5. Y.W. Brans, J.E. Sumners, H.S. Dweck, and G. Cassady: A Noninvasive Approach t o Body Composition in the Neonate: Dynamic Skinfold Measurements. Pediat. Res. 8:215-222, 1974 6. G. Cassady: Plasma Volume Studies in Low B i r t h Weight I n f a n t s . Pediatrics 38: 1020-1027, 1966 7. G. Cassady: Bromide Space Studies in Infants o f Low Birth Weight. Pediar. Res. 4:14-24, 1970 8. G. Cassady and R.R. Milstead: Antipyrine Space Studies and Cell Water Estimates in Infants of Low Birth Weight. Pediat. Res. 5:673-682, 1971
9. J. Gleiss and M. Hermanns: Ektodermale Kriterien zur Klinischen Reifesbestrimmung n e u g e b o r e n e n. A r c h . K i n d a h e i l k . 179:266-283, 1969 10 R. Usher and F. McLean: Intrauterine Growth of Liveborn Caucasian Infants a t Sea Level: Standards Obtained from Measurements in 7 Dimensions of Infants Born between 25 and 44 Weeks o f Gestation. J. Pediat. 74:901-910, 1969 11 A.R. Frisancho and S.M. Garn: Skinfold Thickness and Muscle Size: Implications for Developmental Status and Nutritional Evaluation of Children from Honduras. Am. J. Clin. Nutrition 24:541-546, 1971
FOLATE BINDER IN LEUKOCYTES AND SERUM A macromolecular factor which binds unreduced and partially reduced folate but n o t tetrahydrofolate has been detected in the leukocytes and serum o f patients suffering frotn chronic myelogenous leukemia, pregnant women, and women using oral contraceptives. I t s p h ysiotogical (or pathological) role remains speculative.
Key Words: cell lysates, folic acid, folate binder, macromolecular
A macromolecular factor which binds with unreduced folate and dihydrofolate (FH, but not tetrahydrofolate (FH,) was accidentally discovered by S. P. Rothenberg and co-workersl** in some leukemic cells while assaying the folate reductase activity of those cells using a newly developed radioassay. Trace amounts of H-folic acid are used as the substrate in this assay and the radio-activity in FH, after chromatographic separation of the product is measured. Thus, it obviates the use of high concentrations of substrate, which are required for the conventional spectro-photometric assay. Little or no folate reductase activity could be detected in the cell lysates from four out of seven patients suffering from chronic rnyelogenous leukemia ICML) when tracer concentration (1.14 x 1O'9 MI of the substrate 3H-folic acid was used.
However, when the substrate concentration was raised by the addition of 0.22 x 10-6M stable folic acid, reduction of H-folic acid became apparent. With a further increase in the concentration of stable folate, an isotope dilution effect was evident. In the case o f cell lysates having normal enzyme activity, the curve for isotope dilution could be extrapolated t o zero concentration of cold folate. The peculiar behavior of lysates from CML patients was also apparent when they were mixed with lysates of cells having normal enzyme activity. This suggested the presence of a material in CML cells, which bound the trace amount of labeled folate and made it unavailable for enzymatic reduction. Addition of stable folate displaced the 3H-folate from the binder and reduction was evident. Gel filtration of the cell lysates from CM L patients preincubated with H-folate indicated that the binder was a macromolecule having a molecular weight of NUTRITION REVIEWSIVOL. 33. NO. 1 I JANUARY 1975 9
100,000 t o 200,000. I t was found to be resistant to heating a t 56" C for 30 minutes and was minimally active below a pH of 5.0. It bound folic acid and FH, rapidly and reacted with such reduced folate analogues a s diopterin, pteropterin, and methotrexate. I t s presence could also be detected in the serum, probably due t o liberation from the cells that were dest ro yed. The folate binder could not be detected in a l l leukemic diseases, but that does not rule out i t s presence since endogenous folate may bind it and render it undetectable by the "isotope binding" method. In a more recent paper, M. Da Costa and S. P. Rothenberg3 demonstrated the presence of this factor in the leukocytes of pregnant women as well as of women taking oral contraceptive agents (OCA). It seems t o be induced by the hormonal changes associated with pregnancy and use of OCA. Sixty-four women in the third trimester of pregnancy, ten women taking OCA, and 15 non-pregnant menopausal women were examined. Lysates or sera binding less than 5 percent 3H-folic acid (PGA) were considered t o contain no folate binder. Of the 64 pregnant women studied, 31 had lysates which bound more than 5 percent of the labeled folate, with a mean SEM binding of 38 5 percent. Seven out of ten women on OCA had a detectable binder with a mean of 22 f 8 percent. None of the lysates from non-pregnant women bound 3H-PGA. There was a marked fall in the binding of 3H-PGA by cell lysates after parturition. The hormone induced folate binder was also a macromolecule and could react with the anti-sera for CM L binder. The anti-sera were obtained by immunizing rabbits with cell lysates of CML cells containing a high concentration of the binder. This shows that the folate binder from leukemic cells is immunologically similar t o the hormonally induced binder and that it is not just peculiar t o malignant cells. Levels of several proteins are known t o rise in pregnancy and in women using OCA.
