Mitotane, the only drug approved by the Food and Drug Administration (FDA) for the treatment of adrenocortical carcinoma, is associated with several side effects including neurotoxicity. The aim of our study is to investigate the relationship between mitotane plasma levels and neurological toxicity.We have considered five patients affected by adrenocortical carcinoma treated with mitotane. The neurological assessment included a neurological examination, an electroencephalogram, event-related potentials (P300), and a neuropsychological assessment. All of the patients were first considered at the onset of symptoms of neurotoxicity or when mitotanemia levels were above 18 mg/L, for the second time at mitotanemia normalization and subsequently at its further increase, or in case of persistent neurological abnormalities, some months after normalization.At the first neurotoxicity, four patients showed impaired neurological examination, electroencephalogram, and P300; three patients had impaired neuropsychological assessment; one patient, only P300. At mitotanemia normalization, the neurological examination became normal in all patients and electroencephalogram normalized in one patient, improved in another one, continuing to be altered in the other three. P300 latency and neuropsychological assessment normalized in two patients and persisted altered in the patient experiencing long-term mitotane toxicity. At the third evaluation, in the patient with prolonged mitotane toxicity, the normal mitotanemia in the previous 9 months restored P300 and improved the electroencephalogram but not the neuropsychological assessment. In the two patients experiencing a further rise of mitotanemia, neurological examination was normal but P300 and electroencephalogram were altered.The results of our study highlighted the presence of neurophysiological and neuropsychological abnormalities associated with mitotane values above 18 mg/L. Adrenocortical carcinoma (ACC) is a rare form of endocrine neoplasia with an estimated incidence of 1/1.5–2 million individuals per year. The prognosis of ACC is generally poor especially in the advanced stages, with a 5-year survival <15% for metastatic disease –5. PatieA retrospective/prospective monocentric study has been conducted in patients affected by ACC treated with mitotane. Since it is a rare disease, 10 patients affected by ACC are followed up in our department: three of them have no indication for mitotane therapy while the other seven take mitotane for adjuvant purposes or for metastatic disease. Among these seven patients, two were excluded from the study, since they were recently enrolled and have not yet reached therapeutic levels of mitotanemia nor have they developed neurological symptoms from drug toxicity. In this study, we have analyzed the preliminary retrospective results of the first five patients enrolled.The inclusion criteria for this study were as follows:- Patients diagnosed with ACC receiving mitotane therapy both for adjuvant purposes and in the presence of metastatic disease, associated or not with other anticancer treatments- Patients with suspected symptoms of neurological toxicity and/or detection of mitotanemia levels in the high limits or above the therapeutic range (>18 mg/L)- Patients signed an informed consent formThe exclusion criteria for this study were as follows:- Central nervous system disorders of other nature- Mitotanemia <18 mg/L with absence of neurological signs/symptomsAll of the five patients have been considered for the first time at the onset of signs/symptoms of neurotoxicity and/or when mitotane serum concentration was >18 mg/L. The neurological assessment included a neurological examination (NE), an EEG, event-related potentials (P300), and a neuropsychological assessment (NA).They have been tested a second time just at the restoring of normal mitotane plasma levels and subsequently at the time of a further increase in mitotanemia or, in case of persistent neurological abnormalities, some months after mitotanemia normalization.® service, a free-of-charge service offered by HRA Pharma to European prescribers and associated with the use of Lysodren®. Samples were collected in our center, sent to a centralized laboratory, extracted by precipitation with ethanol, and tested by a standardized gas chromatography/mass spectrometry method.Mitotane concentrations were measured by LysosafeMitotanemia blood levels have been measured routinely, in particular after approximately 30 days from the start of therapy with mitotane, and periodically, every 30–60 days and/or in any case according to clinical judgment. At the time of each mitotanemia assessment, clinical examination and blood laboratory tests including thyroid and adrenal function, liver and kidney function, and electrolytes were performed. These hematic tests were essential to discriminate against the nature of neurological symptoms, if present.EEG is one of the standard methods to measure electrical brain activity in many fields and the main source of signals for noninvasive brain–computer interfaces.In our laboratory, we use electrodes that get mounted at the scalp level with the help of an EEG cap, located in the standard position 10-20 . EEG recP300 is a cognitive potential belonging to long latency auditory evoked potentials. It corresponds to the largest positive wave after the N1-P2 complex registered by surface electrodes placed at the scalp level with international 10-20 system position. The P300 component is elicited by the recognition of a rare random deviant auditory stimulus  within a series of frequent similar auditory stimuli at 80 ± 10 Decibel Sound Pressure level (dB) that should be mentally counted. It can be used to identify the functional integrity, the level of impairment, or the degree of maturation of visual, auditory, somatosensory peripheral nerve pathways and of higher cognitive functions. Normal latency values of our laboratory are from 305 to 320 ms with no difference between the left and right ears , 13.Neurocognitive functions were assessed by a battery of standardized tests lasting approximately 60 min in order to balance brevity and sensitivity . Tests uFor cognitive measures, age-, gender-, and education-corrected scores and equivalent scores were calculated from the raw scores according to Italian normative standards. In order to accomplish data reduction for statistical purposes and following a hierarchical approach, individual test scores were grouped into seven cognitive domains: verbal learning (VL), short-term memory (STM), long-term memory (LTM), executive functions (EFs), abstract reasoning (AR), attention (A), and visuo-constructional abilities (VCAs).Participants were coded as “non-impaired” or “impaired” in each single domain according to a pathological equivalent score corresponding to 1,65 standard deviations below the average score of normative populations.Patient 1 is a 36-year-old woman who started mitotane therapy in August 2014 at the age of 29 after adrenalectomy for ACC. In 2016, she underwent stereotaxic radiation treatment for a lung metastatic lesion without discontinuation of mitotane. At the first neurological evaluation, in February 2021, mitotane blood levels were above the therapeutic threshold (23.9 mg/L) for more than 2 years at dose of 3 g daily. The NE showed an ideomotor slowdown and a mild short-term memory deficit. At the EEG, epileptic abnormalities were found, and the P300 exam showed an increased latency. At the NA, the patient showed a selective visual-constructive deficit and visual memory deficit. At the time of neurological toxicity, thyroid and adrenal function tests were normal, well compensated by replacement therapy with levothyroxine and hydrocortisone. An MRI was performed to exclude metastatic cerebral lesions. After 3 months, at the normalization of mitotane levels (17.4 mg/L) with a reduced dose of 1.5 g, the NE was normal and the NA slightly improved, with persistence of the same visuo-constructive deficit. The EEG and P300 assessment, on the other hand, were confirmed impaired. Only 9 months after mitotanemia normalization, the patient experienced a normal P300 and an improvement of EEG, even though slight bioelectric abnormalities persisted in occipito-temporo-parieto regions. Also, the selective visual-constructive deficit was confirmed at NA. At the last neurological assessment, maintaining mitotane levels within the therapeutic window for 20 months, in January 2023, the NE and P300 were normal, slight abnormalities were confirmed at the EEG as well as the visual-constructive deficit at NA, even milder than at the time of toxicity Figure 1Patient 2 is a 60-year-old woman who underwent a left adrenalectomy in 2017 for ACC and a resection of a liver metastasis in 2018. In 2019, a metastatic lesion in the iliac bone was treated with embolization and local radiotherapy. Mitotane treatment was started in 2018 and discontinued in 2019, then resumed in July 2020. In October 2020, mitotane levels were over the therapeutic threshold (26.8 mg/L) with a daily dose of 4 g, but neurological assessment was performed only after 4 months, in February 2021, when mitotane concentration was 24 mg/L at the dose of 2.5 g. The first NE showed a general slight slowdown, and the EEG was indicative of a slight slowing of the background activity with anomalies on the temporal-parieto-occipital regions. The latency of the P300 exam was at the upper limits of normal, and the NA showed a selective visual-constructive deficit. Thyroid and adrenal function tests were normal, well compensated by replacement therapy. Two months later, at the dose reduction to 1.5 g/day, mitotane levels dropped to normal range (17 mg/L); then, the NE, P300, and NA normalized, but the EEG showed only slight nonspecific anomalies without slowing down of the background activity. In March 2022, when mitotanemia dropped below the normal limit (12 mg/L), neurological assessment was completely normal, including EEG. At the latest neurological evaluation, in December 2022 (mitotanemia 18 mg/L), the NE and P300 were normal, whereas the EEG showed a slight slowdown of background activity with nonspecific anomalies in the temporo-parieto-occipital regions and the NA demonstrated a short-term verbal memory deficit Figure 2Patient 3 is a 50-year-old man who, in December 2019, underwent surgical removal of a 4-cm retroperitoneal mass diagnosed as ACC. Due to the presence of lung metastases at diagnosis, the patient was immediately initiated mitotane treatment (in April 2020) combined with cisplatin and etoposide chemotherapy (started in May 2020). After 8 months of mitotane therapy at a dose until 4.5 g, in November 2020, the patient developed a severe clinical neurological toxicity characterized by spatio-temporal disorientation and memory disturbances. During the NE, the patient presented with a mild disturbance of concentration and bilateral ideomotor apraxia. The EEG showed a diffuse slowing of brain electrical activity with mild diffuse brain distress. Blood levels of mitotanemia were increased (23 mg/L), then a discontinuation of treatment was necessary. A computed tomography scan ruled out the presence of brain metastases. The NA and P300 were not carried out at the time of clinical neurological toxicity. One month after the interruption of mitotane, when mitotanemia levels dropped