The potentiometric measurements were carried out under different conditions of temperature (15 ≤ t/°C ≤ 37). The obtained speciation pattern includes two species for both the investigated systems. More in detail, for system containing ampicillin MLH and ML species, for that containing amoxicillin, MLH2 and MLH ones. The spectrophotometric findings have fully confirmed the results obtained by potentiometry for both the systems, in terms of speciation models as well as the stability constants of the formed species. Enthalpy change values were calculated via the dependence of formation constants of the species on temperature. The sequestering ability of ampicillin and amoxicillin towards Mn2+ was also evaluated under different conditions of pH and temperature via pL0.5 empirical parameter .A potentiometric and UV spectrophotometric investigation on Mn The biological interest is more obvious than the environmental one, however, one must consider that antibiotics are excreted via feces and urine, through domestic and hospital wastewaters. They are thus released into aquatic environments, soils, sediments and many of them persist for long time in the environment [Photosynthesis, proteoglycan biosynthesis and antioxidative actions are the main biochemical functions . Mn2+ isolutions ,4. In biosphates ,6. The mas found . For humas found . As a coas found . Therebyironment . In thisN-acylderivatives of 6-aminopenicillanic acid. Semi-synthetic β-lactam antibiotics have been designed for a wider spectrum of action and better pharmacokinetic properties than natural penicillin. Among semi-synthetic penicillins, ampicillin and amoxicillin have the best oral absorption and they distribute well in body fluids. They are commonly employed as antibiotics owing to the wide spectra mechanisms of action, since they stop bacteria proliferation [2+, Cu2+, Ca2+ and Mg2+, were already studied [Among antibiotics, the aminopenicillins are semi-synthetic penicillins that can be considered as feration . It is kferation ,15,16,17feration . Complex studied ,20,21,222+ with ampicillin (Amp) and amoxicillin (Amox), shown in Amp and Amox with Mn2+ was performed [In this paper the interactions of Mnerformed . The invAmp and Amox was quantified via a Boltzmann type equation where the sum of molar fractions of all Mn2+-ligand complex species were fitted vs. cologarithm of total ligand concentration. Sequestering ability under different conditions of temperature, pH and ionic strength was also evaluated.Sequestering ability of In this investigation, potentiometric and UV spectrophotometric titrations were performed in order to define the best speciation models for both the investigated systems and to obtain reliable formation constants values of the complex species. Formation equilibria of metal cation(M)-ligand(L) species are the following (were the charges are omitted for simplicity):2+ and protonation constants of Amp and Amox, were taken into account in the calculations (2+(M)-Amp(L) system, under all the temperature and ionic strength conditions investigated, was found to contain only 1:1 M:L species, namely MLH and ML.Hydrolysis constants of Mnulations . The elat = 25 °C, CM = 2 mmol L−1, CL = 4 mmol L−1, I = 0.15 mol L−1 in NaCl. It can be observed from the diagram that the MLH species is present in a wide pH range with metal fraction greater than 0.4, the ML one reaches almost 0.4 as a fraction at pH = 8.5. The M2(OH)3 hydrolytic species is significant only starting from pH > 9.2+ and Amp at t = 25 °C and I = 0.15 mol L−1 were performed under different concentration conditions. As an example, experimental spectra recorded on solutions containing Mn2+ and Amp at CM = 0.05 mmol L−1 and CL = 0.05 mmol L−1, were depicted in In order to confirm the speciation model and the formation constants gained via potentiometry by spectroscopic techniques as already established for other systems ,32,33,34Amp species and of Mn2+-Amp complexes at t = 25 °C and I = 0.15 mol L−1 in NaCl are represented in 2 species of Amp.Via the processing of the spectrophotometric experimental data with the HypSpec program the results listed in 2+-Amox solutions has provided the results listed in 2+(M)-Amox(L) system, under all the investigated ionic strength and temperature conditions, includes only complex species with 1:1 M:L ratio, i.e., MLH2 and MLH. The speciation diagram regarding this system at CM = 2 mmol L−1, CL = 4 mmol L−1, t = 25 °C, I = 0.15 mol L−1 in NaCl is depicted in 2 species predominates in the acid pH range, 3 ≤ pH ≤ 6.5, with metal fractions close to 0.6. MLH is the main species in the range 7 ≤ pH ≤ 9, with a maximum metal fraction almost equal to 0.8 at pH = 8.5. M2(OH)3 hydrolytic species does not reach significant formation percentages up to pH = 9.The processing of the potentiometric experimental data carried out on MnAmp, the system containing Mn2+-Amox has been investigated with potentiometry as well as with UV spectrophotometry. Experimental UV spectra at different pH values recorded on solutions containing Mn2+(M) and Amox(L) are shown in M = 0.05 mmol L−1, CL = 0.075 mmol L−1, t = 25 °C, I = 0.15 mol L−1 in NaCl). As known, spectra of solutions containing Amox result more complex than those containing Amp. In the range λ = 220–310 nm the former present intense bands, as expected for ligands having heteroatoms, linked to benzene ring, giving electronic transfer phenomena. Accordingly, the observed absorptions are typical of aromatic compounds with polar substituents, such as phenolic groups, characterized by charge transfer with ε = 5 × 103–16 × 103 L mol−1 cm−1 in the range mentioned above.As for 2+ causes a slight hypsocromic shift of the phenolic band which can be explained by admitting metal-ligand interaction. More in detail, in the UV spectra shown in The UV spectra in terms of absorptivity, shape and band position are influenced by alkyl substitution, which generally causes a bathochromic shift of the phenolic band . The pre2+-Amox species as well as those of Amox are depicted in 3 L mol−1 cm−1, followed by MLH and MLH2 with ε ≅ 21 × 103, 18 × 103 L mol−1 cm−1, respectively at λ = 204 nm. At λ = 229 nm, only ligand protonated species and complex ones present a maximum. LH2 species shows ε ≅ 1 × 103 L mol−1 cm−1 at 229 nm, the interaction with metal causes a slight shift of the maximum. Accordingly, MLH2 and MLH present a maximum at 228 nm with ε ≅ 9 × 103.These absorption maxima show different trends with increasing pH, the maxima at about 204 and 272 nm undergo a hyperchromic effect, on the contrary the maximum at 229 nm has a hypochromic effect. Molar absorbances of MnThe results obtained by processing the spectrophotometric experimental data are listed in 0 is the formation constant at infinite dilution, z* = ∑(charge)2reactants − ∑(charge)2products, C is an empirical parameter. Several examples of these applications are reported in previous papers [C parameter for the dependence on the ionic strength are listed in The experimental results of s papers ,38,39,40I = 0.15 and 1 mol L−1 were obtained, as depicted in By using the experimental results of t = 15, 25, 37 °C and I = 0.15 mol L−1 for both Mn2+-Amp and Mn2+-Amox systems. The formation constant values at the various temperatures investigated are listed in T is the formation constant at a temperature T expressed in Kelvin, logβθ is the formation constant at T = 298.15 K, ΔH0 is expressed in kJ mol−1, R = 8.314472 J K−1 mol−1.Potentiometric titrations were performed at 2+-Amp and Mn2+-Amox systems were determined by using Equation (4). These values, together with those of free energy and entropy changes, are reported in 2+-Amp complexes ΔH values are exothermic for both the species. The trend of the thermodynamic parameters of Mn2+-Amp species is the same of Mg2+, the cation having the most similar biological behaviour to Mn2+, More in detail, for ML species of Mg2+-Amp system ΔH = −31.1 kJ mol−1, TΔS = −12.6 kJ mol−1 (at t = 25 °C and I = 0 mol L−1), fairly similar to ΔH = −21 kJ mol−1, TΔS = −8 kJ mol−1 here reported for the same species containing Mn2+.Enthalpy change values relating to all species of Mn2+-Amox complexes, by considering partial reaction (2), ΔH value is exothermic for MLH species and endothermic for MLH2. Referring to partial reactions, the prevalence of the enthalpy and the entropic contribution for the species containing Amp and Amox, respectively, is not marked at all. For Mnt = 15, 37 °C relating to Mn2+-Amp and Mn2+-Amox systems, respectively. In both systems the change in temperature leads very different formation percentages of complex species, highlighting the need to take into account this parameter for a correct speciation study.0.5, widely tested on multiple metal-ligand systems, was proposed for this purpose. pL0.5 represents the cologarithm of the concentration of the ligand able to bind 50% of the metal cation in traces, under the investigated conditions of pH, temperature and ionic strength. The following Boltzmann equation with asymptotes of 0 for pL→0 and 1 for pL→∞ is used to quantitatively define the sequestering capacity of a ligand against a specific metal cation [0.5, as it depends on the system conditions , considering all competitive reactions of the metal cation and the ligand. Moreover, in the calculation of the sequestering capacity a concentration of metal cation in traces was considered, making this parameter closer to the concentration conditions of many metal cations in real systems, such as body fluids. Under physiological conditions , the sequestering ability values calculated against Mn2+ for Amp and Amox resulted in pL0.5 = 2.01, 3.17, respectively. It emerges that under physiological conditions the binding capacity of Amox is more than one logarithmic unit compared to Amp. These results are comparable with those obtained with another divalent cation, Ca2+, under the same conditions, pL0.5 = 1.82, 2.88 for Amp and Amox, respectively [2+, in this case in the same conditions the sequestering ability is comparable for Amox, while for Amp it is significantly different. More in detail, pL0.5 = 3.16, 2.88 for Amp and Amox, respectively, towards Zn2+ [Amp and Amox towards Mn2+ were calculated under different conditions of pH, temperature and ionic strength. These values are listed in 2+ under physiological conditions. Binding ability of Amox is significantly higher than Amp under these conditions.Often the simple comparison of the formation constant values and the formation percentages of the species is not sufficient to analyze the higher or lesser sequestering capacity of a ligand towards different metal cations or of different ligands towards the same metal cation. In order to assess the sequestering ability, it is necessary to consider all the equilibria in which the ligands and various metal cations participate, such as hydrolysis reactions, ligand protonations, weak interactions of the ligand with the cations of the background salt. Several years ago, an empirical parameter, pLl cation ,43,44,45ectively . Anotherrds Zn2+ . The seq2+-Amp and Mn2+-Amox species. Only formation constant values for Cu2+, Ni2+, Zn2+, Cd2+, Co2+ are found. As far as we know, no thermodynamic data are reported on the complex species formed by Amox with any metal cation, apart from the results already published on Ca2+, Mg2+, Cu2+, Zn2+ cations [2+ with those obtained with other divalent cations. Among these cations, the behaviour of Mn2+ is quite similar to Mg2+ and Ca2+. More in detail, for the system containing Amp, formation constant values for MgLH and MgL species are 2.54, 2.86, respectively (at I = 0.15 mol L−1 and t = 25 °C) [2+ are 2.47, 2.37, respectively, as reported here