Go to research.

Abstract

This work aims to review and showcase the unique properties of rebaudioside M as a natural non-caloric potential sweetener in food and beverage products. To determine the potential of rebaudioside M, isolated from Stevia rebaudiana Bertoni, as a high potency sweetener, we examined it with the Beidler Model. This model estimated that rebaudioside M is 200–350 times more potent than sucrose. Numerous sensory evaluations of rebaudioside M’s taste attributes illustrated that this steviol glycoside possesses a clean, sweet taste with a slightly bitter or licorice aftertaste. The major reaction pathways in aqueous solutions (pH 2–8) for rebaudioside M are similar to rebaudioside A. Herein we demonstrate that rebaudioside M could be of great interest to the global food industry because it is well-suited for blending and is functional in a wide variety of food and beverage products.

Discovery of Rebaudioside M

Work to elucidate the chemical structures of S. rebaudiana sweeteners began in the early twentieth century, but proceeded slowly. The structures 1 and 8 () were not fully determined until 1970 [,,]. During the 1970s, additional sweet components, including rebaudiosides A–E, were isolated from S. rebaudiana leaves and characterized by Osamu Tanaka and co-workers at Hiroshima University in Japan []. Several novel steviol glycosides have been reported from the commercial extracts of the leaves of S. rebaudiana in the last few years [,,,,,,,,]. Recently we reported the structure elucidation and isolation of rebaudioside M from S. rebaudiana Bertoni (7) [,]. In addition, rebaudioside M received a Letter of No Objection concerning its Generally Recognized as Safe (GRAS) status from US FDA [].

2.1. Purification of Rebaudioside M

Rebaudioside M (7) was obtained from Stevia rebaudiana Bertoni cultivar AKH L1 leaves []. Two kilograms of S. rebaudiana leaves were extracted by 40 L water at 40 °C for two hours. The filtrate was separated and treated with a flocculant (calcium oxide) to remove the mechanical particles, proteins, polysaccharides and coloring agents. The resulting precipitate was separated by filtration and the filtrate was deionized by Amberlite FCP22 (H+) and Amberlite FPA53 (OH−) ion-exchange resins. The deionized filtrate was fractionated by seven columns, packed with Diaion HP20 (Mitsubishi Chemical, Japan). The columns with higher rebaudioside M content were eluted using aqueous ethanol. The obtained glycoside eluate was treated with activated carbon, deionized, evaporated and dried in vacuo. The obtained material was recrystallized twice from aqueous methanol to afford about 1.1 g of rebaudioside M with >98% purity by HPLC []. The highly purified stevia extract meets the international Joint Expert Committee on Food Additives (JECFA) purity requirement for steviol glycosides, which requires ≥95% total steviol glycosides [].

2.2. Properties of Rebaudioside M

Pure rebaudioside M (7) (>95%) crystallizes out as a crystalline form which is slightly soluble in water (0.1 g/100 mL at 25 °C in 5 min) and in ethanol. Its amorphous material has a solubility of 1.1%–1.3% in water at 25 °C. The thermodynamic equilibrium solubility in water is 0.26% at 25 °C [].

2.3. Stability of Rebaudioside M

As a dry powder, rebaudioside M (7) is stable for at least one year at ambient temperature and under controlled humidity conditions. In solution, it is most stable in pH 4–8 and noticeably less stable below pH 2. As expected, the stability decreases with increasing temperature. Its stability is very similar to rebaudioside A [].

2.3. Stability of Rebaudioside M

In aqueous solutions (pH 2–8), the major reaction pathways leading to loss of rebaudioside M are as follows (): isomerization of the C-16 olefin to form the C-15 isomer (12), hydration of the C-16 olefin to yield compound 13, hydrolysis of the glycosidyl ester at C-19 to form compound 2, and isomerization of the C-16 olefin to form the C-15 isomer in 2 to form compound 16. All of these compounds (2, 12, 13 and 16) are sweet. Compound 12 is sweeter than compounds 2, 13 and 16, and has similar taste properties as that of compound 7. Upon metabolism in vitro, rebaudioside M (7), just like rebaudioside A (1), is primarily converted to its aglycone steviol (14) and to the glucuronic acid of steviol, generally known as steviol glucuronide (15) [].