*
10
+_
NUTRITION REVIEWSNOL. 33, NO. 1 I JANUARY 1975
It i s possible that this factor is a normal constituent of cells and serum, but remains saturated by the endogenous folate. In folate deficiency or in conditions where the level of the binder increases, i t s presence can be detected by the 3H-folate binding test. The presence of folate binder(s) in the sera of folate deficient subjects has been rePO rted .3 The physiological or pathological implications of this macromolecular factor are not clear. Since it binds FH,, it may have some function in de novo thymineDNA synthesis. Da Costa e t al.4 observed that CML cells containing a high concentration of the folate binder possess reduced capacity for the methylation of deoxyuridylate to thymidylate. Since the factor can also bind with methotrexate (a potent inhibitor of folate reductase), it may affect the treatment of leukemia by methotrexate and may be the basis of drug resistance shown by some leukemic cells. In such cases an appropriate adjunct t o therapy might b e simultaneous administration of folic acid which by virtue of i t s greater a f f i n i t y f o r t h e binder would leave methotrexate t o bind with the enzyme folate reductase. The folate binding factor may have potential value in the treatment o f neoplastic diseases, provided it could enter the cell. Biochemical evidence of folic acid deficiency, as judged by low serum folate levels and elevated FIGLU excretion, is quite common in pregnancy and among women using OCA. Cases of megaloblastic anemia have been reported among these women, particularly during pregnancy. Although Da Costa and Rothenberg3 could not observe a direct correlation between the concentration of folate binder and serum folate or hemoglobin, nor did any of the women studied have megaloblastic anemia, the percent of pregnant women showing serum folate concentration above 5 ng per milliliter was higher in the absence of the binder (63 percent) than in the presence of the binder (38 percent). Based on this, the authors speculate that "increased synthesis of this binder in prolifera-
ting cells such as bone marrow could sequester FH, from a metabolically active pool of folate coenzymes. Under such circumstances this could potentiate the metabolic consequences of inadequate folate intake or increase folate demands."
1. S. P. Rothenberg: A Macromolecular Factor in Some Leukemic Cells Which Binds Folic A c i d . Proc. SOC. Exp. Biol. Med. 133: 428432,1970
2. S. P. Rothenberg and M. Da Costa: Further Observations on the Folate-Binding Factor in some Leukemic Cells. J. Clin. Invest, 50: 719-726, 1971 3. M. Da Costa and S. P. Rothenberg: Appearance o f Folate Binder in Leukocytes and Serum o f Women Who are Pregnant or Taking Oral Contraceptives. J. Lab. Clin. Med. 83: 207-214, 1974. 4. M. Da Costa, S. P. Rothenberg, and B. Kamen: DNA Synthesis in Chronic Myelogenous Leukemic Cells: Comparison of Results in Cells Containing Folate Binding Factor to Replicating Cells without Binder. Blood 39: 621-627, 1972
THE CORRELATION OF SERUM FERRlTlN AND BODY IRON STORES The relationship between serum ferritin levels and total body iron stores is discussed. Recent work is evaluated which correlates these parameters in iron deficiency, liver disease, inflammation, renal failure, and states with increased red cell turnover. Key Words: serum ferritin, body iron stores, hepatocellular disease, chronic inflammation
The assessment of total body iron stores has long lacked an easy method of measurement. The differential desferrioxamine test,' where iron chelated and excreted is thought to be proportional t o body iron stores, requires a fair degree of cooperation from the subject studied and there is evidence that some of the iron chelated i s not derived from iron stores.* The most commonly used method i s assessment of the amount of iron present in the bone marrow but this is an uncomfortable procedure and a t best only semi-quantitative. Quantitative venesection t o the level of incipient iron deficiency and anemia is sensitive3 but impracticable. Certainly none of these methods i s suitable for large scale population surveys. In 1972, G. M. Addison and his colleagues published a sensitive radioimmunoassay for serum ferritin.4 Recently a 'two-
site' immunoassay has also been e ~ o l v e d . ~ In this modification, unlabeled antibody linked t o an immunoadsorbent couples with ferritin which then reacts with labeled antibody. Thus quantitation of label bound is a measure of ferritin concentration. It has become apparent that in many instances serum ferritin concentration is related t o the total body iron Ferritin is a high molecular weight compound composed of a protein shell of apoferritin synthesized by the liver surrounding a ferric hydroxide core, the whole forming an octahedral structure. It is the main storage form of iron in the liver and reticulo-endothelial system either as ferritin itself, or as hemosiderin which is thought t o be a complex aggregate of partially denatured ferritin. In contrast t o the iron stores (1 g in a normal adult male) the amount of ferritin found in the serum by radioimmunoassay is miniscule. The normal range for the two site assay i s generally NUTRITION REVIEWSIVOL. 33, NO. 1 I JANUARY 1975 11
9. J. Gleiss and M. Hermanns: Ektodermale Kriterien zur Klinischen Reifesbestrimmung n e u g e b o r e n e n. A r c h . K i n d a h e i l k . 179:266-283, 1969 10 R. Usher and F. McLean: Intrauterine Growth of Liveborn Caucasian Infants a t Sea Level: Standards Obtained from Measurements in 7 Dimensions of Infants Born between 25 and 44 Weeks o f Gestation. J. Pediat. 74:901-910, 1969 11 A.R. Frisancho and S.M. Garn: Skinfold Thickness and Muscle Size: Implications for Developmental Status and Nutritional Evaluation of Children from Honduras. Am. J. Clin. Nutrition 24:541-546, 1971
FOLATE BINDER IN LEUKOCYTES AND SERUM A macromolecular factor which binds unreduced and partially reduced folate but n o t tetrahydrofolate has been detected in the leukocytes and serum o f patients suffering frotn chronic myelogenous leukemia, pregnant women, and women using oral contraceptives. I t s p h ysiotogical (or pathological) role remains speculative.
