Bernard A. Cooper

Altered vitamin B12 metabolism in fibroblasts from a patient with megaloblastic anemia and homocystinuria due to a new defect in methionine biosynthesis.

Cultured fibroblasts from a recently described patient with homocystinuria and megaloblastic anemia of infancy without methylmalonic aciduria were previously shown to have normal cobalamin uptake and a specific decrease in the proportion of intracellular methylcobalamin. As in control cells but unlike in those from patients with combined homocystinuria and methylmalonic aciduria (cobalamin C and cobalamin D), accumulated 57Co-labeled cobalamin was bound in appropriate amounts and proportion to intracellular binders which are known to be the two vitamin B12-dependent enzymes, methionine synthetase and methylmalonyl-CoA mutase. Despite the association of a normal quantity of intracellular cobalamin with methionine synthetase, the proportion of intracellular cobalamin which was methyl-B12 was below normal and in the range observed in cobalamin C and D cells. This methyl-B12 was decreased by exposure of fibroblasts in culture to nitrous oxide as was observed with control cells. Exposure of control fibroblasts during culture, but not of fibroblasts from this patient, to nitrous oxide significantly reduced the holoenzyme activity of methionine synthetase assayed in cell extracts. In addition, although methionine synthetase activity in cell extracts of control and cells from the patient were similar in the presence of standard assay concentrations of thiols, at low thiol concentrations, methionine synthetase activity in extracts of cells from the patient was much lower than in control extracts. Mixing of control patient extracts corrected this decreased activity in excess of that explained by addition of the individual activities added. The defect of this patient appears to be in a reducing system required for methionine synthesis.

Absorption of Unaltered Folic Acid, from the Gastro‐intestinal Tract in Man

Hepatic portal and systemic plasma folate levels were determined in human subjects during umbilical‐vein catheterization after oral administration of folic acid. Hepatic‐vein blood was obtained during right heart catheterization. Serial folate measurements with L. casei and S. faecalis and TEAE‐cellulose chromatography revealed that folic acid is rapidly absorbed into the portal blood without undergoing metabolic conversion. A delay in rise in folate levels in systemic and hepatic‐vein blood, together with the finding of large amounts of folic acid and methylfolate in blood subsequently leaving the liver, indicates that this organ is the major site of folic‐acid metabolism.

Folate Distribution in Cultured Human Cells: STUDIES ON 5,10-CH2-H4PTEGLU REDUCTASE DEFICIENCY

We have studied the distribution of folate coenzyme forms in cultured human fibroblasts from control lines and from lines derived from nine patients representing all of the published reports of 5,10-CH(2)-H(4)PteGlu reductase deficiency. Based on mobility on DEAE-Sephadex and differential microbiological assay the major folate fractions in extracts of human fibroblasts were 5-CH(3)-H(4)PteGlu, 10-CHO-H(4)PteGlu, and 5-CHO-H(4)PteGlu with smaller fractions, which included 5-CH(3)-H(2)PteGlu, 10-CHO-PteGlu, and H(4)PteGlu. Evidence that the 5-CHO-H(4)PteGlu may have been derived from 5,10-CH=H(4)PteGlu during extraction is presented. In most of the mutant fibroblasts the absolute concentration of 5-CH(3)-H(4)PteGlu was lower than in control cells but the proportion of intracellular folate which was 5-CH(3)-H(4)PteGlu was strikingly lower in mutant cells when determined by chromatography or differential microbiological assay. In both control and mutant cells most of the 5-CH(3)-H(4)-PteGlu was polyglutamate. The proportion of intracellular folate which was polyglutamate was similar in control and mutant cells. A direct relationship was observed between the proportion of cellular folate which was 5-CH(3)-H(4)PteGlu, and both the clinical severity of this disorder and the residual enzyme activity indicating that the distribution of different folates may be an important control of intracellular folate metabolism. These studies indicate that 5,10-CH(2)-H(4)PteGlu reductase is the only significant intracellular pathway for the generation of 5-CH(3)-H(4)PteGlu, that the activity of this enzyme regulates the level of this folate in control and mutant cells under conditions of culture used here, that the majority of intracellular folate is in the polyglutamate form, and that the relative distribution of folates may control folate metabolism by interaction in the various folate reactions.

Intestinal Conversion of Folinic Acid to 5‐Methyltetrahydrofolate in Man

Summary. In three adult subjects undergoing diagnostic umbilical vein catheteriza‐tion, the active stereoisomer of folinic acid (dl,‐5‐formyltetrahydrofolate) was metabolized during absorption and appeared in hepatic portal venous plasma as 5‐methyltetrahydrofolate. A small amount of 5‐formyltetrahydrofolate and probably of 10‐formylfolate was detected in hepatic portal venous plasma early during absorption as well.

