Nephroselmis sp. was previously identified as a species of interest for its antioxidant properties owing to its high carotenoid content. In addition, nitrogen availability can impact biomass and specific metabolites’ production of microalgae. To optimize parameters of antioxidant production, Nephroselmis sp. was cultivated in batch and continuous culture conditions in stirred closed photobioreactors under different nitrogen conditions . The aim was to determine the influence of nitrogen availability on the peroxyl radical scavenging activity  assay) and carotenoid content of Nephroselmis sp. Pigment analysis revealed a specific and unusual photosynthetic system with siphonaxanthin-type light harvesting complexes found in primitive green algae, but also high lutein content and xanthophyll cycle pigments , as observed in most advanced chlorophytes. The results indicated that N-replete conditions enhance carotenoid biosynthesis, which would correspond to a higher antioxidant capacity measured in Nephroselmis sp. Indeed, peroxyl radical scavenging activity and total carotenoids were higher under N-replete conditions and decreased sharply under N-limitation or starvation conditions. Considering individual carotenoids, siphonaxanthin, neoxanthin, xanthophyll cycle pigments, and lycopene followed the same trend as total carotenoids, while β-carotene and lutein stayed stable regardless of the nitrogen availability. Carotenoid productivities were also higher under N-replete treatment. The peroxyl radical scavenging activity measured with ORAC assay (63.6 to 154.9 µmol TE g−1 DW) and the lutein content (5.22 to 7.97 mg g−1 DW) were within the upper ranges of values reported previously for other microalgae. Furthermore, contents of siphonaxanthin ere 6 to 20% higher than in previous identified sources . These results highlight the potential of Nephroselmis sp. as a source of natural antioxidant and as a pigment of interest. Microalgae are recognized as promising sources of natural antioxidant products for nutraceuticals, pharmaceuticals, and cosmetics industries ,2,3,4,5.Dunaliella spp. exposed to N-starvation combined with high light and high salinity [Nitrogen availability can impact the production of biomass and specific metabolites. Indeed, as a major component of proteins, nucleic acids, and chlorophylls, nitrogen (N) is one of the most important elements known to influence the biochemical content of microalgae. In addition, N-starvation is known to induce ROS generation in microalgae cells ,24,25. Isalinity ,30,34. Hsalinity , whereassalinity ,35,36.Nephroselmis sp. It is a chlorophyte species isolated in a tropical lagoon in New Caledonia that was previously identified as a good source of natural antioxidant owing to its high carotenoid content [Nephroselmis sp. contains siphonaxanthin [In this study, we focus on the effects of nitrogen availability on the antioxidant activity and carotenoid content of  content . In addiaxanthin ,38, an uaxanthin , but alsaxanthin , antiobeaxanthin ,42,43, aaxanthin , and apoaxanthin .Nephroselmis sp. [Nephroselmis sp. The aim of this study was to assess the effects of nitrogen availability, combined with high light intensity, on the antioxidant activity and carotenoid content of Nephroselmis sp. in order to optimize these factors.In a previous study, it was found that high light conditions induced an increase of carotenoid contents, including siphonaxanthin, as well as peroxyl radical scavenging activity on lmis sp. . HoweverNephroselmis genus (Nephroselmis sp. (N3C46) is closely related to Nephroselmis rotunda  a f6 ± 0.3 × 106 cell mL−1 (−1 on day 6 (−1 (6 ± 0.5×106 cell mL−1 and a production of biomass of 0.35 ± 0.02 g L−1 (−1) and the C/N ratio (14 ± 0.2) remained higher than in N-replete conditions. At day 28, the cultures were switched back to batch, which induced a decrease of the nitrogen cellular content to its lowest values (~32 fmol N cell−1), whereas the cell concentration, carbon content, and biomass production increased at the beginning of the N-starvation. The growth and carbon content declined when the senescence phase began. Dissolved phosphate persisted in the medium throughout all experiments (During the first batch culture period (day 0 to day 6), nitrate in culture medium was depleted over three days, however cells kept dividing until day 4, reaching a maximum cell concentration of 29 × 10ell mL−1 . N-starvell mL−1 a, i.e., on day 6 . On the ay 6  reached at day 3 was concomitant to the first day of the nitrate starvation in the culture medium. In all conditions with N-limitation or starvation , antioxidant activity was more than 2 times lower than in N-replete cultures. After nutrient resupply on day 7, antioxidant activity increased by 47%, but maximal antioxidant activity observed on day 3 was not restored.Antioxidant activity of E g−1 DW . The higNephroselmis sp. include chlorophyll a (chl a), chlorophyll b (chl b), β-carotene, lycopene, lutein, zeaxanthin, antheraxanthin, violaxanthin, all-trans-neoxanthin, cis-neoxanthin, and siphonaxanthin (see HPLC chromatogram in a (express on dry weight basis or on carbon content basis), chlorophyll b, and total carotenoid (TC) contents , when nitrate was depleted in the culture medium. Once nitrate was depleted, chlorophyll and total carotenoid content quickly decreased from day 3 to day 6 by a factor 2 and by a factor 1.5, respectively. After nutrient resupply (day 7), the chlorophyll and total carotenoid increased by 59% and 26% and then decreased by 22% and 26%, respectively, at steady state. During the second batch period, the pigment content decreased along with the N-starvation, reaching its lowest values  at the end of the experiment. The TC/Chl ratio , and similarly to chlorophyll, siphonaxanthin and neoxanthin drastically declined along with N-starvation, reaching values of 0.74 ± 0.12 and 1.72 ± 0.25 mg g−1 DW, respectively, on day 31. The xanthophyll cycle pigments  and lycopene followed the same trend as siphonaxanthin and neoxanthin, but with less variations of the concentrations. Their respective concentrations varied from 12.14 ± 0.35 mg g−1 DW and 3.32 ± 0.37 mg g−1 on day 3 to 5.24 ± 0.88 mg g−1 DW and 0.95 ± 0.16 mg g−1 DW on day 31.The other carotenoids were present in smaller concentrations. Siphonaxanthin and neoxanthin varied concomitantly with chlorophyll. Their highest concentration was measured on day 3 , but this was attributed to the concomitant variation of chlorophyll with siphonaxanthin and neoxanthin.A correlation analysis was performed to assess a potential relationship between carotenoid content and peroxyl scavenging activity . A good −1 day−1) and in continuous mode at steady state at 0.3 days−1 of renewal (110.6 ± 5.7 mg L−1 day−1). The highest productivities for all individual pigments and total carotenoids were also measured on day 3 .Biomass and carotenoid productivities were calculated during the first batch culture period (day 2 to 6) and at steady state for the continuous culture. The highest biomass productivities were obtained on day 3 (120.8 ± 1.5 mg LNephroselmis sp. After the first nitrate starvation (during the first batch period), the cell concentration of Nephroselmis sp. still increased for one day   a,c. Nitrrivation d. This iDunaliella spp. [−2 s−1 prior to steady-state measurements (day 8) was implemented to avoid light limitation during continuous culture. The pigment contents of Nephroselmis sp. were not significantly impacted, since their values were similar at day 6 and day 28 under identical nitrogen availability and culture method. N-limitation and an important self-shading effect owing to the high biomass that reduces the impact of light availability might explain this absence of difference in pigment contents.High light intensity and N-starvation are known to have cumulative effects on pigment content. Under N-starvation, chlorophyll and primary carotenoids usually decrease further with high light intensity, while secondary carotenoid production is stimulated, such as for β-carotene in lla spp. ,64,65. INephroselmis sp., similarly to the majority of microalgae species, reacted to nitrogen starvation or limitation by reducing its photosynthetic activity. As a result, the pool of photosynthetic pigments decreased. Siphonoxanthin, neoxanthin, lycopene, and xanthophyll cycle pigments followed similar variations as the chlorophyll contents in response to nitrogen availability, owing to their implication in the PSs.Bryopsis corticulans that siphonaxanthin was associated with PSI and chlorophylls a and b in a siphonaxanthin–chlorophyll ab–protein complex, and with the light-harvesting complex (LHC) of PSII with chlorophylls a and b, neoxanthin, and siphonaxanthin esters. The light harvesting complexes with siphonaxanthin are ancestral LHCs that evolved by replacing siphonaxanthin with lutein and xanthophyll cycle pigments in higher plants to adapt to high light environments [Nephroselmis sp.  suggests that the LHCs of Nephroselmis sp. might be similar to LHCs of Bryopsis corticulans. However, unlike Bryopsis, Nephroselmis sp. also contains xanthophyll cycle pigments and a high concentration of lutein; this species might, thus, be at the intermediate stage between primitive chlorophytes with siphonaxanthin-type LHCs and higher plants [The biological role of siphonaxanthin was not clearly described in green microalgae, but its functions were described in previous studies on siphonous green algae. This ketocarotenoid, firstly described by Yokohama , acted aronments . The strr plants ,70.Nephroselmis into five distinct types according to their carotenoid compositions, especially siphonaxanthin and its derivatives. Indeed, siphonaxanthin