under the same conditions. For the system containing Amox, formation constant values for MLH and ML species with Ca2+ are 2.69, 2.59, respectively (at I = 0.15 mol L−1 and t = 25 °C) [2+ are 2.78, 3.20, respectively, as reported here under the same conditions.In literature, in the main databases that report thermodynamic data ,47,48, t cations ,20,21,222+ were prepared by weighing and dissolution of the corresponding salt, manganese(II) chloride tetrahydrate  These solutions were standardized by titration with ethylenediamine tetraacetic acid disodium salt (EDTA) standard solution. Solutions containing ligand were obtained by weighing and dissolution of ampicillin anhydrous and amoxicillin trihydrate , used without further purification. Ligand purity, checked by alkalimetric titration, resulted > 99%. Fresh solutions of ligands were prepared every day. Hydrochloric acid and sodium hydroxide solutions were prepared by dilution of the respective ampoules  and after they were standardized with sodium carbonate  and potassium biphthalate , respectively, previously dried in an oven at 110 °C. Sodium hydroxide solutions were prepared very frequently and were stored in bottles containing soda lime traps. Sodium chloride solutions were obtained by weighing the salt , previously dried at 110 °C. All the solutions were prepared using bidistilled water (conductivity < 0.1 μS cm−1) and grade A glassware.The solutions containing MnPotentiometric titrations were performed by two distinct systems including a Metrohm ) model 809 Titrando potentiometer, a Metrohm LL-Unitrode WOC combined glass electrode, and an automatic dispenser Metrohm Dosino 800. The system just described was connected to a PC and the measurements were acquired by the Metrohm TIAMO 2.2 software, which can control titrant delivery, e.m.f. stability, and data acquisition. Estimated accuracy of this system is ±0.15 mV and ±0.002 mL for e.m.f. and for titrant volume readings, respectively.2+, ligand and a supporting electrolyte (NaCl). For Mn2+(M)-Amp(L) solutions, 1 ≤ CM/mmol L−1 ≤ 3, 1 ≤ CL/mmol L−1 ≤ 6 and concentration ratios 1 ≤ CL/CM ≤ 3. For Mn2+(M)-Amox(L) solutions, 1.5 ≤ CM/mmol L−1 ≤ 2, 2 ≤ CL/mmol L−1 ≤ 4 and concentration ratios 1 ≤ CL/CM ≤ 2. The investigated pH ranges between 2 and 10. The measurements were performed into glass jacket thermostated cells, under different conditions of temperature (15 ≤ t/°C ≤ 37) and ionic strength (0.15 ≤ I/mol L−1 ≤ 1.0), under magnetic stirring and by bubbling pure N2 in order to avoid CO2 and O2 inside the solutions. An independent titration of HCl with standard NaOH was carried out for each measurement to calculate the pKw value and the standard electrode potential E0 under the same experimental conditions of ionic strength and temperature of the measurement itself.During the titration NaOH standard was added to 25 mL of the solution containing Mn2+-Amp and Mn2+-Amox solutions. The instrument was connected to a PC for the acquisition of the UV spectra in the range from 200 to 320 nm. A volume of 25 mL of the solution containing the metal cation and the ligand at different metal/ligand ratios was investigated at t = 25 °C, I = 0.15 mol L−1 in NaCl and at different pH values. For Mn2+(M)-Amp(L) solutions, 0.04 ≤ CM/mmol L−1 ≤ 0.05, 0.05 ≤ CL/mmol L−1 ≤ 0.08 and concentration ratios 1 ≤ CL/CM ≤ 2. For Mn2+(M)-Amox(L) solutions, 0.05 ≤ CM/mmol L−1 ≤ 0.1, 0.075 ≤ CL/mmol L−1 ≤ 0.12 and concentration ratios 1 ≤ CL/CM ≤ 2. The titrations were performed by potentiometric apparatus already described in the 3.2 paragraph. Aliquots of standard NaOH were added to solution containing Mn2+-Amp and Mn2+-Amox in order to reach the maximum absorbance of each complex species.A Varian ) Cary 50 UV-Vis spectrophotometer equipped with an optical fiber probe having a path length equal to 1 cm, was employed for spectrophotometric titrations on Mn0, junction potential). LIANA software was employed in order to obtain the parameters for the dependence of complex formation constants on temperature and ionic strength. Details on software used in the refinement of the experimental data are reported in ref. [Experimental potentiometric data were processed via the BSTAC and STACO programs, in order to gain the best speciation model for the systems under study, the formation constant values of the complex species, and all the parameters of an acid-base titration (analytical concentration of the reagents, standard potential E in ref. . Experim in ref. ,51. HySS in ref. .2+-Amp and Amox solutions were performed by potentiometry under different experimental conditions of temperature and ionic strengths. The results in terms of most reliable speciation model and formation constant values were fully confirmed by UV spectrophotometry for both the systems. More in detail, despite the different metal and ligand concentrations employed with these two techniques, a satisfactory agreement among the stability constants was achieved. The dependence of the stability constants on temperature and on ionic strength was studied. Accordingly, enthalpy changes at t = 25 °C and parameters for the dependence on ionic strength were also calculated.Due to the widespread use of aminopenicillins as antibiotics, knowledge of their complexation behavior and speciation is of fundamental importance for understanding their pharmacokinetics and pharmacodynamics. Speciation studies on MnAmp and Amox with Mn2+, are necessary to evaluate the action mechanism of these ligands and the bioavailability of the metal cation in the presence of the drugs under physiological conditions. The assessment of the sequestering capacity is of fundamental importance as it takes into account the competitive processes of the metal cation and the ligand and facilitates comparisons among several ligands towards the same metal cation or the same ligand towards several cations under the same conditions of pH, temperature and ionic strength. The sequestering ability of Amp and Amox towards Mn2+ was evaluated under different conditions, including physiological ones. Under these conditions Amox has shown a higher binding capacity towards Mn2+ of more than one logarithmic unit compared to Amp.For the correct assessment of the bioavailability of a drug or a trace element in the presence of that drug, it is essential to know the possible interactions occurring in body fluids. Accordingly, the knowledge of speciation and sequestering ability of two drugs employed as antibiotics, such as


Lead is a toxic metal that affects almost every organ in the body. Children are more susceptible to lead toxicity because they ingest non-food items (pica), have oral exploratory habits, absorb more substantial amounts of ingested lead compared to adults, and have a developing central nervous system. This study describes venous blood lead concentrations (BLC) in young children living in Birjand, Iran.A cross-sectional study was performed in 2016 on children 1–7 years of age who were referred to healthcare centers in Birjand City. Demographic information was obtained, and their BLC was tested using atomic absorption spectrometry (AAS).p < 0.01). The risks of an elevated BLC was 61% lower in girls compared to boys (OR (95%CI): 0.39 (0.17–0.92), p = 0.03). Further, per one rate of increase in the BMI, the chance of an elevated BLC was higher (OR (95%CI): 1.13 (1.02–1.24), p = 0.01). Children whose fathers were laborers had higher BLC than those with employee fathers (p = 0.01).Four hundred children were tested. Their mean age was 52.37 ± 23.77 months; their mean BLC was 2.49 ± 2.64 μg/dL (median 1.85 μg/dL). Thirty-two (8%) children had a BLC > 5 μg/dL. A logistic regression model revealed that per one unit of increase in age, the chance of an elevated BLC decreased by 3% (OR (95%CI): 0.97 (0.96–0.99), Of 400 children aged 1–7 years old living in Birjand, Iran, 8% had elevated BLC. BLC correlated with the child ‘s age, gender, body mass index, and father’s occupation. Lead is a toxic environmental metal that influences almost every organ in the body . Lead toIn 1991, CDC highly recommended screening by a blood lead test at least once for all children younger than 2 years of age . Prior sIn this descriptive and analytical study, 400 children with 1–7 years of age in Birjand city were studied during September to December 2016. The primary outcome was elevated blood lead concentrations (BLC ≥ 5 μd/dL) and some potential associated factors of BLCs in children. Birjand city was divided into four parts based on the four major healthcare centers in this city . Primary Health Care Centers is one of the health care networks in Iran run by the Ministry of Health and Medical Education (MOHME). These centers are a far-reaching network of public clinics that provide primary and preventive health care such as nutrition, family planning, hypertension assessment, prenatal care, immunization, and environmental monitoring. These centers are the first level of communication between families and the health system. Children in this study typically go to health centers and use health centers in the province.A total sample size of 400 children was determined based on the study of Wang et al., With a first error level of 5%, a standard deviation of 0.15 and accuracy of 10% of standard deviation error .From each healthcare center, a health base was chosen through simple random sampling from the list of the healthcare bases covered by each center. Then, 1-to-7-year-old children were ascertained through simple random sampling in line with the sample size from each base. The total number of children aged 1–7 years, covered by Birjand Health Centers, is 27,691 people. The selected number of children and the total number of children covered by each Health center of 1–8 in Birjand city were 1790(26), 2676(39), 3395 (49), 2884 (42), 5966 (86), 2799 (40), 3512 (51), 4669 (67), respectively  and sent to Imam Reza Hospital laboratory. The blood containing EDTA was used for performing a complete blood count (CBC) using the Cell Dyn Analyzer. Blood calcium, magnesium, iron, and zinc concentrations were measured using a Selectra automatic device. In order to collect the samples for lead concentrations, the free sodium heparin pink-cap 6-mL Vacuette tubes were applied. Then the samples in ice containing Styrofoam boxes were transported to the laboratory. The lead concentration in blood was measured using an atomic absorption spectrometer , using the standards recommended by the National Institute of Occupational Safety and Health (NIOSH) .Data analysis was conducted through SPSS 19. Descriptive indices, including frequency, percentage, mean, and standard deviation, were calculated. Using the Kolmogorov Smirnov test, the assumption of normality of quantitative variables was tested. Mann-Whitney and Kruskal Wallis tests were used to compare BLC with demographic variables. With a logistic regression model, the factors possibly associated with BLC were inspected. Pearson correlation coefficient tests were employed to investigate the relationship between variables. An alpha level of significance was set at 0.05.n = 1) were followed and received free medical care.The research proposal was approved by the research deputy of Birjand University of Medical Sciences and by the ethics committee (ir.bums. REC.1395.131). Children with elevated lead concentrations who need treatment  that routinely refer to Health Care centers. Their mean age was 52.37 ± 23.78 months (range: 12 to 116 months); 200 (50%) of the children were boys. The mean BLC was 2.49 ± 2.64 μg/dL (range: 0.5–29.40 μg/dL) with a median of 1.85 [IQR: 1.10–2.87] μg/dL (Table p = 0.39). The mean lead concentration in those whose father was