Vitamin B, Metabolism by Human Liver‘ ALFRED H. MERRILL, J R . ~AND J. MICHAEL HENDERSONc bDepartment of Biochemistry CDepartmentof Surgery Emory University School of Medicine Atlanta, Georgia 30322
INTRODUCTION The vitamin B, group is composed of three natural compounds-pyridoxine (pyridoxol, PN), pyridoxamine (PM), and pyridoxal (PL).’ Pyridoxine is encountered in foods in the free form and as the glycoside,2 which is partially hydrolyzed in the intestine (the glycoside can also be a b ~ o r b e d )P~L; and P M are present primarily as the 5’-phosphates, which are hydrolyzed by intestinal pho~phatases.~ The three vitamers are absorbed in the jejunum (which involves metabolic trapping by pho~phorylation)’~~ and cross the basolateral membrane to enter circulation in mainly the nonphosphorylated forms.’ Once in blood, PL is bound by albumin,’ and P N and P L are taken up by erythrocytes, where some PN can be converted to P L and bound by hemoglobin.’ A substantial portion of the absorbed vitamin B, is transported to liver and, in the unphosphorylated forms, enter the hepatocytes by diffusion followed by metabolic trapping.’.’’ The various aspects of vitamin B, metabolism have been reviewed in depth,”-13 and most investigations have built on the elegant early findings of Snell and colleagues (for examples, see Refs. 14-16) (FIG. 1). After phosphorylation by a single k i n a ~ e ,the ’ ~ P N P and P M P are oxidized to P L P by an FMN-dependent oxidase,” and PLP is bound by apo-enzymes or released into plasma (as P L P or after hydrolysis).” The PLP is dephosphorylated by alkaline phosphatase before uptake by other tissues. Most of the P L in excess of tissue needs is oxidized to 4-PA, presumably by the liver and kidney. Because essentially all tissues have PL kinase, but few have significant amounts of the PNP(PMP) oxidase, it is thought that liver is responsible for converting dietary P N and PM to P L (via PLP) and that other tissues take up PL from circulation and convert it to PLP.
ENZYMOLOGY OF VITAMIN B, METABOLISM BY HUMAN LIVER The apparent kinetic properties of these enzymes have been determined with human liver.’’ While a rigorous evaluation of the fluxes through each step of the pathway also requires knowledge of the substrate concentrations in vivo, an approxima‘Supported in part by Public Health Services General Clinical Research Center Grant 5 M 0 1 -RR00039. 110
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tion based on available i n f ~ r m a t i o n is ’ ~given in FIGURE1 . The major features of this scheme follow. 1. The rates of phosphorylation of PN and PL are much greater than that for dephosphorylation. This differs from depictions using the “optimal” activity of the phosphatase,I2 which is higher only at alkaline pH (pH 9). Since PN and PL are efficiently trapped in hepatocytes in the phosphorylated our conclusion that the kinase predominates over the phosphatase” appears valid. Fonda has recently purified a neutral phosphatase” that was not previously considered in the assessment of the enzymes of vitamin B, metabolism; hence, we have assayed liver homogenates under the same conditions used by Dr. Fonda2’ and found higher activities (i.e., about 2 nmol/min per g of liver) than were seen previo~s1y.l~ Nonetheless, PL kinase was still more active than PLP phosphatase under conditions that approximate those in viva
PM
i
PM
PN
PL
4
PN
L
Q)
> .-J -1
PLP
PL
4-PA
FIGURE 1. Metabolism of vitamin B, by human liver. The diagram depicts the estimated rates of the major reactions of vitamin B, metabolism (from Merrill et a1.,I9 except for the higher estimate of 2.0 for the PLP phosphatase, which was determined in this study), and shows the major forms that are taken up and released by liver. The rates are given in nmol/min per g of liver assuming a substrate concentration of 10 p M .
2. The rate of oxidation of PL to 4-PA is similar to that for phosphorylation. This predicts that a substantial portion of PL is rapidly converted to 4-PA, which has been born out by dietary studies2‘ 3. The phosphorylation of PN and PM to the 5’-phosphates is somewhat slower than the conversion of PNP and PMP to PLP. Furthermore, PNP(PMP) oxidase is highly sensitive to product inhibition (not shown) so that the amount of cytosolic PLP probably influences the flux through this step.15~22 This may help avoid PLP accumulation, which could lead to greater wastage (upon dephosphorylation PLP to PL and subsequent oxidation to 4-PA) and/or to nonspecific inhibition of the many enzymes that react with this reagent in a noncoenzymatic manner. It has also been suggested that protein binding helps determine the pyridoxal 5‘-phosphate levels of cells.’’