derivatives were found among all the Nephroselmis species studied by Yoshii et al. [Yoshii et al.  classifii et al. , such asi et al. ), and wei et al. . Thus, wNephroselmis sp. under N-replete conditions  are known to have the ability to accumulate β-carotene in extra-thylakoid lipid droplets when subjected to N-starvation, along with other stressors such as high light intensity [Nephroselmis sp. under stressful conditions; however, to the best of our knowledge there is no report of extra-thylakoid lutein accumulation. Additional investigations are necessary to clarify the localization and the physiological role of lutein in Nephroselmis sp.Unlike the other carotenoids , β-carotene and lutein contents did not follow the same variation as the chlorophyll content. The β-carotene and lutein are known to act as primary carotenoids with photoprotective functions ,77,78. T the PSs . Some chntensity ,32,83. TNephroslemis sp. extract. Indeed, the Nephroselmis sp. extract was twice as active under N-replete conditions than under N-limited or starved conditions . As phenolic and carotenoid contents showed similar responses to nitrogen levels, this suggests that these compounds were the main contributors to the antioxidant activity. Our results showed a clear correlation between the peroxyl radical scavenging activity of the Nephroselmis sp. extract and the total carotenoid content , with different efficiencies depending on the number of conjugated double bonds [As for growth and pigment content, nitrogen availability strongly impacted the antioxidant activity  of the nditions . Those rs et al.  found an content , in agreotenoids ,85,86. Hguration ,85. Despguration ,85,86. Iguration  have shoextracts . These rle bonds ,78,87.Nephroselmis sp. were determined (−1 day−1) and in continuous culture at steady state (110.6 ± 5.7 mg L−1 day−1). However, as the carotenoid content was 62.5% higher in the first batch culture than at steady state, the total carotenoid productivity increased by 80% between the two conditions. The best productivity was also achieved on day 3 for all individual carotenoids. Thus, cultivation under N-replete conditions is essential to obtain the highest carotenoid productivity and radical peroxyl scavenging activity in Nephroselmis sp.In order to estimate the best nitrogen supply conditions for carotenoid production, the biomass and carotenoid productivities in termined . The higNephroselmis sp. a species of interest. Indeed, the peroxyl radical scavenging activity of Nephroselmis sp., at between 63.6 and 154.9 µmol TE g−1 DW, was among the highest values reported previously with ORAC assays of microalgae crude extracts. Banskota et al. [−1 DW in methanol extracts of nine microalgae species . An antiperoxyl radical activity of 61.53 µmol TE g−1 DW was reported in 50% water acetone extract of Phormidium autumnale [−1 DW were measured in 50% water ethanolic extracts of Chlorella vulgaris and Spirulina platensis, respectively [Nephroselmis sp. (45 to 288 µmol TE g−1 DW) in hexane extracts of eleven microalgae species ; however, peroxyl radical scavenging activity was higher in water (60 to 350 µmol TE g−1 DW) and ethyl acetate extracts (169 to 577 µmol TE g−1 DW). Furthermore, the lutein content of Nephroselmis sp. at up to 7.97 ± 0.84 mg g−1 DW was within the upper range values reported for lutein-producing strains, ranging between 0.5 and 9.6 mg g−1 DW [−1 day−1) [Nephroselmis sp. were 6–20% higher than contents reported in green algae [Nephroselmis sp. as a species of interest for siphonaxanthin production.The high total carotenoid contents of up to 5% of the cell’s dry weight with pigments of interest, such as lutein and siphonaxanthin, and the high antioxidant activity make a et al.  found ORutumnale , while Oectively . Ahmed eectively  found vag g−1 DW ,92,93,941 day−1) ,95,96,971 day−1) , antiobe1 day−1) ,43, and 1 day−1) . The sipen algae , indicatNephroselmis sp. N3C46  at a temperature of 26.5 °C.s medium . The cong, 10 min, 4 °C). The pellet was washed twice with TE-NaCl buffer . After overnight incubation with TE-NaCl, the pellet was pretreated with buffer lysis  for 2 h at 40 °C. The extract was purified with equal volumes of phenol/chloroforme/isoamyl alcohol mixture  and centrifuged . The upper aqueous layer was further purified with an equal volume of chloroform and centrifuged . The aqueous phase containing DNA was then pretreated by RNase . After a second step of PCA extraction, DNA was precipitated and washed with isopropanol and 70% ethanol, respectively. Then, the DNA pellet was solubilized in 100 µL of DNase-free water. The concentration and quality of the extracted genomic DNA were measured using a NanoDrop Spectrophotometer . The amplification of the microalgal 18S rRNA was done using a universal primer pair of 18S-F (5′-ACCTGGTTGATCCTGCCAGT-3′) and 18S-R (5′-TCCTTCTGCAGGTTCACCTAC-3′). The PCR reaction was performed at a final volume of 50 μL, which included Green GoTaq® Reaction Buffer , MgCl2 (1.5 mM); dNTPs (0.2 mM), GoTaq G2 , primer forward (1 µM); primer reverse (1 µM), and extracted DNA (0.01 ng µL−1). The PCR amplification was then performed in a thermocycler . The PCR product was then examined in 1% agarose gel and subsequently cloned (band at 1800 bp) with a TOPO TA cloning kit . The clones were sequenced on Sanger ABI at Eurofins Genomics . The DNA sequence was then compared with Basic Local Alignment Search Tool  for taxlarly to ,103 with2 addition. Light was provided on one side of the PBRs using seven adjustable fluorescent light tubes (OSRAM cool 109 daylight HO24W/965). Continuous light was set at 600 µmol m−2 s−1 by measuring the light intensity inside the PBRs. High light irradiance was used on the basis of previous results [Nephroselmis sp. with high light intensity. A Rushton turbine at 60 rpm and 0.2 µm filtered air bubbling were used to homogenize the culture. Before inoculation, PBRs were sterilized for 20 min with a 5‰ peroxyacetic acid solution and rinsed twice with 0.2 µm filtered seawater. PBRs were inoculated with 1 L of inoculum and filled up to 9.5 L with 0.2 µm filtered seawater enriched with 1 mL L−1 of Walne’s medium at 1.18 mM-N to reach an initial concentration of 7 × 105 cell mL−1.Experiments were carried out in two 10 L photobioreactors (PBRs) made of transparent polymethylmethacrylate. The temperature was kept constant at 26.5 °C ± 0.3 and the pH was regulated at 7.75 ± 0.04 by automated CO results  that rep(1)−1 of Walne’s medium at 1.18 mM-N to ensure that the temperature and pH probes remained submerged;A first batch culture period (day 0 to day 6) was applied to study the effects of N-replete to N-starvation conditions. At the end of this period, and owing to sample collections during this first batch culture, PBRs volumes were restored to 9.5 L (day 6) by adding filtered sea water (0.2 µm) enriched with 1 mL L(2)−1 was applied. The culture medium was composed of filtered seawater (0.2 µm) enriched in 1 mL L−1 of Walne’s medium at 1.18 mM-N [−2 s−1 on day 8 to ensure that there was no light limitation owing to the very high cellular concentration (>25 × 106 cell mL−1). Continuous culture was maintained until analyses at steady state ;The PBRs were then switched to a continuous mode of culture in chemostat to study the effects of N-limitation (day 7 to day 27). During this period, a dilution rate of 0.3 day.18 mM-N . The lig(3)Finally, the PBRs were switched back to a second batch culture period (day 28 to day 31) in N-starvation conditions until beginning of senescence.The experiment was separated in 3 successive stages with various nitrogen conditions :(1)A fira absorption and non-pigmented cell compounds absorption, respectively , then filters were dried at 70 °C for 24 h and kept at −20 °C until analysis by a CHN (Carbon Hydrogen Nitrogen) elemental analyzer (SERCON Integra 2). For residual nitrate and phosphate, 10 mL of culture medium was filtered through a 0.2 µm filter and the filtrate was kept at −20 °C until analyses using continuous flow auto analyzer In each PBR, samples of 1 L of culture were collected at different steps of the culture to determine the dry weight, antioxidant activity, and pigment composition. During the first batch culture, two samples were collected during the exponential growth phase (days 2 and 3) and two sample were collected during the stationary phase (days 4 and 6). During continuous culture, one sample was collected after refilling the PBRs (day 7) and two samples were collected and pooled at steady state (days 23 and 24). During the second batch culture, samples were collected daily until senescence .g, 10 min, 4 °C), freeze-dried, and kept at −80 °C until extraction. Dry weight (DW) was determined by weighing the total amount of harvested biomass that was freeze-dried.For all samples, microalgae were harvested by centrifugation . The supernatant was conserved and the pellet was resuspended in 2 mL of ethanol and centrifuged again. The procedure was repeated until the pellet remained colorless (two to three times). The supernatants of each extract were pooled, dried under a stream of nitrogen, and stored under nitrogen atmosphere at −80 °C until analysis. On the basis of a previous study, the oxygen radical absorbance capacity (ORAC) assay was selected to determine the antioxidant activity [Nephroselmis sp. extracts [−1). Then, 10% of the extracts or Trolox solutions were mixed with 90% (v/v) of a diluent solution made of 7% (w/v) of randomly methylated β-cyclodextrin (RMCD) in 50% (v/v) acetone aqueous solution. Then, each extract and Trolox were diluted again with DMSO/diluent solution (10:90 v/v) to obtain 3 different concentrations  and a Trolox concentration range of 