Is low-dose oral cobalamin enough to normalize cobalamin function in older people?

OBJECTIVES: To investigate whether the use of low-dose oral cobalamin (Cbl) supplements by older persons, as frequently found in multivitamin preparations, affects their Cbl serum concentrations and function, determined by measurements of the serum Cbl-related metabolites methylmalonic acid (MMA), homocysteine (HCYS), and methylcitric acid (MCTR). DESIGN: Cross-sectional study. SETTING: Community. PARTICIPANTS: Two hundred forty-two independent, active, community-living, older adult volunteers recruited from community events and activities for seniors. MEASUREMENTS: We systematically collected data on vitamin supplement intake, diet, medications, and medical and surgical history. Serum was obtained for Cbl, MMA, HCYS, and MCTR, and creatinine and hematological parameters. RESULTS: Serum levels of Cbl were significantly higher in subjects on oral Cbl supplements (2-37.5 microg/day). Similarly, serum levels of the metabolites MMA, HCYS, and MCTR were also lower in subjects on Cbl supplementation. Intake of low-dose oral supplements of Cbl significantly reduced the odds of low Cbl levels or high MMA. The relationship between Cbl supplement dosage and the biochemical parameters was dose dependent. CONCLUSION: Oral Cbl (2-37.5 microg/day) intake by community-dwelling healthy older adults is associated with higher serum levels of Cbl and improved or normalized Cbl function, as indicated by lower concentrations of the metabolites MMA, HCYS, and MCTR. Use of low-dose oral Cbl replacement therapy might be sufficient to prevent Cbl deficiency in a large proportion of this population.

Analysis of protein-mediated 3-O-methylglucose transport in rat erythrocytes: rejection of the alternating conformation carrier model for sugar transport.

3-O-Methylglucose (3OMG) transport in rat erythrocytes (RBCs) is mediated by a low-capacity, facilitated diffusion-type process. This study examines whether the characteristics of sugar transport in rat RBCs are consistent with the predictions of two diametric, theoretical mechanisms for sugar transport. The one-site carrier describes a transport mechanism in which sugar influx and efflux substrate binding sites are mutually exclusive. The two-site carrier describes a transport mechanism in which sugar influx and efflux substrate binding sites can exist simultaneously but may interact in a cooperative fashion when occupied by substrate. Michaelis and velocity parameters for saturable 3OMG transport in rat erythrocytes at 24 degrees C were obtained from initial rate measurements of 3OMG transport. The results are incompatible with the predictions of the one-site carrier but are consistent with the predictions of a symmetric two-site carrier, displaying negligible cooperativity between substrate binding sites. This allows reduction of the two-site carrier transport equations to a form containing fewer constants than the one-site carrier equations without limiting their predictive success. While the available evidence does not prove that rat erythrocyte sugar transport is mediated by a two-site mechanism, we conclude that adoption of the formally more complex one-site model for sugar transport in rat erythrocytes is unnecessary and unwarranted. Counterflow experiments have also been performed in which the time course of radiolabeled 3OMG uptake is measured in cells containing saturating levels of 3OMG. The results of these experiments are consistent with the hypothesis [Naftalin et al. (1985) Biochim. Biophys. Acta 820, 235-249] that exchange of sugar between intracellular compartments (cell water and hemoglobin) can be rate limiting for transport under certain conditions.

Clinical application of measurement of glucose transport in human polymorphonuclear leukocytes.

Transport of the non-metabolizable hexose analogue 3-O-methyl-D-glucose (3OMG) was measured at 37 degrees C, pH 7.4, in human polymorphonuclear leukocytes (PMNLs) obtained from 15 ml of fresh venous blood. In the study, 0.05 mM of 3OMG was equilibrated with a half-time of about 10 s, and the rate constant was 0.074/s in PMNLs from a healthy subject (male, 38 years). The coefficient of variation of values assayed on different days was about 7% and the intra-assay variation was about 2%. The transport rate did not differ between the two sexes or between subjects of different ages (23-63 years), but was significantly higher in 23 patients with non-insulin-dependent diabetes than in 29 normal controls (13.3 +/- 3.7 vs. 10.4 +/- 2.5 fl/cell.s, mean +/- SD). In conclusion, the measurement of transport of 3OMG in PMNLs may be useful for the study of glucose transport in clinical investigations.

