Increased Muscle Phosphorylase in Rats Fed High Levels of Vitamin B61 ARTHUR L. BLACK,2 BEVERLY M. GUIRARD,3 ANDESMOND E. SNELL3 Department of Biochemistry, University of California, Berkeley, California 94720 ABSTRACT The present study was undertaken to test the hypothesis that muscle phosphorylase may function as a repository for vitamin B8 in the animal. Since a repository would be expected to accumulate surplus material, one would predict that phosphorylase, which contains stoichiometric amounts of pyridoxal phosphate, would increase in muscle of animals surfeited with the vitamin. Rats were fed a vitamin B6-free diet supple mented with pyridoxine providing levels 10, 1.0 and 0.1 of those recom mended by the National Research Council ( NRG ). At the high intake level, muscle phosphorylase and total muscle vitamin Be increased steadily and in almost constant ratio for at least 6 weeks, whereas both alanine and aspartate transaminase increased initially, but reached a plateau within 2 weeks. At the intermediate level of pyridoxine intake, muscle phosphorylase also in creased, but less rapidly than in rats fed the higher level. When vitamin B6 intake was restricted to 10% of the NRC-recommended level, no increase in phosphorylase concentration occurred during a period of 10 weeks. These results support the hypothesis that muscle phosphorylase acts as a reservoir for vitamin Be in the animal and provide experimental evidence that muscle enzyme content expands as vitamin is accumulated during high dietary intake. J. Nutr. 107: 1962-1968, 1977. vitamin B6 •muscle phosphorylase INDEXING KEY WORDS vitamin reservoir Krebs and Fischer (1) proposed that muscle phosphorylase acts as a repository for vitamin B6 based on experimental data showing high content of the vitamin in muscle. They calculated that 60% of the vitamin present in rat muscle was ac counted for by phosphorylase while, the enzyme accounted for 75% to 96% of the total vitamin content of muscle in mice. Since phosphorylase comprises nearly 5% of the soluble protein in muscle (2) and muscle accounts for about 40% of the body mass, it is apparent that this single enzyme would provide substantial capacity and, consequently, fulfills one essential condi tion for an effective reservoir. Evidence for accessibility of vitamin B6 from phosphorylase was based on studies reporting that enzyme concentration fell to
35% of normal in muscle of rats (3) and mice (4) fed vitamin B6-deficient diets for 8 weeks. Comparable decreases in muscle content of vitamin B6, itself, in rats fed de ficient diets (5) further supported the hypothesis. An important aspect of the reservoir hypothesis that has not been tested pre viously is whether the enzyme content of muscle expands when the animal receives a dietary surplus of the vitamin. The pres ent study was undertaken to examine this
Received for publication April 28, 1977. 1This study was supported in part by NIH grants #AM 01448 and #AI 01575. 2On leave during this study from Department of Physiological Sciences, University of California, Davis, California 95616. » Current address : Department of Microbiology, Uni versity of Texas, Austin, Texas 78712. 1962
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PHOSPHORYLASE RESPONSE TO VITAMIN B,
question and the results reported here sus tain the concept that one function of phosphorylase is to provide a reservoir of vita min B6 in rats. MATERIALS
AND METHODS
Animais. Weanling male rats (50 to 60 g) of the Sprague-Dawley strain4 were housed individually in wire-bottom cages with continuous access to water and food. The animal room had regulated tempera ture (20°) and was lighted for 12 hours daily, beginning at 06:00 hours. Diet. The rats were fed a purified diet to which a known amount of pyridoxine had been added. The basic diet contained 25% vitamin-free casein,5 42% cornstarch,6 21% sucrose,7 5% corn oil,8 5% minerals9 (6), and 2% vitamin mixture 10 containing all vitamins required by the rat except for vitamin B6. The basic diet was enriched by adding pyridoxine11 to provide three concentra tions of the vitamin: a) low level, con tained 0.7 mg vitamin B6/kg; b) normal level, contained 7 mg vitamin Be/kg; c) high level, contained 70 mg vitamin B6/kg. The dietary vitamin levels were based on recommendations of the National Research Council (NRC) committee on laboratory animal nutrition (7). The low level diet provided 10% of the recommended daily intake, while the high diet provided ten times the recommended level. Preparation of tissue for enzyme assay. Rats were decapitated with a guillotine after receiving one of the experimental diets for a specified time. Killing time was approximately 08:00 hours in all experi ments to avoid any influence of diurnal variation in enzyme level. A sample of gastrocnemius and pectoralis muscle was re moved, weighed, and homogenized in 9 volumes of buffer containing 0.1 M Pipes,12 pH = 6.8, 0.5 HIM ethylene diamine-tetraacetate, and 57 HIM ß-mercapto-ethanol. Homogenates were centrifuged at 5°,10* X g, for 10 minutes to produce the super natant used for the enzyme assays. Assay procedures. 1. Phosphorylase (EC 2.4.1.1) was assayed by a modification of the Cori technique (8). The assay solu tion contained 100 mM glucose-1-phosphate, 150 mM NaF, 2mM AMP, and 2% glycogen at pH = 6.8. The homogenate supernatant
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1963
