Division of Gastroenterology, Hepatology, and Nutrition, Department of Pediatrics, University of Arkansas for Medical Sciences and the Arkansas Children's Hospital Research Institute, Little Rock, Arkansas 72202
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ABSTRACT |
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We sought to
determine whether the proliferation of immune cells affects the
cellular uptake of the vitamin biotin. Peripheral blood mononuclear
cells (PBMC) were isolated from healthy adults. The proliferation
of PBMC was induced by either pokeweed lectin, concanavalin A, or
phytohemagglutinin. When the medium contained a physiological
concentration of
[3H]biotin,
nonproliferating PBMC accumulated 406 ± 201 amol
[3H]biotin · 106
cells1 · 30 min
1. For proliferating
PBMC, [3H]biotin
uptake increased to between 330 and 722% of nonproliferating values.
Maximal transport rates of
[3H]biotin in
proliferating PBMC were also about four times greater than those in
nonproliferating PBMC, suggesting that proliferation was associated
with an increase in the number of biotin transporters on the PBMC
membrane. The biotin affinities and specificities of the transporter
for proliferating and nonproliferating PBMC were similar, providing
evidence that the same transporter mediates biotin uptake in both
states. [14C]urea
uptake values for proliferating and nonproliferating PBMC were similar,
suggesting that the increased
[3H]biotin uptake was
not caused by a global upregulation of transporters during
proliferation. We conclude that PBMC proliferation increases the
cellular accumulation of biotin.
concanavalin A; lipoic acid; phytohemagglutinin; pokeweed; transport kinetics
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INTRODUCTION |
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DEFICIENCY OF THE VITAMIN biotin adversely affects the proliferation and metabolism of cells of the immune system. In biotin-deficient rodents, the activities of biotin-dependent carboxylases in spleen lymphocytes are decreased (3); the numbers of spleen cells and circulating B and T lymphocytes are reduced (3, 19); and antibody synthesis is reduced (12, 22). Moreover, incubation of cells from mouse spleen in biotin-deficient media reduces cytotoxic T lymphocyte generation (13). Perhaps as a result of the deficiency of biotin-dependent carboxylases, hereditary abnormalities of biotin metabolism lead to reduced function of humoral and cellular immunity in humans and a tendency to cutaneous fungal infections (6).
For several micronutrients, the normal host response of the immune system, which includes immunocyte proliferation, leads to an increased demand in one or more cell lines and theoretically could lead to a relative state of nutrient deficiency. For example, Wang et al. (29) demonstrated that the accumulation of ascorbic acid in human neutrophils is increased if neutrophils are incubated in the presence of bacteria. These authors proposed that the accumulation of ascorbic acid might be a mechanism to provide neutrophils with self protection against the intracellular reactive oxygen species needed to destroy bacteria. In the present study, we investigated whether the proliferation of peripheral blood mononuclear cells (PBMC) causes an increase in the rate of biotin uptake.
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MATERIALS AND METHODS |
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Chemicals.
[3H]biotin (sp act
2.15 TBq/mmol) was purchased from Dupont (Boston, MA).
[3H]thymidine (sp act
1.29 TBq/mmol) was purchased from ICN Pharmaceuticals (Costa Mesa, CA).
[14C]urea (sp act 0.36 GBq/mmol) was purchased from Sigma (St. Louis, MO). Biotin-free RPMI
1640 containing 25 mM HEPES was prepared by Atlanta Biologicals
(Norcross, GA); the pH was adjusted to 7.4 with
NaHCO3. This medium was filtered
sterilely with a 0.22-µm filter (Millex-GV; Millipore, Bedford, MA).
Concanavalin A, pokeweed lectin (from
Phytolacca
americana), phytohemagglutinin
PHA-P, antibiotics (10,000 IU/ml penicillin plus 10,000 µg/ml
streptomycin), DL--lipoic
acid, and Dulbecco's PBS without calcium chloride and
magnesium chloride were purchased from Sigma.
Isolation of PBMC and induction of proliferation. PBMC were isolated aseptically from human peripheral blood by gradient centrifugation and washed two times with PBS containing 100 IU/ml penicillin and 100 µg/ml streptomycin (31). The PBMC pellet was suspended in medium containing biotin-free RPMI 1640, autologous plasma, and penicillin-streptomycin stock solution (90:10:1 by volume). The cell number per milliliter of medium was determined with a hemocytometer, and the cell number was adjusted to 4 × 106 PBMC/ml by dilution with medium. Then, either the T cell mitogen concanavalin A (final concentrations at 5, 10, or 20 µg/ml), the T cell mitogen phytohemagglutinin (5, 10, or 20 µg/ml), or the T and B cell mitogen pokeweed mitogen (0.5, 1.0, or 2.0 µg/ml) was added to the PBMC suspension. After the addition of mitogens, PBMC suspensions were incubated at 37°C for 3 days in an atmosphere of 95% O2-5% CO2 to induce proliferation; nonproliferating controls were incubated without mitogen. Tissue culture flasks and 96-well plates were used for the incubations. Cell viabilities were assessed by the exclusion of 2.1 mM trypan blue, which was determined by light microscopy at ×160. Five counts of ~60 cells per count were performed for each determination. After incubation for 3 days, cell numbers per milliliter of suspension and cell viability were determined again; the uptake rates of [3H]thymidine, [3H]biotin, and [14C]urea were normalized to 106 viable cells.