Inhibition of hexose transport by adenosine derivatives in human erythrocytes.

The human erythrocyte membrane carriers for hexoses and nucleosides have several structural features in common. In order to assess functional similarities, the effects of adenosine derivatives on hexose transport and cytochalasin B binding sites were studied. Adenosine inhibited zero-trans uptake of 3-O-methylglucose half-maximally at 5 mM, while more hydrophobic adenosine deaminase-resistant derivatives were ten- to 20-fold more potent transport inhibitors. However, degradation of adenosine accounted for very little of this difference in potency. Hexose transport was rapidly inhibited by N6-(L-2-phenylisopropyl)adenosine at 5 degrees C in a dose-dependent fashion (EC50 = 240 microM), to lower the transport Vmax without affecting the Km. A direct interaction with the carrier protein was further indicated by the finding that N6-(L-2-phenylisopropyl)adenosine competitively inhibited [3H]cytochalasin B binding to erythrocytes (Ki = 143 microM) and decreased [3H]cytochalasin B photolabeling of hexose carriers in erythrocyte ghosts. The cross-reactivity of adenosine and several of its derivatives with the hexose carrier suggests further homologies between the carriers for hexoses and nucleosides, possibly related to their ability to transport hydrophilic molecules through the lipid core of the plasma membrane.

Parameters for 3-O-methyl glucose transport in human erythrocytes and fit of asymmetric carrier kinetics.

1. Equilibrium exchanges in the range of 2-40 mM-3-O-methyl glucose at 16 degrees C suggested that the half-saturation concentration for exchange was 22 mM and that the maximum velocity (Vmax) was ca. 149 mmol l-1 min-1. 2. Initial rates of exchange influx from 1, 2, 4 and 8 mM into 76 mM solution gave a half-saturation value of 3.6 mM and a Vmax of 122 mmol-1 min-1. 3. The non-transportable inhibitor 4,6-O-ethylidene-alpha-D-glucopyranose (ethylidene glucose) acting on the outside of the cells inhibited 3-O-methyl glucose exchanges at 16 degrees C with an inhibition constant (KI) of ca. 11 mM. 4. Sen-Widdas exit experiments gave the half-saturation for 3-O-methyl glucose at 16 degrees C as only ca. 2 mM and the KI for ethylidene glucose as ca. 4 mM. 5. Efflux inhibitions by ethylidene glucose are satisfactorily predicted by the asymmetric carrier kinetics of Regen & Tarpley (1974) when using the parameters derived from the exchange experiments but not with parameters from Sen-Widdas exits. 6. Uphill transfer by counterflow experiments and Sen-Widdas exits cannot be fitted by the Regen and Tarpley kinetics (using the same parameters) unless the kinetics are modified to provide for an extra exchange element which replaces some of the net exit component in the equations. 7. At present the modification to the kinetics is only possible in computer simulations and data handling, but with it the fit to experimental results is good. The nature of the modification is described and in the light of it a revised interpretation of the significance of the Km derived from Sen-Widdas exits is discussed.

Comparison of the equilibrium exchange of nucleosides and 3-O-methylglucose in human erythrocytes and of the effects of cytochalasin B, phloretin and dipyridamole on their transport.

Because of similarities in the physical and molecular properties of the nucleoside and sugar transporters of human erythrocytes and the photoaffinity labeling of the sugar transporter by 8-azidoadenosine (Jarvis et al. (1986) J. Biol. Chem. 261, 11077-11085), we have directly compared the equilibrium exchange of uridine and 3-O-methylglucose in these cells as measured by rapid kinetic techniques under identical experimental conditions. Both the Michaelis-Menten constant and maximum velocity were about 100-fold higher for 3-O-methylglucose exchange than for uridine exchange so that the first order rate constants for both transporters were about the same. When calculated on the basis of the number of nucleoside and sugar carriers per red cell estimated by equilibrium binding of nitrobenzylthioinosine and cytochalasin B, respectively, the turnover numbers for the sugar and nucleoside carriers with 3-O-methylglucose and uridine, respectively, as substrates were quite similar. Various sugars up to concentrations of 108 mM had no effect on the exchange of 500 microM uridine or adenosine, and uridine up to a concentration of 50 mM had no effect on the exchange of 10 mM 3-O-methylglucose. Adenosine, on the other hand, inhibited 3-O-methylglucose exchange in a concentration dependent manner, though not very effectively (IC50 approximately equal to 3 mM). Both uridine and 3-O-methylglucose exchange were inhibited in a concentration dependent manner by cytochalasin B, phloretin and dipyridamole, but cytochalasin B and phloretin were 100-times more effective in inhibiting 3-O-methylglucose than uridine exchange, whereas the opposite was the case for the inhibition by dipyridamole.