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There are still many unanswered questions about this pathway, such as the rates of interconversion of protein-bound P M P and P L P in vivo (and the extent to which PNP(PMP) oxidase is utilized to convert the P M P from transamination reactions to PLP), the exact relationship between hepatocyte uptake versus efflux, and the nature of PLP efflux. The coupling between transport and metabolism warrants further consideration, especially since Kozik and McCormick" have shown that the rate of P N uptake by rat hepatocytes at "physiological" concentrations (i.e. puM) is on the same order of magnitude (i.e., 0.2 to 10 nmol/min per g of liver) as these rates of metabolism. In addition, more information is needed about the molecular defects responsible for aberrant vitamin B, metabolism in specific disease^.'^
1
I
m PN
LPN
Before PN supplementation
After PN supplementation
6
12
18
I
24
Hours after PN load FIGURE 2. Representative profiles for the levels of the different forms of vitamin B6 in the plasma of a patient with cirrhosis. The measurements were made after the patient had taken a 25-mg load of pyridoxine. The upper panel depicts the profile at the beginning of the study and the lower panel the results after 28 days of daily supplementationwith 25 mg of pyridoxine.
KINETICS OF PLP APPEARANCE IN PLASMA Plasma P L and PLP rise rapidly after P N intake (FIG.2).23The P L is also cleared rapidly, whereas P L P remains elevated for some time, presumably due to albumin binding. The clearance of PLP depends on its rate of hydrolysis by alkaline phosphatase, as exemplified by the increase in circulating P L P in patients with h y p o p h ~ s p h a t a s i aan , ~ ~inborn error characterized by deficient activity of this enzyme, and in the faster clearance of PLP by patients with liver disease, who typically have elevated hepatic and serum alkaline p h ~ s p h a t a s e . Overall, ~ ~ . ~ ~ it appears that the PL
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released in circulation has access to peripheral tissues first,27before the excess is taken up by liver.
THE FASTING B, VITAMER PROFILE AND RESPONSE TO A PYRIDOXINE LOAD IN NORMAL AND CIRRHOTIC SUBJECTS Several methods are used to assess vitamin B, status, including measuring plasma PLP,’* urinary 4-PA,Zs329stimulation of apo-PLP-dependent enzymes by added c ~ f a c t o r , ~and ’ urinary metabolites of pathways sensitive to depletion of vitamin B,.3’332 Another way to define abnormalities in a metabolic pathway is by a load test, which can accentuate defects. The same principle has been applied to vitamin B,23,33using high-performance liquid ~ h r o m a t o g r a p h yto~resolve ~ the different metabolites. Two profiles depicting the plasma levels of the major forms of vitamin B, after a 25-mg load of P N are shown in FIGURE2. The lower panel most resembles the results obtained with a normal healthy subject. There is a n initial, rapid rise in PN, but this form disappears within one to two hours. Increases in plasma P L and PLP follow, with P L predominating but also disappearing more rapidly (PLP typically persists at the elevated level for over 24 hr). 4-PA appears somewhat more slowly and is also cleared fairly soon thereafter. Cirrhotics typically showed a different profile (as shown in the upper panel), and it changed after P N supplementation for 28 days with 25 mg of P N (cf. the upper and lower panels of FIG. 2). Before supplementation, fasting PLP was lower and the increases in the various forms after consumption of the load were delayed. The blunted responses and the lower areas under these curve^^^.^^ indicate that the patient was using a portion of the P N to replete tissue stores. The more normal profile after supplementation indicates that the patient has achieved a more normal vitamin B, status. The studies that we have conducted on the enzymology of vitamin B, metabolism by liver samples from cirrhotic and noncirrhotic patient^,'^.^' plus the load and supplementation s t ~ d i e s ’ ~have . ~ ~led to several new findings on the altered B, metabolism of cirrhosis. The cirrhotic subjects had normal levels of the enzymes of PLP synthesis2’ and achieved a normal or nearly normal level of plasma P L P within one hour of their P N ingestion and maintained it for 24 hours. This supports the hypothesis that the defect lies in enhanced PLP degradation rather than impaired synthesis. Indeed, many of the patients exhibited elevated levels of PLP pho~phatase.~’ These findings contrast, however, with a number of previous attempts to normalize plasma PLP,34perhaps because the patients constituted a differently stratified population. Cirrhosis represents a broad spectrum of impaired hepatic function and blood flow. Evidence for differences in the handling of vitamin B, by patients with varying degrees of cirrhosis was seen in a correlation between the plasma PLP and standard indices of hepatic function and blood flow.I4It was poorest in the cirrhotic patients with the “best” hepatocyte function and blood flow, which may represent an accelerated degradation of PLP to P L and 4-PA during passage through liver in the early stages of cirrhosis (hence, impaired utilization of this supplement), or an increased delivery of the vitamin to peripheral tissues (hence, a more effective cellular utilization of the dose). A definitive answer to these alternatives can only be given by measuring tissue levels. Whatever the mechanism of the initially low plasma PLP in cirrhotics, supple-
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mentation with 25 mg of P N per day achieved a normal vitamin B, status as reflected in elevated P L P and greater plasma and urinary 4-PA.
