The Journal of Nutrition. First published ahead of print November 18, 2015 as doi: 10.3945/jn.115.219378. The Journal of Nutrition Nutrient Physiology, Metabolism, and Nutrient-Nutrient Interactions
A Vitamin B-6 Antagonist from Flaxseed Perturbs Amino Acid Metabolism in Moderately Vitamin B-6–Deficient Male Rats1–3 Shyamchand Mayengbam,4 Sara Raposo,4 Michel Aliani,4,6 and James D House4–6* 4 6
Department of Human Nutritional Sciences and 5 Department of Animal Science, University of Manitoba, Winnipeg, Canada; and St-Boniface Hospital Research Centre, Winnipeg, Canada
Abstract Downloaded from jn.nutrition.org at TUFTS UNIVERSITY on November 24, 2015
Background: Pyridoxal 5#-phosphate (PLP) plays a crucial role as a cofactor in amino acid metabolism. There is a prevalence of moderate vitamin B-6 deficiency in the population that may be exacerbated through the ingestion of 1-amino D -proline (1ADP), a vitamin B-6 antagonist found in flaxseed. Objective: Given prior evidence of the impact of synthetic 1ADP on indexes of pyridoxine metabolism, the current study was designed to investigate the effects of 1ADP derived from flaxseed on amino acid metabolism in moderately vitamin B-6–deficient rats. Methods: Male weanling rats (n = 8/treatment) consumed a semipurified diet containing either 7 mg pyridoxine hydrochloride/kg diet [optimum vitamin B-6 (OB)] or 0.7 mg pyridoxine hydrochloride/kg diet [moderately vitamin B-6 deficient (MB)], each with 0 or 10 mg vitamin B-6 antagonist/kg diet, in either a synthetic form (1ADP) or as a flaxseed extract (FE), for 5 wk. At the end of the experiment, plasma vitamin B-6 and amino acid concentrations and the activities of hepatic PLP-dependent enzymes were analyzed. Results: Compared with the MB control group, plasma PLP concentrations were 26% and 69% lower, respectively, in the MB+FE and MB+1ADP rats (P # 0.001). In the MB+FE group, the plasma cystathionine concentration was 100% greater and the plasma a-aminobutyric acid and glutamic acid concentrations were 59% and 30% lower, respectively, than in the MB control group. Both synthetic 1ADP and FE significantly (P < 0.001) inhibited the in vitro hepatic activities of 2 PLP-dependent enzymes, cystathionine b-synthase (up to 44%) and cystathionine g-lyase (up to 60%), irrespective of vitamin B-6 concentrations. Because of vitamin B-6 antagonist exposure, observed perturbations in plasma biomarkers and hepatic enzyme activities were not evident or of lesser magnitude in rats consuming adequate vitamin B-6. Conclusion: The current data from a rat model provide evidence that a vitamin B-6 antagonist now prevalent in the human food supply may pose challenges to individuals of moderate vitamin B-6 status. J Nutr doi: 10.3945/jn.115.219378.
Keywords:
vitamin B-6, vitamin B-6 antagonist, 1-amino D-proline, flaxseed, amino acid
Introduction Vitamin B-6 is known for its biological role as a cofactor in myriad metabolic reactions because of its ability to link various carbon and nitrogen enzymatic reactions and its involvement in 1 Supported by the Discovery Grants Program of the Natural Sciences and Engineering Research Council of Canada (NSERC) (to MA and JDH). S Mayengbam was the recipient of an NSERC Collaborative Research and Training Experience (CREATE) - Food Advancement through Science and Training (FAST) scholarship. 2 Author disclosures: S Mayengbam, S Raposo, M Aliani, and JD House, no conflicts of interest. 3 Supplemental Table 1 is available from the ‘‘Online Supporting Material’’ link in the online posting of the article and from the same link in the online table of contents at http://jn.nutrition.org. *To whom correspondence should be addressed. E-mail: james.house@ umanitoba.ca.
the biosynthesis of biogenic amines and one-carbon units (1, 2). Usually, pyridoxal 5#-phosphate (PLP)7, the active form of vitamin B-6, binds to the e-amino group of active lysine residues contained in vitamin B-6–dependent enzymes, thus forming a Schiff base/external aldimine. The latter species then acts as a common central intermediate for all PLP-catalyzed reactions, including decarboxylation, racemization, transamination, b-elimination,
7 Abbreviations used: CBS, cystathionine b-synthase; CGL, cystathionine g-lyase; FE, flaxseed extract; FER, feed efficiency ratio; MB, moderately vitamin B-6 deficient; OB, optimum vitamin B-6; PLK, pyridoxal kinase; PLP, pyridoxal 5#-phosphate; PNHCl, pyridoxine hydrochloride; PNPOx, pyridoxine phosphate oxidase; SHMT, serine hydroxymethyltransferase; 1ADP,1-amino D-proline; 1ADPE, 1-amino D-proline equivalent; 4-PA, 4-pyridoxic acid.
ã 2016 American Society for Nutrition. Manuscript received July 6, 2015. Initial review completed July 31, 2015. Revision accepted October 22, 2015. doi: 10.3945/jn.115.219378.
Copyright (C) 2015 by the American Society for Nutrition
1 of 7
freeze dried. Then, it was finely ground and the amount of total vitamin B-6 antagonist content was quantified with the use of ultra-high– performance liquid chromatography/mass spectrometry, with synthetic 1ADP (Santa Cruz Biotechnology) and in-house linatine as standards (13). The flaxseed extract (FE) containing the concentrated vitamin B-6 antagonist was stored at 220°C until use. Animals and diet. Forty-eight male Sprague-Dawley rats weighing 112 6 9 g were purchased from the University of Manitoba Central Animal Care. They were individually housed in polypropylene cages in a room maintained at a temperature of 20 6 2°C with a 12 h light/dark rhythm at 50–70% relative humidity. After acclimatization for 1wk, rats were randomly divided into 6 groups (n = 8) and fed a semipurified diet (AIN93G, based on vitamin-free casein) containing pyridoxine hydrochloride (PNHCl) at 7 mg/kg diet [optimum vitamin B-6 (OB)] or 0.7 mg/kg diet [moderately vitamin B-6 deficient (MB)], employing a model of moderate vitamin B-6 deficiency, as previously described (15). Each group of rats also consumed ab libitum a 10 mg/kg diet of vitamin B-6 antagonist either in synthetic form (1ADP) (Santa Cruz Biotechnology) or FE or none (control) for 5 wk (Supplemental Table 1). Food intake was monitored daily and body weight was measured every week. At the end of the experiment, plasma and tissue samples were collected after 12 h of fasting for biochemical analyses. Plasma biochemical analyses. Plasma B-6 vitamers including PLP, pyridoxal, pyridoxine, and 4-pyridoxic acid (4-PA) were measured as their semicarbazide derivatives with the use of HPLC and fluorescence detection as previously described (15). Plasma total thiols, including homocysteine and cysteine, were quantified with the use of ammonium 7-fluorobenzo-2-oxa-1,3-diazole-4-sulfonate by HPLC with fluorescence detection (21). Plasma amino acid profile analysis was performed with the use of a commercially available kit (EZ:faast kit, Phenomenex) through the use of a Varian 450-GC couple with Varian 240-MS IT. Hepatic PLP-dependent enzymes activities. Liver samples (0.5 g) were homogenized with 5 mL ice-cold 50 mmol/L potassium phosphate buffer (pH 6.8) and centrifuged at 15,000 3 g and 4°C to collect the supernatant (15). Hepatic CBS enzyme activity was determined with the use of a previously described method with some modifications. The supernatant sample was incubated in a reaction mixture containing a radioactive material, 25 mmol/L of L-[U-14C]serine (;78 000 dpm/mmol) (PerkinElmer), and L-cystathionine (0.15 mmol/L), DL-homocysteine (41.67 mmol/L), S-adenosylmethionine (0.32 mmol/L), DL-propargylglycine (2.08 mmol/L), Tris (125 mmol/L), and EDTA (2.08 mmol/L). After incubation at 37°C, the radioactivity of newly formed 14C-cystathionine was counted to determine the CBS enzyme
Methods Preparation of flaxseed extract. Flaxseed was obtained from a commercial market and was defatted with the use of a Soxhlet apparatus, with hexane as a solvent. The defatted flaxseed was reground to obtain a fine powder. Vitamin B-6 antagonist from the ground, defatted flaxseed was extracted through the use of an ultrasonification method, previously described, with minor modifications, including the use of 40% isopropanol at 25°C with a 10:1 solvent-to-solid ratio for 30 min (13). The extract was concentrated under reduced pressure and 2 of 7
Mayengbam et al.
FIGURE 1 Effect of FE and 1ADP on the growth of rats fed diets containing either 7 mg or 0.7 mg PNHCl/kg diet for 5 wk. Values are means 6 SEMs; n = 8. Within a vitamin B-6 group, labeled means without a common letter differ, P , 0.05. FE, flaxseed extract; MB, moderately vitamin B-6 deficient; OB, optimum vitamin B-6; PNHCl, pyridoxine hydrochloride; 1ADP, 1-amino D-proline.
Downloaded from jn.nutrition.org at TUFTS UNIVERSITY on November 24, 2015
and aldol cleavage, and thus serves to perform diverse functions in amino acid, sugar, and lipid metabolism (2, 3). In certain amino acid metabolic pathways, including those of the trans-sulphuration and remethylation pathways of methionine metabolism, vitamin B-6 is required as a cofactor (4–6). In the trans-sulphuration pathway, PLP acts as a coenzyme for cystathionine b-synthase (CBS), which condenses homocysteine with serine to form cystathionine and cystathionine g-lyase (CGL), which converts cystathionine to cysteine by releasing a-aminobutyric acid as a by-product. In the remethylation pathway, homocysteine is converted back to methionine by coupling several enzymes, including serine hydroxymethyltransferase (SHMT). This PLP-dependent enzyme is involved in the reversible reaction to transfer a -CH2- group from serine to tetrahydrofolate to form 5,10-methylenetetrahydrofolate, with the latter also formed via the glycine cleavage system (4, 5, 7, 8). The bodyÕs vitamin B-6 status is primarily modulated by its dietary supply, and may be perturbed by the ingestion of a vitamin B-6 antagonist (9). Several drugs (isoniazide, cycloserine, penicillamine, and mestranol), as well as natural substances (gyromitrin, canavanine, agaritin, thyophylline, caffeine, and linatine) act as vitamin B-6 antagonists in the body by reducing free PLP and/or inhibiting the activity of pyridoxal kinase (PLK) and pyridoxine phosphate oxidase (PNPOx) enzymes (9–11). Linatine, a naturally occurring dipeptide of glutamic acid and 1-amino D-proline (1ADP), is a vitamin B-6 antagonist present in flaxseed. Chemically, it is 1-[(n-g-L-glutamyl)-amino]-Dproline. After hydrolysis with hydrochloric acid, linatine releases 1ADP, the active form of this vitamin B-6 antagonist (12). Previous studies have documented that the total vitamin B-6 antagonist content, expressed in 1-amino D-proline equivalents (1ADPEs; equal to 1/2 linatine + 1ADP), was 177– 437 mg/g of whole flaxseed depending on the variety sampled (12, 13). 1ADP binds with PLP to form hydrazone complexes, and these complexes can reduce the free in vivo PLP pool or cause hydrazone toxicity in the body (12, 14). We have previously shown that the toxicity of synthetic 1ADP was much more evident in animals that were moderately vitamin B-6 deficient compared with those that were replete. The oral ingestion of synthetic 1ADP at a concentration of 10 mg/kg diet caused severe impairment in homocysteine metabolism in moderately vitamin B-6–deficient rats (15). Consumption of flaxseed has been increasing because of its purported health benefits, including the reduction of risk factors associated with diabetes, cancer, and cardiovascular diseases (16–18). Concurrent ingestion of antinutrients including the vitamin B-6 antagonist 1ADP present in flaxseed by the general population, particularly those who have moderate vitamin B-6 deficiency, may be a matter of concern. Because overt vitamin B-6 deficiency is rare and moderate deficiency is prevalent (19, 20), the current study was designed to evaluate the effect of a vitamin B-6 antagonist from flaxseed on amino acid metabolism in a rodent model of moderate vitamin B-6 deficiency.
