the identity of the DOPA after boiling the dog food in acid by comparing the electrochemical HPLC signal obtained for the DOPA from dog food with an authentic DOPA standard. This study showed that considerable DOPA is present in dog food in bound form and that it did not alter the plasma levels of DOPA. When DOPA is ingested orally, it may be metabolized by oxidative deamination and conjugation, probably before absorption from the gastrointestinal tract. The findings of Hoeldtke et a1.2 are completely different from the observation that dietary dopamine affects both free and conjugated plasma d ~ p a m i n eThus . ~ levels of DOPA in plasma can be considered a useful index of the activity of tyrosine hydroxylase, the rate-limiting enzyme in the biosynthesis of catecholamines. The present study further corroborates the suggestion’ that DOPA in plasma originates

primarily from neurons; finally, it points to the possibility that plasma DOPA can be a source for catecholamine synthesis in tissues that do not contain tyrosine hydroxylase. 1. Goldstein DS, Udelsman R, Eisenhofer G, Stull R, Keiser HR, Kopin IJ. Neuronal source of plasma dihydroxyphenylalanine. J Clin Endocrinol Metab 1987;64:856-61 2. Hoeldtke R, Baliga B, lssenberg P, Wurtman R. Dihydroxyphenylalanine in rat food containing wheat and oats. Science 1972;175:761-2 3. Banwart B, Miller TD, Jones TD, Tyce GM. Plasma dopa and feeding. Proc SOCExp Biol Med 1989; 1911357-61 4. Dousa MK, Tyce GM. Free and conjugated plasma catecholamines, DOPA and 3-0-methyldopa in humans and in various animal species. Proc SOC Exp Biol Med 1988;188:427-34 5. Davidson L, Vandongen R, Beilin LJ. Effect of eating bananas on plasma free and sulfate-conjugated catecholamines. Life Sci 1981;29:1773-8

ZINC AND THE REGULATION OF VITAMIN B, METABOLISM Pyridoxal kinase, a key enzyme in the formation of vitamin 6,coenzymes, requires a zinc-ATP complex as a substrate. Recent findings show that zinc-metallothionein facilitates the formation of the zinc-ATP complex. Thus, the concentration of zincmetallothionein in tissues may serve in the regulation of vitamin 6,metabolism.

Most dietary vitamin B6 is absorbed in nonphosphorylated form and requires enzymatic conversion in various tissues to the primary coenzymic species, pyridoxal-5‘phosphate (PLP). Pyridoxal kinase catalyzes the phosphorylation of pyridoxal, pyridoxamine, and pyridoxine to form PLP, pyridoxamine-5’-phosphate (PMP), and pyridoxine-5‘-phosphate (PNP), respectively. PMP and PNP are converted to PLP by pyridoxamine (pyridoxine)-5’-phosphate oxidase. These two enzymes are the site of interaction of vitamin B, metabolism with that of several other nutrients, including ri-

boflavin (through its coenzymic function as FMN in the oxidase reaction) and zinc (required for activation of pyridoxal kinase). Pyridoxal kinase is not a metalloprotein1s2but has been shown to require a divalent metal ion for a ~ t i v i t yThe . ~ metal ion participates in the kinase reaction by interacting with ATP to form the metal-ATP complex required as a ~ u b s t r a t e In . ~ studies with purified forms of pyridoxal kinase from various mammalian tissues, metal ions including Zn2+, Co2+, Mn2+, Mg2+, and Fe2+ have been shown to support the kinase reaction in vitro. The reported order NUTRITION REVIEWSIVOL 48, NO GIJUNE 1990 255