to 9 mg/L, the NE, EEG, and NA showed no anomalies (P300 not performed). From March to June 2021, the patient underwent a further four cycles of chemotherapy with doxorubicin, cisplatin, and etoposide and, for progression of disease, in March 2022, a second-line systemic treatment with temozolomide, even in combination with mitotane, was started and it is still ongoing. In March 2022, mitotanemia levels increased to 31 mg/L without neurological symptoms. The NE was found normal as well as NA, but EEG showed again a diffuse slowing of electrical activity with mild diffuse distress, and P300 significantly increased. Mitotanemia normalized after 2 months (13 mg/L in May 2022), and a new neurological assessment was performed 7 months after this normalization (December 2022), when temozolomide therapy was ongoing, showing that P300 persistently increased, without anomalies of NE, EEG, and NA Figure 3Patient 4 is a 52-year-old woman who underwent left adrenalectomy and ipsilateral nephrectomy with paraaortic lymphadenectomy in November 2019 for ACC. She started adjuvant mitotane from February 2020 and, in November 2020, the drug blood levels were raised to 21.6 mg/L at a mitotane daily dose of 2.5 g. The NE revealed slower thinking, difficulty in concentration, and confusion; the EEG showed slight nonspecific anomalies; P300 was slightly increased; and at NA, a selective visual-constructive deficit and an impairment of visual memory and executive functions were found. In February 2021, she underwent surgical removal of a relapsing lesion in the left adrenal region. She was reassessed in April 2021, at the second cross-threshold mitotanemia finding (24 mg/L), and the same previous abnormalities at NE, EEG, P300, and NA were confirmed. Mitotanemia normalized in June 2021 and remained in the therapeutic range until January 2022, when the patient completed chemotherapy with cisplatin, doxorubicin, and etoposide that started in July 2021 for progression of disease, and neurological assessment was repeated. A collateral neurotoxicity characterized by cognitive and somatosensorial disturbances, associated with ataxia, deficit of attention and concentration, and bilateral hearing loss were evident at NE. Mitotanemia was 16 mg/L, the EEG showed slight nonspecific anomalies, NA was normal, but P300 was not performed. Due to abdominal disease progression, the patient underwent surgical removal in January 2023 of two massive abdominal recurrences of ACC, temporarily discontinuing mitotane therapy from December 2022 to February 2023. In washout of mitotane and chemotherapy, NE was normal as well as P300 and NA, while at EEG, slight nonspecific anomalies in the bilateral fronto-centro-temporal regions persisted Figure 4Patient 5 is a 33-year-old woman, the youngest patient of this series, who in April 2021 underwent left adrenalectomy for a 6-cm ACC lesion. Due to a possible incomplete resection (fragmented neoplasm and therefore not fully assessable), the patient underwent radiotherapy on the tumor bed and on adjacent lymph nodes and started mitotane treatment in an adjuvant setting in April 2021. Drug blood levels did not reach the therapeutic range until February 2022. In March 2022, mitotanemia reached 18.5 mg/L at a dose of 6 g and NA was performed: NE, EEG, and NA were all normal but P300 was increased. The same abnormalities were demonstrated in June 2022 when mitotanemia levels were further raised to 20 mg/L. In December 2022, when drug blood levels decreased to 10 mg/L, also P300 returned to perfectly normal.The use of mitotane as adjuvant therapy in ACC is still controversial also due to the absence of randomized controlled clinical trials that can support its effectiveness. Since the recurrence rates and the onset of distant metastases are very high, it is often necessary to associate conventional chemotherapy with etoposide, doxorubicin, and cisplatin with mitotane. A recent meta-analysis  includinDespite a long-standing clinical use, the molecular mechanisms underlying mitotane efficacy are still poorly understood. Mitotane exerts its action through a selective damage of the corticoadrenal tissue, particularly of the fasciculated and reticular area, less frequently of the glomerular area . RecentlA recent study found that mitotane and its main metabolite bind to lipid membranes by inserting into the lipid–water interface of the bilayer and causing an alteration of the membrane integrity, leading to an increased permeability for polar molecules. This could help to better explain even the side effects of this drug .Mitotane is a lipophilic drug with a low absorption rate and an extremely long half-life , 26. TheMitotane blood concentrations should be maintained between 14 and 20 mg/L because above this , the drug is toxic . MitotanA significant increase in neurological toxicity has been reported when plasma mitotane levels exceed 20 mg/L. The French National pharmacovigilance database reported from 2004 to 2016 14 cases of neurological adverse events including asthenia, drowsiness, memory disorders, confusion, headaches, and space–time disorientations . There a2/day) and with drug plasma concentration below the therapeutic range . Even patient 4 complained of confusion, dizziness, and instability in walking, but such symptomatology was much more nuanced and fluctuating during the course of therapy. The patient with longer-term mitotane toxicity never complained of neurological symptoms, although at the first evaluation, all of the tests were strongly altered probably due to the chronic excess of mitotanemia (patient 1). Patient 2 and patient 5 did not have any neurological symptoms even with drug blood levels above the threshold. Patient 5 reached the therapeutic window with difficulty and slowly, even with a dose of mitotane up to 6 g daily and at a drug level of 18.5 mg/L, the only impaired test was the P300, immediately reversible after the normalization of mitotanemia.We have confirmed that neurological toxicity can occur at any dose of mitotane, although a more serious and persistent toxicity is associated with higher doses. Patient 3 developed neurological symptoms with a dose of 4.5 g, while patient 1, in whom the most serious and persistent instrumental alterations were found for a long time, had been treated in the last 4 years with daily doses between 4.5 and 3 g.We demonstrated that the NE, generally characterized by disturbance of concentration, confusion, slower thinking, ideomotor slowdown, and mild short-term memory deficit, and in worst case by bilateral ideomotor apraxia, was impaired when mitotanemia levels were well beyond the threshold (from 21 to 24 mg/L) and normalized rapidly, as soon as the drug blood levels returned to normal.The EEG is the most sensitive examination and was found to be altered even for mitotanemia levels at the upper normal limits (18 mg/L). The scale of alteration proved to be proportional to the level of toxicity, since in patient 1, after 7 years of therapy and 2 years of toxicity, the EEG showed even epileptic abnormalities. The EEG alterations were always reversible except in patient 1 in whom the epileptic abnormalities persisted despite the normalization of mitotanemia and only an improvement but not a complete normalization of the EEG was observed even 20 months after the restoration of mitotanemia values in the therapeutic window. In one of the five patients, the EEG was always normal, even in the presence of mitotanemia values over the threshold, and this can be explained by the young age of the patient or related to the difficulty in reaching the therapeutic range of the drug and consequently with a probable low tendency to accumulate in the brain tissues.Among the neurological instrumental assessments, we evaluated for the first time, in patients with neurological mitotane toxicity, the P300. The P300 test was always altered in all of the five patients at drug levels above 20 mg/L and normalized along with mitotanemia except in patient 1 in whom it was restored only 9 months after the normalization of mitotanemia. The P300 resulted increased despite a mitotanemia in range in patient 3, and we can hypothesize a toxic effect of the concomitant therapy with temozolomide.Regarding the neuropsychological aspect, the NAs were altered in three out of five patients, at the time of toxicity and for mitotanemia values >18 mg/L, but normalized with mitotanemia values, except for patient 1, also probably influenced by persistently high levels of drugs in the previous years. Interestingly, and in agreement with previous literature data by Bollen and Lanser , the neuAt baseline assessment, patients struggled to set up the task, adopting a poorly functional copying strategy (element by element), without an evident execution plan and with poor overall results. Interestingly, copying strategies often improved with normalization of mitotanemia values. Improvement was particularly evident from a qualitative point of view, thus suggesting that performance gain could not be based on practice effect alone. Visual memory, executive (patient 4), and short-term verbal memory deficit (patient 2) occurred less frequently.The occurrence of neurological side effects has not been associated with electrolyte disturbances, thyroid function alterations, or hypoadrenalism, all of which are adverse events of mitotane therapy. All patients, in fact, despite the occurrence of iatrogenic hypothyroidism and hypoadrenalism, had normal levels of thyroid and adrenal hormones, well compensated by replacement therapy at every time of neurological assessment. None of the patients was taking drugs interfering with mitotane metabolism, especially those metabolized by cytochrome P450 (three patients were taking rosuvastatin or ezetimibe for mitotane-induced hypercholesterolemia).Although the molecular mechanisms underlying mitotane neurological toxicity are still poorly understood, we can speculate that the lipophilicity of the drug may play a role in facilitating across the blood–brain barrier. Additionally, the central nervous system, rich in lipid, can favor the accumulation of lipophilic molecules such as mitotane, but the amount of this accumulation is not yet currently measurable. Nevertheless, it could be hypothesized that the neurotoxic damage could be due to a direct cytotoxic drug-effect on the myelin sheaths, since it has been demonstrated that mitotane can alter the lipid membrane structure .The major limitation of this study is the low number of patients, which prevented reaching precise conclusions but allowed us to generate hypotheses that provide input to further investigations and studies with a larger number of patients. Unfortunately, not all of the tests were carried out in all of the patients, for logistic reasons, at the time of toxicity and at the time of its resolution. Due to the rarity of the disease, the use of chemotherapeutic agents was not an exclusion criterion, then the neurotoxicity of concurrent chemotherapeutic agents cannot be excluded. We aim to continue this prospective study by enrolling more patients in order to provide additional data regarding the neurological toxicity of mitotane. Mitotane pharmacogenetics could be an additional tool to maintain drug concentrations in the therapeutic range and adjust the right dose to each patient. The detection of the allelic cytochrome CYP2B6 variants could add useful information for both therapy optimization and evaluation of a possible association between them and neurotoxicity; this could be a starting point for further studies.In this study, we confirmed that mitotane toxicity is characterized by neurophysiological and neuropsychological abnormalities.We also demonstrated how the short-term mitotane toxicity results in reversible neurological alterations, while, in cases of years-long toxicity, they can persist for a long time and some of them (as EEG and NA) could be irreversible and significantly affecting the patient’s quality of life.This study confirmed the importance of frequent monitoring of mitotanemia, which is currently the only way to avoid long periods of overtreatment and to prevent neurological toxicity that could be irreversible. We demonstrated that EEG and NA are very sensitive as indicators of neurological damage, since they can be altered at mitotane blood levels >18 mg/L. For this reason, we should consider a new mitotane therapeutic threshold of 18 mg/L in order to minimize the possibility of neurological toxicity. The discovery of these neurological anomalies, particularly the visuo-constructive deficit, could lead to conducting studies that can better investigate the mechanism of action of this drug, still unknown in some respects, and the possible accumulation in lipidic tissues and in specific brain areas.