In one set of experiments, we sought to determine the time course of stimulation of biotin uptake into proliferating PBMC. Also, we sought to determine whether the inhibition of protein synthesis by cycloheximide affects the stimulation of biotin transport into mitogen-induced PBMC. In these experiments, we added either pokeweed mitogen (final concentration 2.0 µg/ml) or pokeweed mitogen plus cycloheximide (final concentration 17.8 µM) to a suspension of PBMC (4 × 106 PBMC/ml). Then PBMC were incubated for up to 168 h; [3H]biotin and [3H]thymidine uptake into these cells was measured before the addition of pokeweed mitogen and cycloheximide (0 h) and 24, 48, 72, 96, and 168 h after addition. Cell viabilities and cell numbers per milliliter of suspension were determined periodically as described above.Uptake of thymidine, biotin, and urea into PBMC. Unless noted otherwise, the uptake of [3H]thymidine, [3H]biotin, and [14C]urea into PBMC was measured after mitogen induction for 3 days; controls without mitogens were also incubated for 3 days before uptake studies. [3H]thymidine uptake into PBMC was measured in triplicate in 0.2-ml aliquots with a Matrix 9600 direct beta counter (Packard Instrument, Meriden, CT) as described by Coligan et al. (4).
[3H]biotin uptake into PBMC was measured at least in triplicate in 1-ml aliquots as described by us previously (31). In previous studies, we demonstrated that PBMC do not catabolize biotin during incubations lasting up to 168 h; hence the presence of 3H in PBMC reflects [3H]biotin uptake rather than uptake plus metabolism. The [3H]biotin concentration (475 pM) that was used in most experiments of the present study was similar to the free biotin concentration in normal plasma (15). In experiments to determine Michaelis-Menten constants and maximal transport rates, the concentration of [3H]biotin in the medium was varied from 238 to 2,850 pM. In all experiments, some unlabeled biotin was present in the medium because of the addition of autologous plasma. This endogenous unlabeled biotin accounted for only 1.5-15% of the total biotin, a conclusion based on the following reasoning. For each experiment, the biotin concentration of autologous plasma was measured by a combination of HPLC and the avidin-binding assay (14). The concentration of unlabeled biotin present in the medium because of the addition of 10% autologous plasma averaged 44 ± 25 pM. Where appropriate, calculations using the specific radioactivity of [3H]biotin in the medium were adjusted for unlabeled biotin. To determine the substrate specificity of the biotin transporter in PBMC, we measured the cellular uptake of [3H]biotin in the presence of lipoic acid at concentrations similar to that of normal plasma (50 nM) and at pharmacological concentrations (5,000 nM) (26, 27). To account for the poor water solubility of lipoic acid, we verified the concentration of lipoic acid in the stock solution by measuring the absorbance at 330 nm, with molar extinction coefficient (Statistics. The significance of differences among groups (e.g., among the three concentrations of a mitogen and the control or among the three different mitogens and the control) was tested by one-way ANOVA. Values were logarithmically transformed before ANOVA to adjust for heterogeneous variances identified by Bartlett's test (2). Dunnett's post hoc procedure was used for post hoc testing; Dunnett's procedure compares the means measured for the treatment groups to the control mean, i.e., that for unstimulated PBMC (1). Paired comparisons were made by using the Wilcoxon signed-rank test. SuperANOVA 1.11 and StatView 4.5 (Abacus Concepts, Berkeley, CA) were used to perform all calculations. Differences were considered significant at P < 0.05.
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RESULTS |
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Biotin uptake.
[3H]biotin uptake into
proliferating PBMC was significantly greater than that into
nonproliferating controls. When the medium contained 475 pM
[3H]biotin (a
physiological concentration), nonproliferating controls accumulated 406 ± 201 amol
[3H]biotin · 106
cells1 · 30 min
1. If proliferation was
induced by pokeweed mitogen (lectin from P.
americana), biotin uptake was
increased with increasing concentration of pokeweed mitogen to between
481 and 722% of the control value (Fig. 1,
left). If proliferation
was induced by concanavalin A, biotin uptake was increased with
increasing concentration of concanavalin A to between 346 and 672% of
the control value (Fig. 1, middle). If proliferation was induced by phytohemagglutinin, biotin uptake was
between 330 and 456% of the control value (Fig. 1,
right). Thus the percent increases
of [3H]biotin uptake
were similar for all three mitogens. However, the mitogens were not
used at equimolar concentrations.
