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Folate compartments during gestational maturation
May 1980
842
TheJournalofPEDIATRICS
Folate compartments during gestational maturation Studies on 41 healthy preterm infants and 53 term infants revealed that almost 70% of the total folate is present in the bound form. Beyond the neonatal period the fraction of bound folate drops to that observed in normal adults." approximately 30%. The increase in bound folate with increasing gestational maturity reflects Jetal demands placed on folate transport and affords an explanation for the loss of folate and corresponding increase in unsaturated binder during pregnancy. With the knowledge of folate biology gained from radioligand data, it is reasonable to postulate that effective folate metabolism depends upon a metabolically active transport mechanism.
Samuel Gross, M.D.,* B a r t o n K a m e n , M.D., Ph.D., Avroy Fanaroff, M.D., and Douglas Caston, Ph.D., C l e v e l a n d , O h i o
S E R U M F O L A T E L E V E L S in both preterm and term infants are often three to four times higher than either maternal or adult values, ~ 2 little is known regarding the mechanism of folate transport or the nature of sequential changes during gestational maturation. A highaffinity folate binder has recently been identified in cord serum 3 with the use of a radioligand system? -6 Comparisons of folate determinations by the radioligand and bioassay methods 7 reveal essentially close agreement, but the latter cannot distinguish free and bound folate. In order to relate the biologic significance of folate binders and transplacental transport with advancing gestational maturity, the radioligand method was utilized to study folate partition in infants ranging in gestational age from 30 weeks to term. We conformed to hospital policy regarding informed consent. ALTHOUGH
METHODS Serum folate partition values were obtained in duplicate on 0.08 ml ofsera according to the method of Kamen and Caston? Bound folate was determined by subtracting the unextracted (unboiled) free folate value from the boiled (extracted) total folate value. The possible presence of unsaturated folate binder was sought by assaying the binding capacity of the unextracted samples utilizing 3H-pteroylglutamic acid (15 to 50 Ci/mM, AmershamFrom the Departments of Pediatrics and Anatomy, Rainbow Babies and Childrens Hospital and Case Western Reserve University School of Medicine. *Reprint address: 2101 Adelbert Road, Cleveland, OH 44106.
Vot 9~ No. 5, pp. 842-844
Searle). If present, such samples would bind more radionuclide than the zero value tube on the standard curve. Subjects included 41 preterm infants (gestational age 30 to 36 weeks; birth weight 1,080 to 1,910 gm). Only uninfected infants and those who did not receive blood products or antibiotics were included. Determinations were obtained from either cord blood or peripheral blood during the first 10 to 12 days of life. In previous studies, no differences were observed during this time interval in randomly compared samples from either source. Control values were obtained from 53 term infants and 25 children between 1 and 6 years of age. The mothers of the study infants allegedly received supplemental folate during pregnancy. Abbreviation used FABP: folic acid binding protein RESULTS The data reveal steady increments in total and bound folate in infants whose birth weights ranged from 1,080 to 1,910 gm (Figure) to evaluate age-weight related differences, the infants were subgrouped below 1,500 gm (A), between 1,500 and 1,700 gm (B), and over 1,700 gm (C). Group D consisted of term infants. The folate values for these groups as well as the older children are shown in Table I. The bound and total folate levels in the more mature newly born infants (C and D) are essentially twice those obtained in the least mature infants (A): P < 0.01. Although the differences between
0022-3476/80/050842 +03500.30/0 9 1980 The C. V. Mosby Co.