Key Words: cell lysates, folic acid, folate binder, macromolecular
A macromolecular factor which binds with unreduced folate and dihydrofolate (FH, but not tetrahydrofolate (FH,) was accidentally discovered by S. P. Rothenberg and co-workersl** in some leukemic cells while assaying the folate reductase activity of those cells using a newly developed radioassay. Trace amounts of H-folic acid are used as the substrate in this assay and the radio-activity in FH, after chromatographic separation of the product is measured. Thus, it obviates the use of high concentrations of substrate, which are required for the conventional spectro-photometric assay. Little or no folate reductase activity could be detected in the cell lysates from four out of seven patients suffering from chronic rnyelogenous leukemia ICML) when tracer concentration (1.14 x 1O'9 MI of the substrate 3H-folic acid was used.
However, when the substrate concentration was raised by the addition of 0.22 x 10-6M stable folic acid, reduction of H-folic acid became apparent. With a further increase in the concentration of stable folate, an isotope dilution effect was evident. In the case o f cell lysates having normal enzyme activity, the curve for isotope dilution could be extrapolated t o zero concentration of cold folate. The peculiar behavior of lysates from CML patients was also apparent when they were mixed with lysates of cells having normal enzyme activity. This suggested the presence of a material in CML cells, which bound the trace amount of labeled folate and made it unavailable for enzymatic reduction. Addition of stable folate displaced the 3H-folate from the binder and reduction was evident. Gel filtration of the cell lysates from CM L patients preincubated with H-folate indicated that the binder was a macromolecule having a molecular weight of NUTRITION REVIEWSIVOL. 33. NO. 1 I JANUARY 1975 9
100,000 t o 200,000. I t was found to be resistant to heating a t 56" C for 30 minutes and was minimally active below a pH of 5.0. It bound folic acid and FH, rapidly and reacted with such reduced folate analogues a s diopterin, pteropterin, and methotrexate. I t s presence could also be detected in the serum, probably due t o liberation from the cells that were dest ro yed. The folate binder could not be detected in a l l leukemic diseases, but that does not rule out i t s presence since endogenous folate may bind it and render it undetectable by the "isotope binding" method. In a more recent paper, M. Da Costa and S. P. Rothenberg3 demonstrated the presence of this factor in the leukocytes of pregnant women as well as of women taking oral contraceptive agents (OCA). It seems t o be induced by the hormonal changes associated with pregnancy and use of OCA. Sixty-four women in the third trimester of pregnancy, ten women taking OCA, and 15 non-pregnant menopausal women were examined. Lysates or sera binding less than 5 percent 3H-folic acid (PGA) were considered t o contain no folate binder. Of the 64 pregnant women studied, 31 had lysates which bound more than 5 percent of the labeled folate, with a mean SEM binding of 38 5 percent. Seven out of ten women on OCA had a detectable binder with a mean of 22 f 8 percent. None of the lysates from non-pregnant women bound 3H-PGA. There was a marked fall in the binding of 3H-PGA by cell lysates after parturition. The hormone induced folate binder was also a macromolecule and could react with the anti-sera for CM L binder. The anti-sera were obtained by immunizing rabbits with cell lysates of CML cells containing a high concentration of the binder. This shows that the folate binder from leukemic cells is immunologically similar t o the hormonally induced binder and that it is not just peculiar t o malignant cells. Levels of several proteins are known t o rise in pregnancy and in women using OCA.
