin E
6.4 3.8 8.8 5.1
.(2.4)t (2.0) r (3.1)t (2.6)
5.6 3.0 6.4 4.4
(2.4)t (1.4) e (3.6)t (2.1)
5.0 2.6 5.1 3.3
(2.2)t (1.2)* (1.8)t (1.4)
p R a n g e < 0.01 to < 0.001 between groups given vitamin E alone (*) and groups indicated by dagger ( ~ ) .
Serum free tocopherol levels a n d values for erythrocyte H20~ fragility are recorded in Fig. 4. Between 6 a n d 10 weeks, those infants in all groups who received vitamin E h a d higher tocopherol levels; these differences
were significant only in B W G Categories A and B (p < 0.001). Serum tocopherol levels during the first ten weeks of life were related not only to vitamin E s u p p l e m e n t a t i o n b u t to gestational age as well. T h e percentage of
Volume 79 Number 4
Vitamin E-dependent anemia
BWG "C"
BWG "B"
575
BWG "A"
150
t25
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Fig. 3. Mean bound serum iron in study groups within each BWG category. Significant differences shown represent a P value <0.01, and are graphically demonstrated by relating (+) to ( i ) . infants with serum tocopherol levels below 0.50 rag. per cent between 4 and 10 weeks was greatest in the lowest gestational age infants, irrespective of whether vitamin E was given (Table I I I ) . This relationship was even more striking, considering that the dosage of vitamin E was the same for all treated patients; i.e., the dosage on a per kilogram basis was greater for the infants of lower birth weight. Also of interest was the pattern of serum tocopherol levels in those infants not given supplementary vitamin E. As seen in Fig. 4, unsupplemented groups in BWG Category C maintained, with the exception of the second week, mean tocopherol levels above 0.50 mg. per cent throughout the study. By contrast, the unsupplemented groups in Categories A and B consistently had mean serum tocopherol levels less than 0.50 mg. per cent until the tenth and eighth weeks of life, respectively. Although the differences were not significant, the mean serum toeopherol levels in all patients who received both iron and vitamin E supplements were regularly lower than
Table I I I . Per cent of infants with vitamin E deficiency (tocopherol < 0.50 rag. per cent) in each BWG category. BWG Category A 1,000-1,500 Gm. 28-32 wk. B
1,501-2,000 Gm. 32-36 wk. C 2,001-2,400 Gm. 36-40 wk.
Vitamin E supplement None
Per cent deficient 83 89 75 78 28
25 units/day 54 60 55 52 None
65 46 48
5
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0
None
46
8
40
22
15
10
0
25 units/day 21 Weeks 2
10 4
5 6
6 8
0 12
those recorded in patients given only vitamin E. Marked differences in red cell H202 fragility developed during the 6 to 10 week interval between infants given vitamin E supplement and those given only iron supplement in Categories A and B. H20= fragility values in the groups given iron were significantly
576
M e l h o r n and Gross
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The Journal of Pediatrics October 1971
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Fig. 4. Mean serum vitamin E (free tocopherol) leveI and red cell H_~O2fragility in study groups within each BWG category. Vitamin E values below the shaded horizontai line (at the 0.50 mg. per cent level) reflect vitamin E deficiency. more elevated (p < 0.001). During this same period of time, differences were also present between infants who received both iron and vitamin E and those who received no supplement (p < 0.01, age 6 weeks). Mean H202 fragility was usually more elevated, although not significantly, in patients given both iron and vitamin E as compared to those who received vitamin E alone. In infants given no supplement, the values for H202 fragility did not differ significantly from those infants given iron alone. An inverse relationship between mean H202 fragility above 30 per cent and serum tocopherol level below 0.50 mg. per cent was seen in therapy groups only after 4 weeks
of age. Groups in all BWG categories had mean red cell H202 fragility values above 30 per cent in the first 2 weeks of life, during which time there was no clear correlation between H202 fragility and serum tocopherol level. Although the association of H202 fragility greater than 30 per cent and serum tocopherol level less than 0.50 mg. per cent was noted after 4 weeks, no relationship was found between the degrees of each. It is evident from the data presented in Table I I I that a significant minority of patients not given supplementary vitamin E in BWG Categories A and B had serum tocopherol levels above 0.50 mg. per cent between 4 and 8 weeks of age. In order to ascertain
Volume 79 Number 4
the effects of iron administration on the vitamin E-deficient infants in Categories A and B, the hemoglobin concentration and reticulocyte counts in vitamin E-deficient patients treated with iron were compared with those of similar vitamin E-deficient infants who were given no iron supplement (Fig. 5). When considered in this fashion, hemoglobin concentration was significantly lower (8.1 versus 9.3 Gm. per cent, p < 0.001) and reticulocyte count higher (10.2 versus 6.4 per cent, p < 0.001) at 6 weeks of age in those vitamin E-deficient infants who were treated with iron. Similar differences were seen at 8 weeks of age (p = 0.01).