Volume 96 Number 5
Folate compartments dur!ng gestational maturation
843
50-
9 Total 9 Bound 40-
F~.J 0
iii IIIIII! ItII IIII I I iiI :iliIII I II
30b-I
20-
U-
io 2
Iii I
I000
~ 1200
~
14~00 BIRTH
i
I i O0
t
' 1800
'
WEIGHT-grams
Figure. Distribution of the folate partition values relative to birth weight in the premature infants. There is a progressive increase in total and bound folate with increasing gestational maturity. Table I. Serum folate partition (ng/ml) relative to birth weight/gestational age
Group A < 1,500 gm n Birth weight (gin) Mean Range Gestational age (wk) Mean Range Free Mean _+ SD Range Bound Mean _+ SD Range Total Mean _+ SD Range Percent bound
Group B 1,500-1,700 gm
Group C 1, 700-2,000 gm
Group D term
19
12
10
53
1,300 !,080-1,490
1,620 1,560-1,690
1,780 1,710-1,910
2,800 2,440-3,2 l0
30 26-32
33 31-34
35 33-36
39 36-41
5.1 _+ 3.3* 1.5-11.0
7.8 _+ 4.5 2.1-14.8
11.0 +_ 9.1" 4.2-28.3
17.8 _+ 7.0 7.3-36.4
16.1 _+ 7.8* 7.5-33.0 68
25.6 +_ 10.5 12.1-43.5 69
1-6 y r
25
8.4 +_ 3.2
5.3 _+ 3.0
23.1 • 13.5" 8.9-38.0
20.2 + 16.6
2.0 • 1.1
34.2 _+ 18.0"
28.6 + 17.3
7.3 _+ 4.2
11.7 +_ 5.0*
2.1-18.0
11.0-48.0
67
71
28
*P < 0.01.
Groups A and B are not significant, comparisons above and below 1,500 gm (A versus B and C) show significance; P = 0.01. This manipulation is intended to show the continued rise in bound and total folate with increasing maturity. The decline in folate values a b o v e 1,700 gm suggests a temporal association with deceleration of the maturation process. As a further index o f folate requirements relative to maturation, the data were subjected to an analysis of individual folate levels per unit of w e i g h t . The results (Table II), in confirming the steady rise with advancing age, also indicate that the peak requirement occurs in the Group C infants (1,700 to 2,000 gm). In all cases the major component ( > 65%) is the bound folate
value. Beyond the newborn period the compartmentalization is reversed (Table I), a p h e n o m e n o n which is temporally related to cessation of active transplacental transfer. In none of the analyses was there free binder. DISCUSSION Recent studies s have shown that serum folate levels in pregnant w o m e n are considerably lower than those in nonpregnant w o m e n and that the opposite is true for the folic acid-binding protein levels. As pregnancy advances into the second trimester, both absolute and relative increases occur in the level of the unsaturated binder, These changes apparently do not extend beyond the
844
Grosset al.
The Journal of Pediatrics May 1980
Table II. ng/ml folate per kg birth weight
Group A [ Group B [ Group C [ Grot,pD Free Bound
3.9 8.5
5. I 10.9
6.2 12.8
2.6 7.6
Total
12.4
16.0
19.0
10.2
second trimester. When Kamen and Caston 3 identified the high-affinity folate binder in cord serum (molecular weight approximately 40,000), they also noted that the binder demonstrated an especially strong affinity for 5-methyltetrahydrofolate, which is the main serum folate. It was reasonable to assume that the binder probably functions as the mechanism whereby the fetus concentrates most of the maternally supplied folate. Suppgrt for that conclusion has b e e n gained from this study which identified both markedly elevated levels as well as a steady increase in bound folate with advancing maturity. The demand for folate decreases as gestational maturity closely approximates term. Both longitudinal and sequential analyses substantiate this finding. In effect, as the incremental weight changes decline, the amo~rtt of bound folate per unit of weight also declines, t~ereby underscorhag the proportional relationship between maturation and need. The need for impressive quantities of folate by the rapidly developing fetus has been well documented by Shojania and Hornady? In comparing the clearance and excretion rates of varying doses of pteroyiglutamic (folic) acid in adults, term infants, and preterm infants, they found that plasma clearance was much more rapid in the newborn infants than in adults and that much less folate was excreted in the urine of neonates. Moreover, the excretion in the preterm infants was approximately half of that recorded in term infants. In helping to establish the biologic role of the folate binder in the neonate, credence is also lent to the nature of changes in FABP during pregnancy? ~ The increase in the unsaturated binder very likely represents the transfer of folate from maternal to infant binder. In effect, the decline of folate values in association with a rise in binding protein during pregnancy, heretofore felt by some to reflect the effects of an increased plasma volume,11very likely is an expression of folate transfer.