TABLE 1 Effect of FE and 1ADP on body weight, feed intake, and the feed efficiency ratio in rats fed diets containing PNHCl at either 7 mg/kg diet or 0.7 mg/kg diet for 5 wk1 Vitamin B-6 group (PNHCl) and treatment OB diet (7 mg/kg diet) OB OB+FE OB+1ADP P MB diet (0.7 mg/kg diet) MB MB+FE MB+1ADP P
Initial body weight, g
Final body weight, g
Total weight gain, g
RLW
Feed intake, g
FER
112 6 6 113 6 3 114 6 3 0.92
456 6 9 456 6 14 430 6 12 0.26
344 6 6 343 6 16 316 6 12 0.2
3.49 6 0.13 3.49 6 0.06 3.57 6 0.07 0.79
930 6 15.3 918 6 24.7 869 6 24.8 0.29
0.37 6 0.01 0.39 6 0.03 0.37 6 0.01 0.56
113 6 3 112 6 2 114 6 2 0.98
383 6 8a 373 6 7a 255 6 8b ,0.001
270 6 10a 260 6 7a 141 6 8b ,0.001
3.44 6 0.08 3.35 6 0.09 3.54 6 0.14 0.72
780 6 15.8a 770 6 18.8a 514 6 19.0b ,0.001
0.35 6 0.01a 0.34 6 0.01a 0.28 6 0.02b 0.001
1 Values are means 6 SEMs; n = 8, except for OB+1ADP, n = 7. Labeled means in a column within a vitamin B-6 group without a common letter differ, P , 0.05. FER = total weight gain/total feed intake. RLW = liver weight 3 100/body weight. FE, flaxseed extract; FER, feed efficiency ratio; MB, moderately vitamin B-6 deficient; OB, optimum vitamin B-6; PNHCl, pyridoxine hydrochloride; RLW, relative liver weight; 1ADP, 1-amino D-proline.
Statistical analysis. Data were sliced based on dietary PNHCl concentration (OB and MB) in order to determine the simple effects of antagonist exposure independent of dietary vitamin B-6 exposure, a statistical procedure used in prior work (15). Statistical analysis was then performed with the use of SPSS 16.0. Normality of the data were tested before analysis with a Shapiro-Wilk test, and, if necessary, the data were normalized by log10 transformation. A general linear model was used to compare the treatment effects within the same vitamin B-6 diet group with the use of BonferroniÕs test by setting the significance at P < 0.05. For all plasma biochemical analytes, we used ANCOVA by taking feed intake as a covariate to control, account for, and equalize the discrepancies that might have occurred because of differences in feed consumption. Otherwise, we used 1-factor ANOVA to compare the treatment effects.
Results Preparation of FE. Upon quantification by ultra-high–performance liquid chromatography/mass spectrometry, the amount TABLE 2 Effect of FE and 1ADP on plasma B-6 vitamers in rats fed diets containing PNHCl at either 7 mg/kg diet or 0.7 mg/kg diet for 5 wk1 Vitamin B-6 group (PNHCl) and treatment OB diet (7 mg/kg diet) OB OB+FE OB+1ADP P MB diet (0.7 mg/kg diet) MB MB+FE MB+1ADP P
B-6 vitamers, nmol/L PN
PLP
4-PA
PL
320 6 41.4 278 6 38.4 266 6 47.4 0.65
731 6 62.2 721 6 50.8 663 6 26.5 0.61
29.5 6 4.3 29.9 6 4.2 25.1 6 2.6 0.64
571 6 64.9a 566 6 58.9a 397 6 42.6b 0.012
164 6 21.4 112 6 6.6 72.3 6 9.4 0.15
63.4 6 3.0a 47.1 6 3.0b 19.8 6 2.0b 0.001
2.6 6 0.9 2.4 6 0.5 3.6 6 1.3 0.62
39.3 6 3.2a 29.7 6 2.3a 9.8 6 1.0b 0.003
1 Values are means 6 SEMs; n = 8, except for OB+1ADP, n = 7. Labeled means in a column within a vitamin B-6 group without a common letter differ, P , 0.05. FE, flaxseed extract; MB, moderately vitamin B-6 deficient; OB, optimum vitamin B-6; PL, pyridoxal; PLP, pyridoxal 5#-phosphate; PN, pyridoxine; PNHCl, pyridoxine hydrochloride; 1ADP, 1-amino D-proline; 4-PA, 4-pyridoxic acid.
of vitamin B-6 antagonist in freeze-dried FE was found to be 305 mg of linatine and 17 mg of 1ADP per 50 mg of sample. According to the molar calculation as mentioned in our previous study (13), the total amount of vitamin B-6 antagonist, expressed as 1ADPEs, present in a 50 mg sample was 169 mg, and this value was used in the preparation of the FE-based diets that contained 10 mg 1ADPEs/kg diet. Performance data. In rats fed the OB diets, those that were exposed to either 1ADP or FE did not show any significant differences in feed intake, final body weight, feed efficiency ratio (FER), or total weight gain compared with those fed the OB control diet (Figure 1 and Table 1). However, in rats fed the MB diets, those in the MB+1ADP group had lower feed intake, FER, and total body weight gain than those in the group fed the MB control diet (P < 0.001). As a result, final body weights were also significantly reduced (P < 0.001). Nevertheless, the rats in the MB+FE group were not different from the control rats in terms of growth performance markers. Additionally, the feeding of 1ADP, irrespective of source, did not change the relative liver weight of rats consuming diets with either optimal or moderately deficient concentrations of PNHCl (Table 1). One mortality from the OB+1ADP group was observed over the course of the study. Death was sudden and the rat did not exhibit anorexia or reduced weight gain; however, post-mortem examination did not reveal the cause of death. Plasma B-6 vitamers. In the rats fed the MB diets, plasma PLP concentrations were reduced by 26% and 69% (P # 0.001) in the rats consuming the MB+FE and MB+1ADP diets, respectively, compared with the MB control group. However, the feeding of a vitamin B-6 antagonist to rats in the OB diet groups did not significantly (P = 0.61) affect plasma PLP concentrations. On the contrary, the rats consuming OB+1ADP and MB+1ADP had a significant reduction in plasma pyridoxal concentrations compared with their respective controls (Table 2). Despite the lowered plasma pyridoxine concentrations in rats fed either vitamin B-6 antagonist, compared with the control rats, there were no significant differences between the treatments (P = 0.15) due to the confounding effect of food intake. Likewise, plasma 4-PA, the by-product of vitamin B-6 catabolism, was not affected when rats were fed either FE or 1ADP, and were stratified according to dietary vitamin B-6 supply. Vitamin B-6 and flaxseed
3 of 7
Downloaded from jn.nutrition.org at TUFTS UNIVERSITY on November 24, 2015
activity (22). The hepatic activity of CGL was determined by an enzymecoupled assay with the use of a spectrophotometric method previously described (23).
TABLE 3 Effect of FE and 1ADP on plasma sulfur-containing amino acid concentrations in rats fed diets containing PNHCl at either 7 mg/kg diet or 0.7 mg/kg diet for 5 wk1 Vitamin B-6 group (PNHCl) and treatment OB diet (7 mg/kg diet) OB OB+FE OB+1ADP P MB diet (0.7 mg/kg diet) MB MB+FE MB+1ADP P
Plasma sulfur-containing amino acids, mmol/L Met
Cys
Hcy
Cth
49.9 6 1.3 49.9 6 1.8 46.3 6 1.2 0.19
282 6 6.0a 284 6 6.9a 256 6 5.0b 0.009
10.2 6 0.7 11.4 6 0.3 12.0 6 0.5 0.09
47.6 6 0.8 48.4 6 1.0 48.0 6 1.9 0.9
280 6 6.2 263 6 6.1 249 6 4.5 0.08
8.5 6 0.5b 9.5 6 0.6b 71.7 6 15.7a ,0.001
1.7 6 0.0c 2.2 6 0.1b 3.4 6 0.2a ,0.001 2.3 6 0.1c 4.6 6 0.9b 32.2 6 5.9a ,0.001
Plasma sulfur containing amino acids. Besides B-6 vitamers, plasma homocysteine and cystathionine concentrations are important biomarkers of vitamin B-6 deficiency (24, 25). In the current study, we found increases in plasma homocysteine and cystathionine concentrations due to 1ADP exposure (P < 0.001). Plasma homocysteine concentrations were elevated by 744% in the MB+1ADP rats compared with the MB control
Other plasma amino acids. The concentrations of other plasma free amino acids are given in Table 4. In the MB diet groups, plasma a-aminobutyric acid (59%; P < 0.001) and glutamic acid (30%; P = 0.017) were significantly reduced in MB+FE rats compared with control, with the greater effect observed in MB+1ADP rats. Additionally, plasma glycine (P = 0.02) concentration was elevated, and plasma serine (P = 0.013) and asparagine (P = 0.016) concentrations were reduced in MB+1ADP rats. We also found a significant reduction (P # 0.013) in the plasma a-aminobutyric acid concentrations of OB+1ADP rats. Other plasma amino acids given in Table 4 were not affected by the oral ingestion of either FE or 1ADP. Hepatic PLP-dependent enzymes activities. In the current study, we found a significant reduction (P < 0.001) in hepatic CBS and CGL enzyme activities (Figure 2) from either FE or
TABLE 4 Effect of FE and 1ADP on major plasma free amino acids in rats fed diets containing PNHCl at either 7 mg/kg diet or 0.7 mg/kg diet for 5 wk1 OB diet (PNHCl: 7 mg/kg diet), mmol/L Plasma amino acid Alanine Sarcosine Glycine a-Aminobutyric acid Valine Leucine Isoleucine Threonine Serine Proline Asparagine 4-Hydroxyproline Glutamic acid Phenylalanine a-Aminoadipic acid Glutamine Ornithine Lysine Histidine Tyrosine Proline-hydroxyproline Tryptophan
OB 619 10.8 285 56.1 184 148 92.7 682 603 171 96.1 64.9 206 64.1 2.0 956 60.5 303 61.8 58.7 23.1 82.3
6 27.1 6 0.1 6 22.2 6 4.4a 6 9.4 6 7.8 6 5.2 6 71.7 6 60.1 6 7.1 6 5.6 6 6.6 6 10.6 6 1.8 6 0.2 6 97.2 6 2.6 6 13.7 6 5.1 6 1.9 6 1.3 6 7.2
OB+FE 706 6 10.9 6 297 6 44.3 6 198 6 158 6 96.0 6 590 6 472 6 168 6 94.8 6 55.7 6 213 6 69.2 6 2.3 6 778 6 66.1 6 271 6 59.4 6 56.5 6 20.3 6 67.7 6
25.2 0.1 19.4 4.4a,b 8.8 8.2 5.2 39.9 38.9 4.4 3.1 4.6 4.9 1.4 0.2 93.4 4.2 7.9 5.9 2.1 0.1 6.2
OB+1ADP 619 10.9 289 33.0 177 143 86.1 444 497 162 97.0 59.7 201 65.0 2.3 867 62.4 273 60.3 52.6 20.5 78.4
6 39.5 6 0.1 6 15.5 6 3.3b 6 6.0 6 6.2 6 4.5 6 38.2 6 45.1 6 6.6 6 4.3 6 6.3 6 20.2 6 1.7 6 0.2 6 114.2 6 2.6 6 12.9 6 4.5 6 1.9 6 0.3 6 8.2
MB diet (PNHCl: 0.7 mg/kg diet), mmol/L P 0.08 0.33 0.8 0.013 0.26 0.42 0.46 0.05 0.2 0.33 0.94 0.56 0.63 0.09 0.65 0.49 0.48 0.2 0.92 0.29 0.05 0.34
MB 565 10.8 442 45.9 188 147 90.6 766 585 183 99.3 65.1 197 62.4 2.5 962 86.5 272 62.7 51.3 21.1 84.4
6 16.2 6 0.1 6 24.2b 6 4.0a 6 8.3 6 6.5 6 4.2 6 90.6 6 49.9a 6 8.5 6 4.7a 6 6.2 6 14.0a 6 1.0 6 0.1 6 119 6 7.4 6 16.1 6 5.3 6 1.5 6 0.3 6 6.9
MB+FE 542 6 10.9 6 511 6 19.0 6 190 6 147 6 92.4 6 648 6 469 6 188 6 87.3 6 59.9 6 152 6 61.6 6 2.9 6 797 6 91.6 6 257 6 65.7 6 52.0 6 20.3 6 71.1 6
29.8 0.1 38.5b 1.9b 7.1 6.4 4.7 64.9 23.5a,b 4.2 5.8a,b 6.3 8.6b 2.2 0.3 92.8 8.2 13.2 6.5 1.9 0.2 5.0
MB+1ADP 494 11.1 899 13.8 151.5 136 82.9 486 353 186 79.1 51.4 129 57.7 2.5 759 87.1 210 84.0 49.1 20.5 72.3
6 30.5 6 0.1 6 74.4a 6 1.1b 6 8.7 6 9.8 6 6.0 6 43.2 6 32.4b 6 10.6 6 4.4b 6 5.6 6 6.0b 6 1.4 6 0.2 6 81.8 6 5.9 6 13.3 6 8.6 6 2.3 6 0.3 6 6.6
P 0.25 0.71 0.02 ,0.001 0.19 0.26 0.23 0.35 0.013 0.89 0.013 0.87 0.017 0.21 0.38 0.26 0.88 0.16 0.4 0.31 0.05 0.33
1 Values are means 6 SEMs; n = 8, except OB+1ADP, n = 7. Labeled means in a row within a vitamin B-6 group without a common letter differ, P , 0.05. FE, flaxseed extract; MB, moderately vitamin B-6 deficient; OB, optimum vitamin B-6; PNHCl, pyridoxine hydrochloride; 1ADP, 1-amino D-proline.
