The glycophospholipid-linked folate receptor internalizes folate without entering the clathrin-coated pit endocytic pathway.

The folate receptor, also known as the membrane folate-binding protein, is maximally expressed on the surface of folate-depleted tissue culture cells and mediates the high affinity accumulation of 5-methyltetrahydrofolic acid in the cytoplasm of these cells. Recent evidence suggests that this receptor recycles during folate internalization and that it is anchored in the membrane by a glycosyl-phosphatidylinositol linkage. Using quantitative immunocytochemistry, we now show that (a) this receptor is highly clustered on the cell surface; (b) these clusters are preferentially associated with uncoated membrane invaginations rather than clathrin-coated pits; and (c) the receptor is not present in endosomes or lysosomes. This receptor appears to physically move in and out of the cell using a novel uncoated pit pathway that does not merge with the clathrin-coated pit endocytic machinery.

Absorption, plasma transport, and cellular retention of cobalamin analogues in the rabbit. Evidence for the existence of multiple mechanisms that prevent the absorption and tissue dissemination of naturally occurring cobalamin analogues.

Analogues of cobalamin (Cbl; vitamin B(12)) are prevalent in nature as a result of bacterial synthesis, and are of additional interest because of their potential use as antimetabolites and chemotherapeutic agents. We have synthesized 14 Cbl analogues containing (57)Co and have compared their gastrointestinal absorption, plasma transport, and cellular retention to that of [(58)Co]Cbl in rabbits. Many of the Cbl analogues were bound with low affinity by intrinsic factor, and none of these [(57)Co]Cbl analogues were taken up by the ileum or absorbed into the body in amounts comparable to that of [(58)Co]Cbl. The Cbl analogues that were bound by intrinsic factor with high affinity were taken up by the ileum but, in many cases, they were retained there in significant amounts. Most of the Cbl analogues were bound by plasma transcobalamin II with high affinity and all of these transcobalamin II-[(57)Co]Cbl analogue complexes were taken up by a variety of tissues in a manner that was indistinguishable from that of transcobalamin II-[(58)Co]Cbl. The few analogues that were bound by transcobalamin II with low affinity were taken up by tissues in lesser amounts, and 20-70% of these analogues was rapidly excreted in the urine as occurs with native Cbl when it is present in plasma in unbound form. All of the Cbl analogues were bound by the granulocyte R-type Cbl-binding protein with high affinity and all of the R-type protein-[(57)Co]Cbl analogue complexes were cleared rapidly from plasma exclusively by hepatocytes as occurs with R-type protein-[(58)Co]Cbl. Some Cbl analogues were released back into the plasma and were disseminated among a variety of tissues via transcobalamin II as occurs with native Cbl. Other Cbl analogues were retained in the liver and eventually excreted in the feces and urine without accumulating in other tissues. These studies indicate that intrinsic factor and the ileum prevent certain Cbl analogues from entering the body and that the granulocyte R-type protein and hepatocytes prevent the dissemination of certain Cbl analogues that may gain entry such as during infections with Cbl analogue-producing bacteria. The fact that transcobalamin II binds and transports a large number of Cbl analogues indicates that these protective mechanisms can be circumvented and supports the feasibility of using Cbl analogues as antimetabolites in vivo.

Presence and formation of cobalamin analogues in multivitamin-mineral pills.

Because the origin of cobalamin (vitamin B12) analogues in animal chows and animal and human blood and tissues is unknown, we investigated the possibility that multivitamin interactions might convert cobalamin to cobalamin analogues. We homogenized three popular multivitamin-mineral pills in water, incubated them at 37 degrees C for 2 h, and isolated the cobalamin. Using paper chromatography we observed that 20-90% of the cobalamin was present as cobalamin analogues. Studies using CN-[57Co]cobalamin showed that these analogues were formed due to the concerted action of vitamin C, thiamine, and copper on CN-cobalamin. These cobalamin analogues are absorbed from the gastrointestinal tract of mice and either fail to stimulate or actually inhibit cobalamin-dependent enzymes when injected parenterally. We conclude that CN-cobalamin can be converted to potentially harmful cobalamin analogues by multivitamin-mineral interactions and that these interactions may be responsible for the presence of cobalamin analogues in animal chows and animal and human blood and tissues.