FAILURE TO IMPROVE AMINO ACID METABOLISM AFTER VITAMIN B, REPLETION IN CIRRHOSIS WITH ORAL PYRIDOXINE Hepatic encephalopathy is one of the major metabolic problems of cirrhosis. Factors that have been suggested to contribute to its pathogenesis are the unusual handling of phenylalanine, tyrosine, tryptophan, methionine and other amino acids (both in fasting measurements and in load studies), hyperammonemia, and accumulation of plasma m e r c a ~ t a n s . ~ Because ~,~’ PLP-requiring enzymes are involved in the metabolism of these compounds, reduced plasma P L P may contribute to the aberrant handling of amino acids. The ability to normalize plasma PLP in the cirrhotic patient enabled studies of the possible benefit of daily oral P N supplementation in this di~ease.’~ Eight subjects were treated with 25 mg of PN for 28 days with assessment of fasting plasma vitamer levels and response to an oral P N load at the beginning and end of this period. Despite repletion of B, stores as evidenced by a significant rise in fasting plasma PLP, a higher excretion of the load as urinary 4-PA, and a more normal post-supplementation area under the plasma concentration versus time curve for PLP, no significant changes were observed in methionine clearance after a load, nor in any other amino acid after a protein load. These findings are consistent with a previous study by Horowitz et who noted that the lack of accumulation of intermediates of the methionine metabolism in cirrhotic patients given a methionine load implied a block very early in the pathway. Thus, it is unlikely that altered amino acid clearance in cirrhosis is due to a functional deficiency of vitamin B,. These studies do not exclude more subtle effects, such as neurological or other complications in this disease, nor the possibility of a beneficial effect in patients with more severe disease. Similarly, it has not addressed the effects of longer-term repletion, but considering that a full month of repletion was achieved in this study, it would seem unlikely that longer repletion would have a major impact.
CONCLUSIONS These studies represent an attempt to rationally evaluate the metabolic defect responsible for aberrant vitamin B, metabolism in a selected disease, to design a way to normalize the currently accepted parameters of vitamin B, status, and to assess the biochemical and clinical response to achieving a more normal B, profile. While it did not find a link between the low plasma P L P in cirrhosis and the defects(s) in amino acid metabolism, it may have served as a model for addressing this type of nutritional question with respect to other disorders in cirrhosis or in other disease. The level of P N supplementation (25 mg) appears to be sensible in such studies since there is little increase in plasma P L P with higher a m o ~ n t s , especially ~’ considering the potential for side effects with megadose particularly when the patients already have compromised liver function.
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SUMMARY The B, vitamers (pyridoxine, pyridoxamine, and pyridoxal) are primarily metabolized in liver to pyridoxal 5’-phosphate (PLP) and the deadend catabolite 4-pyridoxic acid. We have built on the elegant early work of Snell and others to describe the activities of the human liver enzymes responsible for vitamin B, metabolism” and to develop a model of the relative rates of these interconversions in vivo. This model is consistent with changes in plasma B, after a load, the clearance of different vitamers (e.g., pyridoxine versus pyridoxal), and with the low plasma PLP in patients with cirrhosis.25Because cirrhotics were found to be capable of PLP synthesis, we have used oral supplementation with pyridoxine to restore plasma PLP to the normal range, and have evaluated the effects of this intervention on amino acid metabolism.” N o significant differences were observed in plasma or urinary clearance of methionine (or cystathionine) after an oral load, nor in amino acid clearance from circulation after a protein load for cirrhotic patients before and after restoration of normal plasma PLP. Hence, the abnormal metabolism of vitamin B, does not appear to be an important factor in the deranged amino acid metabolism in this disease. Nonetheless, this approach may be generally useful in assessing the importance of PLP in other abnormalities.
ACKNOWLEDGMENTS The authors are very grateful to the students and technicians who have conducted this research (Mark A. Codner, Bettey Hollins, Bill W. McDonald, Steven S. Scott, Elaine Wang, and the staff of the Clinical Research Facility at Emory) and to colleagues who have helped in various ways (Michael H. Kutner, Donald B. McCormick, and William J. Millikan).
REFERENCES 1.
