Validation of the Diagnostic Value of Plasma Pyridoxal S'-Phosphate Measurements in Vitamin B6 Nutrition of the Rat12 LAWRENCE LUMENG,3 MICHAEL P. RYAN ANDTING-KAI LI Indiana University School of Medicine, Departments of Medicine and Biochemistry and the Veterans Administration Hospital, Indianapolis, Indiana 46202 ABSTRACT The relationship of plasma pyridoxal 5'-phosphate (PLP) to PLP content of tissues and activities of PLP-dependent enzymes was examined to establish its value in assessing vitamin Be nutrition. Weanling rats were fed ad libitum for 9 weeks purified diets which supplied 0, 4, 12, 24, and 100 mg of pyridoxine daily. Growth increased with increasing pyridoxine intake, reaching a maximum at 24 ¿ig/day. Liver and brain PLP also increased, attaining maximal values at 12 ^g. By contrast, muscle and plasma PLP did not saturate when vitamin B6 intake was increased to 100 /ig. Erythrocytic holoenzyme activity of aspartate (Asp) aminotransferase became maximal with 24 /tg but that of alanine (Ala) aminotransferase did not. Hepatic holoenzyme activities of Ala, Asp and tyrosine aminotransferases reached maximal values with only 4 /¿gvitamin B6 but that of serine dehydratase became maximal with 12 /*g. Measurement of coenzyme saturability suggested that apoenzyme degradation, coenzyme affinity and PLP transfer determine the activities of these enzymes. It is concluded that plasma and muscle PLP behave as mobilizable storage pools and that plasma PLP is a sensitive and reliable indicator of vitamin B6 nutrition. J. Nutr. JOS: 545-553, 1978. INDEXING KEY WORDS plasma pyridoxal 5'-phosphate •tissue pyridoxal 5'-phosphate •vitamin B6 nutrition •Bc-dependent enzymes The concentration of pyridoxal 5'-phosphate (PLP) in blood plasma can now be measured reproducibly by enzymatic assay with tyrosine decarboxylase apoenzyme (1-3). As a result, plasma PLP content is being employed with increasing frequency as an indicator of the nutritional status of vitamin B8 ( 3-9 ). Past studies in man have established that plasma PLP concentration correlates with the urinary excretion of xanthurenic acid (10), the saturation of erythrocyte aspartate aminotransferase ac tivity and the PLP content of red cells (11). However, this measurement can be re garded as a useful diagnostic test only if it also correlates in some well-defined manner with the coenzyme content of vital
organs, such as liver and brain, and with skeletal muscle which, owing to its mass, contains the largest tissue store of this co enzyme (12). Ideally, it should reflect not only undernutrition of the vitamin but also storage capacity. In the present study, we have validated in the rat the usefulness of plasma PLP measurements through such correlations. In addition, we have compared the activities of several hepatic Received for publication June 21, 1977. 1Supported by a grant from the Veterans Adminis tration Hospital (MRIS 583-5246). 2Part of this work was presented at the 60th Annual Meeting of the Federation of American So cieties for Experimental Biology. Anaheim, Cali fornia, April 1976. Federation Proc. 35, 660 (1976). »Towhom reprint requests should be sent.
545
This
One
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Downloaded from https://academic.oup.com/jn/article-abstract/108/4/545/4770444 by Boston University Medical Center user on 06 July 2018
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LUMENG,
and erythrocytic PLP-dependent enzymes when vitamin B6 intake is varied.