0.5 to 10 µg mL−1 to make a calibration curve. In a black 96-well plate, 35 µL of each sample was placed in the wells. A blank was made with the same volume of DMSO/diluent solution (10:90 v/v). Fluorescein  was added to the wells and the plate was incubated at 37 °C for 10 min with 20 rpm agitation. Then, 50 µL of AAPH (82.4 mM) was added and the fluorescence was measured for 300 min every 2 min at an excitation wavelength of 485 nm and emission wavelength of 528 nm.Extracts for antioxidant activity determination were obtained by maceration in ethanol. In the dark and at room temperature, 50 mg of freeze dried biomass was ground using a pestle and mortar and then suspended in 5 mL of ethanol. The solution was placed at −20 °C in a closed container for 30 min in darkness to limit oxidation, then the extract was centrifuged .For each sample and the blank, the area under the curve (AUC) was calculated with the formula from Huang et al. : (1)AUC=g, 5 min, 6 °C), the supernatant was conserved and the pellet was resuspended using a vortex with 500 µL of ethanol, then centrifuged again. This procedure was repeated twice until the pellet remained colorless. The three supernatants were then pooled and filtered on a 0.2 µm PTFE (polytetrafluoroethylene) filter prior to HPLC analysis. The samples were analyzed following the method of Van Heukelem and Thomas [a and b, purchased from DHI, Denmark). Quantification of siphonaxanthin was done according to fucoxanthin standard as recommended by Roy et al. [For pigment analysis, extraction was performed on 40 mg of fresh dried biomass that was previously ground using a mortar and pestle to obtain a fine powder, then suspended in 4 mL of absolute ethanol. The mixture was homogenized using a vortex, then 0.5 mL was immediately sampled and mixed with 100 mg of 150–400 µm glass beads in a mixer miller (Retsch MM-400) for 10 min at a frequency of 30 Hz. After centrifugation  < 0.05. A Fisher’s least significant difference (LSD) test was then applied to determine which experimental conditions were significantly different. To study the correlation between carotenoid content and antioxidant activity, a Pearson’s correlation test was used. Statistical analyses were carried out using Statgraphics Centurion XV.I .Following an examination of the homogeneity of variance and normal distribution (Kolmogorov–Smirnov test), a one-way ANOVA was performed and differences were considered significant at Nephroselmis sp. revealed a specific composition of the photosynthetic system at the crossroads of primitive green algae and higher plants. The correlation of siphonaxanthin and neoxanthin with chlorophylls suggests the presence of siphonaxanthin-type LHCs, as in primitive green algae. However, the results showed the presence of xanthophyll cycle pigments and lycopene, which are also implied in the PSs, as well as the presence of a large amount of lutein, as in most advanced chlorophytes. Surprisingly, lutein did not seem to be associated with the PSs.Pigment analysis of Nephroselmis sp. highlight that the N-replete condition leads to high peroxyl radical scavenging activity, primary carotenoid contents, and productivities with Nephroselmis sp. Indeed, a 3-fold increase was found for the primary carotenoid content and productivity, while a 2.4-fold increase for antioxidant activity under N-replete conditions. Lutein and β-carotene contents were not influenced by nitrogen availability, but their productivities, which followed biomass productivity, were also higher under N-replete conditions. In addition, the results showed high peroxyl radical scavenging capacity that was linked to the carotenoid content of Nephroslemis sp., in particular to the siphonaxanthin and neoxanthin contents. This high antioxidant activity owing to the high carotenoid content makes Nephroselmis sp. a species of interest for carotenoid production as natural antioxidants, especially lutein and siphonaxanthin. The impacts of other culture parameters such as pH or temperature have not been investigated yet, but it would be interesting to study their effects to further improve the carotenoid content and productivity of Nephroselmis sp.Our results with


Effective second‐line chemotherapy options are limited in treating advanced biliary tract cancers (BTCs). Resminostat is an oral histone deacetylase inhibitor. Such inhibitors increase sensitivity to fluorouracil, the active form of S‐1. In the phase I study, addition of resminostat to S‐1 was suggested to have promising efficacy for pre‐treated BTCs. This study investigated the efficacy and safety of resminostat plus S‐1 in second‐line therapy for BTCs.Patients were randomly assigned to receive resminostat or placebo  and S‐1 group  over a 21‐day cycle. The primary endpoint was progression‐free survival (PFS). Secondary endpoints comprised overall survival (OS), response rate (RR), disease control rate (DCR), and safety.p = 0.502); median OS was 7.8 months vs. 7.5 months, respectively ; the RR and DCR were 6.0% vs. 9.8% and 70.0% vs. 78.4%, respectively. Treatment‐related adverse events (TrAEs) of grade ≥ 3 occurring more frequently (≥10% difference) in the resminostat+S‐1 than in the placebo+S‐1 comprised platelet count decreased (18.0% vs. 2.0%) and decreased appetite (16.0% vs. 2.0%).Among 101 patients enrolled, 50 received resminostat+S‐1 and 51 received placebo+S‐1. Median PFS was 2.9 months for resminostat+S‐1 vs. 3.0 months for placebo+S‐1 . This randomized, placebo‐controlled, double‐blind study investigated the efficacy and safety of resminostat plus S‐1 as a second‐line therapy in patients with BTCs. This was the first randomized study to use S‐1, which is widely used in second‐line treatment for BTC patients in Japan, as an active comparator. The results showed that resminostat plus S‐1 prolonged neither PFS nor OS in comparison with placebo plus S‐1. By acting on the histones in the nucleosome, they modulate the structure of chromatin, regulating the expression of a variety of genes involved in the control of cell survival, proliferation, differentiation, and apoptosis.Fluorouracil, the active form of S‐1, inhibits deoxyribonucleic acid biosynthesis by forming a ternary complex with thymidylate synthase (TS) and a reduced form of folic acid.22.13, platelet count ≥100,000/mm3, aspartate transaminase and alanine transaminase ≤2.5 × the institutional upper limit of normal, serum total bilirubin ≤2.0 mg/dl, serum creatinine ≤1.5 mg/dl, creatinine clearance ≥60 ml/min, and Fridericia‐corrected QT interval <460 msec).The eligibility criteria included the following: unresectable/recurrent BTCs, including cancers of the intra‐ or extrahepatic bile ducts, the gallbladder, and the ampulla of Vater; pathologically confirmed adenocarcinoma; only one prior systemic chemotherapy regimen consisting of gemcitabine and a platinum agent; disease progression confirmed by the investigator based on available imaging reports; at least one measurable tumor lesion according to the Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1; Eastern Cooperative Oncology Group performance status (ECOG PS) of 0 or 1; age 20–79 years; life expectancy of at least 12 weeks; and adequate organ and bone marrow function , prior radiation therapy for BTCs, history of myocardial infarction within 6 months prior to enrollment or cardiovascular complications, ascites requiring treatment, clinically significant bone metastasis, and known or suspected brain metastasis.The protocol of this study received approval from the institutional review board of each participating site. It was performed according to the requirements of Good Clinical Practice and the Declaration of Helsinki. Before enrollment, all patients provided written informed consent.2.2This was a multi‐center, randomized, placebo‐controlled, double‐blind, phase II study . It was performed at 21 sites in Japan. The patients were randomly assigned in a ratio of 1:1 to receive resminostat+S‐1 or placebo+S‐1 by the minimization method, with stratification according to site, primary tumor site , a history of postoperative recurrence (yes vs. no), and ECOG PS (0 vs. 1). Enrollment and assignment were performed using the interactive web response system.The primary endpoint comprised PFS as assessed by the investigator; secondary endpoints were OS, response rate (RR), disease control rate (DCR), and safety.2.32, 100 mg/day for BSA of 1.25 to <1.50 m2, and 120 mg/day for BSA of ≥1.50 m2) was administered twice daily after meals on days 1–14.Each patient was scheduled to receive either resminostat 200 mg or placebo once daily after meals on days 1–5 and 8–12 over a 21‐day cycle. S‐1  occurred, the dose of the drug judged as being the more likely cause of the AE by the investigator was reduced. No dose escalation of resminostat/placebo or S‐1 after reduction was permitted.2.4Tumor response was evaluated by each investigator in accordance with RECIST version 1.1 at before enrollment, at weeks 6, 12, 18, and 24, and then every 8 weeks thereafter until progressive disease. Assessment of AEs was performed according to the Common Terminology Criteria for Adverse Events version 4.0.2.5PFS in placebo+S‐1 was expected to be 3.0 months based on previous studies of S‐1 as second‐line therapy for BTCs and a meta‐analysis of second‐line therapies for BTCs.The safety analysis targeted all patients receiving a minimum of one dose of any of the study drugs. The efficacy analyses were based on the full analysis set comprising all patients meeting the study eligibility criteria in the safety analysis population.The following definitions were set: PFS, time from enrollment date to disease progression or death from any cause; OS, time from enrollment date