The purpose of this study was to evaluate specifically in type 1 diabetes mellitus (DM) individuals the relationship between perifoveal superficial capillary plexus (SCP) parameters assessed by optical coherence tomography angiography (OCTA) and diabetic retinopathy (DR) grade.Cross-sectional analysis of a large scale prospective OCTA trial cohort . A total of 1186 eyes , 956 type 1 DM eyes (478 patients), and 230 control eyes  were included in this study. DR stage was graded according to the International Classification. OCTA imaging was performed with a commercially available device (Cirrus HD-OCT). Vessel density (VD), perfusion density (PD), and foveal avascular zone (FAZ) area, perimeter and circularity measurements were quantified in the SCP and receiver operating characteristic (ROC) curves were constructed for each OCTA parameter.P < 0.001 in all cases) in a multiple regression analysis after controlling by age, gender, signal strength index, axial length, and DM duration. Greater FAZ area and perimeter and conversely lower circularity measurements were observed as DR severity increased in both scanning protocols .VD and PD (in both 3 × 3 and 6 × 6 mm captures) were inversely associated with DR stage (In type 1 DM individuals, OCTA provides an objective, continuous, and reliable method for accurate quantification of VD, PD, and FAZ parameters in the SCP, which ultimately correlate with DR stages.Objective OCTA measurements of the retinal microvasculature could substitute the clinical DR classification in patients with type 1 DM, identify patients at risk of DR progression, and inform treatment decisions to modify the evolution of the disease. Diabetic retinopathy (DR) is a clinically diverse entity originating from the alteration of small retinal vessels. A selective loss of pericytes and endothelial cells, as well as the thickening of the basal membrane in retinal capillaries, occurs as a result of a long period of exposure to elevated glucose levels in blood, damaging the retinal vessels and affecting the retinal flow in a time dependent manner.,OCTA represents an interesting opportunity to evaluate the perifoveal vascular network at different moments of the disease, with the potential of being an excellent tool to identify early changes  in a noninvasive way.With this aim, we designed a prospective research project directed to study the retinal vascular network through OCTA in a large cohort of patients with type 1 DMThe purpose of this specific report is to determine objectively in a cross-sectional analysis the characteristics of the perifoveal capillary network in patients with type 1 DM and to study their relationship with the DR grade compared to those observed in a control group of healthy volunteers. We aim to evaluate the potential of OCTA as a new tool to detect objective parameters of DM status in the perifoveal vascular network in a large series of patients with type 1 DM, which may have direct implications in the systemic management of these patients.A cross sectional, exploratory study was conducted in a large cohort of patients with type 1 DM recruited from the Diabetes Unit of Hospital Clínic in a 24-month period (May 2017–May 2019), with prospective collection of OCTA images and relevant ocular and systemic clinical data. The study protocol has been described elsewhere.Patients with type 1 DM undergoing yearly follow-up visits, as per routine clinical care at the Diabetes Unit of our center, were invited to participate in the study. Those willing to participate were referred for a comprehensive ocular examination. Controls were collected from healthy volunteers recruited in the general population after social media campaigns supported by the Communications department of the hospital. Exclusion criteria include concomitant ocular pathologies, macular edema, presence of macular cysts, previous ocular surgery, previous macular laser, previous ocular treatment, including intravitreal therapies, media opacities, and inability to perform complete ocular examinations, including retinal imaging , as well as inability to give written informed consent to participate in the study. A consolidated standard of reporting trials (CONSORT)-style flow chart describing included and excluded eyes in the study and each individual OCTA analysis is presented in All participants underwent a complete ocular examination. Clinical data collected included: best-corrected visual acuity (BCVA), slit-lamp biomicroscopy, intraocular pressure measurement, retinal fundus examination, and biometry . Diabetic retinopathy stage was graded in all study eyes using the International Clinical Diabetic Retinopathy Disease Severity Scale.Systemic status of the diabetic disease was evaluated in routine clinical care examinations in the Diabetes Unit of the Endocrinology service, and clinical data collected included: age, gender, smoking habit, duration of the disease, type of treatment , concomitant pathology , body mass index (BMI), hemogram, HbA1c, lipid profile , and kidney function tests .All OCTA and OCT images were obtained using the same OCT device . OCTA scanning protocols included 3 × 3 mm and 6 × 6 mm cube scans centered in the fovea by gaze fixation . StructuP values < 0.05 were considered