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: GARRbox.The studies involving humans were approved by the Ethics Committee of IRCCS Regina Elena National Cancer Institute. The studies were conducted in accordance with the local legislation and institutional requirements. The participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.MM, EG, and AP contributed to conception and design of the study. MM, EG, MB, RL, GPu, AT, UA, GS, GPe, and GG collected and interpreted the data. MM wrote the first draft of the article. EG and AT wrote sections of the article. MA reviewed and supervised the article. All authors read and approved the final article.


The obesity prevalence is growing worldwide. There is strong evidence indicating that a disturbance of zinc, copper and magnesium concentrations is associated with the development of obesity and its related diseases. Our aim was to determine the effect of curcumin supplementation on serum zinc, magnesium and copper in obese individuals. In this randomized crossover trial study, thirty obese patients with an age range of 18 to 65 years were randomized to treatment with curcumin 1 g/day or placebo for 30 days. There was then a two-week wash-out period, after which, subjects crossed to the alternate regimen. Serum levels of zinc, copper and magnesium were determined at baseline and at the end of the study.The study groups were similar to each other in base line characteristics. We did not observe significant impacts (p>0.05) of curcumin on Cu, Zn, Mg serum concentrations.Curcumin administration at a dose of 1 g/day for 30 days did not affect serum Cu, Zn, Mg levels in obese subjects. In addition, obesity is known as an independent risk factor for type 2 diabetes, dyslipidemia and cardiovascular diseases (CVDs) . Due to poor nutrition in countries with obesity and overweight, lack of some micronutrients  and vitamins were reported . Studies show the important role of trace elements in human health. Copper and zinc are two essential elements in the diet that are involved in many biological reactions . They act as cofactors in enzymatic reactions and show an essential role in various biochemical and metabolic processes in humans . Also, magnesium is the 4th most abundant cation in the body and acts as a cofactor . Concerning the investigation of obese individuals with suboptimal levels of serum zinc, copper, and magnesium was essential for disease diagnosis. Delays in diagnosis for copper deficiency result in disability of residual neurological in patient . According to these studies, copper as well as zinc and magnesium has an essential role in lipid metabolism . Serum copper level is lower in obese subjects than healthy subjects based on previous studies .During recent decades, obesity has been considered an important threat to public health . Obesity is defined as a body mass index Some studies have reported that zinc, copper and magnesium have antioxidant properties. Also, both zinc and copper are cofactor for metalloenzymes and antioxidant enzymes .Curcumin is a polyphenolic compound and a natural yellow pigment, also called turmeric . Turmeric has the most active natural biological properties include anti-inflammatory, antioxidant, antimicrobial, anti-cancer, anti-diabetic, anti-microbial, fat modifier, anti-arthritis, analgesic, anti-ischemic and anti-depressant activities . Curcumin also exerts antioxidant activities through various mechanisms such as inhibition and elimination of reactive oxygen species (ROS) and modulation of enzymatic antioxidants and non-enzymatic ones . The US Food and Drug Administration (FDA) stated the limits of safe consumption levels to a maximum of 20 mg . Despite its medicinal properties, curcumin is chemically unstable, so researchers are now looking for a suitable formulation to improve its bioavailability .Regarding the association of curcumin with oxidative stress, the study aims to assess the effect of curcumin on zinc, magnesium and copper serum levels in obese individuals.Study Design2. Anthropometric indices were measured using standard protocols for all participants and they completed a questionnaire including demographic information.This study was a randomized clinical trial approved by the Mashhad University of Medical Sciences (ID: 960443) (IRCT2013082914521N1) . Thirty obese individuals aged 18 to 65 years who referred to Nutrition Department of Ghaem Hospital, Mashhad, Iran, were recruited from August 2010 to August 2012. All individuals signed written informed consent. The Exclusion criteria were breast feeding, pregnancy, suffering from systemic diseases. Obesity was defined as a BMI>30 kg/mClinical trialComputer randomization was used to make random distribution to groups. To implement the random allocation sequence, we used sequentially numbered sealed envelopes that was opened by someone not involved in the project. After assignment to intervention, the participants, clinical research staff and statistician were blinded. A non-researcher coded the capsules containers as A and B and this stayed confidential until data analysis. Curcumin capsules included 5 mg piperine plus 500 mg curcuminoids, were used  . The placebo capsules containing 5 mg of piperine were similar in shape and color to the curcumin ones. All participants received curcumin or placebo 1 g/day for a period of 30 days (n=15 in each study group). Then, the subjects were crossed over to the alternative regimen after a two-week wash-out period. In other words, there are two general phases in this project (before and after the wash-out period), in which the two groups participate including “Group 1: The patients received curcuminoids and then crossed over to the placebo; and Group 2: The patients received placebo and then crossed over to the curcuminoids.”Serum biochemical variablesBlood samples were taken four times from every individual after 12-hr fasting at baseline and after a period of 30 days consumption at two general phases; before and after the wash-out period. Serum magnesium, zinc and copper levels were assayed using commercial kits by auto analyzer .Statistical analysisThe power value of the study was 0.72 as measured using R software version 4.0.3. The Mann-Whitney U test and t-test were used for non-normally and normally dispersed factors, respectively. The period, treatment and carry over effects were evaluated for 2X2 cross-over study. A two-sided p-value of <0.05 was considered significant.As This randomized double-blind crossover trial study has been the first trial one that investigated the effect of curcumin on serum Zn, Cu and Mg levels in obese people. Today, obesity is considered a serious threat to health. Studies have shown a negative relationship between serum Mg and Zn levels and obesity . A meta-analysis study suggested that a higher serum copper concentration may be associated with obesity in children and adults . Copper and zinc as two essential trace elements play an important role in biological processes so that they may have a significant impact on the pathogenesis of metabolic diseases. In a study, zinc and copper deficiency were associated with a higher risk of CVDs and diabetes, and an imbalance between Zn and Cu levels led to oxidative stress and insulin resistance . Darroudi et al., demonstrated that serum zinc and copper were altered in Iranian adults who were metabolically obese but in normal weight. They suggested that copper and zinc were the strong risk factor for metabolic syndrome (MetS) in normal weight individuals  .Curcumin is able to exert antioxidant effects by reducing oxygen and nitrogen free radicals or modulating the cellular defense system . Also, curcumin anti-inflammatory and antioxidant properties can prevent the progression of inflammatory reactions . Obesity is considered a low-grade chronic metabolic inflammation, and empirical evidence has shown that curcumin is effective in reducing the incidence of obesity-related diseases .There is little evidence about the effect of curcumin on trace element levels in obese individuals. According to the results of the present study, consumption of 500 mg of curcumin twice a day, with the elimination of confounders, had no significant effect on magnesium, zinc, copper, and zinc to copper ratio in obese people. Consistent to our results, a study on 30 obese patients showed that a daily intake of 1 g/day of curcumin together with black pepper at a dose of 10 g/day for four weeks did not have a significant effect on copper and zinc, however, it was associated with a significant increase in zinc/copper and decrease in copper to zinc ratio . In a double-blind clinical trial on 120 individuals with metabolic syndrome, it was shown that after daily consumption of 1 g of curcumin and phospholipidated curcumin for 6 weeks, Zn concentration and Zn/Cu levels were significant and Zn was increased so that in the phospholipidated curcumin group, it was higher, but there was no significant effect on Cu . We have previously reported that phospholipidated curcuminoids (1 g per day), for a period of 6 weeks, does not have effects on serum cholesteryl ester transfer protein level , or sleep-duration  in subjects with MetS.The difference between our study and other studies can be due to the dose and duration of curcumin intake in obese people. According to studies, doses above 12 g per day are safe and tolerable for humans and only mild side effects have been reported . However, in this study, a dose of 1 g per day was investigated.In this study, we investigated the effect of curcumin on serum Cu, Zn and Mg levels in obese people. Our results demonstrated that curcumin did not affect serum Cu, Zn, Mg concentrations in obese subjects. Since, the evidence about the effect of curcumin on trace elements concentrations in obese individuals is little, it is suggested to investigate the association of these factors in future studies.The authors have declared that there is no conflict of interest.