thought to indicate that physiologic conof activity of assorted divalent cations in centrations of zinc stimulate the activity of pyridoxal kinase reactions has been inconpyridoxal kinase and enhance the formasistent and may reflect differences in the tion of PLP.g Although these levels of concentration dependence for each ion added zinc are comparable with the conand differences between sources of the encentration of total zinc in tissues, this interzyme. For example, human erythrocyte pyrpretation was later shown to be inconsisidoxal kinase exhibited maximal activation tent with the fact that in most tissues zinc is with 330 p~ Zn2+ and 410 p~ Mg2+, reassociated with metal-binding ligands such ~ p e c t i v e l y .When ~ each metal was evaluas metallothionein.1° Metallothionein binds ated at 417 p ~the , order of activation was Co2+ > Mn2+ > Mg2+ > Zn2+ > Cu2+ > zinc extremely tightly, with Kd values of -10-l2 M at neutral pH,” and the actual Ni2+ > When purified pyridoxal kiconcentration of free Zn2+ in many tissues nase from bovine brain was evaluated with is M.IO Thus, the concentration of , order of actieach metal ion at 160 p ~the free Zn2+ in tissues may be much less than vation was Zn2+ > Co2+ > Mn2+ > Mg2+ > the concentration of total zinc required for Fe2+.4Purified pyridoxal kinase from sheep activity in in-vitro studies. The question liver exhibited activation in the order of arises as to the mechanism of formation of Mn2+ > Zn2+ > Mg2+ when each was evalZnATP2- and the activity of pyridoxal ki; the order was uated at 400 p ~ however, nase in vivo in the presence of such low Zn2+ > Mn2+ > Mg2+ when analyzed at 80 concentrations of free zinc. With nanomop ~ . ’Earlier in-vitro studies with partially lar concentrations of free Zn2+, the spontapurified pyridoxal kinase from rat liver and neous formation of ZnATP2- in tissues turkey liver showed that Zn2+ was the sucould not provide the concentration reperior cation at the submaximal concentraquired to support pyridoxal kinase reaction of 200 p ~Thus . it~ has been assumed tions with K,,, values for ZnATP2- in the that zinc is the primary functional cation in range of 20-50 p ~ . the pyridoxal kinase reaction under physiologic conditions. Recently, Churchich et aL2 conducted a The role of zinc ions in this reaction inseries of in-vitro studies to investigate the ability of zinc to function in the pyridoxal volves formation of a complex with ATP. kinase reaction even at very low concentraZinc ions and ATP reversibly form a tions of free Zn2+. Of primary interest was ZnATP2- complex with a dissociation conthe possible mediation of pyridoxal kinase stant (Kd) of 5 p~ at pH 6.4In contrast, the activity by metallothionein, the major formation of ZnHATPl- is approximately metal-binding protein in many tissues. For tenfold less probable in the physiologic pH these experiments, metallothionein and range.4 Such observations led to the aspyridoxal kinase were purified to homogesumption that the primary form of zinc in neity from pig liver and sheep brain, rethe pyridoxal kinase reaction is ZnATP2-, spectively. The two major isoforms of mewhich serves as the phosphate donor in the tallothionein were combined, and portions phosphorylation of vitamin B6 compounds. were converted to the Zn2+ and Co2+ speReported K,,, values for ATP (actually cies. In the absence of metallothionein, ZnATP2-) in pyridoxal kinase reactions in vitro are typically 20-50 p ~ . ~ , ~ , ~ - ~ pyridoxal kinase exhibited a K,,, for ZnATP2- o f 3 0 p ~and , the order of preferThe relevance of these in-vitro observaence of the kinase for metal-ATP comtions with respect to the in-vivo function of plexes (as judged by K,,/K,,, ratios) was pyridoxal kinase is unclear. Ebadi et aL9 exZn2+ > Co2+ > Ca2+ > Mg2+.The catalytic amined the activity of pyridoxal kinase in activity in the presence of MgATP2-, a subsupernatants (40,000g) of rat brain and strate for many other kinases, is -90% less found that the addition of micromolar concentrations of Zn2+ (0.4-16 p ~ increased than that observed with ZnATP2-. ) In experiments with in-vitro reaction mixthe enzymatic a ~ t i v i t y These .~ results were 256 NUTRITION REVIEWSIVOL 48.NO GIJUNE 1990

