Donald W. Horne

High-performance liquid chromatographic determination of the distribution of naturally occurring folic acid derivatives in rat liver☆

Procedures which allow extraction and quantitation of labile, reduced folic acid derivatives in rat liver have been developed. These procedures entail extraction of hepatic folates at 100 degrees C in 2% (w/v) sodium ascorbate, 0.2 M 2-mercaptoethanol, pH 7.85. The extract was treated with conjugase to hydrolyze folate polyglutamates and reverse-phase, ion-pair high-performance liquid chromatography was used to separate the resulting monoglutamates which were measured by microbiological assay using Lactobacillus casei. Experiments with HPLC-purified standard derivatives, so treated, showed excellent stability of tetrahydropteroylglutamic acid (H4PteGlu), 10-formyl-H4PteGlu, 5-formyl-H4PteGlu, 5-methyl-H4PteGlu, and pteroylglutamic acid (PteGlu). Under these conditions, approximately 56% of H2PteGlu was recovered unchanged while about 27% was converted to PteGlu; 5,10-methylene-H4PteGlu was quantitatively recovered as H4PteGlu. These procedures were applied to the task of measuring the distribution of naturally occurring folate cofactors in rat liver. These results indicated that rat liver folates have the following compositions: 5-methyl-H4PteGlu, 37.2%; H4PteGlu, 32.7%; 10-formyl-H4PteGlu, 22.6%; and 5-formyl-H4PteGlu, 7.7%. Experiments with [3H]PteGlu injection showed that all hepatic folates had the same specific radioactivity as determined by radioassay and L. casei assay, indicating that L. casei exhibited the same growth response to all the folates detected in rat liver.

Effect of nitrous oxide inactivation of vitamin B12-dependent methionine synthetase on the subcellular distribution of folate coenzymes in rat liver☆

The effects of nitrous oxide inactivation of the vitamin B12-dependent enzyme, methionine synthetase (EC 2.1.1.13), on the subcellular distribution of hepatic folate coenzymes was determined. In controls, cytosolic folates were 5-methyltetrahydrofolate (45%), 5- and 10-formyltetrahydrofolate (9 and 19%, respectively), and tetrahydrofolate (27%). Exposure of rats to an atmosphere containing 80% nitrous oxide for 18 h resulted in a marked shift in this distribution pattern to 5-methyltetrahydrofolate, 84%; 5- and 10-formyltetrahydrofolate, 2.1 and 9.1%, respectively; and tetrahydrofolate, 4.7%. Activity of the cytosolic enzyme, methionine synthetase, was reduced by about 84% as compared to that of air breathing controls. In controls, mitochondrial folates were 5-methyltetrahydrofolate (7.3%), 5- and 10-formyltetrahydrofolate (11.5 and 33.1%, respectively), and tetrahydrofolate (48.1%). This distribution did not change after exposure to nitrous oxide. These results show that the effects of nitrous oxide inactivation of vitamin B12 are confined to the cytosol, at least in the short term, and suggest that there is little, if any, transport of free folates between the cytosolic and mitochondrial compartments.

A functional, active transport system for methotrexate in freshly isolated hepatocytes

Abstract Methotrexate transport was studied in isolated rat liver cells. The process was found to be saturable with a K t of 2.3 × 10 −3 M and Vmax of 282 nmol/g wet wt. min. The system showed a requirement for sodium ions and it was sensitive to ouabain. Metabolic inhibitors, e.g., 2,4-dinitrophenol, anaerobic conditions, and deprivation of glucose, suppressed the uptake rate. Folic acid, but not folinic acid, was slightly inhibitory. It is suggested that methotrexate is transported in isolated hepatocytes by an active, sodium dependent process.

High-pressure liquid chromatographic separation of the naturally occurring folic acid monoglutamate derivatives

Abstract A high-pressure liquid chromatographic procedure that allows separation and quantitation of the naturally occurring monoglutamate derivatives of folic acid, as well as several oxidation and decomposition products of reduced folates, has been developed. The procedure employs reverse-phase, ion-pair chromatography on a C 18 column eluted with a nonlinear gradient of ethanol. Folate derivatives are detected by their ultraviolet absorbance of 280 nm. The detector response (peak height) is a linear function of the amount of folate derivative injected over a 10-fold range of concentrations. The limiting sensitivity varies from 10 to 25 ng for various folate derivatives.