was diluted with Pipes 12 buffer to obtain about 0.5 units of phosphorylase activity per ml, and 0.4 ml of this diluted homog enate was transferred to a test tube and equilibrated at 25°.The assay was started by adding 0.4 ml of assay solution (equili brated at 25°) sequentially to samples. After exactly 5 minutes, the reaction was stopped by addition of 1.0 ml of 10% trichloroacetic acid to each assay sample. The precipitated protein was centrifuged at 15,000 X g and 0.5 ml of the supernatant volume was assayed for inorganic phos phate by the method of Fiske-Subbarow (9). Enzyme activity is expressed in units of phosphorylase/g fresh weight of muscle ( 1 unit = 1 /¿molePi released per minute at 25°). Each assay was corrected with a blank containing 0.4 ml of diluted homogenate supernatant that had been placed in boil ing water for 15 seconds, to destroy phos phorylase, prior to adding the assay solu tion. Assay results were linear with time and were proportional to the aliquot of homogenate used. 2. Alanine aminotransferase (L-alanine: 2-oxoglutarate aminotransferase, EC 2.6.1.2). This enzyme assayed(10), by using a modifica tion of Segal'swas method phos phate rather than tris buffer. The buffer was 0.1 M phosphate, pH = 7.4, containing a-ketoglutarate (18 mM). To 3.0 ml of buffer were added 50 /J of 15 mM NADH (in 0.12 M NaHCOs) and 5 /J of lactate dehydrogenase 13 (25 units; ammonia-free). After equilibrating at 25°,50 to 150 /J of homogenate supernatant was added, and 1Rats were purchased from Simonsen Laboratories, Gilroy, California. 5ICN Pharmaceuticals, Inc., Cleveland, Ohio. 8American Maize Products Co., Hammond, Indiana. 7Spreckles Sugar Co., San Francisco, California. 8MazólaCo., Englewood Cliffs, New Jersey. 8Rogers & Harper Mineral Mix (6) supplied by ICN Pharmaceuticals, Inc., Cleveland, Ohio. 10Vitamin Mixture contained the following com ponents in g per kg of vitamin mixture : Vitamin A concentrate, containing 80% retinyl palmitate and 20% retinyl acetate (2x10» unlts/g), 4.5; cholecalciferol (4 x 10Bunits/g). 0.25; alpha tocopnerol, 5.0; ascorbic acid, 45 ; inositol, 5 ; choline chloride, 75 ; menadione, 2.25 ; para-aminobenzolc acid, 5.0 ; nlacin, 4.5 ; rlboflavln, 1.0 ; thiamln hydrochlorlde, 1.0 ; cal cium pantothenate, 3.0 ; and the following in mg per kg of vitamin mixture : biotin, 20 ; folle acid, 90 ; vitamin B-12, 1.35. The mixture, triturated In sucrose, was purchased from ICN Pharmaceuticals, Inc., Cleveland, Ohio. "• The pyridoxine-HCl was USP grade, HoffmanLa12Pipes Roche, Inc., New Jersey. Is Nutley, plperazine-N-N'-bis[2-ethane sulfonlc acid], supplied by Calbiochem, La Jolla, California. 13Boehrlnger-Mannhelm, Indianapolis, Indiana.
BLACK, GUIRARD AND SNELL
1964
O
IO
20
3O
40 Days
60
70
Fig. 1 Rats weighing 80 to 90 g, initially, were fed the low B, diet (0.7 mg B,/kg of diet) for 12 days. Half of the rats ( A and A, symbols ) were then fed (first arrow) the high B« diet while the remaining rats serving as controls (O, and •, symbols), were fed only the low B«diet. During the 2 week interval (between arrows ) rats on high B«were pair-fed with control rats but subse quently received food, ad libitum; open and closed symbols represent phosphorylase activity in gastrocnemius and pectoralis muscles, respectively, in rats sacrified throughout the experiment. Each symbol represents the phosphorylase assay of muscle tissue from one rat.
the endogenous rate of NADH oxidation recorded for 2 minutes with a recording spectrophotometer at 340 nm. The assay was completed by adding 400 /ul of l M L-alanine (pH = 7.4), mixing and record ing rate of change in absorbance. The ali quot of homogenate was adjusted in both aminotransferase reactions to obtain lin earity with time for absorbance change dur ing the assay. 3. Aspartate aminotransferase (L-aspartate :2-oxoglutarate aminotransferase, EC 2.6.1.1) was assayed by a modification of the method of Hedrick et al. (11). To 3 ml of phosphate buffer (0.1 M, pH = 7.4, con taining a-ketoglutarate (18 HIM) and L-aspartate (2 HIM) were added 50 ¿Jof NADH (15 HIM in 0.12 M NaHCO3) and 5 /J of malate dehydrogenase (50 units; am monia-free), and the sample was equili brated at 25°.After adding 50 to 150 ,J of homogenate supernatant, the rate of change of absorbance at 340 nm was recorded. The endogenous rate of NADH oxidation, as described above, was subtracted from the
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observed rate. All enzyme activities have been expressed in comparable units ( 1 unit = 1 /Amolé product/minute at 25°). 4. Assay for muscle vitamin B6 content. Total vitamin B6 was determined on aliquots of the gastrocnemius muscle homog enate following hydrolysis in 0.055 N HC1 by assay with S. carlsbergensis (12). Antibody for phosphorylase. Antiserum 14 was prepared in goats by using rabbit muscle phosphorylase-b isolated in the E. G. Krebs laboratory.15 Feeding experiments. Rats had food and v/ater, ad libitum, at all times except during a 2-week period in the first experiment when pair-feeding was used to eliminate food intake as a variable in the response to dietary vitamin Bg. In this experiment, 16 rats, weighing 80 to 90 g, were fed the low B6 diet for 11 days; subsequently half of the rats were changed to the high B6 diet, but restricted in total food intake (i.e., pair fed) to the same level as rats remaining on the low Bg diet. The pair feeding was dis continued after 2 weeks (interval between arrows on fig. 1) and all remaining rats were allowed free access to food until ter minated. RESULTS The time course of change in muscle phosphorylase when rats were fed high or low levels of vitamin Be is shown on figure 1. Rats killed during the initial period of 11 days, when all rats were on the low vitamin intake (0.7 mg vitamin Be/kg diet), had approximately 40 units of phosphorylase/g of gastrocnemius muscle. After switching half of the rats to the high vita min diet (fig. 1, first arrow), there was a rapid increase in phosphorylase concentra tion in gastrocnemius and pectoralis mus cles that was nearly linear in rate for the first 2 weeks. In rats killed at later times, the enzyme concentration appeared to level out in gastrocnemius or increased at a slower rate in pectoralis. Rats that were fed only the low vitamin B«diet main tained the original enzyme concentration throughout the entire experiment, while the muscle phosphorylase in rats fed the "The antlserum was prepared by Antibodies, Inc.. Davis. California. 16The authors are Indebted to Dr. D. A. Walsh for the rabbit phosphorylase-b used to prepare goat antiserum.