Attention-deficit hyperactivity disorder (ADHD) is a neurodevelopmental condition typified by inattention, hyperactivity, and impulsivity. Comorbid psychiatric disorders are common among children and adolescents with ADHD. In this study, it was aimed to examine anxiety and somatic symptoms in children and adolescents with ADHD and the effect of methylphenidate treatment on these symptoms.  Three groups were formed, consisting of 37 children and adolescents diagnosed with ADHD and received methylphenidate treatment, 37 newly diagnosed, treatment-naive children and adolescents with ADHD diagnosis, and 37 children and adolescents without the diagnosis of ADHD. The symptoms of ADHD in children were examined by using the DSM-IV-based child and adolescent behavior disorders screening and rating scale, the symptoms of anxiety were examined by using the screen for child anxiety-related disorders (SCARED), and somatic symptoms were examined by using the DSM-5 level 2 somatic symptom scale.  In the newly diagnosed, treatment-naive with ADHD group, anxiety and somatic symptoms were found to be significantly higher compared to the ADHD group with methylphenidate treatment and the non-ADHD group. It was shown that the symptoms of panic disorder, generalized anxiety, and social phobia were observed more in the newly diagnosed, treatment-naive with ADHD group compared to the treatment group with ADHD.  It was determined that children and adolescents diagnosed with ADHD had more anxiety and somatic symptoms. Anxiety and somatic symptoms increased as the severity of ADHD symptoms increased. Anxiety and somatic symptoms were lower than in ADHD children receiving methylphenidate treatment. Clinicians should keep in mind to evaluate anxiety and somatic symptoms in children and adolescents with ADHD before the treatment. Attention-deficit hyperactivity disorder (ADHD) is identified as a neurodevelopmental disorder typified by symptoms of inattentiveness, hyperactivity, and impulsiveness. These symptoms negatively affect the social, school, and work-related activities . The preComorbid psychiatric disorders accompany 40-80% of children with ADHD . JehnsenThe incidence of anxiety disorder is 6.5% in children and adolescents . The coeSomatic symptoms are common in children and adolescents . It was Psychostimulants are the first choice in the treatment of ADHD . StudiesDue to children and adolescents with AHDH diagnosis having anxiety and somatic symptoms, and to our knowledge, there are limited studies on this issue, it was aimed to examine the frequency and dispartion of anxiety and somatic symptoms in children and adolescents with ADHD. It was also aimed to investigate the methylphenidate treatment effect on anxiety and somatic symptoms in children and adolescents with ADHD.A total of 111 children and adolescents, including 37 newly diagnosed, treatment-naive children and adolescents with ADHD between the ages of 6 and 15, 37 children and adolescents diagnosed with ADHD receiving only methylphenidate (extended-release or immediate-release formulations) treatment for at least six months, and 37 children and adolescents without the diagnosis of ADHD as the control group, participated in the current study. Newly diagnosed, treatment-naive children with ADHD did not use any psychotropic medication. A psychiatric interview based on DSM-5 was held by a child and adolescent psychiatrist with all participants. All participants' parents filled out the DSM-IV-based child and adolescent behavior disorders screening and rating scale in order to assist the diagnosis of ADHD. The sociodemographic data form was given to parents to obtain the sociodemographic data. The parents filled out the screen for child anxiety and related disorder- (SCARED-) parent form to examine the symptoms of anxiety and the DSM-5 level 2 somatic symptom scale-parent form to examine somatic symptoms. Those with a score of 25 and above in the SCARED were excluded. Somatic symptoms were also evaluated by clinician in all participants. The patients who had an IQ score of less than 80, known neurological, genetic, and chronic medical diseases, and used medication related to it were not included. Also, having mental retardation, bipolar disorder, and psychosis were determined as exclusion criteria in all groups. The study was approved by the Trakya University Local Ethics Committee (TÜTF-BAEK 2019/400). All participants were informed about the current study, and their written consent was obtained.It is a 41-item scale developed by converting the DSM-IV diagnostic criteria into a question form. The scale has 9 items examining attention deficit, 6 items examining hyperactivity, 3 items examining impulsivity, 8 items examining ODD, and 15 items examining CD. To confirm the diagnosis of ADHD, at least 6 of 9 items questioning attention deficit should be rated as 2 or 3, or at least 6 of 9 items questioning hyperactivity and impulsivity should be rated as 2 or 3. The Turkish validity and reliability study of this scale has been carried out by Ercan et al. .It consists of 41 items and 5 subscales . It is accepted that a score of 25 and above in the SCARED is a warning for anxiety disorder. Its Turkish validity and reliability study has been carried out by Çakmakçı et al. .The parent form for children aged 6-17 is a Likert-type scale that evaluates the somatic symptoms in the last seven days. The total score is between 0 and 26, and higher scores indicate more severe somatic symptoms. Its Turkish validity and reliability study has been carried out by Sapmaz et al. .U and Kruskal-Wallis tests were used for the analysis of quantitative independent data. For analyzing qualitative independent data, the chi-square test was used. The relationship between the variables was analyzed using the Spearman correlation analysis. IBM SPSS 27.0 was used. The limit of significance was chosen as p < 0.05 for all statistics.Mean, median, minimum and maximum values, standard deviation, and percentage were used in descriptive statistics. The Mann–Whitney p > 0.05). Maternal educational level was lower in the newly diagnosed, treatment-naive with ADHD group (p > 0.05). There was no significant difference between the groups in terms of paternal educational level (p > 0.05). The distribution of the income levels of the families was similar in all groups (p > 0.05)  .p < 0.05). The total score was significantly higher in the newly diagnosed, treatment-naive with ADHD group compared to the treatment group with ADHD (p < 0.05)  .p < 0.05). When subscale scores were compared, the scores for panic/somatic symptoms, generalized anxiety, and school phobia were demonstrated to be higher in the newly diagnosed, treatment-naive with ADHD group compared to the treatment group with ADHD (p < 0.05). No significant difference was indicated in the panic/somatic symptoms, separation anxiety, generalized anxiety, and school phobia subscale scores between the treatment group with ADHD and the non-ADHD group (p > 0.05)  .The total score of SCARED is found to be significantly higher in boys than girls in the newly diagnosed, treatment-naïve with ADHD group. No significant difference was found between girls and boys in the total score of SCARED in the treatment group with ADHD and the non-ADHD group.p < 0.05). No significant difference was indicated between the ADHD group with treatment and the non-ADHD group (p > 0.05)  .No significant difference was found between girls and boys in the total score of the DSM-5 level 2 somatic symptoms scale in all groups.p = 0.002). A significant correlation was found between somatic symptom scale total scores and the SCARED total score (p = 0.001)  .It was observed that the children and adolescents with ADHD had more anxiety and somatic symptoms compared to the non-ADHD children and adolescents in this study which is compatible to the literature. Both anxiety and somatic symptoms were significantly higher in the newly diagnosed, treatment-naive with ADHD group compared to the children and adolescents diagnosed with ADHD receiving methylphenidate treatment. It was observed that anxiety and somatic symptoms increased as the severity of ADHD symptoms increased. In a study conducted with 102 children with ADHD, it was shown that while externalizing symptoms were mostly observed in hyperactive-impulsive type ADHD, anxiety, depression, delinquent behavior, and internalizing symptoms were mostly observed in the combined type. Somatic complaints were more common in boys .The total score of SCARED was significantly higher in the newly diagnosed, treatment-naive with ADHD group compared to the treatment group with ADHD and the non-ADHD group in this study. The symptoms of generalized anxiety, panic disorder, social phobia, separation anxiety, and school phobia were shown to be higher in the newly diagnosed, treatment-naive with ADHD group compared to the non-ADHD group. It was shown that the symptoms of panic disorder, generalized anxiety, and social phobia were observed more in the newly diagnosed, drug-naive ADHD group compared to the ADHD group with treatment. The ADHD group with treatment had more social phobia symptoms compared to the non-ADHD group. Souza et al. demonstrated that 23.5% of the children with ADHD were accompanied by anxiety disorders, and the generalized anxiety disorder (GAD) is the most common accompanying anxiety disorder by 12.8%, followed by social phobia by 3.84%, and separation anxiety by 3.8% in their study . In anotIt was shown that anxiety symptoms increased as the severity of ADHD symptoms increased in our study. Taurines et al. showed that the coexistence of ADHD diagnosis and anxiety disorders is associated with more attention problems, school phobia, mood disorder, and lower social ability compared to anxiety alone or ADHD alone . Most ofIn the current