to death from any cause. The log‐rank test was used to make a comparison of PFS and OS between treatment groups. Median PFS, median OS, and their two‐sided 95% confidence intervals (CIs) for each treatment group were estimated using the Kaplan–Meier method. The hazard ratios (HRs) and their 95% CIs were calculated using Cox regression analysis. Subgroup analyses were preplanned to explore the heterogeneity of PFS and OS in each subgroup according to patient characteristics and baseline tumor size. The RR , the DCR , and each 95% CI was calculated for an inter‐group comparison.The primary analysis was conducted at 6 months following the date on which the last patient was enrolled . All statistical analyses were carried out using SAS version 9.3 or 9.4 (SAS Institute).33.1Between March 2018 and February 2019, 101 patients were enrolled in this study, of which 50 were randomly assigned to resminostat+S‐1 and 51 to placebo+S‐1  in resminostat+S‐1 and 4 (range: 1–21) in placebo+S‐1. The median relative dose intensity of resminostat/placebo was 82.4% (range: 25.0–100.0) in resminostat+S‐1 and 85.2% (range: 28.0–100.0) in placebo+S‐1, while the median relative dose intensity of S‐1 was 80.2% (range: 17.8–100.0) and 80.0% (range: 25.8–100.0), respectively. Eleven patients (10.9%) were still receiving the study treatment (five patients [10.0%] in resminostat+S‐1 and six patients [11.8%] in placebo+S‐1) at the data cut‐off. Disease progression was the most frequent reason for treatment discontinuation in both groups (41 of 45 patients [91.1%] in resminostat+S‐1 and 43 of 45 patients [95.6%] in placebo+S‐1).3.3p = 0.502)  in resminostat+S‐1 and 6.7 months (95% CI: 0.0–14.8) in placebo+S‐1. Median PFS was 2.9 months (95% CI: 2.6–4.5) in resminostat+S‐1 compared with 3.0 months (95% CI: 2.8–4.2) in placebo+S‐1  Figure .p = 0.834)  in resminostat+S‐1 and 9.5 months (95% CI: 6.8–12.3) in placebo+S‐1. Median OS was 7.8 months (95% CI: 6.2–9.2) in resminostat+S‐1 compared with 7.5 months (95% CI: 6.0–11.5) in placebo+S‐1  Figure .p = 0.715), with no patient in either group achieving CR.Three patients  in resminostat+S‐1 and five patients  in placebo+S‐1 achieved PR (p = 0.369) (Table DCR was 70.0% (95% CI: 55.4–82.1) in resminostat+S‐1 and 78.4% (95% CI: 64.7–88.7) in placebo+S‐1 p = 0.69 (Table3.4Treatment‐related AEs (TrAEs) with an incidence of ≥10% in either group are shown in Table No cardiac TrAEs occurred in resminostat+S‐1. Grade 2 electrocardiogram QT prolonged was reported in one patient in placebo+S‐1; this was the only cardiac TrAE reported in this study.Treatment was discontinued due to a TrAE in a total of three patients. The TrAEs that required discontinuation of the study treatment were tumor hemorrhage (one patient [2.0%]) and pneumonitis (one patient [2.0%]) in resminostat+S‐1, and leukoencephalopathy (one patient [2.0%]) in placebo+S‐1.Treatment‐related death occurred in one patient in resminostat+S‐1 due to tumor hemorrhage.4The aim of this randomized study was to determine whether adding the HDAC inhibitor resminostat to oral fluoropyrimidine S‐1 improved survival outcome in BTC patients after failure of gemcitabine plus platinum‐based first‐line therapy. The results demonstrated that resminostat+S‐1 prolonged neither PFS nor OS in comparison with placebo+S‐1 in second‐line treatment for advanced or recurrent BTCs. Placebo+S‐1 showed a median PFS of 3.0 months, which is identical to that expected with S‐1 monotherapy. Resminostat+S‐1 showed a median PFS of only 2.9 months. The result did not support the findings from the phase I studyVarious prognostic factors, such as ECOG PS, history of resection, and tumor markers, have been reported to be associated with survival in patients with BTCs.We expected that combining resminostat with S‐1 would result in a synergistic effect manifesting in suppression of expression of TS. However, tumor resistance to fluorouracil agents was reported to be due to not only up‐regulation of TS but also other mechanisms.In this study, the daily dose of 200 mg resminostat (days 1–5 and 8–12) in combination with S‐1 as the recommended regimen for phase II was selected based on the results of a previous study.HDAC inhibitors have demonstrated clinical benefits in some types of hematological malignancy. Pan‐HDAC inhibitors, including vorinostat and panobinostat, were approved for use in cutaneous T‐cell lymphoma and multiple myeloma. This suggests that differences in molecular background such as the frequency of MYC gene abnormality between hematological malignancies and BTCsGastrointestinal TrAEs  as well as platelet count decreased were observed more frequently in resminostat+S‐1. These toxicities were similar in nature to those reported in the previous phase I studyTo the best of our knowledge, this was the first randomized study to use S‐1, a drug commonly used in second‐line treatment of BTCs in Japan, as an active comparator. We are sure that the outcomes observed in placebo+S‐1 will provide a valuable reference for future studies. Recently, the median OS in an active symptom control plus mFOLFOX6 group was reported to be 6.2 months in the ABC‐06 study.In summary, the results of this study indicated that resminostat plus S‐1 therapy was not an effective second‐line treatment for unresectable or recurrent BTCs. The number of TrAEs, especially gastrointestinal toxicity and platelet count decreased, with resminostat plus S‐1 therapy was higher than those with placebo plus S‐1 therapy. However, the safety profile with this combination therapy was consistent with that in previous studies, and the TrAEs were manageable.MU reports grants and personal fees from Yakult during the conduct of the study; grants and personal fees from Taiho, AstraZeneca, Merck Serono, MSD, Daiichi Sankyo, Ono, personal fees from Nihon Servier, grants from Astellas, Eisai, Sumitomo Dainippon, Incyte outside the submitted work. CM reports grants and personal fees from Taiho, Yakult, Nobelpharma, personal fees from Novartis, AbbVie, Fujifilm, Teijin, grants from Eisai, Pfizer, Ono outside the submitted work. DS reports grants and personal fees from Chugai, grants from Yakult, Ono, Eli Lilly, Daiichi Sankyo, Astellas, Incyte outside the submitted work. YK reports grants and personal fees from Yakult during the conduct of the study; grants and personal fees from Asahi Kasei, Bayer, Daiichi Sankyo, Ono, Taiho, personal fees from Bristol‐Myers Squibb, Chugai, Eli Lilly, Kyowa Kirin, Medical Review, Merck Biopharma, Mitsubishi Tanabe, Moroo, Nipro, Pfizer, Sanofi, Shire, grants from A2 Healthcare, Astellas, Sumitomo Dainippon, Eisai, Mediscience Planning, NanoCarrier, Parexel, Sanofi, Shionogi, Incyte, IQVIA, MSD, Nippon Zoki, Syneos Health Clinical, Sysmex outside the submitted work. YN reports grants and personal fees from Yakult, Taiho outside the submitted work. MO reports grants and personal fees from Yakult during the conduct of the study; grants and personal fees from Taiho, personal fees from Bayer, Pfizer, Novartis, Takeda, Eisai, EA pharma, Mitsubishi Tanabe, grants from Merck, Incyte, ASLAN outside the submitted work. NM reports grants from Yakult during the conduct of the study; grants and personal fees from Novartis, Taiho, AstraZeneca, MSD, personal fees from Yakult, Teijin, grants from Eisai, Sumitomo Dainippon, ASLAN, Incyte, Ono outside the submitted work. MM reports grants and other from Chugai, grants from Taiho, Sysmex, Riken Genesis, Mitsui Knowledge, other from Yakult outside the submitted work. AF reports grants from Yakult during the conduct of the study; grants and personal fees from Taiho, Teijin, personal fees from Nihon Servier, Shire, Yakult, grants from Sumitomo Dainippon, NanoCarrier, Aslan, Incyte Biosciences Japan outside the submitted work. MI reports grants and non‐financial support from Yakult during the conduct of the study; grants and personal fees from Bayer, Eisai, Eli Lilly Japan, Chugai, AstraZeneca, MSD, Takeda, Nano Carrier, ASLAN, personal fees from Sumitomo Dainippon, Taiho, Nihon Servier, Teijin, Mylan, Astellas, EA Pharma, Shire, Otsuka, grants from Novartis, Bristol‐Myers Squibb, Merck Serono, Ono, J‐Pharma, Pfizer outside the submitted work. AT reports grants from Yakult during the conduct of the study; grants and personal fees from Taiho, Chugai, Eli Lilly, Merck Biopharma, Takeda, Sanofi, personal fees from Bristol‐Myers Squibb, grants from Ono, Kyowa Kirin, Eisai, Toray Medical, Daiichi Sankyo, Bayer, Shionogi, Pfizer, Yakult outside the submitted work. TM reports grants and personal fees from Yakult during the conduct of the study; grants and personal fees from Takeda, Taiho, personal fees from Chugai, Eli Lilly, Bayer, Sanofi, Ono, grants from MSD, Eisai outside the submitted work. TK reports personal fees from Chugai, Taiho, Bristol‐Myers Squibb, Merck Biopharma, Kyowa Kirin outside the submitted work. HI reports personal fees from Yakult outside the submitted work. SS reports grants from Yakult during the conduct of the study. NN, SK and KM are employees of Yakult. NN and KM own stock in Yakult. JF reports grants and personal fees from Yakult during the conduct of the study; personal fees from Eisai, Bayer, Taiho, Ono, Novartis, Yakult, Teijin, Shionogi, EA pharma, Eli Lilly, Takeda, Chugai, Mochida, Nihon Servier, Sanofi, Fujifilm Toyama Chemical, Nobel pharma, Pfizer, Sawai, Daiichi Sankyo, Sumitomo Dainippon, Merck Serono, Nippon Kayaku, MSD, Shire, Kyowa Kirin, grants from Ono, MSD, Sumitomo Dainippon, J‐Pharma, Yakult, AstraZeneca, Daiichi Sankyo, Eisai, Bayer, Pfizer, NanoCarrier, Kyowa Kirin, Taiho, Chugai, Sanofi, Takeda, Mochida, Astellas, Eli Lilly outside the submitted work.Table S1Click here for additional data file.