SNELL, E. E. 1986. Pyridoxal phosphate: History and nomenclature. In Vitamin B, Pyridoxal Phosphate: Chemical, Biochemical and Medical Aspects. D. Dolphin, R. Poulson & 0.Avramovic, Eds. Part A. Vol. 1A 1-12. Wiley. New York. GREGORY, J. F. 1988. Methods for determination of vitamin B, in foods and other biological materials: A critical review. J. Food Comp. Anal. 1: 105-123. TRUMBO, P. R. & J. F. GREGORY. 1988. Metabolic utilization of pyridoxine @-glucosidein rats: Influence of vitamin B, status and route of administration. J. Nutr. 118: 1336-1 342. MIDDLETON, H. M. 1986. Intestinal hydrolysis of pyridoxal 5’-phosphate in vitro and in vivo in the rat. Effect of protein binding and pH. Gastroenterology 91: 343-350. HENDERSON, L. M. 1985. Intestinal absorption of B, vitamers. In Vitamin B,: Its Role in Health and Disease. Current Topics in Nutrition and Disease. R. D. Reynolds & J. E. Leklem, Eds. Vol. 13: 25-33. Liss. New York. H. M. 1985. Uptake of pyridoxine by in vivo perfused segments of rat small 6. MIDDLETON, intestine: A possible role for intracellular vitamin metabolism. J. Nutr. 115 1079-1088. 7. DEMPSEY,W. B. & H. N. CHRISTENSEN.1962. The specific binding of pyridoxal 5’phosphate to bovine plasma albumin. J. Biol. Chem. 237: 1113-1 120. H. & L. M. HENDERSON. 8. MEHANSHO, 1980. Transport and accumulation of pyridoxine and pyridoxal by erythrocytes. J. Biol. Chem. 2 5 5 11901-1 1907.
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9. MEHANSHO,H., D. D. Buss, M. W. HAMM& L. M. HENDERSON.1980. Transport and metabolism of pyridoxine in rat liver. Biochim. Biophys. Acta. 631: 112-123. 1984. Mechanism of pyridoxine uptake by isolated rat 10. KOZIK,A. & D. B. MCCORMICK. liver cells. Arch. Biochem. Biophys. 229 187-193. 1984. Vitamin B, metabolism. Ann. Rev. Nutr. 4 45511. INK,S. L. & L. M. HENDERSON. 470. 12. LEKLEM,J. E. 1987. I n Clinical and Physiological Applications of Vitamin B,. Current Topics in Nutrition and Disease. R. D. Reynolds & J. E. Leklem, Eds. Vol. 1 9 3-28. Liss. New York. 1987. Diseases associated with defects in vitamin A. H., JR. & J. M. HENDERSON. 13. MERRILL, B, metabolism or utilization. Ann. Rev. Nutr. 7: 137-156. D. B., M. GREGORY& E. E. SNELL.1961. Pyridoxal phosphokinases. I. 14. MCCORMICK, Assay, distribution, purification, and properties. J. Biol. Chem. 236 2076-2084. 1961. The enzymatic oxidation of pyridoxine and pyridoxamine 15. WADA,H. & E. E. SNELL. phosphate. J. Biol. Chem. 236 2089-2095. E. E. & B. E. HASKELL.1981. The metabolism of vitamin B,. In Comprehensive 16. SNELL, Biochemistry. M. Florkin & E. H. Stotz, Eds. Vol. 21: 41-71. Elsevier/North Holland. Amsterdam. 1975. Rabbit liver pyridoxamine (pyridoxine) M. N. & D. B. MCCORMICK. 17. KAZARINOFF, S’-phosphate oxidase. Purification and properties. J. Biol. Chem. 250 3436-3442. L. & T.-K. LI. 1980. Mammalian vitamin B, metabolism: Regulatory role of 18. LUMENG, protein-binding and the hydrolysis of pyridoxal S’-phosphate in storage and transport. In Vitamin B, Metabolism and Role in Growth. G. P. Tryfiates, Ed.: 27-51. Food and Nutrition Press. Westport, CT. E. WANG,B. W. MCDONALD & W. J. MILLIKAN. A. H. JR., J. M. HENDERSON, 19. MERRILL, 1984. Metabolism of vitamin B, by human liver. J. Nutr. 114: 1664-1674. 20. FONDA,M. 1987. Partial purification and characterization of vitamin B,-P phosphatases from human erythrocytes. In Biochemistry of Vitamin B,. Proceedings of the 7th International Congress on Chemical and Biological Aspects of Vitamin B, Catalysis. T. K. Korpela and P. Christen, Eds. Vol. 2 399-402. Birkhauser Congress Reports, Life Sciences. Birkhauser Verlag. Basel. J. R., J. E. LEKLEM& L. T. MILLER.1980. The metabolism of small doses of 21. WOZENSKI, vitamin B, in men. J. Nutr. 110 275-285. 