What influence the quantity of maternal folate, per se, has in this system is yet to be determined, although it is probable ihat, within the limits of available folate, the high-affinity binder favors transport in the direction of the fetus. Application of these methods to a double-blind study should help resolve questions concerning the need for maternal folate supplementationTM 13 and its subsequent effect on optimal fetal development. REFERENCES
1. Shojania AM, and Gross S: Folic acid deficiency and prematurity, J PEDIATR64:323, 1964. 2. Hibbard ED: Plasma and erythroc3)te folate concentrations in normal mature infants, Arch Dis Child 48:743, 1973. 3. Kamen BA, and Caston JD: Purification of folate binding factor in normal Umbilicalcord serum, Proc Natl Acad Sci 72:426i, 1975. 4. Rothenberg SP, daCosta M, and Rosenberg Z: A radioassay for serum folate: Use of a two-phase sequential-incubation, ligand binding system, N Engl J Med 286:1335, 1972. 5. Waxman S, and Schreiber C: Measurement of serum folate levels and serum folic acid-binding protein by ~H-PGA radjoassay, Blood 42:281, 1973. 6. Kamen BA, and Caston JD: Direct radiochemical assay for serum folate: Competition between 3H-folic acid and 5methyltetrahYdro-folic acid for a folate binder, J Lab Clin Med 83:164, 1974. 7. Waters AH, and Mollin DL: Studies on the folic acid activity of human serum, J Clin Pathol 14:335, 196L 8. Colman/'q, and Herbert V: Total folate binding capacity of normal human plasma, and variations in uremia, cirrhosis, and pregnancy, Blood 48:911, 1976. 9. Shojania AM, and Hornady G: Folate metabolism in newborns and during early infancy. IX. Clearance of folic acid in plasma and excretion of folic acid in urine by newborns, Pediatr Res 4:422, 1970. 10. Areekul S, Yamarat P, and Vongyuthithum M: Folic acid and folate binding protein in pregnancy, J Nutr Sci Vitaminol 23:447, 1977. 11. Hall MH, Pirani BBK, and Campbell D: The cause of the fall in serum folate in normal pregnancy, Br J Obstet Gynecol 83:132, 1976. 12. Hansen H, and Klewesahl-Palm H: Blood folic acid levels and clearance rate of injected folic acid in normal pregnancy and puerperium, Scand J Clin Lab Invest 15 (Suppl 69):78, 1963. 13. Herbert V, Cohnan N, Spivack M, Ocasio E, Chanta V, Kimme! E, Brenner.L, Freundlich J, andScott J: Folic acid deficiency in the United States: Folate assays in a prenatal clinic, Am J Obstet Gynecol 123.:175, 1975.
842
TheJournalofPEDIATRICS
Folate compartments during gestational maturation Studies on 41 healthy preterm infants and 53 term infants revealed that almost 70% of the total folate is present in the bound form. Beyond the neonatal period the fraction of bound folate drops to that observed in normal adults." approximately 30%. The increase in bound folate with increasing gestational maturity reflects Jetal demands placed on folate transport and affords an explanation for the loss of folate and corresponding increase in unsaturated binder during pregnancy. With the knowledge of folate biology gained from radioligand data, it is reasonable to postulate that effective folate metabolism depends upon a metabolically active transport mechanism.
Samuel Gross, M.D.,* B a r t o n K a m e n , M.D., Ph.D., Avroy Fanaroff, M.D., and Douglas Caston, Ph.D., C l e v e l a n d , O h i o
S E R U M F O L A T E L E V E L S in both preterm and term infants are often three to four times higher than either maternal or adult values, ~ 2 little is known regarding the mechanism of folate transport or the nature of sequential changes during gestational maturation. A highaffinity folate binder has recently been identified in cord serum 3 with the use of a radioligand system? -6 Comparisons of folate determinations by the radioligand and bioassay methods 7 reveal essentially close agreement, but the latter cannot distinguish free and bound folate. In order to relate the biologic significance of folate binders and transplacental transport with advancing gestational maturity, the radioligand method was utilized to study folate partition in infants ranging in gestational age from 30 weeks to term. We conformed to hospital policy regarding informed consent. ALTHOUGH
METHODS Serum folate partition values were obtained in duplicate on 0.08 ml ofsera according to the method of Kamen and Caston? Bound folate was determined by subtracting the unextracted (unboiled) free folate value from the boiled (extracted) total folate value. The possible presence of unsaturated folate binder was sought by assaying the binding capacity of the unextracted samples utilizing 3H-pteroylglutamic acid (15 to 50 Ci/mM, AmershamFrom the Departments of Pediatrics and Anatomy, Rainbow Babies and Childrens Hospital and Case Western Reserve University School of Medicine. *Reprint address: 2101 Adelbert Road, Cleveland, OH 44106.