Influence on immunoreactive folate-binding proteins of extracellular folate concentration in cultured human cells.

The influence of extracellular folate concentration on cellular levels of the folate transport protein and its soluble product was studied directly in cultured human nasopharyngeal carcinoma (KB) cells. As determined by radioimmunoassay, levels of the folate transport protein and the soluble folate-binding protein were 58 +/- 17 (mean +/- SD) and 5 +/- 2 pmol/mg cell protein, respectively, in KB cells maintained in standard medium (containing 2,300 nM folic acid). These levels significantly increased to 182 +/- 34 and 26 +/- 6 pmol/mg cell protein, respectively, in KB cells serially passaged in low folate medium (containing 2-10 nM 5-methyltetrahydrofolate). Increases in folate-binding protein levels occurred more rapidly in KB cells serially passaged in very low folate medium containing less than 2 nM folate and were prevented by the addition of 100 nM 5-methyltetrahydrofolate or 0.1-1 microM 5-formyltetrahydrofolate to this medium. When KB cells which had been passaged in low folate medium were passaged back into either standard medium or low folate medium supplemented with reduced folates, the levels of both folate-binding proteins fell linearly towards the levels in KB cells continuously maintained in standard medium. The folate transport protein was identified in and underwent similar changes in human and mouse mammary tumor cells. These studies indicate that the folate transport system is probably regulated by the extracellular folate concentration through changes in intracellular metabolite levels.

Nitrous oxide has multiple deleterious effects on cobalamin metabolism and causes decreases in activities of both mammalian cobalamin-dependent enzymes in rats.

In man, use of the general anesthetic nitrous oxide, N2O, is associated with hematologic and neurologic abnormalities that mimic those seen in cobalamin (Cbl, vitamin B12) deficiency. We have measured a number of aspects of Cbl metabolism in rts exposed to various concentrations of N2O for various periods of time. As little as 2% N2O given for 15 h resulted in 30% inhibition of methionine synthetase (MS) in rat liver. With 50% N2O, inhibition of 70% occurred with 1 h and did not change during the next 48 h. Under these conditions, no inhibition of methylmalonyl-CoA mutase (MMCoAM) was observed. The recovery of MS activity was slow and was only 80% of control values 72 h after N2O was stopped. Studies employing rats previously injected with [57Co]Cbl showed that N2O displaced [57Co]Cbl from MS in a manner that temporally and quantitatively paralleled the loss of MS activity. Recovery of MS activity paralleled the reappearance of [57Co]Cbl on MS. N2O also caused the hepatic content of CH3-[57Co]Cbl to decrease by 20-60%. When [57Co]-Cbl was extracted from liver and analyzed by paper chromatography, [57Co]Cbl analogues were present (10-40% of total [57Co]Cbl) in rats exposed to N2O, but not in control animals. When rats were exposed to 50% N2O for 33 d, the total of endogenous Cbl and Cbl analogues in liver decreased to 35% of control values and endogenous Cbl decreased to 10% of control values. At this time, MS activity was 15% of control values and MMCoAM was only 26% of control values. We conclude that N2O causes multiple defects in Cbl metabolism that include the following: (a) rapid inhibition of MS activity with a slow recovery when N2O is stopped; (b) displacement of Cbl from MS; (c) decreased CH3-Cbl; (d) conversion of Cbl to Cbl analogues; (e) the gradual development of Cbl deficiency and (f) an eventual decrease in MMCoAM activity with a further decrease in MS activity.

Folate and methotrexate interactions in the rat kidney.