PHOSPHORYLASE
RESPONSE TO VITAMIN B,
high B6 diet reached a level 3 to 4 times as great. The first experiment was designed to ex clude several variables that might affect phosphorylase response. All rats were the same strain ( Sprague-Dawley ) and sex (male), and had the same birth date, to avoid differences in tissue development due to age. Furthermore, during the first 2 weeks that rats were on the high vitamin intake, their daily food was restricted to the same level (i.e., pair-fed) as rats fed only the low vitamin diet ( interval between arrows on fig. 1), which avoided the com plication that would result if differences in energy intake affected glycogen deposi tion and, consequently, phosphorylase levels. In the presence of these controls, it seems reasonable to conclude that the greater intake of vitamin B6, per se, was responsible for the increasing level of mus cle phosphorylase that occurred. The next experiment examined the uniqueness of the phosphorylase response after placing weanling rats on high vitamin Be intake. For this purpose, the activity of two other pyridoxal phosphate enzymes, alanine and aspartate aminotransferase, was measured in the same tissue samples used to assay for phosphorylase. The rats used in this experiment were younger and smaller (50-60 g) than those used in the
Fig. 2 Rats weighing 50 to 60 g, initially, were fed the high vitamin B«diet throughout experiment 2. The concentration in gastrocnemius muscle of phosphorylase, A; aspartate amino transferase, O; and alanine aminotransferase, • (values for latter enzyme multiplied X 10), is shown during 7 weeks of consuming the diet. When two rats were killed on the same day, re sults are given as double symbols connected by a bar to show range of variation between rats. Each symbol represents the enzyme assay of gas trocnemius muscle from one rat.
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1965
first experiment (80-90 g), and their initial level of muscle phosphorylase, 30 units/g of gastrocnemius, was only 75% of the level in experiment 1 ( see fig. 1 ). This dif ference in initial enzyme level between the two experiments presumably reflects changes that occurred during the 1-week period between weaning and the start of the first experiment, an interval during which those rats had received a stock diet containing 4.3 mg of vitamin B6/kg of diet. As shown in figure 2, all three enzymes increased initially in gastrocnemius muscle of the rats. The concentration of aspartate aminotransferase reached a plateau within the first few days while alanine amino transferase, which increased somewhat less rapidly, leveled off within 2 weeks. Mean while, phosphorylase continued to increase for at least 6 weeks and while the last 450 -
Weeks Fig. 3 The total units of phosphorylase in gastrocnemius muscle was calculated from en zyme activity (units phosphorylase/g) X combined weight of both gastrocnemius muscles for each rat used in experiment 2. Rats were fed the high vitamin B«diet throughout the experiment. Each symbol represents total phosphorylase in gastrocne mius muscle from one rat. When two rats were killed on the same day the symbols are connected by a bar to show the range of values between rats.
1966
BLACK, GUIRARD AND SNELL
samples, taken after consuming the high B6 diet, indicate a reduced rate of enzyme accumulation, this conclusion is incorrect, as will be shown. The traditional means for expressing en zyme activity as concentration in the tissue sampled (units of activity/g of muscle) can be misleading, especially in growing animals. For example, if the rate of increase of muscle tissue is greater than the rate of phosphorylase increase, then the concen tration of the enzyme will fall even though the total amount of enzyme in muscle con tinues to increase. To avoid this ambiguity, phosphorylase activity was calculated for total gastrocnemius muscle ( units/g X com bined weight of both gastrocnemius mus cles) and these values are plotted on figure 3 for the 7-week period of experiment 2. These data clearly show that rats fed a high Be diet accumulated phosphorylase in gas trocnemius muscle at a rate that was nearly linear throughout most of the experimental period. According to principles enunciated by Schimke et al. (13), this response sug gests that the presence of surplus vitamin B6 may have protected the enzyme against degradation while enzyme synthesis con tinued unabated. Thus vitamin B6 could function to stabilize phosphorylase in a manner analogous to tryptophan which acts to protect tryptophan pyrrolase from degradation. In contrast to the latter en zyme (13), which was shown to accumu late during a period of 9 to 16 hours, the effect of vitamin B6 on phosphorylase ac cumulation appears to extend almost undiminished for weeks, and even months. Until additional studies are completed, however, it will not be possible to predict the duration of the vitamin BR effect nor define the mechanism responsible for phos phorylase accumulation. It was important to exclude the possi bility that the increase in phosphorylase might be an artifact due to the abnormally high levels of vitamin B6 used ( 10 X the NRC recommended level). For this pur pose, eight rats which had received a vita min Bß-freediet for 11 days after weaning, were divided into two groups. The first group of four was transferred to a diet containing the NRC-recommended vitamin level (7 mg/kg of diet), while the second group received the high vitamin diet (70
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mg/kg of diet). After 3 weeks, pairs of rats, including one from each group, were killed every 2 days. The results, expressed as mean ±SEfor all four rats in each group killed during the interval between 3 and 4 weeks, were 111.7 ±1.2 and 122.6 ±3.2 units phosphorylase/g of gastrocnemius for rats fed normal and high vitamin B6, respec tively. In both groups, the concentration of phosphorylase was two to three times the initial level (about 40 units/g gas trocnemius). The rats given surplus vita min had significantly higher phosphorylase, but the value was only 10% greater than the average concentration observed in the normal group. If the NRC-recommended level is correct (and our data provide no basis for judging this point) then normal intake of the vitamin stimulates phosphoryl ase accumulation and the response to very high levels that we have used was not artifactual. The data on figure 4 show that the vita min B6 content of muscle, measured with S. carlsbergensis (12), was positively corre lated with phosphorylase activity and, thereby, support the conclusion that changes in muscle phosphorylase reflect changes in "reservoir" content. This con clusion was further strengthened by im6r
y o o 4 o O
P 3 >;.
