Children with attention deficit hyperactivity disorder (ADHD) have more visits to the emergency department (ED) due to injuries than those without ADHD. However, no study has investigated whether children with ADHD have more ED visits or hospitalizations due to infectious diseases (IDs) and whether methylphenidate (MPH) treatment may reduce the risk.HRs) in the population level and relative risks for the self-controlled case series design, respectively.The incidence of ID-related ED visits or hospitalizations was defined as the main outcome. The Cox regression and conditional Poisson regression models were calculated to estimate hazard ratios  and hospitalizations  due to IDs than those without ADHD. In the ADHD subgroup, those who received MPH treatment have a reduced risk of emergency visits  and hospitalizations , compared to those without treatment. The risk of ID-related emergency visits decreased to 0.21 (95% CI: 0.21~0.22); and hospitalizations decreased to 0.71 (95% CI: 0.69~0.73). Within self-controlled analysis, it is demonstrated that compared with non-MPH exposed period, children with ADHD had significantly decreased risks for infection-related emergency visits  or hospitalizations  during MPH-exposed periods.Children with ADHD had higher rates of emergency visits (This is the first study that reported an increased risk of ID-related healthcare utilizations in children with ADHD compared to those without, and that such risks may be significantly reduced in ADHD children that received MPH treatment. Attention deficit and hyperactivity disorder (ADHD) is one of the most common neurodevelopmental disorders in childhood . ADHD isEmerging evidence has supported the treatment effects of methylphenidate (MPH) for ADHD symptoms or related adverse behavioral outcomes –9, as weReductions in the prevalence of infectious diseases (IDs) in children has contributed to the decrease in childhood mortality in the 20th century , 22. YetUsing a large population-based dataset, we investigated whether higher rates of IDs-related emergency visits or hospitalizations were found in children with ADHD compared to those without. Between-subject comparisons of ADHD children with or without MPH treatment, and within-comparison analysis comparing subsequent risks of IDs-related emergency visits and hospitalizations between MPH-exposed or unexposed periods were performed to elucidate the treatment effects of MPH.In this retrospective cohort study, we used data from the Taiwan National Health Insurance Research Database (NHIRD) under the aegis of the National Health Research Institute, which includes data on outpatient, ambulatory, and hospital inpatient care. Taiwan launched a single-payer National Health Insurance (NHI) program on March 1, 1995. The NHI covers the delivery of all healthcare services to over 99.5% of the national population . To avoiThe ADHD cohort was selected from NHIRD. We selected individuals born between 1997 and 2005 because we applied data from NHIRD from 1997 to 2013 and the age counterparts without ADHD were selected from the Longitudinal Health Insurance Database 2005 (LHID). LHID 2005 contains data for 1,000,000 enrollees randomly sampled from the 2005, thus enrollees in the LHID 2005 would not be born after 2005. The distribution of sex and age of the sampled enrollees in the LHID 2005 did not differ significantly from that of the general population . We idenThe main outcome of this study was IDs-related visits of the ED or hospitalizations. Participants were followed-up for the incidence of IDs-related ED visits or hospitalizations as an outcome, or until the end of 2013. IDs-related diagnoses pediatricians in Taiwan often use indicating the clinical infectious conditions that required visits to the ED or hospitalizations included: meningitis (ICD-9-CM codes: 320.xx~323.xx), conjunctivitis (372.xx), acute otitis media (382.xx), upper respiratory infections, pharyngitis, laryngitis, bronchitis (461.xx ~ 466.xx), pneumonia (480.xx ~ 490.xx), gastritis, duodenitis (535.xx), urinary tract infection (599.xx), fever (780.6), and abdominal pain (789.xx). In addition, we reported the top three most frequently primary diagnosis for IDs-related ED visits and hospitalizations.We ascertained the MPH treatment status by dispensed prescriptions recorded in the prescribed drug register. MPH  was the only stimulant approved for treating ADHD in Taiwan, and was regarded as the first-line treatment for ADHD by the Taiwan National Insurance. Indications approved in the reimbursement system for prescribing short-acting MPH HCL are ADHDs and narcolepsy; and could be prescribed by doctors of all specialty in Taiwan. Further criteria for prescribing MPH HCL Extended Release were limited to patients between 6 and 18 years old diagnosed with ADHD according to DSM or ICD criteria, and those who cannot tolerate the side effects or gained beneficial effects from short acting MPH HCL . For those who meet the requirements, have been treated with MPH, and still need to take the medication after the age of 18, their medical history and reasons for use must be recorded in detail in their medical records to be reimbursed. Since 2017, atomoxetine (ATX), a non-stimulant, received approval for ADHD treatment in Taiwan. Both medications were approved for patients with age of 6 or older. However, compared with MPH, there is a much lower rate of prescription of ATX  . ATX is Several covariates were selected, such as sex, age, and psychiatric comorbidity comprising autism spectrum disorder , oppositional defiant disorder  or conduct disorder , major depressive disorder , tic disorders (ICD-9 code: 307.2), epilepsy (ICD-9 code: 345), and intellectual disabilities (ICD-9 codes: 317–319) at any time during the study period.2) test was used to compare the characteristics between the ADHD and control groups. This study performed two different analyses: between subject comparison and within subject comparison. We used the one-to-one propensity score matching to draw a comparison cohort by exact matching for gender and greedy matching for age group and comorbidity. We matched subjects on the logit of the propensity score using a caliper of a width of 0.001. Standardized mean differences were used to evaluate the difference of matching variables between the ADHD and non-ADHD groups, and the MPH user and non-MPH user groups among ADHD. A standardized mean difference of 0.2 or greater indicates a notable difference between the two groups  and R 4.0.2 . To describe the distribution of the study population, a chi-square  model, in which time was divided into periods similar to the conditional Poisson regression model, with each patient as a separate stratum . Individual's record of MPH medication was used from the entire study period (by the end of 2013). The results are presented as relative incidences (RRs) with 95% CIs. The RR estimated from the SCCS model indicates the risk of emergency visits or hospitalizations during the period an individual took MPH compared with the period when they did not take MPH. The SCCS model automatically adjusts for all time-invariant factors  for the same patient before and during the follow-up, and we further adjusted for time-varying variables .For between-subject comparison, the adjusted risk of emergency visits or hospitalizations between those with ADHD and without was estimated using a robust Cox proportional hazard model to take the propensity score matching strata into account. The results are presented as adjusted hazard ratios (aHRs) with 95% The effect of exposure to MPH was defined as 3 months after the completion of each MPH treatment, and the length of effect period for MPH was based on the suggestion of previous studies . We furtHR = 1.25, 95% CI: 1.23~1.27), IDs-related emergency visits , all hospitalizations (aHR = 1.28 (1.26~1.31), and IDs-related hospitalizations  after controlling for demographics and psychiatric comorbidities using propensity score matching. Results from our subanalyses differentiating subtypes of inattentive or hyperactivity revealed similar patterns as our main analyses  for IDs-related emergency visits; and 0.71 (95% CI: 0.69~0.73) for IDs-related hospitalizations. Those taking MPH for ≥90 days had an aHR of 0.12 (95% CI 0.09~0.16) for IDs-related emergency visits and 0.72 (95% CI: 0.63~0.83) for IDs-related hospitalizations. The results for the trend test were significant (p < 0.01). In addition, all subgroup analyses of different demographics or comorbidities revealed similar patterns , 30 to 60 , and 60 to 90 days . An average pooled estimate of these effect periods indicated an RR of 0.73(95% CI: 0.68~0.78). Significant reductions in the incidence of IDs-related hospitalizations were observed with an average pooled estimate of these effect periods at an RR of 0.19 (95% CI: 0.17~0.21).We have performed additional analyses comparing results separating the short- and long-acting MPH subgroups in This is the first population-based study to report that children with ADHD had increased risks of IDs, and that there is an overall consistent pattern of MPH use and decreased likelihood of IDs among children with ADHD. In addition, using the self-controlled case series analysis, we found that risks of IDs-related emergency visits and hospitalizations decreased 36~81% in the MPH exposed periods compared with the non-exposed periods after adjusting for time-invariant covariates.Our finding that children with ADHD had higher risks of subsequent IDs than children without ADHD was similar to a recent cohort study reporting significantly increased rates of sexually transmitted infections (STI) in children with ADHD . In linePrevious research examining the effect of MPH on the risk of IDs-related healthcare utilizations among children with ADHD is lacking. Man et al. described the protective effects of MPH on trauma-related emergency admissions and not on non-trauma ones. In our study, significant reductions in emergency visits or hospitalizations due to upper or lower respiratory or gastrointestinal infections were found in the MPH treated subgroup or exposed periods. Our findings are in agreement with the previous research that reported reduced risks of subsequent STI under short-term or long-term use of ADHD medications in children with ADHD . In addiRisks of IDs-related hospitalizations from the within-subject analysis did not show a decreased pattern in gradient as the length of exposure to MPH increased. Reasons may be the low overall long-term treatment rates (only 16% of children with ADHD received long-term medication treatments) , or the Strengths of this study include, first, the use of a long-term population-based dataset with records of diagnoses and health utilizations that provide detailed information without recall bias. A sample size large enough for sufficient statistical power is an advantage. Second, the within-subject