Vot 9~ No. 5, pp. 842-844
Searle). If present, such samples would bind more radionuclide than the zero value tube on the standard curve. Subjects included 41 preterm infants (gestational age 30 to 36 weeks; birth weight 1,080 to 1,910 gm). Only uninfected infants and those who did not receive blood products or antibiotics were included. Determinations were obtained from either cord blood or peripheral blood during the first 10 to 12 days of life. In previous studies, no differences were observed during this time interval in randomly compared samples from either source. Control values were obtained from 53 term infants and 25 children between 1 and 6 years of age. The mothers of the study infants allegedly received supplemental folate during pregnancy. Abbreviation used FABP: folic acid binding protein RESULTS The data reveal steady increments in total and bound folate in infants whose birth weights ranged from 1,080 to 1,910 gm (Figure) to evaluate age-weight related differences, the infants were subgrouped below 1,500 gm (A), between 1,500 and 1,700 gm (B), and over 1,700 gm (C). Group D consisted of term infants. The folate values for these groups as well as the older children are shown in Table I. The bound and total folate levels in the more mature newly born infants (C and D) are essentially twice those obtained in the least mature infants (A): P < 0.01. Although the differences between
0022-3476/80/050842 +03500.30/0 9 1980 The C. V. Mosby Co.
Volume 96 Number 5
Folate compartments dur!ng gestational maturation
843
50-
9 Total 9 Bound 40-
F~.J 0
iii IIIIII! ItII IIII I I iiI :iliIII I II
30b-I
20-
U-
io 2
Iii I
I000
~ 1200
~
14~00 BIRTH
i
I i O0
t
' 1800
'
WEIGHT-grams
Figure. Distribution of the folate partition values relative to birth weight in the premature infants. There is a progressive increase in total and bound folate with increasing gestational maturity. Table I. Serum folate partition (ng/ml) relative to birth weight/gestational age
Group A < 1,500 gm n Birth weight (gin) Mean Range Gestational age (wk) Mean Range Free Mean _+ SD Range Bound Mean _+ SD Range Total Mean _+ SD Range Percent bound
Group B 1,500-1,700 gm
Group C 1, 700-2,000 gm
Group D term
19
12
10
53
1,300 !,080-1,490
1,620 1,560-1,690
1,780 1,710-1,910
2,800 2,440-3,2 l0
30 26-32
33 31-34
35 33-36
39 36-41
5.1 _+ 3.3* 1.5-11.0
7.8 _+ 4.5 2.1-14.8
11.0 +_ 9.1" 4.2-28.3
17.8 _+ 7.0 7.3-36.4
16.1 _+ 7.8* 7.5-33.0 68
25.6 +_ 10.5 12.1-43.5 69
1-6 y r
25
8.4 +_ 3.2
5.3 _+ 3.0
23.1 • 13.5" 8.9-38.0
20.2 + 16.6
2.0 • 1.1
34.2 _+ 18.0"
28.6 + 17.3
7.3 _+ 4.2
11.7 +_ 5.0*
2.1-18.0
11.0-48.0
67
71
28
*P < 0.01.
Groups A and B are not significant, comparisons above and below 1,500 gm (A versus B and C) show significance; P = 0.01. This manipulation is intended to show the continued rise in bound and total folate with increasing maturity. The decline in folate values a b o v e 1,700 gm suggests a temporal association with deceleration of the maturation process. As a further index o f folate requirements relative to maturation, the data were subjected to an analysis of individual folate levels per unit of w e i g h t . The results (Table II), in confirming the steady rise with advancing age, also indicate that the peak requirement occurs in the Group C infants (1,700 to 2,000 gm). In all cases the major component ( > 65%) is the bound folate
value. Beyond the newborn period the compartmentalization is reversed (Table I), a p h e n o m e n o n which is temporally related to cessation of active transplacental transfer. In none of the analyses was there free binder. DISCUSSION Recent studies s have shown that serum folate levels in pregnant w o m e n are considerably lower than those in nonpregnant w o m e n and that the opposite is true for the folic acid-binding protein levels. As pregnancy advances into the second trimester, both absolute and relative increases occur in the level of the unsaturated binder, These changes apparently do not extend beyond the
844
Grosset al.
