COMPARATIVE
HEMATOLOGIC
RESPONSE
TO
IRON
FORTIFICATION
OF
A
MILK
FORMULA
FOR
INFANTS
By
A.
Marsh, M.D., H. Long, and E. Stierwalt, R.N. Department of Pediatrics, University of Kansas Medical Center404
PEDIATRIcs, September 1959
D
ESPITE current knowledge of nutritional needs and the advances in technology, iron deficiency anemia in infants persists at a high incidence. Woodruff’ calls “shock-ing” the continued occurrence of hypo-chromic anemia, “a preventable disease,” with an incidence approaching“30%
of in-fants in the second year of life in at least one large pediatric clinic.” Sturgeon” 2acon-eludes that “some degree of iron deficiency exists in a significant fraction of the normal infant population.” In premature infants, Schulman’ states, after 50 to 130 days, the iron made available for hemoglobin forma-tion by the destruction of erythrocytes must be supplemented from exogenous sources. Schulz and Smith4 point out that “dietary intake of iron insufficient to satisfy iron re-quirements during growth is the single most important cause of iron deficiency in in-fancy.” They also state that “a disturbing incidence of dietary iron deficiency [is] by far the most common nutritional problem in early life today, . . . and the question of
iron supplementation for all infants must be carefully considered.”
Because iron deficiency is most frequent from 6 months to 2 years of age, addition of iron to milk would be the surest method of assuring adequate intake. To date, data on absorption of iron when given in associa-tion with milk are not conclusive, providing the opportunity for a wide range of opin-ion. A standard pediatric textbook states that iron should not be administered with milk because of the interaction of phos-phates with the iron, precluding absorption. Radioactive tracer studies indicated that iron was absorbed in lesser quantity when administered with milk than separately.
Josephs8 states that iron administered in
(Accepted NIay 22, 1959; submitted April 25.) ADDRESS: (AM.) Kansas City 12, Kansas.
milk is well absorbed.
The present study was designed to test the efficacy of iron added to milk formula, during tile first 9 months of life, with care-ful observation for possible side reactions. Although a cereal of very low iron content was permitted, no other iron-containing supplements were permitted.
STUDY GROUPS AND METHODS
Full-term and premature infants were ob-served from birth to 9 months of age. All sub-jects were born at the University of Kansas Medical Center between January 1, 1956 and November 1, 1957, and were considered
healthy at birth. The’ were selected at random
from patients eligible for clinic care, whose parents were considered likely to adhere to the
dietary regimen and return them for follow-up
visits. Seventy-four full-term and 42 premature
infants were observed for the full 9 months. Infants were assigned shortly after birth to one of three feeding regimens, as follows,
fur-nishing one experimental and two control
groups:
Group 1 . A prepared milk formula#{176} was
augmented by the manufacturer with ferrous
sulfate. When diluted to contain 20 calories
per ounce, the iron content was 12. milligrams
in 32 ounces. In order to assure use of this mixture, the milk was delivered to this group.
Group 2. The same prepared milk without
added iron was delivered to this group.
Group 3. Evaporated milk with added water
and sucrose, at the same caloric concentration,
was prescribed for this group. This milk was
not furnished initially. Because of failure to obtain the prolonged period of observation in a large proportion of this group, a prepared evaporated milk formula was provided during
the last months of the study.
Distribution of the infants as to feeding
ARTICLES 405
TABLE I
STUDY GRoups, FULL-TERM INFANTS
.
.
reeaing uegzmen vegro 7 .
n’ iute u ate remate Iotat
.
Mean Birth
Wt. (gm)
Mean Weight at 9 uon1ns
(gm)
Group 1-Prepared formula, iron-fortified 21 9 15 15 30 3,231 8,515
Group 2-Prepared formula 15 9 10 14 24* 3,268 8,772
Group 3-Evap. milk with CHO formula 16 4 7 13 20* 3,129 8,068
Total 52 22 32 42 74
* Includes one infant changed to group 1 feeding regimen at 7 months of age.
regimen, race and sex is shown for the full-term and premature infants in Tables I and II, respectively. Mean birth weight and weight at 9 months of age is also shown.