4 of 7
Mayengbam et al.
Downloaded from jn.nutrition.org at TUFTS UNIVERSITY on November 24, 2015
Values are means 6 SEMs; n = 8, except for OB+1ADP, n = 7. Labeled means in a column within a vitamin B-6 group without a common letter differ, P , 0.05. Cth, cystathionine; Cys, cysteine; FE, flaxseed extract; Hcy, homocysteine; MB, moderately vitamin B-6 deficient; Met, methionine; OB, optimum vitamin B-6; PNHCl, pyridoxine hydrochloride; 1ADP, 1-amino D-proline.
1
group, but no changes were observed in the MB+FE rats (Table 3). However, plasma homocysteine concentrations were not significantly affected by exposure to a vitamin B-6 antagonist (either FE or 1ADP) in the OB diet groups. In the MB diet groups, there were profound increases in plasma cystathionine concentrations in rats consuming MB+FE (100%) or MB+1ADP (1300%). A similar pattern was also exhibited for rats in the OB diet groups (Table 3). Somewhat unexpectedly, a significant reduction (P = 0.005) in plasma cysteine concentration was observed in rats in the OB+1ADP group compared with control, but this was not the case in the MB diet groups. There was no significant effect of vitamin B-6 antagonist exposure on plasma methionine concentrations (Table 3).
1ADP treatment compared with the respective controls. Hepatic CGL enzyme activity was reduced relative to the MB group by ;48% in MB+FE and MB+1ADP rats, whereas the reduction was 26% and 60% for OB+FE and OB+1ADP rats, respectively, in the OB diet group. In the case of hepatic CBS enzyme activity, MB+1ADP rats had a higher degree of enzyme inhibition (44% reduction) than did the MB+FE rats (26% reduction). However, in the OB diet groups, the reductions were of a similar magnitude, being reduced by ;31% for both the OB+FE and OB+ 1ADP rats.
Discussion
FIGURE 2 Effect of FE and 1ADP on liver CBS (A) and CGL (B) enzyme activities in rats fed diets containing either 7 mg or 0.7 mg PNHCl/kg diet for 5 wk. Values are means 6 SEMs; n = 8.Within a vitamin B-6 group, labeled means without a common letter differ, P , 0.05. CBS, cystathionine b-synthase; CGL, cystathionine g-lyase; FE, flaxseed extract; MB, moderately vitamin B-6 deficient; OB, optimum vitamin B-6; PNHCl, pyridoxine hydrochloride; 1ADP, 1-amino D-proline.
Vitamin B-6 and flaxseed
5 of 7
Downloaded from jn.nutrition.org at TUFTS UNIVERSITY on November 24, 2015
Dietary vitamin B-6 has a substantial impact on the concentration of plasma PLP (9, 26). In humans, a plasma PLP concentration of 20–30 nmol/L or a vitamin B-6 intake of 0.7 mg/d indicates marginal vitamin B-6 status (4, 27). Within the general population, vitamin B-6 inadequacy is prevalent in 24% of the American population and 21% of the Canadian elderly (>50 y) population (19, 28). Inadequate vitamin B-6 status has also been implicated in the development of many
common diseases, including chronic inflammation, colorectal cancer, and cardiovascular diseases (29, 30). The consumption of compounds possessing vitamin B-6 antagonist activity concurrent with marginal vitamin B-6 deficiency may therefore increase the risk of the development of metabolic defects or disease associated with low vitamin B-6 status (14, 31). As such, the current study was designed to investigate the potential responses in biomarkers elicited by nutritional co-insults from the oral consumption of a flaxseed-derived component in a model of moderate vitamin B-6 deficiency. Vitamin B-6 deficiency normally causes reductions in food intake, weight gain, and FER in growing animals, signifying overt defects in intermediary metabolism, as well as nitrogen retention during suboptimal vitamin B-6 status (15, 32, 33). In the present study, rats in the MB+1ADP group exhibited a significant reduction in FER as well as weight gain compared with other groups. These data support previous findings from a study that examined the impact of feeding a vitamin B-6 antagonist, 4-deoxypyridoxine, which led to significantly lower body weights in rats of low vitamin B-6 status than in their pairfed counterparts (34). Tissue concentrations of the classic vitamin B-6 markers, including PLP, pyridoxal, pyridoxine, and 4-PA, primarily reflect the nutritional supply of pyridoxine, but can be affected by the ingestion of vitamin B-6 inhibitors (9, 10, 35). In the current study, the significant alteration of major vitamin B-6 markers— developed as a result of vitamin B-6 antagonist administration— is in line with the findings of previous studies (15, 31, 36). Our data showed that FE reduced plasma PLP concentrations, with significant evidence in rats from the MB diet groups, indicating a potential binding ability of naturally occurring 1ADP to endogenous B-6 vitamers. Additionally, the synthetic 1ADP indicated a stronger and wider affinity to B-6 vitamers over naturally occurring ones, because it had significantly reduced both plasma PLP and pyridoxal concentrations. Therefore, the current study provided evidence that oral ingestion of a vitamin B-6 antagonist, either in the form of synthetic 1ADP or as an FE, reduced the overall B-6 vitamer pool in the body. One of the main reasons for lowering in vivo B-6 vitamers in the current study might be the result of the formation of hydrazone complexes of 1ADP with freely available endogenous PLP and pyridoxal. These findings, also suggested by others, were more pronounced during low vitamin B-6-status (10, 14). Other potential mechanisms include a vitamin B-6 antagonist–induced inactivation of vitamin B-6 salvage enzymes such as PLK and PNPOx (11) and increased urinary excretion of B-6 vitamers during vitamin B-6 antagonist exposure (37). It is well known that the PLK and PNPOx enzymes play a crucial role in maintaining the homeostasis in vivo of B-6 vitamers through their phosphorylation and interconversion (11, 38). Interestingly, their activities have been shown to be inhibited by several vitamin B-6 antagonists, such as isoniazid, cycloserine, and dopamine, which have a similar mode of action as 1ADP, causing in vivo PLP deficiency (11). However, further work is needed to characterize the effect of this vitamin B-6 antagonist on in vivo vitamin B-6 homeostasis and its catabolism. A deficiency of vitamin B-6 alters the plasma amino acid profile because of the involvement of this cofactor in numerous PLP-dependent enzymes, particularly those involved in transamination, trans-sulphuration, and decarboxylation reactions (6). For instance, administration of the vitamin B-6 antagonists DL penicillamine, thiosemicarbazide, and semicarbazide-HCl significantly reduced g-aminobutyric acid content in the mouse brain (39). Additionally, an intraperitoneal injection of 1ADP in
6 of 7
Mayengbam et al.
flaxseed on a population presenting with moderate vitamin B-6 deficiency. However, the bioefficacy of naturally occurring bioactive compounds largely depends on their bioavailability. For example, synthetic drugs tend to have better liberation, absorption, distribution, and metabolism than do naturally occurring bioactive components (48). The major vitamin B-6 antagonist factor present in flaxseed is the dipeptide linatine, which must liberate free 1ADP via the hydrolysis of the gglutamyl bond attached to glutamic acid in the gastrointestinal tract in order to induce in vivo vitamin B-6 antagonist toxicity. Incomplete hydrolysis and/or reduced absorption and metabolism of the linatine contained in FE might be responsible for the lowered potency of flaxseed-derived 1ADP compared with the synthetic 1ADP. Nevertheless, flaxseed-derived 1ADP did demonstrate an ability to perturb certain markers of amino acid and vitamin B-6 metabolism in the current study. To conclude, the present study provided evidence that the oral ingestion of vitamin B-6 antagonist, either from flaxseed or a synthetic source, lowered free in vivo B-6 vitamers potentially because of hydrazone formation, thus impairing amino acid metabolism. The most intriguing finding of this study was the ability of FE to inhibit the activities of PLP-dependent enzymes. However, further investigations are necessary to better understand the mechanism of action of 1ADP and the protective role of vitamin B-6 during vitamin B-6 antagonist toxicity. Additional opportunities also exist to investigate the long-term effect of this naturally occurring vitamin B-6 antagonist linked to flaxseed on other metabolic pathways. Acknowledgments We thank Haifeng Yang for technical assistance. SM and JDH designed the research; SM and SR conducted the research and analyzed the data; SM wrote the manuscript; MA and JDH reviewed the manuscript; and JDH had primary responsibility for the final content. All authors read and approved the final manuscript.
References 1.
Christen P, Mehta PK. From cofactor to enzymes. The molecular evolution of pyridoxal-5#-phosphate-dependent enzymes. Chem Rec 2001;1:436–47. 2. Hellmann H, Mooney S. Vitamin B6: A molecule for human health? Molecules 2010;15:442–59. 3. Toney MD. Reaction specificity in pyridoxal phosphate enzymes. Arch Biochem Biophys 2005;433:279–87. 4. Gregory III JF, Park Y, Lamers Y, Bandyopadhyay N, Chi YY, Lee K, Kim S, da Silva V, Hove N, Ranka S, et al. Metabolomic analysis reveals extended metabolic consequences of marginal vitamin B-6 deficiency in healthy human subjects. PLoS One 2013;8:e63544. 5. Stipanuk MH. Sulfur amino acid metabolism: Pathways for production and removal of homocysteine and cysteine. Annu Rev Nutr 2004;24:539–77. 6. Bowling FG. Pyridoxine supply in human development. Semin Cell Dev Biol 2011;22:611–8. 7. Medici V, Peerson JM, Stabler SP, French SW, Gregory, 3rd JF, Virata MC, Albanese A, Bowlus CL, Devaraj S, Panacek EA, et al. Impaired homocysteine transsulfuration is an indicator of alcoholic liver disease. J Hepatol 2010;53:551–7. 8. House JD, Jacobs RL, Stead LM, Brosnan ME, Brosnan JT. Regulation of homocysteine metabolism. Adv Enzyme Regul 1999;39:69–91. 9. Spinneker A, Sola R, Lemmen V, Castillo MJ, Pietrzik K, Gonzalez´ Gross M. Vitamin B6 status, deficiency and its consequences - An overview. Nutr Hosp 2007;22:7–24. 10. Klosterman JH. Vitamin B6 antagonists of natural origin. Journal of Agricultural and Food Chemistry. 1974;22–1:13–6.
