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Abstract

The genus Nicotiana (Solanaceae) includes over 70 species, with a long history of traditional use; many of them are nowadays used in bioengineering, biosynthesis, molecular biology, and other studies, while common tobacco, N. tabacum L., is one of the most economically important industrial crops worldwide. Although Nicotiana species have been extensively investigated, relatively less research has focused on flowers, especially research related to obtaining aromatic products for cosmetic and perfumery use. On the other hand, there is evidence that Nicotiana flowers accumulate various secondary metabolites with a distinct aroma and biological activities, and the flowers represent a biomass available in sufficient quantities. Therefore, this study aimed to determinate the chemical composition (by GC-MS) and the olfactory profiles of a specific type of natural aromatic product (concrete), obtained from the flowers of four Nicotiana species, in a direct comparison between them. The yields of extracted concrete were sufficiently high, varying between the species, 1.4% (N. rustica L.), 2.5% (N. glutinosa L.), 1.6% (N. alata Link&Otto genotype with white flowers), 2.7% (N. alata genotype with pink flowers), 3.2% (N. tabacum, Oriental type), and 5.2% (N. tabacum, Virginia type). The major components of the obtained concretes belonged to different chemical classes: N. rustica and N. tabacum (OR), the hydrocarbons n-tetratriacontane (14.5%; 15.0%) and n-triacontane (12.1%; 13.3%), and 3-methyl-pentanoic acid (11.1%; 12.2%); N. glutinosa, the diterpenes sclareol (25.9%), 3-α-hydroxy-manool (16.3%), and 13-epimanool (14.9%); N. alata (WF), the phenylpropanoid terephthalic acid and di(2-ethylhexyl) ester (42.9%); N. alata (PF), the diterpene tributyl acetylcitrate (30.7%); and N. tabacum (FCV), the hydrocarbons n-hexacosane (12.9%) and n-pentacosane (12.9%). Each of the flower concretes revealed a characteristic odor profile. This is the first report about Nicotiana species as a source for obtaining flower concretes; these initial results about the concrete yield, olfactory profile, and chemical composition are a prerequisite for the possible processing of Nicotiana flowers into new aromatic products for use in perfumery and cosmetics. The study provides new data in favor of the potential of the four Nicotiana species as aromatic plants, as well as a possible alternative use of flowers, a valuable, but discarded, plant material in other applications.

2. Results

In the concrete obtained from N. rustica flowers, 24 components (99.5% of the total content) were identified; only one of them had a concentration under 1% As seen from , the major constituents (over 3%) of the extract were: n-tetratriacontane (14.6%), n-triacontane (12.1%), pentanoic acid, 3-methyl (11.1%), tributyl acetylcitrate (9.1%), n-octacosane (5.5%), n-nonacosane (4.4%), n-dotriacontane (4.3%), n-hexatriacontane (3.7%), and (Z,Z)-linoleic acid (3.4%). The dominant group of substances in the concrete was that of aliphatic hydrocarbons (65.2%), and the rest of the identified components represented oxygenated aliphatics (29.4%), alkaloids (2.9%), and phenyl propanoids (2.4%).

Abstract

Honeybees produce royal jelly (RJ) from their cephalic glands. Royal jelly is a source of nutrition for the queen honey bee throughout its lifespan and is also involved in fertility and longevity. Royal jelly has long been considered beneficial to human health. We recently observed that RJ delayed impairment of motor function during aging, affecting muscle fiber size. However, how RJ affects skeletal muscle metabolism and the functional component of RJ is as of yet unidentified. We demonstrate that feeding mice with RJ daily prevents a decrease in myofiber size following denervation without affecting total muscle weight. RJ did not affect atrophy-related genes but stimulated the expression of myogenesis-related genes, including IGF-1 and IGF receptor. Trans-10-hydroxy-2-decenoic acid (10H2DA) and 10-hydroxydecanoic acid (10HDAA), two major fatty acids contained in RJ. After ingestion, 10H2DA and 10HDAA are metabolized into 2-decenedioic acid (2DA) and sebacic acid (SA) respectively. We found that 10H2DA, 10HDAA, 2DA, and SA all regulated myogenesis of C2C12 cells, murine myoblast cells. These novel findings may be useful for potential preventative and therapeutic applications for muscle atrophy disease included in Sarcopenia, an age-related decline in skeletal muscle mass and strength.