The mothers were carefully instructed not to give solid foods. By inquiry at the time of
Clillic examination and of visit to the home
by
a nurse, faithfulness in following instructions was checked. If a mother insisted on offering solid foods, a specially-prepared cereal#{176} un-enriched in iron was provided and permitted ad libitum. If a mother violated the “no other solid food” rule, her infant was dropped from the study. Groups 1 and 2 received n supple-mental vitamins other than already provided in the milk formula, and group 3 was given 0.6 ml of a common vitamin preparation. I
Initial observations were made in the hospi-tal when the infants were 3 days of age, and
* Cooked farina: 0.1 mg iron per 100 gm. I Tni-Vi-Sol#{174}, containing vitamins A, C, and D.
subsequent data were collected during well-baby clinic visits at the ages of 6 weeks, 3, 33k, 4, 43k, 5, 53k, 6, 7, 8, and 9 months. Each in-fant had been subjected to a series of 12 groups of observations and tests by the time he was 9 months of age. The data collected related to : 1) History-food intake, gastrointestinal
up-set, sleep habits, illness, stooling, etc.; 2) rou-tine physical examination, including measure-ments of height, weight and head circumfer-ence; 3) laboratory data-hemoglobin,
hemato-crit, reticulocvte count, total and differential
leukocyte counts, serum iron (for which ap-proximately 5 ml of blood were taken).
Almost all clinical information was gathered by one of the authors (AM). In addition to the contact with the doctor who saw the patients at clinic visits, a full-time nurse visited all homes shortly after the infant was discharged from the hospital and subsequently, to assure follow-up visits and adherence to conditions
of the study.
TABLE II
STUDY GROUPS, PREMATURE INFANTS
Feeding Regimen Negro White Male Female Total Mean Birth Wt. (gm)
Mean Weight at 9 Months
(gm)
Group 1-Prepared formula, iron-fortified 15 1 3 18 16 ,O18 7,862
Group 2-Prepared formula 11 4 6 9 15 2,156 8,076
Group 8-Evap. milk with CHO formula 11 0 5 6 lit 2, 114 6,841
Total 37 5 14 28 42
406 IRON FORTIFICATION
Hemoglobin values were determined by the
evanmethemoglobin method, as standardized
and described by the U. S. Armed Forces.’ Hematocnits were done in microhematocnit tubes by the Cuest centrifuge method.1#{176} Serum
iron determinations were performed on 2 ml of serum. The protein was precipitated with dilute HCI, the ferric iron reduced to the ferrous form
by means of ascorbic acid and treated with o-phenanthroline to form an orange red com-plex, which was read against a known
stand-and in a Beckman spectrophotometer.11
It was decided to provide supplements of iron in all cases before advanced iron deficiency could cause an anemia that might be harmful to the infant. Arbitrarily, a serum iron below 50 ,.g/100 ml or a hemoglobin below 8 gm/100 ml was considered to be evidence of
develop-ing iron deficiency anemia. If both of these indices became low, the feeding regimen was changed, and subsequent data was evaluated
separately. Such infants in groups 2 and 3
were then fed the iron-fortified formula. Since none of the infants of group 1 developed these indices of iron deficiency, all remained on the initial diet for the full period of observation.
RESULTS
Certain mothers felt that omission of solid foods from the diet imposed a hard-ship on the infant or the household. Al-though Tables I and II indicate serial ob-servations on only 74 full-term and 42 pre-mature infants, there were many others from whom data were collected until grand-mother or helpful neighbors prevailed upon the mothers to feed their children solid foods because “anyone knows that you should feed your baby solid foods.” To add weight in numbers, the statistical evalu-ation was done not only upon data collected from infants who completed the 9-month observation period, but also from other infants until tile time when they were fed solid foods and the data became invalid. Data were, in fact, analyzed both with and without data from infants who later broke the “no solid food” rule, and little difference was noted in results between the t\V() meth-ods of analysis.