Folates in plasma and bile of man after feeding folic acid—3H and 5-formyltetrahydrofolate (folinic acid)

During the 1st hr after feeding folic acid-(3)H ((3)H-PteGlu) to fasting human volunteers, plasma S. faecalis and (3)H activity were elevated to an equivalent degree, whereas after this, the (3)H activity exceeded S. faecalis activity, which suggests gradual conversion of folic acid-(3)H to methyltetrahydrofolate-(3)H (5-CH(3)H(4) PteGlu). The increase of L. casei activity exceeded the increase of S. faecalis and (3)H activity, which is consistent with flushing of endogenous methyltetrahydrofolate from the tissues by the administered folic acid-(3)H. Feeding of 5-formyltetrahydrofolate (+/-5CHOH(4)PteGlu) produced a large increase of plasma L. casei activity and only a slight increase of S. faecalis and P. cerevisiae activity, which is consistent with very rapid conversion of folinic acid to methyltetrahydrofolate. Bile folate concentration determined microbiologically was 2.3-9.8 times plasma folate. 40-80% of the bile folate was S. faecalis-active and 20-35% P. cerevisiae-active. Chromatography of bile folates on TEAE-cellulose showed several folates including four tentatively identified as 10-formyltetrahydrofolate (10-CHO-H(4)PteGlu), 10-formylfolate (10-CHO-PteGlu), and/or 10-formyldihydrofolate (10-CHOH(2)PteGlu), methyltetrahydrofolate, and possibly a triglutamate folate. After folate ingestion bile folate concentration increased rapidly. The distribution of bile folates measured by microbiological assay was similar after either folic or folinic acid feeding. Most of the (3)H label of folic acid-(3)H appeared in the biological folates of bile rather than in the folic acid fraction, which shows that the administered folic acid was rapidly transformed to other folates. Folate polyglutamate deconjugating enzyme activity was found to be much less than in serum. Polyglutamates of the type found in yeast were not found in bile. It is suggested that biliary folate may reflect the hepatic intracellular oligoglutamate folate pool rather than the folate as it appears in the hepatic portal blood.

Inherited disorders of cobalamin metabolism.

2. Structure. distribution and transport of cobalamin. 2.1. Structure of cobalamin 2.2. Distribution and dietary sources. 2.3. Absorption and transport of cobalamin ..... 2.3.1. R binders. 2.3.2. Intrinsic factor (IF). 2.3.3. Transcobalamin II (TCII). ........ 2.3.4. Alternative Cbl transport. ......... 2.35. Intracellular Cbl metabolism. ...... 2.4. Mitochondrial Cbl uptake

Lysosomal cobalamin accumulation in fibroblasts from a patient with an inborn error of cobalamin metabolism (cblF complementation group): Visualization by electron microscope radioautography

Cobalamin (Cbl, vitamin B12) bound to transcobalamin II (TCII) enters cultured fibroblasts by receptor-mediated endocytosis. Following degradation of the TCII, Cbl is subsequently found in either the cytoplasm bound to methionine synthase or in the mitochondria bound to methylmalonyl CoA mutase. In fibroblasts from patients belonging to the cblF complementation group, Cbl is found free in the cell and is not transferred to the above two target enzymes. Quantitative EM radioautography was utilized to visualize intracellular Cbl in fibroblasts from cblF patients and from normal subjects. In cblF cells, 60% of all silver grains were assigned to lysosomes, with only 12.6% over cytoplasm and 1.2% over mitochondria. In contrast, in control cells, only 4.7% were assigned to lysosomes, with 47% to cytoplasm and 23.4% to mitochondria. Subcellular fractionation showed that in cblF cells, the majority of label was associated with clearly recognizable lysosomes. These studies conclusively demonstrate that secondary lysosomes accumulate Cbl in cblF disease.

Solubilized receptor for vitamin B12-intrinsic factor complex from human intestine.

Summary. A specific receptor for intrinsic factor‐vitamin B12 complex has been prepared in soluble form from human intestinal mucosal cell membranes employing Triton X‐100. The extraction procedure has not detectably altered its characteristics relative to membrane‐bound receptor. It appears to be macromolecule containing critical peptide and disulphide bonds.

An immunologic basis for acquired resistance to oral administration of hog intrinsic factor and vitamin B12 in pernicious anemia.

In pernicious anemia, vitamin B12 deficiency develops owing to the failure of gastric intrinsicfactor secretion. Patients with this disease absorb vitamin B12 from the gastrointestinal tract when it is fed together with an extract of hog pyloric mucosa. Some patients have relapsed during the course of oral therapy with this mixture (1-3). These subjects absorb vitamin B1.2 administered with normal human gastric juice but do not absorb it when it is administered with hog preparations possessing intrinsic-factor activity (3). These patients have become refractory to the hog preparations. It has been reported that sera obtained from most of these refractory patients (4), and from a minority of nonrefractory patients and normal subjects (5, 6), possess intrinsic factor-neutralizing properties. Such observations have directed attention to a possible immunologic mechanism for the refractory state. This study was undertaken to determine whether the refractory state might be due to antibodies against the hog intrinsic-factor preparation, which could be detected in vitro.

Endogenous folate of normal fibroblasts using high-performance liquid chromatography and modified extraction procedure

The endogenous levels of the various folate monoglutamate compounds in cultured human fibroblasts were determined using high-performance liquid chromatography for the separation of folate monoglutamate. Endogenous folates were converted to monoglutamate forms using conjugase enzyme present in rat serum and incubation was carried out at pH 6.5. This minimized folate coenzyme interconversion during processing. Using methanol for precipitation of protein instead of heat minimized degradation of labile folates. Recovery of all folates except 10-formyltetrahydrofolic acid (10-CHO H4PteGlu) using this procedure was more than 90%. Disruption of cells by boiling appeared to cause less postextraction changes of cell folates than did freezing and thawing or sonication. When heat to release endogenous folate, conjugase treatment with rat serum at pH 6.5, and precipitation of protein with methanol were used, more than half of the intracellular folate of normal fibroblasts in confluent growth was 5-methyltetrahydrofolic acid (5-CH3 H4PteGlu), and 10-CHO H4PteGlu and tetrahydrofolic acid (H4PteGlu) comprised 29 and 6%, respectively.