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Transport kinetics.
We investigated the mechanism mediating the observed increase in
transport. We hypothesized that the increased uptake was caused either
by an increased density of biotin transporters on the cell surface or
by an increased affinity of biotin transporters for biotin (or both).
PBMC proliferation was induced by 2.0 µg/ml pokeweed mitogen
(controls lacked pokeweed mitogen), and biotin uptake was measured at
biotin concentrations in the medium varying from 238 to 2,850 pM.
Lineweaver-Burk plots of these data revealed an intersection near the
x-axis for the regression lines of
proliferating and nonproliferating PBMC (Fig.
2). This finding is consistent with the
hypothesis that the transporter density on the cell surfaces of
proliferating PBMC was greater than that for controls and that the
affinities for biotin in proliferating PBMC and in control PBMC were
similar. The maximal transport rates
(Vmax) of the
biotin transporter in proliferating PBMC and in controls were 9,126 ± 6,641 and 2,328 ± 1,627 amol · 106
cells1 · 30 min
1, respectively
(P < 0.05 by Wilcoxon signed-rank
test). The Michaelis-Menten constants for the biotin transporter in
proliferating PBMC and in controls were 2.4 ± 1.7 and 3.7 ± 3.4 nM, respectively (P = 0.86, not
significant). These findings suggest that increased biotin uptake was
mediated by an increased density of biotin transporters on the cell
surface rather than by an increased affinity of biotin transporters for
biotin.
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Transporter specificity. Theoretically, it seemed possible that increased biotin uptake was achieved by increased synthesis of a transporter other than the specific biotin transporter described by us in nonproliferating PBMC (31). Other biotin transporters exist. For example, certain mammalian cells use a less specific biotin-lipoic acid-pantothenic acid transporter to accumulate biotin (21). Potentially, proliferating PBMC might synthesize this "multivitamin" transporter at an increased rate rather than the specific biotin transporter.
To investigate this possibility, we examined the effect of lipoic acid on biotin transport. Extracellular lipoic acid did not significantly reduce [3H]biotin uptake into nonproliferating PBMC (P = 0.73; Fig. 4), confirming results from our previous studies (31). Likewise, in proliferating PBMC, lipoic acid did not inhibit biotin uptake. [3H]biotin uptake values in the presence of physiological (50 nM) and pharmacological (5,000 nM) concentrations of lipoic acid were 113 ± 47 and 89 ± 11%, respectively, of the uptake in lipoic acid-free medium (P = 0.15). These data provide evidence that the transporter in both proliferating and nonproliferating PBMC is the structurally specific biotin transporter.
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Thymidine uptake.
Uptake of the DNA constituent
[3H]thymidine is a
well-established marker of cell proliferation (25). In the present
study, we measured
[3H]thymidine uptake
into PBMC to confirm that mitogen-induced cells were indeed
proliferating. In every experiment, the uptake of [3H]thymidine was
significantly greater in mitogen-induced PBMC than in controls. As a
typical example, Fig. 5 provides the
[3H]thymidine uptake
data from experiments in which proliferation was induced by either 2.0 µg/ml pokeweed mitogen, 20 µg/ml concanavalin A, or 20 µg/ml
phytohemagglutinin.
[3H]thymidine uptake
was at least 20 times greater than control values for each mitogen
(P < 0.01 for each).
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Urea uptake. To determine whether the effect of PBMC proliferation is relatively specific to biotin uptake, [14C]urea uptake into PBMC was also measured. Urea was chosen because of its structural similarity to biotin (ureido group in the heterocyclic ring of biotin). Despite the structural similarity of urea and biotin, these compounds likely enter cells via different transporters (16, 28, 31). In ureotelic organisms such as humans, urea is a terminal product of intermediary metabolism (8); hence the quantitation of cellular uptake is not confounded by urea catabolism.
When PBMC proliferation was induced by either 2.0 µg/ml pokeweed mitogen, 20 µg/ml concanavalin A, or 20 µg/ml phytohemagglutinin, [14C]urea uptake was 86 ± 33, 119 ± 21, or 98 ± 30% of control values, respectively (P = 0.59, 0.44, or 0.93, respectively; all not significant). Values were similar when proliferation was induced by pokeweed mitogen at concentrations of 0.5 or 1.0 µg/ml or by concanavalin A or phytohemagglutinin at concentrations of 5 or 10 µg/ml (data not shown). Together, these data provide evidence that the increase of transport rates for biotin in proliferating PBMC is not simply part of a global, nonspecific increase in transport. ![]() |
DISCUSSION |
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This study provides evidence that biotin uptake into proliferating PBMC increases about three- to sevenfold compared with uptake into nonproliferating controls. This effect is probably not specific to a particular mitogen because biotin transport was increased by each of three mitogens: pokeweed mitogen, concanavalin A, and phytohemagglutinin. Maximal activation of biotin transport was achieved 48-72 h after the addition of mitogens to the medium.
