Transport and metabolism of pyridoxine in rabbit iris-ciliary body

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1 Transport and metabolism of pyridoxine in rabbit iris-ciliary body Jon J. Dutton, Theodore Krupin, Carol Fritz, and Bernard Becker Isolated rabbit iris -ciliary body preparations were found to accumulate 3 H-pyridoxine by a mechanism that was time- and temperature-dependent, saturable in part, but not altered by omission of oxygen or specific ions. Tissue accumulation toas only partially energy-dependent, and metabolic inhibitors had only small effects. Other B 6 vitamers markedly blocked accumulation. Metabolism of 3 H-pyridoxine within the tissue was extensive. After 60 min incubation with a H-pyridoxine, about 65% of the radioactivity in the iris-ciliary body was associated with phosphorylated vitamers which did not efflux from the tissue as readily as did the nonphosphorylated forms. Unaltered pyridoxine accounted for only 16% of intracellular 3 H-B 6, and this did not represent accumulation against a concentration gradient. A similar saturable uptake process occurred in vivo. These data were consistent with the hypothesis that pyridoxine was accumulated by facilitated diffusion with intracellular trapping of phosphorylated metabolites. Key words: vitamin B 6, pyridoxine, rabbit, facilitated transport, metabolism, iris-ciliary body, red blood cell volume itamin B 6 is a required co-factor for a large number of enzymatic reactions. ls 2 Most animal tissues are capable of converting the various forms of the vitamin into active vitamers, and such activity is especially high in liver, spleen, kidney, and brain. 1 This vitamin cannot be synthesized de novo by mammalian tissues and must be taken up from the blood after ingestion in the diet. 3 In animals vitamin B 6 exists in at least six forms: From the Department of Ophthalmology and the Oscar Johnson Institute, Washington University School of Medicine, St. Louis, Mo. Supported in part by Research grant EY and a Research Career Development Award EY (Dr. Krupin) from the National Eye Institute, Bethesda, Md. Dr. Dutton received a Postdoctoral Research Fellowship ( ) from Fight For Sight, Inc., New York City. Submitted for publication Oct. 10, Reprint requests: Theodore Krupin, M.D., Department of Ophthalmology, Washington University School of Medicine, 660 South Euclid Ave., St. Louis, Mo pyridoxine (PN), the alcohol; pyridoxal (PL), the aldehyde; pyridoxamine (PM), the amine; and their respective 5'-phosphorylated vitamers. 4 The three nonphosphorylated vitamers are collectively represented as Pyr, the three phosphorylated forms as Pyr-P, and all six vitamers as B 6. PN is the major form of the vitamin in many plants such as grains, and after consumption by animals, it is rapidly metabolized to more active compounds. Pyridoxal phosphate (PL-P) and pyridoxamine phosphate (PM-P) are the principal active metabolites formed from PN within animal tissues. Although PL-P is subsequently released by the liver and constitutes more than 25% of the plasma B 6, its dephosphorylated analogue PL appears to be the major transport form into cells. 5 ' 6 The brain and choroid plexus contain levels of B 6 25 to 50 times higher than does plasma, but cerebrospinal fluid contains levels comparable to those in plasma. 7> 3 The choroid plexus appears to serve a major role in regulating B 6 entry into the cerebrospinal /81/ $00.70/ Assoc. for Res. in Vis. and Ophthal., Inc.