Downloaded from jn.nutrition.org at TUFTS UNIVERSITY on November 24, 2015
rats led to a significant increase in plasma a-aminoadipic acid, citrulline, and cystathionine concentrations (14), whereas perfusion of 1ADP into an isolated rat liver significantly reduced the synthesis of carnitine from lysine residues (40), thus signifying major perturbations in amino acid metabolism. Several studies have demonstrated significantly higher plasma homocysteine and cystathionine concentrations in both animal models and humans when subjected to a vitamin B-6–restricted diet (15, 24, 25, 26). In the current study, the significant increase in plasma homocysteine that was observed in response to oral 1ADP exposure likely was due to the inhibition of hepatic CBS activity in the trans-sulphuration pathway, a condition that was more pronounced under low vitamin B-6 status. The latter mechanism is supported by the evidence that plasma homocysteine concentration is higher (40-fold) in mice lacking CBS activity than in wild-type mice (41). Additionally, hepatic CGL enzyme activity also was inhibited because of the feeding of a vitamin B-6 antagonist from either flaxseed or synthetic 1ADP, leading to increases in plasma cystathionine and a significant reduction in plasma a-aminobutyric acid concentrations. A similar pattern of inhibition of CBS and CGL enzyme activities has been reported in the livers of vitamin B6–deficient animals (24, 26, 42). The current findings support the contention that moderate vitamin B-6 deficiency as well as oral exposure to vitamin B-6 antagonists were able to independently inhibit activities of PLP-dependent enzymes, including hepatic CBS and CGL, in rats. Glycine decarboxylation through the glycine cleavage system and the conversion of glycine to serine by SHMT in one-carbon metabolism also require PLP. An inhibition of SHMT and/or the glycine cleavage system have been speculated to be the mechanisms explaining the shifts in plasma glycine-to-serine ratios during low vitamin B-6 status (25, 26, 43). Therefore, one of the reasons for the elevation of plasma glycine and reduction of plasma serine concentrations in the current study might be the impairment of these 2 enzymes, which led to reduced transformation of in vivo glycine to serine. Additionally, reductions in plasma asparagine and glutamic acid concentrations as a result of 1ADP feeding during low vitamin B-6 status might be related to the impairment of other PLP-dependent enzymes involved in the transamination and interconversion of amino acids. Our data are supported by a previous study by Swendseid et al. (43), who found higher concentrations of cystathionine and glycine and lower concentrations of serine and total glutamine and asparagine in the plasma of vitamin B-6–deficient rats. Responses induced by dietary vitamin B-6 deficiency or the exposure to vitamin B-6 antagonists on the amino acid profile can exhibit variability, likely related to the duration or severity of the imposed deficiency (4, 14, 44). Gregory et al. (4) recently showed that marginal vitamin B-6 deficiency alone can cause minor perturbations in metabolic indexes, including in vivo amino acid profiles, and a concurrent exposure to synthetic vitamin B-6 antagonist further exacerbated vitamin B-6 status (15). Interestingly, the current data provide evidence of a perturbation of certain vitamin B-6 markers due to the co-insult of a flaxseed-derived vitamin B-6 antagonist during moderate vitamin B-6 deficiency when provided at a dose of 0.5 mg 1ADPEs kg21 body weight d21. Consumption of 25–30 g/d of flaxseed, a serving size recommended by some (45–47), will result in the intake of 0.15 mg 1ADPEs kg21 body weight d21 (assuming a body weight of 65 kg), a concentration within the same order of magnitude as that provided in the current study. As such, our data serve to highlight the potential deleterious effects of the oral exposure to a vitamin B-6 antagonist linked to
30. Larsson SC, Orsini N, Wolk A. Vitamin B6 and risk of colorectal cancer: A meta-analysis of prospective studies. JAMA 2010;303:1077–83. 31. Chang SJ. Vitamin B6 antagonists alter the function and ultrastructure of mice endothelial cells. J Nutr Sci Vitaminol (Tokyo) 2000;46:149–53. 32. Wolfson M, Kopple JD. The effect of vitamin B6 deficiency on food intake, growth, and renal function in chronically azotemic rats. JPEN J Parenter Enteral Nutr 1987;11:398–402. 33. Kirchgessner M, Kosters WW. Influence of vitamin B 6 deficiency in early weaned piglets on the digestibility and conversion of protein and energy. Arch Tierernahr 1977;27:299–308. 34. Coburn SP, Mahuren JD, Schaltenbrand WE, Wostmann BS, Madsen D. Effects of vitamin B-6 deficiency and 4#- deoxypyridoxine on pyridoxal phosphate concentrations, pyridoxine kinase and other aspects of metabolism in the rat. J Nutr 1981;111:391–8. 35. Lumeng L, Lui A, Li TK. Plasma content of B6 vitamers and its relationship to hepatic vitamin B6 metabolism. J Clin Invest 1980;66: 688–95. 36. Jaffe IA. Antivitamin B6 effect of D-penicillamine. Ann N Y Acad Sci 1969;166:57–60. 37. Kajiwara T, Matsuda M. Effects of penicillamine on distribution of B6 vitamens in rat urine. J Nutr Sci Vitaminol (Tokyo) 1978;24:1–7. 38. McCormick DB, Chen H. Update on interconversions of vitamin B-6 with its coenzyme. J Nutr 1999;129:325–7. 39. Abe M, Matsuda M. Effect of antivitamin B6 on regional GABA metabolism in mouse brain and its relation to convulsions. J Nutr Sci Vitaminol (Tokyo) 1979;25:459–68. 40. Dunn WA, Aronson NN, Jr., Englard S. The effects of 1-amino-Dproline on the production of carnitine from exogenous protein-bound trimethyllysine by the perfused rat liver. J Biol Chem 1982;257:7948– 51. 41. Watanabe M, Osada J, Aratani Y, Kluckman K, Reddick R, Malinow MR, Maeda N. Mice deficient in cystathionine beta-synthase: animal models for mild and severe homocyst(e)inemia. Proc Natl Acad Sci USA 1995;92:1585–9. 42. Finkelstein JD, Chalmers FT. Pyridoxine effects on cystathionine synthase in rat liver. J Nutr 1970;100:467–9. 43. Swendseid ME, Villalobos J, Friedrich B. Free amino acids in plasma and tissues of rats fed a vitamin B6-deficient diet. J Nutr 1964;82:206–8. 44. da Silva VR, Rios-Avila L, Lamers Y, Ralat MA, Midttun O, Quinlivan EP, Garrett TJ, Coats B, Shankar MN, Percival SS, et al. Metabolite profile analysis reveals functional effects of 28-day vitamin B-6 restriction on one-carbon metabolism and tryptophan catabolic pathways in healthy men and women. J Nutr 2013;143:1719–27. 45. Edel AL, Aliani M, Pierce GN. Supported liquid extraction in the quantitation of plasma enterolignans using isotope dilution GC/MS with application to flaxseed consumption in healthy adults. J Chromatogr B Analyt Technol Biomed Life Sci 2013;912:24–32. 46. Caligiuri SP, Aukema HM, Ravandi A, Guzman R, Dibrov E, Pierce GN. Flaxseed consumption reduces blood pressure in patients with hypertension by altering circulating oxylipins via an alpha-linolenic acid-induced inhibition of soluble epoxide hydrolase. Hypertension 2014;64:53–9. 47. Wu H, Pan A, Yu Z, Qi Q, Lu L, Zhang G, Yu D, Zong G, Zhou Y, Chen X, et al. Lifestyle counseling and supplementation with flaxseed or walnuts influence the management of metabolic syndrome. J Nutr 2010;140:1937–42. 48. Rein MJ, Renouf M, Cruz-Hernandez C, Actis-Goretta L, Thakkar SK, da Silva Pinto M. Bioavailability of bioactive food compounds: a challenging journey to bioefficacy. Br J Clin Pharmacol 2013;75:588–602.
Vitamin B-6 and flaxseed
7 of 7
Downloaded from jn.nutrition.org at TUFTS UNIVERSITY on November 24, 2015
11. di Salvo ML, Contestabile R, Safo MK. Vitamin B6 salvage enzymes: Mechanism, structure and regulation. Biochim Biophys Acta 2011;1814:1597–608. 12. Klosterman HJ, Lamoureux GL, Parsons JL. Isolation, characterization, and synthesis of linatine. A vitamin B6 antagonist from flaxseed (Linum usitatissimum). Biochemistry 1967;6:170–7. 13. Mayengbam S, Yang H, Barthet V, Aliani M, House JD. Identification, characterization, and quantification of an anti-pyridoxine factor from flaxseed using ultrahigh-performance liquid chromatography-mass spectrometry. J Agric Food Chem 2014;62:419–26. 14. Sasaoka K, Ogawa T, Moritoki K, Kimoto M. Antivitamin B6 effect of 1 aminoproline on rats. Biochim Biophys Acta 1976;428:396–402. 15. Mayengbam S, Raposo S, Aliani M, House JD. Oral exposure to the anti-pyridoxine compound 1-amino D-proline further perturbs homocysteine metabolism through the transsulphuration pathway in moderately vitamin B6-deficient rats. J Nutr Biochem 2015;26:241–9. 16. Carraro JCC, de Souza Dantas MI, Espeschit ACR, Martino HSD, Ribeiro SMR. Flaxseed and human health: Reviewing benefits and adverse effects. Food Rev Int 2012;28:203–30. 17. Prasad K. Flaxseed and cardiovascular health. J Cardiovasc Pharmacol 2009;54:369–77. 18. Hall, 3rd C, Tulbek MC, Xu Y. Flaxseed. Adv Food Nutr Res 2006;51:1–97. 19. Morris MS, Picciano MF, Jacques PF, Selhub J. Plasma pyridoxal 5#-phosphate in the US population: The National Health and Nutrition Examination Survey, 2003–2004. Am J Clin Nutr 2008;87:1446–54. 20. Haller J, Lowik MR, Ferry M, Ferro-Luzzi A. Nutritional status: blood vitamins A, E, B6, B12, folic acid and carotene. Euronut SENECA investigators. Eur J Clin Nutr 1991;45 Suppl 3:63–82. 21. Pfeiffer CM, Huff DL, Gunter EW. Rapid and accurate HPLC assay for plasma total homocysteine and cysteine in a clinical laboratory setting. Clin Chem 1999;45:290–2. 22. Taoka S, Ohja S, Shan X, Kruger WD, Banerjee R. Evidence for hememediated redox regulation of human cystathionine b- synthase activity. J Biol Chem 1998;273:25179–84. 23. Stipanuk MH. Effect of excess dietary methionine on the catabolism of cysteine in rats. J Nutr 1979;109:2126–39. 24. Zhang Z, Kebreab E, Jing M, Rodriguez-Lecompte JC, Kuehn R, Flintoft M, House JD. Impairments in pyridoxine-dependent sulphur amino acid metabolism are highly sensitive to the degree of vitamin B6 deficiency and repletion in the pig. Animal 2009;3:826–37. 25. Lamers Y, Williamson J, Ralat M, Quinlivan EP, Gilbert LR, Keeling C, Stevens RD, Newgard CB, Ueland PM, Meyer K, et al. Moderate dietary vitamin B-6 restriction raises plasma glycine and cystathionine concentrations while minimally affecting the rates of glycine turnover and glycine cleavage in healthy men and women. J Nutr 2009;139:452–60. 26. Martinez M, Cuskelly GJ, Williamson J, Toth JP, Gregory, III JF. Vitamin B-6 deficiency in rats reduces hepatic serine hydroxymethyltransferase and cystathionine b-synthase activities and rates of in vivo protein turnover, homocysteine remethylation and transsulfuration. J Nutr 2000;130:1115–23. 27. Mashiyama ST, Hansen CM, Roitman E, Sarmiento S, Leklem JE, Shultz TD, Ames BN. An assay for uracil in human DNA at baseline: effect of marginal vitamin B6 deficiency. Anal Biochem 2008;372:21–31. 28. Statistics Canada . Vitamin B6: Usual intakes from food. 2004. Report No.: Cycle 2.2, Nutrition. 29. Lotto V, Choi SW, Friso S. Vitamin B6: a challenging link between nutrition and inflammation in CVD. Br J Nutr 2011;106:183–95.