DISCUSSION Relationships between vitamin E deficiency and hematologic changes. Subsequent to the observation that vitamin E deficiency is common early in the life of the premature infant, z~ a search had been carried out to identify pathologic conditions associated with this lack. Our results confirm those of previous studies 1, z, 3 with regard to the relatlonship of vitamin E deficiency to abnormally elevated in vitro red cell H202 fragility, anemia, and increased reticulocytosis in significantly premature infants during the second month of life. The correlation between lack of vitamin E, increased in vitro H.oO2 red cell hemolysis, and lower hemoglobin seen here again suggests that a mild hemolytic process is present and may reflect increased peroxide stress. Red ceil survival studies were not performed in the present study, but shortened RBC lifespan under similar circumstances has been documented previously?, s Hydrogen peroxide fragility during the first 4 weeks of life in infants whose gestatational age was less than 36 weeks, and during the first 2 weeks in infants above 36 weeks' gestation, was not consistently related to serum tocopherol level. During this initial period of life H202 fragility was often elevated ( ~ 30 per cent hemolysis) in patients with vitamin E levels above 0.50 rag. per cent and also occasionally indicated less
Vitamin E-dependent anemia
577
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Fig. 5. Mean hemoglobin concentration and reticulocyte count between 4: and 8 weeks in vitamin E deficient infants in BWG categories A and B who did not receive vitamin E supplement. Study groups receiving only iron had significantly lower hemoglobin concentration and higher reticulocyte counts (P<0.001) than groups receiving no supplements. than 30 per cent hemolysis when tocopherol levels were below 0.50 rag. per cent. It would be tempting to relate abnormal RBC response to peroxidative stress to conditions commonly occurring in the early life of the premature infant, particularly the respiratory distress syndrome associated with prolonged oxygen therapy. In the selection of infants for this study, patients with significant respiratory distress were equally distributed among therapy groups; consequently, the evaluation of these relationships was not feasible. It is also possible that the early increased H202 fragility represents deficiencies in other cellular mechanisms that ordinarily protect against peroxidafion, e.g., decreased levels of glutathione peroxidase31 In noting the relationships between vitamin E deficiency and anemia in premature infants, Ritehie and associatess recorded a coincident thrombocytosls during the period of vitamin E lack and relative anemia. In the present study, platelet counts in groups not
578
Melhorn and Gross
given vitamin E were consistently higher than those occurring in infants receiving vitamin E during the period between 4 and 10 weeks. However, platelet count elevations did not approximate the degrees of thrombocytosis observed by Ritchie and associates. The small number of infants studied by Ritchie and associates had birth weights below 1,500 Gm. and were selected because of the combination of unusually low hemoglobin concentrations and vitamin E deficiency. A similar group of infants, not included in analysis of the present data, is considered in the second part of this study. 14 Relationships between iron and vitamin E in the premature infant. Among the recognized nutritional requirements which must be met for the premature infant during the first year of life is the increased need for utilizable iron. Although the gestationally immature infant is born with iron stores which approximate those of the full-term infant when measured in terms of milligrams per kilogram of body weight, the premature infant's body mass (and consequently blood volume and hemoglobin mass) normally increases at a more rapid rate than does that of the full-term infant during the first year. Since dietary sources of iron are often limited, outstripping of iron stores possessed at birth frequently results in significant iron deficiency in the premature infant during the second 6 months of life. For this reason, supplementation of the premature infant's diet with iron has been recommended. 7 It has been suggested that 1.5 to 3.0 mg. of elemental iron per kilogram per day is adequate to maintain iron sufficiency in the premature infant during the first year of life. Implicit in this calculation is the assumption that the infant will continue to receive such supplementation throughout the first year. Because medical follow-up for the premature infant may be erratic or incomplete, it has become common practice to provide relatively large amounts of iron during the early weeks of life while the infant is in the premature nursery. The effort is made to supply iron requirements for the entire first year of life while the patient is