analysis with SCCS was able to help control for the unmeasured time-invariant confounders. Key limitations include, first, diagnoses were obtained from a database established for administrative rather than research purposes. Although this may reflect a naturalistic clinical setting, the underdiagnosis of ADHD or other psychiatric comorbidities might still occur. Second, as for estimating medication exposures, we only have information of prescriptions. There might still be a gap we were not able to identify between medication prescribed and doses that patients actually consumed. Third, although the SCCS design has helped controlled for unmeasured covariates that is constant overtime, there are still time-variant confounders, such as body weight, nutrition status, or non-pharmacological treatments, we were not able to control for. Another possible confounder was the proficiency of parenting skills, such as parents' own standards for cleanliness, protection of health conditions of children, or supervision and correction of hygiene of children, may also affect whether children may be infected. Although this aspect might be seen as a time-invariant confounder, and might be controlled by our SCCS model; with more sufficient information, such as explorations of parenting skills or parental ID conditions within the same period of the infection of child, it may be possible to assess how parenting skills may affect the risk of infection in the child. Last, our results were analyzed from an Asian population with a healthcare system of near-universal coverage, generalizations to other nations may be limited.Results of our study suggested that MPH treatment was able to significantly decrease IDs-related emergency visits or hospitalizations, either compared with non-MPH treated children with ADHD, or MPH unexposed periods within the same study subject. Future research on possible pathways of MPH and its protective effects on infections in daily environment of children with ADHD might be explored. Psychoeducation and provisions of early and appropriate MPH treatment are not only important for the traditional academic concerns but might also be helpful in preventing the infection-related premature mortality in this vulnerable population.Datasets being analyzed, results being generated and reported in this article can be obtained from the National Health Research Institute of the Ministry of Health and Welfare in Taiwan. Restrictions applied to these data, which were used under license for our study, and so are not publicly available for duplication. Data can be requested only from the Ministry of Health and Welfare.VC, S-IW, Y-LC, and K-LK designed the study and wrote the protocol. Y-LC undertook the statistical analysis. All authors contributed to the writing and have approved the final manuscript.This study was supported by grants from the Chiayi Chang Gung Memorial Hospital, Chiayi, Taiwan (Grant Number: CMRPG6E0271). S-IW is part-funded by the Department of Medical Research, Mackay Memorial Hospital .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. 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Internet gaming Disorder (IGD) constitutes a recently proposed clinical disorder . The present study examined if IGD is best conceptualized as categorical (present/absent), or dimensional (severity ranging from low to high), or both .Ratings of the nine DSM-5 IGD symptoms, as presented in the Internet Gaming Disorder Scale 9-Short Form , from 738 gamers, aged 17 to 72 years, were collected. Confirmatory factor analysis (CFA), latent class analysis (LCA), and factor mixture modelling analysis (FMMA) procedures were applied to determine the optimum IGD model.Although the findings showed most support for a FFMA model with two classes and one factor, there was also good statistical and substantive support for the one-factor CFA model, and the LCA model with three classes.It was concluded that while the optimum structure of IGD is most likely to be a hybrid model , a uni-dimensional model and/or a three-class categorical model are also plausible.The online version contains supplementary material available at 10.1186/s12888-021-03148-8. International Classification of Diseases  = p > .05).A convenience online community sample comprised 738 adult Australian online gamers, with ages varying from 17 to 72 years .The IGDS9-SF was used to assess DSM-5  IGD sympα = .85 and α = .77 respectively).The ASRS comprises the 18 DSM-5 ADHD symptoms: nine assessing ADHD-IA symptoms and nine assessing ADHD-HI symptoms. An example ADHD-IA item is: “How often do you make careless mistakes when you have to work on a boring or difficult project?” An example ADHD-HI symptom is: “How often do you fidget or squirm with your hands or feet when you have to sit down for a long time?” For all items, participants indicate how often they have experienced each symptom over the past 6 months on a scale with options of 0 (“never”), 1 (“rarely”), 2 (“sometimes”), 3 (“often”), and 4 (“very often”). Item scores are added per dimension resulting in a scoring range between 0 and 36, with lower scores indicating lower symptom levels. In the present study, the internal reliabilities for the ADHD-IA and ADHD-HI dimensions were good to very good .Depression, anxiety and stress were assessed using the 21-item DASS-21. The DASS-21 comprises three seven-question sub-scales for depression, anxiety, and stress . An exam2 deviation from zero), with an effect size (f2) of .15, an alpha (α) error probability of .05, and power of .95, indicated that a minimum sample size of 138 participants were required. The DASS-21 and the ASRS responses included in the data have been previously used by Gomez, Stavropoulos and Griffiths  regarding the distribution of the two genders across the classes. Table There were 226 (60.4%), 126 23.4%) and 23 (6.1%) males in the low, intermediate, and high endorsement classes, respectively; and there were 219 (60.2%), 110 (30.2%) and 35 (9.6%) females in the low, intermediate, and high endorsement classes, respectively. The results of the chi-square test indicated no significant difference).Similar to some existing data , the oneThe results and conclusions made in the present study should be evaluated in the light of certain limitations. First, the study utilized ratings of the IGD criteria collected utilizing the IGDS9-SF . TherefoAdditional file 1: Supplementary Table S1. Frequencies and descriptive statistics of background variables collected in the study for all participants together. Supplementary Table S2. Frequencies of Endorsements of the Different Response Categories for the Nine Items of the IGDS9-SF. Supplementary Table S3. Description of the Different Variants of FMMA Models.Additional file 2.


To conclude, BXs in plasma, including metabolites not previously analyzed, were quantified. BX metabolites were significantly higher after rye vs refined wheat consumption. Four BX-related metabolites were inversely associated with PSA, which merits further investigation.Prostate cancer (PC) is a common cancer among men, and preventive strategies are warranted. Benzoxazinoids (BXs) in rye have shown potential against PC in vitro but human studies are lacking. The aim was to establish a quantitative method for analysis of BXs and investigate their plasma levels after a whole grain/bran rye vs refined wheat intervention, as well as exploring their association with PSA, in men with PC. A quantitative method for analysis of 22 BXs, including novel metabolites identified by mass spectrometry and NMR, was established, and applied to plasma samples from a randomized crossover study where patients with indolent PC (n = 17) consumed 485 g whole grain rye/rye bran or fiber supplemented refined wheat daily for 6 wk. Most BXs were significantly higher in plasma after rye (0.3–19.4 nmol/L in plasma) vs. refined wheat (0.05–2.9 nmol/L) intake. HBOA-glc, 2-HHPAA, HBOA-glcA, 2-HPAA-glcA were inversely correlated to PSA in plasma ( In particular, the disease has high prevalence in the Nordic countries, partly due to increased incidence during -90ies, as a likely result of introduction of prostate specific antigen (PSA) screening1. The high prevalence calls for research efforts to develop preventive strategies adaptable to the Nordic region. The causes of prostate cancer are yet to be explained and there are only a few well-established risk factors, such as age, race, family history of prostate cancer and some genetic variants2. Lifestyle, including diet, has been suggested as potential modifiable risk factor and there is strong evidence that overweight, or obesity increase the risk of advanced prostate cancer3. However, for specific dietary factors the evidence is limited and controversial4. Rye-based foods or foods rich in components from rye are of high relevance in the Nordic region and have attracted interest for prevention and mitigation of prostate cancer6. Rye is rich in bioactive compounds and is the cereal with highest dietary fibre content6. Rye-based foods have been suggested to reduce risk of developing prostate cancer in epidemiological studies as well as to reduce initial tumor progression and reduce prostate-specific antigen (PSA) in animal model studies and in a few small human studies14. The potential mechanism for the preventive effect has been suggested to be related to lowered insulin secretion caused by rye14, high intake of lignans15, and more recently, due to benzoxazinoids (BXs) and their metabolites. BXs represent a group of compounds with high bioactive potential present in rye and to some extent in wheat and maize19. In planta, BXs are frequently found as conjugated forms, most typically with glucose and are produced for defense and allelopathic purposes20. The most common chemical structures of BXs include hydroxamic acids , lactams  and benzoxazolinones . Hydroxamic acids and lactams typically occur as sugar- or double sugar conjugates.Prostate cancer is the second most common cancer in men worldwide with high mortality in many Western countries in Europe and in the US19. BXs have been hypothesized to affect growth of prostate cancer cells, which could be one mechanism partly explaining the observed effects of rye on biomarkers of prostate cancer progression12. Several in vitro studies have found an inhibitory effect on prostate cancer cells for the BXs DIBOA and HMBOA24. Although a few small dietary intervention studies have shown promising suggestive results including lower PSA and slower initial tumor growth after rye vs control25, no study has been reported where the effects have been linked to BX intake or BX metabolite concentrations in biological fluids. In a small pilot study with 10 prostate cancer patients, it was shown that rye-enriched diet rich in BXs for 1 week prior to prostatectomy resulted in detectable BXs concentrations in prostate tissue (0.15–10.6 ng/g