2.4. Sensory Panel for Rebaudioside M and Other Sweeteners

To determine the sensory attributes of rebaudioside M (7) we compared it to rebaudioside A (1), which possesses well defined characteristics. In addition, blending techniques with rebaudioside M and other sweetener were examined to demonstration the potential of this sweetener. Herein we describe our sensory panel testing. Concentration-response (C/R) function work was done with 60 in-house trained panelists (Two-Alternative Forced Choice, 2-AFC method), with the panelists specifying which of the two samples was perceptually sweeter. Sweetener blend work was done with 8–10 descriptive analysis (DA) panelists that have been highly trained. DA panelists go through 10–12 weeks of extensive descriptive training. The panel was calibrated at the beginning of each test session. In addition the panel also received anchor references representing early (sucrose), middle (aspartame) and late (thaumatin) AT every three test samples. Samples were given to the panel members sequentially and coded with triple digit numbers. The order of sample presentation was randomized to avoid the order of presentation bias. Water and unsalted crackers were provided in order to cleanse the palate. The panel members were asked to rate different attributes including sweetness onset, total sweetness, rounded sweetness, bitterness, acidity, leafy note, licorice, astringency, mouthfeel, mouth coating, sweet lingering, and bitter lingering. Samples were rated on a scale of zero (0) to ten (10), with zero indicating immediate onset, no intensity, watery/low viscosity, or very sharp peak, and ten indicating very delayed onset, high intensity, thick/high viscosity, or very round peak. One-way single factor ANOVA was used to analyze sensory results, where α = 0.05.

3.1. Rebaudioside M Sensory Attributes

In water solution, rebaudioside M (7) showed (A) the reduced perception of bitterness, astringency, bitter lingering compared to rebaudioside A (1), and similar sweetness intensity. A much higher sweetness perception of rebaudioside M than rebaudioside A in acidified water was established in B. Rebaudioside M displayed faster sweetness onset, reduced non-sweet taste (bitterness, sour, astringency) and bitterness lingering.

3.1. Rebaudioside M Sensory Attributes

To expand the descriptive taste profile of rebaudioside M (7) a set of trained panelist () examined 10% sucrose, aspartame at 531 mg/L, and rebaudioside M at 563 mg/L in water at 4 °C. Unlike other steviol glycosides including rebaudioside A (1), trained descriptive panel did not detect any significant bitter or licorice off taste when evaluated rebaudioside M in water at approximately 10% SE levels (). Rebaudioside M had a slightly more intense sweet aftertaste then aspartame. Based on our study rebaudioside M and aspartame have similar high potency sweetener profiles.

3.1. Rebaudioside M Sensory Attributes

With these samples in hand panelist examined the AT and ET of the three sweeteners described in . The sweetness temporal profiles of aspartame at 531 mg/L, rebaudioside M at 563 mg/L, and sucrose at 10% in water at room temperature were compared over a period of 3 min (samples were swallowed at 5 s). The AT maximum was the shortest for sucrose, slightly longer for aspartame and longest for rebaudioside M. The ET was longest for rebaudioside M, followed by aspartame and then sucrose.

3.2. Blending

To study the interactions between rebaudioside M (7) and other natural ingredients, blends were made with rebaudioside A (1), rebaudioside D (4), rebaudioside B (2), and erythritol at various concentrations in acidified water and sensory evaluation were performed.

3.2. Blending

illustrates the average response from sensory panelists that tasted the acidified solution with rebaudioside M (300 ppm) and rebaudioside A (100 ppm) or rebaudioside D (100 ppm) blends. The di-blends showed improvement in total sweetness, overall sweetness profile (peak), and leafy note. The blend with rebaudioside M and rebaudioside D showed higher improvement in sweetness intensity, overall sweetness profile, bitter lingering and sweet lingering.

3.2. Blending

The average response from sensory panelists that tasted the acidified solution with rebaudioside M and rebaudioside B blends (100 ppm or 50 ppm) is presented in . The rebaudioside M and rebaudioside B di-blend exhibited additional rounded sweetness profile with slight improvement in sweetness intensity, onset and bitterness perception.