RYAN AND LI
same day the samples were prepared. Plasma samples were extracted as de scribed previously (3) and frozen until MATERIALS AND METHODS analysis, usually within 72 hours. The whole brain, which included cere Animals and diets. Forty-two male wean bellum, medula oblongata, cerebral cortex ling rats of the Sprague-Dawley strain, and mesencephalon-diencephalon, was each weighing about 50 g, were fed ad quickly removed after decapitation, libitum purified liquid diets which supplied weighed and homogenized in 20 ml of 0, 4, 12, 24, and 100 ^g of pyridoxine (or water and 1.7 ml of 10% metaphosphoric 0, 4.9, 14.6, 29.2, and 122.6 /¿gof pyri acid (14) with a Potter-Elvehjem tissue doxine HC1) daily for 9 weeks. There were grinder. The brain homogenate was then 6 rats in each of the groups supplied with heated at 80°for 15 minutes and centri 0 and 4 ng pyridoxine and 10 rats in each fuged at 17,500 X g for 15 minutes. The of the groups on 12, 24, and 100 /tg pyri supernatant was diluted doxine per day. One rat fed the pyridoxine- deproteinated 1:10 with water and assayed for PLP. deficient diet died before completion of Samples of liver and skeletal muscle the study. The basic liquid diet4 was pre (right gastrocnemius), approximately 0.1 pared as described by Lieber and DeCarli (13) except that pyridoxine was omitted. g in size, were weighed and put individu It contained 47r/o of the total energy as ally into 10 ml of cold sodium phosphate buffer (pH 7.4, 80 HIM). These samples carbohydrates, 18% as protein and 35% as were immediately homogenized.5 Aliquots fats. The use of this basic liquid diet fa of the homogenate, 0.5 ml, were added to cilitated accurate daily adjustment of the 1.3 ml of water and 0.2 ml of 757o tricontent of pyridoxine in the diet for each chloroacetic acid. This mixture was then of the experimental groups. Based on the centrifuged at 17,500 X g for 15 minutes volume of liquid diet consumed on the preceding day, an appropriate amount of and the supernatant was extracted with pyridoxine was added to the basic liquid ether and assayed for PLP (3). For the measurement of serine dehydiet on the test day. The rats were housed individually in wire-bottom cages under dratase (EC 4.2.1.13; SDH) and tyrosine conditions of constant temperature (24°) aminotransferase (EC 2.6.1.5; TAT) ac and regulated lighting (12-hour light and tivities, livers were homogenized in 9 vol 12-hour dark schedule). There was no umes of 0.25 M sucrose containing 1 HIM other source of water than that in the dithiothreitol and 1 mM EDTA (pH 7.4) liquid diet. The rats were weighed twice and then centrifuged at 100,000 X g for 60 minutes. Aliquots of the resultant soluble weekly. were used in the assay of these Preparation of plasma and tissue extracts. fractions enzymes. Asp-AT and Ala-AT activities At the end of 9 weeks, the rats were fasted were measured in sonicated liver homogeovernight and decapitated. Blood was col lected from the cervical blood vessels in nates prepared in 0.25 M sucrose. The were sonicated 3 times in tubes containing 0.1 ml of 0.2 M ethyl ene homogenates 20-second pulses at 4°. diaminetetraacetic acid, pH 7.4. The blood Analyses. PLP was determined enzysamples were centrifuged at 600 x g for matically with tyrosine decarboxylase apo20 minutes in order to separate the plasma enzyme (3). The recoveries of authentic from the buffy coat and red cells. The buffy coat was discarded and the red cells PLP added to plasma and to tissue ho were washed twice with cold normal mogenates of brain, liver and skeletal mean ± saline and hemolyzed with twice their muscle were 90 ±8% (n=10, so), 76 ±5% (n = 5), 104 ±Q% (n = 8), packed cell volume of distilled, deionized and 111 ±2% (n = 5), respectively. Erythwater. Erythrocyte aspartate aminotransferase (EC 2.6.1.1; Asp-AT) and alanine aminotransferase (EC 2.6.1.2; Ala-AT) « Bio-Mix Diet #711 without pyridoxine, Bio-Serv Frenchtown. New Jersey. activities were determined on the hemoly- Inc..5 Polytron PT20 homogenizer, at a power setting sates with and without added PLP on the of 0. for 30 seconds.