tissue)12. Occurrence of BXs and BX-derived metabolites in human and animal plasma has been reported before31 but the isolation of mammal BX-derived metabolites from biofluids and structural elucidation by NMR has never been reported before and thus quantification of BX-metabolites in plasma have therefore not yet been reported. Establishment of a quantitative method of BX and their metabolites in human samples is an important step for further investigations of the role of BX in human health.BXs have been reported in various in vitro studies to possess antimicrobial, anticancer, reproductive system stimulatory, central nervous system stimulatory, and immunoregulatory activitiesThe aim of our current study was to establish a quantitative method for comprehensive analysis of BX and their common metabolites in plasma samples from humans. The aim was further to apply the method to evaluate the effect of a diet high in whole grain/bran rye-based foods rich in BXs vs fiber supplemented refined wheat on the structural and quantitative variety of BXs and BX-derived metabolites in plasma samples after regular consumption, and to investigate whether the concentrations of any of such metabolites correlated with PSA levels in men with indolent prostate cancer.Chemicals used for sample preparation included: Methanol (Fisher Scientific), Milli-Q water , formic acid, hydrochloric acid, ammonium hydroxide, trifluoro acetic acid, acetic acid .HPLC-grade solvents, methanol , acetonitrile , 2-propanol (Fisher Scientific) and Milli-Q water  were used in the LC-MSMS analysis.32. Group 2 (compounds known as phase 1 metabolites of BXs): 2-HPAA, HPMA, 2-HHPAA, and HMPAA were purchased from AKOS Consulting and Solutions, Germany. Group 3 (phase 2 metabolites of BXs): HBOA-glcA, 2-HPAA-glcA, 2-HHPAA-glcA, 2-HPAA-sulfate, 2-HHPAA-sulfate, and DIBOA-sulfate were isolated from urine as described below. Group 4 : 4-HPAA-sulfate (known as acetaminophen-sulfate) and 4-HPAA-glcA (known as acetaminophen-glucuronic acid) were purchased from Sigma-Aldrich. The internal standard d3-MBOA was obtained as described by Etzerodt et al.33.Reference standards used for the analysis are listed in Table 12. The post- intervention urine samples were combined into one lot and stored at − 20 °C until initiation of the purification work. The combined urine was passed through MF-Millipore Membrane Filters  to remove precipitated matter. The precipitate was discarded after verifying with HPLC–MS that it did not contain significant amounts of BX metabolites.For structural determination of novel BXs using data dependent acquisition-mass spectrometry (DDA-MS) and nuclear magnetic resonance (NMR), samples of high concentration are needed. Therefore, urine samples from an already performed clinical trial with rye was used. Ten volunteers had a diet rich in rye, comprising six slices of rye bread a day or equivalent for one week. Before and after the rye intervention, 24 h urine samples were collected from the 10 participants34 . The sample was applied through the column at approx. 1 drop per sec. to allow maximum interaction of the sample with the column material. At the end of the process, vacuum was applied to the column until no more drops appeared. The cartridge was eluted 2*20 ml water, then with 3*20 ml 5% methanol (aq) followed by 3*20 ml 20% methanol (aq) at 1 drop per s. The cartridge was emptied by vacuum between each fraction. During the loading procedure four consecutive fractions of 50 ml were collected; subsequent portions were collected individually and analyzed for the content of BX related metabolites using the HPLC–MS DDA-EMS-EPI and DDA- multiple reaction monitoring (MRM)-EPIThe fractions from the HLB-SPE containing the BX compounds were dried and reconstituted in 20 ml 2.5% formic acid in water and loaded onto a cationic exchange SPE column  put in series before a weak anion exchange column . They were conditioned and equilibrated according to vendor recommendations prior to loading of the reconstituted sample. Further details can be found in Supplementary Text Semi-preparative HPLC was applied to the dried ad reconstituted SPE fractions. An Agilent HPLC equipped with a PDA detector 1100), a fraction collector (1260 infinity), pump (1260 infinity) was used for the isolation. Four UV-tracks  were used for generating and evaluating the chromatogram and the fraction collector was set to collect by time in 30 s time windows. The separations were performed using a Synergy Fusion column . The eluents used were A: 0.5% trifluoracetic acid (aq) B: 95% methanol (aq). The chromatographic method was adjusted individually for the fractions; a typical example is the following: 0–9 min 12% B, at 21 min 35% B, at 25–28 min 100% B, 28.5–35 12% B. Collected fractions were analyzed for BX-related metabolites using the DDA-MS methods described in Supplementary Text 100, a fr3-MBOA was added to each Eppendorf and the mixture was thoroughly shaken. The samples were placed at − 20 °C for 30 min for optimization of the protein precipitation, followed by centrifugation at 10.000 g for 30 min. The supernatant was carefully removed and stored in glass vials at − 20 °C until analysis, when the samples were thawed, diluted 1:3 with Milli-Q water, filtered and placed in the autosampler in a dedicated vial.After fraction collection of the target BX-metabolites from the urine samples, a method for their quantitative analysis in plasma was established. Plasma samples (see below) were taken from the − 80 °C freezer and thawed at 4 °C. After thorough mixing of each individual sample, 200 µl were taken from each sample and transferred to an Eppendorf tube and randomized into 6 batches and frozen at − 80 °C. One batch (approx. 40 samples) at a time (day) was thawed at 4 °C. All solvents and samples were kept in ice-bath throughout the whole procedure. Methanol (400 µl) with 4 ng/ml dThe LC–MS system setup was similar as for qualitative analysis in urine  with a BMI of 27.5 ± 4.6 kg/m2. The study was carried out with approval from the Regional Ethics Review Board in the Uppsala Region (DnR 2005/002). The study was conducted according to relevant guidelines, informed consent was obtained from all participants. During the study, participants were instructed to keep to their habitual diet except for bread, porridge, muesli, and other fiber- or lignan-rich foods and table spreads, which were replaced by products provided by the study (intervention foods). During the interventions, participants were provided isocaloric, dietary fiber adjusted intakes of whole grain/bran rye foods (RF) or refined wheat products with added cellulose derived from cereal straws (WP). Participants were asked to include intervention foods as part of their breakfast, lunch, dinner, and 2–3 snacks/day during the intervention weeks. In brief, the intervention foods consisted of 300 g/d soft bread (5 pieces), 100 g/d crisp bread (10 pieces), 50 g/d breakfast cereals, 35 g/d porridge (uncooked), and 58 g/d table spread. The bran that was added to the soft bread and crisp bread were sourdough fermented before addition. The exact nutritional composition of intervention food products has been described elsewhere14. The fiber intake from the experimental foods were very high, 58 g/day during the rye regime and 57 g/day during the endosperm fiber supplemented wheat regime14. BX contents of the intervention foods was not analyzed since there was no material left when the current study was initiated. For qualitative comparisons, we have estimated the contents based on previous human studies with similar products35. Blood samples were collected at baseline and after 2, 4, and 6 weeks in each period. Samples were processed immediately after collection as described elsewhere and were stored at − 80 °C until BX analysis14.The method for quantitative analysis of BXs and their metabolites in plasma was applied on samples from a randomized crossover study with two 6-wk intervention periods separated by a 2-wk washout period in men with indolent prostate cancer. Participants and study design have been described in detail elsewherep < 0.05). Pairwise comparisons were performed for overall effects. Data is presented as mean and 95% confidence interval. Rank-transformed variables were presented as median and interquartile range.The plasma levels of BX metabolites between treatments were evaluated by mixed linear model. Identity was included as a random factor and diet, week, and period as fixed factors. Initially, the interaction term diet x time was included but was not significant and therefore removed. There was no clear trend of a time effect, thus data was average for each id and treatment. Normality of the residual distribution of each variable was tested by visually inspecting the Q-Q plot and Residuals versus Fitted plot. Data where residuals were not normally distributed was log-transformed. If still not normally distributed data was rank transformed. Log-transformed values were back-transformed to the original scale. Overall effects were investigated with type III tests adjusted with the false discovery rate method . Partial spearman correlation was performed on variables significant between diets and included total PSA and metabolites adjusted for id, including all 8 data points . For each variable, the correlation with total PSA was based on complete data, excluding missing values. The analyses were performed in the programming software R (version 4.0.1), packages used were lme4 (1.1–27.1), car (3.0–11), emmeans (1.7.0) and Hmisc (4.6–0).Principal component analysis (PCA) was conducted on data averaged for each treatment and identity. Imputation of data for metabolites in the PCA was performed using LOD/2, the LOD was estimated by averaging the 3 lowest values for each variable divided by 2. The PCA was visualized with the R package rdCV  can be seen, thus supporting the annotation. DIBOA-sulfate   and the neutral loss of 80 corresponding to SO3 in 2-HPAA-sulfate ), presented in descending order of highest concentration in the whole grain/bran rye intervention. The effect of the interventions on the metabolites is visualized in a PCA in Fig. The established quantitative method for determination of 22 BX metabolites 41. In our study, participants were supplied with foods containing wholegrain rye with fermented rye bran, i.e. containing both whole grains and added bran. The high concentration of 2-HPAA-glcA is likely related to the fermented rye bran. Food processing have a central role in the concentration and composition of BX molecules due to enzymatic activities or microbial processes28.The metabolite at highest concentration after the rye diet was 2-HPAA-glcA, followed by HBOA-glc. Previous animal14. We investigated the correlation between PSA and the different measurable BX compounds in the circulation and interestingly, we found that HBOA-glc, HBOA-glcA, 2-HHPAA