Inhibition of 3-O-methylglucose transport in human erythrocytes by forskolin.

The effect of forskolin, an activator of adenylate cyclase, was investigated on glucose transport in human erythrocytes. Forskolin was found to be a potent inhibitor of 3-O-methylglucose (3-O-MG) influx in human erythrocytes. The inhibition of 3-O-MG transport was instantaneous and reversible. The inhibitory effect of forskolin was concentration-dependent, having an IC50 value of 7.5 microM. Forskolin caused a decrease in Vmax of carrier-mediated 3-O-MG transport from 35.32 to 1.56 mumol/ml of cell X min in the presence of 50 microM forskolin. Inhibition of influx was not reversed at high concentrations of 3-O-MG. In addition, forskolin inhibited the influx of other carbohydrates including galactose, ribose, and fructose. In contrast, forskolin was without effect on adenosine transport. To unravel the underlying mechanism responsible for the inhibitory action of forskolin, the possible involvement of cyclic AMP in controlling glucose transport was examined. Erythrocytes treated with 50 microM forskolin exhibited an increase in cyclic AMP content from the basal levels of 258 fmol/ml of cell to 334 fmol/ml of cell within 10 s after forskolin exposure. However, erythrocytes in which cyclic AMP was allowed to accumulate in excess of 10,000 times the basal level, by means of preincubation with exogenous cyclic AMP, displayed 3-O-MG transport indistinguishable from that of cyclic AMP-poor control cells. In view of the finding that cyclic AMP plays no discernible role in the erythrocyte 3-O-MG transport, it is suggested that the forskolin inhibition is mediated by a mechanism other than by stimulating adenylate cyclase activity. Moreover, forskolin appears to directly inactivate the 3-O-MG transport system since glucose-sensitive cytochalasin B binding to erythrocyte membranes is virtually abolished by 50 microM forskolin.

Characterization of sugar transport in the pigeon red blood cell.

Sugar transport in pigeon red blood cells is mediated by two pathways. One is saturable, shows competition between sugars, is inhibited by phloretin and cytochalasin B, and shows many of the properties of 'carrier-mediated' transport, characterized in the human red blood cell. The other is not saturable, and shows no competition between sugars. The saturable pathway is virtually absent from freshly drawn cells, but may be stimulated by pre-incubation with 2 mM-NaCN, or by Ca and the ionophore A 23187. The non-saturating pathway is stimulated only slightly by CN, but considerably by Ca and A 23187. The inhibition of sugar transport by cytochalasin B is antagonized competitively by sugars acting at the inner surface of the membrane. External sugars have no effect, as in the human red blood cell (Widdas, 1980). The binding of cytochalasin B to the cells shows a limited number of high-affinity sites. These are unrelated to inhibition of sugar transport as binding, but not transport, is prevented by the presence of cytochalasin E.

The role of calcium in the regulation of sugar transport in the pigeon red blood cell.

The saturable, 'carrier-mediated' pathway for sugar transport in pigeon red blood cells may be stimulated by metabolic depletion or by loading the cells with Ca by means of a high-voltage discharge or the ionophore A 23187. All of these methods for stimulating the saturable pathway of sugar transport also cause a drop in cellular ATP levels. The relationship between the stimulation of transport and the fall in ATP is very similar in metabolically depleted or Ca-loaded cells, but cells treated with Ca and A23187 show a greater stimulation of transport than would be expected from the decline in ATP. Altering free Ca2+ levels during metabolic depletion has little or no effect on stimulation of the saturable pathway. Conversely, metabolic depletion of fresh cells in Ca-free solutions has no detectable effect on intracellular free Ca2+ levels. These results suggest that Ca2+ ions are not involved in regulation of this pathway. The non-saturable pathway for sugar transport is stimulated by a rise in cell Ca. This process is probably stimulated half-maximally by about 10 microM-free Ca2+.