Microbiological assay of folates in 96-well microtiter plates

Publisher Summary This chapter presents the microbiological assay of folates in 96-well microtiter plates and outlines the problem inherent to clear-wall plates and turbidimetric assays, which results in overestimation of folate concentration of samples in the two perimeter rows of 96-well plates. Rat pancreas cytosol is used in the chapter as an example. Male Sprague-Dawley rats are anesthetized with ketamine and xylazine and the pancreas of each is removed, trimmed of fat, weighed, and homogenized in 3 vol of ice-cold 0.25 M sucrose, 10 m M HEPES (pH 7.4), 10 m M 2-mercaptoethanol, and 10 m M sodium ascorbate. For routine assay of total folates by the microbiological assay, tissue is extracted by mincing in 1× extract buffer, heating in a boiling water bath for 10 min, homogenizing using a Polytron, microcentrifuging, and treating with conjugase. For liquid samples 0.2 vol of 5× extract buffer is added and is heated in boiling water bath for 5 rain followed by microcentrifuge to remove precipitated proteins. Serum and plasma contain only the monoglutamate derivatives; therefore, conjugase treatment is not necessary. These extraction procedures have been shown to preserve the reduced folates present in tissues and to lead to minimal interconversion of individual derivatives.

Effect of human milk folate binding protein on folate intestinal transport

The present study examined the effect of human milk folate binding protein (FBP) on the intestinal transport of 5-methyltetrahydrofolate (5-CH3H4PteGlu). This was performed by examining the transport of radiolabeled 5-CH3H4PteGlu bound to FBP using everted sacs of rat intestine. In the jejunum at pH 6, transport of 27 nM bound 5-CH3H4PteGlu was linear with time for 30 min of incubation. Transport of 13 nM bound 5-CH3H4PteGlu was higher in the jejunum than in the ileum at both pH 6 (2.1 +/- 0.3 and 0.36 +/- 0.03 pmol/g wet wt/25 min, respectively) and pH 8 (1.9 +/- 0.3 and 0.32 +/- 0.02 pmol/g wet wt/25 min, respectively). In the jejunum, transport of 13 nM bound 5-CH3H4PteGlu at pH 6 was less than transport of an equimolar concentration of free 5-CH3H4PteGlu (2.1 +/- 0.3 and 5.1 +/- 0.5 pmol/g wet wt/25 min, respectively) but was similar at pH 8 (1.9 +/- 0.3 and 2.47 +/- 0.3 pmol/g wet wt/25 min, respectively). In the ileum transport of bound and free 5-CH3H4PteGlu was similar at pH 6 (0.36 +/- 0.03) and 0.41 +/- 0.06 pmol/g wet wt/25 min, respectively) and pH 8 (0.32 +/- 0.02 and 0.43 +/- 0.1 pmol/g wet wt/25 min, respectively). The transport process of bound 5-CH3H4PteGlu in the jejunum was energy, temperature, and Na+ dependent, but not pH dependent, and was competitively inhibited by sulfasalazine. Ninety-two percent of the transport substrate that appeared in the serosal compartment following incubation with bound 5-CH3H4PteGlu was found to be free (unbound) 5-CH3H4PteGlu. These results show that human milk FBP decreases the rate of transport of 5-CH3H4PteGlu in the jejunum and suggest that FBP-bound 5-CH3H4PteGlu may utilize the same transport system as free 5-CH3H4PteGlu. The results also suggest a role for human milk FBP in regulating the nutritional bioavailability of folate.