The Journal of Pediatrics October 1971
"available." Thus the dose of elemental iron used in the present study exceeded "prophylactic" requirements and was utilized in order to study the effects of the administration of a relatively large amount of iron during the early life of the premature infant. In light of the correlation between vitamin E deficiency and anemia early in the life of the premature infant, the data presented here must raise questions concerning the propriety of administration of large amounts of iron to such infants during the initial weeks of life. The lowest hemoglobin concentrations and highest reticulocyte counts in infants whose gestational age was less than 36 weeks were seen in vitamin E-deficient infants receiving iron. Conversely, infants receiving vitamin E supplement without additional iron maintained the highest hemoglobin concentrations throughout the same period. There is ample experimental evidence that valent iron increases in vitro and in vivo RBC hemolysis when the antiperoxidant protection of the RBC membrane is inadequate. Golberg and associates22 demonstrated an increased accumulation of lipid peroxides in the tissues of vitamin E-deficient rats given overloads of iron. In other studies, vitamin E-deficient mice developed a hemolytic anemia associated with elevated red cell H202 fragility and abnormally increased lipid peroxidation2 The mechanism by which iron increases lipid peroxidation is apparently a function of its ability to act as a catalyst in the nonenzymatic autooxidation of unsaturated fatty acids2 It is clear that such an agent as valent iron, known to enhance the rate of lipid peroxidation, might function adversely in this regard when the normal cellular mechanisms for detoxification of peroxidants are deficient. The function of iron in enhancing peroxidation appears to depend upon its valence. In experiments with in vitro RBC lysis by iron-dextran, Fielding 2~ presented evidence that increased hemolysis was the result of relatively small amounts of valent iron in the preparation. This observation is relevant to the present study, in which oral ferrous
Volume 79 Number 4
sulfate was the form of exogenous iron which is transported in the valent state loosely bound to transferrin. In contrast, exogenous iron given parenterally in the form of irondextran remains almost entirely in the complexed state while in circulation. 24 It may therefore be worthwhile to consider future studies utilizing iron-dextran in investigating such relationships between iron and vitamin E in the premature infant. I t is not the intention of this report to suggest that the practice of administering supplemental iron to the premature infant is unwarranted; the justification for such nutritional supplementation is well established. T h e present study did not evaluate the effects of small amounts of iron administered in iron-supplemented (approximately 10 rag. per quart) low solute formulas, and it is not known whether such supplemented formulas would satisfy the premature infant's need for this mineral without producing the effects of large iron doses noted above. Both conditions would presumably be met, however, only if infants remained on such formulas during the entire first year, and only if vitamin E sufficiency could be maintained during the early weeks of life. Other alternative approaches to administration of iron to the premature infant are suggested. In infants for whom appropriate medical attention during the first year of life seems assured, delay in iron supplementation until the third month of life is reasonable. Since the addition of vitamin E supplement during the period of iron administration early in life appears to negate many of the adverse effects of oral iron, the combination of these supplements may be helpful, but only when the infant is capable of intestinal absorption of tocopherol. Data presented elsewhere ~* clearly indicate that the absorption of vitamin E is, at best, erratic during the first three months in low-gestational-age infants; for these infants it may be necessary to explore alternate routes of forms of vitamin E administration. In any event, the data presented above should caution against overzealous attempts to meet the iron requirements of the prema-
Vitamin E-dependent anemia
5 79