Mean hemoglobin values for full-term and premature infants are graphically
repre-sented in Figures 1 and 2, respectively. After the initial observation in the hospital, the range of variation of hemoglobin values in each group from the group average is consistently small for both full-term and premature infants. Standard deviations are generally less than 1.5 gm/100 ml.
Mean hemoglobin values were higher after 3-3% months for all infants who re-ceived iron-fortified formula. At this age, full-term infants who had received addi-tional iron (group 1) had an average of 0.62 gm/100 ml more hemoglobin than those in group 2, and 0.96 gm more than those in group 3. At 5% months the differ-ences averaged 1.28 and 1.40 gm, respec-tively. By 9 months of age there was a 2 to 3 gm difference (12.69 vs. 10.46 vs. 9.67 gm).
The standard deviation for group 1 in-fants is 0.95 gm/100 ml at 9 months of age; for group 2, 1.43; for group 3, 1.51 gm. At 2 S.D., or the 95% level of proba-bility, the lower limit of mean hemoglobin concentration for group 1 is 10.79 gm/100 ml; for group 2, 7.60; for group 3, 6.65 gm. Only among the iron-fed infants (group 1) is the lower level at 2 S.D. above the criterion used for determining iron do-ficiency anemia in this study (8.0 gm/100 ml).
In the premature infants the hemoglobin values became significantly higher in tile iron-supplemented group at 3 months of age, and the divergence was greater tilan among the full-term infants. By 9 months there was a 3 to 4 gin difference (12.49 vs. 9.40 vs. 8.55 gin). In groups 2 and 3, as will be pointed out later, the mean hemo-globin levels would obviously have been lower Ilad those infants been retained who were changed to an iron-supplemented formula because of the development of anemia.
E a
0 E
C 0 0) 0
E
I
Age (months)
4 41 5 51 6 7
Group 1 vs. 3
p = 0.001
p = <0.001
p = <0.001
20
18
16
14
2
l0
8
6
FIG. 2. Mean values for hemoglobin of prenlature infants receiving tIIrce feeding reIIInens. I)ifferences IJy the Student t test at age levels A, B, C:
Group 1 vs. 2 Group 1 vs. 3
A) = 0.025 1’= 0.20
B) = 0.005 p = < 0.001
C) p = < 0.001 p = < 0.001
The number of observations at A for groups 1, 2 and 3, respectively, are 23, 26 and 21. They are 19, 11 and 13 at 6 months.
5
Age (months)
FI(;. 1. Mean for hemoglobin values of full-ternl infants receiving three feeding
regimens. Differences by the Student t test at age levels A, B, C:
Group 1 vs. 2
A) p = 0.02
B) p = < 0.001
C) p = < 0.001
p = 0.05 or less considered significant. The number of observations at A for groups
i, 2 and 3, respectively, are 27, 36 and 28. They arc 25, 22 and 20 at B. See text for
explanation of group differences.
E
a
a
E
U) C
-a
0
a)
0
E
‘1
408 IRON FORTIFICATION
Age (months)
=. 120
E
100
80
60
C 0
-
4020
Fic. 3. Mean values for serum iron of full-term infants receiving three feeding regimens. Differences by the Student t test at age levels A, B, C:
Group 1 vs. 2 Group 1 vs. 3
A) p=O.lO p=0.025
B) p = 0.005 p = < 0.001
C) p = 0.005 p = < 0.001
120
l00
80
60
40
20
I 3 3 4 4 5 5 6 7 8 9
Age (months)
Group 1 vs. 2
A) p = < 0.001
B) p = < 0.001 C) p = <0.001
Group I vs. 3
p = < 0.001
p = < 0.001
p= <0.001
E
0 0
0)
E
‘-I) C C 0
ARTICLES 409
difference did not appear between group 1 and group 2 until 5 months of age. In addition to this, the curves of the two con-trol groups siiow three isolated points (4, 43i and 6 months) which suggest differences between tile control groups. The possibility that an infant or two in group 2 was fed some iron-containing food without the mothers’ reporting it, cannot be discarded. The hemoglobin values did not, however, reflect this. The range of variation of values represented in any one mean value for serum iron is rather wide, the standard deviations being in the magnitude of 15 to 25 tg/109 ml.