Downloaded from https://academic.oup.com/jn/article-abstract/108/4/545/4770444 by Boston University Medical Center user on 06 July 2018
VALIDATION
OF PLASMA PYRIDOXAL 5'-P MEASUREMENTS
rocytic and hepatic Asp-AT and Ala-AT activities were assayed on hemolysates and sonicated homogenates with and without added PLP, 0.2 HIM, as described previ ously (7). Hepatic SDH activity was de termined in the presence and absence of 0.4 HIM PLP by the method of Ishikawa et al. (15) and TAT activity with and without 0.05 HIM PLP by the method of Diamondstone (16). Activity measured in the presence of added PLP is defined as total enzyme activity; that measured in the absence of PLP, holoenzyme activity. The numerical difference between the two was the apoenzyme activity. Protein concentra tion was measured by the method of Lowry et al. (17). Student's i-test (18) was used to determine the significance of the dif ference between mean values.
3 0.
547
20-1
B H
a io-
-0.5
i
O
r
24
4
PYRIDOXINE INTAKE, jig/d
Fig. 2 Effect of pyridoxine intake on the PLP content of liver and brain. Each value repre sents the mean ±SE. Values, denoted by * ( P < 0.05) and "° (P < 0.001) were signiBcantly lower than those shown by rats which were fed 100 fig pyridoxine/day.
RESULTS respectively. They are not significantly Growth response to the different levels different from each other and are similar of intake of pyridoxine. Figure 1 shows the to those reported by others (20, 21). How growth curves of the five groups of rats ever, the growth curves of the groups on on daily intakes of 0, 4, 12, 24, and 100 /¿g 0, 4, and 12 /xg pyridoxine per day devi of pyridoxine. The average initial body ated significantly from linearity when log weight was 52.2 ±1.2 (mean ±SE). The W was plotted vs 1/i. By the ninth postgrowth curves of the rats on 24 and 100 /¿g weaning week, the mean (^SE) body pyridoxine per day fit Zuckers' post-wean weights of the rats receiving 24 and 100 fj.g pyridoxine per day were 584 ±12 and ing growth equation (19) and the loga 598 ±157c of the initial weights. The rithm of the body weights (W) was weight gains of groups on 0, 4, and 12 /¿g linearly related to reciprocal time (1/i) in weeks. The slopes of these lines (k pyridoxine per day were 342 ±7, 451 ± values) exhibited by these groups were 28, and 535 ±9%, respectively. All the 3.06 ±0.04 and 3.10 ±0.04 (mean ±SE), 140 700— 600—
O—O PYRIDOXINE lOOug/d "
11
24 12
4
120 loo
50080 400—
6°
300-
40
• PLASMA O MUSCLE X »SE
o
e»
20
o i WEEKS AFTER WEANING
Fig. 1 Effect of varying daily intake of pyri doxine on body weight gain. The mean body weight at weaning was 52.2 g. Growth curves, indicated by " (P < 0.005) and "" (P < 0.001), were significantly lower than that exhibited by rats which were fed 100 /tg pyridoxine/day.
Downloaded from https://academic.oup.com/jn/article-abstract/108/4/545/4770444 by Boston University Medical Center user on 06 July 2018
12
ä •10 5
24
PYRIDOXINE INTAKE. |ig/d
Fig. 3 Effect of pyridoxine intake on the PLP content of plasma and skeletal muscle. The values, denoted by " (P
organs, such as liver and brain, and with skeletal muscle which, owing to its mass, contains the largest tissue store of this co enzyme (12). Ideally, it should reflect not only undernutrition of the vitamin but also storage capacity. In the present study, we have validated in the rat the usefulness of plasma PLP measurements through such correlations. In addition, we have compared the activities of several hepatic Received for publication June 21, 1977. 1Supported by a grant from the Veterans Adminis tration Hospital (MRIS 583-5246). 2Part of this work was presented at the 60th Annual Meeting of the Federation of American So cieties for Experimental Biology. Anaheim, Cali fornia, April 1976. Federation Proc. 35, 660 (1976). »Towhom reprint requests should be sent.