The mechanisms controlling the renal retention and urinary output of methotrexate and folates were studied in rats. 125I-Labeled folic acid administered i.v. was shown to be retained in the kidney through a system that could be inhibited by either folic acid or 5-methyltetrahydrofolate. Methotrexate also inhibited this system but required concentrations 50- to 100-fold greater than that required for folic acid and 5-methyltetrahydrofolate. Extracts from solubilized kidneys were shown to contain a folate binder with the same relative affinities for folates and methotrexate as the in vivo system. Methotrexate was shown to cause an increase in the urinary output of endogenous folates in rats when administered as equivalent doses to those used in treating human disease. Conversely, [3H]methotrexate administered i.v. was shown to be retained in the kidney through an additional system that could be inhibited by unlabeled methotrexate, but not by folates. This system was not demonstrable in solubilized kidney. These data demonstrate two systems for the renal retention of methotrexate.

Changes in plasma methionine and total homocysteine levels in patients receiving methotrexate infusions.

Methotrexate reduces intracellular pools of 5-methyltetrahydrofolate and could result in reduced conversion of homocysteine to methionine by methionine synthetase. This study was designed to investigate the effects of moderate dose to very high dose methotrexate on methionine and total homocysteine as reflections of methotrexate induced intracellular events. Methionine and total homocysteine were measured prior to, during, and following twenty-six 24-h i.v. infusions of 33.6 g/m2 methotrexate (very high dose methotrexate) in 16 children with acute lymphocytic leukemia and seven 4-h i.v. infusions of 8 g/m2 methotrexate (high dose methotrexate) in 5 children with osteogenic sarcoma. Amino acids were measured by gas chromatography/mass spectrophotometry. Mean methionine levels decreased by 70.0 +/- 3.1% (SE) with very high dose methotrexate and 72.6 +/- 5.9% with high dose methotrexate at 24 and 4.5 h, respectively, after beginning methotrexate infusions. Mean total homocysteine levels increased by 61.7 +/- 3.1% with very high dose methotrexate and 55.6 +/- 17.5% with high dose methotrexate at 36 and 24 h, respectively, after beginning methotrexate infusions. No consistent or significant changes were noted in levels of total cysteine, leucine, isoleucine, or valine. Similar changes did not occur in patients receiving prednisone, vincristine, daunomycin, and intrathecal methotrexate as therapy for acute lymphocytic leukemia. These changes in homocysteine and methionine may reflect biological effects of methotrexate that may predict cytotoxicity of methotrexate.

Nonmyeloablative allogeneic hematopoietic stem cell transplantation for congenital sideroblastic anemia.

Congenital sideroblastic anemia (CSA) is a dyserythropoietic disorder that leads to transfusion dependency and subsequent iron overload. Nonmyeloablative allogeneic hematopoietic stem cell transplantation (NST) was performed for a patient with CSA, who had contraindications to conventional allografting. Conditioning was fludarabine, low-dose total body irradiation and antithymocyte globulin, followed by peripheral blood stem cell transplant. Cyclosporine and mycophenolate mofetil were used for graft-versus-host disease prophylaxis. Complete donor chimerism was observed day +131. Early after transplant, the patient became transfusion independent, allowing a regular phlebotomy program. On day +190, refractory lactic acidosis followed by fatal cardiovascular collapse developed, without evidence of infection. Data from this case demonstrates that NST may correct the erythropoietic defect of CSA.

Serum xylose analysis by gas chromatography/mass spectrometry.

A gas chromatography/mass spectrometric (GC/MS) isotope dilution assay for xylose was developed using tertbutyldimethylsilyl-derivatized xylose and [13C]1xylose, and applied to human serum samples. A calibration curve in serum using this assay showed < 3% variation (< 10 mg/L) for any given point. The correlation coefficient for xylose measurements made on 27 sera between a colorimetric method performed by a national commercial reference laboratory and the GC/MS method developed here was .952. However, xylose determinations of 10 of 27 samples differed by > 10% (up to 150 mg/L) when colorimetric values were compared to GC/MS. Two of these samples had borderline-low xylose values by GC/MS, but were well within the normal range by colorimetric analysis. gas chromatography/mass spectrometric isotope dilution assay appears to be an accurate method to measure xylose in serum. These data also suggest that further prospective studies comparing GC/MS to colorimetric methods are indicated for subjects undergoing oral xylose testing.