Dotinurad, a novel selective urate reabsorption inhibitor, exerts a serum uric acid-lowering effect by selectively inhibiting urate transporter 1 (URAT1) in patients with hyperuricemia. It is generally known that the progression of renal dysfunction is associated with a reduction in the serum uric acid-lowering effects of uricosuric drugs. We, therefore, investigated the pharmacokinetics (PK), pharmacodynamics (PD), and safety of dotinurad in subjects with renal dysfunction.2) or moderate  renal dysfunction or normal  renal function.This was a parallel-group, open-label, single-dose clinical pharmacology study. Dotinurad (1 mg) was administered once, orally to subjects with mild  were significantly lower in the moderate renal dysfunction group than in the normal renal function group. However, other PD parameters were not significantly different among the groups. No notable adverse events or adverse drug reactions were observed in this study.The time-course of mean plasma concentration of dotinurad had similar profiles across the groups. Regarding PK, there was no significant difference between the renal dysfunction groups and normal renal function group. Regarding PD, the maximum reduction rate in serum uric acid levels and the fractional uric acid excretion (FE) ratio  contains supplementary material, which is available to authorized users. Hyperuricemia, defined in Japan as a serum uric acid level > 7.0 mg/dL , 2, is kLifestyle modification is considered vital for treating hyperuricemia. However, because patients with recurrent gouty arthritis or gouty tophi find it difficult to alleviate their symptoms by lifestyle modification alone, they are advised to maintain a serum uric acid level of less than 6.0 mg/dL using antihyperuricemic agents. Hyperuricemia is mainly classified into three types: excess uric acid production “overproduction type”; reduced uric acid excretion “underexcretion type”; and a mixture of both “combined type”. The basic principle of hyperuricemia treatment in the Japanese guidelines for the management of hyperuricemia and gout is the use of xanthine oxidase inhibitors (XOIs)  for “overproduction type” and uricosuric drugs  for “underexcretion type” . HoweverIn recent cohort study, it has been reported that hyperuricemia is associated with the progression of renal dysfunction ; many hyIn this context, it was considered necessary to investigate the pharmacokinetics (PK), pharmacodynamics (PD), and safety of dotinurad in subjects with renal dysfunction.2. Moderate renal dysfunction was defined as an eGFR of ≥ 30 to < 60 mL/min/1.73 m2. Normal renal function was defined as an eGFR of ≥ 90 mL/min/1.73 m2. The subjects were admitted to the institution on Day−1, where they remained until completion of the study, 48 h after the administration of dotinurad (Day 3). After subjects fasted for at least 10 h, dotinurad (1 mg) was administered once, orally on the morning of Day 1 with 180 mL of water. Follow-up testing was performed 5–10 days after the administration of dotinurad.This parallel-group, open-label, single-dose clinical pharmacology study was conducted to examine the PK, PD, and safety of dotinurad in subjects with renal dysfunction. After informed consent was obtained, a screening test was conducted to confirm subject eligibility within 30 days prior to dotinurad administration. During the screening, confirmation of medical history, physical examination, clinical laboratory tests, electrocardiogram (ECG), abdominal ultrasonography, and abdominal X-ray were performed and the estimated glomerular filtration rate (eGFR) was calculated. The eGFR of subjects judged to be eligible at the time of screening was calculated again on Day−1 (the day before dotinurad administration) and the subjects were assigned to one of three groups , according to the eGFR. Mild renal dysfunction was defined by an eGFR of ≥ 60 to < 90 mL/min/1.73 mThe inclusion criteria for this study were as follows: male Japanese subjects aged 20 years or older at the time of informed consent; assignment of subjects to the same group—one of normal renal function, mild renal dysfunction or moderate renal dysfunction—at the time of the screening test and on Day−1; variation in serum creatinine of no more than 0.2 mg/dL; absence of contraindications to inclusion in this study as a result of examination of the following items, performed within 30 days before administration of the investigational drugs: (1) findings on medical examination, (2) physical examination, body mass index (BMI) and assessment of vital signs, (3) ECG, (4) abdominal ultrasonography, plain abdominal x-ray, and (5) laboratory tests; subjects with a BMI of not less than 18.5 and less than 30.0.Exclusion criteria were as follows: disorders of the digestive tract, heart, or liver; severe renal dysfunction; history of surgery involving the digestive organs  or kidneys, excluding surgery not considered to affect drug absorption (such as appendectomy and hemorrhoidectomy); renal calculus or clinical manifestations such as hematuria or back pain that might be due to a urinary calculus as assessed by abdominal ultrasonography or plain abdominal X-ray in the screening period; consumption of St. John’s wort or foods containing St. John’s wort within 4 weeks of administration of the dotinurad; consumption of grapefruit or foods containing grapefruit within 7 days of dotinurad administration; use of other drugs  within 7 days of dotinurad administration; alcohol consumption within 3 days of dotinurad administration; use of investigational drugs other than dotinurad within 12 weeks of dotinurad administration (or 4 weeks in the case of patch tests);previous participation in a study of dotinurad with administration of the investigational drug.Blood samples for PK and PD analyses were collected 12 times, namely before administration and 0.5, 1, 2, 3, 4, 6, 8, 12, 24, 36, and 48 h after administration. Urine samples for PK and PD analyses were collected three times at 24-h intervals from 24 h before administration to 48 h after administration.The dotinurad and its metabolites’ concentration in plasma and urine were measured using liquid chromatography–tandem mass spectrometry (LC–MS/MS) at the research institute, Fuji Yakuhin Co., Ltd.The Agilent 1100 series  was used for the liquid chromatography; API3000  was used for the mass spectrometry; and Inertsil ODS-3  was used for the analysis column. Measurements were made using 5 mmol/L ammonium acetate solution (pH 4.0)/methanol (50:50) in the mobile phase. The lower limit of quantification for dotinurad and its metabolites in human plasma and urine was 1 ng/mL.The PK samples were analyzed at Fuji Yakuhin Co., Ltd .Tmax), maximum observed plasma concentration (Cmax), area under the plasma concentration-time curve (AUC), AUC from zero to infinity (AUC0–inf), apparent total body clearance (CL/F), terminal elimination half-life (T1/2), apparent volume of distribution (Vd/F), elimination rate constant (kel), and mean residence time from time zero to the last measurable sample point (MRTt0–). Urinary PK parameters included the amount of unchanged drug excreted in urine over each collection interval (Ae) and fraction of dose excreted in urine expressed as a percentage (fe). These PK parameters were derived using WinNonlin Professional, version 6.2 .The PK parameters of dotinurad and its metabolites were estimated using a non-compartmental method. Plasma PK parameters included time to reach maximum plasma concentration (max) and mean time-matched change in serum uric acid levels from baseline (∆AUEC). Urinary PD parameters included the amount of uric acid in urine ; renal clearance of uric acid (CLR); creatinine clearance (Ccr), which was calculated as Ae divided by plasma uric acid AUC over the same time interval, and fractional excretion of uric acid (FE), which was calculated as (CLR/Ccr) × 100.Serum and urinary uric acid concentrations were measured at the clinical trial institution and PD parameters were calculated from the measurement results. Serum PD parameters included the maximal change in serum uric acid levels from baseline . All AEs were classified according to the system organ class and preferred term  and evaluated in terms of their potential causality with the study drug, severity, and seriousness. AEs judged to have a causal relationship with the study drug were defined as adverse drug reactions (ADRs).P < 0.15 denoted statistical significance. The other statistical tests and confidence intervals were two-sided and P < 0.05 denoted statistical significance.All statistical analyses were performed using SAS software . All subjects who received dotinurad and who had evaluable PK and PD data, excluding those who did not meet the inclusion and exclusion criteria, were included in PK and PD analysis sets. Safety analysis sets consisted of all subjects who received dotinurad. Descriptive statistics for PD and safety population data included number of subjects, arithmetic mean, minimum, maximum, and standard deviation (SD). Statistical tests for baseline characteristics were two-sided and Cmax, AUC, and AUC0–inf.Summary statistics are provided for PK and PD parameters in each group. Regarding the PK and PD parameters of dotinurad, to evaluate the effect of renal dysfunction, Dunnett’s test was used to compare the mild and moderate renal dysfunction groups with the normal renal function group. Differences of means with their corresponding 90% confidence intervals and estimates of geometric mean ratios via anti-logarithmic transformation with their corresponding 90% confidence intervals were generated for In this study, 18 adult male subjects were enrolled with six subjects assigned to each of the three groups. All subjects received dotinurad and completed this study. However, because one subject in the moderate renal dysfunction group deviated from the study protocol (use of other drugs in the 7 days before administration of dotinurad), data for this subject were excluded from the PK and PD analyses. Therefore, 17 subjects were included in the PK and PD evaluation and 18 subjects were included in the safety evaluation.Demographic and other baseline characteristics of each group are shown in Table Cmax 85.67 ± 10.65, 88.73 ± 22.74, and 88.38 ± 14.39 ng/mL, respectively; AUC0–inf 1157.32 ± 269.46, 1366.57 ± 427.94, and 1428.54 ± 379.58 ng h/mL, respectively; CLtot/F 0.91 ± 0.25, 0.81 ± 0.33, and 0.75 ± 0.25 L/h, respectively; fe0–24 0.97 ± 0.35%, 0.88 ± 0.32%, and 0.52 ± 0.16%, respectively. No urinary excretion of dotinurad was observed after 24 h of administration in any group. The mean plasma and urine PK parameters of dotinurad were comparable among the groups and no significant differences of parameters  were observed between the renal dysfunction groups and normal renal function group.The time-course of mean plasma concentration of dotinurad in each group is shown in Fig. Cmax of glucuronate and sulfate conjugates in each group were less than 2.0 and 4.0 ng/mL, respectively. The AUC0–inf of both glucuronate and sulfate conjugates in each group were not calculated. In contrast, the urinary excretion rates (0–24 h) of glucuronate (mean ± SD) for the normal renal function and mild and moderate renal dysfunction groups were 27.80 ± 1.30%, 25.29 ± 4.05%, and 19.06 ± 3.81%, respectively, and those of sulfate-conjugates were 19.45 ± 5.85%, 12.78 ± 5.84%, and 11.53 ± 7.06%, respectively.The plasma and urine PK parameters of dotinurad and its major metabolites (glucuronate and sulfate conjugates) in each group are shown in Table max, ΔAUEC0–48, and maximum reduction rate) in the normal renal function and mild and moderate renal dysfunction groups were as follows: ΔECmax – 2.07 ± 0.34, − 2.07 ± 0.53, and − 1.42 ± 0.43 mg/dL, respectively; ΔAUEC0–48 − 73.41 ± 12.99, − 79.12 ± 27.23, and − 49.49 ± 14.59 mg·hr/dL; maximum reduction rate 35.25 ± 7.24%, 33.94 ± 6.05%, and 21.31 ± 10.40%, respectively. The maximum reduction rate was significantly different between the normal renal function and moderate renal dysfunction groups, but no significant differences were observed for other PD parameters in the renal dysfunction groups, in comparison with those in the normal group.The time-course of mean serum uric acid concentrations and summary statistics in each group are shown in Fig. 0–24, ua and summary statistics in each group are shown in Fig. 0–24, ua, CLR0-24, FE0-24/FE−24–0) in the normal renal function and mild and moderate renal dysfunction groups were as follows: Ae0-24, ua 980.67 ± 131.71, 982.83 ± 100.22, and 811.75 ± 81.23 mg, respectively; CLR0–24 16.05 ± 2.92, 15.80 ± 4.38, and 9.96 ± 3.86 mL/min, respectively; and FE0–24/FE−24-0 3.34 ± 0.51%, 2.86 ± 0.50%, and 2.46 ± 0.36%, respectively. FE0–24/FE−24–0 was significantly different between the normal renal function and moderate renal dysfunction groups, whereas no differences were observed for other urine PD parameters in renal dysfunction groups in comparison with those in the normal group.The time-course of AeAEs were not observed in the normal renal function and mild renal dysfunction groups; however, six AEs were observed in two subjects in the moderate renal dysfunction group Table . No seriThis was the first pharmacological study to investigate the PK, PD, and safety of dotinurad in subjects with renal dysfunction.max and ΔAUEC0-48 in the renal dysfunction groups were not significantly different from those in the normal renal function group. Conversely, some PD parameters (the maximum reduction rate in serum uric acid levels and FE0–24/FE−24–0) were significantly lower in the moderate renal dysfunction group than in the normal renal function group. Regarding safety, gouty arthritis was observed as an ADR in the moderate renal dysfunction group alone (one event in one subject). Otherwise, no safety issues were observed related to clinical examination, vital signs, and ECGs. In addition, in a subgroup analysis based on renal function  in phase II and phase III studies of patients with hyperuricemia with or without gout, the serum uric acid-lowering effect and safety of dotinurad were barely affected by mild or moderate renal dysfunction . From these results, it is undeniable that dotinurad may be affected by moderate renal dysfunction, but this seemed to have no significant effect. Therefore, dose adjustment may not be necessary when administering dotinurad to patients with mild-to-moderate renal dysfunction.Regarding PK, we observed that dotinurad concentration profiles and PK parameters of dotinurad and its major metabolites in the mild and moderate renal dysfunction groups were not significantly different from those in the normal renal function group. These findings suggest that the PK of dotinurad is not affected by mild or moderate renal dysfunction. Regarding PD, dotinurad increased urinary uric acid excretion and concomitantly reduced serum uric acid levels, even in subjects with moderate renal dysfunction. ΔECSupplementary file1 (DOCX 41 kb)Below is the link to the electronic supplementary material.