and 2-HPAA-glcA were significant inverse partially correlated with PSA (Table 9 and cell line studies42, where an effect of rye or specific BXs had an inhibitory effect on prostate cancer cells. The specific BXs tested were DIBOA and DIMBOA42. Future studies should investigate the effect of an extended spectrum of BX molecules, including the ones correlated with PSA in our study, and preferably on several prostate cancer cell lines. A human pilot study suggested that high intake of rye bran caused apoptosis of prostate tumors, but BX molecules were not analyzed25. In another human trial, one-week with whole grain rye consumption in men with prostate cancer resulted in higher BXs concentrations detected in plasma and urine but also in prostate tissue biopsies. Together these results suggest that a high rye intake leads to quantifiable levels of BX in the circulation and in the prostate tissue12 and that circulating BX levels are associated with biomarkers of prostate cancer. Often phase II metabolites, i.e. conjugated substances, are assumed to be less biological active, but there are exceptions. For example morphine-6-glucuronide has a higher analgesic effect than the non-glucuronide form43. The result from our study suggests that at least some of the conjugated forms may be biologically active or could later have been de-conjugated and distributed further in the organism. Further studies in humans are needed to verify this.The total PSA was lower in plasma after intervention with whole grain/bran rye compared to fiber supplemented wheatThe fact that rye products treated with different processing techniques result in variations in the content of BXs and BX related molecules creates possibilities for industry to tailor compound profiles by optimizing the processes to bring more healthy foods to the market.46 and indirect metabolic effects on insulin/C-peptide44. Such compounds and processes may exert beneficial effects in parallel with BXs. However, BX has so far only been investigated in a few human studies and was therefore the focus here. A major strength of this study is that we managed to isolate and identify novel BX compounds from urine as well as integrating them in the first quantitative/semiquantitative method for determination of these compounds in human plasma samples. Another strength is the cross over design, were the between-participant variation can be eliminated when evaluating the effect of the diets. The risk for carry over is low, due to short half-life of BXs, shown in the fact there were no differences in concentration between weeks. A third strength is that we were able to quantify many BX and BX-metabolites in plasma for the first time in human plasma and their identities were confirmed by NMR.Our study has some limitations but also strengths. A limitation of this study is the lack of synthetic reference standards for all identified BX compounds which prevents absolute quantification of all compounds. However, we used calibration curves of the most similar metabolites where such were available for semi-quantitation. Another limitation is the lack of analysis of the BXs content in the intervention foods. However, comparing our results to previous studies with similar foods consumed, could give us a rough estimation of the intake. A third limitation is the small sample size, increasing the risk of chance findings. Our results should therefore be interpreted with caution and needs to be verified in independent studies. We acknowledge that rye-based foods contain many other compounds than BX that may be of relevance to prevention/mitigation of prostate cancer, such as lignansWe have successfully established a new method of comprehensive, quantitative analysis of BXs, including compounds not quantified before, in plasma. In total seven BXs and seven BX metabolites were significantly higher in plasma after a diet with whole grain/bran rye foods compared to a diet with refined wheat supplemented with a pure fiber in men with indolent prostate cancer. The BXs HBOA-glc, HBOA-glcA, 2-HHPAA and 2-HPAA-glcA were significantly inversely correlated to the total PSA level, suggesting a potential association with prostate cancer growth. These findings warrant further investigations in larger clinical trials with defined BX intakes.Supplementary Information 1.Supplementary Information 2.


Numerous studies showed that adults with attention deficit hyperactivity disorder (ADHD) suffer from impairments in a range of cognitive functions when compared to healthy controls. However, only little is known about the neuropsychological functions when compared to various clinical control groups and whether a distinct neuropsychological profile can be identified for adult ADHD.n = 78) or to one of two clinical comparison groups, depending on whether they show indications (n = 71) or no indications (n = 50) for the presence of psychiatric disorders other than ADHD. All individuals performed a comprehensive neuropsychological test battery.This retrospective study examined data of 199 outpatients referred for clinical evaluation of adult ADHD, allocated either to an ADHD group (Data analysis revealed impairments in a range of cognitive functions in a substantial number of patients of all three groups. However, profiles of neuropsychological impairments were similar between groups. Furthermore, significant small- to medium-sized correlations between basic and higher-order cognitive functions were revealed in the ADHD group and the clinical comparison group with indications for psychiatric disorders other than ADHD.Neuropsychological impairments are prominent in psychiatric outpatients seeking a clinical evaluation of adult ADHD but are not specific for ADHD. It is concluded that neuropsychological test performance may have limited incremental value to support the psychiatric differential diagnosis. Furthermore, a clinical trajectory may need to take into account that deficits in a range of higher-order cognitive functions can be substantially explained by deficits in basic cognitive functions. Attention deficit hyperactivity disorder (ADHD) is one of the most prevalent neurodevelopmental childhood disorders that persists into adulthood in a large proportion of cases , who all performed a comprehensive test battery consisting of a broad range of measures, which was specifically composed for the neuropsychological assessment of adult ADHD. In this study, we aim to reveal differences in cognitive functions between individuals who receive a diagnosis of ADHD and individuals who have been referred for clinical assessment because of an assumed ADHD, but who actually did not fulfill the diagnostic criteria of an ADHD. We expect that adults diagnosed with ADHD perform significantly poorer in several aspects of attention and executive function than individuals not reaching diagnostic criteria for ADHD. However, we expect that effect sizes of impairments between groups differ across functions and will not exceed small to medium size , conclusions about what cognitive impairments are most characteristic for ADHD are difficult to draw. Thus, in order to further elucidate the role of a neuropsychological assessment in the clinical evaluation of adult ADHD, the present study employs a large sample of clinically referred individuals to an ADHD outpatient clinic  and were therefore referred for a diagnostic assessment to the ADHD outpatient clinic of the Department of Psychiatry and Psychotherapy, University of Duisburg-Essen, Germany. All individuals underwent a comprehensive diagnostic assessment by trained psychologists or psychiatrists. The diagnosis of ADHD was established based on the criteria as outlined in the Diagnostic and Statistical Manual of Mental Disorders, 5th Edition , the Clinical Comparison Group  and the Cinical Comparison Group-Not Diagnosed . Participants of the CCG showed evidence for one or more psychiatric disorders other than ADHD, including mood disorders (n = 50), addiction disorders (n = 22), anxiety disorders (n = 5), personality disorders (n = 3), eating disorders (n = 3), adjustment disorders (n = 2), schizoaffective disorders (n = 2), obsessive–compulsive disorders (n = 1), conduct disorders (n = 1), and intellectual developmental disorders (n = 1). With regard to symptom presentations of ADHD, 66 patients with ADHD were diagnosed with the combined presentation and nine patients with the predominantly inattentive presentation, whereas the symptom presentation of three other patients with ADHD was not reported. Moreover, 31 of the 78 patients with ADHD showed evidence for one or more comorbid psychiatric disorders, including mood disorders (n = 19), addiction disorders (n = 5), adjustment disorders (n = 5), anxiety disorders (n = 3), obsessive–compulsive disorders (n = 2), personality disorders (n = 1), oppositional defiant disorders (n = 1), intellectual developmental disorders (n = 1), and autistic disorders (n = 1). Table F(2) = 7.026, p = 0.001, sex, χ2(2) = 6.553, p = 0.038, education level, χ2(8) = 16.718, p = 0.033, childhood ADHD symptoms, F(2) = 24.486, p < 0.001, and current ADHD symptoms, F(2) = 12.060, p < 0.001. Compared to the CCG-ND, patients with ADHD had a significantly lower female-to-male ratio, and scored significantly higher on childhood and current ADHD symptoms. Compared to the CCG, patients with ADHD were on average significantly younger, more individuals attained a relatively low level of education, and obtained significantly higher scores in both scales for ADHD symptom severity. The CCG only differed significantly from the CCG-ND with regard to a higher score for childhood ADHD symptoms.Forty-nine of the 248 participants were excluded from the present study, i.e., 47 participants were excluded because the diagnostic process was not completed or did not allow a formal diagnostic decision, and two participants were not considered because the neuropsychological assessment was not or only partly administered, leaving a sample of 199 participants for inclusion in the final data set that entered data analysis. All participants in this sample were allocated to one of three diagnostic groups, i.e., the ADHD group  was administered to assess childhood ADHD symptoms retrospectively . Participation was voluntary, unpaid, and it was stressed that the agreement to take part in research did not affect their clinical assessment or treatment. All participants were asked to complete a set of questionnaires at home prior to the diagnostic interview. The clinical evaluation started with the diagnostic interview, and continued with the neuropsychological assessment (cognitive testing) at the same or another day of convenience for the examinee. The neuropsychological assessment using cognitive tests took about two hours to administer, and was led by a trained psychologist or neuropsychological test assistant under close supervision. Participants