A Vitamin B-6 Antagonist from Flaxseed Perturbs Amino Acid Metabolism in Moderately Vitamin B-6–Deficient Male Rats1–3 Shyamchand Mayengbam,4 Sara Raposo,4 Michel Aliani,4,6 and James D House4–6* 4 6
Department of Human Nutritional Sciences and 5 Department of Animal Science, University of Manitoba, Winnipeg, Canada; and St-Boniface Hospital Research Centre, Winnipeg, Canada
Abstract Downloaded from jn.nutrition.org at TUFTS UNIVERSITY on November 24, 2015
Background: Pyridoxal 5#-phosphate (PLP) plays a crucial role as a cofactor in amino acid metabolism. There is a prevalence of moderate vitamin B-6 deficiency in the population that may be exacerbated through the ingestion of 1-amino D -proline (1ADP), a vitamin B-6 antagonist found in flaxseed. Objective: Given prior evidence of the impact of synthetic 1ADP on indexes of pyridoxine metabolism, the current study was designed to investigate the effects of 1ADP derived from flaxseed on amino acid metabolism in moderately vitamin B-6–deficient rats. Methods: Male weanling rats (n = 8/treatment) consumed a semipurified diet containing either 7 mg pyridoxine hydrochloride/kg diet [optimum vitamin B-6 (OB)] or 0.7 mg pyridoxine hydrochloride/kg diet [moderately vitamin B-6 deficient (MB)], each with 0 or 10 mg vitamin B-6 antagonist/kg diet, in either a synthetic form (1ADP) or as a flaxseed extract (FE), for 5 wk. At the end of the experiment, plasma vitamin B-6 and amino acid concentrations and the activities of hepatic PLP-dependent enzymes were analyzed. Results: Compared with the MB control group, plasma PLP concentrations were 26% and 69% lower, respectively, in the MB+FE and MB+1ADP rats (P # 0.001). In the MB+FE group, the plasma cystathionine concentration was 100% greater and the plasma a-aminobutyric acid and glutamic acid concentrations were 59% and 30% lower, respectively, than in the MB control group. Both synthetic 1ADP and FE significantly (P < 0.001) inhibited the in vitro hepatic activities of 2 PLP-dependent enzymes, cystathionine b-synthase (up to 44%) and cystathionine g-lyase (up to 60%), irrespective of vitamin B-6 concentrations. Because of vitamin B-6 antagonist exposure, observed perturbations in plasma biomarkers and hepatic enzyme activities were not evident or of lesser magnitude in rats consuming adequate vitamin B-6. Conclusion: The current data from a rat model provide evidence that a vitamin B-6 antagonist now prevalent in the human food supply may pose challenges to individuals of moderate vitamin B-6 status. J Nutr doi: 10.3945/jn.115.219378.
Keywords:
vitamin B-6, vitamin B-6 antagonist, 1-amino D-proline, flaxseed, amino acid
Introduction Vitamin B-6 is known for its biological role as a cofactor in myriad metabolic reactions because of its ability to link various carbon and nitrogen enzymatic reactions and its involvement in 1 Supported by the Discovery Grants Program of the Natural Sciences and Engineering Research Council of Canada (NSERC) (to MA and JDH). S Mayengbam was the recipient of an NSERC Collaborative Research and Training Experience (CREATE) - Food Advancement through Science and Training (FAST) scholarship. 2 Author disclosures: S Mayengbam, S Raposo, M Aliani, and JD House, no conflicts of interest. 3 Supplemental Table 1 is available from the ‘‘Online Supporting Material’’ link in the online posting of the article and from the same link in the online table of contents at http://jn.nutrition.org. *To whom correspondence should be addressed. E-mail: james.house@ umanitoba.ca.
the biosynthesis of biogenic amines and one-carbon units (1, 2). Usually, pyridoxal 5#-phosphate (PLP)7, the active form of vitamin B-6, binds to the e-amino group of active lysine residues contained in vitamin B-6–dependent enzymes, thus forming a Schiff base/external aldimine. The latter species then acts as a common central intermediate for all PLP-catalyzed reactions, including decarboxylation, racemization, transamination, b-elimination,
7 Abbreviations used: CBS, cystathionine b-synthase; CGL, cystathionine g-lyase; FE, flaxseed extract; FER, feed efficiency ratio; MB, moderately vitamin B-6 deficient; OB, optimum vitamin B-6; PLK, pyridoxal kinase; PLP, pyridoxal 5#-phosphate; PNHCl, pyridoxine hydrochloride; PNPOx, pyridoxine phosphate oxidase; SHMT, serine hydroxymethyltransferase; 1ADP,1-amino D-proline; 1ADPE, 1-amino D-proline equivalent; 4-PA, 4-pyridoxic acid.
ã 2016 American Society for Nutrition. Manuscript received July 6, 2015. Initial review completed July 31, 2015. Revision accepted October 22, 2015. doi: 10.3945/jn.115.219378.
Copyright (C) 2015 by the American Society for Nutrition
1 of 7
freeze dried. Then, it was finely ground and the amount of total vitamin B-6 antagonist content was quantified with the use of ultra-high– performance liquid chromatography/mass spectrometry, with synthetic 1ADP (Santa Cruz Biotechnology) and in-house linatine as standards (13). The flaxseed extract (FE) containing the concentrated vitamin B-6 antagonist was stored at 220°C until use. Animals and diet. Forty-eight male Sprague-Dawley rats weighing 112 6 9 g were purchased from the University of Manitoba Central Animal Care. They were individually housed in polypropylene cages in a room maintained at a temperature of 20 6 2°C with a 12 h light/dark rhythm at 50–70% relative humidity. After acclimatization for 1wk, rats were randomly divided into 6 groups (n = 8) and fed a semipurified diet (AIN93G, based on vitamin-free casein) containing pyridoxine hydrochloride (PNHCl) at 7 mg/kg diet [optimum vitamin B-6 (OB)] or 0.7 mg/kg diet [moderately vitamin B-6 deficient (MB)], employing a model of moderate vitamin B-6 deficiency, as previously described (15). Each group of rats also consumed ab libitum a 10 mg/kg diet of vitamin B-6 antagonist either in synthetic form (1ADP) (Santa Cruz Biotechnology) or FE or none (control) for 5 wk (Supplemental Table 1). Food intake was monitored daily and body weight was measured every week. At the end of the experiment, plasma and tissue samples were collected after 12 h of fasting for biochemical analyses. Plasma biochemical analyses. Plasma B-6 vitamers including PLP, pyridoxal, pyridoxine, and 4-pyridoxic acid (4-PA) were measured as their semicarbazide derivatives with the use of HPLC and fluorescence detection as previously described (15). Plasma total thiols, including homocysteine and cysteine, were quantified with the use of ammonium 7-fluorobenzo-2-oxa-1,3-diazole-4-sulfonate by HPLC with fluorescence detection (21). Plasma amino acid profile analysis was performed with the use of a commercially available kit (EZ:faast kit, Phenomenex) through the use of a Varian 450-GC couple with Varian 240-MS IT. Hepatic PLP-dependent enzymes activities. Liver samples (0.5 g) were homogenized with 5 mL ice-cold 50 mmol/L potassium phosphate buffer (pH 6.8) and centrifuged at 15,000 3 g and 4°C to collect the supernatant (15). Hepatic CBS enzyme activity was determined with the use of a previously described method with some modifications. The supernatant sample was incubated in a reaction mixture containing a radioactive material, 25 mmol/L of L-[U-14C]serine (;78 000 dpm/mmol) (PerkinElmer), and L-cystathionine (0.15 mmol/L), DL-homocysteine (41.67 mmol/L), S-adenosylmethionine (0.32 mmol/L), DL-propargylglycine (2.08 mmol/L), Tris (125 mmol/L), and EDTA (2.08 mmol/L). After incubation at 37°C, the radioactivity of newly formed 14C-cystathionine was counted to determine the CBS enzyme
Methods Preparation of flaxseed extract. Flaxseed was obtained from a commercial market and was defatted with the use of a Soxhlet apparatus, with hexane as a solvent. The defatted flaxseed was reground to obtain a fine powder. Vitamin B-6 antagonist from the ground, defatted flaxseed was extracted through the use of an ultrasonification method, previously described, with minor modifications, including the use of 40% isopropanol at 25°C with a 10:1 solvent-to-solid ratio for 30 min (13). The extract was concentrated under reduced pressure and 2 of 7
Mayengbam et al.
FIGURE 1 Effect of FE and 1ADP on the growth of rats fed diets containing either 7 mg or 0.7 mg PNHCl/kg diet for 5 wk. Values are means 6 SEMs; n = 8. Within a vitamin B-6 group, labeled means without a common letter differ, P , 0.05. FE, flaxseed extract; MB, moderately vitamin B-6 deficient; OB, optimum vitamin B-6; PNHCl, pyridoxine hydrochloride; 1ADP, 1-amino D-proline.
Downloaded from jn.nutrition.org at TUFTS UNIVERSITY on November 24, 2015
and aldol cleavage, and thus serves to perform diverse functions in amino acid, sugar, and lipid metabolism (2, 3). In certain amino acid metabolic pathways, including those of the trans-sulphuration and remethylation pathways of methionine metabolism, vitamin B-6 is required as a cofactor (4–6). In the trans-sulphuration pathway, PLP acts as a coenzyme for cystathionine b-synthase (CBS), which condenses homocysteine with serine to form cystathionine and cystathionine g-lyase (CGL), which converts cystathionine to cysteine by releasing a-aminobutyric acid as a by-product. In the remethylation pathway, homocysteine is converted back to methionine by coupling several enzymes, including serine hydroxymethyltransferase (SHMT). This PLP-dependent enzyme is involved in the reversible reaction to transfer a -CH2- group from serine to tetrahydrofolate to form 5,10-methylenetetrahydrofolate, with the latter also formed via the glycine cleavage system (4, 5, 7, 8). The bodyÕs vitamin B-6 status is primarily modulated by its dietary supply, and may be perturbed by the ingestion of a vitamin B-6 antagonist (9). Several drugs (isoniazide, cycloserine, penicillamine, and mestranol), as well as natural substances (gyromitrin, canavanine, agaritin, thyophylline, caffeine, and linatine) act as vitamin B-6 antagonists in the body by reducing free PLP and/or inhibiting the activity of pyridoxal kinase (PLK) and pyridoxine phosphate oxidase (PNPOx) enzymes (9–11). Linatine, a naturally occurring dipeptide of glutamic acid and 1-amino D-proline (1ADP), is a vitamin B-6 antagonist present in flaxseed. Chemically, it is 1-[(n-g-L-glutamyl)-amino]-Dproline. After hydrolysis with hydrochloric acid, linatine releases 1ADP, the active form of this vitamin B-6 antagonist (12). Previous studies have documented that the total vitamin B-6 antagonist content, expressed in 1-amino D-proline equivalents (1ADPEs; equal to 1/2 linatine + 1ADP), was 177– 437 mg/g of whole flaxseed depending on the variety sampled (12, 13). 1ADP binds with PLP to form hydrazone complexes, and these complexes can reduce the free in vivo PLP pool or cause hydrazone toxicity in the body (12, 14). We have previously shown that the toxicity of synthetic 1ADP was much more evident in animals that were moderately vitamin B-6 deficient compared with those that were replete. The oral ingestion of synthetic 1ADP at a concentration of 10 mg/kg diet caused severe impairment in homocysteine metabolism in moderately vitamin B-6–deficient rats (15). Consumption of flaxseed has been increasing because of its purported health benefits, including the reduction of risk factors associated with diabetes, cancer, and cardiovascular diseases (16–18). Concurrent ingestion of antinutrients including the vitamin B-6 antagonist 1ADP present in flaxseed by the general population, particularly those who have moderate vitamin B-6 deficiency, may be a matter of concern. Because overt vitamin B-6 deficiency is rare and moderate deficiency is prevalent (19, 20), the current study was designed to evaluate the effect of a vitamin B-6 antagonist from flaxseed on amino acid metabolism in a rodent model of moderate vitamin B-6 deficiency.