3.1. pRJ Attenuates Denervation-Induced Skeletal Muscle Atrophy

To examine the effect of pRJ on skeletal muscle atrophy, C57BL/6 mice were fed on control or pRJ diets for four weeks, and then muscle atrophy was induced by denervation. Daily feeding of pRJ for 1 month had no significant effect on total body weight (A) or loss of total tibialis anterior muscle weight induced by denervation (B). However, pRJ prevented the decrease in skeletal muscle fiber diameter following denervation (C,D). In order to determine the mechanism by which pRJ prevents the decrease in muscle fiber size, we next compared the expression levels of atrophy, proliferation, or skeletal muscle differentiation genes. qPCR and Western blotting analysis showed that pRJ did not alter the expression of catabolic genes such as Atrogin-1 (A and 2K), Muscle ring finger protein 1 (MuRF1) (B), or Forkhead box O-1 (Foxo-1) (C). However, pRJ increased the expression of proliferation and differentiation-related genes such as Cyclin E1 (D), Cyclin A2 (E), or Myogenin (G). Protease-treated RJ had no significant effect on the upregulation of Mych1 (H), Myhc2 (), Myhc4 (), or Myhc7 (). Protease-treated RJ stimulated the upregulation of IGF-1 (I) and IGF receptor (IGFR) (J) and phosphorylation of AMPK (K).

3.2. pRJ Stimulates Myoblast Proliferation

Protease-treated royal jelly treatment for 48 hours increased the number of cells (A,B) as well as the expression of cell-cycle-related genes (C–F). Furthermore, Ki67 immunostaining showed that pRJ treatment increased the number of Ki67-positive proliferative cells (G,H). Trans-10-hydroxy-2-decenoic acid and 10-hydroxydecanoic acid are two fatty acids specifically occurring in RJ. Trans-10-hydroxy-2-decenoic acid and 10-hydroxydecanoic acid can be metabolized into 2DA and SA, respectively []. To determine whether RJ-derived fatty acids and/or their metabolic products affect proliferation, C2C12 cells were treated with the indicated compounds for 24, 48, and 72 hours, after which cell numbers were quantified by WST-8. As shown in A, 10H2DA, 10HDAA, 2DA, and SA all significantly increased cell proliferation (A). These RJ-related fatty acids also increased the protein levels of cell cycle genes such as Cyclin D1 and Cyclin A2 (B).

3.3. pRJ Stimulates Myoblast Differentiation

Finally, we examined the effect of pRJ and RJ-related fatty acid products on myoblast differentiation. C2C12 cells were induced to differentiate in the presence or absence of pRJ. Myosin heavy-chain immunostaining showed that pRJ treatment led to an increase in myotube formation compared to control treatment cells (A,B). Furthermore, pRJ treatment elevated the expression level of muscle differentiation genes such as MyoD, Myogenin, Mck, or Myhc1 (C–H). C3H10T1/2 cell model is a mouse embryonic fibroblast cell line with myogenic potential. C3H10T1/2 cells, however, do not normally express MyoD, a master regulator of myogenesis []. To evaluate the effect of pRJ on MyoD function, we overexpressed MyoD in C3H10T1/2 cells and then treated the cells with or without pRJ. qPCR analysis revealed that pRJ treatment enhanced the induction of Myogenin and Myhc1 induced by MyoD (I,J). Treatment with 10H2DA, 10HDAA, 2DA, and SA increased myotube formation (A,B). The treatment of cells with these RJ-related fatty acids increased the protein levels of myogenic differentiation marker genes such as Myhc and myogenin (C).