Tile differences in mean values for serum iron between groups are much more ap-parent among the premature infants. In the group who received additional iron, there were significantly higher values for serum iron from 3 months of age on.
No full-term or premature infant in group 1 had to be given iron in addition to that provided in the formula, i.e. none had a hemoglobin of 8.0 gm/100 ml or less and a serum iron of less than 50 g/100 ml. It was not unusual, however, to find values for serum iron of 30 to 40 p.g/100 ml, in premature infants, associated with hemo-globin values of 11 to 13 gm/100 ml. One full-term infant in each of the control groups, and
7
and 9 premature infants in groups 2 and 3, respectively, became anemic and required treatment by a change to the group 1 feeding regimen. This treatment became necessary at7
months for the full-term infants and at 43-53 months for the premature infants. Of 10 infants in groups 2 and 3 with birthweights of less than 2,000 gm, 8 needed treatment; the 2 infants not requiring treatment were lost from the study at 5 and7
months of age. Half of the premature infants who required treatment had birthweights between 2,000 and 2,500grams.
Data collected subsequent to onset of treatment had to be considered separately. Hematologic differences between experi-mental and control groups, as shown in Figures 1-4, would have been greater had
it not been necessary to exclude the data from the infants showing anemia before the ninth month. Response to treatment was prompt and satisfactory in all instances. The average hemoglobin value when started on iron-containing milk was 7.5 gm/100 ml. Mothers were asked to bring infants back for follow-up every 2 weeks, but some were not able to keep every appointment. At 2 to 3 weeks, 10 infants showed an average in-crease in hemoglobin of 1.3 gm/100 ml (range 0.3 to 3.3 gm/100 ml); at 4 weeks, 7 infants showed an increase of 3.1 gm/100 ml (range 1.1 to 4.8 gm); at 6 weeks, 7 infants showed an increase of 3.3 gm/100 ml (range
1.5 to 5.2 gm).
Analysis of hematocrit values shows re-suits similar to those found with hemoglobin and serum iron (Figs. 5 and 6). The reticu-locyte counts did not show significant dif-ferences among the group mean values, since they were universally under 2% after 3 days of life. None of the infants who were receiving iron treatment returned for ex-amination sooner than 2 weeks after onset of treatment, and some not until a month later. Consequently, the typical reticulocyte response to treatment was not seen.
Although the nurse had the subjective impression that infants who were receiving iron in the diet were happier, healthier and more alert than those in groups 2 and 3, this could not be substantiated by any calibrated criteria. While this study was not designed to measure accurately differences in growth or relative susceptibility to ill-ness, gross observation revealed no signifi-cant differences. Respiratory illness is classi-fled as mild, moderate and severe, and tabu-lated separately by number of episodes in 3-month periods of time. Mild respiratory illnesses in the 3 to 6-month age period in all groups were quite frequent. There were few severe illnesses and no deaths.