were not informed about their diagnostic status at the time of the neuropsychological assessment.Missing values occurred in 5.3% of the data due to administrative errors and were not replaced. Test data of all three groups are presented in descriptive statistics. Furthermore, neuropsychological test data are interpreted based on norm scores as provided by the test publisher, i.e., to derive the number of individuals having impairment in each of the functions assessed. An impairment is defined as an individual test performance that is equal or below the 16th percentile  of the representative test norms as provided by the test publisher  were not met in several variables, nonparametric statistical analyses were performed. Per test score, the ADHD group was compared with the CCG and CCG-ND, respectively, using Mann–Whitney ct Cohen .r = 0.1), medium (r = 0.3), and large (r = 0.5).In order to investigate the effect of basic on complex cognitive functions, functional domain scores were created representing different aspects of basic  and complex  cognitive functions  were computed to determine performance differences between the ADHD group and both the CCG and the CCG-ND. Test statistics of Table r = 0.31, r = 0.34, and r = 0.26, for CCG-ND, CCG, and ADHD, respectively.Nonparametric group comparisons (Mann–Whitney r = 0.28). Differentiating between different aspects of complex cognitive functions, a significant association of medium size was obtained with inhibition/interference control (r = 0.36) and a significant association of small size with fluency (r = 0.29). For the CCG, a significant and medium-sized effect was found for the total compound score of complex cognitive functions (r = 0.34), with a significant small-sized effect to inhibition/interference control (r = 0.26) and a significant medium-sized effect to fluency (r = 0.32). Finally, a small and nonsignificant association was found between basic cognitive functions and the compound score of complex cognitive functions of the CCG-ND (r = 0.15). Per domain of complex cognitive functions, a significant effect (medium size) was revealed only for the association with fluency (r = 0.45).Spearman’s rank correlation coefficients between basic and complex aspects of cognitive functions in the three groups are presented in Table This study aimed to explore neuropsychological functioning of individuals at clinical evaluation of adult ADHD, and examine the associations between basic and higher-order cognitive functions in this population. An analysis of neuropsychological test performance revealed that individuals with ADHD exhibit impairments in several of the neuropsychological functions assessed in this study. Considerable rates of impairment, as determined by use of test norms, were shown in adults with ADHD in selective attention, vigilance, inhibition, and interference control . This is in line with the results of numerous previous studies showing impairments in adults with ADHD in various cognitive functions , with a peak at two to four impairments. The observation of similar patterns of neuropsychological functions between the three groups is consistent with the view of ADHD as dimensional construct, with ADHD-like symptoms and impairments occurring in large parts of the population, including the general psychiatric population (Sergeant, Geurts and Oosterlaan Finally, findings of earlier research (Butzbach et al. This study needs to be seen in the context of several limitations. First, the group of patients with ADHD is a selected sample, with the majority being diagnosed with the combined symptom presentation and various comorbid psychiatric disorders. It is, therefore, difficult to evaluate how representative the present data are for the population of adults with ADHD when compared to clinical control groups, and whether the observed effects would hold in clinical samples with different characteristics.Second, because the clinical assessment was designed mainly to determine the presence of adult ADHD, only clinical indications, but no verified diagnoses, could be given for the differentiation between other clinical conditions. Thus, the differentiation between individuals presumably having or not having psychiatric conditions, and subsequent group comparisons, must be interpreted with caution.Third, the missing of more differences between groups may have been caused by similarities in group characteristics. For example, a similar range of psychiatric disorders is observed both in the ADHD group and the CCG. Further, given this context of an ADHD outpatient clinic, it must be considered that also individuals not being diagnosed with ADHD may suffer from a similar clinical pattern which may just not reach diagnostic threshold for ADHD.Fourth, even though the neuropsychological assessment using cognitive tests was not part of the standard diagnostic routine of clinicians, results of the cognitive assessment were accessible to patients and clinicians, and may have guided clinical decision-making. However, this may even support the notion that a neuropsychological assessment using cognitive tests may not contribute substantially to a differential diagnostic process of psychiatric disorders, if one takes into account that the neuropsychological assessment was not completely independent from the diagnostic assessment and still the ADHD group does not largely differ in neuropsychological functions from the two other clinical groups.This study demonstrates that individuals seeking a clinical evaluation of adult ADHD show marked impairments in several aspects of cognitive functions, irrespectively from whether they fulfill diagnostic criteria for ADHD or not. This is underlined by group comparisons indicating no meaningful differences in cognitive functions between patients with ADHD, the clinical comparison group, and the clinical comparison group with no diagnostic status. We conclude that cognitive deficits are prominent in patients of this setting, but are not specific for ADHD. And a neuropsychological assessment using cognitive tests may not provide the clinician with incremental information for the differential diagnostic process of adult ADHD. Furthermore, we conclude and support earlier work that deficits in a range of cognitive domains can be substantially explained by deficits in lower-order cognitive functions, such as processing speed and basic aspects of attention and distractibility.


According to the scientific literature, 50 to 70% of individuals with autism spectrum disorder (ASD) also present with comorbid attention deficit hyperactivity disorder (ADHD). From a clinical perspective, this high rate of comorbidity is intriguing. What is the real significance of this dual diagnosis? Is ADHD in fact always present in such cases? Might the attentional impairment reported among our ASD patients actually be a distinct trait of their ASD—namely, impaired joint attention—rather than an ADHD attention deficit? Could their agitation be the consequence of this joint attention impairment or related to a physical restlessness etiologically very different from the agitation typical of ADHD? The neurobiological reality of ASD-ADHD comorbidity is a subject of debate, and amphetamine-based treatment can have paradoxical or undesirable effects in the ASD population. Consequently, does a dual diagnosis, notwithstanding its currency in the literature, prevent us from shedding sufficient light on major physiopathologic questions raised by the clinical picture of ASD? The semiology of autism spectrum disorder (ASD) and attention deficit hyperactivity disorder (ADHD) presented in current nosography, which helps clinicians to identify these disorders, makes it clear that they are different entities, affecting children and their developmental histories in ways that are clearly distinct. In the first case, we are primarily describing distracted children who pay little attention in academic settings, lose their belongings, and have difficulty sustaining mental effort. In the second case, we are talking about children who seldom associate with others, have a hard time interacting and communicating, and may display unique motor or verbal behaviors, including stereotypies, echolalia, and idiosyncratic language. Whereas, children with ADHD tend to be relatively boisterous and talkative, and eager rather than apprehensive of interactions with peers or adults, autistic children may be distinguished by their repetitive and less coordinated motor function, difficulty communicating, emotions in sync with their sensory reality more than with their social setting, and uniform behaviors that keep the unpredictable at bay.Epidemiologically, these two disorders also differ in their incidence. Their diagnoses are made at different ages. Children with ASD can be identified before they are 3 years old, while ADHD is diagnosed later on. Both fall into the wider category of neurodevelopmental disorders, within which “comorbidities” are considered relatively frequent.The prevalence of ADHD in people with ASD ranges from 50 to 70%, according to the literature . Where dThis figure is the product of meta-analyses. It is important to note the variability of findings between studies—more than a mean prevalence, a precise yet illusory value reflecting the view that this comorbidity is a clearly measurable entity—because it admits an alternative interpretation of the phenomenon in question. Indeed, reported rates of comorbidity range from 10 to 90%. In the logic of meta-analysis, variation is often explained through methodological arguments: studies diverging from the mean are said to describe different populations, apply less valid methods of measurement, or gather their data in atypical ways. Yet, in part, what may be described as “bias” could betray an unsound theoretical foundation, accommodating a multitude of experimental paradigms measuring what is thought to be the same phenomenon.This article thus aims to recall the attentional features inherent to ASD; to describe and analyze the variability of data reported in the literature; and finally, to draw on studies of factors related to attention, such as memory, sensorimotor function, executive functioning, and intellectual disability, for a better understanding of the role these variables play in the expression of cognitive capacity within the ASD population.Attention is defined as a process of selection applied to the product of perception, and may even be directed toward memories. To direct attention on a perceptual or internally represented entity, our working memory must be trained on a goal for the duration of the task at hand. Attention encompasses essential elements of the executive functions and can be further broken down into sustained attention; focused attention; visual search, whereby the target of attention is defined using a template stored in working memory; voluntary