4. Discussion

In this study, we demonstrate that feeding mice daily with pRJ prevents a decrease in myofiber size following denervation. In a previous study, we showed that pRJ affects muscle fiber size in elderly mice without changing total muscle weight []. Our current findings did not conflict with this study. Muscle weight during pathological conditions such as obesity, type 2 diabetes, or age-related Sarcopenia can be affected by the infiltration of adipose and/or connective tissue []. In our study, we did not quantify the infiltration of adipose or connective tissue into the muscle tissue, even though pRJ treatment tended to increase muscle weight. It may be interesting to explore the effect of RJ on the infiltration of muscle by adipose and connective tissue. Our data showed that daily feeding with pRJ did not decrease the expression levels of atrophy-related genes such as Atrogin-1, Murf1, or Foxo-1 although our previous work showed that pRJ feeding decreases atrophy-related gene expression during aging []. This discrepancy might be explained by differences in the relative extent of atrophy induced by denervation in comparison to aging. Nevertheless, differences between the two experimental models may be helpful to clarify the effect of RJ on skeletal muscle metabolism.

4. Discussion

We observed that RJ treatment stimulated both proliferation and differentiation. RJ stimulates cell proliferation and increases the size of Myhc positive fibers in primary satellite cells isolated from aged mice via upregulation of IGF-1 and IGFR []. In this study, RJ treatment significantly increased expression of IGF-1 and IGFR, suggesting that the IGF1-Akt pathway may contribute to the phenotype. In in vivo experiments, RJ treatment did not increase the expression levels of cyclin D1, whereas RJ treatment in vitro strongly increased cyclinD1. Currently, it is difficult to explain this discrepancy. Furthermore, we could not reveal which cell types are proliferating and differentiating in vivo following RJ treatment. This will be an important issue to resolve in future studies.

Abstract

The king scallop Pecten maximus retains the amnesic shellfish poisoning toxin, domoic acid (DA), for a long time. Most of the toxin is accumulated in the digestive gland, but this organ contains several cell types whose contribution to the accumulation of the toxin is unknown. Determining the time-course of the depuration by analyzing whole organs is difficult because the inter-individual variability is high. A sampling method, using biopsies of the digestive gland, has been developed. This method allows for repetitive sampling of the same scallop, but the representativeness of the samples obtained in this way needs to be validated. In this work, we found that the distribution of DA in the digestive gland of the scallops is mostly homogeneous. Only the area closest to the gonad, and especially its outer portion, had a lower concentration than the other ones, probably due to a transfer of the toxin to the intestinal loop. Samples obtained by biopsies can therefore be considered to be representative. Most of the toxin was accumulated in large cells (mostly digestive cells), which could be due to differences during the toxin absorption or to the preferential depuration of the toxin from the small cells (mostly secretory).

2.2. Distribution of Domoic Acid between Cellular Types

Domoic acid was preferentially accumulated into large (mostly digestive) cells (A, p = 0.0039 by Wilcoxon paired samples test). The measured concentration of this toxin in small cells (basophilic, mostly secretory) was less than 25% of that in large cells.

3. Discussion

The variability of the toxin concentration among areas of the digestive gland of the scallops is moderate-low. It is lower than the usual inter-individual variation in scallops [,] and other bivalves [,,]. Most sectors of the digestive gland had very similar concentrations, with the only significant exception being the sector adjacent to the gonad. In addition, in this sector the outer region was shown to have a lower concentration than the inner one. The most likely cause for this difference is the migration of the toxin to the intestinal loop located in the gonad. In previous studies [], it has been observed that gonadal growth during depuration produces an increase in DA burden in this organ. The origin of the toxin incorporated by the intestinal loop should be the digestive gland, which is where the DA in the scallops is mainly stored. The mechanism involved, nevertheless, is unknown. A possibility could be the reabsorption of the toxin excreted by the digestive gland by the intestinal cells. It has been shown that those cells can significantly absorb different substances [], but the very low depuration rate of the scallops [,] suggests that the amount of DA that could be reabsorbed is very low. In any case, this mechanism would not be expected to affect some parts of the gonad differently. A second possibility could be the transfer of the toxin from the digestive gland cells to the newly generated intestinal ones. Transfers of DA between organs have been documented but are significant only in exceptional situations, such as post-spawning periods [,,]. This mechanism could or could not have an effect on the distribution of DA in the different areas of the digestive gland. The third possibility is that the intestinal loop cells which contain the toxin derive from cells from the digestive gland. If the new cells of the intestinal loop are generated in the nearby area of the digestive gland, a reduction in the toxin concentration in that area would be expected. Intestinal cells are mostly epithelial and the presence of immature cells, as in the digestive gland, has not been observed []. It is therefore possible that the intestinal cells derive from immature cells in the digestive gland and consequently inherit the toxin they contain. This would explain the lower DA concentration in the sector closest to the gonad and also the greater reduction in the outer region relative to the inner one, because the cells in this region are the closest to the intestinal loop. The confirmation of this hypothesis requires additional studies.