laborer was 3.20 ± 3.96 with a median of 1.80 [IQR:1.20–3.30] μg/dL, and in drivers, it was 1.79 ± 0.87 with a median of 1.70 [IQR: 1.00–2.30] μd/dL. The results of the Kruskal Wallis test indicated that the lead concentration was not significantly different between fathers with different occupations . Twelve (3%) family members had a history of drug abuse. The mean BLC in subjects whose family members had a history of drug use was 2.43 ± 1.53 μg/dL (median 2.1 [IQR: 1.17–3.12] μg/dL). The mean BLC in the parents using opium was 2.95 ± 2.10 μg/dL with a median of 2.45 [IQR: 1.25–3.18] ug/dL, in industrial drugs of abuse, it was 1.67 ± 0.61 with the median of 1.65 [IQR: 1.02–2.86] μd/dL. The mean BLCs in children exposed to paint was 2.92 ± 3.32 μg/dL (median 2.10 [IQR: 1.20–3.30] μg/dL)   had anorexia, 3 (9.4%) had persisted vomiting, 2 (6.3%) had constipation and abdominal pain, and 8 (25%) were asymptomatic. The results of the Chi-Square test showed that there was no significant relationship between BLC and clinical symptoms   Fig. .Fig. 2Ip < 0.01). Further, per one rate of increase in the BMI, the chance of an elevated BLC was higher (OR (95%CI): 1.13 (1.02–1.24), p = 0.01). In children aged 12–24 months, the chance of an elevated BLC was 3.87 times that of children older than 72 months (OR (95%CI):3.87 (1.39–10.79), p = 0.01). The risks of an elevated BLC was 61% lower in girls compared to boys (OR (95%CI): 0.39 (0.17–0.92), p = 0.03). Finally, in children whose fathers were laborers, the chance of high lead concentrations was 5.56 times higher than those whose fathers were employees (p = 0.01) : 0.97 (0.96–0.99),  1.02–1.2, p = 0.02 can predict high blood lead concentrations (≥5 μg/dL)  tested children with anemia exposed to lead and reported a significant negative correlation between lead and iron concentration . The relIn this study, the mean BLC of the children was 2.49 μg/dL, well below the CDC reference concentration. However, 8% of studied children had an elevated BLC above 5 μg/dL. Since this age group is most vulnerable to the adverse effects of lead on the nervous system and IQ, preventive interventions should be taken. The reason for the selection of 5 μg/dl as the amount of lead was that the CDC offered the reference BLC as 5 μg/dL or less for children . FaranouThe difference observed between different regions, and countries can be attributed to the different food, environmental conditions, and differences in the use of traditional drugs as well as cosmetics (such as Kohl), smoking, and drug abuse. All of these studies highlight the need to constantly investigating the BLC in this vulnerable population.In this study, we tried our best to investigate the associated factors of elevated BLCs in young children living in Birjand, Iran. But some other potential sources of lead may be involved that were not assessed in this study. This warranted further studies. Since our study investigated the risk factors and consequences concurrently because of its cross-sectional design, this prevented precise investigation of the precedence between them. Prospective studies with a larger sample size are suggested to discover potential factors of elevated blood lead concentrations among children and guarantee the generalizability of the findings to an unselected sample. Also, we did not assess the birth history of included children. To improve the accuracy of results, more extensive data sets, and enhanced data collection methods are suggested in the future. A comparative study for evaluating BLCs in different groups of children aged 1–7 years, infants and older children with considering clinical features and treatment is needed in the future. A follow-up study can be useful to evaluate the trend of lead concentration in the whole blood of children.The mean BLC of 1–7-year-old children in Birjand city was 2.49 μg/dL; around 8% of studied children had elevated BLC. Young age, male gender, and low BMI significantly increased the chance of an elevated BLC. These results can provide information for parents with high-risk children to have more attention and doing countermeasures. In children whose fathers were laborers, the risk of high BLC was more significant compared to those children of employee fathers, suggesting ‘take-home’ lead sources at work. Exposure to toxic materials in the workplace is a substantial concern for the families of laborers employed in polluting industries, needing educating workers, and conducting preventive measures. Knowing the BLC in children offers necessary information to health policymakers for public health measures, preventive interventions in the community, and health promotion of children. It also provides baseline data for future researches.Additional file 1.


Tetraena qataranse. Young seedlings were irrigated with 0, 25, 50, and 100 mg/L Pb every 48 h for seven weeks under greenhouse conditions. A phytotoxicity test showed that at the lowest treatment concentration, Pb stimulates growth. However, at 100 mg/L (1600 mg/kg Pb in the growth medium at harvest), the metal disrupted healthy growth in T. qataranse, particularly root development. Metal accumulation in the root was higher (up to 2784 mg/kg) than that of the shoot (1141.6 mg/kg). Activity assays of superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), guaiacol peroxidase (GPX), and glutathione reductase (GR) showed a progressive increase in enzymatic activities due to Pb treatment. Together, the results of this study suggest that T. qataranse is a Pb hyperaccumulator. Increased antioxidant enzyme activity was essential to maintaining cellular homeostasis and assisted in the arid plant’s tolerance to Pb stress.Lead (Pb) is the second most toxic metal on Earth and is toxic to humans and other living things. In plants, Pb commonly inhibits growth when it is at a concentration in the soil of 30 mg/kg or more but several Pb tolerant plants have been reported. However, few studies have focused on plant response to Pb exposure, particularly at concentrations higher than 30 mg/kg. The assessment and evaluation of metal dose-dependent plant responses will assist in future phytoremediation studies. Therefore, this work documents the Pb concentration-dependent antioxidative response in  In 2015, Pb was listed as the number one heavy metal on Earth. It is generally toxic to most plants at a soil concentration higher than 30 mg/kg4 .Environmental pollution is one of the most challenging sustainable development agendas today. Toxic metals, including Pb, are some pollutants that are most dangerous to human health. This is especially true in countries witnessing rapid industrialization6. Our group was among the first to conduct an assessment of the capacity of various local plants to accumulate different toxic metals, including Pb, under field conditions7. Though metal concentrations in the soil were relatively low, our study found Tetraena qataranse to be the most promising for phytoremediation. While the use of plants to remove toxic metals dates back decades and hundreds of plants have been reported thus far, climatic conditions and soil environments differ between regions and countries around the world5. Arid and semi-arid climates are known to be harsh for the majority of plant species.The state of Qatar is located in the Arabian Peninsula desert and has a growing human population and increased anthropogenic activities. Evidence of the occurrence of some environmental pollutants, including toxic metals, has been documented. However, only a handful of studies have investigated the phytoremediation of toxic metals, including Pb, using native and invasive plants or plant responses to metal toxicity for optimized remediation35.Further, the scale-up of such a promising strategy still needs to be achieved, partly due to a poor understanding of the mechanism governing the toxic metal phytoremediation process. Studying promising plant's responses to toxic metal exposure will help in overcoming such a limitation. In this regard, high metal accumulation in plant tissues provides the perfect condition for evaluating such responses. To date, several Pb accumulating and tolerant plants have been reported. However, only a few hyperaccumulators exist. Additionally, most studies noted that heavy metals, like Pb, preferentially accumulate in roots compared to other plant parts8. PCs are induced by phytochelatins synthase (PCS), an enzyme that is triggered when metal ions are present26. MTs are low molecular weight gene-encoded, cysteine-rich proteins. Numerous studies sugeest that these molecules play important roles in Pb sequestration26. From antioxidative system perspectives, toxic metals, including Pb, interferes with cellular homeostasis by generating reactive oxygen species (ROS) in plant system. This negatively affects plant health, and consequently, may lead to death8. A more recent discussion on Pb accumulation and suggested tolerance and detoxification mechanisms can be found in a crical review by Kumar and Prasad13.Several physiological, biochemical, and molecular mechanisms enable plants toxic metals tolerance. Transporters, and metal-binding proteins such as phytochelatins (PCs), and metallothioneins (MTs) are most notable in aiding toxic metal sequestration in plants6. SOD is the first line of defense; it sequesters noxious superoxide ions and breaks them into less harmful hydrogen peroxide (H2O2) and oxygen molecules (O2). CAT assists in further detoxification by converting H2O2 into water (H2O) and oxygen molecules (O2). GR combats oxidative stress by balancing reduced (GSH) and oxidized glutathione (GSSG)10. Following heavy metal (e.g. Pb) exposure and uptake, determining the activities of antioxidative enzymes is vital to understanding plant response to metal toxicity from a biochemical and, in part, molecular perspective. Therefore, this study aims to further explore T. qataranse Pb tolerance by investigating its antioxidative response to varying concentrations  of Pb under greenhouse conditions.The antioxidant system is essential for shielding plants against heavy metal toxicity and does so by creating changes in the activities of critical enzymes including catalase (CAT), superoxide dismutase (SOD), ascorbate peroxidase (APX), glutathione reductase (GR), and guaiacol peroxidase (GPX)11. Therefore, the evaluation of seedling growth is vital in assessing metal toxicity in plant systems. The effects of Pb concentration on total chlorophyll content, fresh weight, root, and shoot length were evaluated and the results are shown in Fig. p ≤ 0.05) in T. qataranse fresh weight was observed under 25 and 50 mg/L Pb treatments  for the 25 mg/L treatment and an increase for both shoot and root in the 50 mg/L treatment compared to the control, the length of shoots and roots in the 100 mg/L treatment were not significantly different from the control treatment. While no effect occurred for leaf chlorophyll content in either the 25 or 50 mg/L treatments, a significant decline occurred in the 100 mg/L treatment compared with the control treatment  and translocation factor (TF) are indices that are essential in the evaluation of metal bioaccumulation and translocation in plant tissuesT. qataranse when compared to the control in the order SOD > CAT > GPX > APX > GR . Our result is consistent with the reports of many similar studies. For instance, known plant species, including Nerium oleander L. and Brassica juncea, accumulate higher Pb concentrations in their roots than other tissue parts16. Also, in a study involving different plants, Finster, et al.17 determined that the roots always accumulate more Pb than other plant parts, including the fruits, where only traces