1978. Evidence for the regulation of A. H., K. HORIIKE& D. B. MCCORMICK. 22. MERRILL, pyridoxal S’-phosphate formation in liver by pyridoxamine (pyridoxine) 5’-phosphate oxidase. Biochem. Biophys. Res. Commun. 83: 984-990. J. M., M. A. CODNER,B. HOLLINS,M. H. KUTNER& A. H. MERRILL.1986. 23. HENDERSON, The fasting B, vitamer profile and response to a pyridoxine load in normal and cirrhotic subjects. Hepatology 6 464-471. L. A. VRABEL& S. P. COBURN.1985. Markedly increased 24. WHYTE,M. P., J. D. MAHUREN, circulating pyridoxal 5’-phosphate levels in hypophosphatasia. J. Clin. Invest. 76: 752756. E. WANG,M. A. CODNER,B. HOLLINS& W. J. 25. MERRILL,A. H., J R . , J. M. HENDERSON, MILLIKAN. 1986. Activities of the hepatic enzymes of vitamin B, metabolism for patients with cirrhosis. Am. J. Clin. Nutr. 44: 461467. 26. ANDERSON,B. B., H. OBRIEN,G. E. GRIFFIN& D. L. MOLLIN.1980. Hydrolysis of pyridoxal S’-phosphate in plasma in conditions with raised alkaline phosphatase. Gut 21: 192-194. 27. LUMENG, L., S. SCHENKER, T.-K. LI, R. E. BRASHEAR & M. C. COMFTON. 1984. Clearance and metabolism of plasma pyridoxal S’-phosphate in the dog. J. Lab. Clin. Med. 103 59-69. 28. LUI,A., L. LUMENG, G. R. ARONOFF & T.-K. LI. 1985. Relationship between body store of vitamin B, and plasma pyridoxal-P clearance: Metabolic balance studies in humans. J. Lab. Clin. Med. 106 491-497. 29. SCHUSTER, K., L. B. BAILEY,J. J. CERDA& J. F. GREGORY. 1984. Urinary 4-pyridoxic acid excretion in 24-hour versus random urine samples as a measurement of vitamin B, status in humans. Am. J. Clin. Nutr. 3 9 466-470.
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30. WESTERHUIS, L. W. J. J. M. & J. C. M. HAFKENSCHEID. 1983. Apoenzyme content of serum aminotransferases in relation to plasma pyridoxal 5’-phosphate concentration. Clin. Chem. 2 9 789-792. 31 STURMAN, J. A. 1986. In Vitamin B, Pyridoxal Phosphate: Chemical, Biochemical and Medical Aspects. Part B (Vol. IB). D. Dolphin, R. Poulson & 0.Avramovic, Eds.: 507572. Wiley. New York. E. A. 1986. Nutritional aspects of vitamin B,. In Pyridoxal Phosphate: Chemical, 32. DONALD, Biochemical and Medical Aspects. Part B (Vol. IBA). D. Dolphin, R. Poulson & 0. Avramovic, Eds.: 477-505. Wiley. New York. J. M., S. S. SCOTT,A. H. MERRILL,B. HOLLINS& M. H. KUTNER.1989. 33. HENDERSON, Vitamin B, repletion in cirrhosis with oral pyridoxine: Failure to improve amino acid metabolism. Hepatology 9 582-588. 1986. Analysis of B, vitamers in plasma by reversed34. HOLLINS,B. & J. M. HENDERSON. phase column liquid chromatography. J. Chromatogr. 16 157-168. 35. LABADARIOUS, D., J. E. Rossouw, J. B. MCCONNELL, M. DAVIS& R. WILLIAMS. 1977. Vitamin B, deficiency in chronic liver disease-evidence for increased degradation of pyridoxal5’-phosphate. Gut 1 8 23-27. 36. SCHENKER, S., K. J. BREEN& A. M. HOYUMPA. Hepatic encephalopathy: Current status. Gastroenterology 6 6 121-1 51. 37. MCCLAJN,C. J., ZIEVE,L. & W. M. DOIZAKI.1980. Blood methanethiol in alcoholic liver disease with and without hepatic encephalopathy.Gut 21: 318-323. J. H., E. B. RYPINS,J. M. HENDERSON, S. B. HEYMSFIELD, S. D. MOFFITT,et 38. HOROWITZ, al. 1982. Evidence for impairment of transsulfuration pathway in cirrhosis. Gastroenterology 81: 668-675. P. J. BECKER& L. S. DE VILLIERS.1987. Effect of 39. UBBINK,J. B., W. J. SERFONTEIN, different levels of oral pyridoxine supplementation on plasma pyridoxal 5’-phosphate and pyridoxal levels and urinary vitamin B, excretion. Am. J. Clin. Nutr. 4 6 78-85. N. VICK,S. RASMUS,D. PLEASURE& H., J. KAPLAN,A. WINDEBANK, 40. SCHAUMBERG, M. J. BROWN.1983. Sensory neuropathy from pyridoxine abuse: A new megavitamin syndrome. N. Engl. J. Med. 309445-447. 41 PARRY,G. J. & D. E. BREDESEN.1985. Sensory neuropathy with low-dose pyridoxine. Neurology 3 5 1466-1468.