Kinetic mechanism of chlorpromazine inhibition of erythrocyte 3-O-methylglucose transport.

The kinetic mechanism of chlorpromazine inhibition of erythrocyte hexose transport was investigated using the non-metabolizable glucose analog 3-O-methylglucose. It was found that chlorpromazine added to the external medium is a non-competitive inhibitor of both equilibrium exchange and net 3-O-methylglucose transport at pH 7.8, 15 degrees C. The Ki for equilibrium exchange is 76 +/- 21 microM. When net efflux and equilibrium exchange were measured on the same population of cells the equilibrium exchange was 2.5-times the maximum net efflux. The percent reduction of 3-O-methylglucose flux by chlorpromazine is dependent upon chlorpromazine concentration and not 3-O-methylglucose concentration as expected for a non-competitive inhibitor. Equilibrium exchange and net efflux show the same extent of inhibition at each concentration of chlorpromazine evaluated. These results suggest that exchange and net efflux of 3-O-methylglucose in the human erythrocyte may share a common transport system.

Glucose transport carrier of human erythrocytes. Radiation target size measurement based on flux inactivation.

Intact human erythrocytes frozen in the presence of cryoprotective reagents and irradiated with an electron beam retained their diffusion barrier to L-glucose. The carrier-mediated flux of D-glucose, on the other hand, was inactivated as a simple exponential function of the radiation dose. Classical target size analysis of this data yielded a molecular size of 185,000 daltons for the carrier. This represents the first measurement of the functional size of a transport protein based directly on flux inactivation.

On the influence of pentoxifylline on the permeability of rat erythrocytes for methyl-O-glucose.

In a series of experiments it was established that in the presence of pentoxifylline (Trental), methyl-O-glucose permeates rat erythrocyte membrane significantly more than in untreated control experiments. Pentoxifylline itself, apparently does not permeate erythrocyte membrane. It is possible that some of the therapeutic effects of pentoxifylline can be related to the facilitation of red blood cell membrane permeability for glucose.

Sugar transport in reversibly hemolyzed avian erythrocytes.

The technique of reversible hemolysis represents one approach which may be used to study transport regulation in nucleated red cells. After 1 h of incubation at 37 degrees C, 88% of the ghosts regained their permeability barrier to L-glucose. In these ghosts, the carrier-mediated rate of entry of 3-O-methylglucose was more than 10-fold greater than the rate in intact cells. Glyceraldehyde-3-phosphate dehydrogenase prevented ghosts from resealing when it was present at the time of hemolysis. Albumin, lactic dehydrogenase and peroxidase did not have this effect. Sugar transport rate could not be tested in the unsealed ghosts. Two possible mechanisms for the effect of hypotonic hemolysis on sugar transport rate were discussed: (1) altered membrane organization and (2) loss of intracellular compounds which bind to the membrane and inhibit transport in intact cells.

Isolation of membrane glycoproteins by affinity chromatography in the presence of detergents.

Wheat germ agglutinin has been used in a one-step preparative method to isolate the major sialoglycoprotein (glycophorin A) from the human erythrocyte membrane. The conditions for isolation and purification of the sialoglycopeptide included low concentration of sodium dodecyl sulfate in the presence of relatively high salt concentration. This medium caused complete solubilization of the membrane but still allowed almost quantitative binding of the sialoglycopeptide to wheat germ agglutinin-Sepharose. The eluted protein from such affinity systems was found to be chemically comparable to glycophorin A, as prepared by other procedures.

The interaction of 3-deoxy-3-fluoro-D-glucose with the hexose-transport system of the human erythrocyte.

1. By using an optical method the kinetic parameters of hexose transport across the human erythrocyte membrane were determined for several sugars. The series of half-saturated constants is as follows: 3-deoxy-3-fluoro-d-glucose = 3-O-methyl-d-glucose