of the metal translocate the shoot. Our result is also in agreement with the work of Langley-Turnbaugh and Belanger18. Kumar, et al.19 and Pourrut, et al.20 conducted several critical reviews of Pb toxicity in plants and determined that several factors contribute to restricted metal translocation in plants. Of such, Casparian strip endodermis restriction is by far the most limiting for Pb. Notheless, the ability of T. qataranse to accumulate more than 1000 mg/kg Pb suggests that it is a Pb hyperaccumulator21.Pb accumulates differently in plant tissue parts, especially in the rootT. qataranse sequestrate Pb from growth medium that contains up to 1600 mg/kg Pb. However, it was optimal in the 50 mg/kg treatment. At this concentration, the growth medium had up to 800 mg/kg Pb. The TF under all treatments was less than 1  is affected by Pb toxicity25.Various response mechanisms enable plants to withstand metal toxicity, of such, metal avoidance and uptake are the most common. Before compartmentalization, Pb is translocated to a degree that can be described by the TFT. qataranse antioxidative defense. Having the highest enzymatic activity be in the root changes root organic constituents due to Pb complexation. This indicates that, as suggested in our previous work, as an uptake mechanism, Pb ions bind to T. qataranse root by complexation through cationic exchange with hydroxyl and carboxyl functional groups7. This is a well-established complexation mechanism of transition metals, including Pb1. In comparison, GR demonstrated the least activity  or oxidized form (GSSG), acts as an antioxidant and chelating bioligand majorly accountable for metals detoxification. Enzymes involved in glutathione metabolism mediates detoxification Glutathione-S-transferases (GSTs) are a major phase II GSH-dependent ROS scavenging enzymes. They play significant roles in GSH conjugation with exogenous and endogenous species found during oxidative stress, including H2O2 and lipid peroxides10. Consistent with our findings, a more recent review by Kumar and Prasad14 discussed several other studies, some of which include the use of model species, Arabidopsis thaliana, and Oryza sativa, all of which support our findings.The antioxidant system is one mechanism used by plants for protection against metal toxicity. In this study, the result of the SOD, CAT, APX, GPX, and GR assay show increased activity of all five enzymes. SOD activity was the highest, up to ten times higher than the control (0 mg/kg Pb), particularly in the root Fig. a, suggesity Fig. e. Such d20. Further, we recognize that proteins regulate ROS signaling and the expression of such proteins changes due to Pb exposure11. Due to metal stress in plants, increased protein synthesis is essential in cellular metabolic processes. Mitogen-activated protein (MAP) kinase pathways regulate such processes, serving as a signaling system against oxidative stress. Signaling occurs through multiple stages of the reaction, modifying gene expression and, ultimately, protein synthesis4. Therefore, our future work will focus on the differential expression of proteins, particularly those that are related to stress responses, such as the heat shock protein family, due to Pb exposure in T. qataranse.It is worth mentioning that part of the discussion presented in this work is limited to the perspective of deciphering Pb tolerance and uptake mechanisms from metal translocation and plant antioxidative systems. However, both molecular and biochemical mechanisms play significant roles in toxic metals detoxification, including Pb. For instance, glutathione metabolism is known to regulates the biosynthesis of phytochelatins (PC), which bind Pb and transports it to vacuoles where detoxification can occur. Additionally, genes, such as glutamate cysteine ligae 1 (GSH1), glutamate cysteine ligae 1 (GSH2), phytochelatins synthase 1 (PCS1), and phytochelatins synthase 2 (PCS2), are actively involved in GSH-dependent PCs synthesis. Other primary and secondary metabolites that act as antioxidants, such as Tocols, flavonoids, anthocyanidins, and ascorbic acid, are essential to protecting plants against oxidative damage as well. The functions of these metabolites are well documentedT. qataranse. However, while the plant demonstrates good Pb accumulation capacity, further studies are required to ascertain its suitability for practical phytoremediation application. Additionally, our results showed that T. qataranse copes with high levels of Pb via the antioxidant system by synthesizing and increasing the activities of critical enzymes, CAT, SOD, APX, GPX, and GR, which are capable of reducing and manipulating free radicals. However, to sufficiently elucidate the mechanisms of Pb tolerance and uptake, further biochemical and molecular studies are imperative.In this study, we showed that Pb stimulates growth and accumulates in high concentration in T. qataranse seedling and control replicate was planted into a pot containing approximately 300 g of the media for individual treatment. Before Pb treatment, the young seedlings were irrigated with distilled water for three weeks under greenhouse conditions.Young seedlings that were approximately one week old were collected from Qatar University campus (25° 22′ 21.60″ N 51° 29′ 45.28″ E) Doha-Qatar, during October and November 2018. The young seedlings were planted in pots containing regular soil amended with peat moss in a ratio of 3:1. Before transferring the mixture into the pots, the pH and total organic content (TOC) were measured. Each young 2) to create varying Pb concentrations of 25, 50, and 100 mg/L Pb. Only the modified Hoagland nutrient solution (0 mg/L Pb), was used for the control plot29. Treatment concentrations were chosen based on Pb soil concentrations in Qatar7 and the hyperaccumulation threshold.A modified Hoagland nutrient with and without Pb for treatment and controls, respectively, was prepared as the base solution for seedling irrigation. For the treated plots, seedlings were irrigated with a solution prepared using lead chloride  in a randomized manner and irrigated with 200 mL of solution every 48 h for seven weeks. During irrigation, no leaching of the Pb treatment solutions occurred. After seven weeks, growth parameters were evaluated as described in Usman, et al.T. qataranse tissues was performed using inductively coupled plasma optical emission spectroscopy (ICP-OES). All samples were in three replicates and were measured against Standard Reference Materials (SRMs): soil 2709a and apple leaves 151534.Harvested plant biomass was prepared by digestion in nitric acid and hydrogen peroxide before analysis. This was accomplished using a large capacity Environmental Express SC154 HotBlock® digestion system at alternating temperatures until solutions were clear. Standard linear curves were obtained using five different concentrations from 1.0 to 500 µg/L. One standard and quality check analysis was performed and a good correlation coefficient of > 0.999 was obtained. Quantitation of Pb accumulation in 7.The bioconcentration factor  and translocation factor  were evaluated, as previously reported in Usman et al.26. Briefly, 0.2 g of a fresh tissue sample was homogenized in an ice-cooled mortar using 5 mL 10 mM potassium phosphate buffer (pH 7.0), 1% (w/v) polyvinylpyrrolidone, and 0.1 mM Ethylenediaminetetraacetic acid (EDTA). This was followed by a centrifugation of the homogenate at 15,000g for 15 min at 4 °C. Enzymatic activities were determined and expressed as Unit/mg of protein.Enzyme extraction followed the method of Mishra et al.31. A total of 3 mL of assay mixture containing 40 mM phosphate buffer (pH 7.8), 13 mM methionine, 75 µM NBT, 2 µM riboflavin, 0.1 mM EDTA, and an aliquot of enzyme extract was used for the reaction. After 15 min under illumination, absorbance was recorded at 560 nm. The non-illuminated mixture was used as the control.To measure SOD (EC 1.15.1.1) activity, nitroblue tetrazolium (NBT) photochemical reduction inhibition was determined by Beauchamp and Fridovich32. A total reaction mixture of 3 mL was prepared. The reaction mixture was comprised of 50 mM sodium phosphate buffer (pH 7.0), 20 mM H2O2, and an extract of the enzyme. Changes in the absorbance of the solution were measured at 240 nm for 4 min.The CAT (EC 1.11.1.6) assay was performed according to Zhang et al.33. Briefly, 3 mL of reaction mixture containing 50 mM phosphate buffer (pH 7.0), 0.1 mM H2O2, 0.5 mM sodium ascorbate, 0.1 mM EDTA, and the enzyme extract were used. The changes in absorbance were monitored at 290 nm.The determination of APX (EC 1.11.1.7) activity followed the ascorbic acid oxidation rate, as described in Nakano and Asada34. The reaction mixture consisted of 10 mL 1% guaiacol (v/v), 10 mL 0.3% H2O2, 80 mL 50 mM phosphate buffer (pH 6.6), and the enzyme extract.GPX (EC 1.11.1.9) activity following guaiacol oxidation was measured by measuring an increase in absorbance at 470 nm as described in Egley et al.35. The reaction mixture contained 100 mM potassium phosphate buffer (pH 7.8), 2 mM EDTA, 0.2 mM NADPH, 0.5 mM GSSG, and the enzyme extract.GR activity was determined by measuring absorbance at 340 nm due to NADPH oxidation, following the method of Rao et al.p ≤ 0.05. ANOVA was also performed to study the significance of Pb treatments on oxidative stress enzyme activities. The Shapiro–Wilk test was used to test the normality of the data and Tukey’s test was used to separate the means at p ≤ 0.05.Analysis of variance (ANOVA) was performed to study the significant differences in growth changes between the Pb-treated plants and control plants, at


Triterpenoid biosynthesis is generally anaerobic in bacteria and aerobic in Eukarya. The major class of triterpenoids in bacteria, the hopanoids, is different to that in Eukarya, the lanostanoids, and their 4,4,14-demethylated derivatives, sterols. In the deep sea, the prokaryotic contribution to primary productivity has been suggested to be higher because local environmental conditions prevent classic photosynthetic processes from occurring. Sterols have been used as trophic biomarkers because primary producers have different compositions, and they are incorporated in primary consumer tissues. In the present study, we inferred food supply to deep sea, sponges, cnidarians, mollusks, crustaceans, and echinoderms from euphotic zone production which is driven by phytoplankton eukaryotic autotrophy. Sterol composition was obtained by gas chromatography and mass spectrometry. Moreover, we compared the sterol composition of three phyla  collected between a deep and cold-water region and a shallow tropical area. We hypothesized that the sterol composition of shallow tropical benthic organisms would better reflect their photoautotrophic sources independently of the taxonomy. Shallow tropical sponges and cnidarians from environments showed plant and zooxanthellae sterols in their tissues, while their deep-sea counterparts showed phytoplankton and zooplankton sterols. In contrast, echinoids, a class of echinoderms, the most complex phylum along with hemichordates and chordates (deuterostomes), did not show significant differences in their sterol profile, suggesting that cholesterol synthesis is present in deuterostomes other than chordates. In the deep sea , light and temperature diminish while pressure increases with depth . In addi2S) into organic products that can be utilized by the rest of the community [13C of −50‰ to −100‰ [Euphotic zone primary production is driven by eukaryotic autotrophy by phytoplankton and, to a lesser extent, by macroalgae and vascular plants from marine and terrestrial sources. In the deep sea, the prokaryotic