statistically significant. The statistical package R Studio (version 2.5) was used for the statistical analyses.In order to describe the qualitative variables, absolute frequencies and percentages were used. The description of quantitative variables was performed using the mean, standard deviation (SD), median, and quartiles. The Kolmogorov-Smirnov test was used to assess the normality of distributions. Clinical variables, OCTA, and structural parameters according to the DR grade were compared using a Generalized Estimating Equation (GEE) in order to control the potential bias of bilaterality and adjusted by age, gender, SSI, axial length, and DM disease duration from diagnosis. ANOVA or Kruskal-Wallis test  with Bonferroni correction was used in multiple comparisons between groups in the case of quantitative variables. The analysis at the patient level was analyzed using linear regression for continuous variables or Mantel-Hansel test for categorical variables. Receiver operating curves (ROC) were constructed to evaluate the area under the curve (AUC) of each OCTA parameter by subgroups . For all the tests, A total number of 1186 of eyes of 593 individuals were originally included in the study. Four hundred seventy-eight patients with type 1 DM (956 eyes) and 115 healthy controls (230 eyes) underwent a complete ocular examination during the predetermined timeframe. Exclusion criteria were applied and 181 eyes were excluded due to previous ocular surgery (93 eyes), previous ocular treatment (24 eyes), previous macular laser (21 eyes), macular edema (9 eyes), or concomitant ocular pathology (71 eyes), such as glaucoma (19 eyes), amblyopia (16 eyes), myopia magna, or axial length > 27 mm (8 eyes), and other causes (28 eyes), such as retinal vein occlusions or uveitis. OCTA images from 1009 eyes were analyzed. A detailed description of OCTA images excluded due to artifacts, poor OCTA image quality (defined as SSI < 7), or incorrect FAZ delineation by the built-in software for each OCTA parameter analysis is presented in n = 539) of the examined eyes, mild non-proliferative diabetic retinopathy (NPDR) in 21.2% (n = 214), moderate NPDR in 3.3% (n = 34), severe NPDR in 0.4% (n = 5), and finally proliferative DR  in 1.3% (n = 14) of the study eyes. All baseline characteristics of study eyes according to DR stage distribution are summarized in Mean age for patients with diabeteswas 40 ± 12.4 (standard deviation [SD]) years and the mean disease duration was 20 ± 10.8 years. Mean age for healthy controls was 43 ± 14.1 years. All demographic characteristics are shown in P < 0.001), after adjusting data by age, gender, SSI, axial length, and duration of DM disease. Differences between each different DR groups are detailed in P < 0.001 in all cases) between the DR stage groups with lower PD in greater levels of DR severity. Similar findings were observed with the 6 × 6 mm captures, as shown in In 3 × 3 mm captures, a significant association was observed between VD and DR stage, showing lower VD with greater levels of DR stage and statistically significant differences between the different disease severity groups and controls (P = 0.029), FAZ perimeter (P < 0.001), and FAZ circularity (P < 0.001) between controls and the different DR severity groups. Statistically significant differences (P < 0.05) were found in 2 × 2 comparisons between controls and some of the DR groups, as well as between some of the DR groups, as detailed in P = 0.005), FAZ perimeter (P = 0.002), and FAZ circularity (P < 0.001). Statistically significant differences were found in 2 × 2 comparisons between controls and some of the DR groups (P < 0.05), as well as between some of the different DR groups. All these results are summarized in FAZ parameters showed different results in 3 × 3 and 6 × 6 mm scans, as shown in P < 0.001) and PD  for these 3 subgroups in a lineal regression model adjusted for age, gender, SSI, axial length, and DR grade. With regard to the FAZ parameters, no differences were observed for FAZ area  or FAZ perimeter , but significant differences were observed in FAZ circularity . Similar results were observed in the 6 × 6 mm scans, and differences were observed for VD , PD  for these 3 subgroups, in a lineal regression model adjusted for age, gender, SSI, axial length, and DR grade. No differences were observed for FAZ area  and FAZ perimeter  but FAZ circularity showed significant differences between groups . All these results are presented in Subgroup analysis was performed by duration of DM and groups were defined as A (< 5 years), B (5–15 years), and C (> 15 years). Significant differences were observed in the 3 × 3 mm scans for VD . The ROC curves for OCTA parameters in the 3 × 3 mm scans are presented in This study highlights the role of OCTA as an objective tool to obtain reliable measurements of the foveal vessel status specifically in patients with type 1 DM, the rarest type of disease. These objective, quantitative, and continuous OCTA-derived measurements ultimately correlate with DR severity, showing gradually lower VD and PD, and greater FAZ changes in the SCP as DR progresses to greater levels of disease.