INTRODUCTION The vitamin B, group is composed of three natural compounds-pyridoxine (pyridoxol, PN), pyridoxamine (PM), and pyridoxal (PL).’ Pyridoxine is encountered in foods in the free form and as the glycoside,2 which is partially hydrolyzed in the intestine (the glycoside can also be a b ~ o r b e d )P~L; and P M are present primarily as the 5’-phosphates, which are hydrolyzed by intestinal pho~phatases.~ The three vitamers are absorbed in the jejunum (which involves metabolic trapping by pho~phorylation)’~~ and cross the basolateral membrane to enter circulation in mainly the nonphosphorylated forms.’ Once in blood, PL is bound by albumin,’ and P N and P L are taken up by erythrocytes, where some PN can be converted to P L and bound by hemoglobin.’ A substantial portion of the absorbed vitamin B, is transported to liver and, in the unphosphorylated forms, enter the hepatocytes by diffusion followed by metabolic trapping.’.’’ The various aspects of vitamin B, metabolism have been reviewed in depth,”-13 and most investigations have built on the elegant early findings of Snell and colleagues (for examples, see Refs. 14-16) (FIG. 1). After phosphorylation by a single k i n a ~ e ,the ’ ~ P N P and P M P are oxidized to P L P by an FMN-dependent oxidase,” and PLP is bound by apo-enzymes or released into plasma (as P L P or after hydrolysis).” The PLP is dephosphorylated by alkaline phosphatase before uptake by other tissues. Most of the P L in excess of tissue needs is oxidized to 4-PA, presumably by the liver and kidney. Because essentially all tissues have PL kinase, but few have significant amounts of the PNP(PMP) oxidase, it is thought that liver is responsible for converting dietary P N and PM to P L (via PLP) and that other tissues take up PL from circulation and convert it to PLP.
ENZYMOLOGY OF VITAMIN B, METABOLISM BY HUMAN LIVER The apparent kinetic properties of these enzymes have been determined with human liver.’’ While a rigorous evaluation of the fluxes through each step of the pathway also requires knowledge of the substrate concentrations in vivo, an approxima‘Supported in part by Public Health Services General Clinical Research Center Grant 5 M 0 1 -RR00039. 110
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tion based on available i n f ~ r m a t i o n is ’ ~given in FIGURE1 . The major features of this scheme follow. 1. The rates of phosphorylation of PN and PL are much greater than that for dephosphorylation. This differs from depictions using the “optimal” activity of the phosphatase,I2 which is higher only at alkaline pH (pH 9). Since PN and PL are efficiently trapped in hepatocytes in the phosphorylated our conclusion that the kinase predominates over the phosphatase” appears valid. Fonda has recently purified a neutral phosphatase” that was not previously considered in the assessment of the enzymes of vitamin B, metabolism; hence, we have assayed liver homogenates under the same conditions used by Dr. Fonda2’ and found higher activities (i.e., about 2 nmol/min per g of liver) than were seen previo~s1y.l~ Nonetheless, PL kinase was still more active than PLP phosphatase under conditions that approximate those in viva
PM
i
PM
PN
PL
4
PN
L
Q)
> .-J -1
PLP
PL
4-PA
FIGURE 1. Metabolism of vitamin B, by human liver. The diagram depicts the estimated rates of the major reactions of vitamin B, metabolism (from Merrill et a1.,I9 except for the higher estimate of 2.0 for the PLP phosphatase, which was determined in this study), and shows the major forms that are taken up and released by liver. The rates are given in nmol/min per g of liver assuming a substrate concentration of 10 p M .
2. The rate of oxidation of PL to 4-PA is similar to that for phosphorylation. This predicts that a substantial portion of PL is rapidly converted to 4-PA, which has been born out by dietary studies2‘ 3. The phosphorylation of PN and PM to the 5’-phosphates is somewhat slower than the conversion of PNP and PMP to PLP. Furthermore, PNP(PMP) oxidase is highly sensitive to product inhibition (not shown) so that the amount of cytosolic PLP probably influences the flux through this step.15~22 This may help avoid PLP accumulation, which could lead to greater wastage (upon dephosphorylation PLP to PL and subsequent oxidation to 4-PA) and/or to nonspecific inhibition of the many enzymes that react with this reagent in a noncoenzymatic manner. It has also been suggested that protein binding helps determine the pyridoxal 5‘-phosphate levels of cells.’’