tures containing ATP, pyridoxal, and metallothionein complexed with either Zn2+ or Go2+, pyridoxal kinase exhibited catalytic activity.2 With 300 p~ Zn2+-metallothionein, kinase activity was -50% of that obtained with 300 p~ free Zn2+ in the absence of metallothionein. Go2+-metallothionein exhibited -75% of the activity obtained with Zn2+-metallothionein. The activation of pyridoxal kinase by these metallothionein species was concentration dependent and increased over the range of 50-300 p ~ . Further experiments by Churchich et a1.2 studied the mechanism of pyridoxal kinase activation by metallothionein. The possibility of direct interaction between pyridoxal kinase and metallothionein proteins was examined by fluorescence spectroscopy; the fluorescein isothiocyanate derivative metallothionein was used. By monitoring changes in fluorescence emission anisotropy of the metallothionein derivative, the extent of its interaction with pyridoxal kinase in mixtures containing increasing concentration of the enzyme could be determined. With this technique little or no interaction was detected be) s80 p~ tween,metallothionein (10 p ~ and pyridoxal kinase. Because the concen tration of pyridoxal kinase in brain and pre' ~ resumably other tissues is -2 p , ~ , these sults indicate that direct interaction between metallothionein and pyridoxal kinase is probably not the mechanism of activation. These investigators also examined the equilibrium between Zn2+-metallothionein and ATP using equilibrium-dialysis techniques2 They found that ATP promoted the release of Zn2+ from metallothionein. It thus appears that Zn2+-metallothionein acts as a mediator of pyridoxal kinase activity by providing a reservoir of transferrable Zn2+ that serves as a source of ZnATP2- for the kinase reaction even at low concentrations of free Zn2+. In view of the inducible nature of metallothionein and its apparent role in providing ZnATP2- for pyridoxal kinase activity, a number of interesting questions of nutri-

tional relevance arise. Since the concentration of metallothionein responds to dietary intake of zinc and other metals in animals13 and humans,14it would be of interest to determine the role of zinc status in metabolism of vitamin B., Limited evidence of such an interaction between these nutrients has been observed in studies of alcoholic pellagra in rats and humans.15.16 Further, the induction of metallothionein during stress and the acute inflammatory response17suggests that vitamin B, metabolism might also be affected by these nonnutritional factors that influence the concentration of metallothionein in tissue.




11. 12.

Karawya E, Fonda ML. Physical and kinetic properties of sheep liver pyridoxine kinase. Arch Biochem Biophys 1982;216:170-7 Churchich JE, Scholz G, Kwok F. Activation of pyridoxal kinase by metallothionein. Biochim Biophys Acta 1989;996:181-6 McCormick DB, Gregory ME, Snell EE. Pyridoxal phosphokinases. I. Assay, distribution, purification and properties. J Biol Chem 1961;256:207684 Neary JT, Diven WF. Purification, properties, and a possible mechanism for pyridoxal kinase from bovine brain. J Biol Chem 1970;245:5585-93 Chern CJ, Beutler E. Purification and characterization of human erythrocyte pyridoxine kinase. Clin Chim Acta 1975;61:353-65 Karawya E, Fonda ML. Purification, assay, and kinetic properties of sheep liver pyridoxal kinase. Anal Biochem 1978;90:525-33 Kwok F, Churchich JE. Brain pyridoxal kinase. Mobility of the substrate pyridoxal and binding of inhibitors to the nucleotide site. Eur J Biochem 1979;93:229- 35 Kerry JA, Rohde M, Kwok F. Brain pyridoxal kinase. Purification and characterization. Eur J Biochem 1986;158:581-5 Ebadi M, ltoh M, Bifano J, Wendt K, Earle A. The role of Zn2- in pyridoxal phosphate-mediated regulation of glutamic acid decarboxylase in brain. Int J Biochem 1981;13:1107-12 Magneson GR, Puvathingal JM, Ray WJ. The concentrations of free Mg2+ and free Zn2+ in equine blood plasma. J Biol Chem 1987;262: 11140-8 Hamer DH. Metallothionein. Annu Rev Biochem 1986;55:913-51 Kwok F, Churchich JE. Brain pyridoxal kinase. Purification, substrate specificities, and sensi-