545
This
One
LWBN-Z75-EJ73
Downloaded from https://academic.oup.com/jn/article-abstract/108/4/545/4770444 by Boston University Medical Center user on 06 July 2018
546
LUMENG,
and erythrocytic PLP-dependent enzymes when vitamin B6 intake is varied.
RYAN AND LI
same day the samples were prepared. Plasma samples were extracted as de scribed previously (3) and frozen until MATERIALS AND METHODS analysis, usually within 72 hours. The whole brain, which included cere Animals and diets. Forty-two male wean bellum, medula oblongata, cerebral cortex ling rats of the Sprague-Dawley strain, and mesencephalon-diencephalon, was each weighing about 50 g, were fed ad quickly removed after decapitation, libitum purified liquid diets which supplied weighed and homogenized in 20 ml of 0, 4, 12, 24, and 100 ^g of pyridoxine (or water and 1.7 ml of 10% metaphosphoric 0, 4.9, 14.6, 29.2, and 122.6 /¿gof pyri acid (14) with a Potter-Elvehjem tissue doxine HC1) daily for 9 weeks. There were grinder. The brain homogenate was then 6 rats in each of the groups supplied with heated at 80°for 15 minutes and centri 0 and 4 ng pyridoxine and 10 rats in each fuged at 17,500 X g for 15 minutes. The of the groups on 12, 24, and 100 /tg pyri supernatant was diluted doxine per day. One rat fed the pyridoxine- deproteinated 1:10 with water and assayed for PLP. deficient diet died before completion of Samples of liver and skeletal muscle the study. The basic liquid diet4 was pre (right gastrocnemius), approximately 0.1 pared as described by Lieber and DeCarli (13) except that pyridoxine was omitted. g in size, were weighed and put individu It contained 47r/o of the total energy as ally into 10 ml of cold sodium phosphate buffer (pH 7.4, 80 HIM). These samples carbohydrates, 18% as protein and 35% as were immediately homogenized.5 Aliquots fats. The use of this basic liquid diet fa of the homogenate, 0.5 ml, were added to cilitated accurate daily adjustment of the 1.3 ml of water and 0.2 ml of 757o tricontent of pyridoxine in the diet for each chloroacetic acid. This mixture was then of the experimental groups. Based on the centrifuged at 17,500 X g for 15 minutes volume of liquid diet consumed on the preceding day, an appropriate amount of and the supernatant was extracted with pyridoxine was added to the basic liquid ether and assayed for PLP (3). For the measurement of serine dehydiet on the test day. The rats were housed individually in wire-bottom cages under dratase (EC 4.2.1.13; SDH) and tyrosine conditions of constant temperature (24°) aminotransferase (EC 2.6.1.5; TAT) ac and regulated lighting (12-hour light and tivities, livers were homogenized in 9 vol 12-hour dark schedule). There was no umes of 0.25 M sucrose containing 1 HIM other source of water than that in the dithiothreitol and 1 mM EDTA (pH 7.4) liquid diet. The rats were weighed twice and then centrifuged at 100,000 X g for 60 minutes. Aliquots of the resultant soluble weekly. were used in the assay of these Preparation of plasma and tissue extracts. fractions enzymes. Asp-AT and Ala-AT activities At the end of 9 weeks, the rats were fasted were measured in sonicated liver homogeovernight and decapitated. Blood was col lected from the cervical blood vessels in nates prepared in 0.25 M sucrose. The were sonicated 3 times in tubes containing 0.1 ml of 0.2 M ethyl ene homogenates 20-second pulses at 4°. diaminetetraacetic acid, pH 7.4. The blood Analyses. PLP was determined enzysamples were centrifuged at 600 x g for matically with tyrosine decarboxylase apo20 minutes in order to separate the plasma enzyme (3). The recoveries of authentic from the buffy coat and red cells. The buffy coat was discarded and the red cells PLP added to plasma and to tissue ho were washed twice with cold normal mogenates of brain, liver and skeletal mean ± saline and hemolyzed with twice their muscle were 90 ±8% (n=10, so), 76 ±5% (n = 5), 104 ±Q% (n = 8), packed cell volume of distilled, deionized and 111 ±2% (n = 5), respectively. Erythwater. Erythrocyte aspartate aminotransferase (EC 2.6.1.1; Asp-AT) and alanine aminotransferase (EC 2.6.1.2; Ala-AT) « Bio-Mix Diet #711 without pyridoxine, Bio-Serv Frenchtown. New Jersey. activities were determined on the hemoly- Inc..5 Polytron PT20 homogenizer, at a power setting sates with and without added PLP on the of 0. for 30 seconds.