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There are still many unanswered questions about this pathway, such as the rates of interconversion of protein-bound P M P and P L P in vivo (and the extent to which PNP(PMP) oxidase is utilized to convert the P M P from transamination reactions to PLP), the exact relationship between hepatocyte uptake versus efflux, and the nature of PLP efflux. The coupling between transport and metabolism warrants further consideration, especially since Kozik and McCormick" have shown that the rate of P N uptake by rat hepatocytes at "physiological" concentrations (i.e. puM) is on the same order of magnitude (i.e., 0.2 to 10 nmol/min per g of liver) as these rates of metabolism. In addition, more information is needed about the molecular defects responsible for aberrant vitamin B, metabolism in specific disease^.'^
1
I
m PN
LPN
Before PN supplementation
After PN supplementation
6
12
18
I
24
Hours after PN load FIGURE 2. Representative profiles for the levels of the different forms of vitamin B6 in the plasma of a patient with cirrhosis. The measurements were made after the patient had taken a 25-mg load of pyridoxine. The upper panel depicts the profile at the beginning of the study and the lower panel the results after 28 days of daily supplementationwith 25 mg of pyridoxine.
KINETICS OF PLP APPEARANCE IN PLASMA Plasma P L and PLP rise rapidly after P N intake (FIG.2).23The P L is also cleared rapidly, whereas P L P remains elevated for some time, presumably due to albumin binding. The clearance of PLP depends on its rate of hydrolysis by alkaline phosphatase, as exemplified by the increase in circulating P L P in patients with h y p o p h ~ s p h a t a s i aan , ~ ~inborn error characterized by deficient activity of this enzyme, and in the faster clearance of PLP by patients with liver disease, who typically have elevated hepatic and serum alkaline p h ~ s p h a t a s e . Overall, ~ ~ . ~ ~ it appears that the PL
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released in circulation has access to peripheral tissues first,27before the excess is taken up by liver.
THE FASTING B, VITAMER PROFILE AND RESPONSE TO A PYRIDOXINE LOAD IN NORMAL AND CIRRHOTIC SUBJECTS Several methods are used to assess vitamin B, status, including measuring plasma PLP,’* urinary 4-PA,Zs329stimulation of apo-PLP-dependent enzymes by added c ~ f a c t o r , ~and ’ urinary metabolites of pathways sensitive to depletion of vitamin B,.3’332 Another way to define abnormalities in a metabolic pathway is by a load test, which can accentuate defects. The same principle has been applied to vitamin B,23,33using high-performance liquid ~ h r o m a t o g r a p h yto~resolve ~ the different metabolites. Two profiles depicting the plasma levels of the major forms of vitamin B, after a 25-mg load of P N are shown in FIGURE2. The lower panel most resembles the results obtained with a normal healthy subject. There is a n initial, rapid rise in PN, but this form disappears within one to two hours. Increases in plasma P L and PLP follow, with P L predominating but also disappearing more rapidly (PLP typically persists at the elevated level for over 24 hr). 4-PA appears somewhat more slowly and is also cleared fairly soon thereafter. Cirrhotics typically showed a different profile (as shown in the upper panel), and it changed after P N supplementation for 28 days with 25 mg of P N (cf. the upper and lower panels of FIG. 2). Before supplementation, fasting PLP was lower and the increases in the various forms after consumption of the load were delayed. The blunted responses and the lower areas under these curve^^^.^^ indicate that the patient was using a portion of the P N to replete tissue stores. The more normal profile after supplementation indicates that the patient has achieved a more normal vitamin B, status. The studies that we have conducted on the enzymology of vitamin B, metabolism by liver samples from cirrhotic and noncirrhotic patient^,'^.^' plus the load and supplementation s t ~ d i e s ’ ~have . ~ ~led to several new findings on the altered B, metabolism of cirrhosis. The cirrhotic subjects had normal levels of the enzymes of PLP synthesis2’ and achieved a normal or nearly normal level of plasma P L P within one hour of their P N ingestion and maintained it for 24 hours. This supports the hypothesis that the defect lies in enhanced PLP degradation rather than impaired synthesis. Indeed, many of the patients exhibited elevated levels of PLP pho~phatase.~’ These findings contrast, however, with a number of previous attempts to normalize plasma PLP,34perhaps because the patients constituted a differently stratified population. Cirrhosis represents a broad spectrum of impaired hepatic function and blood flow. Evidence for differences in the handling of vitamin B, by patients with varying degrees of cirrhosis was seen in a correlation between the plasma PLP and standard indices of hepatic function and blood flow.I4It was poorest in the cirrhotic patients with the “best” hepatocyte function and blood flow, which may represent an accelerated degradation of PLP to P L and 4-PA during passage through liver in the early stages of cirrhosis (hence, impaired utilization of this supplement), or an increased delivery of the vitamin to peripheral tissues (hence, a more effective cellular utilization of the dose). A definitive answer to these alternatives can only be given by measuring tissue levels. Whatever the mechanism of the initially low plasma PLP in cirrhotics, supple-
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mentation with 25 mg of P N per day achieved a normal vitamin B, status as reflected in elevated P L P and greater plasma and urinary 4-PA.