3.2. Blending

The taste description of rebaudioside M and erythritol is displayed in based on the average response from sensory panelists that tasted the acidified solution di-blend. The blend with erythritol helps in reducing acidity, bitterness, astringency and bitter lingering. At a higher level (above 1%, 100 ppm) erythritol contributes additional sweetness, rounded sweet profile and earlier onset.

Abstract

Due to their particular properties, detergents are widely used in household cleaning products, cosmetics, pharmaceuticals, and in agriculture as adjuvants tailoring the features of pesticides or other crop protection agents. The continuously growing use of these various products means that water soluble detergents have become one of the most problematic groups of pollutants for the aquatic and terrestrial environments. Thus it is important to identify bacteria having the ability to survive in the presence of large quantities of detergent and efficiently decompose it to non-surface active compounds. In this study, we used peaty soil sampled from a surface flow constructed wetland in a wastewater treatment plant to isolate bacteria that degrade sodium dodecyl sulfate (SDS). We identified and initially characterized 36 Pseudomonas spp. strains that varied significantly in their ability to use SDS as their sole carbon source. Five isolates having the closest taxonomic relationship to the Pseudomonas jessenii subgroup appeared to be the most efficient SDS degraders, decomposing from 80 to 100% of the SDS present in an initial concentration 1 g/L in less than 24 h. These isolates exhibited significant differences in degree of SDS degradation, their resistance to high detergent concentration (ranging from 2.5 g/L up to 10 g/L or higher), and in chemotaxis toward SDS on a plate test. Mass spectrometry revealed several SDS degradation products, 1-dodecanol being dominant; however, traces of dodecanal, 2-dodecanol, and 3-dodecanol were also observed, but no dodecanoic acid. Native polyacrylamide gel electrophoresis zymography revealed that all of the selected isolates possessed alkylsulfatase-like activity. Three isolates, AP3_10, AP3_20, and AP3_22, showed a single band on native PAGE zymography, that could be the result of alkylsulfatase activity, whereas for isolates AP3_16 and AP3_19 two bands were observed. Moreover, the AP3_22 strain exhibited a band in presence of both glucose and SDS, whereas in other isolates, the band was visible solely in presence of detergent in the culture medium. This suggests that these microorganisms isolated from peaty soil exhibit exceptional capabilities to survive in, and break down SDS, and they should be considered as a valuable source of biotechnological tools for future bioremediation and industrial applications.

Abstract

Due to their particular properties, detergents are widely used in household cleaning products, cosmetics, pharmaceuticals, and in agriculture as adjuvants tailoring the features of pesticides or other crop protection agents. The continuously growing use of these various products means that water soluble detergents have become one of the most problematic groups of pollutants for the aquatic and terrestrial environments. Thus it is important to identify bacteria having the ability to survive in the presence of large quantities of detergent and efficiently decompose it to non-surface active compounds. In this study, we used peaty soil sampled from a surface flow constructed wetland in a wastewater treatment plant to isolate bacteria that degrade sodium dodecyl sulfate (SDS). We identified and initially characterized 36 Pseudomonas spp. strains that varied significantly in their ability to use SDS as their sole carbon source. Five isolates having the closest taxonomic relationship to the Pseudomonas jessenii subgroup appeared to be the most efficient SDS degraders, decomposing from 80 to 100% of the SDS present in an initial concentration 1 g/L in less than 24 h. These isolates exhibited significant differences in degree of SDS degradation, their resistance to high detergent concentration (ranging from 2.5 g/L up to 10 g/L or higher), and in chemotaxis toward SDS on a plate test. Mass spectrometry revealed several SDS degradation products, 1-dodecanol being dominant; however, traces of dodecanal, 2-dodecanol, and 3-dodecanol were also observed, but no dodecanoic acid. Native polyacrylamide gel electrophoresis zymography revealed that all of the selected isolates possessed alkylsulfatase-like activity. Three isolates, AP3_10, AP3_20, and AP3_22, showed a single band on native PAGE zymography, that could be the result of alkylsulfatase activity, whereas for isolates AP3_16 and AP3_19 two bands were observed. Moreover, the AP3_22 strain exhibited a band in presence of both glucose and SDS, whereas in other isolates, the band was visible solely in presence of detergent in the culture medium. This suggests that these microorganisms isolated from peaty soil exhibit exceptional capabilities to survive in, and break down SDS, and they should be considered as a valuable source of biotechnological tools for future bioremediation and industrial applications.