mU 2 E o — l
50
100
Phosphorylase, units/gram
150
Gastrocnemius
Fig. 4 Vitamin Be content of gastrocnemius muscle, determined by yeast assay (see Methods), plotted against phosphorylase activity measured in the same muscle samples. Each symbol repre sents analyses from one rat.
PHOSPHORYLASE RESPONSE TO VITAMIN B,
munological analysis. Addition of antibody to muscle homogenate removed approxi mately 15 units of phosphorylase activity per ml of antiserum added. The same titer was obtained in samples containing as little as 25 units phosphorylase/g of muscle and those containing 140 units phosphorylase/g of muscle. Thus, accumulation of phos phorylase activity does reflect greater mass of enzyme and accumulation of vitamin B6 in the tissue. An estimate of the proportion of total muscle B6 that is present in the pyridoxal phosphate (PLP) in phosphorylase can be determined from data in figure 4, if we as sume that rat enzyme has the same specific activity as purified rabbit muscle phos phorylase. The latter gives 54 units activity per mg by our assay procedure. Thus rat muscle with 150 units of activity (upper values, fig. 4) would contain: 150 = 2.8 mg oi phosphorylase i, -=jEach monomeric subunit (M.W. = IO5) of phosphorylase ( 2 ) contains one molecule of vitamin B6 (M.W. = 169); consequently 2.8 mg of phosphorylase would contain: ICQ
2.8 X -
= 4.7
of vitamin B6
This value accounts for 94% of the 5 ^g of vitamin B6 found in that muscle tissue. That level is greater than Krebs and Fischer (1) calculated for rat muscle but corre sponds to the upper value they estimated for mouse tissue. It appears that most of the vitamin B6 in gastrocnemius muscle is present in phosphorylase which conforms with a reservoir role for the enzyme. DISCUSSION
Variations in the amount of muscle phos phorylase among animals have been re ported by several investigators, but these differences were attributed to factors other than dietary vitamin B6. Krebs and Fischer (14) reported heavier rabbits had more phosphorylase/g of muscle than lighter ones, while Sevilla and Fischer ( 15 ) noted that muscle phosphorylase was two to three times as great in 350 g rats as in 60 g rats. Both of these reports attributed the in creased phosphorylase to the larger size of
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1967
the animals. Burleigh and Schimke (16), conducted a comparative study involving mice, rats, and rabbits and concluded from their results that muscle phosphorylase con centration increased with age. It is recog nized that several enzymes undergo devel opmental change and that age as well as body size may be involved in some of these changes (17, 18). Our first experiment precluded body size as the cause of greater concentration of muscle phosphorylase. It is true that rats fed the high B6 diet gained body weight more rapidly (5.16 ±0.20 g/day; M ±SE for 12 rats) than rats fed the low B6 diet (3.48 ±0.34 g/day; M ±SE for 12 rats) and the concentration of muscle phos phorylase increased threefold in the former group. However, rats fed the low B6 diet tripled their body weight during the ex periment (from 80-90 g initially to 270 g after nearly 10 weeks ) without appreciable increase in muscle phosphorylase concen tration (See fig. 1). If body size were a causative factor in phosphorylase accumu lation, an increase should have occurred in the latter group of rats as well, as they gained weight. Furthermore, rats fed the high B6 diet had doubled their muscle phos phorylase concentration within the first 10 days, while being pair-fed to the control group and after attaining a body weight (about 200 g) that was only 70% of the final weight of rats fed the low B6 diet. Thus phosphorylase accumulation must result from some factor or factors other than body size (14, 15). Our experiments also exclude age as the causative factor in phosphorylase accumu lation. The rats used in experiment 1 were 4 weeks of age initially and 14 weeks of age when the experiment was concluded. This corresponds to the same age interval over which Burleigh and Schimke reported a rapid increase of muscle phosphorylase in their mice, rats, and rabbits ( 16). Among our rats that continued to consume the low vitamin Bfi diet no substantial increase oc curred in either gastrocnemius or pectoralis phosphorylase, but the group receiving sur plus En, during the same age interval, underwent a threefold increase in muscle enzyme. Dietary information is sparce in the referenced studies (14-16), but it seems reasonable to assume that the ani-
1968
BLACK, GUIRARD
mais used were fed a stock diet, which contained 4 to 7 mg of vitamin B6/kg of diet. If that assumption is correct, then, in accordance with our results in rats fed high, as well as normal vitamin levels (7 mg/kg), one would predict an accumulation of mus cle phosphorylase, as reported in the ref erenced studies (14-16) in response to dietary vitamin B6. We conclude that the dietary intake of vitamin B6 is the causa tive factor leading to muscle phosphorylase accumulation, and that neither age nor body size, per se, are directly involved. The present study does not answer the important question of whether the accumu lation of phosphorylase in response to high dietary intake of vitamin B6 is an intrinsic function of the enzyme molecule acting as a reservoir for the vitamin. However, when considered together with the evidence cited earlier showing extensive storage capacity for the vitamin (1, 2) and its mobilization during vitamin B6 deficiency ( 3, 4 ), the present experiments, demonstrat ing that the enzyme pool expands in rats surfeited with the vitamin, add consider able support to the reservoir hypothesis. It is apparent that a vitamin B8 reservoir would have physiological significance in view of the widespread role of the pyridoxal 5'-phosphate as a coenzyme in metab olism. For example, several amino acids are precursors of compounds required for normal function of the nervous system ( catecholamines, choline, serotonin,