3. Discussion

Domoic acid was preferentially retained in large cells, mostly absorptive (digestive cells) []. The concentration measured in small cells, mostly secretory and immature cells, was substantially lower than in the digestive ones. In terms of toxin burden, the importance of large cells was even greater, because they are found in a larger amount in the digestive gland (more biomass). The difference in concentration opens two possibilities regarding the methods of depuration not taken into account to date: (a) the secretory cells depurate the DA they have accumulated during the intoxication phase (which could be the case if both cell types accumulate the toxin to the same extent); and (b) DA is transferred from absorptive to secretory cells, which excrete it. Unfortunately, no information about the distribution of DA during the intoxication phase exists and the transfer between cellular types has not been documented, so new studies would be required to shed some light on the subject. However, in our opinion, neither of the two ways is very likely. Firstly, it seems unlikely that absorptive and secretory cells would absorb and accumulate DA during the intoxication at the same rate, because of their main functions. Secondly, it is difficult to explain how the toxin could pass the cellular membrane of the absorptive cells to go to the secretory ones but not to be excreted (the depuration rate is very low [,]) or transferred to other tissues [,].

Abstract

The aim of the present study was to assess the effects of l-buthionine-(S,R)-sulfoximine (BSO), a glutathione (GSH) synthesis inhibitor, and GBR 12909, a dopamine reuptake inhibitor, administered alone or in combination to Sprague-Dawley rats during early postnatal development (p5–p16), on the levels of reactive oxygen species (ROS), lipid peroxidation (LP) and the activities of antioxidant enzymes: superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and glutathione disulfide reductase (GR) in peripheral tissues (liver, kidney) and selected brain structures (prefrontal cortex, PFC; hippocampus, HIP; and striatum, STR) of 16-day-old rats. The studied parameters were analyzed with reference to the content of GSH and sulfur amino acids, methionine (Met) and cysteine (Cys) described in our previous study. This analysis showed that treatment with a BSO + GBR 12909 combination caused significant decreases in the lipid peroxidation levels in the PFC and HIP, in spite of there being no changes in ROS. The reduction of lipid peroxidation indicates a weakening of the oxidative power of the cells, and a shift in balance in favor of reducing processes. Such changes in cellular redox signaling in the PFC and HIP during early postnatal development may result in functional changes in adulthood.

2.1. Animals and Treatment

To create the neurodevelopmental model of schizophrenia, pregnant Sprague-Dawley females were delivered to our laboratory by the Charles River Company (Sulzfeld, Germany). They were kept in individual cages, with free access to standard laboratory food and tap water under standard laboratory conditions i.e., at room temperature (22 °C) and under an artificial light/dark cycle (12/12 h). On the day of delivery, the sex of pups was determined. Only male pups which were left with their mothers were treated on the postnatal days p5–p16, with the selective inhibitor of GCL, compound l-buthionine-(S,R)-sulfoximine (BSO) and the DA reuptake inhibitor GBR 12909, alone or in combination. BSO at a dose of 3.8 mmol/kg s.c., was administered once daily, while GBR 12909 at a dose of 5 mg/kg s.c., every second day. Both model compounds were dissolved in 0.9% NaCl. Under the combined treatment regimen, BSO administration proceeded GBR 12909 injection. Control rats instead of the model compounds received a 0.9% NaCl once daily. Every day, pups were weighed and the injected volumes of the studied model compounds were adjusted accordingly with the actual body weight. On postnatal day p16, the rats were killed by decapitation 4 h after the last doses of model compounds, and their liver, kidney and selected brain structures (PFC, HIP, STR) were dissected, immediately frozen on dry ice and stored at −80 °C until further biochemical analysis.