2 Volume 20 Number 4 Pyridoxine metabolism 451 < or 7 IEDIUN UJ O CO y TIME (minutes) Fig. 1. Time course for uptake of 3 H-PN (100 nm) by isolated rabbit iris-ciliary body at 37 C. Values are means ± S.E.M. for eight experiments at each time. fluid across the blood-brain barrier and in maintaining its homeostasis in the central nervous system. 9 Our preliminary studies showed that vitamin B 6 is present in the rabbit aqueous humor at levels close to that of the plasma, but that the iris-ciliary body contains at least 20 to 25 times that concentration. Because of this and the known anatomic and physiologic similarities between the choroid plexus and ciliary body, we undertook to determine whether the rabbit iris-ciliary body preparation is capable of accumulating and metabolizing vitamin B 6 in similar fashion. Materials and methods In vitro uptake studies. Male albino rabbits, weighing 1.5 to 2 kg, were maintained on a normal diet of Purina Laboratory Rabbit Chow (4.5 mg/kg PN) prior to the experiments. Rabbits were sacrificed by intracardiac air embolism. Eyes were promptly enucleated and opened posteriorly, the lens and vitreous were carefully dissected free, and the iris-ciliary body ring was removed in one piece. This tissue was incubated in 2 ml of Tyrode's solution adjusted to ph 7.4 by bubbling with 95% O 2 and 5% CO 2. Labeled (G) 3 H-PN (sp. act Ci/mmol; Amersham Corp.) was added to afinalconcentration of 100 nm. Nonradioactive materials were also added to some samples at various concentrations. For specific ion-free media, choline chloride was substituted for sodium chloride in sodium-free Tyrode's, sodium chloride was substituted for calcium chloride in calcium-free Tyrode's, and potassium chloride was omitted without substitution in potassium-free Tyrode's. The contralateral ciliary body served as the control for the inhibitor studies. All incubations were carried out in subdued light at 37 C for 60 min under air except as noted. After incubation, tissues were blotted dry between pieces of filter paper and weighed. Tissues were hydrolyzed in 0.5 ml of IN sodium hydroxide at 65 C for 30 min and neutralized with 0.5 ml of IN hydrochloric acid. Samples (100 /i,l) of tissue hydrolysate and media, taken both before and after incubation, were placed in 5 ml of Biofluor scintillation fluid (New England Nuclear) and counted on a Packard TriCarb Model 3375 liquid scintillation spectrometer with external standard correction for quenching. Tissue-to-medium (T/M) ratios were calculated as the ratio of counts per milligram of

3 452 Dutton et al. Invest. Ophthalmol. Vis. Sci. April 1981 < rr < A (3) o LLJ UJ ID CO CO ( (4) z 2 (3) 2 S (4) " ,-5 10" Concentration of PN (M) Fig. 2. Inhibition of 3 H-PN (100 nm) uptake by increasing concentrations of nonlabeled PN added to the incubation medium. Values are means ± S.E.M. for eight experiments at each concentration. tissue water to counts per microliter of medium. Tissue water was found to be 85% by weighing the tissue before and after drying, and all values were corrected to a tissue water basis. The contribution of nonspecific binding to total apparent uptake of radioactivity was determined on tissue specimens which were homogenized and sonicated in an Artek Sonic Dismembrator. The homogenate was sealed into 1 cm diameter Spectropore dialysis tubing with 1 ml oftyrodes solution. The tubing was incubated in medium containing :i H-PN at 37 C for 6 hr. The entire homogenate sample, as well as a 100 fx portion of medium, was counted for radioactivity. Thin-layer chromatography was performed on iris-ciliary body preparations after 60 min incubation with :i H-PN. The tissue was homogenized and deproteinized in cold 0.12M metaphosphoric acid in order to quantitatively maintain the phosphorylated B 6 vitamers. 9 The homogenate was centrifuged with the supernatant, and appropriate standards were streaked onto precoated Eastman silica gel chromogram sheets (No ) under subdued red light. Plates were developed in the dark in a solvent of n-amyl alcohol: acetone: water: diethyl amine (40:35:20:5) for 3 hr. 10 Standards were located under ultraviolet absorption at 265 nni, and corresponding spots were cut from each column. Spots were placed in 10 ml of scintillation fluid and counted for radioactivity. Chromatographic analyses were also performed on incubation media and homogenate samples from the tissue-binding experiments described above. Efflux studies were performed on iris-ciliary body preparations after prior incubation for 1 hr with 3 H-PN. Tissues were gently blotted between pieces of filter paper and transferred serially to four vials of fresh Tyrode s solution for 15 min each under red light. The tissue was weighed and hyrolyzed, and 100 /x samples of tissue hy-