or reflexive orienting and disengagement; attentional filtering; and expectation .In people with ASD, these components of attention have a characteristic profile: sustained and focused attention is stronger than in normal subjects; visual search is also, though this seems to reflect the quality of perceptual processing more than attention; and the ability to orient attention toward non-social stimuli is deficient, as are reflexive and voluntary disengagement, in certain settings. Attentional filtering is not compromised in ASD patients having no intellectual disability. Higher-functioning ASD patients have a specific attentional impairment not observed among other ASD patients . Joint aCan the comorbidity of ASD and ADHD truly be established by considering executive functions alone, as several studies suggested ? The devWhile the DSM-4 and ICD-10 give mutually exclusive diagnoses of ADHD and ASD, the DSM-5 mentions each condition in its description of the other, admitting the possibility of comorbidity.Sprenger et al.  concludeSome studies have reported structural differences in attentional functions between ASD and ADHD patients, while others suggest these disorders present identical deficiencies , 8.Barnard-Brak  reportedHochhauser et al.  describeMayes et al.  demonstrIs observed agitation then a sign of the prefrontal inhibitory deficit underlying ADHD or rather a state of restlessness, a minimal expression of cerebellar dysfunction, characterizing an etiologically distinct entity? The association of ASD with alterations in specific brain regions is becoming increasingly clearer. The affected regions include the orbitofrontal cortex, superior temporal sulcus, fusiform gyrus, amygdala, and cerebellum , and theADHD and ASD are described as frequently co-occurring, sharing certain cognitive phenotypes. However, it is important to be able to trace these shared features back to a common physiopathology and identify the physiopathologic characteristics of comorbidity, which may present additional neurofunctional deficits. Chantiluke et al.  comparedAs shown by Lau-Zhu et al. , ASD andResearch applying quantitative EEG has demonstrated atypical profiles for ADHD, principally concerning theta and beta frequency bands , 19, andTo better understand the distinct neuropsychological profiles of ADHD and ASD patients, it seems appropriate to consider visuospatial exploration strategies. In terms of visuospatial abilities, ADHD patients in a study by Cardillo et al.  exhibiteIt has often been stated in the literature that ASD and ADHD are difficult to distinguish when making a diagnosis. Mayes et al. , consideMayes et al. also found no significant difference between children of normal intelligence with ASD, ADHD-Combined type, or ASD-Inattentive type in terms of performance on neuropsychological tests that evaluated attention, working memory, processing speed, and graphomotor skills.N = 43) and three groups of individuals clinically diagnosed with ADHD (N = 21), oppositional defiant disorder (N = 26), and ASD (N = 41), respectively—on go-no-go and switch tasks, and detected deficits shared by the ADHD, oppositional defiant disorder, and ASD groups, such as increased reaction time variability. After controlling for symptoms of ADHD and oppositional defiant disorder, differences in reaction time variability between groups were no longer significant. For cognitive flexibility, as evaluated by the switch task, there was also no observed difference between groups. The ASD group alone exhibited impaired response inhibition and premature responsiveness, relative to the typically developing group. Carter Leno et al. thus concluded that executive functioning impairments specifically described as present in ADHD are also found in ASD. This stresses the need to explore and precisely define the characteristics of attentional impairment in autism. These characteristics, their causes, and their consequences should be included in the definition of ASD, to more accurately represent its specificities.Can comorbidity of ASD and ADHD truly be established by considering executive functions alone, as suggested by some studies ? Carter These findings are supported by those of Rosello et al. , who repThe attentional impairment best described for ASD is diminished joint attention. It is also said to give rise to later social communication impairments, especially in connection with oromotor skills –29. ReceWith respect to psychomotor agitation, it has been posited that ASD patients have atypical arousal systems, their state of overexcitement reflecting both excessive and fluctuating cortical activation by the brain stem. Dopaminergic hyperactivity along the nigrostriatal (explaining stereotypies) and mesolimbic  pathways is a hypothesis gaining wider support. It would explain the perceived effectiveness of dopamine antagonists, vs. agonists, on all symptoms of autism. The above confirms the need to include the characteristics of hyperfocus among the diagnostic criteria for autism. Hyperfocus is a dimension also suggested by MEG studies  pointingMundy et al.  explain To date, the mixed findings of etiologic ASD-ADHD comorbidity studies do not permit a clinical description of the physiopathologic comorbidity of these disorders. When speaking of comorbidity, are we not rather describing the severity of an attentional trait, with disabling functional effects, present in all ASD children?These studies themselves demonstrate that children diagnosed with a comorbid disorder have more severe ASD. This suggests that—rather than an ASD-ADHD comorbidity, as authors conclude—the causes and consequences of a major attentional deficit typical of ASD are responsible for these patients' profiles. If so, this attentional trait should be included in the clinical definition and description of ASD. Instead of an ADHD explanation, we might then adopt an alternative etiologic perspective on these neurocognitive peculiarities and more fully account for the various brain impairments observed. As we have shown, children with ASD are affected much more by heightened attentional abilities than by a primary attention deficit, although the latter may be mistakenly (and dangerously) suggested by their clinical presentation. Therefore, we highlight the urgent need to develop new clinical and electrophysiological instruments to best fractionate and analyze these important neuropsychological features. This would be of crucial interest in particular for those modeling aspects of the co-morbidity in IPSC lines or animal models but also for the optimization of treatments use.The attentional specificities observed in ASD, and their consequences, which are a direct reflection of unique brain functioning again challenge the validity of polythetic diagnoses in psychiatry. Attention deficits are key behavioral phenotypes of a considerable number of neurological and genetic diseases characterized by complex psychiatric disorders. Might the error lie in conflating such deficits with the unique, and very different, attentional traits of autism? In other words, are the attentional characteristics of disorders ignored by making an erroneous generalization?Though further description of these characteristics is necessary, are they not key clinical symptoms of ASD in their own right—justifying their inclusion in the definition of this disorder—and potentially sufficient for its diagnosis?The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. 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Surfactants are essential in the manufacture of polymeric nanoparticles by emulsion formation methods and to preserve the stability of carriers in liquid media. The deposition of non-ionic surfactants at the interface allows a considerable reduction of the globule of the emulsion with high biocompatibility and the possibility of oscillating the final sizes in a wide nanometric range. Therefore, this review presents an analysis of the three principal non-ionic surfactants utilized in the manufacture of polymeric nanoparticles; polysorbates, poly, and poloxamers. We included a section on general properties and uses and a comprehensive compilation of formulations with each principal non-ionic surfactant. Then, we highlight a section on the interaction of non-ionic surfactants with biological barriers to emphasize that the function of surfactants is not limited to stabilizing the dispersion of nanoparticles and has a broad impact on pharmacokinetics. Finally, the last section corresponds to a recommendation in the experimental approach for choosing a surfactant applying the systematic methodology of Quality by Design. Surface active agents, commonly known as “surfactants”, are molecules that decrease surface and interfacial tension at the interfaces between solids, liquids, and gases, acting as dispersants, wetting agents, emulsifiers, and detergents . FurtherA dispersed system consists of one substance distributed (dispersed phase) in discrete units in a second substance (continuous phase). Most of the nanoparticle manufacturing methods in the biomedical field involve forming a liquid/liquid stable dispersed system with the contribution of surfactant agents to produce a new colloidal type solid/liquid dispersed system. The initial globule size of emulsified dispersed systems is greater than the colloidal particle size at the end of the manufacturing process, combined with the presence of a high surface free energy and, therefore, the tendency of resulted nanoparticles (NP) to flocculate and coagulate can be observed . At the The presence of a surfactant affects the particle size, polydispersity index (PDI), drug loading, zeta potential value, and correlation with apparent physical stability . For thiPolymeric NP are carriers that predominate in biomedical applications, while non-ionic surfactants confer high biocompatibility in most methodologies. For several decades, most nanoparticle formulations have included one of the following three excipients as a surfactant: polysorbates (PS), poly (PVA), or poloxamers. This broad trajectory of study has allowed an abundant exploration of technological benefits and formulation limitations.This work is a tribute to the principal non-ionic surfactants utilized to manufacture polymeric NP in the biomedical field. It focuses on the three leading excipients for stabilization: PS, PVA, and poloxamers. The purpose of this work is to offer an overview of each type of non-ionic surfactant, including a general description of the types of molecules in the family, general applications, obtention of derivatives in search of novel properties, and formulation of NP with tables that condense aspects of the physicochemical parameters of the NP according to the type of stabilizer. Moreover, we mention a brief section on toxicity aspects, a critical section on advantages and disadvantages, and a section on drawbacks and