35% of normal in muscle of rats (3) and mice (4) fed vitamin B6-deficient diets for 8 weeks. Comparable decreases in muscle content of vitamin B6, itself, in rats fed de ficient diets (5) further supported the hypothesis. An important aspect of the reservoir hypothesis that has not been tested pre viously is whether the enzyme content of muscle expands when the animal receives a dietary surplus of the vitamin. The pres ent study was undertaken to examine this
Received for publication April 28, 1977. 1This study was supported in part by NIH grants #AM 01448 and #AI 01575. 2On leave during this study from Department of Physiological Sciences, University of California, Davis, California 95616. » Current address : Department of Microbiology, Uni versity of Texas, Austin, Texas 78712. 1962
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PHOSPHORYLASE RESPONSE TO VITAMIN B,
question and the results reported here sus tain the concept that one function of phosphorylase is to provide a reservoir of vita min B6 in rats. MATERIALS
AND METHODS
Animais. Weanling male rats (50 to 60 g) of the Sprague-Dawley strain4 were housed individually in wire-bottom cages with continuous access to water and food. The animal room had regulated tempera ture (20°) and was lighted for 12 hours daily, beginning at 06:00 hours. Diet. The rats were fed a purified diet to which a known amount of pyridoxine had been added. The basic diet contained 25% vitamin-free casein,5 42% cornstarch,6 21% sucrose,7 5% corn oil,8 5% minerals9 (6), and 2% vitamin mixture 10 containing all vitamins required by the rat except for vitamin B6. The basic diet was enriched by adding pyridoxine11 to provide three concentra tions of the vitamin: a) low level, con tained 0.7 mg vitamin B6/kg; b) normal level, contained 7 mg vitamin Be/kg; c) high level, contained 70 mg vitamin B6/kg. The dietary vitamin levels were based on recommendations of the National Research Council (NRC) committee on laboratory animal nutrition (7). The low level diet provided 10% of the recommended daily intake, while the high diet provided ten times the recommended level. Preparation of tissue for enzyme assay. Rats were decapitated with a guillotine after receiving one of the experimental diets for a specified time. Killing time was approximately 08:00 hours in all experi ments to avoid any influence of diurnal variation in enzyme level. A sample of gastrocnemius and pectoralis muscle was re moved, weighed, and homogenized in 9 volumes of buffer containing 0.1 M Pipes,12 pH = 6.8, 0.5 HIM ethylene diamine-tetraacetate, and 57 HIM ß-mercapto-ethanol. Homogenates were centrifuged at 5°,10* X g, for 10 minutes to produce the super natant used for the enzyme assays. Assay procedures. 1. Phosphorylase (EC 2.4.1.1) was assayed by a modification of the Cori technique (8). The assay solu tion contained 100 mM glucose-1-phosphate, 150 mM NaF, 2mM AMP, and 2% glycogen at pH = 6.8. The homogenate supernatant
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1963
was diluted with Pipes 12 buffer to obtain about 0.5 units of phosphorylase activity per ml, and 0.4 ml of this diluted homog enate was transferred to a test tube and equilibrated at 25°.The assay was started by adding 0.4 ml of assay solution (equili brated at 25°) sequentially to samples. After exactly 5 minutes, the reaction was stopped by addition of 1.0 ml of 10% trichloroacetic acid to each assay sample. The precipitated protein was centrifuged at 15,000 X g and 0.5 ml of the supernatant volume was assayed for inorganic phos phate by the method of Fiske-Subbarow (9). Enzyme activity is expressed in units of phosphorylase/g fresh weight of muscle ( 1 unit = 1 /¿molePi released per minute at 25°). Each assay was corrected with a blank containing 0.4 ml of diluted homogenate supernatant that had been placed in boil ing water for 15 seconds, to destroy phos phorylase, prior to adding the assay solu tion. Assay results were linear with time and were proportional to the aliquot of homogenate used. 2. Alanine aminotransferase (L-alanine: 2-oxoglutarate aminotransferase, EC 2.6.1.2). This enzyme assayed(10), by using a modifica tion of Segal'swas method phos phate rather than tris buffer. The buffer was 0.1 M phosphate, pH = 7.4, containing a-ketoglutarate (18 mM). To 3.0 ml of buffer were added 50 /J of 15 mM NADH (in 0.12 M NaHCOs) and 5 /J of lactate dehydrogenase 13 (25 units; ammonia-free). After equilibrating at 25°,50 to 150 /J of homogenate supernatant was added, and 1Rats were purchased from Simonsen Laboratories, Gilroy, California. 5ICN Pharmaceuticals, Inc., Cleveland, Ohio. 8American Maize Products Co., Hammond, Indiana. 7Spreckles Sugar Co., San Francisco, California. 8MazólaCo., Englewood Cliffs, New Jersey. 8Rogers & Harper Mineral Mix (6) supplied by ICN Pharmaceuticals, Inc., Cleveland, Ohio. 10Vitamin Mixture contained the following com ponents in g per kg of vitamin mixture : Vitamin A concentrate, containing 80% retinyl palmitate and 20% retinyl acetate (2x10» unlts/g), 4.5; cholecalciferol (4 x 10Bunits/g). 0.25; alpha tocopnerol, 5.0; ascorbic acid, 45 ; inositol, 5 ; choline chloride, 75 ; menadione, 2.25 ; para-aminobenzolc acid, 5.0 ; nlacin, 4.5 ; rlboflavln, 1.0 ; thiamln hydrochlorlde, 1.0 ; cal cium pantothenate, 3.0 ; and the following in mg per kg of vitamin mixture : biotin, 20 ; folle acid, 90 ; vitamin B-12, 1.35. The mixture, triturated In sucrose, was purchased from ICN Pharmaceuticals, Inc., Cleveland, Ohio. "• The pyridoxine-HCl was USP grade, HoffmanLa12Pipes Roche, Inc., New Jersey. Is Nutley, plperazine-N-N'-bis[2-ethane sulfonlc acid], supplied by Calbiochem, La Jolla, California. 13Boehrlnger-Mannhelm, Indianapolis, Indiana.