Lactobacillus strains capable of producing 1,2-propanediol and short-chain fatty acids on maize silage aerobic stability improvement. The research showed that during 90-day ensilage, the applied preparations significantly reduced the content of DM, water-soluble carbohydrates (WSCs), pH and DM recovery (P < 0.05). The concentration of lactic acid (LA), acetic acid (AA) and propionic acid (PA) in the inoculated samples increased significantly (P < 0.05). 1,2-Propandiol and 1-propanol were not found in control silages (without additives). The addition of L1 and L2 significantly (P < 0.05) increased the concentration of these substances. The L1 and L2 mixtures significantly extended (P < 0.05) the silage aerobic stability.The aim of this study is to determine the influence of a commercial bacterial inoculant (L1) and a preparation (L2) containing three  According to the results, this heterofermentative strain improves the silage aerobic stability. Apart from that, it anaerobically degrades lactic acid to acetic acid and 1,2-propanediol. Probably, 1,2-propanediol is an intermediate metabolite, which becomes degraded into 1-propanol and propionic acid by Lactobacillus diolivorans [Lactobacillus reuteri can synthesise cobalamin, which is a coenzyme for diol dehydratase – the enzyme catalysing 1,2-propanediol conversion into 1-propanol and propionic acid [Lactic acid-fermenting bacteria are used as a major microbiological modifier that could improve the silage chemical composition . These blivorans . Lactobanic acid . During nic acid .L. buchneri strains on 1,2-propanediol and 1-propanol, the chemical composition and aerobic stability of maize silages.The aim of this study is to determine the influence of a commercially available bacterial preparation and a mixture of 22.1Zea mays L.) SAN cultivar (FAO 240) from the Hodowla Roślin in Smolice Ltd/Sp. z o.o. IHAR Group was ensilage. Type of use: medium-early three-line hybrid (TC) with advantages of a grain hybrid for CCM and silage. The features of maize were as follows: high yields of total dry matter and dry matter of cobs, high resistance to Fusarium stem rot and root lodging, tolerant to smut, long-lasting green leaves and stems and the height of 270 cm. Plant density is 150,000 per ha. Maize was harvested in October at the end of silage maturity (BBCH 83). It was cut/harvested at a height of 40 cm. Before ensiling, it was cut into 2–3 cm chaff. Maize was grown in monoculture.Maize . A dose of 5 g t−1 of ensiled material was used. The concentration of bacteria in 1 g of the mixture was 109 cfu g−1.L2 is a mixture (lyophilisate) of three strains: 2.33 equipped in a closure allowing removal of gaseous products. The average temperature during ensiling was 20 ± 1°C. During the aerobic stability test, samples were aerated for 7 days at 20°C. After this period, changes in microorganism counts and silage selected chemical parameters were investigated. After 90-day ensilage, moist samples weighing 85 g were removed from micro silos and placed in 500 mL plastic containers with 4 mm holes enabling air circulation. The temperature was measured with a temperature reader  every 5 min at 2 h intervals. Stability was defined as the time necessary to raise silage temperature by ≥2°C relative to the ambient temperature. The number of replications was 5.Silages were prepared in PVC laboratory micro silos with a capacity of 4 dm2.4Lactic fermentation bacteria were cultured on MRS Agar (Oxoid). Incubation time was 48 h at 35°C. Yeasts and moulds were cultured on OGYE Agar (Oxoid) with oxytetracycline (oxytetracycline-glucose-yeast-extract agar). Incubation time was 5 days at 25°C.3PO4 filling and Varian 8200 CX autosampler. Hydrogen was used as the carrier gas (flow 30 cm3 min−1) with oven temperature of 120°C, injection temperature of 250°C and detector temperature of 300°C. Fluka acid standards were used.Lactic acid, acetic acid, propionic acid, ethanol, 1-propanol and 1,2-propanediol concentrations were measured with a gas chromatograph equipped with an FID detector, 2 m long 80/100 Chromosorb® WAW glass column (Supelco), I.D. 2 mm with GP 10% SP–1,200/1% H3 of demineralised water, which was homogenised for 10 min.The basic composition of feeds was determined in accordance with AOAC . pH valu2.5The GLM SAS procedure package was used for statistical calculations . Differe3Basic chemical composition and counts of lactic acid bacteria, yeast and mould in the ensilaged maize forage are presented in P < 0.05) than in the control sample. The silages with Lactobacillus strains had significantly (P < 0.05) lower pH values and a lower DM recovery index. Also, concentrations of LA, AA and PA were significantly (P < 0.05) higher in the inoculated samples. The inoculation did not have a significant effect (P > 0.05) on the content of ethanol and CP in silages. Mixtures of Lactobacillus strains caused a significant (P < 0.05) increase in the LAB population and a decrease (P < 0.05) in the population of yeasts and moulds. There was no 1,2-propandiol or 1-propanol found in control silage samples. The addition of L1 and L2 significantly (P < 0.05) increased concentrations of these substances. The content of 1,2-propandiol and 1-propanol in the combination with L2 was relatively 62% and 75%, respectively, greater than in the combination with L1.P < 0.05) reduced the growth of pH in silages. AA and PA concentrations in the samples with Lactobacillus strains were significantly (P < 0.05) greater than in the control sample, but the LA content was significantly (P < 0.05) reduced. LAB counts in the inoculated silages were significantly (P < 0.05) greater, whereas yeast and mould counts were lower (P < 0.05) than in the control samples. No 1,2-propandiol or 1-propanol was found in the control samples. The content of 1,2-propandiol and 1-propanol in the combinations with L1 and L2 was the same as in silages before the aerobic stability test.P < 0.05) extended the aerobic stability. The silage temperature in control samples increased by 2°C within 72 h. The inoculated silages were characterised by longer stability, i.e., 103 h for L1 and 102 h for L2.4According to the study by Rezende et al. , when siLactobacillus can be used to improve the silage aerobic stability of maize. In our experiment, the effect of a commercially available preparation was compared with a preparation containing heterofermentative strains, namely, L. buchneri, L. dioliovorans and L. reuteri. Zielińska et al. [Lactobacillus strains and their improvement of silage stability. It seems very important that these strains can metabolise 1,2-propandiol into propionic acid and 1-propanol. According to the scientific reports, L. buchneri [L. dioliovorans [L. reuteri [Many strains of a et al.  and Mucka et al.  describebuchneri , L. dioliovorans  and L. r reuteri ,19,20 exL. buchneri strains increased acetic acid and 1,2-propandiol concentrations and decreased lactic acid content in silages. Our research findings were similar. However, it is noteworthy that too high concentrations of acetic acid may affect the silage taste.Driehuis et al.  and JungLactobacillus strains were applied to silages, they reduced pH values and WSC concentrations. The same observations were made in our research. However, contrary to the results of our research, Oliveira et al. also found a lower concentration of acetic acid in samples with Lactobacillus strains. Similar results were recorded when the concentration of lactic acid in the inoculated samples was greater than in the control samples. At the same time, these authors concluded that the observed effects depended on the type of plant ensiled.Oliveira et al.  observed9 cfu g−1). Basso et al. [5 cfu g−1. According to the study by Ranjit and Kung [Lactobacillus can be continued during exposure to oxygen. Acetic acid concentration tended to increase, whereas the content of lactic acid was found to decrease in inoculated silage samples subjected to aerobic incubation. In consequence, pH decreased because acetic acid exhibited higher pKa values than lactic acid [L. buchneri strains and noted an increase in the CP level compared with that in the control sample. Similar results were reported by Bumbieris et al. [Lactobacillus strains used in our study reduced yeast and mould counts. The L2 preparation exhibited a stronger fungistatic effect. It is noteworthy that the production of substances inhibiting fungal growth, including acetic and propionic acid, may largely depend on the phase of their growth as well as temperature, chemical composition and pH of the substrate [When the aerobic stability of silages was checked, the concentration of acetic acid was found increased in inoculated samples (concentration of bacteria in 1 g of preparation was 10o et al.  noted siand Kung , acetatetic acid  used L. s et al. , who obsubstrate .5Lactobacillus strains revealed better aerobic stability than control samples due to higher acetic and propionic acid concentrations, which reduced pH as well as yeast and mould counts. At the same time, silages inoculated with heterofermentative strains of Lactobacillus had a higher content of 1,2-propandiol and 1-propanol. This fact may indicate that mixtures of these bacterial strains are excellent inoculants that improve the aerobic stability of silage.The performed research showed that silages inoculated with