contribution to primary productivity has been suggested to be higher because local environmental conditions prevent classic photosynthetic processes from occurring . In reduommunity . FurtherPanaeus monodon [The lipid signature approach, including lipid class, fatty acid, and sterol composition has been used successfully to help understand marine trophodynamics ,7. Typic monodon ,19.As triterpenoid biosynthesis is generally anaerobic in bacteria and aerobic in Eukarya, the major class of triterpenoids in bacteria, the hopanoids, is different to that in Eukarya, the lanostanoids, and their 4,4,14-demethylated derivatives, sterols. There is a further bifurcation in the pathway of sterol biosynthesis in that photosynthetic organisms  form primarily cycloartenol as the first cyclization product, whereas nonphotosynthetic organisms  form mostly lanosterol. There are exceptions to this general pattern of triterpenoid distribution, with hopanoids being found in some Eukarya  and sterols being found in certain methanotrophic bacteria and in cyanobacteria. It is possible that the two pathways are very primitive and that one or other of these pathways has, for functional reasons, been subsequently selected in different groups of organisms . HoweverThe pathway of sterol synthesis has been thoroughly studied in vertebrates, fungi, and terrestrial plants, with cholesterol and ergosterol at the end of the synthesis pathway in vertebrates and fungi, respectively, and a higher variety of sterols in plants such as campesterol, sitosterol, and stigmasterol . In contTo assess the euphotic contribution from phytodetritus to deep-sea sponges, corals, gastropods, decapods, and echinoids, we used sterols, most of which are absent in prokaryotes, with the exception of modified lanosterol products  probablyDaphnia magna than cholesterol , while others were less efficient , indicating substantial differences in the assimilation efficiency and further processing within the body for these dietary sterols [Sterol biosynthesis in invertebrates does not take place or else proceeds at a slow rate; therefore, they should be provided by diet . Certain sterols . TherefoHere, we evaluated sterol composition of five phyla, Porifera, Cnidaria, Mollusca, Arthropoda, and Echinodermata, from the deep and cold northeast Atlantic Ocean to infer their food sources. We then compared three phyla, Porifera, Cnidaria , and Ech−1 wet weight in Porifera to 37.7 ± 12.1 mg·g−1 in Echinodermata. While the number of sterols ranged from five, in the arthropod Pasiphaea. tarda, to 17, in the sponge Thenea muricata, no hopanoids were detected. Porifera had the lowest proportion of cholesterol (22.8% ± 1.8%), but significantly higher proportions of phytoplankton sterols, such as 24-methylencholesterol (12.2% ± 0.4%) and episterol (11.4% ± 1.6%), the phytoplankton and red algae sterol brassicasterol (12.1% ± 0.5%), the terrestrial plant sterol β-sitosterol (8.9% ± 0.8%), and a zooplankton sterol 24-nordehydrocholesterol (6.1% ± 0.8%). Furthermore, from the deep, cold-water site, Porifera was the only phylum showing sterols previously reported in diatoms and brown algae such as poriferasterol (2.0% ± 0.6%) and fucosterol (3.5% ± 0.7%), and specifically in brown algae such as 4-24 dimethyl 5,7-dien-3β-ol (2.0% ± 0.6%), compared to the other phyla studied. The cnidarian Pseudoanthomastus agaricus had more cholesterol than sponges with 68.8% ± 0.3%; in second place was occelasterol (11.6% ± 0.7%) probably from zooplankton, followed by brassicasterol (8.0% ± 2.6%) from phytoplankton and red algae. The mollusk Buccinum sp. and the arthropod P. tarda had similar sterol composition with cholesterol at 91.7% ± 1.0% and 95.3% ± 2.0%, respectively, with low contributions of occelasterol from zooplankton at 1.9% ± 0.9% and 2.2% ± 0.9%, respectively, and a detritus contribution detected with stigmasterol at 0.6% ± 0.2% and 1.5% ± 0.6%, respectively. The echinoid Phormosoma placenta had cholesterol at 73.9% ± 11.0%, similar to cnidarians, with sterols previously reported in zooplankton [Species systematics, sample size, depth, and water temperature of animals collected in the deep Northwest Atlantic off Newfoundland and in the coral reef system of Veracruz are shown in plankton  such as t = 8.68, Markov chain probability (p(MC)) = 0.001, Montastraea cavernosa had significantly higher proportions of campesterol and gorgosterol from zooxanthellae, while the deep-water cnidarian P. agaricus had a higher proportion of cholesterol, occelasterol, and brassicasterol, thus indicating zooplankton and phytoplankton sources  = 0.001, Aplysina sp., from the shallow, tropical site, showed high proportions of plant related sterols β-sitosterol and stigmasterol, as well as campesterol and cholesterol. On the other hand, the deep-water sponge T. muricata showed significantly higher proportions of phytoplankton sterols such as 24-methylenecholesterol and episterol  = 0.001, t = 2.09, p(MC) = 0.082, Aplysina sp. had 15.4 ± 5.4 mg·g−1 wet weight, around fivefold more lipids than T. muricata with 2.6 ± 1.2 mg·g−1, while the cnidarian M. cavernosa had 7.2 ± 3.6 mg·g−1, almost twice the content of P. agaricus (4.1 ± 2.2 mg·g−1). A similar pattern was detected in echinoids, with Echinometra lucunter having 12.6 ± 1.2 mg·g−1 of lipids and P. placenta having 6.0 ± 3.1 mg·g−1  have significantly decreased growth and survival rates in early stages [The mollusk y stages . There iP. placenta had a greater variety of sterols including phytoplankton sterols such as occelasterol and zooplankton-related sterols such as 24-nordehydrocholesterol [13C of −50‰ to −100‰ [Lastly, the echinoid lesterol , a lowerp = 0.001). Furthermore, between cnidarians there were 56 permutations allowing detection of significant differences (p = 0.001). In contrast, echinoids with 35 permutations did not show a significant difference (p = 0.082) because sterol profiles of echinoids from shallow and deep sites were quite similar.Small sample sizes were not a statistical issue due to small variation and clear differences among species and sites. Permutational multivariate analysis of variance (PERMANOVA) allows multivariate comparisons of compositional data such as sterol profiles with data with or without a normal distribution of residuals. Here, 996 permutations were performed in the main test, and, in those species with small sample sizes such as sponges, 10 permutations allowed detecting significant differences , poriferasterol , and fucosterol  E-dien-3β-ol) were present in sponges from both environments, suggesting they could be synthesized by sponges [Tropical, shallow benthic organisms with a lower complexity such as Porifera and Cnidaria reflected the available food supply in their sterol composition. The most obvious difference was detected in cnidarians because  Bahamas , whereasat 145 m . The deeon Atoll . Howeverlesterol . We did  sponges  or their sponges .13C of −10‰ ± 0.1‰ from macroalgae and their δ15N of 3.3‰ ± 0.3‰ [13C of −14.3‰ ± 0.9‰, indicating bentho-pelagic carbon sources, and higher δ15N ratios of 12.3‰ ± 0.3‰ [E. lucunter had a high contribution of red algae Galaxaura sp. [Galaxaura sp. [P. placenta suggests that echinoderms can actually synthesize cholesterol. Lathosterol has been recorded in other cold, deep-water echinoderms such as the holoturian Cucumaria japonica [Echinoids from the tropical, shallow site were herbivores, as their δ‰ ± 0.3‰  suggest ‰ ± 0.3‰ , indicataura sp. , coincidaura sp. . The secjaponica . This sujaponica  is also −3 [−3 [Lastly, as lipids are the storage reservoir of energy in animals , the rat−3  in compa−3 [−3  in simila ranged from 1.24 mg·m−3 in November to 2.23 mg·m−3 in April [The geographical location and depth profile of the northwest Atlantic and southwest Gulf of Mexico sites are shown for comparison in in April , and thein April ,45,46. D−3 total plankton wet biomass [−3 [In contrast, the tropical shallow samples were collected in the Veracruz reef system national park , which is located off the State of Veracruz, Mexico, adjacent to the cities of Boca del Rio and Anton Lizardo (19°02′24″ to 19°15′27″ north (N), 96°12′01″ to 95°46′46″ west (W)). It is part of a larger coral reef system in the Caribbean and Gulf of Mexico. A group of 13 reefs is located near Veracruz and Boca del Rio, and another group of 15 reefs with larger structures is located near Antón Lizardo; the two reefs are separated by the Río Jamapa and delimited to the north by Río La Antigua and to the south by the Río Papaloapan. During the summer, under sustained southward winds, a cyclonic eddy develops off Veracruz Port, which enhances productivity in the area reaching 2.3 ± 1.1 g·m biomass  and a chmass [−3 .In the cold, deep site, animals belonging to five phyla were opportunistically collected within 7 days in November–December 2013, during one of the annual multispecies bottom-trawl surveys conducted by Fisheries and Oceans (DFO), Canada. Individuals were sampled onboard the CCGS Teleost, from a total of 23 tows inside a 100 km radius and a depth range of 313 to 1407 m. The gear used to collect the samples (Campelen 1800 shrimp trawl) included a 16.9 m wide net with four panels of polyethylene twine. Once on board, individuals were immediately vacuum-packed and frozen at −20 °C to minimize lipid oxidation and hydrolysis. Individuals were identified to the lowest possible taxonomic level, from direct observation and through photo-identification. A total of 34 deep-sea organisms, belonging to five species from different phyla, were weighed, and samples of 0.7 ± 0.2 g wet weight were collected from each individual and processed for lipid analysis at the Core Research Equipment and Instrument Training Aquatic Research Cluster (CREAIT-ARC) Facility at Memorial University . The folSponges, cnidarians, and echinoids sterols from the deep, cold-water site were compared to sterols of sponges and cnidarians  and echiSamples were weighed wet, and around 0.7 ± 0.2 g was immersed in 4 mL of chloroform, sealed under nitrogen gas, and stored in a freezer (−20 °C). Lipids were extracted by adding 2 mL of methanol and 1.8 mL of distilled water, mixed with a vortex, and centrifuged to separate polar and nonpolar phases. The lower phase was recovered in a lipid-cleaned tube; an aliquot was separated into lipid classes by Chromarod thin-layer chromatography (TLC) . Chromar+) which provides information on number of carbons and double bonds [+, (M-R-2H)+, (M-H2O)+, (MR-H2O)+, (M-R-2H-H2O)+, and (M-CH3-H2O)+. Other fragments indicated the position of double bonds. For instance, the lathosterol–TMS ether has a MW = 458, suggesting that the molecule has 27 carbons and one double bond. We should expect a signal at m/z = 213 indicating a double bond at C7. For the stigmasterol–TMS ether, double bonds at C5 and C22 are shown by ions with m/z = 129 and m/z = 255 [Neutral lipid, galactoglycerolipid, and phosphoglycerolipid fractions were transmethyl esterified with Hilditch reagent, and then silylated with bis trimethylsilyl trifluoroacetamide (BSTFA) (Supelco: 3-2024) . Sterolsle bonds ,50. SomeSpecies differences of individual (univariate) sterols were tested with one-way ANOVA using the Fisher statistic (F). Residual analyses were used to test assumptions of normality and equal variance. The effect of tropical shallow vs. cold deep waters on sponge, cnidarian, and echinoderm sterols was tested with multivariate analyses of sterol profile (>2% in a least one sample) using PRIMER 6.1.16 software and PERMANOVA + 1.0.6 . Nonmetric multidimensional scaling was conducted on the basis of the Bray–Curtis similarity coefficient.p(MC) values because the sample size was n < 30.No transformation was used to avoid artificial weighting of sterols that gave only trace contributions to their respective profiles. Similarities among species and depth were investigated using the similarity percentages function (SIMPER), and statistical differences were tested with two-way analysis, for depth and phyla, using permutational multivariate analysis of variance (PERMANOVA), which allows multivariate comparisons with data with or without a normal distribution of residuals. The method of permutation of residuals under a reduced model was used, and significant differences were tested with Markov chain probability A. dufresnii, with differences in their total concentration from intact to spawned states explained by gamete concentrations [In the deep cold sites, gastropods and arthropods had cholesterol as their main sterol. Other extraction systems such as acetic acid and methanol could facilitate finding bioactive sterol metabolites for use against diabetes and oxidative stress-induced inflammatory diseases . Spongestrations . As gonatrations , sex and