*
10
+_
NUTRITION REVIEWSNOL. 33, NO. 1 I JANUARY 1975
It i s possible that this factor is a normal constituent of cells and serum, but remains saturated by the endogenous folate. In folate deficiency or in conditions where the level of the binder increases, i t s presence can be detected by the 3H-folate binding test. The presence of folate binder(s) in the sera of folate deficient subjects has been rePO rted .3 The physiological or pathological implications of this macromolecular factor are not clear. Since it binds FH,, it may have some function in de novo thymineDNA synthesis. Da Costa e t al.4 observed that CML cells containing a high concentration of the folate binder possess reduced capacity for the methylation of deoxyuridylate to thymidylate. Since the factor can also bind with methotrexate (a potent inhibitor of folate reductase), it may affect the treatment of leukemia by methotrexate and may be the basis of drug resistance shown by some leukemic cells. In such cases an appropriate adjunct t o therapy might b e simultaneous administration of folic acid which by virtue of i t s greater a f f i n i t y f o r t h e binder would leave methotrexate t o bind with the enzyme folate reductase. The folate binding factor may have potential value in the treatment o f neoplastic diseases, provided it could enter the cell. Biochemical evidence of folic acid deficiency, as judged by low serum folate levels and elevated FIGLU excretion, is quite common in pregnancy and among women using OCA. Cases of megaloblastic anemia have been reported among these women, particularly during pregnancy. Although Da Costa and Rothenberg3 could not observe a direct correlation between the concentration of folate binder and serum folate or hemoglobin, nor did any of the women studied have megaloblastic anemia, the percent of pregnant women showing serum folate concentration above 5 ng per milliliter was higher in the absence of the binder (63 percent) than in the presence of the binder (38 percent). Based on this, the authors speculate that "increased synthesis of this binder in prolifera-
ting cells such as bone marrow could sequester FH, from a metabolically active pool of folate coenzymes. Under such circumstances this could potentiate the metabolic consequences of inadequate folate intake or increase folate demands."
1. S. P. Rothenberg: A Macromolecular Factor in Some Leukemic Cells Which Binds Folic A c i d . Proc. SOC. Exp. Biol. Med. 133: 428432,1970
2. S. P. Rothenberg and M. Da Costa: Further Observations on the Folate-Binding Factor in some Leukemic Cells. J. Clin. Invest, 50: 719-726, 1971 3. M. Da Costa and S. P. Rothenberg: Appearance o f Folate Binder in Leukocytes and Serum o f Women Who are Pregnant or Taking Oral Contraceptives. J. Lab. Clin. Med. 83: 207-214, 1974. 4. M. Da Costa, S. P. Rothenberg, and B. Kamen: DNA Synthesis in Chronic Myelogenous Leukemic Cells: Comparison of Results in Cells Containing Folate Binding Factor to Replicating Cells without Binder. Blood 39: 621-627, 1972
THE CORRELATION OF SERUM FERRlTlN AND BODY IRON STORES The relationship between serum ferritin levels and total body iron stores is discussed. Recent work is evaluated which correlates these parameters in iron deficiency, liver disease, inflammation, renal failure, and states with increased red cell turnover. Key Words: serum ferritin, body iron stores, hepatocellular disease, chronic inflammation
The assessment of total body iron stores has long lacked an easy method of measurement. The differential desferrioxamine test,' where iron chelated and excreted is thought to be proportional t o body iron stores, requires a fair degree of cooperation from the subject studied and there is evidence that some of the iron chelated i s not derived from iron stores.* The most commonly used method i s assessment of the amount of iron present in the bone marrow but this is an uncomfortable procedure and a t best only semi-quantitative. Quantitative venesection t o the level of incipient iron deficiency and anemia is sensitive3 but impracticable. Certainly none of these methods i s suitable for large scale population surveys. In 1972, G. M. Addison and his colleagues published a sensitive radioimmunoassay for serum ferritin.4 Recently a 'two-
site' immunoassay has also been e ~ o l v e d . ~ In this modification, unlabeled antibody linked t o an immunoadsorbent couples with ferritin which then reacts with labeled antibody. Thus quantitation of label bound is a measure of ferritin concentration. It has become apparent that in many instances serum ferritin concentration is related t o the total body iron Ferritin is a high molecular weight compound composed of a protein shell of apoferritin synthesized by the liver surrounding a ferric hydroxide core, the whole forming an octahedral structure. It is the main storage form of iron in the liver and reticulo-endothelial system either as ferritin itself, or as hemosiderin which is thought t o be a complex aggregate of partially denatured ferritin. In contrast t o the iron stores (1 g in a normal adult male) the amount of ferritin found in the serum by radioimmunoassay is miniscule. The normal range for the two site assay i s generally NUTRITION REVIEWSIVOL. 33, NO. 1 I JANUARY 1975 11