study, somatic complaints were observed to be significantly higher in the newly diagnosed, treatment-naïve children and adolescents with ADHD. Somatic complaints were observed to be less in the ADHD treatment group compared to the newly diagnosed, treatment-naive with ADHD group in the current study. Ahmann et al. demonstrated that irritability, staring, daydreaming, nail-biting, and anxiety were reduced by short-acting methylphenidate treatment. In the same study, appetite disturbance, stomachache, insomnia, headache, and dizziness increased with methylphenidate . BarkleyThere is no significant difference between girls and boys in terms of somatic symptoms in this study. In a study, it was reported that generalized anxiety, phobia, and being a girl were risk factors for somatic symptoms in children with ADHD, and children with ADHD and somatic symptoms had more anxiety symptoms . Also, cThe small sampling is the first limitation of this study for affecting the generability of the results. More studies conducted with larger sampling are needed. The data were obtained only from the parents. The data could be obtained from children and their teachers to increase the data diversity, so this is the second limitation. Making comparisons between three groups is the strength of the study. In conclusion, it was shown that children and adolescents with ADHD had more anxiety and somatic symptoms than children and adolescents without ADHD, and children and adolescents with newly diagnosed, treatment-naive with ADHD had more anxiety and somatic symptoms in this study. Less anxiety and somatic symptoms in children with ADHD who receive methylphenidate treatment may show the protective effect of ADHD treatment from comorbid conditions. Although it is known that somatic side effects are observed with psychostimulant treatment, the clinician should keep in mind that somatic symptoms may also be present before the initiation of the treatment.Based on the findings of current research, clinicians should evaluate and consider anxiety and somatic symptoms in children and adolescents with ADHD while planning and monitoring methylphenidate treatment. Future studies with larger sampling are needed to investigate anxiety and somatic symptoms in children and adolescents with ADHD and the effect of methylphenidate treatment on these symptoms due to mentioned limitations of this study.


Objectives: To assess the incidence of prenatally diagnosed isolated single umbilical artery (iSUA) and its impact on major pregnancy outcomes, as well as to investigate potential risk factors. Materials and methods: A prospective study of singleton pregnancies, undergoing routine anomaly scans at 20+0–24+0 weeks of gestation, was carried out from 2018 to 2022. The effect of sonographically detected iSUA on small-for-gestational-age neonates (SGA) and preterm delivery (PTD) was evaluated using parameterized Student’s t-test, nonparametric Mann–Whitney U test and the chi-square test. Multivariable logistic regression models were implemented to assess the independent association between iSUA and the main outcomes, as well as with potential risk factors, while adjusting for specific confounders. Results: The study population included 6528 singleton pregnancies and the incidence of prenatally diagnosed iSUA was 1.3%. Prenatally diagnosed iSUA had a statistically significant association with both SGA neonates  and PTD , while no association was identified between this sonographic finding and preeclampsia. With regard to risk factors, conception via assisted reproductive technology (ART) was associated with increased risk of iSUA , while no other independent predictor for the development of this anatomical variation was identified. Conclusions: Prenatally diagnosed iSUA seems to be associated with a higher incidence of SGA and PTD and is more common in pregnancies following ART, which constitutes a novel finding. Single umbilical artery (SUA) is a variation of the umbilical cord anatomy where there is only one artery around the fetal bladder and in the fetal umbilical cord , whereasWhile an apparently isolated SUA (iSUA) is not strongly correlated with chromosomal abnormalities, the coexistence of one or more anatomical fetal defects increases the risk of aneuploidy, especially trisomy 18 . NotablyTherefore, the aim of this prospective study was to investigate the impact of diagnosing iSUA during the prenatal period; we aimed to assess the risk factors and possible adverse pregnancy outcomes of these cases in a well-designed setting and offer exact and trustworthy effect estimates for potential associations.A prospective study was carried out from June 2018 to June 2022, at the Third Department of Obstetrics and Gynecology, School of Medicine, Aristotle University of Thessaloniki, Greece. The study’s design and reporting adhered to the STROBE checklist .+0–24+0 gestational weeks were eligible to participate in the study. Pregnancies with aneuploidy or congenital anomalies, as well as with miscarriage or termination of pregnancy and those with missing data were excluded from the analysis. Maternal–fetal medicine specialists of the Department performed all the scans. Pregnancies diagnosed with iSUA were compared to those with a three-vessel cord, serving as study and control groups, respectively. The diagnosis of SUA was performed by direct visualization of the umbilical cord, or by tracking the umbilical arteries around the fetal bladder with color Doppler, as recommended by the International Society of Ultrasound in Obstetrics and Gynecology [All women experiencing a singleton pregnancy and undergoing routine anomaly scan at 20necology ; all theAll the participants authorized the use of their anonymized files for the study via a written consent form. The bioethics committee of the School of Medicine at the Aristotle University of Thessaloniki, Greece, approved the study protocol (3.188/2-5-2018). It should be noted that there were no benefits offered for participating in the study.In the context of this study, medical and obstetric parameters were collected. All the relevant covariables used in our analysis, including sociodemographic characteristics , were collected during the recording of the maternal history or directly through our database.Our study’s analyses focused on SGA, defined as a birthweight below 10 st., and preterm delivery (PTD), defined as birth between 22 and 37 weeks of gestation. We also investigated the impact of iSUA on preeclampsia, as well as the possible risk factors that could be correlated with the presence of iSUA.During the routine anomaly scan, uterine artery pulsatility index z-score (UtA PI z-score) was recorded as per protocol , as wellt-test and nonparametric Mann–Whitney U test for continuous variables and the chi-square test for the categorical ones. The continuous variables are described as means and standard deviation when normally distributed, and otherwise as medians and interquartile ranges. Population and percentages are used to represent categorical variables.Comparisons between groups were carried out using the parameterized Student’s Taking into consideration the known and available confounders, associations with the two primary outcomes, SGA and PTD, were investigated using both univariate and multivariable logistic regression models ,14. Of nTo account for confounding and provide more accurate effect estimates, a risk factor analysis using a multivariable logistic regression with the iSUA as the dependent variable was carried out. The backward stepwise selection method was used to identify the risk factors with a significant prognostic value for iSUA.The TRIPOD recommendations were applied while conducting and reporting the statistical analysis . All anan = 132), miscarriage (n = 14), pregnancy termination (n = 24) and aneuploidies or congenital anomalies (n = 45) (p = 0.025) and higher rates of SGA  and PTD . Moreover, iSUA was associated with lower median values for gestational age  and lower median birthweight . Regarding preeclampsia, 6 women out of 83 in the study group (7.2%) and 480 women out of 6445 in the control group (7.4%) were diagnosed with it; no association between iSUA and preeclampsia was detected (p = 1.0)  . Prenatap = 1.0) .The univariate logistic regression model found that iSUA was associated with twice the risk for SGA neonates compared to singleton pregnancies with a three-vessel cord . The association persisted in the multivariable model, pointing to a 1.9 times higher probability for SGA . Increased BMI and multiparity were important confounders, reducing the risk of SGA, while current smoking and an increased UtA PI-z score had a positive association with SGA. A higher probability for PTD was also identified in the study group compared to the controls, both in the univariate  and the multivariable analysis . Regarding PTD, six variables were identified as significant confounders; increased BMI, current smoking, ART, increased UtA PI z-score, previous history of 1st trimester miscarriage and previous history of PTD, all having a positive association with the risk of PTD .Regarding risk factor analysis, conception via ART was found to be the only important independent predictor, as it increases the probability of iSUA by 2.2 times according to the multivariable analysis  .The variance inflation factor was calculated among all the variables in every model; each value was close to 1, ruling out the possibility of multicollinearity. There were also no influential observations that could affect our results.The main findings of this study were the following: (1) the incidence of prenatally diagnosed iSUA among singleton pregnancies is 1.3%, (2) there is a statistically significant association between iSUA and both SGA and PTD, suggesting that the prenatal diagnosis of this anatomical