Gastrointestinal disturbances, including regurgitation, vomiting and colic, occurred no more frequently among the infants fed
U 0
E
I
60
50
40
30
20
I0
3 3 4
Age (months)
4 5 5 6 7 8 9
A) p = 0.90 B) p = 0.20
C) p=<O.OOl
feeding regimens.
p = 0.40
p = < 0.001
p = < 0.001
70
60
50
.-t
40
U 0
: #{176}
20
l0
I 3
34:
44
5
si
6 7 8 9Age (months)
A) p = 0.01
B) p = 0.05 C) p=<O.OO1
p = 0.20
p = < 0.001
p = <0.001
70
Ftc. 5. Mean values for hematocrit of full-term infants receiving three Differences by the Student t test at age levels A, B, C:
Group 1 vs. 2 Group 1 vs. 3
Ftc. 6. Mean values for hematocrit of premature infants receiving three feeding regimens. 1)ifferences h the Student t test at age levels A, B, C:
ARTICLES
411healthy infants, as observed in clinical prac-tice. The pattern of defecation did not vary among the three groups, as established by information elicited from the mothers. In-cidence of loose stools and constipation approximated that found in any normal in-fant population.
DISCUSSION
It is immediately apparent that the sup-plementary iron provided in the milk formula was absorbed by these infants. Others2 5, 7, S 12 have found that iron
ad-ministered to infants at prophylactic levels, in milk, food or drop dosage, is readily absorbed. Schulman et al.’ state that “dun-ing the first three to four months of life, more iron exists in the body than is utilized for hemoglobin synthesis” and that iron administration before this date is not mdi-. cated. Yet Reedy et al.14 gave iron to pre-mature infants for the first 3 months of life and then discontinued it. Until the sixth month of life, these infants showed higher levels of hemoglobin than untreated con-trols, but some of them subsequently de-veloped anemia. Consequently, while iron fed in the first months of life sustains hemo-globin levels for a time, it is unable to prevent later anemia in premature infants continued on diets low in iron. Studies by Hammond and Murphy1’ show that ex-ogenous iron may be utilized by the pre-mature infant as early as the second month of life, preferentially “for hemoglobin for-mation at a time when body iron stores are presumably not depleted.” The present study again illustrates that the initial drop in hemoglobin after birth is not prevented, but it probably demonstrates that utiliza-tion of exogenous iron does begin between 6 weeks and 3 months of life.
Schulz and Smith4 and Sharpe et al.#{176}state that iron absorption shows an inverse re-lationship to tile total bulk of the feeding,
regardless of otiler factors involved. We can perhaps conclude from their findings that it is better to add iron to milk formulas of in-fants, and to delay the addition of solid foods to the diet until solid foods are more
readily taken. The evidence from tile pres-ent study indicates that delaying the pro-vision of solid foods until 9 months of age is not harmful from a nutritional point of view when a formula including vitamins is fortified with iron.
It is not clear what significance can be at-tributed to the concentrations of iron found in the serums of these infants. Tile usual level in adults is 100 to 130 .tg/100 mi.’7’ ‘
Serum iron remained low by adult stand-ards, even in the infants receiving iron and maintaining normal hemoglobin values.
Currently available evidence indicates that premature infants need therapeutic supplemental iron after the third month, as do some full-term infants. The first 2 years of life is the period during which dietary iron is most likely to be insufficient; the critical period is 3 to 20 months. Smith and Schulz’ conclude that oral supplementation is preferable, administered in some “food form of added iron suitable for routine use in infants.” Sturgeon2’ 2a has shown that
long-term iron supplementation in small prophylactic oral doses increases hemo-globin concentration. At 6 months of age,
8 to 12 mg/day provides optimal response. Addition of iron to milk used in infant feeding offers several advantages. It assures adequate intake; it avoids supplements to the infant diet which the mother can neg-lect; it avoids the potential dangers of over-dosage of medicinal iron; and it eliminates the availability of medicinal iron for ac-cidental ingestion in toxic doses by siblings. It also avoids the discomforts and hazards of intramuscular administration.
SUMMARY AND CONCLUSIONS
Seventy-four full-term and 42 premature infants were studied from birth to 9 months of age in relation to intake of iron. All were maintained on a vitamin-supplemented milk diet, and approximately a third had iron supplementation of 12 mg in 32 fluid ounces of milk intake.