future. Finally, this review presents an analysis of the influence of surfactants on the interaction with biological barriers and a narrative and comprehensive description of the main variables involved in the methodological selection of surfactants through the Quality by Design strategy (QbD).Surfactants are crucial excipients in the synthesis of NP; they are amphiphilic molecules characterized by a hydrophilic head group (ionic or non-ionic) and a hydrophobic tail a. The amSoap  is formulated from anionic surfactants, and the first records of its manufacture date back to 2800 B.C. in ancient Babylon ,20. HoweThe physical stability of NP mainly depends on electrostatic, steric, entropic, and Van der Waals forces . The DLVMore stable dispersions can be obtained when the system contains oppositely charged NP and surfactants, such as anionic NP and cationic surfactants or vice versa. The dominant mechanisms are electrostatic interactions and hydrogen bonding . In elecN-erucamidopropyl-N,N-dimethyl ammonium) propane sulfonate and coordinated water in trans-[FeCl2(H2O)4] Cl structure; this masks the ionic forces of repulsion between the head groups of surfactants x [CH(CH3)CH2O]y [CH2CH2O]xOH, where y is higher than 14. Depending on the x and y values, these copolymers present different amphiphilic properties based on their HLB values. The physicochemical characteristics of poloxamers could be modified by changing the POE and PPO relation mass, obtaining different sizes, lipophilicity, and hydrophilicity. Regularly, the molar mass ratio between POE and PPO blocks ranges from 8:2 to 9:1, derived from the coexistence of hydrophilic and hydrophobic sections in the poloxamers structure. These copolymers present a high temperature-dependent self-assembling characteristic as well as thermo-reversible properties. Solutions of poloxamers with concentrations higher than their CMC produce gels at temperatures above their sol-gel transition temperature. Additionally, PPO blocks dehydrate at high temperatures, and it is less soluble in water, which triggers the formation of the micelles with the dehydrated PPO core and hydrated POE shell. Consequently, depending on the main properties needed for the application, appropriate physical and chemical modifications could be applied in the poloxamer synthesis. Interestingly, several reports demonstrated that poloxamers could decrease the cell membrane microviscosity (membrane fluidization) due to the presence of hydrophobic PPO chains [Poloxamers are synthesized by sequential polymerization of ethylene oxide and PPO units in sodium hydroxide and potassium hydroxide. Their chemical formula is HO[CHO chains ,228. FurO chains ,229,230.® known as L121 (poloxamer 401) has a liquid presentation with above 2,000 Da of molecular weight and HLB of 3; meanwhile, Pluronic® F68 (poloxamer 188) has an average molecular weight of 8,400 Da, a percentage of POE around 80%, and an HLB value of 29 with flake morphology [® P123 (poloxamer 403) is a paste with an HLB value of 8 and an average molecular weight of 5,750 Da [As we mentioned, poloxamers are polymorph materials covering a range of gelation states from liquid to paste and solid, depending on the molar mass ratios between the POE and PPO blocks . This clrphology . On the 5,750 Da ,233.The structural versatility of poloxamers makes them particularly attractive emulsifying, solubilizing, and dispersing ingredients for pharmaceutical formulation ,235. It The synthesis of chemically cross-linkable poloxamers has also been analyzed to enhance their mechanical properties. The chemical structure of poloxamers presents only reactive groups available for the modification at the end of the chains; thus, chemically cross-linkable groups can only be used to end-cap the triblock chain. Ethoxylsilane and methacrylate/acrylate are two groups employed for the crosslink of end-capping groups . To intrPoloxamers have been widely applied in nanotechnology as a stabilizer and nanoparticle shell component for different applications. In 2019, Del Prado et al. developed a system of PCL NP stabilized by poloxamer 188 . The resSeveral studies have reported the use of poloxamers-based or -coated NP in cell culture, finding that the toxicity of these surfactants is relatively low . For ins® copolymers presented low cytotoxicity and, remarkably, weak immunogenicity in topical and systemic administration. It is known that POE-PPO-POE copolymers are non-degradable; however, molecules with a molecular weight of 15 kDa or less are usually filtered by the kidney and cleared in urine [As mentioned, poloxamers exhibit characteristics that are very useful for the pharmaceutical and biomedical fields. For instance, thermosensitivity, high capacity to solubilize drugs, drug release properties, and the absence of toxicity in mucous membranes ,280. Furin urine . On the in urine ,284. Autin urine . Howeverin urine . ConsequThe impact of surfactants on the physicochemical parameters of NP is not limited to stability phenomena. There is a significant influence of surface phenomena driven by surfactants and interactions at the cellular level ,286 Fig. TherefoThe BBB is a highly sophisticated brain barrier with tight junctions between endothelial cells and a foreign substance detection system; therefore, it represents a challenge for drug passage. Interestingly, Voigt et al.  conducteHistorically, the oral route has been the preferred drug administration route due to patient comfort, ease of application, and low treatment costs. However, there are different limitations inherent to the gastrointestinal region that produce variations in the bioavailability of drugs. The use of NP is desirable to ensure adequate bioavailability, drug stability, and even control in the sustained release system. The presence of non-ionic surfactants can increase mucus penetration, reduce recognition and clearance, enhance plasma circulation times, and promote drug accumulation. PEG is one of the predominant strategies, and it is also a passive mucopenetrating excipient that reduces interactions with luminal components and mucus in the gut .®) and poloxamer 188 were also desirable [Nasal administration is intended for local or systemic action. Some advantages of the nasal region include a large surface area, low enzymatic activity, vascularized subepithelial layer with direct passage to the systemic circulation, and evasion of the first-pass metabolism in the liver. On the other hand, some challenges include low membrane permeability of polar drugs and rapid clearance . In thisesirable .Pulmonary drug delivery allows local and systemic effects. The lung has advantages such as avoiding the gastrointestinal environment and reducing the first-pass metabolism of drugs. However, its main barriers in the absorption process are the epithelial and capillary cell barrier and a surfactant layer. Strategically, one of the tools in drug delivery is the decrease in particle size and surfactants at the interface . In thisThe pharmaceutical industry is working hard to achieve robust and high-quality drug products. The QbD, which the International Conference of Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use defines in the harmonized tripartite guideline for the pharmaceutical development Q8(R2) , is “a sIn the current Quality by Test (QbT) system, product quality is ensured by following a sequence of steps, including raw material testing, fixed drug product manufacturing process, and end-product testing. If the specifications or other standards are met, the product may be kept in the manufacturing or incorporated into the market. Otherwise, it will have to be reprocessed . Due to Based on the above, it can be considered that the search for a successful nanoparticle-based product results from the effect of several variables. One of these variables is associated with the selection of the stabilizer since it has been shown in several studies that the stabilizer, which is generally a surfactant, has a significant impact on several response variables of the NP; for example, on the efficiency of encapsulation, particle size, charge, among others. Through the employment of QbD, Saha et al.  indicateOn the other hand, Patel et al.  evaluateWith the above, we can see that three of the most common CMAs in the evaluation by QbD concerning the stabilization of NP are the concentration of surfactant, the type of surfactant, and the surfactant ratio . Of the As mentioned, the establishment of CMAs related to NP stabilizers is carried out through risk analysis. The risk analysis to identify and evaluate the type and concentration of the stabilizer as CMAs, during the manufacture of NP, has been carried out mainly through strategies such as Cause and Effect Diagram , Failure® and Pluronic® make it challenging to distinguish structurally from the type of stabilizer. The new trends outline the lines of research in the search for surfactants of natural origin that allow a high interaction in the interfaces but with high biocompatibility and biodegradation. There may even be modifications of naturally occurring surfactants with synthetic surfactant fragments.As described in previous sections, the diversity of surfactant types is vast, and the possibility of derivatization towards new applications is also possible. It is one of the trends in the search for environmentally friendly surfactants. We detected that although there are different innovations in new structures, there is a predominance of the three mentioned stabilizers, PS, PVA, and poloxamers. However, there is not a complete description of the stabilizer properties in most studies in the case of PVA; while with the PS, some aspects persist about the possible toxic effect in biological models. Concerning poloxamers, the trade names for LutrolNon-ionic surfactants offer a wide versatility of applications in the different manufacturing methods of polymeric nanoparticles, highlighting their high biocompatibility and moderate interaction with biological barriers. Reports of significant toxicity are scarce, while the physicochemical parameters of nanoparticles are widely modulated, from particle size to encapsulation capacity. Although some PS, PVA, and poloxamers derivatives exist, the traditional use of primary structures predominates due to the high stability conferred to dispersed systems. The stability-biointeraction balance is necessary to have an adequate performance of the formulation. At the same time, the systematic approach of QbD in the choice of a surfactant is a route that has marked for some years a new and reliable experimental strategy. The outlook in the manufacture of polymeric nanoparticles for biomedical applications seems to indicate that the use of non-ionic surfactants will continue to predominate in the following years due to their ease of application, broad utility, and extensive biosafety background.


