143-148 (1992)

Lack of Hormonal Stimulation of Pyridoxine Metabolism in Isolated Rat Hepatocytes KENNETH


*Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907; and Department Pharmacology and Nutrition, University of Southern California, School of Medicine, Los Angeles, California !20033


Received May 21, 1992 Isolated hepatocytes obtained from Sprague-Dawley rats (145-175 g) were incubated for 15 min at 30°C in Krebs-Henseleit bicarbonate buffer, pH 7.4, containing 0.5 mM concentration of each of the 20 natural amino acids and either 4.5 or 23 PM [U-‘4C]pyridoxine. Pyridoxine, pyridoxal, pyridoxal phosphate, and pyridoxic acid separated by an anionexchange chromatographic technique were quantified using a phosphate analyzer and a liquid scintillation counter. The conversion of [U-‘%]pyridoxine to its metabolites was more than doubled by increasing the amount of pyridoxine (4.5 to 23 PM) in the incubation medium. Insulin (10 mu/ml), glucagon (1 nM), or epinephrine (10 PM) did not have any significant effect on the conversion of [‘%Z]-pyridoxine to pyridoxal, pyridoxal phosphate, or pyridoxic acid. Our earlier observations of a large decrease in serum pyridoxal phosphate in the diabetic rat cannot be explained by any direct hormonal effects on pyridoxine metabolism. 0 1992 Academic Press, Inc.

Our earlier studies (1) showed significantly lower levels of pyridoxal phosphate in plasma and in liver mitochondria of H-week streptozotocin diabetic rats. A significant (53%) diminution in the activity of mitochondrial aspartate aminotransferase, a pyridoxal phosphate-dependent enzyme, was also observed in these diabetic animals. An increase in pyridoxal phosphate-dependent cytosolic enzymes in tissues of diabetic animals (1-S) reported in the literature indicates an increased requirement of pyridoxal phosphate for the gluconeogenesis pathway in the diabetic state. Matsuzawa (9) suggested that any lack of pyridoxal phosphate results in the destruction of pyridoxal-dependent enzymes by cellular proteases and may lead to the accumulation of substrates. This is also supported by the observations of Walsh et al. (10) who showed that rats on a vitamin B,-deficient diet accumulate more 3-hydroxykynurenine and 3-hydroxyanthranilic acid in their urine, which could be due to the diminution in the activity of the pyridoxal phosphate-dependent kynureninase (11). Pyridoxal phosphate is metabolized through a series of related enzyme reactions to give the final catabolite 4-pyridoxic acid (12,13). Figure 1 143 0885-4505192 $5.00 Copyright 0 1992 by Academic Press, Inc. AI1 rights of reproduction in any fomt reserved.



Pyridoxfne PhO$phOtaS*









1. Major reactions of pyridoxine metabolism in liver are shown.

summarizes the major reactions involved in the metabolism of pyridoxine. A rise in plasma pyridoxal phosphate indicates active vitamin B6 metabolism by the liver, since that is the only organ secreting pyridoxal phosphate into plasma (14). In this study isolated rat hepatocytes were used to investigate the effects of insulin, glucagon, and epinephrine on the metabolism of pyridoxine. MATERIALS AND METHODS Male Sprague-Dawley rats (145-175 g) maintained on ad libitum lab chow and water were anesthetized by an intraperitoneal injection of sodium pentobarbital (45 mg/kg body wt). Sodium heparin (500 U/kg body wt) was injected into the femoral vein. Liver perfusion and the isolation of hepatocytes were carried out by the methods described earlier (15-17). Hepatocytes were washed and centrifuged twice in icecold Krebs-Henseleit bicarbonate (KHB) buffer, pH 7.4. Incubations were carried out in 25 ml Erlenmeyer flasks containing 1.6 ml of KHB buffer, pH 7.4, containing all 20 natural amino acids, each to a final concentration of 0.5 mM; 0.4 ml of hepatocyte suspension (8-10 mg protein); 2 insulin (10 mu/ml; Sigma, Bovine Crystalline); f glucagon (1 nM, Sigma); f epinephrine (10 PM, Sigma); and either 4.5 or 23 PM [U - 14C]pyridoxine chloride (sp act, 90 $Zi/mg). After addition of the isotope, the flasks were stoppered with rubber stoppers and placed in a Dubnoff metabolic shaker (60 oscillations/min) at 30°C. The reaction was terminated by the addition of 0.6 ml of 3 N perchloric acid into the reaction flasks. The contents of the flasks were centrifuged and the pH of the supematant was adjusted to 6 by using 10 M potassium hydroxide and