412 IRON FORTIFICATION
The infants fed supplemental iron had higher values for hemoglobin, hematocrit and serum iron, after 3 to 3% months of age, and these values continued to be signifi-cantly higher throughout the 9-month pe-nod of observation.
Among the infants who received no iron supplement, 2 full-term and 16 premature infants developed evidence of anemia, which responded quickly when the infants were changed to the iron-fortified formula.
No signs of toxicity or difficulty were noted in the iron-supplemented group, nor of lack of acceptability by the infants of
any formula used in the study.
Acknowledgment
Dr. Daniel C. Darrow gave valuable advice and assistance throughout this study.
REFERENCES
1. Woodruff, C. W. : Multiple causes of iron deficiency in infants. J.A.M.A., 167:715, 1958.
2. Sturgeon, P. : Studies of iron requirements in infants and children, in Iron in Clini-cal Medicine, Walierstein, R. 0., and Mettier, S. R., eds. Berkeley, Univ. Cali-fornia Press, 1958, p. 183.
2a. Sturgeon, P. : Studies of iron requirements in infants and children. III. Influence of supplemental iron during normal preg-nancy on mother and infant. B. The in-fant. Bnit.
J.
Haematol., 5:45, 1959. 3. Schulman, I. : Ill Aspects of the care ofnewborn and premature infants (ab-stract). PErnATlucs, 22:180 (See erratum
p. 237), 1958.
4. Schulz,
J.,
and Smith, N.J.:
A quantita-tive study of the absorption of food iron in infants and children. A.M.A.J.
Dis. Child., 95:109, 1958.5. Smith, N.
J.,
and Schulz,J.:
The absorp-tion of iron in infants and children, in Iron in Clinical Medicine, Wallerstein,R. 0., and Mettier, S. R., eds. Berkeley, Univ. California Press, 1958, p. 65. 6. Nelson, W. E., ed. : Textbook of
Pedi-atrics, 6th Ed. Philadelphia, Saunders, 1954.
7. Josephs, H. W. : Iron metabolism and the hypochromic anemia of infancy. Medi-cine, 32:126, 1953.
8. Idem: Iron metabolism in infancy. Bull. Johns Hopkins Hosp., 65: 145, 1939. 9. Crosby, W. H., Munn,
J.
I., and Furth,F. W. : Standardizing a method for clinical hemoglobinometry. U.S. Armed Forces NI.
J.,
5:693, 1954.10. Guest, C. M., and Suer, V. E. : A centri-fuge method for the determination of the volume of cells in the blood.
J.
Lab.& Clin. Med., 19:757, 1934.
11. Fister, H.
J.:
Standardized Procedures for Spectrophotometric Chemistry; Iron Method 1-26.1. New York, Standard Scientific Supply Corp., 1950.12. Niccum, W. L., Jackson, R. L., and Stearns, C. : Use of fernic and ferrous iron in the prevention of hvpochromic anemia in infants. Am.
J.
Dis. Child., 86:553, 1953.13. Schulman, I., Smith, C. H., and Steam, C. S.: Studies on the anemia of
prema-tunitv. Am.
J.
Dis. Child., 88:567, 1954. 14. Reeds’, M. E., Schwartz, S. 0., andPlatt-nem, E. B. : Anemia of the premature.
J.
Pediat., 41:24, 1952.15. Hammond, D., and Murphy, A. : The in-fluence of exogenous iron on hemoglobin formation in the premature infant. Clin. Res., 7:53, 1959.
16. Sharpe, L. M., Peacock, W. C., Cook, R., and Harris, R. S. : Effect of phytate and
other food factors in iron absorption.
1.
Nutrition, 41:433, 1950.17. Smith, C. H., Schulman, I., and Morgen-thau,
J.
E. : Iron metabolism in infants and children. Advances Pediat., 5:197, 1952.18. Sturgeon, P.: Iron metabolism-a review with special consideration of iron re-quirements during normal infancy.