variation has clinical significance and should probably prompt closer surveillance in the third trimester, and (3) the only independent risk factor for the development of iSUA, constituting a novel finding, is conception via ART.p < 0.001) and SGA  [p  = 0.009) [Prenatally diagnosed iSUA affects approximately 1 in 100 singleton pregnancies, according to the findings of the present study; this is in accordance with the incidence calculated in a meta-analysis including 11 studies, which was equal to 1.2% . Our stu< 0.001) , and a c= 0.009) . With re= 0.009) . Another= 0.009) ,31. An o= 0.009) . In addi= 0.009) . A study= 0.009) . However= 0.009) . Further= 0.009) .p = 0.002), rendering this sonographic finding an independent risk factor for PTD  [Regarding PTD, the iSUA group had an almost doubled likelihood of having PTD compared to the controls. This finding is consistent with the existing literature; the most recent systematic review and meta-analysis showed that prenatally diagnosed iSUA carries a higher risk of PTD . Several= 0.002) . Another= 0.002) . Possibl= 0.002) ,38. Thusp = 0.0006) [p = 0.05) [As for preeclampsia, in the present study, no statistically significant association was identified between this pregnancy complication and iSUA. This finding is quite intriguing and in conflict with the most recently published meta-analysis on this topic, which found that iSUA is associated with an increased risk of pregnancy-induced hypertension (PIH)  . Another = 0.05) . It is n = 0.05) .With regard to risk factors for iSUA, the only important individual risk factor was conception via ART. As maternal age becomes more advanced and subsequently the use of ART increases, the importance of this finding is clearly outlined. To date, no other study investigating the association between ART and iSUA has been found. Notably, a retrospective cohort investigated non-isolated SUA in singletons and ART and reported a statistically significant association . The in To date, this is the first prospective cohort investigating the association of prenatally diagnosed iSUA with pregnancy outcomes, as well as possible risk factors. The greatest strength of this study lies in its design and methodology. The multivariable models that were carried out and reported according to the TRIPOD statement and the latest suggestions of the bibliography offer trustworthy and unbiased estimates. By further dealing with non-linearity and confirming the fulfilment of the regression assumptions, we took the extra step to ensure the robustness of our results. Additionally, the investigated pregnancy complications were meticulously selected based on their well-known correlation with adverse maternal and neonatal outcomes. More specifically, PTD is the main contributor to prolonged antenatal hospitalization, as well as the most significant cause of neonatal morbidity worldwide due to its association with intraventricular hemorrhage, retinopathy, necrotizing enterocolitis, acute respiratory distress syndrome and long-term neurologic impairment of the offspring, while it is also the second cause of infant mortality, following pneumonia . With reIsolated SUA is associated with an increased risk for SGA and PTD. This finding may justify an alteration in the routine obstetric management to include scanning of the umbilical cord vessels as a compulsory component and upon the diagnosis of iSUA a closer monitoring of fetal wellbeing in the third trimester may be warranted. ART, for the first time, is identified an independent predictor of the development of this anatomical variation. Further research into the detected associations and their pathophysiology is required.


This in vitro study aimed to evaluate the color stability of microhybrid and nanohybrid restorative composites after exposure to immersion media common in Yemen for different periods.n = 5/group; diameter, 10 mm; thickness, 2 mm; shade A2) of each restorative material were immersed for 1 week in distilled water, qat solution, Yemeni coffee, traditional Yemeni coffee (qishr), red tea, and Dilsi cola. Color changes were evaluated by colorimetry. The color data and pH were measured before and 1, 3, and 7 days after immersion. The data were statistically analyzed.Two composite materials, nanohybrid Tetric N-Ceram and microhybrid Te-Econom Plus, were investigated. Six groups of 30 cylindrical specimens (p < 0.05). The role of low pH in discoloration depended on the colorant.Tetric N-Ceram showed lesser discoloration than did Te-Econom Plus. Qat, coffee, and red tea caused highly significant discoloration than did Dilsi cola and distilled water (Nanohybrid Tetric N-Ceram composites are more resistant to discoloration than are microhybrid Te-Econom Plus composites. Qat and coffee have the highest effect on composite discoloration.These findings will aid in selecting composite materials and patient instruction. Developments in conservative restorative materials and adhesive systems have revolutionized dental practice. The possibility of bonding the restorative material to the dental surface and innovations in esthetic procedures have enabled more conservative processes in tooth preparation. The outcome of a restoration is positively correlated with the quality of the filling material . Resin cDiscoloration, the most common reason for composite restoration replacement, is generally caused by dietary products, such as coffee, tea, and nicotine . RestoraMany factors affect the color stability of resin composite restoration materials; these include patient habits, such as beverage consumption, smoking, or qat chewing, as well as colorants that may be incorporated into food and drinks. These staining media lead to the absorption of pigments, which affects the color of the material. Many factors affect the speed of the degradation reaction, including pH, matrix, type of chemical bond filler, water uptake, and copolymer composition. In particular, low pH is an undesirable factor for hydrophilic resins owing to its impact on degradation rates during catalysis.Catha edulis) are put inside the cheek and chewed for more than 4 h for amphetamine-like effects 1/2, where ΔE represents the total color difference and ΔL*, Δa*, and Δb* represent the changes in lightness, red-green coordinates, and yellow-blue coordinates, respectively.The color of the specimens was assessed at four-time points: before and 1, 3, and 7 days after immersion in the media. After sample preparation and immersion in distilled water, color measurements were taken for each specimen using a colorimeter. During color measurement, a white background was used as a baseline. The colorimeter measured the specimen color based on the CIELAB L*a*b* color space system, which allows three-dimensional determination of color. The color differences (ΔE) among the color coordinates were calculated by applying the formula to compare values before and after immersion: ΔE = [(ΔL*)After 24 h, 3 days, and 7 days, color measurements were performed for two different positions of the specimen, and the average was calculated for each specimen.To be familiar with the color measurement, immersion solutions, and colorimeter device, a pilot experiment was performed using six specimens of each composite type before the actual experiment.p < 0.05.Data were collected and analyzed using the Excel computer software package  and Statistical Package for the Social Sciences . The Shapiro–Wilk test was used to analyze normality, followed by paired sample t-test and three-way analysis of variance (ANOVA). One-way ANOVA with Scheffé test was applied to analyze statistically significant differences in the test samples and multiple comparisons for adjusting the level of significance to decrease the error rates. The significance level was set at Qat is a cultural plant in many countries including Yemen. Its use is not illegal, and no permission is needed to collect or consume it. and it is available to everyone in many markets across the country.p > 0.05). A paired sample t-test was used to evaluate the reliability, and the value was 0.97, indicating high internal consistency.All measurement data  were normally distributed according to the Shapiro–Wilk test . The Te-Econom Plus surfaces exhibited brownish discoloration, with the highest difference after immersion in red tea for 3 days, followed by coffee, Dilsi cola, and qishr to the same degree. Qat caused the least change in the a* axis. The most brownish discoloration of Tetric N-Ceram surfaces appeared after immersion in red tea and coffee for 3 days, followed by qishr, Dilsi cola, water, and qat. The brightness of Te-Econom Plus specimens, represented by L*, was most reduced by distilled water. A yellow-to-green (greenish) discoloration appeared on the surface of all Te-Econom Plus specimens; the most yellow-to-green discoloration was due to qat, followed by coffee, red tea, and qishr; Dilsi cola and distilled water resulted in the lowest. The color changes increased with time, but Tetric N-Ceram specimens showed the most yellow-to-green discoloration after immersion in the decreasing order: Qat>coffee>qishr>red tea>Delsi>distilled water. The color changes increased with increasing immersion time. In general, changes on the b* axis started after 24 h and increased after 3 days, reaching the highest point after 7 days. Changes on the a* and b* axes were clearer than those on the L* axis due to changes in color. In addition, compared with immersion in other staining solutions, immersion in qat resulted in the highest differences for both tested composites.p < 0.05).We performed pairwise comparison of ΔE values at all measured times between all staining media for the two composites. Only the qat solution, coffee, and red tea produced color changes that were clinically perceptible (ΔE > 3.3). Among all different immersion