Carol Schreiber

[60] Determination of folate by use of radioactive folate and binding proteins☆

Publisher Summary This chapter discusses the use of radioactive folate and binding proteins for the determination of folate. The concentration of folate in the serum and red cells is an important clinical tool in the diagnosis of megaloblastic anemia and for the evaluation of nutrition. This chapter defines the folate radioassay methodology, and discusses the advantages and disadvantages of the modifications introduced related to this methodology. The folate radioassay methodology is a proven and reliable procedure. It utilizes the commercially available high specific activity tritiated pteroylglutamic acid as the radioactive tracer, methyltetrahydrofolate as the standard for serum folate assays, and utilizes heat extraction only when there is an elevation of unsaturated serum folate-binding protein present. It is rapid and reproducible, and in an emergency a single sample can be assayed without the necessity for a new standard curve. In contrast to the microbiologic assay, it is not affected by antibiotics or turbidity and only minimally so by the antineoplastic drugs other than the folate antagonists. The use of gamma-labeled folates simplifies the radioassay methodology by eliminating liquid scintillation spectrometry. l25 I-labeled folic acid, as either the monotyramide or the histamine conjugate, and 75 Se-labeled folate, as the nonphysiologic pteroyl- L -methylselenocysteine, are now available.

The isolation and characterization of the folate binding protein from goat milk

It is now known that there are specific folate binding proteins (FABP) in various tissues such as serum, milk and intestinal mucosa [2,3,4]. Whereas the FABP have been purified from cow milk [5], human milk [6] and umbilical cord serum [7] the study of the physical characteristics and physiologic functions has been hampered by the small quantities of FABP present. Goat milk, which contains a low concentration of folic acid, was recently reported to contain high levels of unsaturated FABP [8]. Affinity chromatography has been used successfully to separate the FABP from large quantities of goat milk and the present communication describes its physical and immunological characteristics.

The Role of Folic Acid Binding Proteins (FABP) in the Cellular Uptake of Folates

Summary HeLa cells and human lymphocytes were grown in complete media or conditioned in folate deficient media and were used to study the physiology of folic acid binding proteins (FABP). The uptake of tritiated folic acid (3HPGA) and N-5-methyl tetrahydrofolic acid (3H methyl-THFA) by folate replete HeLa cells increased for 3 hr, was temperature dependent, was greater for 3H methyl-THFA than 3HPGA and was not influenced by preincubation with Dilantin or ethanol. HeLa cells conditioned in folate deficient media after 1 week exhibited deranged DNA synthesis (abnormal deoxyuridine suppression of 3H thymidine into DNA and a decreased growth curve) which was progressive with degree of folate deficiency. The uptake of 3HPGA and 3H methyl-THFA was greater (five and three times, respectively) in the folate deficient HeLa cells than in the folate replete HeLa cells. 3HPGA or 3H methyl-THFA bound to the FABP of folate deficient sera, some uremic sera, human or cows milk was less available for the uptake by the HeLa cells. The percent uptake of 3HPGA by HeLa cells was inversely related to the amount of FABP in several sera tested. FABP added to the culture medium following a 1 hr pulse of 3HPGA did not decrease the cellular uptake of 3HPGA. Evidence was found for an energy dependent, active mechanism for efflux of 3HPGA from the HeLa cell.

The isolation of the folate binding protein from commercially purified bovine beta lactoglobulin

Commercially prepared bovine beta lactoglobulin contains binding determinants for both reduced and oxidized mono and polyglutamates [2] and therefore has been utilized as a stable binder in the radioisotopic assays for serum and whole blood folates [3,4]. However, because of the comparatively large amount of the crystalline product needed to bind 50% of a tracer amount of tritiated pteroylglutamic acid ( [3 H] PGA), (0.25 ng [3 HI PGA bound/0.1 mg) it was felt that the folate binding moiety was not the beta lactoglobulin per se but rather a separate protein purifying very closely to the beta lactoglobulin itself. As part of a study to identify the folate binding protein (FABP) of cows milk we fractionated mixed beta lactoglobulin into its phenotypic alleles and in so doing effectively separated the folate binder from the composite whole. The present report describes the isolation of FABP from the beta lactoglobulin fraction of cows milk in sufficient quantity to enable its preliminary characterization.