Our data provide evidence that Vmax increased about fourfold in proliferating PBMC, consistent with an increased number of transporters per cell. Our observation that cycloheximide inhibited the mitogen-induced increase of biotin transport is consistent with the hypothesis that proliferating PBMC increase biotin transport by synthesizing new transporters. Theoretically, PBMC might also increase biotin uptake by increasing the affinity of the transporter for biotin (e.g., by phosphorylation or dephosphorylation of the transporter). However, the Michaelis-Menten constants of the transporter for biotin in nonproliferating and proliferating PBMC were similar, suggesting that biotin affinity is not altered in proliferating PBMC.
Theoretically, the observed increase of biotin uptake into proliferating PBMC might also be caused by the activation or increased transcription of a different transporter such as the biotin-lipoic acid-pantothenic acid transporter of rat placenta (20). However, two of our findings suggest that this is not the case. First, the Michaelis-Menten constants of biotin transport for proliferating and nonproliferating PBMC were similar, suggesting that the same transporter accounts for biotin uptake in both cells. Second, lipoic acid did not inhibit biotin transport into either proliferating or nonproliferating PBMC, suggesting similar substrate specificities in both cells. Lipoic acid is a competitive inhibitor of biotin transport into various other cell types (21, 23). Together, these observations provide evidence that proliferating PBMC increase the transport of biotin by increasing the density of the structurally specific biotin transporter on the cell surface.
Proliferating PBMC probably increase biotin uptake specifically rather than as part of a global activation of transport. Consistent with this hypothesis is the observation that the levels of uptake of urea in proliferating and nonproliferating PBMC were similar. Teleologically, one might propose that proliferating PBMC specifically upregulate the transport of those substances that are needed for proliferation, e.g., biotin and thymidine. If so, what might be generating the demand for biotin? First, cell proliferation requires the synthesis of fatty acids for cell membranes. Biotin is a coenzyme for acetyl-CoA carboxylase (E.C. 6.4.1.2), which is the key enzyme for fatty acid synthesis (8). Hence the increased rate of fatty acid synthesis during proliferation might increase the cellular requirement for acetyl-CoA carboxylase and for its coenzyme, biotin.
Second, proliferating PBMC might require increased biotin for the biotinylation of histones. Hymes and co-workers (10, 11) have shown that biotinidase (E.C. 3.5.1.12) has biotinyl transferase activity, with histones acting as specific biotinyl acceptors. The physiological role of these biotinylated histones is uncertain. Some clues come from the observation that histones dissociate from DNA in biotin-deficient rats, suggesting that biotinylated histones may play a role in the packaging of DNA (18). Biotin deficiency is also associated with decreased phosphorylation and methylation of histones and increased acetylation of histones (17). Moreover, even if proliferation does not alter the molar ratio of biotin to histones, an increased mass of histones in proliferating cells might account for increased biotin requirements during proliferation. In support of this possibility is the observation that the mass of histone H2B in cell membranes increased from 6.2 to 31.9 µg/mg membrane protein (from 0.9 to 6.7 µg/108 cells) when PBMC proliferation was induced by phytohemagglutinin (30).
Third, as a mechanism of host defense, proliferating PBMC might
accumulate biotin to sequester plasma biotin from invading microorganisms. By analogy, an antimicrobial effect of biotin withdrawal has been proposed to operate in eggs. Egg white contains the
protein avidin (0.05% of total protein), which binds biotin tightly;
the dissociation constant of the biotin-avidin complex is
1015 M (9). The tight
binding of biotin to avidin prevents the diffusion of biotin to the
microbes, and avidin is resistant to a variety of proteases (thus
preventing microbes from releasing biotin from avidin). An additional
analogy comes from a hypothesis offered to explain the dramatic
decrease in the concentration of zinc in serum associated with stress
(7). This has been proposed to represent an attempt by the body to
withhold zinc as a nutrient during the stress of sepsis (5).
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ACKNOWLEDGEMENTS |
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We thank Melain Raguseo for technical assistance with HPLC and the avidin-binding assay.
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FOOTNOTES |
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-36823.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: D. M. Mock, Arkansas Children's Hospital, Dept. of Pediatrics (GI), 800 Marshall St., Little Rock, AR 72202-3591 (E-mail: mockdonaldm{at}exchange.uams.edu).
Received 30 November 1998; accepted in final form 29 January 1999.
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