media, the qat solution induced the highest discoloration level. The least discoloration was found with distilled water. Generally, Te-Econom Plus showed more color change than did Tetric N-Ceram. As confirmed by three-way ANOVA, a significant interaction between the two resin composites, six staining media, and different immersion times was observed (p < 0.05) were found when the immersion time and staining media were fixed (Table p > 0.05) in either composite when the material and staining media were fixed. The b* value was the most affected factor in discoloration. All immersion media had acidic pH; Dilsi cola and qat solution were the most acidic. With the increase in immersion time, the solutions became more acidic and discoloration increased; however, the effect of pH indirectly depends on the colorant. The change in color after 168 h of immersion was clear  from the highest to the lowest were as follows: qat>coffee>red tea>qishr>Dilsi cola>distilled water Fig. . Signified Table . No signWe observed that only qat and qishr media produced gases with foam. When opening the closed tube before 24 h was complete, an acidulous smell was noticed for these two media, more than that for Dilsi cola, which is highly carbonated.Both composite types had physical variations rather than discoloration when immersed in Dilsi cola, and instances of surface roughness were observed Fig. .Fig. 6ThPerceptible color changes may compromise the clinical acceptability of a composite restoration . In the Color changes of both restorative materials depended upon the type of the restorative material, time of immersion, and type of pigmenting media. This result is in agreement with those of Nuaimi and Ragab , Özdaş eIn this study, both resin materials were discolored, but the severity of discoloration depended on the type of immersion media. This finding is in accordance with those of Gupta et al.  which suAnother finding in this study is that the nanohybrid composite was more resistant to pigmentation than was the microhybrid composite. We conclude that this is due to the size of particles, although many factors affect the color stability of resin composites, including extrinsic and intrinsic factors. The extrinsic factors include patient habits of consuming beverages and food containing colorants that lead to the absorption of pigments and affect the color of the material .Intrinsic factors of the composite also affect color stability. The staining ability of the composite is related to the resin matrix, percentage of filler particles, adsorption and absorption mechanisms of stains, and chemical interactions between composites and the stains. The composition of the resin materials and the relative amount of resin and filler content presence influence the color change of a resin material. Resin materials that have lower filler content and higher resin content tend to absorb more water at the resin–filler interface, leading to hydrolytic degradation of the filler. Thus, resin-based materials with lower filler content have poor color stability . TherefoAdditionally, water sorption is mostly due to direct absorption in the resin matrix. Glass filler particles cannot absorb water, yet they can contribute to water adsorption at the surface of the material. The level of water sorption is a function of the resin content of the material and the strength of the resin–filler interface. Excessive water sorption causes plasticization and expansion of the resin, which leads to reduced hydrolysis of saline and longevity of the composite resin, which in turn creates micro-cracks. Thus, the interfacial gaps or micro-cracks at the interface between the matrix and filler allow penetration of staining materials and discoloration .Qat caused the highest color changes in both materials in all time periods in this study; the mean ΔE ranged between 18.1 and 28.0. This finding is in agreement with the results of Al-Anesi et al.  which inThe increased discoloration was associated with increasing immersion time. Small values of color changes were observed in the first week, which increased; this explains why most qat chewers who use qat for long periods every day have more discolored teeth than do those who chew only occasionally.The discoloration by staining media affects the teeth without the loss of enamel luster. Stains associated with teeth are caused by the presence of chromophores (colored agents). Chromophores arise from two chemical sources: organic compounds  and inorganic transition metal ions  . In the Red tea was shown to cause the third-largest color change in both materials in all time periods; the mean ΔE ranged between 6.6 and 10.2. Red tea came after coffee in discoloration, and this finding is in agreement with those of Gupta et al.  and Al-AThe mechanism behind the color alteration of composite restorations after immersion in various liquids has been the subject of great interest in many studies. The absorption and adsorption of colorants with different polarities found in beverages, such as coffee and tea, on the surface of composite restorations have been implicated .With tea and coffee immersion for 30 d, Nuaimi and Ragab  also fouThe results of our study showed that the effects of coffee, tea, and Dilsi cola decreased in that order. This is inconsistent with the findings of Gupta et al.,  who noteThe results of this study prove that both Tetric N-Ceram and Te-Econom Plus underwent discoloration after immersion in staining solutions . The microhybrid was more discolored than was the nanohybrid; this result contradicts the results reported by Nuaimi and Ragab , who conp < 0.01) of immersion in different beverages.Time is one of the multiple factors that affect the discoloration of composite restorations. For Tetric N-Ceram and Te-Econom Plus, time played an important role in discoloration, and the color became more saturated, with discoloration increasing with increasing time of immersion. The longer the colorant is in contact with the surface of restoration, the greater the discoloration observed. This result is in agreement with that of Özdaş et al.  who obseBefore and 24 h, 3 days, and 7 days after immersion, the pH of Yemeni coffee was 6.4, 5.9, 5.7, and 5.4 while that of red tea was 6.6, 6.1, 5.7, and 5.8, respectively. This is in agreement with the results of Özdaş et al.  who showBefore immersion, Dilsi cola showed the lowest pH among all beverage immersion media, and the acidity increased with increasing immersion time. The negative effect of lower pH was seen in the wear of the composite materials and erosion of the polymer. The acidity in the beverage caused roughness on the surface, which helped accelerate discoloration. Dilsi cola had the most acidity but the lowest ∆E because Dilsi cola contains a weak colorant component, caramel; even though the surface was rough, the weak colorant led to low discoloration.This result is in agreement with those of Gupta et al.  who showThe effects of Dilsi cola pH could be attributed to the susceptibility of the composite resin to chemical erosion of the resin matrix due to the low pH; this results in the hydrolytic breakdown of the filler particles and chemical degradation of the silane coupling agent, which can further be responsible for their discoloration . FurtherIn this study, the pH of Dilsi cola before and 7 days after immersion was 3.1 and 2.8, respectively; it was the most acidic among all immersion media. The pH of water was 7.1 . Choi et al.  also shoDiscoloration was not associated with the pH value in this study. Qat was the highest discoloring agent with a 5.1 pH; although Dilsi cola was more acidic than was qat, it produced lesser discoloration. This result is in agreement with the results of Catelan et al.  who concThis study had some limitations. First, the specimens had flat surfaces. In clinical settings, various anatomical features, including grooves and pits, may be present, and this may complicate the polishing procedure, and provisional restorations will have an irregular shape with convex and concave surfaces. This limitation could affect the results because it does not mimic reality; different anatomical features show different degrees of discoloration on the same tooth, unlike the flat surface of our samples, which had the same degree of discoloration on the entire surface.Second, the concentration of solutions used as daily beverages is highly variable depending on the type of cuisine and individual preference. Therefore, the concentrations used in this study provide only a general estimation. Third, no artificial saliva was incorporated to mimic the oral environment in this study. Other factors could also influence the degree of total color change, including thermocycling processes and abrasion. Fourth, no comparisons were made between Yemeni coffee and other universal types since more precision would be required, such as the type of coffee, degree of roasting, whether the beans were processed or natural, the concentration of coffee in each cup, addition of sugar, as well as the difficulty of obtaining all types of Yemeni and international coffee.The nanohybrid composite was more color-stable than was the microhybrid composite. In composite resins, a smaller filler particle size increases the resistance to discoloration; therefore, the microhybrid composite (Te-Econom plus) was more susceptible to discoloration than was the nanohybrid composite (Tetric N-cream).Qat caused greater discoloration of both materials than did other staining solutions and resulted in the most undesirable discoloration. Dilsi cola caused abrasion of the composite specimen, and this could facilitate discoloration. Time plays an important role in discoloration, as increasing time increases the ability of staining agents to cause discoloration (with the indirect role of pH).