FAILURE TO IMPROVE AMINO ACID METABOLISM AFTER VITAMIN B, REPLETION IN CIRRHOSIS WITH ORAL PYRIDOXINE Hepatic encephalopathy is one of the major metabolic problems of cirrhosis. Factors that have been suggested to contribute to its pathogenesis are the unusual handling of phenylalanine, tyrosine, tryptophan, methionine and other amino acids (both in fasting measurements and in load studies), hyperammonemia, and accumulation of plasma m e r c a ~ t a n s . ~ Because ~,~’ PLP-requiring enzymes are involved in the metabolism of these compounds, reduced plasma P L P may contribute to the aberrant handling of amino acids. The ability to normalize plasma PLP in the cirrhotic patient enabled studies of the possible benefit of daily oral P N supplementation in this di~ease.’~ Eight subjects were treated with 25 mg of PN for 28 days with assessment of fasting plasma vitamer levels and response to an oral P N load at the beginning and end of this period. Despite repletion of B, stores as evidenced by a significant rise in fasting plasma PLP, a higher excretion of the load as urinary 4-PA, and a more normal post-supplementation area under the plasma concentration versus time curve for PLP, no significant changes were observed in methionine clearance after a load, nor in any other amino acid after a protein load. These findings are consistent with a previous study by Horowitz et who noted that the lack of accumulation of intermediates of the methionine metabolism in cirrhotic patients given a methionine load implied a block very early in the pathway. Thus, it is unlikely that altered amino acid clearance in cirrhosis is due to a functional deficiency of vitamin B,. These studies do not exclude more subtle effects, such as neurological or other complications in this disease, nor the possibility of a beneficial effect in patients with more severe disease. Similarly, it has not addressed the effects of longer-term repletion, but considering that a full month of repletion was achieved in this study, it would seem unlikely that longer repletion would have a major impact.
CONCLUSIONS These studies represent an attempt to rationally evaluate the metabolic defect responsible for aberrant vitamin B, metabolism in a selected disease, to design a way to normalize the currently accepted parameters of vitamin B, status, and to assess the biochemical and clinical response to achieving a more normal B, profile. While it did not find a link between the low plasma P L P in cirrhosis and the defects(s) in amino acid metabolism, it may have served as a model for addressing this type of nutritional question with respect to other disorders in cirrhosis or in other disease. The level of P N supplementation (25 mg) appears to be sensible in such studies since there is little increase in plasma P L P with higher a m o ~ n t s , especially ~’ considering the potential for side effects with megadose particularly when the patients already have compromised liver function.
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SUMMARY The B, vitamers (pyridoxine, pyridoxamine, and pyridoxal) are primarily metabolized in liver to pyridoxal 5’-phosphate (PLP) and the deadend catabolite 4-pyridoxic acid. We have built on the elegant early work of Snell and others to describe the activities of the human liver enzymes responsible for vitamin B, metabolism” and to develop a model of the relative rates of these interconversions in vivo. This model is consistent with changes in plasma B, after a load, the clearance of different vitamers (e.g., pyridoxine versus pyridoxal), and with the low plasma PLP in patients with cirrhosis.25Because cirrhotics were found to be capable of PLP synthesis, we have used oral supplementation with pyridoxine to restore plasma PLP to the normal range, and have evaluated the effects of this intervention on amino acid metabolism.” N o significant differences were observed in plasma or urinary clearance of methionine (or cystathionine) after an oral load, nor in amino acid clearance from circulation after a protein load for cirrhotic patients before and after restoration of normal plasma PLP. Hence, the abnormal metabolism of vitamin B, does not appear to be an important factor in the deranged amino acid metabolism in this disease. Nonetheless, this approach may be generally useful in assessing the importance of PLP in other abnormalities.
ACKNOWLEDGMENTS The authors are very grateful to the students and technicians who have conducted this research (Mark A. Codner, Bettey Hollins, Bill W. McDonald, Steven S. Scott, Elaine Wang, and the staff of the Clinical Research Facility at Emory) and to colleagues who have helped in various ways (Michael H. Kutner, Donald B. McCormick, and William J. Millikan).
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