Lactococcus lactis strains were identified from different naturally fermented Brazilian sausages. Ion exchange and reversed-phase chromatographies were used to purify the bacteriocins from culture supernatant of the five strains. Mass spectrometry (MALDI-TOF/TOF) showed that the molecular masses of the bactericoins from L. lactis ID1.5, ID3.1, ID8.5, PD4.7, and PR3.1 were 3330.567 Da, 3330.514 Da, 3329.985 Da, 3329.561 Da, and 3329.591 Da, respectively. PCR product sequence analysis confirmed that the structural genes of bacteriocins produced by the five isolates are identical to the lantibiotic nisin Z. Optimal nisin Z production was achieved in tryptone and casein peptone, at pH 6.0 or 6.5. The most favorable temperatures for nisin Z production were 25°C and 30°C, and its production was better under aerobic than anaerobic condition. The type of carbon source appeared to be an important factor for nisin Z production. While sucrose was found to be the most efficient carbon source for nisin Z production by four L. lactis isolates, fructose was the best for one isolate. Lactose was also a good energy source for nisin Z production. Surprisingly, glucose was clearly the poorest carbon source for nisin Z production. The five isolates produced different amounts of the bacteriocin, L. lactis ID1.5 and ID8.5 isolates being the best nisin Z producers. DNA sequence analysis did not reveal any sequence differences in the nisZ and nisF promoter regions that could explain the differences in nisin Z production, suggesting that there should be other factors responsible for differential nisin Z production by the isolates.In this study, five bacteriocin-producing  Lactococcus lactis strains and include nisins A, F, H, J, Q, U, and Z [Lactic acid bacteria (LAB) constitute a diverse group of bacteria that produce lactic acid as a major end-product of hexose fermentation. They are widely used as starter cultures in the production of many fermented foods . ApproprU, and Z –13.Lactococcus lactis is applied in fermented foods (mostly dairy products), and it is generally recognized as safe. Nisin A was the first bacteriocin approved and commercially employed as food preservative [nisABTCIPRKFEG) organized into three operons [nisRK is constitutive, whereas the nisABTCIP and nisFEG promoters are controlled by the two-component regulatory system NisRK [Nisin-producing ervative . The gen operons . Transcrem NisRK . The Nisem NisRK .In view of the widespread use of this bacteriocin, an important factor to consider for its application is the cost of production. It is well known that bacteriocin production in fermentation systems is influenced by many factors, such as type of carbohydrate, nitrogen source, pH, temperature, and other nutritional and physiochemical properties , 17–19. L. lactis strains have been isolated from cheese, raw milk, grain, fish, and fermented vegetable [Pediococcus and Lactobacillus species [Most nisin-producing egetable , 20–22.  species –26.In our previous studies, bacteriocinogenic LAB were isolated during the natural fermentation of sausage  processed in two industries located in the cities of Imbituva and Prudentópolis  . The purLactococcus lactis strains were isolated from naturally fermented sausage  (21). The susceptible strain Micrococcus luteus ATCC 10240 was used as an indicator strain for bacteriocin activity. It was grown at 30°C in basal media containing (per liter): animal peptone (10 g), yeast extract (4 g), meat extract (8 g), NaCl (5 g), Na2HPO4 (2.5 g), and glucose (10 g).All LAB used in this study were grown at 30°C in LAPT broth . Bacteri5′CCGAATTCGACAACAGAGTTTGATCCTGGCTCAG3ʹ and md-5ʹCCCGGGATCCAAGCTTAAGGAGGTGA-TCCAGCC3ʹ [The nisin-producing isolates were identified to species level by partial 16S rRNA gene sequence analysis. Genomic DNA was isolated with Wizard Genomic DNA purification Kit . The PCR was performed by using combinations of primers, fd-CCAGCC3ʹ , under tnisA structural gene and nisA, and nisF promoters . The PCR thermal cycle program included an initial denaturation at 94°C for 2 min, followed by 35 cycles of denaturation at 94°C for 1 min, annealing at 40°C, at 48°C, and at 68°C for 30 s for the primers sets nqf5ʹCGTTCGAAGGAACTACAAAATAAATT3ʹ/naqzr5ʹACAGACCAGCATTATATTTCTGC3ʹ, pnisAf5ʹTTGAGTCTTAGACATACTTGAAC3ʹ/pnisAr5ʹCAATGACAAGTTGCTGTTTTCA3ʹ, and pnisFf5ʹTCCTCAAAAAGGTGGGGCAGAAGT3ʹ/pnisFr5ʹGCCTCGATTAAGGCTCCAAGT3ʹ, respectively [Nucleotide sequencing was performed on the PCR products obtained from the amplifications of genomic DNA with primers specific to L. lactis strains are located on plasmid. L. lactis strains were grown in LAPT broth and 10 μg mL−1 of ethidium bromide at 30°C for 24 h. After incubation, the same procedure was repeated several times. Cultures, which survived were diluted, plated on LAPT agar, and incubated at 30°C for 24 h. Colonies were further screened for bacteriocin production. The plasmids of the wild type and variants were checked by EZ N. A. ™Plasmid Spin Protocol .Plasmid curing was done in order to determine whether or not the genetic determinants for bacteriocin production in the five −1) was defined as the reciprocal of the highest dilution that showed a zone of inhibition of at least 5 mm in diameter.Quantitative determination of the antimicrobial activity of the bacteriocins was performed by using agar well diffusion assay . PreparaL. lactis strains using ion exchange and revered-phase chromatography. The bacteriocins were precipitated by using ammonium sulfate (40%), dissolved in water, and the pH was adjusted to 3.5. The preparation was then passed through SP Sepharose Fast Flow  equilibrated with 10 mmol·L−1 acetic acid. The column was eluted with a stepwise gradient consisting of 10 mL of each 0.1, 0.3, and 1.0 mol·L−1 NaCl at 1 mL·min−1 flow rate. The fractions that showed the highest bacteriocin activity were fractionated on a reversed-phase column  equilibrated with 0.1% (v/v) trifluoroacetic acid (TFA) in water, using Äkta Purifier Fast Protein Liquid Chromatography System. Elution was performed by using a linear gradient from 0 to 100% isopropanol containing 0.1% (v/v) TFA. The fractions with activities were mixed 1 : 1 with a solution of 15 mg α-cyano-4-hydroxycinnamic acid in 50% acetonitrile, 49.9% ethanol, and 0.1% TFA, and deposited on a ground steel Matrix-Assisted Laser Desorption Ionization target. Mass spectra were recovered in a positive reflector mode with an Ultra Flex TOF/TOF  by using a pulsed ion extraction duration of 40 ns and an acceleration voltage of 25 kV.Bacteriocins were purified from the culture supernatant of the five −1. Cultures standardized (OD600nm = 0.6) were grown in LAPT broth with glucose, lactose, sucrose, or fructose and incubated at 30°C without agitation for 24 h. Samples were examined every hour for bacterial growth (OD600nm), changes in culture pH, and bacteriocin production.The carbon sources tested were glucose, lactose, sucrose, and fructose at the concentration of 10 g·L−1). Influence of the pH of the culture medium on bacteriocin production was determined by adjusting the initial pH of the LAPT broth to pH 4.5, 5.0, 5.5, 6.0, 6.5, and 7.0 with sterile 1 N HCl. The effect of temperature on bacteriocin production was determined by growing bacteriocinogenic cultures in LAPT medium at temperatures of 20, 25, 30, 35, and 40°C for 24 h. Bacteriocinogenic cultures were also grown in LAPT medium under anaerobic  and aerobic conditions (with agitation). All tested broths were inoculated with standardized (OD600nm = 0.6) bacteriocinogenic cultures for 24 h. After the incubation, the final pH, bacteriocin production, and bacterial growth (OD at 600 nm) were determined for all conditions described above.To study the effect of different nitrogen sources on bacteriocin production, the LAPT medium was supplemented with each different nitrogen source  (data not shown), indicating that the bacteriocin-producing isolates are L. lactis.In this study, we report the identification and production of bacteriocins produced by LAB isolated from sausage . BLAST analysis of the partial sequence of 16S rRNA gene (approximately 1500 nucleotides) of each isolate showed 99% nucleotide identity to the 16S rDNA sequence of L. lactis strains, three steps of purification were carried out from the cell free supernatant. The highest active fraction of the bacteriocin was eluted with 30% to 40% isopropanol for all strains (data not shown). The molecular masses of the purified fractions from L. lactis ID1.5, ID3.1, ID8.5, PD4.7, and PR3.1 were 3330.567 Da, 3330.514 Da, 3329.985 Da, 3329.561 Da, and 3329.591 Da, respectively  (The PCR products obtained from the amplifications of genomic DNA of the five B375441) , which hL. lactis strains confirmed that all the promoters of the structural nisin genes were 100% identical to the published sequences of the nisZ and nisA promoters (GenBank: Y13384.1 and Z18947.1)  . The pro of nisZ , which c8-TCT motif present at position −107 to −94, which was also confirmed in this work   . The seq Figures . The nis Figures .L. lactis (data not shown). It was observed that for all strains cured derivatives were able to produce bacteriocin, suggesting that the genes encoding the bacteriocin are located on the chromosome. Various researchers have found that nisin genes are present on a number of plasmids or also on the chromosome [Plasmids were found in the five strains of romosome , 37, 38.romosome , 38.L. lactis isolates are presented in Figures L. lactis ID 3.1 was supplemented with fructose  did not influence the growth of the five nisin-producing strains, but resulted in reduced nisin Z production. Temperature can affect the stability of the peptide by interfering in posttranslational modification, adsorption to cells, and proteolysis of the bacteriocin . In addinisF and nisZ promoter sequences because these promoters are controlled in the same manner; however, the sequences are similar in all strains, demonstrating that there should be other factors responsible for differential production of nisin Z.As demonstrated in this work, the structural gene of nisin Z was identified in the five strains, which produced nisin Z at variable mounts. We analyzed L. lactis strains are identical to those of nisin Z, indicating that the bacteriocin produced by each of these strains is nisin Z. Maximum nisin Z production in batch culture was achieved by two isolates L. lactis ID1.5 and ID8.5 (not ID3.1), at initial pH of 6.0 and 6.5, at incubation temperatures of 25°C and 30°C, under aerobic condition, and when sucrose was used as a sole carbon source. Supplementation of LAPT broth with tryptone and casein peptone increased the production of nisin Z by the five L. lactis strains. These parameters are important for the optimization of nisin Z production, which is essential for the use of these strains or their bacteriocins as biopreservation agents.In conclusion, the structural genes and molecular masses of the bacteriocins produced by five L. lactis isolated from fermented sausage in Brazil. Our study is also the first study that showed the production of identical bacteriocins at different levels and under diverse conditions, by different wild strains of L. lactis isolated from same environment. We suggest that identification of autochthonous strains producing higher amounts of the antimicrobials would lead to their application as starters in preservation of foods and may further help to reinforce originality of traditional foods.To our knowledge, this is the first report of nisin Z production by