To evaluate the clinical value of systemic immune-inflammation index (SII) based on peripheral blood neutrophil, lymphocyte, and platelet count in evaluating the subtype and severity of depression in patients with depressive disorder.This retrospective cohort study was conducted in the Third People’s Hospital of Fuyang City from January 1, 2020 to December 31, 2022. The data included sociodemographic information at admission, clinical data, discharge diagnosis and inflammatory markers. Patients were divided into low SII group and high SII group according to the optimal threshold of SII determined by receiver operating characteristic curve (ROC curve). Binary logistic regression was used to analyze the correlation between moderate/major depression and SII level.χ2 = 7.663, p = 0.006), more smokers , more moderate/major depression patients , and a higher proportion of patients with accompanying somatic symptoms . In the final logistic regression model, after controlling for confounding factors, SII at admission was significantly associated with moderate/major depression . Patients with high SII scores were 3.614 times more likely to have moderate/severe depression than those with low SII scores. We propose a cut-off value of SII =540.78 (sensitivity = 36.4% and specificity = 80.3%) according to the maximum Youden index.Compared to the low SII group, the high SII group had a higher age level (Our research indicates that SII may be a useful, repeatable, convenient, and affordable index to identify moderate/major depression in depressive disorder. In 2008, the World Health Organization ranked depression as the third leading cause of disease burden worldwide and predicts that by 2031, depression will rank first  and inflammatory factors have been reported . ExperimThe baseline systemic immune-inflammation index (SII) is an indicator of immune response and systemic inflammation based on peripheral blood platelet, lymphocyte, and neutrophil counts, and is a simple way to objectively reflect the balance between inflammatory and immune responses , 13. SIIThe purpose of this study is to explore the clinicopathological characteristics of depressed patients with high SII, to explore the potential of the SII index in the assessment of diagnostic subtypes in patients with depressive disorders, and to provide a simpler diagnostic assessment tool for the clinic from a new perspective.2.2.1.th revision Diagnostic Criteria for Mental Behavior Disorders of International Statistical Classification of Diseases and Related Health Problems (ICD-10). The Hamilton Depression Scale (HDRS) is used to assess the severity of depressive symptoms. Hamilton total score was evaluated as follows: 8–16: mild depression; 17–23: moderate depression; and ≥24: major depression  and was performed in accordance with the ethical standards of the Declaration of Helsinki. Patients who met the criteria had signed a written informed consent and were allowed to voluntarily withdraw from this study at any time. A total of 1,478 patients were initially enrolled in this study, of whom 102 met the exclusion criteria and 64 had insufficient baseline clinical information, and 1,312 patients were finally included in the study as study subjects.2.2.Baseline demographic characteristics , clinical manifestations , discharge diagnosis, and hematological data on admission  were collected from the medical records. Blood samples were obtained from the cephalic vein of participants after fasting for at least 8 h between 6:00 and 8:00 a.m., and were collected in EDTA tubes. The complete blood counts, which included measurements of total white blood cells, neutrophils, lymphocytes, monocytes, and platelets, were analyzed using a Mindray BC-5380/BC-5180 Hematology Analyzer . Subsequently, SII, NLR, MLR, and PLR were calculated using the appropriate formulas. SII = platelet count 2.3.U test were used to compare demographic and clinicopathological features among each group. A receiver operating characteristic (ROC) curve analysis was performed to determine the cut-off point for biomarkers to detect moderate/major depression. The optimal cut-off point is the point on the ROC curve that corresponds to the maximum Youden index. The area under the ROC curve (AUC) was performed to determine the predictive value of various indicators for moderate/major depression. A binary logistic regression model (enter) was estimated to determine the independent factors associated with moderate/major depression in patients with depressive disorder. The level of statistical significance was set at α = 0.05 (two-sided).All data were statistically analyzed using SPSS 26.0 software. Data are presented as frequencies and percentages for categorical variables and as mean (standard deviation) for numeric variables. Analysis of variance (ANOVA) or chi-square test or Mann–Whitney 3.3.1.A total of 1,312 patients with depressive disorder were enrolled according to the inclusion and exclusion criteria. On average, the age of the entire sample was 41.69 years old , most patients were female , besides, more than half of patients were unmarried  and most had no history of smoking . SII mean was 462.83 . Among them, 591 patients (45%) were moderate or major depression patients, and 721 patients (55%) were non-moderate or major depression patients, according to the admission diagnosis. 3.2.p < 0.01). The optimal cut-off point is the point on the ROC curve that corresponds to the maximum Youden index. The optimal cut-off point of SII to determine moderate/major depression in patients with the depressive disorder was 540.78, the sensitivity was 0.364, the specificity was 0.803, and the Youden’s index was 0.167. Patients were grouped according to this optimal cutoff value: 357 (27%) patients in the high SII group (SII ≥ 540.78) and 955 (73%) patients in the low SII group (SII < 540.78). When NLR, MLR and PLR were used for ROC analysis, the areas under the curve were 0.54, 0.517 and 0.527, respectively , more smokers , more moderate/major depression , and a lower number of relapse , and the proportion of patients with somatic symptoms was higher , the difference was statistically significant. The levels of neutrophils, leukocytes, platelets, SII, NLR, MLR, PLR were significantly higher in the high SII group compared to the low SII group, with statistical significance . There were no statistically significant differences in gender, age, education level and whether there were accompanying psychiatric symptoms between the two groups , as shown in Compared with the low SII group, the high SII group had a higher age of onset of depressive disorder , concomitant psychotic symptoms , relapse , age , SII level  was significantly associated with moderate/major depression. SII ≥ 540.78 was a risk factor for moderate/major depression in patients with depressive disorder (p < 0.001), as shown in Moderate/major depression were used as the dependent variable , marital status , age classification , gender , education level , smoking , somatic symptoms , psychotic symptoms , recurrent depressive disorder , SII  as independent variable, binary multi-factor logistic regression analysis  was perf4.Depressive disorder is a disease with high morbidity and disability rate. This study showed that patients with depressive disorder in the high SII group were older, had a higher odds of a history of smoking history, had a higher proportion of moderate/major depression, and had a higher proportion of accompanying somatic symptoms. At the same time, high SII is a risk factor for moderate/major depression in patients with depressive disorder.This study demonstrated the potential application value of SII index on admission to determine the severity of depression. The SII index objectively reflects the balance between the inflammatory and immune responses of the body. It provides a simple, rapid, and inexpensive way to measure the level of inflammation. The SII index can be calculated by the simple parameters in the blood routine upon admission. For patients with high SII depression, the occurrence of moderate/major depression can be warned at an early stage. The incidence of suicide, somatic, and psychotic symptoms in patients with moderate/major depression are relatively high. Therefore, individualized treatment programs including psychological and physical therapy can be actively adopted in the follow-up treatment to reduce the incidence of suicide and improve the clinical cure rate . On the p < 0.05) (p = 0.02). The researchers further validated the relationship between SII and diabetic depression by using propensity matching analysis, and SII may be an accessible and cost-effective strategy for identifying depression in diabetic patients , and neutrophil/lymphocyte ratio (NLR) increased at admission, in particular, increased SII was significantly associated with the occurrence of post-ischemic stroke depression one month later, which may provide some prognostic clues for early detection of post-ischemic stroke depression . In the  < 0.05) . A studypatients . Data frpatients .More and more animal models and clinical studies of depression suggest that immunoinflammatory and neuroinflammatory pathways are involved in the pathophysiological processes of depression. Studies on immune inflammation and depression animal models have shown that systemic inflammatory response induced by lipopolysaccharide, tumor necrosis factor α and other pro-inflammatory factors can lead to depression-like behavior in mice . ComparePlatelets are a specific inflammatory indicator. Platelets are increasingly recognized to play an important role in inflammation and as a putative bridge between mental illness and inflammatory response. Platelet overactivation has been observed in patients with depressive disorders, and platelet parameters have potential predictive power for major depression and bipolar disorder . InflammPeripheral blood neutrophil/lymphocyte ratio (NLR) is a component of the SII formula and a simple measure to evaluate the inflammatory status and can economically and easily detect the activation of inflammatory systems. More and more studies have shown that the level of NLR in patients with depressive disorder is higher than that in healthy control volunteers, and is closely related to the degree of depression. For example, one study showed significantly higher levels of NLR in patients with affective disorders, and may assess the course of affective disorders . AnotherThe novelty of this study lies in the identification of SII index as a potential predictor for moderate/severe depression. This suggests that monitoring SII levels could help in identifying individuals who are at risk for developing moderate/severe depression, and potentially enable early intervention and treatment. This finding also adds to the growing body of literature on the complex relationship between inflammation and mental health, and highlights the potential role of SII as a novel biomarker for depression risk assessment.This study has the following deficiencies: (1) there is a lack of information on potential confounders, such as whether the patient was taking any psychiatric medications at the time of admission, especially antidepressants, including the dose and duration of treatment, as previously stated in the literature, antidepressants have anti-inflammatory effects , 38. (2)Although this study has some limitations, our study confirms that high SII on admission is an independent risk factor for symptoms reaching moderate/major depression in patients with depressive disorder. In future clinical work, for depressed patients with high SII value upon admission, emphasis should be placed on early depression assessment, early psychological intervention and prevention, which will have a good impact on the prognosis of patients. On the other hand, rapid identification of people with high SII may lead to improvements in subsequent treatments, such as combining conventional treatments with treatments that reduce inflammation levels to treat major depression.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Ethics Committee of the Third People’s Hospital of Fuyang City . Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.SC, HL, and YL: conception and design. SC, YL, and GY: administrative support. SC, JL, YW, ZL, LiS, and LoS: collection and assembly of data. SC, GY, JL, and HL: data analysis and interpretation. SC, JL, YL, GY, YW, ZL, LiS, LoS, and HL: manuscript writing. All authors contributed to the article and approved the submitted version.This study was funded by the Fuyang Municipal Health Commission Research Project (FY2020xg14) and Clinical Medical Key Specialty Construction Project of Anhui Province of China [(2021)273].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. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.