Transport of Folates and Antifolates in Liver

Abstract The characteristics and mechanisms of hepatic transport of folates and antifolate cancer drugs, for example, methotrexate, have been studied in perfused liver, isolated hepatocytes (in both freshly isolated cells and in primary cell culture), and membrane vesicles isolated from the basolateral membrane. Both naturally occurring folates and antifolates are taken up by the perfused liver and secreted into bile by apparently active processes, since these compounds are concentrated in liver and bile compared with the perfusate. Transport of the naturally occurring folate 5-methyltetrah-ydrofolate in isolated hepatocytes and basolateral membrane vesicles is via cotransport with hydrogen ions, is electroneutral, and is inhibitable by other reduced folates and by methotrexate. Transport of methotrexate is by a multispecific anion carrier, is electrogenie, and is not inhibitable by reduced folates (e.g., 5-methyl- and 5-formyltetrahydrofolate). Thus, the hepatocyte has separate systems for uptake of the naturally occurring, reduced folates and for the 4-amino-substituted antifolates. [P.S.E.B.M. 1993, Vol 202]

Studies on the transport mechanism of 5-methyltetrahydrofolic acid in freshly isolated hepatocytes: effect of ethanol.

Abstract The effect of ethanol on the transport of 5-methyltetrahydrofolate in freshly isolated hepatocytes in vitro resulted in about a 30% increase in accumulation of substrate. It was shown that this was not due to differences in metabolism, nor to an inhibition of efflux. Preincubation with 40 m m ethanol for 45 min resulted in a significantly increased rate of entry of 5-methyltetrahydrofolate into the cells. The stimulatory effect was specific to 5-methyltetrahydrofolate since ethanol inhibited uptake of folate and methotrexate. The increased uptake was due to metabolism of ethanol as shown by studies with pyrazole. Also, the n -alkanols, propanol through pentanol, and sorbitol but not methanol were stimulatory. Anaerobiosis and sodium azide stimulated uptake of 5-methyl-tetrahydrofolate but were inhibitory to methotrexate uptake. These data, taken together, suggest that the ethanol effect is due to increased entry of 5-CH 3 -H 4 PteGlu into the cells possibly as the result of an increased cellular NADH NAD ratio.

Effect of nitrous oxide inactivation of vitamin B12 on the levels of folate coenzymes in rat bone marrow, kidney, brain, and liver

The effects of nitrous oxide inactivation of the vitamin B12-dependent enzyme, methionine synthetase, on the distribution of folic acid derivatives in rat bone marrow cells, kidney, brain, and liver were determined. Methionine synthetase activity was decreased by about 90% in bone marrow cells, kidney, and brain and by 83% in liver. The proportion of 5-methyltetrahydrofolate (5-CH3-H4PteGlu) in N2O-exposed rats increased from 1.4- to 1.9-fold depending on the tissue examined. This increase was at the expense of a decrease in different folate derivatives in different tissues--in bone marrow cells, kidney, and liver 5-HCO-H4PteGlu, 10-HCO-H4PteGlu, and H4PteGlu decreased; in brain only H4PteGlu decreased significantly. Total endogenous folates, as measured by Lactobacillus casei after conjugase treatment, were unchanged in all tissues after nitrous oxide exposure. The results are interpreted as direct support of the methyl trap hypothesis as the explanation of the interrelationship of folate and vitamin B12 metabolism in bone marrow cells, kidney, and brain, as well as in liver.

[43] High-performance liquid chromatographic separation of the naturally occurring folic acid derivatives

Publisher Summary This chapter highlights high performance liquid chromatographic separation of the naturally occurring folic acid derivatives. High-performance liquid chromatography (HPLC) offers high resolution along with the speed and reproducibility. Another important aspect of this analytical methodology is a tissue extraction procedure which protects easily oxidized folates. Microbiological assays of folates are performed as described by Wilson and Horne with certain modifications. HPLC separation of folates is performed by eluting folates from a Beckman–Altex Ultrasphere I.P. column with a concave ethanol gradient using tetrabutylammonium phosphate (TBAP) as an ion-pair reagent. An alternative procedure employs a Spectra-Physics SP8700 Solvent Delivery System equipped with a 2-ml injection loop. Solvents employed consist of water, 25% ethanol (v/v), and 80% methanol (v/v). All standard folates are baseline resolved, a fact which makes quantitation of each derivative unambiguous. The elution position of each standard is confirmed by UV spectroscopy. Some of the standard folic acid derivatives include pteroylglutamic acid (PteGlu, folic acid), H 4 PteGlu, 5-HCO-H 4 PteGlu, and H 2 PteGlu, which are available from Sigma.