4 Volume 20 Number 4 Pyridoxine metabolism 453 Table I. Thin-layer chromatography on iris-ciliary body extracts after incubation at 37 C with 3 H-PN (100 /xm) Percent total radioactivity as Incubation time (min) T/M ratio 3 H-Pyr 3 H-Pyr-P 3 H-PN 3 H-PM-P ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 6.7 Values are mean ± S.E.M., with 6 to 8 tissue preparations per experiment. drolysate, incubation medium, and each efflux medium were counted. Thin-layer chromatography was also performed on the efflux media. In vivo uptake studies. Six rabbits were injected intravenously with :1 H-PN, 100 fxci (80 nmol) in 1 ml of isotonic saline, through a marginal ear vein. In six additional animals, nonlabeled PN, 0.21 mg (1 /xmol), was added to the injection solution. Animals were sacrificed after 2.5 hr. Aliquots (100 /xl) of plasma and aqueous humor, and the entire ciliary body, were counted for residual radioactivity. The contribution of red blood cells to the irisciliary body volume and therefore to PN metabolism and uptake was determined by a modified method of Glass. n 5l Cr-sodium chromate (sp. act. 10 mci/mmol; New England Nuclear), 50 /xci in 0.5 ml of isotonic saline, was slowly added to lightly packed red blood cells from 10 ml of whole blood and incubated at 25 C for 15 min. The red cells were washed in isotonic saline, resuspended in 2 ml of saline, and replaced into the same animals through a marginal ear vein. The animals were sacrificed after 20 min, and radioactivity was determined in whole blood and the iris-ciliary body. The counts in the blood were corrected for hematocrit, and the data were used to calculate the volume of red blood cells in each iris-ciliary body. Endogenous PL levels in blood, aqueous humor, and ciliary body-iris extracts were determined by Bio-Science Laboratories, St. Louis, utilizing the enzymatic assay of Rose. 12 Results Ciliary body-iris preparations incubated with 100 /u-m 3 H-PN were found to accumulate radioactivity as a function of time as shown in Fig. 1. The addition of unlabeled PN at increasing concentrations from 1 /xm to 10 mm decreased the apparent T/M ratio to a low of 1.8 at 1 mm, after which no further saturation of uptake could be achieved (Fig. 2). Assuming a Michaelis-Menten model for accumulation of 3 H-B 6, subtracting the nonsaturable component from the mean T/M ratios resulted in a linear plot with an apparent Km of 2.7 (xm and a Vmax of 22.5 /xmol/mg of tissue per 60 min for uptake at ph 7.4 and 37 C. These values are estimates of total tritium accumulation only. They represent a complex interaction between unaltered PN and its intracellular metabolites, some of which diffuse across cell membranes. Addition to the incubation media of other nonlabeled B 6 vitamers depressed ciliary body-iris accumulation of 3 H-B 6. Concentrations of 10 /xm PL, PN, or PM were sufficient to decrease the T/M ratio by 70% to 75% compared to the ratio from the contralateral control preparations. The phosphorylated vitamers PL-P and PM-P gave similar results at equivalent concentrations. The PN antimetabolite, 4'-deoxypyridoxine, inhibited uptake by 60% at 10 /xm. Addition of 10 /xm 4-pyridoxic acid, the principal excretory form of the vitamin, on the other hand, resulted in a nearly 40% increased accumulation of 3 H-B 6, but no effect was seen at 100 /xm concentration. The rate of accumulation of 3 H-PN was sensitive to decreased temperature, with 60% reduction in 60 min accumulation at 25 C and 87% at 1 C. The omission of oxygen, sodium, or calcium from the incubation medium had little effect on accumulation. Glucose-free media depressed uptake by only about 30%. At 100 /xm concentration para-aminohippurate, prostaglandin E 2, pro-