Measurement of Red Cell Folate Levels by 3H-Pteroylglutamic Acid (3H-PteGlu) Radioassay

Summary. A siniple, rapid radioassay is described for the determination of red cell folate activity utilizing commercially available beta lactoglobulin as thc folate binding protein. The standard curve is prepared with a pteroylpolyglutamate and 3H‐pteroylglutamic acid. The test is performed with a maximum of 20 nicrolitres of whole blood and multiple samples can be processed within 1 hr. Values of red cell folate in normal subjects range from 200 to 875 ng/ml packed cells giving good correlation with established microbiological procedures. Red cell folate levels were lowest in patients whose folate deficiency was accompanied by anaemia. This assay may be useful for measurement of pteroylpolyglutamates in other tissues since an equimolar amount of N‐5‐methyl tetrahydrofolate is barely measurable under the conditions of this assay.

The Significance of Folate Binding Proteins in Folate Metabolism

Various aspects of folate metabolism in the microorganism and in the multicellular organism call for functional, specific folate binding proteins. These folate binding protein functions would include extracellular, intercellular, and intracellular folate transport, distribution, and storage.

Phosphate-induced phosphoribosylpyrophosphate elevations to assess deranged folate and purine nucleotide metabolism.

Abstract Phosphoribosylpyrophosphate (PRPP) levels increase several-fold in HL-60 cells adapted to folate deficiency either by continuous passage in folate-deficient medium or by short-term incubation with 10−8 M methotrexate (MTX). The addition of folic acid (PteGlu) or 5-formyltetrahydrofolic acid (5-CHO-H4PteGlu) in the form of Leucovorin normalizes this effect. The reactions for measuring PRPP levels are time and temperature dependent and are influenced by PRPP-reacting substances in undialyzed serum. Inorganic phosphate (PO4), when added to the assay, markedly stimulates PRPP levels in HL-60 cells and can be used to stress folate-dependent PRPP utilization for purine synthesis. The integrity of the folate-dependent pathways of purine-synthesizing cells can be sensitively assessed by measurement of PRPP levels during a 2-hr assay in the presence of PO4 in medium free of folate but containing dialyzed serum. In HL-60 cells that are folate deficient or in the presence of MTX (as low as 2 × 10−9 M), PO4-stimulated PRPP levels remain elevated due to ineffective utilization unless folate is added to the incubation mixture. The sensitivity of this PRPP assay to metabolically assess the integrity of folate-dependent reactions in purine synthesis is comparable to that of the deoxyuridine suppression assay. Inorganic phosphate can also be used to stimulate the incorporation of purine analogs, such as 6-mercaptopurine, into intact red blood cells which may have therapeutic implications for targeting drug delivery.

Varying effects of deoxynucleosides on the incorporation of thymidine into the dna of monolayer and suspension cultures.

Summary Deoxyuridine will normally suppress the incorporation of subsequently added tritiated thymidine into the DNA thy-mine of cells grown in suspension culture to less than 10%. Conversely, deoxyuridine appears to stimulate thymidine incorporation, at times approaching thousandfold excess, when the cells are maintained as mon-olayers. This stimulation is time, temperature, and, to a lesser extent, pH dependent, but is not directly related to the density of the monolayer. Disruption of the monolayer reverts the cells to normal deoxyuridine suppression of thymidine incorporation into DNA. This study extends earlier evidence of variations in de novo and salvage DNA synthesis between suspension and monolayer cell cultures. We caution the equating of data obtained with suspension cell systems to solid tumor analysis.