BLACK, GUIRARD AND SNELL
1964
O
IO
20
3O
40 Days
60
70
Fig. 1 Rats weighing 80 to 90 g, initially, were fed the low B, diet (0.7 mg B,/kg of diet) for 12 days. Half of the rats ( A and A, symbols ) were then fed (first arrow) the high B« diet while the remaining rats serving as controls (O, and •, symbols), were fed only the low B«diet. During the 2 week interval (between arrows ) rats on high B«were pair-fed with control rats but subse quently received food, ad libitum; open and closed symbols represent phosphorylase activity in gastrocnemius and pectoralis muscles, respectively, in rats sacrified throughout the experiment. Each symbol represents the phosphorylase assay of muscle tissue from one rat.
the endogenous rate of NADH oxidation recorded for 2 minutes with a recording spectrophotometer at 340 nm. The assay was completed by adding 400 /ul of l M L-alanine (pH = 7.4), mixing and record ing rate of change in absorbance. The ali quot of homogenate was adjusted in both aminotransferase reactions to obtain lin earity with time for absorbance change dur ing the assay. 3. Aspartate aminotransferase (L-aspartate :2-oxoglutarate aminotransferase, EC 2.6.1.1) was assayed by a modification of the method of Hedrick et al. (11). To 3 ml of phosphate buffer (0.1 M, pH = 7.4, con taining a-ketoglutarate (18 HIM) and L-aspartate (2 HIM) were added 50 ¿Jof NADH (15 HIM in 0.12 M NaHCO3) and 5 /J of malate dehydrogenase (50 units; am monia-free), and the sample was equili brated at 25°.After adding 50 to 150 ,J of homogenate supernatant, the rate of change of absorbance at 340 nm was recorded. The endogenous rate of NADH oxidation, as described above, was subtracted from the
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observed rate. All enzyme activities have been expressed in comparable units ( 1 unit = 1 /Amolé product/minute at 25°). 4. Assay for muscle vitamin B6 content. Total vitamin B6 was determined on aliquots of the gastrocnemius muscle homog enate following hydrolysis in 0.055 N HC1 by assay with S. carlsbergensis (12). Antibody for phosphorylase. Antiserum 14 was prepared in goats by using rabbit muscle phosphorylase-b isolated in the E. G. Krebs laboratory.15 Feeding experiments. Rats had food and v/ater, ad libitum, at all times except during a 2-week period in the first experiment when pair-feeding was used to eliminate food intake as a variable in the response to dietary vitamin Bg. In this experiment, 16 rats, weighing 80 to 90 g, were fed the low B6 diet for 11 days; subsequently half of the rats were changed to the high B6 diet, but restricted in total food intake (i.e., pair fed) to the same level as rats remaining on the low Bg diet. The pair feeding was dis continued after 2 weeks (interval between arrows on fig. 1) and all remaining rats were allowed free access to food until ter minated. RESULTS The time course of change in muscle phosphorylase when rats were fed high or low levels of vitamin Be is shown on figure 1. Rats killed during the initial period of 11 days, when all rats were on the low vitamin intake (0.7 mg vitamin Be/kg diet), had approximately 40 units of phosphorylase/g of gastrocnemius muscle. After switching half of the rats to the high vita min diet (fig. 1, first arrow), there was a rapid increase in phosphorylase concentra tion in gastrocnemius and pectoralis mus cles that was nearly linear in rate for the first 2 weeks. In rats killed at later times, the enzyme concentration appeared to level out in gastrocnemius or increased at a slower rate in pectoralis. Rats that were fed only the low vitamin B«diet main tained the original enzyme concentration throughout the entire experiment, while the muscle phosphorylase in rats fed the "The antlserum was prepared by Antibodies, Inc.. Davis. California. 16The authors are Indebted to Dr. D. A. Walsh for the rabbit phosphorylase-b used to prepare goat antiserum.