Glutathione oxidation in real time by thermospray liquid chromatography-mass spectrometry.

The oxidation and reduction of glutathione and oxidized glutathione were studied in real time by liquid chromatography-mass spectrometry during exposure to hydrogen peroxide and mercaptoethanol. By mass spectrometry mixed disulfides and both reversible and irreversible oxidations of sulfur to higher states (sulfinic and sulfonic acids) were directly observed during exposure to hydrogen peroxide. The irreversible oxidation of glutathione to glutathione sulfonic acid could be detected after 30 min exposure of glutathione to 40 mM H2O2 at 20 degrees C. A peak consistent with glutathione-sulfinic acid was transiently present, suggesting this compound behaved as an oxygen consuming antioxidant. Liquid chromatography-mass spectrometry appears to be an excellent method to study oxidation and reductions of sulfur containing peptides and amino acids.

Unpredictable intra-individual variations in serum homocysteine levels on folic acid supplementation.

OBJECTIVE: To determine if changes in serum homocysteine values during folic acid supplementation can identify objectively healthy subjects with subclinical folate deficiency. DESIGN: Blood drawn and processed in a regimented fashion from fasting subjects. Serum homocysteine values determined by gas chromatography-mass spectrometry twice before and on days 5 and 8 of daily folic acid supplementation. SETTING: Outpatient University Hospital Clinical Research Center, Denver, Colorado. SUBJECTS: Subjectively healthy adults with normal hematologic and biochemical screening tests. INTERVENTION: Folic acid 1 mg daily for eight consecutive days. RESULTS: Homocysteine values of the group fell significantly during folic acid supplementation. Values pre supplementation were 7.8 +/- 1.8 and 7.4 +/- 2.4 mumol/L while values on days 5 and 8 of supplementation were 6.5 +/- 2.2 and 6.3 +/- 2.2 mumol/L. However, the homocysteine values of any given individual varied up to 60% (rises up to 7 mumol/L and falls of 5 mumol/L) during folic acid supplementation despite the controlled circumstances of blood handling, and an assay coefficient of variation of 8%. CONCLUSIONS: Although group values of serum homocysteine fall during folic acid supplementation, intraindividual variation is so great that subjects with subclinical folate deficiency can not be identified using this study design. Furthermore, these data suggest than an individuals homocysteine values vary enough that single values must be interpreted with caution.

A mechanism of inhibition of short-wavelength ultraviolet light-induced deamination of pyrimidine bases by ascorbic acid.

The relation of ascorbic acid to the ultraviolet light-induced deamination of cytosine (to uracil) and 5-methylcytosine (to thymine) was examined using gas chromatography-mass spectrometry. There was an inverse relation between the uracil content measured in solutions of cytosine after exposure to short wavelength (254 nm) ultraviolet light and the ascorbic acid concentration of the solution indicating inhibition of deamination of cytosine by ascorbic acid. Ascorbic acid also inhibited ultraviolet light-induced deamination of 5-methylcytosine. The ascorbic acid was only partially consumed during the exposure suggesting that protection from deamination was not due entirely to the antioxidant properties of ascorbic acid. Mercaptoethanol did not prevent ultraviolet light-induced deamination of either cytosine or 5-methylcytosine. However, near identical protection from ultraviolet light-induced deamination was provided by 4-aminobenzoic acid and ascorbic acid at equivalent ultraviolet light absorbing concentrations. This observation suggests that ascorbic acid prevents short wavelength ultraviolet light-induced deamination through absorbance of ultraviolet light rather than through antioxidant mechanisms.

Are neuropsychiatric manifestations of folate, cobalamin and pyridoxine deficiency mediated through imbalances in excitatory sulfur amino acids?