Background: Attention deficit hyperactivity disorder (ADHD) is associated with depression among college students, while perceived social support is also associated with depression, especially among young adults. This study aimed to examine to what extent perceived social support mediated the relationship between ADHD symptoms and depressive symptoms. Methods: In total, 124 first year medical students completed the Adult ADHD Self-Report Scale Screener (ASRS), the Patient Health questionnaire-9 and the revised Thai Multidimensional scale of perceived social support reflecting ADHD symptoms, depressive symptoms, and perceive social support, i.e., family members, friends and other significant people, respectively. Structural equation modeling was used to investigate the hypothesized mediation model. Results: ADHD symptoms exhibited a significant indirect effect on depressive symptoms via perceived social support. ADHD symptoms initially had a direct effect on depression; thereafter, it reduced to a non-significance effect after perceived social support was added. The total variance explained by this model was 35.2%. The mediation model with family support as a mediator showed the highest effect size. Conclusions: The study highlighted the importance of perceived social support, particularly family support, on depressive symptoms among young medical students experiencing ADHD symptoms. The model suggests promising relationships for further research on ADHD-related depression and potential treatment in the future. Attention deficit/hyperactivity disorder (ADHD) is a neuropsychiatric condition affecting preschoolers, children, adolescents, and adults worldwide. ADHD is characterized by a pattern of diminished sustained attention and increased impulsivity or hyperactivity. Patients with a diagnosis of ADHD might have trouble finishing tasks, sitting still or keeping track of things, appointments or details ,2. The pThe association between ADHD and depression has been well-documented ,13. HaviAnother hypothesis is related to social rejection; significant covariates for depressed mood resulted from disruptive behavior problems ,22. ChilMoreover, the parent–child relationship may mediate depression risk among children with a diagnosis of ADHD. Related studies have revealed that those children may perceive both parent–child rejection and hostility due to parental cognitions about child behaviors and parenting stress. Children with a diagnosis of ADHD require tremendous parental efforts, which may disrupt normative parenting behaviors. Parent–child relationships were vulnerable to disruption, and these parent–child problems increased the risk of depression ,25. In aSocial support is a well-known protective factor for depression in a variety of populations and clinical settings ,29,30,31The aforementioned evidence as well as related studies have revealed the association of perceived social support, mostly from the parents among children with a diagnosis of ADHD. Long-term studies in children with a diagnosis of ADHD have demonstrated that ADHD symptoms remain over time, at least into the emerging adult period ,34. One This survey was conducted among 124 first year medical students in Chiang Mai University, Thailand in 2017. Each participant provided written informed consent before completing the questionnaires, which included sociodemographic data and records related to parents. Inclusion criteria for eligible students were (1) age range from 16 to 21 years old; and (2) first year medical students. The only exclusion criterion was a refusal of the informed consent. They also completed questionnaires concerning Adult ADHD, perceived social support, and depressive symptoms. Ethics approval was obtained from the Faculty of Medicine, Chiang Mai University, Thailand, before taking any further steps in the research .The self-reported questionnaires consisted of sociodemographic and health-related characteristics including sex, age, and underlying diseases including mental health difficulties. Moreover, information on the parents’ education and parents’ occupation was collected.ASRS, developed by Kessler et al., is a six-item self-rated questionnaire evaluating ADHD symptoms using the DSM-IV-TR among adults. Among six items, four items measure inattention (items 1–4), and two measure hyperactivity  or a mediation model within the structural equation modeling (SEM) framework. The advantage of SEM over the path model is that SEM reduces random measurement errors, which is absent using the standard regression method . In thisBefore SEM analysis, data were examined for skewness, kurtosis, and multivariate normality. All variables were shown to have skewness and kurtosis within the acceptable range of <±3 ; the ordBased on the following determined input, anticipated effect size, 0.3; desired statistical power, 0.8; number of latent variables, 3; number of observed variables, 13–14 (including the covariates of age and sex). The probability level was 0.05, which yielded a sample size of 119 .MSPSS divided the sources of support into three sources. Referring to overall social support may not give sufficient information because each source may have a different impact at different stages of life. In particular, to children and adolescents, family support is important, while adults may find other significant people such as doctors and bosses important as well . We testFor each latent construct, four manifest indicators were considered (except Model 1). The items of the ADHD and PHQ-9 questionnaire were reduced to four parcels. The parcels were constructed according to Little et al. , whereup2/DF); the root mean square error of approximation (RMSEA); the comparative-fit index (CFI); and the Tucker–Lewis Index (TLI). For a good model fit, the ratio χ2/DF should be <3, and CFI and TLI should be higher than 0.95 [p < 0.05. All statistical analyses were performed using the program IBM SPSS, 22.0 and AMOS, Version 18 .To test for model fitness, the following model fit indices were employed: the minimum ratio of chi-square divided by its degrees of freedom . Other details are shown in Of 124 participants, most were female, with a mean age of 18.17 ± 0.7 years. One-third of the participants reported having allergies as an underlying disease. None reported having any mental health or psychiatric illnesses, including history of ADHD. Slightly over half of the participants (52.4%) were firstborn. A quarter of participants had ADHD symptoms based on the ASRS screener criteria. No significant difference between the ADHD symptoms and non-ADHD symptoms groups was observed, except for the number of siblings (p = 0.008), which then reduced  after MSPSS was added to the SEM. p = 0.152, Chi-square/df = 1.19. CFI = 0.971, TLI = 0.960, and RMSEA = 0.040 (90%CI = 0.000–0.071).Initially, the latent variable of ASRS had a significantly direct effect on the latent variables of PHQ-9 .For Model 2, the significant indirect effect of the latent variable of ASRS through overall perceived family members’ support (β = 0.116 (95% CI 0.037–0.256)); the variance explained by this model was 24.4%. The fit statistics of Model 1 were as follows: Chi-square = 74.352, degrees of freedom = 69, p = 0.111, Chi-square/df = 1.21. CFI = 0.977, TLI = 0.969 and RM = 0.042 (90%CI = 0.000–0.070).For Model 3, the non-significant indirect effect of the latent variable of ASRS through overall perceived friends’ support (β = 0.068 (95% CI 0.000–0.084)); the variance explained by this model was 25.3%. The fit statistics of Model 1 were as follows: Chi-square = 83.619, degrees of freedom = 69, p < 0.001, Chi-square/df = 1.67. CFI = 0.931, TLI = 0.908 and RM = 0.074 (90%CI = 0.049–0.097).For Model 4, the non-significant indirect effect of the latent variable of ASRS through overall perceived significant people’s support (β = 0.050 (95% CI 0.000–0.110)); the variance explained by this model was 19.8%. The fit statistics of Model 1 were as follows: Chi-square = 114.02, degrees of freedom = 68, This study aimed to examine whether perceived social support was able to mediate the relationship between ADHD symptoms and depressive symptoms among young adults, i.e., first year medical students, and the results supported our hypotheses. Notably, perceived family support was the only significant mediator compared with the rest, highlighting the importance of perceived family support among these emerging adults. Other sources of support existed even though they were non-significant. These findings support related studies conducted by Meinzer and colleagues regarding the significant role of parents . Their rIn fact, in older age participants such as in the present study, other sources of support, especially friends, should come into play, but this was not the case. The reason perceived as to why family support prevailed, despite the participants being emerging adults, may have been the fact that the participants live in a collectivistic society and are influenced by Thai family tradition; this influence is present in many cultures, especially in Asian countries ,51,52. CBased on related studies, however, perceived family support was vital for Thai medical students, especially first year students, implying that they maintained strong ties with their families ,55. BeinMoreover, based on psychological development, the significance of perceptions of friends and other significant people should be more pronounced in later years of study. Whether those supports have a sufficient effect to mediate the relationship between ADHD symptoms and depressive symptoms as does family support remains unknown, and this should be a subject of further investigation.A concern that should be raised here is what action to take when some students reported high scores on ADHD or depressive symptoms. No doubt these symptoms impact students’ wellbeing as well as their academic performance. The researcher is normally not allowed to provide direct interpretation according to the participant’s confidentiality . However, this concern is mitigated as students are usually informed about consultation services when they are made aware of having problems, instigated by those questionnaires.Perceived social support being associated with decreased depressive symptoms is possibly an innovative finding of this study. The strength of the present study is that it provides some insight about the different impacts of each source of social support.Some limitations should be noted here. (1) The sample size was relatively small, even though it met the minimal requirements for conducting SEM, and (2) it included only first year medical students. Further replication studies either within or outside medical students are strongly encouraged. (3) Some items of the ASRS scale could give a false-positive result, e.g., item 6 (high level of energy). If this self-report is to be used again, other psychiatric inventories should be added to help discriminate symptoms, e.g., hypomanic symptom. Notably, the study was conducted using the DSM-IV-TR because it represents the only validated version. However, the new ASRS screener using DSM-5 appears to have similar items. (4) This study did not have data to rule out the effect of psychoactive substance use, which can mimic inattention and hyperactivity symptoms. In addition, other related factors left unstudied include sleep problems. These issues should also be explored in further studies. (5) We cannot conclude any causal relationship from this cross-sectional design, so a longitudinal study in this population should provide more robust evidence in that regard.Lastly, this study represents only the relationship of ADHD symptoms and depressive symptoms, without definite diagnosis of ADHD and depressive disorders; we cannot confidently determine the magnitude of the mediated effect. Moreover, a further study should be conducted to examine the scale of ADHD, comorbid depressive disorders and its relationship among adolescents as well as Thai medical students.To the best of our knowledge, the present study has emphasized the importance of perceived family support among young adults assumed to be carried over from adolescence, as demonstrated before by related studies. The present study also adds some findings related to sources of support other than family members. This may be one of the earliest studies among medical students experiencing ADHD symptoms. Interestingly, however, exploring to what extent the ADHD symptoms would change and how the three sources of support would impact the relationship between ADHD symptoms and depressive symptoms, especially in later years in medical education in the longer term, is encouraged.