1. Introduction

We tested this hypothesis by administering DCIns to wild-type female mice for three weeks, approximately corresponding to five ovulatory cycles, and analyzing the effects on ovarian histology/function, serum testosterone levels and expression of the ovarian enzyme aromatase. To this end, we employed three different DCIns formulations: 250 mg/kg/day, 500 mg/kg/day and 1000 mg/kg/day. These doses provide approximate daily amounts of 5 mg, 10 mg and 20 mg DCIns/mouse, which are comparable to human doses of 1200, 2400 and 4800 mg/day, respectively [].

2.3. Gross Morphology of Uteri/Ovaries and Histology of Ovaries at the End of the Treatment

Ovaries from DCIns 250-treated mice had apparently normal primary and secondary follicles but also cystic tertiary follicles, some of which contained an atretic oocyte, strongly resembling those found in human polycystic ovaries (B1,B2; C). Ovaries of letrozole-treated mice were similar but contained larger cystic follicles characterized by the absence of the oocyte (C1,C2; D). In some cases, secondary and tertiary follicles from these ovaries had a clearly atretic oocyte with cytosolic vacuolization, suggesting the progression of an apoptotic process.

2.3. Gross Morphology of Uteri/Ovaries and Histology of Ovaries at the End of the Treatment

Ovaries from DCIns 500- and DCIns 1000-treated mice had some primary and secondary follicles, a very limited number of tertiary follicles, no follicles at more advanced stages and no cystic follicles (D1,D2; E1,E2). These ovaries had large follicles/areas with diffused, aberrant cell proliferation (E). The typical structure of the ovary was lost, especially under the DCIns 1000 dose, and the presence of follicles was difficult to assess in many cases (F). In some sections, the presence of oedema foci was observed.

2.6. Testosterone Levels at the End of the Treatment

Under the DCIns 250 dose, levels of testosterone (1.42 ± 0.107 ng/mL) (mean ± SD) increased above those typical of negative control mice (0.37 ± 0.11 ng/mL) (p < 0.05, one-way ANOVA), similarly to those of letrozole-administered mice (1.29 ± 0.22 ng/mL). Levels of testosterone in the serum of DCIns 500- and DCIns 1000-treated mice were similar to or lower than those of negative control mice, being respectively 0.14 ± 011 and 0.27 ± 0.37 ng/mL.

2.7. Presence and Relative Amounts of Aromatase at the End of the Treatment

From a qualitative point of view, a single band of approximately 55 kDa molecular mass was detected in protein extracts from control mouse ovaries, as already reported []. A band of similar size was detected after all treatments, but additional bands were detected, as follows: protein extracts from DCIns 250-treated mice contained a band of smaller size (approximately 52 kDa), those from letrozole-treated mice had a band of closely smaller size (approximately 54 kDa), and extracts from DCIns 500- and DCIns 1000-treated mice had a band of much smaller size (approximately 50 kDa). Induction of different aromatase isoforms by various treatments in mouse ovarian cells appears reasonable in light of the observation that the control of aromatase expression in humans involves a complex mechanism of different promoter and downstream exons []. Densitometric measurements of the amounts of aromatase bands relative to GAPDH, showed a reduction in DCIns 250-ovaries (0.16 ± 0.02 relative units, RU) (mean ± SD) from those of negative control-ovaries (0.34 ± 0.03 RU) (p < 0.05); on the contrary, an increase was observed in letrozole-ovaries (0.56 ± 0.06 RU) (p < 0.05). No variations in the amounts of aromatase were observed in DCIns 500-ovaries (0.45 ± 0.07 RU) and DCIns 1000-ovaries (0.40 ± 0.08 RU) from either negative control- or letrozole-ovaries.