Abstract

Bone remodeling and repair require osteogenic cells to reach the sites that need to be rebuilt, indicating that stimulation of osteoblast migration could be a promising osteoanabolic strategy. We showed that purified δ-tocotrienol (δ-TT, 10 μg/mL), isolated from commercial palm oil (Elaeis guineensis) fraction, stimulates the migration of both MC3T3-E1 osteoblast-like cells and primary human bone marrow mesenchymal stem cells (BMSC) as detected by wound healing assay or Boyden chamber assay respectively. The ability of δ-TT to promote MC3T3-E1 cells migration is dependent on Akt phosphorylation detected by Western blotting and involves Wnt/β-catenin signalling pathway activation. In fact, δ-TT increased β-catenin transcriptional activity, measured using a Nano luciferase assay and pretreatment with procaine (2 µM), an inhibitor of the Wnt/β-catenin signalling pathway, reducing the wound healing activity of δ-TT on MC3T3-E1 cells. Moreover, δ-TT treatment increased the expression of β-catenin specific target genes, such as Osteocalcin and Bone Morphogenetic Protein-2, involved in osteoblast differentiation and migration, and increased alkaline phosphatase and collagen content, osteoblast differentiation markers. The ability of δ-TT to enhance the recruitment of BMSC, and to promote MC3T3-E1 differentiation and migratory behavior, indicates that δ-TT could be considered a promising natural anabolic compound.

2.2. Effect of δ-TT on MC3T3-E1 and BMSC Cell Migration

We then studied the effects of δ-TT on cell migration, performing a scratch assay. MC3T3-E1 cells were exposed to δ-TT 24 h before and 24 h after the wound gap has been applied. As shown in , MC3T3-E1 cells treated with δTT exhibited a faster wound healing rate compared to that of control untreated cells at 18 and 24 h, reaching a statistical significance at 24 h.

2.2. Effect of δ-TT on MC3T3-E1 and BMSC Cell Migration

Lastly, we examined the effects of δ-TT on BMSC cell migration, performing a micro-chemotaxis assay in a Boyden chamber. BMCS exposed to δ-TT 24 h before and during micro-chemotaxis assay, showed a significant increase in cell migration (170%) in comparison to the control-treated cells ().

Abstract

Domoic acid (DA), the main toxin responsible for Amnesic Shellfish Poisoning, frequently affects the marine resources of Chile and other countries across the South Pacific, thus becoming a risk for human health. One of the affected resources is the scallop Argopecten purpuratus. Even though this species has a high commercial importance in Northern Chile and Peru, the characteristics of its DA depuration are not known. In this work, the DA depuration was studied by means of two experiments: one in controlled (laboratory) and another in natural conditions. All organs of A. purpuratus depurated the toxin very quickly in both experiments. In some organs, an increase or a very small decrease of toxin was detected in the early depuration steps. Several models were used to describe this kinetics. The one that included toxin transfer between organs and independent depuration from each organ was the model that best fit the data. It seems, therefore, that the DA in this species is quickly transferred from the digestive gland to all other organs, which release it into the environment. Physiological differences in the two experiments have been shown to have some effect on the depuration from each organ but the actual reasons are still unknown.

2. Results

Two DA depuration experiments were carried out using scallops which had fed on a naturally occurring bloom of toxic Pseudo-nitzschia australis. In the first experiment, scallops were removed from the field, held in the laboratory, and fed non-toxic Isochrysis galbana (Haptophyta). Changes in body weight and tissue-specific DA concentration were then measured for 12 days. In the second, experiment the scallops were maintained in the field and allowed to feed naturally on a post-bloom phytoplankton population containing only a relatively small population of Pseudo-nitzschia australis.