TABLE 1 Effect of FE and 1ADP on body weight, feed intake, and the feed efficiency ratio in rats fed diets containing PNHCl at either 7 mg/kg diet or 0.7 mg/kg diet for 5 wk1 Vitamin B-6 group (PNHCl) and treatment OB diet (7 mg/kg diet) OB OB+FE OB+1ADP P MB diet (0.7 mg/kg diet) MB MB+FE MB+1ADP P
Initial body weight, g
Final body weight, g
Total weight gain, g
RLW
Feed intake, g
FER
112 6 6 113 6 3 114 6 3 0.92
456 6 9 456 6 14 430 6 12 0.26
344 6 6 343 6 16 316 6 12 0.2
3.49 6 0.13 3.49 6 0.06 3.57 6 0.07 0.79
930 6 15.3 918 6 24.7 869 6 24.8 0.29
0.37 6 0.01 0.39 6 0.03 0.37 6 0.01 0.56
113 6 3 112 6 2 114 6 2 0.98
383 6 8a 373 6 7a 255 6 8b ,0.001
270 6 10a 260 6 7a 141 6 8b ,0.001
3.44 6 0.08 3.35 6 0.09 3.54 6 0.14 0.72
780 6 15.8a 770 6 18.8a 514 6 19.0b ,0.001
0.35 6 0.01a 0.34 6 0.01a 0.28 6 0.02b 0.001
1 Values are means 6 SEMs; n = 8, except for OB+1ADP, n = 7. Labeled means in a column within a vitamin B-6 group without a common letter differ, P , 0.05. FER = total weight gain/total feed intake. RLW = liver weight 3 100/body weight. FE, flaxseed extract; FER, feed efficiency ratio; MB, moderately vitamin B-6 deficient; OB, optimum vitamin B-6; PNHCl, pyridoxine hydrochloride; RLW, relative liver weight; 1ADP, 1-amino D-proline.
Statistical analysis. Data were sliced based on dietary PNHCl concentration (OB and MB) in order to determine the simple effects of antagonist exposure independent of dietary vitamin B-6 exposure, a statistical procedure used in prior work (15). Statistical analysis was then performed with the use of SPSS 16.0. Normality of the data were tested before analysis with a Shapiro-Wilk test, and, if necessary, the data were normalized by log10 transformation. A general linear model was used to compare the treatment effects within the same vitamin B-6 diet group with the use of BonferroniÕs test by setting the significance at P < 0.05. For all plasma biochemical analytes, we used ANCOVA by taking feed intake as a covariate to control, account for, and equalize the discrepancies that might have occurred because of differences in feed consumption. Otherwise, we used 1-factor ANOVA to compare the treatment effects.
Results Preparation of FE. Upon quantification by ultra-high–performance liquid chromatography/mass spectrometry, the amount TABLE 2 Effect of FE and 1ADP on plasma B-6 vitamers in rats fed diets containing PNHCl at either 7 mg/kg diet or 0.7 mg/kg diet for 5 wk1 Vitamin B-6 group (PNHCl) and treatment OB diet (7 mg/kg diet) OB OB+FE OB+1ADP P MB diet (0.7 mg/kg diet) MB MB+FE MB+1ADP P
B-6 vitamers, nmol/L PN
PLP
4-PA
PL
320 6 41.4 278 6 38.4 266 6 47.4 0.65
731 6 62.2 721 6 50.8 663 6 26.5 0.61
29.5 6 4.3 29.9 6 4.2 25.1 6 2.6 0.64
571 6 64.9a 566 6 58.9a 397 6 42.6b 0.012
164 6 21.4 112 6 6.6 72.3 6 9.4 0.15
63.4 6 3.0a 47.1 6 3.0b 19.8 6 2.0b 0.001
2.6 6 0.9 2.4 6 0.5 3.6 6 1.3 0.62
39.3 6 3.2a 29.7 6 2.3a 9.8 6 1.0b 0.003
1 Values are means 6 SEMs; n = 8, except for OB+1ADP, n = 7. Labeled means in a column within a vitamin B-6 group without a common letter differ, P , 0.05. FE, flaxseed extract; MB, moderately vitamin B-6 deficient; OB, optimum vitamin B-6; PL, pyridoxal; PLP, pyridoxal 5#-phosphate; PN, pyridoxine; PNHCl, pyridoxine hydrochloride; 1ADP, 1-amino D-proline; 4-PA, 4-pyridoxic acid.
of vitamin B-6 antagonist in freeze-dried FE was found to be 305 mg of linatine and 17 mg of 1ADP per 50 mg of sample. According to the molar calculation as mentioned in our previous study (13), the total amount of vitamin B-6 antagonist, expressed as 1ADPEs, present in a 50 mg sample was 169 mg, and this value was used in the preparation of the FE-based diets that contained 10 mg 1ADPEs/kg diet. Performance data. In rats fed the OB diets, those that were exposed to either 1ADP or FE did not show any significant differences in feed intake, final body weight, feed efficiency ratio (FER), or total weight gain compared with those fed the OB control diet (Figure 1 and Table 1). However, in rats fed the MB diets, those in the MB+1ADP group had lower feed intake, FER, and total body weight gain than those in the group fed the MB control diet (P < 0.001). As a result, final body weights were also significantly reduced (P < 0.001). Nevertheless, the rats in the MB+FE group were not different from the control rats in terms of growth performance markers. Additionally, the feeding of 1ADP, irrespective of source, did not change the relative liver weight of rats consuming diets with either optimal or moderately deficient concentrations of PNHCl (Table 1). One mortality from the OB+1ADP group was observed over the course of the study. Death was sudden and the rat did not exhibit anorexia or reduced weight gain; however, post-mortem examination did not reveal the cause of death. Plasma B-6 vitamers. In the rats fed the MB diets, plasma PLP concentrations were reduced by 26% and 69% (P # 0.001) in the rats consuming the MB+FE and MB+1ADP diets, respectively, compared with the MB control group. However, the feeding of a vitamin B-6 antagonist to rats in the OB diet groups did not significantly (P = 0.61) affect plasma PLP concentrations. On the contrary, the rats consuming OB+1ADP and MB+1ADP had a significant reduction in plasma pyridoxal concentrations compared with their respective controls (Table 2). Despite the lowered plasma pyridoxine concentrations in rats fed either vitamin B-6 antagonist, compared with the control rats, there were no significant differences between the treatments (P = 0.15) due to the confounding effect of food intake. Likewise, plasma 4-PA, the by-product of vitamin B-6 catabolism, was not affected when rats were fed either FE or 1ADP, and were stratified according to dietary vitamin B-6 supply. Vitamin B-6 and flaxseed
3 of 7
Downloaded from jn.nutrition.org at TUFTS UNIVERSITY on November 24, 2015
activity (22). The hepatic activity of CGL was determined by an enzymecoupled assay with the use of a spectrophotometric method previously described (23).
TABLE 3 Effect of FE and 1ADP on plasma sulfur-containing amino acid concentrations in rats fed diets containing PNHCl at either 7 mg/kg diet or 0.7 mg/kg diet for 5 wk1 Vitamin B-6 group (PNHCl) and treatment OB diet (7 mg/kg diet) OB OB+FE OB+1ADP P MB diet (0.7 mg/kg diet) MB MB+FE MB+1ADP P
Plasma sulfur-containing amino acids, mmol/L Met
Cys
Hcy
Cth
49.9 6 1.3 49.9 6 1.8 46.3 6 1.2 0.19
282 6 6.0a 284 6 6.9a 256 6 5.0b 0.009
10.2 6 0.7 11.4 6 0.3 12.0 6 0.5 0.09
47.6 6 0.8 48.4 6 1.0 48.0 6 1.9 0.9
280 6 6.2 263 6 6.1 249 6 4.5 0.08
8.5 6 0.5b 9.5 6 0.6b 71.7 6 15.7a ,0.001
1.7 6 0.0c 2.2 6 0.1b 3.4 6 0.2a ,0.001 2.3 6 0.1c 4.6 6 0.9b 32.2 6 5.9a ,0.001
Plasma sulfur containing amino acids. Besides B-6 vitamers, plasma homocysteine and cystathionine concentrations are important biomarkers of vitamin B-6 deficiency (24, 25). In the current study, we found increases in plasma homocysteine and cystathionine concentrations due to 1ADP exposure (P < 0.001). Plasma homocysteine concentrations were elevated by 744% in the MB+1ADP rats compared with the MB control
Other plasma amino acids. The concentrations of other plasma free amino acids are given in Table 4. In the MB diet groups, plasma a-aminobutyric acid (59%; P < 0.001) and glutamic acid (30%; P = 0.017) were significantly reduced in MB+FE rats compared with control, with the greater effect observed in MB+1ADP rats. Additionally, plasma glycine (P = 0.02) concentration was elevated, and plasma serine (P = 0.013) and asparagine (P = 0.016) concentrations were reduced in MB+1ADP rats. We also found a significant reduction (P # 0.013) in the plasma a-aminobutyric acid concentrations of OB+1ADP rats. Other plasma amino acids given in Table 4 were not affected by the oral ingestion of either FE or 1ADP. Hepatic PLP-dependent enzymes activities. In the current study, we found a significant reduction (P < 0.001) in hepatic CBS and CGL enzyme activities (Figure 2) from either FE or
TABLE 4 Effect of FE and 1ADP on major plasma free amino acids in rats fed diets containing PNHCl at either 7 mg/kg diet or 0.7 mg/kg diet for 5 wk1 OB diet (PNHCl: 7 mg/kg diet), mmol/L Plasma amino acid Alanine Sarcosine Glycine a-Aminobutyric acid Valine Leucine Isoleucine Threonine Serine Proline Asparagine 4-Hydroxyproline Glutamic acid Phenylalanine a-Aminoadipic acid Glutamine Ornithine Lysine Histidine Tyrosine Proline-hydroxyproline Tryptophan
OB 619 10.8 285 56.1 184 148 92.7 682 603 171 96.1 64.9 206 64.1 2.0 956 60.5 303 61.8 58.7 23.1 82.3
6 27.1 6 0.1 6 22.2 6 4.4a 6 9.4 6 7.8 6 5.2 6 71.7 6 60.1 6 7.1 6 5.6 6 6.6 6 10.6 6 1.8 6 0.2 6 97.2 6 2.6 6 13.7 6 5.1 6 1.9 6 1.3 6 7.2
OB+FE 706 6 10.9 6 297 6 44.3 6 198 6 158 6 96.0 6 590 6 472 6 168 6 94.8 6 55.7 6 213 6 69.2 6 2.3 6 778 6 66.1 6 271 6 59.4 6 56.5 6 20.3 6 67.7 6
25.2 0.1 19.4 4.4a,b 8.8 8.2 5.2 39.9 38.9 4.4 3.1 4.6 4.9 1.4 0.2 93.4 4.2 7.9 5.9 2.1 0.1 6.2
OB+1ADP 619 10.9 289 33.0 177 143 86.1 444 497 162 97.0 59.7 201 65.0 2.3 867 62.4 273 60.3 52.6 20.5 78.4
6 39.5 6 0.1 6 15.5 6 3.3b 6 6.0 6 6.2 6 4.5 6 38.2 6 45.1 6 6.6 6 4.3 6 6.3 6 20.2 6 1.7 6 0.2 6 114.2 6 2.6 6 12.9 6 4.5 6 1.9 6 0.3 6 8.2
MB diet (PNHCl: 0.7 mg/kg diet), mmol/L P 0.08 0.33 0.8 0.013 0.26 0.42 0.46 0.05 0.2 0.33 0.94 0.56 0.63 0.09 0.65 0.49 0.48 0.2 0.92 0.29 0.05 0.34
MB 565 10.8 442 45.9 188 147 90.6 766 585 183 99.3 65.1 197 62.4 2.5 962 86.5 272 62.7 51.3 21.1 84.4
6 16.2 6 0.1 6 24.2b 6 4.0a 6 8.3 6 6.5 6 4.2 6 90.6 6 49.9a 6 8.5 6 4.7a 6 6.2 6 14.0a 6 1.0 6 0.1 6 119 6 7.4 6 16.1 6 5.3 6 1.5 6 0.3 6 6.9
MB+FE 542 6 10.9 6 511 6 19.0 6 190 6 147 6 92.4 6 648 6 469 6 188 6 87.3 6 59.9 6 152 6 61.6 6 2.9 6 797 6 91.6 6 257 6 65.7 6 52.0 6 20.3 6 71.1 6
29.8 0.1 38.5b 1.9b 7.1 6.4 4.7 64.9 23.5a,b 4.2 5.8a,b 6.3 8.6b 2.2 0.3 92.8 8.2 13.2 6.5 1.9 0.2 5.0
MB+1ADP 494 11.1 899 13.8 151.5 136 82.9 486 353 186 79.1 51.4 129 57.7 2.5 759 87.1 210 84.0 49.1 20.5 72.3
6 30.5 6 0.1 6 74.4a 6 1.1b 6 8.7 6 9.8 6 6.0 6 43.2 6 32.4b 6 10.6 6 4.4b 6 5.6 6 6.0b 6 1.4 6 0.2 6 81.8 6 5.9 6 13.3 6 8.6 6 2.3 6 0.3 6 6.6
P 0.25 0.71 0.02 ,0.001 0.19 0.26 0.23 0.35 0.013 0.89 0.013 0.87 0.017 0.21 0.38 0.26 0.88 0.16 0.4 0.31 0.05 0.33
1 Values are means 6 SEMs; n = 8, except OB+1ADP, n = 7. Labeled means in a row within a vitamin B-6 group without a common letter differ, P , 0.05. FE, flaxseed extract; MB, moderately vitamin B-6 deficient; OB, optimum vitamin B-6; PNHCl, pyridoxine hydrochloride; 1ADP, 1-amino D-proline.