Helicobacter pylori (HP) infection was more prevalent in infertile populations. HP infection could lead to decreased sperm parameters, and treating the HP infection could improve the quality of sperm. However, studies investigating the relationship between infertility and HP infection are still limited, and more evidence is required. Therefore, we performed the present study to investigate the impact of HP infection on sperm quality in males and on ovarian reserve in females.It has been reported that 13/14C-urea breath HP test were included in this retrospective cross-sectional study. Among them, 565 had performed sperm analysis or ovarian reserve tests in the past three months and were involved for further analyses. Sperm parameters were examined with a computer-assisted sperm analysis system, and serum anti-Müllerian hormone (AMH) and sex hormones were tested with an electrochemiluminescence method.A total of 16,522 patients who visited the Second Hospital of Zhejiang University from January 2016 to June 2019 due to abdominal discomfort and underwent a P > 0.05). Further subgroup analyses stratified into 5-year age groups confirmed that there was no significant difference in sperm parameters (P > 0.05). When pooled with previously published data, no significant difference in sperm concentration or motility was found (P > 0.05). Meanwhile, this study found that the serum AMH level was similar between the HP+ and HP− groups (P > 0.05). Further subgroup analyses confirmed that there was no significant difference in serum AMH level (P > 0.05).Among 363 patients who underwent the sperm test, 136 (37.47%) had HP infection. Among 202 patients who underwent the AMH test, 55 (27.23%) had HP infection. There was no difference in sperm concentration and motility between the HP+ and HP− groups (There were no differences in sperm parameters and AMH levels based on history of HP infection among Chinese patients. Helicobacter pylori (HP) may be related to a series of extragastric diseases, including cardiovascular, neurologic, respiratory, hematologic, metabolic, dermatologic, obstetric, autoimmune, and kidney diseases [Currently, it is widely accepted that diseases , 2. Amondiseases . Ten yeadiseases . In a cydiseases . RecentlThe first study from Italy reported a lower sperm quality in HP-infected patients with idiopathic infertility than in HP-uninfected patients. In CagA-positive patients, both sperm motility and fertility index are reduced , 7. It hIn females, there is a possible association between HP infection and polycystic ovarian syndrome (PCOS). A study from Turkey reported that the proportion of HP seropositivity was almost doubled in the PCOS population . It is sOverall, there appears to be an association between HP infection and infertility, but available support is not sufficient and thus requires further validation. The purpose of the present study is as follows: (1) to investigate the correlation between HP infection and sperm quality in males and (2) to explore the association between HP infection and ovarian reserve in females.From January 2016 to June 2019, patients aged 20–50 years who came to the Second Hospital of Zhejiang University School of Medicine due to abdominal discomfort and underwent HP testing were included in this study. Among them, 565 had plans for pregnancy and had performed sperm analysis or ovarian reserve tests in the past three months, who were involved for further analyses  or 14C-UBT was used to examine HP infection. Two breath samples were collected before and after ingestion of a 13C-urea  or 14C-urea  reagent dissolved in water. For the 13C-urea breath test, a change over baseline value greater than 4.0 delta over baseline (DOB) was taken as a positive result ( HP+). For 14C-UBT, a result greater than 100 DPM was taken as a positive result ( HP+).The Sperm samples were collected with sterile containers by masturbation after 2–7 days of sexual abstinence. After liquefaction at 37 °C for 30 min, routine parameters including sperm concentration and motility were examined with a computer-assisted sperm analysis (CASA) system  according to World Health Organization guidelines . Sperm m2), 12.6% at a level of 0.37 nmol/L for testosterone (T), 2.7% at a level of 4.2 IU/L for luteinizing hormone (LH), 3.9% at a level of 6.9 IU/L for follicle-stimulating hormone (FSH), 4.8% at a level of 69.9 mIU/L for prolactin (PRL), and 12.7% at a level of 3.8 nmol/L for progesterone (P). The measuring ranges were 43.6–11,010 pmol/L for E2, 0.24–52.05 nmol/L for T, 0.07–200 IU/L for LH, 0.3–200 IU/L for FSH, 6.4–4240 mIU/L for PRL, and 0.67–190.8 nmol/L for P.Serum AMH was tested with the electrochemiluminescence method by an Elecsys ® AMH from Roche Diagnostics on a Roche Cobas e602 analyzer. The total imprecision for the assays was 1.2% at a level of 1.19 ng/mL with a measuring range of 0.01–23 ng/mL. Serum sex hormone levels were detected with the electrochemiluminescence method by kits from Siemens Healthcare Diagnostics Inc.. The total imprecision for the assays was 3.0% at a level of 10,585 pmol/L for estradiol (EHelicobacter pylori" OR "Campylobacter pylori"). Meanwhile, we hand-searched the references listed in the achieved papers to obtain additional studies.To search for studies investigating the correlation between HP and sperm parameters, two reviewers independently searched the studies published in English via three databases, including PubMed, Embase, and Cochrane CENTRAL, until June 30, 2019. Articles were identified through computerized searches using the keywords as follows:  AND .P value of < 0.05 was considered statistically significant.Analyses were performed by using the SPSS 19.0 statistics package . Continuous variables are expressed as the mean values ± standard deviation (SD). Student’s t test was used for comparisons between two groups. Pearson correlation analysis was performed to analyze the relationship between two variables. A P value ≥ 0.1 by the Q test), a fixed-effects model (FEM) was applied for calculation, and if there was serious heterogeneity, a random-effects model (REM) was applied [Data from our hospital and previously published results were pooled and calculated together by Review Manager Software (RevMan Version 5.3). When the mean and SD were not provided in the published article, we used formulas to estimate them –14. The  applied .As shown in Fig. P > 0.05). Similarly, in the analysis of AMH and HP, no significant difference was found in age, weight, height, or BMI between the HP+ and HP− groups (P > 0.05).As shown in Table 6 Sp/mL and 53.90 × 106 Sp/mL in the HP+ and HP− groups, respectively, with no significant difference (P > 0.05). Sperm PR was also similar between the HP+ and HP− groups (39.39% vs. 39.92%), with no significant difference (P > 0.05). There was no difference in either normal sperm morphology percentage or sperm head defects (P > 0.05).As shown in Table P > 0.05). As shown in Fig. P > 0.05).To further exclude the impact of age, we divided the population into subgroups of 20–24, 25–29, 30–34, 35–39, 40–44, and 45–50 years of age. As shown in Fig. P > 0.05). No difference was found between HP+ and HP− groups in serum E2, T, LH, FSH, PRL, or P levels (P > 0.05).As shown in Table P > 0.05). Meanwhile, AMH correlated significantly negatively with age .As shown in Fig. Since 2010, six studies investigated the correlation between HP infection and sperm parameters, as listed in Table P > 0.05 for both). In the sperm motility analysis between HP+ and HP−, since serious heterogeneity (P < 0.01) was found, a REM was applied and suggested no significant difference in PR . FEM analysis was applied to compare sperm PR between CagA+ and CagA− groups, which suggested that PR was 16.18% lower in the CagA+ group than in the CagA− group .Five studies including 703 participants were pooled to compare HP+ and HP− groups, and five studies including 210 participants were pooled to compare CagA+ and CagA− groups. As shown in Fig. In the present study, no difference was found in sperm concentration or sperm motility between HP+ and HP− groups. Further subgroup analyses confirmed that there was no significant difference in sperm parameters between HP+ and HP− groups. Furthermore, we pooled our data and those of previous studies and found no significant difference in sperm concentration or motility, indicating that in the Chinese population, HP infection does not disturb spermatogenesis.The results of previous studies were not consistent. Some suggested that sperm concentration and motility were reduced in HP+ patients  and that13C- and 14C-UBT were used to detect HP infection. UBT is the best noninvasive method for patients without gastric resection or proton pump inhibitor (PPI) treatment, with both high positive predictive value and negative predictive value [13C-UBT, 0.92 for 14C-UBT, and 0.84 for serology tests. UBT showed a higher diagnostic accuracy than the serology test for HP infection diagnosis [This is the first study that used UBT to detect HP infection and to investigate its relationship with sperm quality. In previous studies, HP infection was detected with a serology test by enzyme-linked immunosorbent assay (ELISA) and confirmed with western blotting (WB) , 16–18, ve value –21. A meve value , 23. Anoiagnosis , 25. TheMoreover, serology tests cannot distinguish between active and inactive infections . In a leCagA is the major virulence factor in HP, encoding the CagA protein in the cag pathogenicity island . HP infeThe prevalence of the CagA genotype in HP infection varies significantly among different regions. In Western countries, CagA+ strains comprise 50–60% of the HP+ population, and in the Chinese population, CagA+ strains occupy nearly 100% of the HP+ population , 33. StuP = 0.001) [The variability of sperm parameters after HP treatment is an interesting question. It was reported that after the treatment of HP, seminal HP IgA level decreased significantly, and meanwhile progressive sperm motility, nonprogressive sperm motility, and sperm normal forms increased significantly (= 0.001) . In the In the present study, there was no difference in serum AMH level between HP+ and HP− groups, which was confirmed with further age-divided subgroup analyses. Published results of the relationship between PCOS and HP infection are inconsistent. Yavasoglu et al. found that HP antibody positivity was significantly more common in the PCOS group than in the age-matched control group . The posThis is the first observation investigating the impact of HP infection on ovarian reserve, which found that HP infection was not related to the serum ovarian reserve biomarker AMH. In general, HP infection is not a crucial factor affecting sperm parameters or ovarian reserve.