2.3.1. Depuration of Domoic Acid in the Natural Environment

Two types of models were fitted to the experimental data of the toxin burden. The first one was first-order exponential decrease of each organ, without toxin transfer between organs, and the second included toxin transfers from DG to all other organs, which depurated in the same way than in the first type of model. When fitted to the data from the experiment carried out in natural conditions (the complete dataset) the model without transfers of DA from the digestive gland to other organs was unable to correctly describe the depuration, especially because of the increase of toxin burden in the second day of the experiment in some organs. When the transfer between organs was included, the model fits the observed data much better (). The simplest model of this type, with only a common transfer rate to all organs and a common depuration rate, described the general shape of the kinetics but did not fit well the levels of DA in several organs, especially in DG (with levels noticeably underestimated).

2.3.2. Depuration of Domoic Acid in Laboratory

The same model fitted to the data obtained in the laboratory gave different estimates, but the amount of useful observations was very low because of the steep toxin burden descent (). The model estimates some transfer of toxin, but the organs to which the toxin transfer was estimated to be higher did not coincide with those in the experiment carried out in natural conditions (). Some of the observed differences could be explained if the depuration of the different organs does take place totally or partially through the kidney; unfortunately, it was not separated from the adductor muscle in any of the experiments. This may account for the increase of DA in Mu (adductor muscle plus kidney) during the initial steps of the laboratory experiment, in which the depuration rate was very high.

3.2. Domoic Acid Depuration

There were important differences in the apparent depuration rate between the two experiments. Scallops in laboratory depurated faster than in natural conditions (0.91 vs. 0.27 day−1, respectively). Most of the difference is probably due to some degree of reintoxication in natural conditions, because the toxic algae populations had not completely disappeared (). The two experiments also differed at least in temperature and food availability, and both could have contributed to the observed difference. The difference in the temperature at which the experiments were developed (16 °C in the laboratory and 13–14 °C at natural conditions) could have contributed to the difference in depuration rate, since temperature seems to accelerate the DA depuration in other species, like the king scallop Pecten maximus [] or the detoxification of paralytic shellfish toxins in the visceral mass of the surf clam Spisula solidissima []. The magnitude of the recorded difference seems, nevertheless, too high to be the only factor, as the estimated Q10 (temperature coefficient) is near 6, while, for most physiological rates in bivalves it is between 2 and 3 [,]. Food availability would not seem to be primarily responsible because in sight of its likely negative effect on depuration in the king scallop, Pecten maximus []. Nevertheless, its effect cannot be ruled out because the physiological differences between the two species could be important.

Abstract

This review considers the results of recent studies on marine excitatory amino acids, including kainic acid, domoic acid, dysiherbaine, and neodysiherbaine A, known as potent agonists of one of subtypes of glutamate receptors, the so-called kainate receptors. Novel information, particularly concerning biosynthesis, environmental roles, biological action, and syntheses of these marine metabolites, obtained mainly in last 10–15 years, is summarized. The goal of the review was not only to discuss recently obtained data, but also to provide a brief introduction to the field of marine excitatory amino acid research.

1. Introduction

Throughout the 55-year history of the G.B. Elyakov Pacific Institute of Bioorganic Chemistry (PIBOC), Far Eastern Branch, Russian Academy of Science, a vast variety of new marine-derived natural compounds including different glycosides, polar steroids, alkaloids, lipids, and other metabolites have been isolated from echinoderms and sponges, ascidians, algae, fungi, and marine bacteria and thoroughly studied. Many of the discovered new natural compounds proved to have a protective action and were repeatedly mentioned in well-known series of review articles published in Natural Products Reports []. To protect themselves against predators and pathogens, marine organisms use a variety of structural groups of toxins. The latter are synthesized by lower plants, including microalgae [,,], and then accumulated by some invertebrates that transfer them up to higher trophic levels. Marine excitatory amino acids (MEAAs) that belong to this category and play an important role in marine communities have found application in pharmacology. To date, there are only three known groups of marine-derived excitatory amino acids: (1) kainic acid and related compounds, (2) domoic acid and some its derivatives, (3) sponge-derived dysiherbaine and neodysiherbaine A, which specifically bind and activate the kainate-type of glutamate receptors (GluRs).