Abstract

The aim of this work was to study the chemical composition of Allium obliquum L., A. senescens L. subsp. montanum (Fries) Holub, and A. schoenoprasum L. subsp. schoenoprasum. Sulphur-containing compounds analysis was performed by an LC-MS method, the identification and quantification of polyphenolic compounds through a HPLC-UV-MS method, and the presence of five sterols was simultaneously assessed by HPLC-MS-MS. Alliin was identified only in A. obliquum and A. senescens subsp. montanum extracts, whilst allicin was present in all extracts, with higher amounts in A. schoenoprasum and A. obliquum. The pattern of phenol carboxylic acids shows the presence of p-coumaric and ferulic acids in all species. Isoquercitrin was identified in A. obliquum and A. schoenoprasum, and rutin in A. senescens subsp. montanum and A. schoenoprasum. Luteolin and apigenin were identified only in A. obliquum. All three species contain glycosides of kaempferol and quercetol. β-Sitosterol and campesterol were identified in all species. The results obtained showed significant differences in the composition of the three Allium species.

2.1. The Analysis of Polyphenols

Luteolin and apigenin were determined only in A. obliquum, before (172.35 μg/100 g, and 149.03 μg/100 g, respectively), and after acid hydrolysis (280.74 μg/100 g, and 277.06 μg/100 g, respectively). Kaempferol and quercetol were present in both non-hydrolysed and hydrolysed sample of A. schoenoprasum, and only in hydrolysed extracts of A. obliquum and A. senescens subsp. montanum. The richest species in quercetol derivatives was A. senescens (993.90 μg/100 g), and in kaempferol derivatives was A. schoenoprasum (1563.46 μg/100 g).

Abstract

Specialized pro‐resolving mediators (SPMs), such as resolvin D5 (7S,17S‐dihydroxydocosahexaenoic acid, 7S,17S‐DiHDHA; RvD5) and protectin DX (10S,17S‐DiHDHA; PDX), are critical in resolving inflammation in humans. In this study, a unique double‐oxygenating 15S‐lipoxygenase (15S‐LOX) from the alga Chlamydomonas incerta was identified and characterized for its ability to simultaneously produce RvD5 and PDX from docosahexaenoic acid (DHA). Recombinant Escherichia coli expressing the C. incerta 15S‐LOX demonstrated enhanced RvD5 and PDX production under the following optimized reaction conditions: pH 8.0, 25°C, 0.5 g dry cells/L, 7.0 mM DHA, 2.0% (w/v) PVP, 2.0% (v/v) DMSO, and 200 mM cysteine used as a reductant. This one‐step biocatalytic process produced 2.91 mM (1.05 g/L) RvD5 and 2.18 mM (0.78 g/L) PDX in 90 min, with a total of 5.09 mM (1.83 g/L) and a total conversion yield of 79.6% (w/w). Compared to previously reported two‐step biocatalytic processes, this one‐step process significantly enhanced the production of particular PDX with improved productivity and simplicity. Structural analysis identified residues Phe667, Ile705, and Leu713 as regioselectivity modulators for the second oxygenation step. This study demonstrates the efficiency and industrial potential of the double‐oxygenating LOX as a biocatalyst for simultaneously producing RvD5 and PDX.

2.2. EBC Was Attractive to Non-Viruliferous N. Virescens, while 2-Heptanol Was Repellent to Viruliferous N. Virescens

Behavioral assays revealed that 2-heptanol was not attractive or repellent to non-viruliferous N. virescens at any of the three dosages tested (C, 0.01 μg μL−1, χ2 = 4.832, P = 0.185, n = 38; 0.1 μg μL−1, χ2 = 0.270, P = 0.966, n = 40; 1 μg μL−1, χ2 = 1.215, P = 0.749, n = 41). However, 2-heptanol was significantly repellent to viruliferous N. virescens at the three dosages tested (D, 0.01 μg μL−1, χ2 = 58.304, p < 0.001, n = 36; 0.1 μg μL−1, χ2 = 44.367, p < 0.001, n = 36; 1 μg μL−1, χ2 = 82.395, p < 0.001, n = 37). The walking paths of N. virescens are shown in (2-heptanol, 0.1 μg μL−1).