PHOSPHORYLASE
RESPONSE TO VITAMIN B,
high B6 diet reached a level 3 to 4 times as great. The first experiment was designed to ex clude several variables that might affect phosphorylase response. All rats were the same strain ( Sprague-Dawley ) and sex (male), and had the same birth date, to avoid differences in tissue development due to age. Furthermore, during the first 2 weeks that rats were on the high vitamin intake, their daily food was restricted to the same level (i.e., pair-fed) as rats fed only the low vitamin diet ( interval between arrows on fig. 1), which avoided the com plication that would result if differences in energy intake affected glycogen deposi tion and, consequently, phosphorylase levels. In the presence of these controls, it seems reasonable to conclude that the greater intake of vitamin B6, per se, was responsible for the increasing level of mus cle phosphorylase that occurred. The next experiment examined the uniqueness of the phosphorylase response after placing weanling rats on high vitamin Be intake. For this purpose, the activity of two other pyridoxal phosphate enzymes, alanine and aspartate aminotransferase, was measured in the same tissue samples used to assay for phosphorylase. The rats used in this experiment were younger and smaller (50-60 g) than those used in the
Fig. 2 Rats weighing 50 to 60 g, initially, were fed the high vitamin B«diet throughout experiment 2. The concentration in gastrocnemius muscle of phosphorylase, A; aspartate amino transferase, O; and alanine aminotransferase, • (values for latter enzyme multiplied X 10), is shown during 7 weeks of consuming the diet. When two rats were killed on the same day, re sults are given as double symbols connected by a bar to show range of variation between rats. Each symbol represents the enzyme assay of gas trocnemius muscle from one rat.
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1965
first experiment (80-90 g), and their initial level of muscle phosphorylase, 30 units/g of gastrocnemius, was only 75% of the level in experiment 1 ( see fig. 1 ). This dif ference in initial enzyme level between the two experiments presumably reflects changes that occurred during the 1-week period between weaning and the start of the first experiment, an interval during which those rats had received a stock diet containing 4.3 mg of vitamin B6/kg of diet. As shown in figure 2, all three enzymes increased initially in gastrocnemius muscle of the rats. The concentration of aspartate aminotransferase reached a plateau within the first few days while alanine amino transferase, which increased somewhat less rapidly, leveled off within 2 weeks. Mean while, phosphorylase continued to increase for at least 6 weeks and while the last 450 -
Weeks Fig. 3 The total units of phosphorylase in gastrocnemius muscle was calculated from en zyme activity (units phosphorylase/g) X combined weight of both gastrocnemius muscles for each rat used in experiment 2. Rats were fed the high vitamin B«diet throughout the experiment. Each symbol represents total phosphorylase in gastrocne mius muscle from one rat. When two rats were killed on the same day the symbols are connected by a bar to show the range of values between rats.
1966
BLACK, GUIRARD AND SNELL
samples, taken after consuming the high B6 diet, indicate a reduced rate of enzyme accumulation, this conclusion is incorrect, as will be shown. The traditional means for expressing en zyme activity as concentration in the tissue sampled (units of activity/g of muscle) can be misleading, especially in growing animals. For example, if the rate of increase of muscle tissue is greater than the rate of phosphorylase increase, then the concen tration of the enzyme will fall even though the total amount of enzyme in muscle con tinues to increase. To avoid this ambiguity, phosphorylase activity was calculated for total gastrocnemius muscle ( units/g X com bined weight of both gastrocnemius mus cles) and these values are plotted on figure 3 for the 7-week period of experiment 2. These data clearly show that rats fed a high Be diet accumulated phosphorylase in gas trocnemius muscle at a rate that was nearly linear throughout most of the experimental period. According to principles enunciated by Schimke et al. (13), this response sug gests that the presence of surplus vitamin B6 may have protected the enzyme against degradation while enzyme synthesis con tinued unabated. Thus vitamin B6 could function to stabilize phosphorylase in a manner analogous to tryptophan which acts to protect tryptophan pyrrolase from degradation. In contrast to the latter en zyme (13), which was shown to accumu late during a period of 9 to 16 hours, the effect of vitamin B6 on phosphorylase ac cumulation appears to extend almost undiminished for weeks, and even months. Until additional studies are completed, however, it will not be possible to predict the duration of the vitamin BR effect nor define the mechanism responsible for phos phorylase accumulation. It was important to exclude the possi bility that the increase in phosphorylase might be an artifact due to the abnormally high levels of vitamin B6 used ( 10 X the NRC recommended level). For this pur pose, eight rats which had received a vita min Bß-freediet for 11 days after weaning, were divided into two groups. The first group of four was transferred to a diet containing the NRC-recommended vitamin level (7 mg/kg of diet), while the second group received the high vitamin diet (70
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mg/kg of diet). After 3 weeks, pairs of rats, including one from each group, were killed every 2 days. The results, expressed as mean ±SEfor all four rats in each group killed during the interval between 3 and 4 weeks, were 111.7 ±1.2 and 122.6 ±3.2 units phosphorylase/g of gastrocnemius for rats fed normal and high vitamin B6, respec tively. In both groups, the concentration of phosphorylase was two to three times the initial level (about 40 units/g gas trocnemius). The rats given surplus vita min had significantly higher phosphorylase, but the value was only 10% greater than the average concentration observed in the normal group. If the NRC-recommended level is correct (and our data provide no basis for judging this point) then normal intake of the vitamin stimulates phosphoryl ase accumulation and the response to very high levels that we have used was not artifactual. The data on figure 4 show that the vita min B6 content of muscle, measured with S. carlsbergensis (12), was positively corre lated with phosphorylase activity and, thereby, support the conclusion that changes in muscle phosphorylase reflect changes in "reservoir" content. This con clusion was further strengthened by im6r
y o o 4 o O
P 3 >;.
mU 2 E o — l
50
100
Phosphorylase, units/gram
150
Gastrocnemius
Fig. 4 Vitamin Be content of gastrocnemius muscle, determined by yeast assay (see Methods), plotted against phosphorylase activity measured in the same muscle samples. Each symbol repre sents analyses from one rat.