Abstract

A liquid chromatography–high resolution mass spectrometry (LC-HRMS) method for the direct determination of various saturated hydroxy fatty acids (HFAs) in milk was developed for the first time. The method involves mild sample preparation conditions, avoids time-consuming derivatization procedures, and permits the simultaneous determination of 19 free HFAs in a single 10-min run. This method was validated and applied in 17 cow milk and 12 goat milk samples. This work revealed the existence of various previously unrecognized hydroxylated positional isomers of palmitic acid and stearic acid in both cow and goat milk, expanding our knowledge on the lipidome of milk. The most abundant free HFAs in cow milk were proven to be 7-hydroxystearic acid (7HSA) and 10-hydroxystearic acid (10HSA) (mean content values of 175.1 ± 3.4 µg/mL and 72.4 ± 6.1 µg/mL in fresh milk, respectively). The contents of 7HSA in cow milk seem to be substantially higher than those in goat milk.

2.1. LC-ESI-MS Data

Nineteen SHFAs were used in the present study. Eight of them were regio-isomers of hydroxy palmitic acid [2-hydroxypalmitic acid (2HPA), 3-hydroxypalmitic acid (3HPA), 7-hydroxypalmitic acid (7HPA), 8-hydroxypalmitic acid (8HPA), 9-hydroxypalmitic acid (9HPA), 10-hydroxypalmitic acid (10HPA), 11-hydroxypalmitic acid (11HPA), 16-hydroxypalmitic acid (16HPA)], and seven were regio-isomers of stearic acid [2-hydroxystearic acid (2HSA), 3-hydroxystearic acid (3HSA), 7-hydroxystearic acid (7HSA), 8-hydroxystearic acid (8HSA), 9-hydroxystearic acid (9HSA), 10-hydroxystearic acid (10HSA), 12-hydroxystearic acid (12HSA)]. In addition, four 3-hydroxy FAs [3-hydroxycapric acid (3HCA), 3-hydroxylauric acid (3HLA), 3-hydroxymyristic acid (3HMA), 3-hydroxypentadecanoic acid (3HPDA)] were included in the set of compounds tested. The high-resolution mass spectra of these HFAs were recorded in electrospray ionization (ESI) negative mode. The structures of the SHFAs used along with the exact masses of the deprotonated molecules and the retention times observed for each one of them in the chromatographic method are presented in . The MS/MS spectra of all the 19 analytes in negative ESI mode are also presented in the .

2.3. Analysis of Samples

Seventeen cow milk samples and 12 goat milk samples were purchased from the local market and analyzed. The extracted ion chromatograms (EICs) of a cow milk sample (A) and a goat milk (B) sample are shown in . The simultaneous determination of 19 SHFAs was achieved in a single 10-min run. The contents of the 19 analytes in milk samples (in triplicate) are summarized in , and they are expressed as the µg of HFA per mL of milk. The integration of the peak areas was performed manually using MultiQuant 3.0.2. As Hill et al. discussed [], in particular cases a manual baseline may be employed for the quantification of the peak areas. The same integration parameters were used in all cases.

Abstract

Thiamin deficiency, or beriberi, is an increasingly re-recognized cause of morbidity and mortality in the developing world. Thiamin status has traditionally been measured through the erythrocyte activation assay (ETKA) or basal transketolase activity (ETK), which indirectly measure thiamin diphosphate (TDP). Thiamin diphosphate can also be measured directly by high-performance liquid chromatography (HPLC), which may allow a more precise estimation of thiamin status. We compared the direct measurement of TDP by HPLC with basal ETK activity and ETKA in 230 patients with Plasmodium falciparum malaria in rural southern Laos without overt clinical beriberi, as part of a trial of thiamin supplementation. Admission thiamin status measured by basal ETK activity and ETKA (α) were compared with thiamin status assessed by the measurement of TDP by HPLC. 55% of 230 included patients were male, and the median age was 10 (range 0.5–73) years. Using α ≥ 25% as the gold standard of thiamin deficiency, the sensitivity of TDP < 275 ng/gHb as a measure of thiamin deficiency was 68.5% (95% CI: 54.4–80.5%), with specificity of 60.8 (95% CI: 53.2–68.1%). There was a significant inverse correlation between the results of the two tests (Kendall’s tau = −0.212, P < 0.001). Basal ETK activity was also significantly positively correlated with TDP levels (Kendall’s tau = 0.576, P < 0.001). Thiamin diphosphate measurement may have a role in measuring thiamin levels in clinical settings. Further studies evaluating TDP concentration in erythrocytes with basal ETK activity and ETKA (α) in beriberi patients would help establish comparative values of these assays.