2.2. EBC Was Attractive to Non-Viruliferous N. Virescens, while 2-Heptanol Was Repellent to Viruliferous N. Virescens

Behavioral assays revealed that 2-heptanol was not attractive or repellent to non-viruliferous N. virescens at any of the three dosages tested (C, 0.01 μg μL−1, χ2 = 4.832, P = 0.185, n = 38; 0.1 μg μL−1, χ2 = 0.270, P = 0.966, n = 40; 1 μg μL−1, χ2 = 1.215, P = 0.749, n = 41). However, 2-heptanol was significantly repellent to viruliferous N. virescens at the three dosages tested (D, 0.01 μg μL−1, χ2 = 58.304, p < 0.001, n = 36; 0.1 μg μL−1, χ2 = 44.367, p < 0.001, n = 36; 1 μg μL−1, χ2 = 82.395, p < 0.001, n = 37). The walking paths of N. virescens are shown in (2-heptanol, 0.1 μg μL−1).

Abstract

Patients with rheumatoid arthritis have subnormal vitamin B6 status, both quantitatively and functionally. Abnormal vitamin B6 status in rheumatoid arthritis has been associated with spontaneous tumor necrosis factor (TNF)-α production and markers of inflammation, including C-reactive protein and erythrocyte sedimentation rate. Impaired vitamin B6 status could be a result of inflammation, and these patients may have higher demand for vitamin B6. The aim of this study was to determine if daily supplementation with 50 mg of pyridoxine for 30 days can correct the static and/or the functional abnormalities of vitamin B6 status seen in patients with rheumatoid arthritis, and further investigate if pyridoxine supplementation has any effects on the pro-inflammatory cytokine TNF-α or IL-6 production of arthritis. This was a double-blinded, placebo-controlled study involving patients with rheumatoid arthritis with plasma pyridoxal 5'-phosphate below the 25th percentile of the Framingham Heart Cohort Study. Vitamin B6 status was assessed via plasma and erythrocyte pyridoxal 5'-phosphate concentrations, the erythrocyte aspartate aminotransferase activity coefficient (αEAST), net homocysteine increase in response to a methionine load test (ΔtHcy), and 24 h urinary xanthurenic acid (XA) excretion in response to a tryptophan load test. Urinary 4-pyridoxic acid (4-PA) was measured to examine the impact of pyridoxine treatment on vitamin B6 excretion in these patients. Pro-inflammatory cytokine (TNF-α and IL-6) production, C-reactive protein levels and the erythrocyte sedimentation rate before and after supplementation were also examined. Pyridoxine supplementation significantly improved plasma and erythrocyte pyridoxal 5'-phosphate concentrations, erythrocyte αEAST, urinary 4-PA, and XA excretion. These improvements were apparent regardless of baseline B6 levels. Pyridoxine supplementation also showed a trend (p < 0.09) towards a reduction in post-methionine load ΔtHcy. Supplementation did not affect pro-inflammatory cytokine production. Although pyridoxine supplementation did not suppress pro-inflammatory cytokine production in patients with rheumatoid arthritis, the suboptimal vitamin B6 status seen in rheumatoid arthritis can be corrected by 50 mg pyridoxine supplementation for 30 days. Data from the present study suggest that patients with rheumatoid arthritis may have higher requirements for vitamin B6 than those in a normal healthy population.

Study interventions

Qualified subjects started taking the study treatment within one week of plasma pyridoxal 5'-phosphate analysis. These subjects were randomly assigned through the NEMC pharmacy to receive either active vitamin B6 (B6 group) or placebo (placebo group) tablets in double-blinded fashion for 30 days. To minimize the potential confounding effect of methotrexate and prednisone treatment on the functional tests, subjects were stratified by prednisone and methotrexate treatment, and then the subjects in each group were randomized to receive either active or placebo treatment. The randomization procedure was under guidance of a statistician and performed by registered pharmacists not directly involved in the present study.