1 M potassium bicarbonate. Bromothymol blue was used as an indicator. Samples were kept frozen ( -20°C) in the dark until analysis. Chromatographical analysis. Separation of pyridoxine and its derivatives was carried out by anion-exchange chromatography for phosphate compounds as described earlier (16,18). The effluent from the column was collected in 3-ml fractions, and counted in a Searle Delta 300 liquid scintillation counter. Authentic compounds of pyridoxine chloride, pyridoxal phosphate, pyridoxamine phosphate, pyridoxamine, and 4-pyridoxic acid were chromatographed to provide elution times (volumes) for comparison. They were located by ultraviolet detection with a Shimadzu spectrophotometer equipped with a recorder. The percentages of compounds in individual peaks were calculated by comparison to the total counts in each chromatogram after correction for background counts. Picomolar quantities of each derivative were calculated knowing the cpm/pmol of [U - 14C]pyridoxine added to the incubation medium. Calculations and statistical analysis of data were performed with an IBM-compatible PC and STATA statistical program (19). A two-tailed paired Student’s t test was used to ascertain the levels of significance. Probability values of 0.05 or less were considered significant. RESULTS


The data on the effects of insulin, glucagon, and epinephrine on the conversion of [U - 14C]pyridoxine to pyridoxal, pyridoxal phosphate, and 4-pyridoxic acid in isolated rat hepatocytes are summarized in Table 1. The K,,, for pyridoxine uptake by hepatocytes has been reported to be 28 + 8 PM (20). Hence, in our studies 4.5 and 23 PM concentrations of pyridoxine were used. Increasing the concentration of pyridoxine in the incubation medium from 4.5 to 23 PM also increased the utilization of pyridoxine by a factor of 4. Insulin, glucagon, or epinephrine had no significant effect on the amount of pyridoxine used or on the formation of pyridoxal, pyridoxal phosphate, or pyridoxic acid. However, all hepatocyte preparations were responsive to insulin, glucagon, and epinephrine action as seen by their effects on the Krebs cycle oxidation reactions (data not shown, cf. Ref. (16)). Approximately 70% of the carbons - 14 of pyridoxine were recovered in pyridoxal, pyridoxal phosphate, and pyridoxic acid. Radioactivity recovered in other derivatives of pyridoxine was near background levels and was not used in comparisons. Nevertheless, some important results can be identified. The amount of pyridoxal formed was proportional to the pyridoxine concentration in the incubation medium and accounted for about 58 + 5% of the pyridoxine consumed in the reaction. A fourfold increase in the amount of pyridoxine in the incubation medium did not increase the formation of pyridoxal phosphate by a factor of 4. The pyridoxic acid formation was about fivefold greater when 23 PM pyridoxine was added to the medium indicating that pyridoxine concentrations of 23 FM exceeded the capacity of the hepatic tissue to utilize pyridoxal. A higher rate of conversion of pyridoxal to pyridoxic acid has been shown earlier (21). In our study pyridoxic acid accounted for only about 3% of the pyridoxine consumed at lower level (4.5 PM) as compared to 6% at higher levels of pyridoxine (23 PM).


ROGERS ET AL. TABLE 1 Lack of Hormonal Stimulation of [U-‘4C]Pyridoxine


Pyridoxine consumed, pmol/mg protein

Metabolism in the Rat Hepatocyte

Pyridoxal formed, pmol/mg protein

Pyridoxal phosphate formed, pmol/mg protein

Pyridoxic acid formed, pmol/mg protein

21 36 28 2824

5 16 7 923

Control H/13 11114 11116

99 230 278 202 f 53

33 99 120 84 r 26

11/13 11/14 11/16

142 151 282 192 f 45

87 57 110 85 -+ 15

H/13 11/14 11/16

162 259 189 203 k 29

49 182 95 109 2 39

11/13 11/14 H/16

132 252 244 209 k 39

69 163 57 96 ze 34



22 25 38 28 f 5

3 8 6?3

22 27 12 20 2 4

8 6 8 7+1

23 27 39 30 f 5

2 5 8 5?2



’ Values presented were means f standard error of the mean. Incubation concentration of radioactive pyridoxine was 4.5 pM.