The Journal of Pediatrics May 1980
Table II. ng/ml folate per kg birth weight
Group A [ Group B [ Group C [ Grot,pD Free Bound
3.9 8.5
5. I 10.9
6.2 12.8
2.6 7.6
Total
12.4
16.0
19.0
10.2
second trimester. When Kamen and Caston 3 identified the high-affinity folate binder in cord serum (molecular weight approximately 40,000), they also noted that the binder demonstrated an especially strong affinity for 5-methyltetrahydrofolate, which is the main serum folate. It was reasonable to assume that the binder probably functions as the mechanism whereby the fetus concentrates most of the maternally supplied folate. Suppgrt for that conclusion has b e e n gained from this study which identified both markedly elevated levels as well as a steady increase in bound folate with advancing maturity. The demand for folate decreases as gestational maturity closely approximates term. Both longitudinal and sequential analyses substantiate this finding. In effect, as the incremental weight changes decline, the amo~rtt of bound folate per unit of weight also declines, t~ereby underscorhag the proportional relationship between maturation and need. The need for impressive quantities of folate by the rapidly developing fetus has been well documented by Shojania and Hornady? In comparing the clearance and excretion rates of varying doses of pteroyiglutamic (folic) acid in adults, term infants, and preterm infants, they found that plasma clearance was much more rapid in the newborn infants than in adults and that much less folate was excreted in the urine of neonates. Moreover, the excretion in the preterm infants was approximately half of that recorded in term infants. In helping to establish the biologic role of the folate binder in the neonate, credence is also lent to the nature of changes in FABP during pregnancy? ~ The increase in the unsaturated binder very likely represents the transfer of folate from maternal to infant binder. In effect, the decline of folate values in association with a rise in binding protein during pregnancy, heretofore felt by some to reflect the effects of an increased plasma volume,11very likely is an expression of folate transfer.
What influence the quantity of maternal folate, per se, has in this system is yet to be determined, although it is probable ihat, within the limits of available folate, the high-affinity binder favors transport in the direction of the fetus. Application of these methods to a double-blind study should help resolve questions concerning the need for maternal folate supplementationTM 13 and its subsequent effect on optimal fetal development. REFERENCES
1. Shojania AM, and Gross S: Folic acid deficiency and prematurity, J PEDIATR64:323, 1964. 2. Hibbard ED: Plasma and erythroc3)te folate concentrations in normal mature infants, Arch Dis Child 48:743, 1973. 3. Kamen BA, and Caston JD: Purification of folate binding factor in normal Umbilicalcord serum, Proc Natl Acad Sci 72:426i, 1975. 4. Rothenberg SP, daCosta M, and Rosenberg Z: A radioassay for serum folate: Use of a two-phase sequential-incubation, ligand binding system, N Engl J Med 286:1335, 1972. 5. Waxman S, and Schreiber C: Measurement of serum folate levels and serum folic acid-binding protein by ~H-PGA radjoassay, Blood 42:281, 1973. 6. Kamen BA, and Caston JD: Direct radiochemical assay for serum folate: Competition between 3H-folic acid and 5methyltetrahYdro-folic acid for a folate binder, J Lab Clin Med 83:164, 1974. 7. Waters AH, and Mollin DL: Studies on the folic acid activity of human serum, J Clin Pathol 14:335, 196L 8. Colman/'q, and Herbert V: Total folate binding capacity of normal human plasma, and variations in uremia, cirrhosis, and pregnancy, Blood 48:911, 1976. 9. Shojania AM, and Hornady G: Folate metabolism in newborns and during early infancy. IX. Clearance of folic acid in plasma and excretion of folic acid in urine by newborns, Pediatr Res 4:422, 1970. 10. Areekul S, Yamarat P, and Vongyuthithum M: Folic acid and folate binding protein in pregnancy, J Nutr Sci Vitaminol 23:447, 1977. 11. Hall MH, Pirani BBK, and Campbell D: The cause of the fall in serum folate in normal pregnancy, Br J Obstet Gynecol 83:132, 1976. 12. Hansen H, and Klewesahl-Palm H: Blood folic acid levels and clearance rate of injected folic acid in normal pregnancy and puerperium, Scand J Clin Lab Invest 15 (Suppl 69):78, 1963. 13. Herbert V, Cohnan N, Spivack M, Ocasio E, Chanta V, Kimme! E, Brenner.L, Freundlich J, andScott J: Folic acid deficiency in the United States: Folate assays in a prenatal clinic, Am J Obstet Gynecol 123.:175, 1975.