Study interventions

The placebo tablet, made specifically for the study, was identical in appearance as well as ingredients to the active tablet, except that the active tablet (Nutro Laboratories, South Plainfield, NJ, USA) contained 50 mg of pyridoxine hydrochloride and the placebo did not (Tishcon Corp., Westbury, NY, USA). Both tablets contained microcrystalline cellulose, croscarmellose sodium, calcium phosphate, stearic acid, and magnesium stearate, ingredients commonly found in over-the-counter vitamin B6 supplements. Each phase 2 participant was asked to take one assigned tablet daily throughout the 30 day period. To assure compliance with the treatment regimen, each subject was given a personal study calendar with the 30 supplement days highlighted. The subject was asked to record the time of ingestion of each tablet on the calendar. In addition, the study coordinator made phone calls to remind each subject to take the tablets during the 30 day supplement period. The subjects were asked to return the bottle for a tablet count at the end of the 30 day treatment. To test the efficacy of the vitamin B6 supplementation, each subject went through the same 2 day testing procedure described above at the end of the 30 day supplementation period.

Results

Indicators of vitamin B6 status before and after treatment are shown in Table . All markers of vitamin B6 status improved significantly in the B6 group after supplementation, except for net increase in total homocysteine concentration, which only showed a modest trend towards improvement. None of the vitamin B6 status parameters showed significant improvements after treatment in the placebo group. Analysis of co-variance further demonstrated that initial levels of plasma pyridoxal 5'-phosphate, ΔtHcy, post-load urinary XA, αEAST (Table ) and CRP and erythrocyte sedimentation rate (ESR) (Table ) in phase 1 were strong predictors of those indicators after treatment in phase 2. After adjusting for the initial levels in (before treatment), the vitamin B6 supplementation significantly improved plasma and erythrocyte pyridoxal 5'-phosphate concentrations, αEAST, post-load XA and 24 h 4-PA excretion. We found a trend for normalization of plasma ΔtHcy in the vitamin B6 treatment versus placebo group (p = 0.086, ANCOVA). In patients who had abnormal ΔtHcy (above 15 μmol/l) before treatment (n = 22/28), the effect of vitamin B6 treatment was significant (p < 0.02). Plasma pyridoxal 5'-phosphate and ΔtHcy levels were related to CRP in patients with rheumatoid arthritis [], thus CRP could be a potential targets for vitamin B6 supplementation. The correlations between CRP and plasma pyridoxal 5'-phosphate and ΔtHcy disappeared in the B6 group after supplementation, whereas the relationships remained in the placebo group after the 30 day treatment (CRP versus plasma pyridoxal 5'-phosphate, r = -0.61, p = 0.02; CRP versus ΔtHcy, r = 0.48, p = 0.098) (n = 14). We found that vitamin B6 supplementation had no effect on inflammatory cytokines, plasma CRP, ESR, or rheumatoid factor levels in these patients (Table ).

Cell culture & proliferation assays

Ishikawa cells were obtained from the European Collection of Cell Cultures (Cat. No. 99040201) [] and were grown in DMEM/F-12 Ham medium (Cat. No. D6421, Sigma-Aldrich, UK) supplemented with L-glutamine and 10% fetal calf serum in 96-well plates (proliferation assays), 6-well plates (FACS analysis, luciferase reporter assays) or cell culture flasks (RNA extraction, tumour explant preparation). Cells were cultured at 37°C and 5%CO2 with varying doses of fenofibrate (Cat. No. F6020), 9-cis retinoic acid (9-cRA) (Cat. No. R4643) or all-trans retinoic acid (ATRA) (Cat. No. R6265, all Sigma-Aldrich, UK) dissolved in DMSO (Sigma-Aldrich). Control cells were treated with DMSO and the concentration of DMSO was kept the same throughout each experiment, to a maximum of 1% v/v. A minimum of five replicates per dose was performed for automated assays (BrdU and MTS) and all other cell culture experiments were performed independently in duplicate or triplicate. Cell proliferation was measured by the uptake of 5-bromo-2'-deoxyuridine (BrdU) using the BrdU Labelling and Detection Kit (Cat. No. 1444611, Roche, UK) according to the manufacturer's instructions. Relative cell abundance was measured either by cell counting, or using an MTS assay (CellTiter 96 Aqueous Cell Proliferation Assay, Cat. No. TB169, Promega, UK). Absorbance values for each well were measured using a microtiter plate reader (Anthos Labtech Instruments, Salzburg, Austria)