RESULTS

Using α ≥ 25% as the gold standard of biochemical thiamin deficiency, the sensitivity of erythrocyte TDP < 275 ng/gHb as a measure of thiamin deficiency was 68.5% (95% CI: 54.4–80.5%) with specificity of 60.8 (95% CI: 53.2–68.1%). There was a significant inverse correlation between the tests (Kendall’s tau = −0.212, P < 0.001). Inverse correlation was also observed between the two assays when adults () (78 patients, Kendall’s tau = −0.227, P = 0.003) and children () (152 patients, Kendall’s tau = −0.176, P = 0.001) were analyzed separately.

DISCUSSION

Limitations of this study include the fact that no patients in the cohort manifested clinical signs of thiamin deficiency, so it is not possible to correlate results with clinical thiamin deficiency. It is possible that some patients had subclinical deficiency. Determining a clinical endpoint for thiamin deficiency is challenging, as beriberi seems to occur not only when thiamin deficiency exists but also when additional exacerbating factors such as infections are present. The distinction between those who do and do not show clinical symptoms of beriberi at different biochemical assay levels may also be confounded by other factors. Importantly, these data cannot be used to inform biochemical diagnosis of infantile beriberi as children aged < 6 months were not included.

Abstract

Cytochrome P450 monooxygenases (CYPs/P450s) are well known for their role in organisms’ primary and secondary metabolism. Among 20 P450s of the tuberculosis-causing Mycobacterium tuberculosis H37Rv, CYP128A1 is particularly important owing to its involvement in synthesizing electron transport molecules such as menaquinone-9 (MK9). This study employs different in silico approaches to understand CYP128 P450 family’s distribution and structural aspects. Genome data-mining of 4250 mycobacterial species has revealed the presence of 2674 CYP128 P450s in 2646 mycobacterial species belonging to six different categories. Contrast features were observed in the CYP128 gene distribution, subfamily patterns, and characteristics of the secondary metabolite biosynthetic gene cluster (BGCs) between M. tuberculosis complex (MTBC) and other mycobacterial category species. In all MTBC species (except one) CYP128 P450s belong to subfamily A, whereas subfamily B is predominant in another four mycobacterial category species. Of CYP128 P450s, 78% was a part of BGCs with CYP124A1, or together with CYP124A1 and CYP121A1. The CYP128 family ranked fifth in the conservation ranking. Unique amino acid patterns are present at the EXXR and CXG motifs. Molecular dynamic simulation studies indicate that the CYP128A1 bind to MK9 with the highest affinity compared to the azole drugs analyzed. This study provides comprehensive comparative analysis and structural insights of CYP128A1 in M. tuberculosis.

2.6. CYP128A1 Protein Model Has High Affinity to Menaquinone-9

In P450 enzyme systems, substrates and azole drugs are known to coordinate with the ferric core of the heme group. In the case of MK9, coordination takes place at the end of its hydrophobic tail, consistent with omega hydroxylation of the molecule [,]. For azole drugs, coordination occurs via the imidazole ring. Importantly, docking of MK9 and azole drugs was performed in a non-covalent way in the present paper. Therefore, no coordination was predicted for the tested ligands, which would require more thorough investigation, especially considering changes in electronic density. However, since non-covalent binding is a critical step in the establishment of covalent interactions, we believe the present results still provide a good indication of how azoles drugs can interact with CYP128A1 and how they rank in terms of affinity.

3.11. Redocking of Compounds

MK9 and the azoles compounds were re-docked on their corresponding set of equilibrated structures using the MOE’s dock (six structures for MK9, 10 structures for each of the azole drugs). The same options as in the first docking step were used (see ).