ture infant. At a time when many are urging greater supplementation of proprietary infant formulas and other foodstuffs with iron, 25 the potentially adverse effects of larger doses of iron must be considered. The authors wish to express appreciation for the technical assistance of Miss Carol Luckey and Mrs. Sophie Smith. The cooperation of the nursing service at Babies and Chitdrens Hospital in the premature nursery and premature outpatient clinic divisions requires special thanks, particularly the efforts of Mrs. Mary Herkner, R.N., and Miss Ann Noe, R.N. The counsel of Drs. Howard Gruber, Avroy Fanaroff, and Marshall Klaus of the Neonatology Service was also of benefit. Finally, the study could not have been carried out without the assistance of the pediatric house officers at University Hospitals. REFERENCES
1. Oski, F. A., and Barness, L. A.: Vitamin E deficiency: A previously unrecognized cause of hemolytic anemia in the premature infant, J. P~DIAT. 70: 211, 1967. 2. Hassan, H., Hashim, S. A., Van Itallle, T. B, and Sebrell, W. H.: Syndrome in premature infants associated with low plasma vitamin E levels and high polyunsaturated fatty acid diet, Amer. J. Clin. Nutr. 19: 147, 1966. 3. Ritchie, J. H., Fish, M. B., McMasters, V., and Grossman, M.: Edema and hemolytic anemia in premature infants, New Eng. J. Med. 27: 1185, 1969. 4. Horwitt, M. K.: Vitamin E and lipid metabolism in man, Amer. J. Clin. Nutr. 8: 451, 1960. 5. Jacob, H. S., and Lux, S. E., IV: Degradation of membrane phospholipids in peroxide hemolysis: Studies in vitamin E deficiency, Blood 32: 549, i968. 6. Binder, H. J., Herring, D. C., Hurst, V., Finch, S. C., and Spiro, H. M.: Tocopherol deficiency in man, New Eng. J. Med. 273: 1289, 1965. 7. Hammond, D., and Murphy, A.: The influence of exogenous iron on formation of hemoglobin in the premature infant, Pedlatrias 25: 362, 1960. 8. Smith, G. J., and Dunkley, W. L.: Initiation of lipid peroxidatlon by a reduced metM ion, Arch. Biochem. 98: 46, 1962. 9. Smith, K. A., and Mengel, C. E.: Association of iron-dextran induced hemolysis and lipid peroxldation in mice, J. Lab. Clin. Med. 72: 505, I968. 10. Amiel-Tison, C.: Neurologic evaluation of the maturity of newborn infants, Arch. Dis. Child. 43: 89, 1968, 11. Usher, R., McLean, F., and Scott, K.: Judgment of fetal age. II. Clinical signifi-
580
12.
13.
14.
15.
16.
17.
18.
Melhorn and Gross
cance of gestational age and an objective method for its assessment, Pedlar. Clin. N. Amer. 13" 835, 1966. Lubchenko, L. O., Hanssman, C., Dressier, M., and Boyd, E.: Intrauterine growth as estimated from liveborn birthweight data at 24 to 42 weeks of gestation, J. PEDXAT. 71: 159, 1967. Rudolph, A. J., Desmond, M. M., and Pineda, R. G.: Clinical diagnosis of respiratory difficulty in the newborn, Pediat. Clin. N. Amer. 13: 669, 1966. Melhorn, D. K., and Gross, S.: Vitamin E-dependent anemia in the premature infant. II. Relationships between gestational age and absorption of vitamin E, J. P ~ A T . 79; 581, 1971. Gordon, H. H., Nitowsky, H. M., and Cornblath, M.: Studies of tocopherol deficiency in infants and children. I. Hemolysis of erythrocytes in hydrogen peroxide, Amer. J. Dis. Child. 92-" 164, 1956. Quaife, M. L., Scrimshaw, N. S., and Lowry, O. H.: Micromethod for assay of total tocopherols in blood serum, J. Biol. Chem. 188 1229, 1949. Schade, A. L., Oyama, J., Relnhart, 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. M~tulsky, A., and Campbell-Kraut, J. M.: Population genetics of glucose-6-phosphate
The Journal of Pediatrics October 1971
19.
20.
21.
22.
23. 24. 25.
dehydrogenase deficiency of the red ceil, in Blumberg, B., editor: Proceedings of the Conference on genetic polymorphisms and geographic variations in disease, New York, 1962, Grune & Stratton, Inc., pp. 159. Zinkham, W. H., Lenhard, R. E., Jr., and Childs, B.: Deficiency of glucose-6-phosphate dehydrogenase activity in erythrocytes from patients with favlsm, Bull. Hopkins Hosp. 102: 169, 1958. Wright, S. W., Filer, L. J., Jr., and Mason, K. E.: Vitamin E blood levels in premature and full term infants, Pediatrics 7: 386, 1951. Gross, R. T., Bracci, R., Rudolph, N., Schroeder, E., and Kochen, J. A.: Hydrogen peroxide toxicity and detoxification in the erythrocytes of newborn infants, Blood 29: 481, 1967. Golberg, L., Martin, I. E., and Batchelor, Ao: Biochemical changes in the tissues of animals injected with iron. III. Lipid peroxidation, Biochem. J. 83: 291, 1962. Fielding, J.: The hemolytic activity in an iron-carbohydrate complex, J. Clin. Path. 16: 12, 1962. Henderson, P. A., and Hillman, R. S.: Characteristics of iron-dextran utilization in man, Blood 34: 357, 1969. Smith, N. J., editor: Iron nutrition in infancy. Report of the 62nd Ross Conference on Pediatric Research, Columbus, 1970, Ross Laboratories, p. 45.