4 of 7
Mayengbam et al.
Downloaded from jn.nutrition.org at TUFTS UNIVERSITY on November 24, 2015
Values are means 6 SEMs; n = 8, except for OB+1ADP, n = 7. Labeled means in a column within a vitamin B-6 group without a common letter differ, P , 0.05. Cth, cystathionine; Cys, cysteine; FE, flaxseed extract; Hcy, homocysteine; MB, moderately vitamin B-6 deficient; Met, methionine; OB, optimum vitamin B-6; PNHCl, pyridoxine hydrochloride; 1ADP, 1-amino D-proline.
1
group, but no changes were observed in the MB+FE rats (Table 3). However, plasma homocysteine concentrations were not significantly affected by exposure to a vitamin B-6 antagonist (either FE or 1ADP) in the OB diet groups. In the MB diet groups, there were profound increases in plasma cystathionine concentrations in rats consuming MB+FE (100%) or MB+1ADP (1300%). A similar pattern was also exhibited for rats in the OB diet groups (Table 3). Somewhat unexpectedly, a significant reduction (P = 0.005) in plasma cysteine concentration was observed in rats in the OB+1ADP group compared with control, but this was not the case in the MB diet groups. There was no significant effect of vitamin B-6 antagonist exposure on plasma methionine concentrations (Table 3).
1ADP treatment compared with the respective controls. Hepatic CGL enzyme activity was reduced relative to the MB group by ;48% in MB+FE and MB+1ADP rats, whereas the reduction was 26% and 60% for OB+FE and OB+1ADP rats, respectively, in the OB diet group. In the case of hepatic CBS enzyme activity, MB+1ADP rats had a higher degree of enzyme inhibition (44% reduction) than did the MB+FE rats (26% reduction). However, in the OB diet groups, the reductions were of a similar magnitude, being reduced by ;31% for both the OB+FE and OB+ 1ADP rats.
Discussion
FIGURE 2 Effect of FE and 1ADP on liver CBS (A) and CGL (B) enzyme activities in rats fed diets containing either 7 mg or 0.7 mg PNHCl/kg diet for 5 wk. Values are means 6 SEMs; n = 8.Within a vitamin B-6 group, labeled means without a common letter differ, P , 0.05. CBS, cystathionine b-synthase; CGL, cystathionine g-lyase; FE, flaxseed extract; MB, moderately vitamin B-6 deficient; OB, optimum vitamin B-6; PNHCl, pyridoxine hydrochloride; 1ADP, 1-amino D-proline.
Vitamin B-6 and flaxseed
5 of 7
Downloaded from jn.nutrition.org at TUFTS UNIVERSITY on November 24, 2015
Dietary vitamin B-6 has a substantial impact on the concentration of plasma PLP (9, 26). In humans, a plasma PLP concentration of 20–30 nmol/L or a vitamin B-6 intake of 0.7 mg/d indicates marginal vitamin B-6 status (4, 27). Within the general population, vitamin B-6 inadequacy is prevalent in 24% of the American population and 21% of the Canadian elderly (>50 y) population (19, 28). Inadequate vitamin B-6 status has also been implicated in the development of many
common diseases, including chronic inflammation, colorectal cancer, and cardiovascular diseases (29, 30). The consumption of compounds possessing vitamin B-6 antagonist activity concurrent with marginal vitamin B-6 deficiency may therefore increase the risk of the development of metabolic defects or disease associated with low vitamin B-6 status (14, 31). As such, the current study was designed to investigate the potential responses in biomarkers elicited by nutritional co-insults from the oral consumption of a flaxseed-derived component in a model of moderate vitamin B-6 deficiency. Vitamin B-6 deficiency normally causes reductions in food intake, weight gain, and FER in growing animals, signifying overt defects in intermediary metabolism, as well as nitrogen retention during suboptimal vitamin B-6 status (15, 32, 33). In the present study, rats in the MB+1ADP group exhibited a significant reduction in FER as well as weight gain compared with other groups. These data support previous findings from a study that examined the impact of feeding a vitamin B-6 antagonist, 4-deoxypyridoxine, which led to significantly lower body weights in rats of low vitamin B-6 status than in their pairfed counterparts (34). Tissue concentrations of the classic vitamin B-6 markers, including PLP, pyridoxal, pyridoxine, and 4-PA, primarily reflect the nutritional supply of pyridoxine, but can be affected by the ingestion of vitamin B-6 inhibitors (9, 10, 35). In the current study, the significant alteration of major vitamin B-6 markers— developed as a result of vitamin B-6 antagonist administration— is in line with the findings of previous studies (15, 31, 36). Our data showed that FE reduced plasma PLP concentrations, with significant evidence in rats from the MB diet groups, indicating a potential binding ability of naturally occurring 1ADP to endogenous B-6 vitamers. Additionally, the synthetic 1ADP indicated a stronger and wider affinity to B-6 vitamers over naturally occurring ones, because it had significantly reduced both plasma PLP and pyridoxal concentrations. Therefore, the current study provided evidence that oral ingestion of a vitamin B-6 antagonist, either in the form of synthetic 1ADP or as an FE, reduced the overall B-6 vitamer pool in the body. One of the main reasons for lowering in vivo B-6 vitamers in the current study might be the result of the formation of hydrazone complexes of 1ADP with freely available endogenous PLP and pyridoxal. These findings, also suggested by others, were more pronounced during low vitamin B-6-status (10, 14). Other potential mechanisms include a vitamin B-6 antagonist–induced inactivation of vitamin B-6 salvage enzymes such as PLK and PNPOx (11) and increased urinary excretion of B-6 vitamers during vitamin B-6 antagonist exposure (37). It is well known that the PLK and PNPOx enzymes play a crucial role in maintaining the homeostasis in vivo of B-6 vitamers through their phosphorylation and interconversion (11, 38). Interestingly, their activities have been shown to be inhibited by several vitamin B-6 antagonists, such as isoniazid, cycloserine, and dopamine, which have a similar mode of action as 1ADP, causing in vivo PLP deficiency (11). However, further work is needed to characterize the effect of this vitamin B-6 antagonist on in vivo vitamin B-6 homeostasis and its catabolism. A deficiency of vitamin B-6 alters the plasma amino acid profile because of the involvement of this cofactor in numerous PLP-dependent enzymes, particularly those involved in transamination, trans-sulphuration, and decarboxylation reactions (6). For instance, administration of the vitamin B-6 antagonists DL penicillamine, thiosemicarbazide, and semicarbazide-HCl significantly reduced g-aminobutyric acid content in the mouse brain (39). Additionally, an intraperitoneal injection of 1ADP in
6 of 7
Mayengbam et al.
flaxseed on a population presenting with moderate vitamin B-6 deficiency. However, the bioefficacy of naturally occurring bioactive compounds largely depends on their bioavailability. For example, synthetic drugs tend to have better liberation, absorption, distribution, and metabolism than do naturally occurring bioactive components (48). The major vitamin B-6 antagonist factor present in flaxseed is the dipeptide linatine, which must liberate free 1ADP via the hydrolysis of the gglutamyl bond attached to glutamic acid in the gastrointestinal tract in order to induce in vivo vitamin B-6 antagonist toxicity. Incomplete hydrolysis and/or reduced absorption and metabolism of the linatine contained in FE might be responsible for the lowered potency of flaxseed-derived 1ADP compared with the synthetic 1ADP. Nevertheless, flaxseed-derived 1ADP did demonstrate an ability to perturb certain markers of amino acid and vitamin B-6 metabolism in the current study. To conclude, the present study provided evidence that the oral ingestion of vitamin B-6 antagonist, either from flaxseed or a synthetic source, lowered free in vivo B-6 vitamers potentially because of hydrazone formation, thus impairing amino acid metabolism. The most intriguing finding of this study was the ability of FE to inhibit the activities of PLP-dependent enzymes. However, further investigations are necessary to better understand the mechanism of action of 1ADP and the protective role of vitamin B-6 during vitamin B-6 antagonist toxicity. Additional opportunities also exist to investigate the long-term effect of this naturally occurring vitamin B-6 antagonist linked to flaxseed on other metabolic pathways. Acknowledgments We thank Haifeng Yang for technical assistance. SM and JDH designed the research; SM and SR conducted the research and analyzed the data; SM wrote the manuscript; MA and JDH reviewed the manuscript; and JDH had primary responsibility for the final content. All authors read and approved the final manuscript.
References 1.
Christen P, Mehta PK. From cofactor to enzymes. The molecular evolution of pyridoxal-5#-phosphate-dependent enzymes. Chem Rec 2001;1:436–47. 2. Hellmann H, Mooney S. Vitamin B6: A molecule for human health? Molecules 2010;15:442–59. 3. Toney MD. Reaction specificity in pyridoxal phosphate enzymes. Arch Biochem Biophys 2005;433:279–87. 4. Gregory III JF, Park Y, Lamers Y, Bandyopadhyay N, Chi YY, Lee K, Kim S, da Silva V, Hove N, Ranka S, et al. Metabolomic analysis reveals extended metabolic consequences of marginal vitamin B-6 deficiency in healthy human subjects. PLoS One 2013;8:e63544. 5. Stipanuk MH. Sulfur amino acid metabolism: Pathways for production and removal of homocysteine and cysteine. Annu Rev Nutr 2004;24:539–77. 6. Bowling FG. Pyridoxine supply in human development. Semin Cell Dev Biol 2011;22:611–8. 7. Medici V, Peerson JM, Stabler SP, French SW, Gregory, 3rd JF, Virata MC, Albanese A, Bowlus CL, Devaraj S, Panacek EA, et al. Impaired homocysteine transsulfuration is an indicator of alcoholic liver disease. J Hepatol 2010;53:551–7. 8. House JD, Jacobs RL, Stead LM, Brosnan ME, Brosnan JT. Regulation of homocysteine metabolism. Adv Enzyme Regul 1999;39:69–91. 9. Spinneker A, Sola R, Lemmen V, Castillo MJ, Pietrzik K, Gonzalez´ Gross M. Vitamin B6 status, deficiency and its consequences - An overview. Nutr Hosp 2007;22:7–24. 10. Klosterman JH. Vitamin B6 antagonists of natural origin. Journal of Agricultural and Food Chemistry. 1974;22–1:13–6.