Objectives:

We investigated whether lowering of fasting homocysteine concentrations, either with folic acid or with betaine supplementation, differentially affects vascular function, a surrogate marker for risk of cardiovascular disease, in healthy volunteers. As yet, it remains uncertain whether a high concentration of homocysteine itself or whether a low folate status—its main determinant—is involved in the pathogenesis of cardiovascular disease. To shed light on this issue, we performed this study.

Design:

This was a randomized, placebo-controlled, double-blind, crossover study.

Participants:

Participants were 39 apparently healthy men and women, aged 50–70 y.

Interventions:

Participants ingested 0.8 mg/d of folic acid, 6 g/d of betaine, and placebo for 6 wk each, with 6-wk washout in between.

Results:

Folic acid supplementation lowered fasting homocysteine by 20% (−2.0 μmol/l, 95% confidence interval [CI]: −2.3; −1.6), and betaine supplementation lowered fasting plasma homocysteine by 12% (−1.2 μmol/l; −1.6; −0.8) relative to placebo. Mean (± SD) FMD after placebo supplementation was 2.8 (± 1.8) FMD%. Supplementation with betaine or folic acid did not affect FMD relative to placebo; differences relative to placebo were −0.4 FMD% (95%CI, −1.2; 0.4) and −0.1 FMD% (−0.9; 0.7), respectively.

Conclusions:

Folic acid and betaine supplementation both did not improve vascular function in healthy volunteers, despite evident homocysteine lowering. This is in agreement with other studies in healthy participants, the majority of which also fail to find improved vascular function upon folic acid treatment. However, homocysteine or folate might of course affect cardiovascular disease risk through other mechanisms.

Methodology/Principal Findings

Herein we report the expression and purification of Yersinia pestis Dam and the development of a continuous fluorescence based assay for DNA adenine methyltransferase activity that is suitable for determining the kinetic parameters of the enzyme and for high throughput screening against potential Dam inhibitors. The assay utilised a hemimethylated break light oligonucleotide substrate containing a GATC methylation site. When this substrate was fully methylated by Dam, it became a substrate for the restriction enzyme DpnI, resulting in separation of fluorophore (fluorescein) and quencher (dabcyl) and therefore an increase in fluorescence. The assays were monitored in real time using a fluorescence microplate reader in 96 well format and were used for the kinetic characterisation of Yersinia pestis Dam, its substrates and the known Dam inhibitor, S-adenosylhomocysteine. The assay has been validated for high throughput screening, giving a Z-factor of 0.71±0.07 indicating that it is a sensitive assay for the identification of inhibitors.

Introduction

To kinetically characterise Dam and its substrates or inhibitors, it was essential to develop an assay that reported the initial rate of reaction. To do this, we have used a hemimethylated break light oligonucleotide which upon turnover yields the fully methylated product. This is cleaved in situ by the restriction enzyme DpnI, resulting in an increase in fluorescence. Having established a direct relationship between the observed fluorescence increase and the methylation activity of Dam with this hemimethylated substrate, we have used the assay to determine the K M and V max of Y. pestis Dam for S-adenosylmethionine and the oligonucleotide substrate. To demonstrate the convenience of this assay, we have used it to determine the K i of the known Dam inhibitor, S-adenosylhomocysteine with Y. pestis Dam.

Measurement of K i of S-adenosylhomocysteine for Y. pestis Dam

The effect of a known Dam inhibitor, S-adenosylhomocysteine on Y. pestis Dam was investigated using the break light assay. From the data shown in , the K i was calculated to be 6.93±2.01 µM and is lower than the reported value for the E. coli enzyme (K i = 41.6±10.4 µM). These experiments demonstrate the application of the assay to the detailed kinetic analysis of a single inhibitor.