Mycotoxins (MTs) are secondary toxic metabolites that can contaminate food, impacting quality and safety, leading to various negative health effects and serious pathological consequences conferring urgent need to evaluate and validate the currently standard methods used in their analysis. Therefore, this study was aimed to validate ELISA and VICAM immunoaffinity fluorometric, the two common methods used to monitor the level of MTs according to the Egyptian Organization for Standardization and Quality Control. A total of 246 food samples were collected and tested for Aflatoxins (196 samples), Ochratoxin A (139), Zearalenone (70), and Deoxynivalenol (100) using both analytical methods. Results showed that aflatoxins exceeded limits in 42.9, 100, and 13.3% of oily seeds, dried fruits, and chili and spices, respectively. For ochratoxin A, 3.9% of Gramineae and 8% of spices and chili  exceeded the limits, while 17.6% of imported pasta and noodles exceeded the limits for deoxynivalenol. Significant differences for the aflatoxins and ochratoxin A detection among different categories of chocolate, dried fruits, and oily seeds . No zearalenone contamination was detected in the exported, imported, and locally sourced categories. No deoxynivalenol contamination was detected in the tested Gramineae category. In contrast, for pasta and noodles, the imported samples exhibited the highest contamination rate (above the upper limit of 750 µg/kg) with 17.6% of the samples testing positive for deoxynivalenol with no significant difference among different sample categories of Gramineae, pasta, and noodles . In conclusion, our study found no significant differences between the ELISA and immunoaffinity fluorometric analysis in the detection of the respective MTs in various food categories and therefore, they can substitute each other whenever necessary. However, significant differences were observed among different food categories, particularly the local and imported ones, highlighting the urgent need for strict and appropriate control measures to minimize the risk of MTs adverse effects.The online version contains supplementary material available at 10.1186/s13568-023-01629-5. Screening and quantitative estimation of four major MTs in various food matrices to ensure food safety and quality have been done according to standard guidelines.Our findings revealed that some of the tested MTs exceed limits and the possible attributed factors have been discussed and highlighted in the manuscript.Results obtained proved non-significant differences between the ELISA and immunoaffinity fluorometric analysis in the detection of Aflatoxins, Ochratoxin A, Zearalenone, and Deoxynivalenol in various food categories.The online version contains supplementary material available at 10.1186/s13568-023-01629-5. Food quality and safety are crucial to ensure that the market is not harmed by selling subpar or hazardous food. The biggest threat to the safety of human food is posed by food-borne microorganisms, followed by MTs. additionally, MTs constitute the greatest risk when it comes to animal feeds  is a cancer that is brought on by AFs in both humans and animals, which increased curiosity about MTs research  in Tunisia, Balkan endemic nephropathy (BEN), and kidney tumors in various Balkan Peninsula endemic locations  and immunoaffinity columns (IAC) are the most often used techniques for mycotoxin cleanup because they are effective, repeatable, and provide a wide range of selectivity  and VICAM immunoaffinity column (IAC) fluorometric methods (quantitative) for the detection and estimation of four clinically relevant MTs in various food matrices and categories . The food samples were randomly collected from the Egyptian market and were based on standard screening protocol and sample size standard guidelines for detection and estimation of the levels of aflatoxins (AFs), Ochratoxin A (OTA), Zearalenone, and Deoxynivalenol.http://www.aoacofficialmethod.org/index.php?main_page=product_info&products_id=2065 (accessed on 20 August 2022) .A total of 246 representative samples of various food samples and different categories , imported (73), and exported (19) were obtained from the Egyptian market in 2022 according to AOAC 977.16 . For AFs (Art. Nr. R4701), OTA (Art. No. R1312), ZEN (Art. No.: R1401), DON (Art. No.: R5906). All reagents required for the enzyme immunoassay, were included in the test kits. Ready to use standards were included in the test kits with concentrations 0 µg/L, 0.05 µg/L, 0.15 µg/L, 0.45 µg/L, 1.35 µg/L, 4.05 µg/L (1.3 mL each) for AFs; 0 µg/L, 0.03 µg/L, 0.1 µg/L, 0.3 µg/L, 1 µg/L, 3 µg/L (1.3 mL each) for OTA; 0 ng/L, 50 ng/L, 150 ng/L, 450 ng/L, 1350 ng/L, 4050 ng/L (1.3 mL each) for ZEN and 0 µg/L, 3.7 µg/L, 11.1 µg/L, 33.3 µg/L, 100 µg/L (1.3 mL each) for DON. diluted ECO extractor (10x concentrate) was included in the test kit of OTA to be used in extraction (dilution was done 1:10 with distilled or deionized water at 2 to 8 ℃). In the test kit of ZEN, Buffer 1 (50 mL) was included. A microtiter plate spectrophotometer was required for semi-quantification (screening). Special software, RIDASOFT® Win.NET (Art. No. Z9996FF) was used in the screening process. Filter paper: Whatman No. 1 or its equivalent was purchased from VICAM (https://www.vicam.com/category/aflatoxin-testing-solutions (accessed on 12 August 2023). Methanol  was purchased from Sigma Aldrich . Ultra-pure water was purchased and products by the Milli-Q purification system .Screening for the four different MTs was done using an ELISA Test kit (RIDASCREENg) and 25 mL of 70% methanol were mixed for 10 min at room temperature by vortexing and filtered through a Whatman No. 1 filter or centrifuged (10 min / 3500 g / room temperature). Then, 100 µL of the filtrate/supernatant was diluted with 600 µL distilled water. The wells were then filled with 50 µL of standard or sample in duplicate together with 50 µL of the conjugate. Then, 50 µL of the antibody was added to each well, and the plate was gently shaken for mixing and then allowed to sit at room temperature (20 to 25 ℃) in the dark for 30 min. After incubation, the well contents were discarded and the microwell holder was tapped upside down strongly (three times) on absorbent paper. Then wells were washed with 250 µL wash buffer 3 times after which 100 µL of substrate/chromogen was added to each well, gently mixed by hand shaking the plate, and incubated for 15 min at room temperature (between 20 and 25 ℃) in the dark. The stop solution (100 µL) was then pipetted into each well followed by manual shaking of the plate. After 30 min, the extinction was determined at 450 nm.Ground samples (5 g). Subsequently, 1 mL of the supernatant was diluted with 1 mL of wash buffer. An aliquot of 50 µL of standard or sample was then used to fill the wells in duplicate. Each well then received 50 µL of the conjugate which was gently combined by manually shaking the plate for 30 min at room temperature (20 to 25 ℃) in the dark. After that, the well contents were discarded, and the assay was continued as mentioned previously for AFs.An aliquot of 10 gm ground sample was weighed and 50 mL of diluted ECO extractor was added followed by vortexing (10 s). The sample was then shaken violently for 5 min  followed by centrifugation for 5 min at room temperature  were added. Then, vigorous shaking  was carried out for three min. The extracts were centrifuged  or filtered. Following that, sample dilution buffer (buffer 1) was used to dilute the filtrates or supernatants 1:7 (100 µL supernatant or filtrate + 600 µL buffer 1). Then 50 µL of standard or sample were used to fill the wells in duplicate. The ZEN enzyme conjugate (diluted 1:11 in buffer) was added in 50 µL portions to each well. After that, the plate was gently stirred by handshaking and incubated for 2 h at room temperature (20 to 25  ℃) in the dark. After that, the well contents were discarded, and the assay was continued as mentioned previously for AFs.Aliquots of 5 g of ground samples were weighed into a suitable container and 25 mL of distilled water was added and shaken for three min. Whatman No. 1 filter was used to filter the extract. Then 50 µL of standard or sample were added to the wells in duplicate. Then each well received 50 µL of the conjugate. Subsequently, each well received 50 µL of the anti-DONantibody, which was carefully mixed by hand shaking the plate and incubated for 30 min at room temperature (20 to 25 ℃) in the dark. After that, the well contents were discarded, and the assay was continued as mentioned previously for AFs.Aliquots of 5 g)  Zinc acetate powder, and AlCl3 powder were purchased from Sigma Aldrich. A commercial Blender with a Stainless Steel Container  was used. Using a Milli-Q filtration system , ultra-pure water was created. AFs standard product , OTA standard product (product number CRM46912 Lot no. LRAD1407 with concentration approximately 50 ng/µL in benzene: acetic acid (99:1), ampoule of 1 mL) and ZEN standard product  were purchased from Supelco .AflaTest Columns (25 per box), OchraTest Columns (25 per box), zearalaTest Columns (25 per box), Microfiber Filters, filter paper Whatman no.1, 1.0 m, 9 cm (100), Tween-20 (50 mL), 10X Concentrate of 0.01% Tween/PBS (150 mL), 5X Concentrate of 2% Tween/PBS (300 mL), 10X Concentrate of 0.1% Tween/PBS (150 mL), 10x concentrate of PBS , AflaTest Developer (50 mL), OchraTest Eluting Solution (50 mL)and Zearalatest developer were purchased from VICAM A., WATERS, USA. Methanol , ACS Grade Salt  were prepared to extract AFs out of the samples. AflaTest Developer solution was prepared by mixing 45 mL of filtered water and 5 mL of AflaTest Developer concentrate. 10% Tween-20, 10X concentrate PBS, 10X concentrate 0.01% Tween-20 and 10X concentrate 0.1% Tween-20 solutions were prepared by adding 10 mL from each to 90 mL distilled water. 5 X concentrate 2% Tween-20 solution was prepared by adding 20 mL to 80 mL of distilled water. ZnCl2/AL(C2H3O2) 3 was prepared by adding 25 https://www.vicam.com/category/aflatoxin-testing-solutions (accessed on 12 August 2023).). Briefly, a 25 g ground sample was weighed with 5 g salt (NaCl) for paprika, chili, spices, oily seeds, nuts, Gramineae, cereals, chocolate, and cocoa and placed in a blender jar. For green coffee, a 50 g ground sample was weighed with 5 g salt (NaCl). For dried fruits, dried figs, and dates, 25 g ground samples were weighed with no NaCl added. Then 100 mL methanol: water (80%) for chili, paprika, spices, Gramineae, cereals, and green coffee, 125 mL methanol: water (60%) for oily seed and nuts, 100 mL methanol: water (70%) for dried fruits, dried figs and dates and 100 mL of absolute methanol for chocolate and cocoa were added to the jar. The blender jar was then covered and blended for one minute at a high speed. 