Downloaded from https://academic.oup.com/jn/article-abstract/108/4/545/4770444 by Boston University Medical Center user on 06 July 2018
VALIDATION
OF PLASMA PYRIDOXAL 5'-P MEASUREMENTS
rocytic and hepatic Asp-AT and Ala-AT activities were assayed on hemolysates and sonicated homogenates with and without added PLP, 0.2 HIM, as described previ ously (7). Hepatic SDH activity was de termined in the presence and absence of 0.4 HIM PLP by the method of Ishikawa et al. (15) and TAT activity with and without 0.05 HIM PLP by the method of Diamondstone (16). Activity measured in the presence of added PLP is defined as total enzyme activity; that measured in the absence of PLP, holoenzyme activity. The numerical difference between the two was the apoenzyme activity. Protein concentra tion was measured by the method of Lowry et al. (17). Student's i-test (18) was used to determine the significance of the dif ference between mean values.
3 0.
547
20-1
B H
a io-
-0.5
i
O
r
24
4
PYRIDOXINE INTAKE, jig/d
Fig. 2 Effect of pyridoxine intake on the PLP content of liver and brain. Each value repre sents the mean ±SE. Values, denoted by * ( P < 0.05) and "° (P < 0.001) were signiBcantly lower than those shown by rats which were fed 100 fig pyridoxine/day.
RESULTS respectively. They are not significantly Growth response to the different levels different from each other and are similar of intake of pyridoxine. Figure 1 shows the to those reported by others (20, 21). How growth curves of the five groups of rats ever, the growth curves of the groups on on daily intakes of 0, 4, 12, 24, and 100 /¿g 0, 4, and 12 /xg pyridoxine per day devi of pyridoxine. The average initial body ated significantly from linearity when log weight was 52.2 ±1.2 (mean ±SE). The W was plotted vs 1/i. By the ninth postgrowth curves of the rats on 24 and 100 /¿g weaning week, the mean (^SE) body pyridoxine per day fit Zuckers' post-wean weights of the rats receiving 24 and 100 fj.g pyridoxine per day were 584 ±12 and ing growth equation (19) and the loga 598 ±157c of the initial weights. The rithm of the body weights (W) was weight gains of groups on 0, 4, and 12 /¿g linearly related to reciprocal time (1/i) in weeks. The slopes of these lines (k pyridoxine per day were 342 ±7, 451 ± values) exhibited by these groups were 28, and 535 ±9%, respectively. All the 3.06 ±0.04 and 3.10 ±0.04 (mean ±SE), 140 700— 600—
O—O PYRIDOXINE lOOug/d "
11
24 12
4
120 loo
50080 400—
6°
300-
40
• PLASMA O MUSCLE X »SE
o
e»
20
o i WEEKS AFTER WEANING
Fig. 1 Effect of varying daily intake of pyri doxine on body weight gain. The mean body weight at weaning was 52.2 g. Growth curves, indicated by " (P < 0.005) and "" (P < 0.001), were significantly lower than that exhibited by rats which were fed 100 /tg pyridoxine/day.
Downloaded from https://academic.oup.com/jn/article-abstract/108/4/545/4770444 by Boston University Medical Center user on 06 July 2018
12
ä •10 5
24
PYRIDOXINE INTAKE. |ig/d
Fig. 3 Effect of pyridoxine intake on the PLP content of plasma and skeletal muscle. The values, denoted by " (P