Folate, cobalamin and pyridoxine deficiency are associated with psychiatric or neurological symptomatology. Disturbances in sulfur amino acid metabolism leading to accumulation of homocysteine occurs in all three conditions as the metabolism of homocysteine depends on enzymes requiring these vitamins as cofactors. Oxidation products of homocysteine (homocysteine sulfinic acid and homocysteic acid) and cysteine (cysteine sulfinic acid and cysteic acid) are excitatory sulfur amino acids and may act as excitatory neurotransmitters, whereas taurine and hypotaurine (decarboxylation products of cysteic acid and cysteine sulfinic acid) may act as inhibitory transmitters. Homocysteic acid and cysteine sulfinic acid have been considered as endogenous ligands for the N-methyl-D-aspartate (NMDA) type of glutamate receptors. The profile of these sulfur amino acid neurotransmitters could be altered in a similar fashion in states of decreased availability of folate, cobalamin or pyridoxine. It is proposed that the mechanism of neuropsychiatric manifestations in all three conditions result from a combination of two insults to homocysteine catabolism in the brain.

Ascorbate and dehydroascorbate measurements in aqueous solutions and plasma determined by gas chromatography-mass spectrometry.

The tert-butyldlmethylsllyl derivatives of ascorbic acid and dehydroascorbic acid were characterized by gas chromatography-mass spectrometry, and an isotope dilution assay for ascorbate and dehydroascorbate was developed using [13C6]ascorbic acid and [13C6]- and [6,6-2H2]dehydroascorbate. This assay was used to monitor ascorbic acid loss and the resulting rise of dehydroascorbic acid in aqueous solutions and plasma. Ascorbic acid was shown to rapidly decompose in aqueous solutions containing transition metal ions or when exposed to oxygen. Ethylenedlaminetetraacetic acid chelation did not prevent ascorbic acid degradation in aqueous solution, and ascorbate in ethylenedlaminetetraacetic acid chelated plasma was converted to dehydroascorbate on freezing. Gas chromatography-mass spectrometry appears to be a satisfactory method for determining the ascorbate and dehydroascorbate content of solutions including human blood plasma, whether or not there is ongoing oxidation of ascorbate in those solutions.

Structural specificity of mechanisms controlling the hepatic uptake and biliary output of methotrexate in the rat.

Using an in vivo model with systemic administration of compounds, the hepatic uptake from blood and hepatic release into bile of [3H]methotrexate [( 3H]MTX) are shown to involve structurally distinct and specific mechanisms. The hepatic uptake of [3H]MTX from blood is shown to proceed through two separate mechanisms: one inhibitable by the bile salt cholic acid, and the other inhibitable by either unlabeled MTX or folic acid, but not the lipophilic antifol, trimetrexate. The biliary output of [3H]MTX was shown to be related to the cholic acid-sensitive mechanism of hepatic uptake of [3H]MTX. In contrast, the biliary output of [3H]MTX was shown to be markedly stimulated by either unlabeled MTX or trimetrexate but not folic acid, demonstrating structural specificity for the biliary output of [3H]MTX distinct from the structural specificity shown for the hepatic uptake of [3H]MTX.

Recognition of two intracellular cobalamin binding proteins and their identification as methylmalonyl-CoA mutase and methionine synthetase.