Our data indicate that when excess pyridoxine is presented to the hepatocyte preparation the major response is the production of pyridoxal. This is in agreement with the in viva work of Schaeffer et al. (22) who showed that feeding excess pyridoxine (1.4 g vs 7 mg/kg diet) to female rats increased liver pyridoxal content from 1.6 to 2.6 nmol/g tissue with only trivial changes in liver pyridoxal phosphate or pyridoxic acid. This may be of significant biological importance as the activity of pyridoxal phosphate-dependent enzymes will not fluctuate in an erratic manner during any short-term deficiency of vitamin Bg. We must conclude that our earlier observations (1) of a large decrease in serum pyridoxal phosphate in the diabetic rat with a concomitant reduction in liver mitochondrial pyridoxal phosphate cannot be explained by a direct hormonal action on hepatic pyridoxine metabolism. A decrease in serum pyridoxal phosphate in the diabetic state may be due to the elevated levels of alkaline phosphatase (8) that would enhance the conversion of pyridoxal phosphate to pyridoxal(21,23). ACKNOWLEDGMENT We express our appreciation for the free gift of U-‘% Arnold Liedman, and Peter Sorter, Hoffman-LaRoche,

pyridoxine received from Drs. Yu-Ying Liu, Nutley, New Jersey.



Lack of Hormonal Stimulation

TABLE 2 of [U-%]Pyridoxine

Pyridoxine consumed, pmol/mg protein

Pyridoxal formed, pmol/mg protein

11/13 11/14 11/16

595 851 418 621 + 126

283 478 248 336 2 72

11/13 11/14 11/16

794 826 810 810 + 9

380 575 692 549 + 91

11/13 H/l4 H/16

977 765 753 832 + 73

532 446 484 487 + 25

11/13 H/14 H/16

408 927 1078 804 f 203

90 737 1255 694 2 337




Metabolism in the Rat Hepatocyte Pyridoxal phosphate formed, pmol/mg protein

Pyridoxic acid formed, pmol/mg protein

40 59 51 50 2 6

26 62 55 48 + 11

45 42 55 47 2 4

34 64 49 49 4 9

46 30 49 42 f 6

38 47 50 45 -1- 4

31 38 49 39 2 5

32 43 42 39 2 4





y Values presented were means f standard error of the mean. Incubation concentration of radioactive pyridoxine was 23 PM.

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18. Geiger PJ, Ahn S, Bessman SP. Separation and automated analysis of phosphorylated metabolic intermediates. In Methods in Carbohydrate Chemistry (Whistler RL, BeMiller JN, Eds.). New York: Academic Press, 1980, Vol. 8, Chap. 3. 19. Edwards AL. Statistical Analysis. New York: Rinehart. 1958, p 127; Statistical Analysis Program, “Stata,” available from Computing Resources Center, Santa Monica, CA. 20. Kozik A, McCormick DB. Mechanism of pyridoxine uptake by isolated rat liver cells. Arch Biockem




21. Merrill AH, Henderson JM. Vitamin B, metabolism by human liver. In Vitamin B6 (Dakshinamurti K, Ed.). New York: Annals of the New York Academy of Science, 1990, Vol. 585, pp 110-117. 22. Schaeffer MC, Sampson DA, Skala JH, Gietzen DW, Grier RE. Evaluation of vitamin B, status and function of rats fed excess pyridoxine. J Nutr 119:1392-1398, 1989. 23. Bode W, van den Berg H. Pyridoxal-5’-phosphate and pyridoxal biokinetics in male Wistar rats fed graded levels of vitamin B,. J Nutr l21:1738-1745, 1991.

Lack of hormonal stimulation of pyridoxine metabolism in isolated rat hepatocytes.

Isolated hepatocytes obtained from Sprague-Dawley rats (145-175 g) were incubated for 15 min at 30 degrees C in Krebs-Henseleit bicarbonate buffer, pH...
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