5 454 Dutton et al. Invest. Ophthalmol. Vis. Sci. April 1981 Table II. In vivo accumulation of 3 H-PN (80 nmol) by rabbit iris-ciliary body and aqueous humor after intravenous injection Aqueous humor Irisciliary body Intravenous solution 3 H-PN (80 nmol) 0.76 ± ± H-PN (80 nmol) + PN ( ± ± 0.08 Six animals were studied with and without the addition of nonlabeled PN (1 /iinol). Values are expressed as aqueous humor- or tissue-to-plasma ratios for total radioactivity, mean ± S.E.M. benecid, or ouabain failed to alter the accumulation of 3 H-PN. The metabolic inhibitors iodoacetate, dinitrophenol, and cyanide each decreased accumulation by approximately 25% to 35% at 100 /AM. Uptake studies using homogenized and dismembrated tissues separated from 3 H-PN by a dialysis membrane gave T/M ratios of slightly less than 1. Thin-layer chromatography demonstrated metabolism of PN by homogenated tissue; however, binding of the metabolites did not occur. These findings suggested lack of nonspecific tissue binding by PN and its metabolites. Thin-layer chromatography of media containing 3 H-PN found nearly 95% of the radioactivity as nonphosphorylated B 6, of which 80% migrated at the PN spot. There was no difference in medium composition before and after incubation with tissue. However, after 15 min of incubation with exogenous 3 H-PN at 100 fim, half of the radioactivity within the tissue preparation was present as phosphorylated vitamers ( 3 H-Pyr-P) (Table I). Nearly two thirds of the tissue label was associated with these phosphorylated vitamers after 30 min of incubation. This proportion was unchanged at 60 min. The T/M tritium ratio was 8.5 after 60 min, but only 16% of the intracellular tritium was present as PN. Correction of the T/M ratio for PN alone with the use of uptake and thin-layer chromatography data resulted in a value of 1.4. In contrast, the T/M ratio for 3 H-Pyr-P was 5.6. The distribution of 3 H-label within the tissue showed that there were equal amounts of PM-P and PL-P at 15 min, with increasing proportions of label being found as PL-P at 30 and 60 min (Table I). Efflux studies demonstrated significant tissue release of accumulated 3 H-B 6. Of the iris-ciliary body accumulated 3 H, 43% was effluxed after 60 min into the fresh medium. Nearly 75% of this label was in the form of unphosphorylated B 6 vitamers. Experiments in which red blood cells were labeled with 51 Cr-sodium chromate showed that red blood cells made up 3.5% ± 0.4 of the iris-ciliary body preparation. This was higher than the 0.5% reported for human ciliary processes alone by Wistrand and Garg. 13 Endogenous PL-P was 14 ng/ml in the serum and 11 ng/ml in the aqueous humor. The iris-ciliary body had a level of ng/gm. By 2.5 hr after the intravenous injection of 3 H-PN (80 nmol), radioactivity in the irisciliary body was 3.4 times greater than that in plasma (Table II). Radioactivity in the aqueous humor was 0.76 of plasma levels. The addition of nonlabeled PN to the intravenous injection caused a significant (paired t test, p < 0.001) decrease in accumulation by the iris-ciliary body and in the aqueous humor. Discussion The rabbit iris-ciliary body in vitro and in vivo accumulates 3 H-PN against a concentration gradient. In vitro accumulation is timeand temperature-dependent, is partially saturable, and is inhibited by both nonphosphorylated and phosphorylated forms of the vitamin. Uptake is not dependent on oxygen and is only partially dependent on energy. Nonspecific tissue binding does not occur. Red blood cells undoubtedly participate in some of the apparent uptake. Rabbit red blood cells accumulate B 6 with T/M ratios of 8.7 at 15 min and 12.6 at 30 min. 7 Human red cells have more than 90% uptake of PN in 60 min. 5 However, our results show that red blood cells are only 3.5% of the iris-ciliary body tissue. This percentage could account