Downloaded from jn.nutrition.org at TUFTS UNIVERSITY on November 24, 2015
rats led to a significant increase in plasma a-aminoadipic acid, citrulline, and cystathionine concentrations (14), whereas perfusion of 1ADP into an isolated rat liver significantly reduced the synthesis of carnitine from lysine residues (40), thus signifying major perturbations in amino acid metabolism. Several studies have demonstrated significantly higher plasma homocysteine and cystathionine concentrations in both animal models and humans when subjected to a vitamin B-6–restricted diet (15, 24, 25, 26). In the current study, the significant increase in plasma homocysteine that was observed in response to oral 1ADP exposure likely was due to the inhibition of hepatic CBS activity in the trans-sulphuration pathway, a condition that was more pronounced under low vitamin B-6 status. The latter mechanism is supported by the evidence that plasma homocysteine concentration is higher (40-fold) in mice lacking CBS activity than in wild-type mice (41). Additionally, hepatic CGL enzyme activity also was inhibited because of the feeding of a vitamin B-6 antagonist from either flaxseed or synthetic 1ADP, leading to increases in plasma cystathionine and a significant reduction in plasma a-aminobutyric acid concentrations. A similar pattern of inhibition of CBS and CGL enzyme activities has been reported in the livers of vitamin B6–deficient animals (24, 26, 42). The current findings support the contention that moderate vitamin B-6 deficiency as well as oral exposure to vitamin B-6 antagonists were able to independently inhibit activities of PLP-dependent enzymes, including hepatic CBS and CGL, in rats. Glycine decarboxylation through the glycine cleavage system and the conversion of glycine to serine by SHMT in one-carbon metabolism also require PLP. An inhibition of SHMT and/or the glycine cleavage system have been speculated to be the mechanisms explaining the shifts in plasma glycine-to-serine ratios during low vitamin B-6 status (25, 26, 43). Therefore, one of the reasons for the elevation of plasma glycine and reduction of plasma serine concentrations in the current study might be the impairment of these 2 enzymes, which led to reduced transformation of in vivo glycine to serine. Additionally, reductions in plasma asparagine and glutamic acid concentrations as a result of 1ADP feeding during low vitamin B-6 status might be related to the impairment of other PLP-dependent enzymes involved in the transamination and interconversion of amino acids. Our data are supported by a previous study by Swendseid et al. (43), who found higher concentrations of cystathionine and glycine and lower concentrations of serine and total glutamine and asparagine in the plasma of vitamin B-6–deficient rats. Responses induced by dietary vitamin B-6 deficiency or the exposure to vitamin B-6 antagonists on the amino acid profile can exhibit variability, likely related to the duration or severity of the imposed deficiency (4, 14, 44). Gregory et al. (4) recently showed that marginal vitamin B-6 deficiency alone can cause minor perturbations in metabolic indexes, including in vivo amino acid profiles, and a concurrent exposure to synthetic vitamin B-6 antagonist further exacerbated vitamin B-6 status (15). Interestingly, the current data provide evidence of a perturbation of certain vitamin B-6 markers due to the co-insult of a flaxseed-derived vitamin B-6 antagonist during moderate vitamin B-6 deficiency when provided at a dose of 0.5 mg 1ADPEs kg21 body weight d21. Consumption of 25–30 g/d of flaxseed, a serving size recommended by some (45–47), will result in the intake of 0.15 mg 1ADPEs kg21 body weight d21 (assuming a body weight of 65 kg), a concentration within the same order of magnitude as that provided in the current study. As such, our data serve to highlight the potential deleterious effects of the oral exposure to a vitamin B-6 antagonist linked to
30. Larsson SC, Orsini N, Wolk A. Vitamin B6 and risk of colorectal cancer: A meta-analysis of prospective studies. JAMA 2010;303:1077–83. 31. Chang SJ. Vitamin B6 antagonists alter the function and ultrastructure of mice endothelial cells. J Nutr Sci Vitaminol (Tokyo) 2000;46:149–53. 32. Wolfson M, Kopple JD. The effect of vitamin B6 deficiency on food intake, growth, and renal function in chronically azotemic rats. JPEN J Parenter Enteral Nutr 1987;11:398–402. 33. Kirchgessner M, Kosters WW. Influence of vitamin B 6 deficiency in early weaned piglets on the digestibility and conversion of protein and energy. Arch Tierernahr 1977;27:299–308. 34. Coburn SP, Mahuren JD, Schaltenbrand WE, Wostmann BS, Madsen D. Effects of vitamin B-6 deficiency and 4#- deoxypyridoxine on pyridoxal phosphate concentrations, pyridoxine kinase and other aspects of metabolism in the rat. J Nutr 1981;111:391–8. 35. Lumeng L, Lui A, Li TK. Plasma content of B6 vitamers and its relationship to hepatic vitamin B6 metabolism. J Clin Invest 1980;66: 688–95. 36. Jaffe IA. Antivitamin B6 effect of D-penicillamine. Ann N Y Acad Sci 1969;166:57–60. 37. Kajiwara T, Matsuda M. Effects of penicillamine on distribution of B6 vitamens in rat urine. J Nutr Sci Vitaminol (Tokyo) 1978;24:1–7. 38. McCormick DB, Chen H. Update on interconversions of vitamin B-6 with its coenzyme. J Nutr 1999;129:325–7. 39. Abe M, Matsuda M. Effect of antivitamin B6 on regional GABA metabolism in mouse brain and its relation to convulsions. J Nutr Sci Vitaminol (Tokyo) 1979;25:459–68. 40. Dunn WA, Aronson NN, Jr., Englard S. The effects of 1-amino-Dproline on the production of carnitine from exogenous protein-bound trimethyllysine by the perfused rat liver. J Biol Chem 1982;257:7948– 51. 41. Watanabe M, Osada J, Aratani Y, Kluckman K, Reddick R, Malinow MR, Maeda N. Mice deficient in cystathionine beta-synthase: animal models for mild and severe homocyst(e)inemia. Proc Natl Acad Sci USA 1995;92:1585–9. 42. Finkelstein JD, Chalmers FT. Pyridoxine effects on cystathionine synthase in rat liver. J Nutr 1970;100:467–9. 43. Swendseid ME, Villalobos J, Friedrich B. Free amino acids in plasma and tissues of rats fed a vitamin B6-deficient diet. J Nutr 1964;82:206–8. 44. da Silva VR, Rios-Avila L, Lamers Y, Ralat MA, Midttun O, Quinlivan EP, Garrett TJ, Coats B, Shankar MN, Percival SS, et al. Metabolite profile analysis reveals functional effects of 28-day vitamin B-6 restriction on one-carbon metabolism and tryptophan catabolic pathways in healthy men and women. J Nutr 2013;143:1719–27. 45. Edel AL, Aliani M, Pierce GN. Supported liquid extraction in the quantitation of plasma enterolignans using isotope dilution GC/MS with application to flaxseed consumption in healthy adults. J Chromatogr B Analyt Technol Biomed Life Sci 2013;912:24–32. 46. Caligiuri SP, Aukema HM, Ravandi A, Guzman R, Dibrov E, Pierce GN. Flaxseed consumption reduces blood pressure in patients with hypertension by altering circulating oxylipins via an alpha-linolenic acid-induced inhibition of soluble epoxide hydrolase. Hypertension 2014;64:53–9. 47. Wu H, Pan A, Yu Z, Qi Q, Lu L, Zhang G, Yu D, Zong G, Zhou Y, Chen X, et al. Lifestyle counseling and supplementation with flaxseed or walnuts influence the management of metabolic syndrome. J Nutr 2010;140:1937–42. 48. Rein MJ, Renouf M, Cruz-Hernandez C, Actis-Goretta L, Thakkar SK, da Silva Pinto M. Bioavailability of bioactive food compounds: a challenging journey to bioefficacy. Br J Clin Pharmacol 2013;75:588–602.
Vitamin B-6 and flaxseed
7 of 7
Downloaded from jn.nutrition.org at TUFTS UNIVERSITY on November 24, 2015
11. di Salvo ML, Contestabile R, Safo MK. Vitamin B6 salvage enzymes: Mechanism, structure and regulation. Biochim Biophys Acta 2011;1814:1597–608. 12. Klosterman HJ, Lamoureux GL, Parsons JL. Isolation, characterization, and synthesis of linatine. A vitamin B6 antagonist from flaxseed (Linum usitatissimum). Biochemistry 1967;6:170–7. 13. Mayengbam S, Yang H, Barthet V, Aliani M, House JD. Identification, characterization, and quantification of an anti-pyridoxine factor from flaxseed using ultrahigh-performance liquid chromatography-mass spectrometry. J Agric Food Chem 2014;62:419–26. 14. Sasaoka K, Ogawa T, Moritoki K, Kimoto M. Antivitamin B6 effect of 1 aminoproline on rats. Biochim Biophys Acta 1976;428:396–402. 15. Mayengbam S, Raposo S, Aliani M, House JD. Oral exposure to the anti-pyridoxine compound 1-amino D-proline further perturbs homocysteine metabolism through the transsulphuration pathway in moderately vitamin B6-deficient rats. J Nutr Biochem 2015;26:241–9. 16. Carraro JCC, de Souza Dantas MI, Espeschit ACR, Martino HSD, Ribeiro SMR. Flaxseed and human health: Reviewing benefits and adverse effects. Food Rev Int 2012;28:203–30. 17. Prasad K. Flaxseed and cardiovascular health. J Cardiovasc Pharmacol 2009;54:369–77. 18. Hall, 3rd C, Tulbek MC, Xu Y. Flaxseed. Adv Food Nutr Res 2006;51:1–97. 19. Morris MS, Picciano MF, Jacques PF, Selhub J. Plasma pyridoxal 5#-phosphate in the US population: The National Health and Nutrition Examination Survey, 2003–2004. Am J Clin Nutr 2008;87:1446–54. 20. Haller J, Lowik MR, Ferry M, Ferro-Luzzi A. Nutritional status: blood vitamins A, E, B6, B12, folic acid and carotene. Euronut SENECA investigators. Eur J Clin Nutr 1991;45 Suppl 3:63–82. 21. Pfeiffer CM, Huff DL, Gunter EW. Rapid and accurate HPLC assay for plasma total homocysteine and cysteine in a clinical laboratory setting. Clin Chem 1999;45:290–2. 22. Taoka S, Ohja S, Shan X, Kruger WD, Banerjee R. Evidence for hememediated redox regulation of human cystathionine b- synthase activity. J Biol Chem 1998;273:25179–84. 23. Stipanuk MH. Effect of excess dietary methionine on the catabolism of cysteine in rats. J Nutr 1979;109:2126–39. 24. Zhang Z, Kebreab E, Jing M, Rodriguez-Lecompte JC, Kuehn R, Flintoft M, House JD. Impairments in pyridoxine-dependent sulphur amino acid metabolism are highly sensitive to the degree of vitamin B6 deficiency and repletion in the pig. Animal 2009;3:826–37. 25. Lamers Y, Williamson J, Ralat M, Quinlivan EP, Gilbert LR, Keeling C, Stevens RD, Newgard CB, Ueland PM, Meyer K, et al. Moderate dietary vitamin B-6 restriction raises plasma glycine and cystathionine concentrations while minimally affecting the rates of glycine turnover and glycine cleavage in healthy men and women. J Nutr 2009;139:452–60. 26. Martinez M, Cuskelly GJ, Williamson J, Toth JP, Gregory, III JF. Vitamin B-6 deficiency in rats reduces hepatic serine hydroxymethyltransferase and cystathionine b-synthase activities and rates of in vivo protein turnover, homocysteine remethylation and transsulfuration. J Nutr 2000;130:1115–23. 27. Mashiyama ST, Hansen CM, Roitman E, Sarmiento S, Leklem JE, Shultz TD, Ames BN. An assay for uracil in human DNA at baseline: effect of marginal vitamin B6 deficiency. Anal Biochem 2008;372:21–31. 28. Statistics Canada . Vitamin B6: Usual intakes from food. 2004. Report No.: Cycle 2.2, Nutrition. 29. Lotto V, Choi SW, Friso S. Vitamin B6: a challenging link between nutrition and inflammation in CVD. Br J Nutr 2011;106:183–95.