5 mL of filtered extract was then diluted with 20 mL purified water in case of paprika, chili, Gramineae, cereals, or green coffee or diluted with 20 mL 10% tween 20 solution in case of spices and mixed well.The assay was done according to the VICAM international standard guidelines  for chili and paprika, but for the other types of samples, 10 mL were passed through the column. Then,10 mL of methanol: water (20%) was passed through the column at a rate of about 1–2 drops/second in the case of paprika, chili, chocolate, and cocoa or 10 mL of distilled water was passed in case of spices, oily seeds, nuts, Gramineae and cereals, green coffee. The previous step was repeated once more until air went through the column. For dried fruits, dried figs, and dates, 10 mL of 0.01% Tween/PBS solution was passed through the column at a rate of 1–2 drops/second then 10 mL of purified water was passed through the column. After that, a glass cuvette was placed under the column and 1 mL HPLC grade methanol was added into a glass syringe barrel. By allowing methanol to run through the AflaTest column and collecting all the samples eluate in a glass cuvette, the column was eluted at a rate of 1 drop/second or slower. 1 mL of AflaTest Developer solution was added to eluate in the cuvette and thoroughly mixed after that. Then the cuvette was immediately placed in a calibrated fluorometer Series 4EX Fluorometer 110 V, U.S.A. (Part Number/N G8001) and 220 V, International (P/N G8002). Total AF concentration was read after 60 s.For oily seeds and nuts, 20 mL of filtered extract was diluted with 20 mL of purified water. For dried fruits, dried figs, and dates, 5 mL of filtered extract was diluted with 20 mL 0.01% Tween/PBS solution and mixed well. For chocolate and cocoa, 5 mL of filtered extract was mixed with 20 mL of ZnCl2/Al(C2H3O2)3 solution. Then, using marks on the barrel to quantify 4 mL, the diluted extract was filtered through a 1.5 m microfiber filter into a clean vessel or straight into a glass syringe barrel. At a rate of approximately 1 drop/second, 4 mL of filtered diluted extract was completely passed through an AflaTest column (4 mL = 0.2 https://www.vicam.com/category/ochratoxin-testing-solutions (accessed on 12 August 2023). The remaining solutions were prepared as mentioned previously for AFs. The assay was done according to the manufacturer’s manual  as follows: 50 g ground samples were weighed with 5 g salt (NaCl) for paprika, chili, and spices and placed in the blender jar. But for Gramineae and cereals, 50 g ground samples were weighed with no NaCl added. For green coffee, roasted coffee, and Nescafe, 25 g ground samples were weighed with no NaCl added. Aliquots of 100 mL methanol: water (80%) for paprika, chili, spices, Gramineae, and cereals, 50 mL methanol:1% sodium bicarbonate (70%) for green and roasted coffee were added to the jar. The blender jar was covered and blended for 1 min at high speed, then 5 mL of filtered extract was diluted with 20 mL purified 10% tween 20 in case of paprika and spices and mix well. For Gramineae and cereals, 5 mL of filtered extract was diluted with 20 mL of purified 10% PBS solution. For green roasted coffee and Nescafe, 5 mL of the filtered extract was diluted with 20 mL 2% Tween-20/PBS. After that, the diluted extract was filtered through a 1.5 m microfiber filter into a clean vessel or straight into a glass syringe barrel using markings to measure 10 mL on the barrel. Then, 10 mL of filtered diluted extract were entirely run through the Ochratest column at a rate of around 1 drop/second until air went through the column .Methanol/water solutions  were prepared to extract OTA out of the samples. Methanol: 1% Sodium bicarbonate solution was also prepared as previously reported. This test was performed using the VICAM international standard guidelines  was placed under the OchraTest column and the glass syringe barrel was filled with the OchraTest Elution Solution. The column was eluted at a rate of one drop per second and all the sample eluate (1.5 mL) was collected in the glass cuvette. The cuvette was mixed and then placed into the previously mentioned fluorometer. OTA concentration was read after 60 s.https://www.vicam.com/category/zearalenone-testing-solutions (accessed on 12 August 2023). Methanol/water solution (80%) and acetonitrile/water solution (90%) were prepared to extract ZEN from the sample. To prepare Dilute ZearalaTest Developer Solution, aluminum chloride hexahydrate was dissolved in 50 mL of HPLC Grade-methanol prior to use. The dissolved zearalatest developer was stored at room temperature for up to one month. The remaining solutions were prepared as mentioned previously for AFs and OTA. For Gramineae, 20 g ground samples were weighed with 2 g salt (NaCl) and put in a blender jar. Then 50 mL of either acetonitrile: water (90%) or methanol: water (80:20%) were added to the jar. The blender jar was then covered and blended for 2 min at high speed. 5 mL of filtered extract were then diluted with 20 mL 0.1% tween PBS buffer and then mixed well. Following that, the diluted extract was filtered through a 1.5 m microfiber filter into a clean container or straight into a glass syringe barrel utilizing markings on the barrel to measure 10 mL. These 10 mL of filtered diluted extract were entirely run through a zearalatest column at a rate of around 1 drop/second until air passed through the column . The column was then cycled through with 10 mL of 0.1% tween PBS buffer at a rate of around 1–2 drops/second until air flowed through the column then, 10 mL of deionized, or distilled water was run through it at a rate of around 2 drops per second. Finally, 1.0 mL of HPLC-grade methanol was injected into a glass syringe barrel while a glass cuvette  was positioned beneath the ZearalaTest column. All the sample eluate (1 mL) from the column was collected in a glass cuvette after it was eluted at a rate of 1 drop/second. To eluate in the cuvette, 1.0 mL of ZearalaTest Developer solution was added. After thoroughly mixing the cuvette, it was immediately placed into the previously mentioned fluorometer, and the ZEN concentration was measured after 300 s.This test was performed using the VICAM international standard guidelines Using certified Iso17034 and traceable to NIST  standards, the AFs mix standard solution was prepared at 10 µg/kg while OTA standard was prepared at 20 µg/kg. ZEN standard solution was prepared at 50 µg/kg. In addition, blank (samples known to be zero MTs) samples were spiked with those prepared standard solutions at each run to be employed as quality control to guarantee accurate assessment of the data quality for all targeted MTs in regular sample analysis.p-values were considered significant at α < 0.05. Qualitative variables have been tested using X2 test, The General Linear Model’s quantitative variables were evaluated using the Student’s t test and One Way ANOVA. Post hoc analysis using Tukey was done after ANOVA where groups sharing similar letters had no significant differences. Bar charts were graphed showing percentages of each level within each category using Excel 365.Data was analyzed using Excel 365, Minitab 20 and SPSS 26. Count and percentage were calculated for qualitative variables while for quantitative variables, mean, SD, SE, median, first quartile, third quartile, and inter quartile range were determined as descriptive statistics. Before performing any statistical analyses, the data was cleaned. Missing information and typographical issues have been examined. Inferential statistics has been used to find correlations and differences among different food and beverage categories or type and source . All parametric assumptions for quantitative variables have been examined, and when necessary, the best approach was used to apply the Box-Cox transformation for non-normal dependent variables. The various models matched the data well, and following data transformation, all analyses had linear attitudes in the normal residual probability plots. Table https://www.eos.org.eg/en/standard/12561, (accessed on 8 august 2023) for total AFs. Tables A total of 196 samples were screened using ELISA followed by quantitative determination of AFs using VICAM Aflatest IAC then fluorometric method. The results of the total AFs-positive samples are presented in Table Concerning OTA, a total of 139 samples were analyzed using ELISA followed by quantitative determination of OTA using VICAM Ochratest IAC and then the fluorometric method. The number and percentage of positive samples for different food types and categories are summarized in Table The results of ZEN contamination analysis of 70 Gramineae samples using ELISA followed by quantitative determination of ZEN using VICAM Zeralatest IAC then fluorometric method are summarized in Table The results of DON contamination analysis in different food categories by applying the ELISA technique only, including Gramineae and pasta/noodles, are presented in Table MTs pose a significant risk to the public’s health when they contaminate food, according to JECFA (the Joint FAO/WHO Expert Committee on Food Additives). The World Health Organization (WHO) designated MTs as priority food pollutants in the System/Food Monitoring Evaluation Program (GEMS/Food). Foods and feeds for humans and animals are invariably exposed to fungal invasion from crop planting through harvest, transportation, storage, and even into the grocery store, restaurant, and home, where the product will be ready for the consumer’s final use , spices, chili, and dried fruits were highly contaminated with total AFs highlighting the need for comprehensive monitoring and control strategies. Previous studies revealed how storage conditions affected AF levels in dried fruits, nuts (oily seeds) spices . The humid and hot weather circumstances in Pakistan throughout the summer may be the cause of the elevated incidence levels of DON in wheat and wheat product samples (Iqbal et al. Regarding DON analysis, our results showed high contamination levels in pasta and noodles. In previous studies, DON was found in 180 samples of Chinese wheat, with varying levels from 14.52 to 41157.13 For the use of the ELISA method of analysis, previous studies proved that demand has increased for the creation of a method that is sensitive, accurate, quick, simple, and reliable for detection at low concentrations, such as those observed in milk samples. ELISA technique was judged to be sufficiently meeting the requirements for official control purposes for this purpose (Kos et al. Additional file 1: Table S1. One Way ANOVA results of ZEN concentrations using ELISA among different categories of each type. Table S2. One Way ANOVA results of ZEN concentrations using Fluorometer among different categories of each type. Table S3. One Way ANOVA results of DON concentrations using ELISA among different categories of each type.