The granulocyte R-type cobalamin binding protein delivers cobalamin (Cbl) exclusively to hepatocytes, and transcobalamin II delivers Cbl to various mammalian cells. Both protein-Cbl complexes enter cells by pinocytosis, and the protein moieties are rapidly degraded in lysosomes. The liberated Cbl is subsequently bound to a high-molecular-weight intracellular cobalamin binding protein (ICB). The nature of ICB-Cbl is unknown but appears important because ICB-[57Co]Cbl is missing from cultured fibroblasts of a group of patients whose cells take up CN-[57Co]Cbl normally but do not convert it to either of its coenzyme forms. We have examined supernatants of sonicated rabbit livers and have found that 65% of the total endogenous Cbl elutes from Sephadex G-150 as ICB-Cbl and that this fraction also contains the two mammalian Cbl-dependent enzymes, methylmalonyl-CoA mutase (methylmalonyl-CoA CoA-carbonylmutase;EC 5.4.99.2) and methionine synthetase (tetrahydropteroylglutamate methyltransferase; 5-methyltetrahydropteroyl-L-glutamate:L-homocysteine-S-methyltransferase; EC 2.1.1.13). Gradient elution from DEAE-Sephadex reveals that 90--95% of the ICB--Cbl elutes with methylmalonyl-CoA mutase and 5--10% elutes with methionine synthetase. ICB--[57Co]Cbl first appears 2 hr after the intravenous injection of CN[57Co]Cbl bound to granulocyte R-type protein. This ICB-[57Co]Cbl is associated with either methylmalonyl-CoA mutase or methionine synthetase although the latter appears to be formed at a relatively faster rate. Our studies indicate that mammalian cells contain two ICBs, that these proteins are methylmalonyl-CoA mutase and methionine synthetase, and that the primary abnormality in the group of patients mentioned above lies at a step that is common to the formation of both Cbl coenzymes and that precedes the stable binding of Cbl to both methylmalonyl-CoA mutase and methionine synthetase.

Identification and perturbation of mutant human fibroblasts based on measurements of methylmalonic acid and total homocysteine in the culture media.

Human and mammalian cells contain two cobalamin-dependent enzymes. Methylmalonyl-CoA mutase isomerizes L-methylmalonyl-CoA to succinyl-CoA in the propionyl-CoA pathway while methionine synthase catalyzes the transfer of the methyl group of 5-methyltetrahydrofolate to homocysteine to form methionine. Decreased activity of mutase leads to an increased methylmalonic acid in the serum of humans while decreased activity of methionine synthase leads to increased homocysteine in the serum of humans. In current studies of cultured fibroblasts, methylmalonic acid levels were specifically increased in media of normal fibroblasts exposed to propionate or in fibroblasts with mutations involving mutase. Homocysteine levels were specifically increased in media of normal fibroblasts exposed to reduced folate concentrations or in fibroblasts involving mutations of methionine synthase. In addition, exposure of normal cells to inhibitory cobalamin analogues resulted in an increase of both methylmalonic acid and homocysteine in the media. This method of analysis appears to be both specific and sensitive for reduced activity of these two enzymes in tissue culture.

Presence of cobalamin analogues in animal tissues.

Cobalamin (Cbl, vitamin B-12) has been extracted and isolated from a number of animal tissues by using (i) reverse-affinity chromatography on R protein-Sepharose followed by adsorption to and elution from charcoal-coated agarose and (ii) paper chromatography. Radioisotope dilution assays showed that only 75-97% of the Cbl chromatographed in the position of crystalline Cbl. The remaining 3-25% was present in a number of slower and faster moving fractions. This suggested that Cbl analogues are present in animal tissues because appropriate controls ruled out the possibility that this material was artifactually derived from Cbl during the extraction and purification procedures. With a large-scale isolation from rabbit kidney, the material in five such fractions contained cobalt and had absorption spectra that were similar to but different from the spectrum of Cbl, indicating that they were Cbl analogues. Compared to Cbl, these Cbl analogues had decreased but definite affinities for Cbl-binding proteins with the following order of strength of binding: R protein > transcobalamin II > intrinsic factor. Compared to Cbl, they also had decreased but definite growth-promoting activity for two microorganisms, Euglena gracilis and Lactobacillus leichmannii, which require Cbl for growth. These Cbl analogues differed from each other and from 18 synthetic Cbl analogues, including the most common Cbl analogues synthesized by microorganisms, in at least one of the above features. These studies indicate that animal tissues contain a number of Cbl analogues whose origins, structures, and biologic activities remain to be determined.