6 Volume 20 Number 4 Pyridoxine metabolism 455 for not more than 5% to 10% of the total observed uptake. Iris-ciliary body accumulation of unaltered PN is less than the accumulation of the active metabolites. Two thirds of the intracellular tritium after 60 min of incubation is associated with the phosphorylated vitamers PL-P and PM-P. This is similar to the results reported for brain tissue, where 80% to 90% of exogenous PN is metabolized to PL-P and PM-P. 4 In brain tissue either PM-P 14 or PL-P 15 is reported to be the predominant form. In the present study PL-P and PM-P are present in equal amounts in the iris ciliary body after 15 min incubation with exogenous PN. However, by 30 min PL-P predominates over PM-P in a ratio of 1.5:1, and by 60 min it is nearly four times as abundant (Table I). This suggests a slow conversion of PN-P and possibly PM-P into PL-P. Human red blood cells rapidly convert plasma PN to PL-P and PL. On the basis of the measured tissue content of 3.5% red cells, red blood cell metabolism would be expected to convert approximately 18% to 20% of the exogenous PN into PL-P and PL after 60 min. In the present study we find 65% conversion to PL-P and PM-P and another 20% to PM and PL. Therefore it seems likely that the majority of the observed metabolism of 3 H-PN results from enzymatic activity within the iris-ciliary body and not from the red cells. There does not appear to be any significant extracellular metabolism. Our studies on 3 H-PN accumulation by the rabbit iris-ciliary body do not support an active transport process. They are consistent with the hypothesis that 3 H-PN enters the cell by facilitated diffusion with trapping by intracellular phosphorylation. We cannot definitely rule out diffusion with intracellular binding of metabolites to specific receptors within intact cells. Although the homogenated cellular components are capable of metabolizing PN, binding of PN or its metabolites does not occur. This is presumptive evidence against nonspecific binding of PN metabolites. The major enzymatic pathway for vitamin B 6 involves first PL kinase, a widely distributed enzyme capable of converting all three Pyr vitamers to their corresponding 5 -phosphates, followed by oxidation of PN-P by PL-P oxidase. 16 All these vitamers are interconvertible to PM and PM-P. Accumulation of phosphorylated B 6 compounds in iris-ciliary body suggests the presence of PL kinase activity. This enzyme in other tissues is depleted by 4-deoxypyridoxine, requires ATP, and is also inhibited by 2,4-dinitrophenol, 9 which is consistent with our findings in the rabbit iris ciliary body. Efflux studies show that despite the predominance of 3 H-Pyr-P within the cells, 75% of the eluate consists of 3 H-Pyr. This suggests intracellular trapping of the poorly diffusible phosphorylated compounds but not of Pyr. A similar mechanism for B 6 accumulation has been proposed for brain and choroid plexus. 7 " 9 The choroid plexus releases B 6 into efflux medium predominantly as 3 H-Pyr-P. 7 In contrast, the iris-ciliary body effluxes mainly 3 H-Pyr and thus is more like brain tissue 7 and red blood cells. 5 This is an unexpected finding in view of the otherwise close physiologic similarities between choroid plexus and ciliary body. In our in vitro experiments the Pyr-P vitamers inhibit uptake as effectively as do the Pyr forms. Although brain and choroid plexus do accumulate phosphorylated B 6 from the medium, uptake of these vitamers is slower than for the nonphosphorylated vitamers, 9 ' 15 and Pyr-Ps are significantly less effective as inhibitors of B 6 uptake. However, in red cells, non-protein-bound PL-P and PN-P enter cells readily, both directly and after hydrolysis by membrane phosphatase. 6 Intracellular Pyr-P appears to be an effective inhibitor of PL-P oxidase activity which converts PN-P to PL-P. 16 Although similar mechanisms may be operating here, the exact mode of action for inhibition of uptake by phosphorylated vitamers in iris-ciliary body remains unclear. Rabbit cerebrospinal fluid and plasma levels of vitamin B 6 are equivalent, whereas concentrations in the choroid plexus and brain are 25 to 50 times greater. 8 Within these tissues and the cerebrospinal fluid most