tized photodestruction of an essential histidine. J Biol Chem. 1979;254:6489-95 13. McCormick CC, Menard MP, Cousins RJ. Induction of hepatic metallothionein by feeding zinc to rats of depleted zinc status. Am J Physiol 1981; 240zE414-21 14. Grider A, Cousins RJ. Zinc-induced changes in human erythrocyte metallothionein. FASEB J 1989;3:A1078

15. Vannucchi H, Kutnink M, Sauberlich HE. Interaction among niacin, vitamin B, and zinc in rats receiving ethanol. Int J Vitam Nutr Res 1986;56: 355-62 16. Vannucchi H, Moreno FS. Interaction of niacin and zinc metabolism in patients with alcoholic pellagra. Am J Clin Nutr 1989;50:364-9 17. Dunn MA, Blalock TL, Cousins RJ. Metallothionein. Proc SOCExp Biol Med 1987;185:107-19

AMlNOCARNlTlNE AND RELATED COMPOUNDS AS INHIBITORS OF CARNlTlNE TRANSFERASES: PHYSIOLOGIC IMPLICATIONS p-Aminocarnitine and its N-acetyl and N-palmitoyl amides were examined as inhibitors of carnitine acetyltransferase, carnitine palmitoyltransferase, and of fatty-acid oxidation in whole animals, tissues, and hepatic microsomal systems. Results were consistent with subsequent findings that aminocarnitine and palmitoylcarnitine have significant antiketogenic and hypoglycemic effects in experimental animals.

The close interaction between metabolism of glucose and that of fatty acids in diabetes has long been recognized. Thus, in insulin deficiency, fatty-acid oxidation is increased, with a concomitant rise in ketone bodies in the blood. Conversely, utilization of glucose is slowed, gluconeogenesis is accelerated, and hyperglycemia develops. It is not surprising that considerable research has been directed to discovery of compounds that might inhibit the oxidation of free fatty acids (which are abnormally increased in concentration) and that would have some application in the treatment of diabetes. In this context, recent research from laboratories in Japan'f2 and the United States3r4 that has led to the independent discovery and properties of p-amino analogs of carnitine is of particular interest. In the Japanese study,' a bioautographic screening procedure (similar to antibiotic screening techniques) was devised in a search for metabolic inhibitors of fatty-acid oxidation in microorganisms. The yeast Candida albicans was seeded heavily in minimal agar medium at 45°C; the medium 258 NUTRITION REVI€WS/VOL 48. NO GIJUNE 1990

contained either oleic acid or glucose as the sole source of carbon. After cooling and solidification of these media, paper chromatograms of microbial extracts were placed on the agar plates. After incubation only zones of inhibition observed on oleic acid-containing plates were considered. Such zones of inhibition should have derived from microbial products interfering specifically with oleate catabolism (oxidation), a process essential for growth of C. albicans. By this technique it was found that Emericella quadrilineata produced an inhibitor termed emericedin, which has the structure (R)-3-acetylamino-4-(trirnethylammonio)-butyric acid. Hydrolysis of emericedin gave the free base emeriarnine. CH3


CH3- N+ - CH2- CH - CHZ- COO-





R (Emericedin: R = -COCH3; Emeriamine: R = -H)

Zinc and the regulation of vitamin B6 metabolism.

Pyridoxal kinase, a key enzyme in the formation of vitamin B6 coenzymes, requires a zinc-ATP complex as a substrate. Recent findings show that zinc-me...
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