The formulation was optimized by the addition of low-viscosity hypromellose and mannitol. The final formulation was approved for the bioequivalence trial. Calculated Cmax were 1.92 ± 0.77 and 1.92 ± 0.63 [μg/mL], AUC0-t were 27.46 ± 8.39 and 29.96 ± 9.09 [μg∙h/mL], and AUC0-∞ were 28.22 ± 8.91 and 30.82 ± 9.41 [μg∙h/mL] for the originator and test formulations, respectively. The 90% confidence intervals of all primary pharmacokinetic parameters fell within the 80–125% range. In summary, biorelevant dissolution tests supported successful development of a generic trazodone ER formulation pharmaceutically equivalent with the originator under fed conditions. Employment of biorelevant dissolution tests may decrease the risk of failure in bioequivalence trials of ER formulations.Development of generic extended-release (ER) formulations is challenging. Especially under fed conditions, the risk of failure in bioequivalence trials is high because of long gastric residence times and susceptibility to food effects. We describe the development of a generic trazodone ER formulation that was aided with a biorelevant dissolution evaluation. Trazodone hydrochloride 300-mg monolithic matrix tablets were dissolved both in USP and EMA compliant conditions and in the StressTest device that simulated both physicochemical and mechanical conditions of the gastrointestinal passage. The final formulation was tested against the originator, Trittico XR 300 mg, in a randomized cross-over bioequivalence trial with 44 healthy volunteers, in agreement with EMA guidelines. Initially developed formulations dissolved trazodone similarly to the originator under standard conditions (fThe online version of this article (10.1208/s12249-020-01662-8) contains supplementary material, which is available to authorized users. Additioid (SIF)  or a Fedid (SIF)  can effeid (SIF) .in vivo can cause irregular drug release and increase pharmacokinetic variability  Class II drug is sparingly soluble in water, and its experimentally evaluated logP is 2.9 . It is aout 6.74 . TrazodoThe novelty of the study is an intensive use of bio-predictive, physiology-mimicking dissolution tests during the formulation development process, leading to a successful bioequivalence trial under fed conditions. Until now, the usefulness of the StressTest device was used for the development of novel, pressure-sensitive dosage forms  or for aGeneric trazodone hydrochloride ER tablets (Trazodon XR 300 mg), containing 300 mg of the active ingredient , were manufactured by a direct compression method. The dissolution profiles of the active substance were tested along with the brand name product—Trittico XR 300 mg (Aziende Chimiche Riunite Angelini Francesco A.C.R.A.F. S.p.A.). Six batches, labeled A to F, were tested in biorelevant conditions. The qualitative composition of each analyzed batch is available in Table Biorelevant.com Ltd., London, UK) in a concentration corresponding to a fed state. All of the reagents were of analytical grade.In the development process, both standard and biorelevant media were used. Standard media included 0.1 M hydrochloric acid with 2.92 g/L sodium chloride (pH = 1.2) and 50 mM phosphate buffer pH = 6.0. Biorelevant media were 50 mM phosphate buffer at pH ranging from 4.5 down to 1.8 (with the addition of HCl), and 50 mM phosphate buffer pH = 6.5 with FaSSIF /FeSSIF /FaSSGF (Fasted State Simulated Gastric Fluid) powder  with a constant paddle rotation at 150 rpm, according to “Guideline on quality of oral modified release products” issued by European Medicines Agency (EMA) . The tabuideline . The f2 The dissolution stress test device was first introduced by G. Garbacz and W. Wetschies . The corDissolution tests were designed to simulate the fed intake conditions of the bioequivalence trial. First, the medium was 1100 mL 50 mM phosphate buffer at pH 4.5. The pH of the medium was gradually decreased to 1.8 by addition of 5 M hydrochloric acid; then, after 5 h the pH was adjusted to pH 6.5 by the addition of 40% sodium hydroxide, and 40 mL of a FaSSIF/FeSSIF/FaSSGF concentrate solution in a 50 mM phosphate buffer concentrate was added . The final concentration of the FaSSIF/FeSSIF/FaSSGF in the simulated intestinal fluid was 11.2 g/L, corresponding to the fed state. The media change pattern was aligned with the simulation of the mechanical aspects of the GI tract. The stress program was set up. Figure λ = 313 nm for trazodone signal and λ = 450 nm for the background, respectively.The amount of the drug dissolved was determined every 10 min using online closed-loop UV-Vis spectroscopy. The samples were filtered through a PES (polysulfone) filter with a pore size of 1 μm  and analyzed in a flow-through mode. Flow-through quartz cuvettes of a 5-mm path length  were used with an Agilent 8543 spectrophotometric system  with the photometers set to a differential mode at two wavelengths of The bioequivalence study was a single-center, single-dose, open-label (laboratory blinded), randomized, four-period, four-way cross-over study, according to the “Guideline on the pharmacokinetic and clinical evaluation of modified release dosage forms” (EMA/CPMP/EWP/280/96 Corr1) . The proSustained and predictable release of the drug from the ER dosage is more challenging in fed conditions than in a fasted state, both in dissolution studies and in the proper setup of the clinical study protocol. Therefore, in this paper, we will present the results of the bioequivalence trial performed under fed conditions only.et al.  or the brand name product (Trittico XR 300 mg), both containing 300 mg trazodone hydrochloride, was administered by the oral route. The administration was within 30 min after the start of a standardized high-fat meal, with 250 mL of still, room temperature water. No other fluid intake was allowed from 2 h before until 1-h post-dosing. Additionally, fluids were administered as follows: 250 mL of fluid with breakfast, 150 mL of still water at 1, 2, and 3 h post-administration, 200 mL fluid with meals at 4 and 12 h post-administration, and 100 mL at 5, 6, 7, 8, 9, 10, and 11 h post-administration. Standardized lunch and supper were served at about 4 and 12 h after drug administration.From each subject 4 mL of full blood was collected before drug administration (time “0”), and at 1, 2, 3, 4, 5, 6, 8, 9, 10, 11, 12, 13, 14, 15, 16, 20, 24, 28, 32, 36, 42, 48, and 72 h after the intake of the drug. The blood samples were taken before the intake of fluids. Full blood was collected through heparinized capillaries into a vacuum blood collection system. Immediately after collection, the samples were centrifuged, and the plasma was transferred into separate tubes, stored in dry ice, and shipped into the laboratory for trazodone determination. Any deviations from the sampling protocol were noted, and the actual sampling times were reported for further analysis.Plasma samples were shipped in dry ice to an external, certified laboratory for the determination of trazodone. The drug concentrations were determined with a high-performance liquid chromatography mass spectrometry method (LC-MS), in a multiple reaction monitoring mode (MRM). The method was validated according to the EMA guidelines in terms of selectivity, accuracy, and precision . The low0-t), area under time-concentration curve extrapolated to infinity (AUC0-∞), and maximum concentration obtained directly from the measured concentrations (Cmax). Additional secondary parameters included time to Cmax (Tmax) and plasma half-life (T1/2) that was calculated from 0.693/Kel, where Kel represents the elimination rate constant determined through a linear regression. Of note, only the actual sampling times were used. The parameters were calculated by the linear-log trapezoidal rule in Phoenix WinNonlin software, build 8.1.0.3530 .The pharmacokinetic evaluation was planned in agreement with the guideline CPMP/EWP/QWP/1401/98 Rev. 1/Corr**  and EMA/α = 0.05) level of significance.Bioequivalence after a single dose of trazodone 300 mg under fed conditions was established upon a ratio of test and reference product parameters. Before the analysis, the parameters were log-transformed. The 90% confidence interval for the ratio should be contained within 80.00–125.00%. The pharmacokinetic parameters were analyzed with ANOVA test, with factors for sequence, subject within sequence, period, and treatment. Also, the Schuirmann’s two one-sided parametric T tests were calculated with the null hypothesis of bioinequivalence at the 5% . Also, the type of formulation (test product or the brand name product) influenced the exposure to trazodone, expressed as AUC0-t, and AUC0-∞ . For Cmax this effect was not statistically important (p = 0.5955).ANOVA test showed that the sequence and period effects were negligible for all primary parameters. One of the statistically significant factors was a joint subject and sequence effect. These parameters affected all three primary parameters  as the originator. It becomes challenging if the exposure to the drug differs significantly between fasted and fed states. This phenomenon is observed for trazodone. Karhu et al.  showed tConsequently, the fed conditions can be considered as more difficult with respect to the development of generics. Therefore, the present paper aims at the description of the product performance under fed conditions. The strict criteria for primary pharmacokinetic parameters obtained in the bioequivalence trial result in setting up requirements for the pharmaceutical development and characterization of oral medicines in the preclinical stage. Such characterization, especially when performed with biorelevant methodology, may reduce the risk of failure and accelerate the development process.et al. , as well as hydration and erosion processes of MR matrices . These cet al. ,28.in vivo studies performed using telemetric capsules  consisted mainly of high-viscosity hydroxypropyl methylcellulose . In the dissolution study performed according to USP, they released the drug steadily, reaching almost 95% of the dose labeled within 24 h. Subsequent batches, D and E, were characterized by a noticeably faster drug release than batches A–C. In these two batches, the amount of Hypromellose 100000 was decreased to less than 10% of the total tablet mass, and a low viscosity Hypromellose 4000 was introduced; also, batch E included a small amount of mannitol. Final batch F, which differed from batch E only by a presence of polyvinyl coating, had a comparable dissolution profile. Increased release of the drug was also achieved by increasing the amount of microcrystalline cellulose. Properties of hypromellose may explain observed dissolution performance. Of note, all of the batches tested in the USP apparatus had different dissolution kinetics as compared with the originator, Trittico XR 300 mg. As revealed later in the test under biorelevant conditions, the batches with the dissolution profiles that were most similar to the originator performed worse, especially after the transition to media simulating intestinal fluids.et al.  was much lower than in pure water . Theref8 mg/mL, . Contram8 mg/mL, , but at 8 mg/mL, . Also, t  was shown to be significant for AUC0-t and AUC0-∞ in the latin-square ANOVA. However, all of the primary pharmacokinetic parameters fell within the assumed confidence intervals. Therefore, the formulation effect may be considered as negligible, and the two formulations may be concluded as bioequivalent under fed conditions. Besides the development of a formulation with an optimal release profile, the design of the study also contributed to the success in the clinical trial. According to the protocol, not only meals but also fluid intake were tightly scheduled. In the proposed regimen all subjects received a specific liquid volume every hour after administration. A recently established consortium in Understanding Gastrointestinal Absorption-related Processes (UNGAP) investigated the food-drug interactions that may influence the absorption of orally administered drugs