7 456 Dutton et al. Invest. Ophthalmol. Via. Sci. April 1981 of the B 6 exists as Pyr-P. Since choroid plexus releases Pyr-P but brain does not, it is concluded that the source of most cerebrospinal fluid B 6 is the choroid plexus. We find that endogenous rabbit aqueous humor levels of B 6, measured as PL-P, are about 0.80 of the plasma level. After intravenous administration of 3 H-PN we find a similar aqueous/ plasma ratio of radioactivity at 2.5 hr (0.73). The PL-P concentration in the iris-ciliary body is at least 25 times higher than that of plasma. Although this vitamer diffuses poorly out of tissue, its high concentration in the iris-ciliary body must serve as a source for some of the B 6 in the aqueous humor, the remainder being derived from plasma. The rabbit ciliary body accumulates 3 H-PN after intravenous administration of the radioisotope. This probably is by a saturable process. Such uptake is clearly far more complex because significant metabolism by liver and red cells as well as other tissues would release PL-P and PL into the plasma. 4 ' 5 These vitamers may then inhibit ciliary body uptake as demonstrated above, but the exact mechanism of inhibition is not clear from our data. Vitamin B 6 is necessary for the normal functioning of the nervous system, where it participates in essential lipid and amino acid metabolism. Deficiency states lead to a number of neurologic disturbances. 17 " 19 Deficiencies have been suggested to cause corneal abnormalities, optic neuropathy, and retrobulbar neuritis. 20 The mechanisms of cellular uptake, metabolism, and storage in iris-ciliary body preparations appear to be similar to those of many other mammalian tissues. Implications as to the functioning of the ciliary body of these in vitro studies and the in vivo accumulation of this essential vitamin remain unknown. REFERENCES 1. McCormick DB, Gregory ME, and Snell EE: Pyridoxal phosphokinases. I. Assay, distribution, purification, and properties. J Biol Chem 236:2076, Colombini CE and McCoy EE: Vitamin B 6 metabolism. The utilization of ( H C) pyridoxine by the normal mouse. Biochemistry 9:533, Thiele VF and Brin M: Availability of vitamin B 6 vitamers fed orally to Long-Evans rats as determined by tissue transaminase activity and vitamin B 6 assay. J Nutr 94:237, Johansson S, Lindstedt S, and Tiselius H-B: Metabolic interconversions of different forms of vitamin B 6. J Biol Chem 249:6040, Anderson BB, Flulford-Jones CE, Child JA, Beard MEJ, and Bateman CJT: Conversion of vitamin B 6 compounds to active forms in the red blood cell. J Clin Invest 50:1901, Lumeng L, Brashear RE, and Li T-K: Pyridoxal 5'- phosphate in plasma: source, protein-binding, and cellular transport. J Lab Clin Med 84:334, Spector R: Vitamin B 6 transport in the central nervous system: in vitro studies. J Neurol 30:889, Spector R: Vitamin B 6 transport in the central nervous system: in vivo studies. J Neurol 30:881, Spector R and Greenwald LL: Transport and metabolism of vitamin Be in rabbit brain and choroid plexus. J Biol Chem 253:2373, Smith MA and Dietrich LS: Preparative thin-layer chromatography for the separation of the various forms of vitamin B 6 in tissues. Vitamin B 6 content of chick embryo liver during the midperiod of development. Biochim Biophys Acta 230:262, Glass HE: Standard techniques for the measurement of red-cell and plasma volume (report by the International Committee for Standardization in Haematology (ICSH), Panel on Diagnostic Applications of Radioisotopes in Haematology). Br J Haematol 25:801, Rose DR: Assessment of tryptophan metabolism and vitamin B 6 nutrition in pregnancy and oral contraceptive users. In Biochemistry of Women: Methods for Clinical Investigation, Curry AS and Hewitt JV, editors. Cleveland, 1974, CRC Press. 13. Wistrand PJ and Garg LC: Evidence of a highactivity C type of carbonic anhydrase in human ciliary processes. INVEST OPHTHALMOL Vis Sci 18: 802, Loo YH and Mack K: Subcellular distribution of B 6 vitamers in cerebral cortex. J Neurochem 18:499, Tiselius H-B: Metabolism of tritium-labelled pyridoxine and pyridoxine 5'-phosphate in the central nervous system. J Neurochem 20:937, Wada H and Snell EE: The enzymatic oxidation of pyridoxine and pyridoxamine phosphates, f Biol Chem 236:2089, Tower DB: Neurochemical aspects of pyridoxine metabolism and function. Am J Clin Nutr 4:329, Coursin DB: Effects of vitamin B 6 on the central nervous activity in childhood. Am J Clin Nutr 4:354, Coursin DB: Vitamin Be and brain function in animals and man. Ann NY Acad Sci 166:7, McLaren DS: Nutritional aspects of the eye. In Biochemistry of the Eye, Graymore CN, editor. New York, 1970, Academic Press, Inc.