PHOSPHORYLASE RESPONSE TO VITAMIN B,
munological analysis. Addition of antibody to muscle homogenate removed approxi mately 15 units of phosphorylase activity per ml of antiserum added. The same titer was obtained in samples containing as little as 25 units phosphorylase/g of muscle and those containing 140 units phosphorylase/g of muscle. Thus, accumulation of phos phorylase activity does reflect greater mass of enzyme and accumulation of vitamin B6 in the tissue. An estimate of the proportion of total muscle B6 that is present in the pyridoxal phosphate (PLP) in phosphorylase can be determined from data in figure 4, if we as sume that rat enzyme has the same specific activity as purified rabbit muscle phos phorylase. The latter gives 54 units activity per mg by our assay procedure. Thus rat muscle with 150 units of activity (upper values, fig. 4) would contain: 150 = 2.8 mg oi phosphorylase i, -=jEach monomeric subunit (M.W. = IO5) of phosphorylase ( 2 ) contains one molecule of vitamin B6 (M.W. = 169); consequently 2.8 mg of phosphorylase would contain: ICQ
2.8 X -
= 4.7
of vitamin B6
This value accounts for 94% of the 5 ^g of vitamin B6 found in that muscle tissue. That level is greater than Krebs and Fischer (1) calculated for rat muscle but corre sponds to the upper value they estimated for mouse tissue. It appears that most of the vitamin B6 in gastrocnemius muscle is present in phosphorylase which conforms with a reservoir role for the enzyme. DISCUSSION
Variations in the amount of muscle phos phorylase among animals have been re ported by several investigators, but these differences were attributed to factors other than dietary vitamin B6. Krebs and Fischer (14) reported heavier rabbits had more phosphorylase/g of muscle than lighter ones, while Sevilla and Fischer ( 15 ) noted that muscle phosphorylase was two to three times as great in 350 g rats as in 60 g rats. Both of these reports attributed the in creased phosphorylase to the larger size of
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1967
the animals. Burleigh and Schimke (16), conducted a comparative study involving mice, rats, and rabbits and concluded from their results that muscle phosphorylase con centration increased with age. It is recog nized that several enzymes undergo devel opmental change and that age as well as body size may be involved in some of these changes (17, 18). Our first experiment precluded body size as the cause of greater concentration of muscle phosphorylase. It is true that rats fed the high B6 diet gained body weight more rapidly (5.16 ±0.20 g/day; M ±SE for 12 rats) than rats fed the low B6 diet (3.48 ±0.34 g/day; M ±SE for 12 rats) and the concentration of muscle phos phorylase increased threefold in the former group. However, rats fed the low B6 diet tripled their body weight during the ex periment (from 80-90 g initially to 270 g after nearly 10 weeks ) without appreciable increase in muscle phosphorylase concen tration (See fig. 1). If body size were a causative factor in phosphorylase accumu lation, an increase should have occurred in the latter group of rats as well, as they gained weight. Furthermore, rats fed the high B6 diet had doubled their muscle phos phorylase concentration within the first 10 days, while being pair-fed to the control group and after attaining a body weight (about 200 g) that was only 70% of the final weight of rats fed the low B6 diet. Thus phosphorylase accumulation must result from some factor or factors other than body size (14, 15). Our experiments also exclude age as the causative factor in phosphorylase accumu lation. The rats used in experiment 1 were 4 weeks of age initially and 14 weeks of age when the experiment was concluded. This corresponds to the same age interval over which Burleigh and Schimke reported a rapid increase of muscle phosphorylase in their mice, rats, and rabbits ( 16). Among our rats that continued to consume the low vitamin Bfi diet no substantial increase oc curred in either gastrocnemius or pectoralis phosphorylase, but the group receiving sur plus En, during the same age interval, underwent a threefold increase in muscle enzyme. Dietary information is sparce in the referenced studies (14-16), but it seems reasonable to assume that the ani-
1968
BLACK, GUIRARD
mais used were fed a stock diet, which contained 4 to 7 mg of vitamin B6/kg of diet. If that assumption is correct, then, in accordance with our results in rats fed high, as well as normal vitamin levels (7 mg/kg), one would predict an accumulation of mus cle phosphorylase, as reported in the ref erenced studies (14-16) in response to dietary vitamin B6. We conclude that the dietary intake of vitamin B6 is the causa tive factor leading to muscle phosphorylase accumulation, and that neither age nor body size, per se, are directly involved. The present study does not answer the important question of whether the accumu lation of phosphorylase in response to high dietary intake of vitamin B6 is an intrinsic function of the enzyme molecule acting as a reservoir for the vitamin. However, when considered together with the evidence cited earlier showing extensive storage capacity for the vitamin (1, 2) and its mobilization during vitamin B6 deficiency ( 3, 4 ), the present experiments, demonstrat ing that the enzyme pool expands in rats surfeited with the vitamin, add consider able support to the reservoir hypothesis. It is apparent that a vitamin B8 reservoir would have physiological significance in view of the widespread role of the pyridoxal 5'-phosphate as a coenzyme in metab olism. For example, several amino acids are precursors of compounds required for normal function of the nervous system ( catecholamines, choline, serotonin,