––In the last few years, many OCTA studies have been directed to assess the status of the perifoveal capillary network in DM. However, the vast majority of studies have been performed in patients with type 2 DM,––n = 188 vs. 60); however, Niestrata-Ortiz et al. described that FAZ area was different between diabetic and healthy children (n = 112 vs. 30).,Very few papers have studied this topic specifically in patients with type 1 DM,,–,,,–One of the key issues when interpreting quantitative analysis of OCTA images is which retinal plexus is being evaluated, a feature that is strongly related to the OCT device used for the image capture and the segmentation algorithm used by the software of the machine. In this report, we have used an OCT device that allows measurements only in the SCP with the commercial built-in software. The role of the different plexuses in the pathophysiology of DR is also a controversial topic, and there are discrepancies in the literature. Again, for the reasons mentioned above, most of the current evidence about which plexus and OCTA parameter reflects better the status of the macula of patients with diabetes needs to be taken from type 2 DM studies. Agemy et al. reported that VD measurements were different in the SCP and DCP of diabetic eyes, meanwhile Coscas et al. did not find any significant differences between these two plexuses in patients with diabetes.–,FAZ alterations are commonly seen in patients with diabetes.The main strength of this study is the large number of type 1 DM eyes included, which to date is the largest OCTA cohort published in these patients. Second, the fact of obtaining the data with the device built-in commercial software and no manual corrections present two additional benefits: to obtain reliable and objective data of the analyzed parameters and to be reproducible and generalizable to other groups, without the need to use a research software, unpractical in daily practice, and unavailable to all medical teams. Third, the inclusion of a relatively large number of controls also add significant value to the OCTA analysis performed and constitutes another strong point of this study. As previously mentioned, a potential limitation of the study is the inability of the commercial software of the device used to quantify vascular flow parameters of the DCP, which, according to some authors, may be affected earlier in the development of DR. Future analysis of our dataset using an automated research software will shed some light on this specific issue. Nevertheless, our results have already detected flow alterations in the SCP indicating that this plexus has a determining role in the DR pathophysiology from early stages of the disease. Another possible limitation of the study may be the small number of patients with DM with severe NPDR, due to the efficacy of the public DR screening program in Spain. As the majority of the study cohort presented the milder forms of the disease, the results of this study are especially representative of early DR stages with a relatively spared macular anatomy. This may have had a positive effect in the reliability of the OCTA-derived metrics obtained, with a low rate of artifacts compared to other reported series in pathological eyes.In conclusion, OCTA is able to detect microvascular changes in type 1 DM diabetic eyes, which are correlated with the DR stage independently of age and duration of DM disease. Quantitative parameters as VD, PD, and FAZ area, circularity, or perimeter may be objectively quantified in diabetic eyes with this noninvasive technology. This automated analysis reveals details of the perifoveal vascular network status in an objective way, translating subjective qualitative findings into objective data, a significant step forward to allow ophthalmologists the integration of meaningful retinal vascularity data in daily clinical practice. These findings suggest a major role of OCTA in the next future, as these microvascular changes may be detected in their preclinical status, before the first clinical signs of DR appear in direct fundus examination. Future studies will focus on the possibility of defining OCTA-derived parameter ranges for DR severity stage classification, risk assessment, and, maybe, response to treatment and monitorization based on these objective grounds.


Moreover, the combined coronary artery volumes correlated with patient body surface area . Inter-observer variability was assessed and intraclass correlation coefficient of the total coronary artery volume demonstrated a low variability of 3D QCA . Dedicated 3D QCA facilitates reproducible coronary artery volume estimation and allows discrimination of normal and diseased vessels.Non-atherosclerotic abnormalities of vessel calibre, aneurysm and ectasia, are challenging to quantify and are often overlooked in qualitative reporting. Utilising a novel 3-dimensional (3D) quantitative coronary angiography (QCA) application, we have evaluated the characteristics of normal, diabetic and aneurysmal or ectatic coronary arteries. We selected 131 individuals under 50 years-of-age, who had undergone coronary angiography for suspected myocardial ischaemia between 1st January 2011 and 31st December 2015, at the Bristol Heart Institute, Bristol, UK. This included 42 patients with angiographically normal coronary arteries, 36 diabetic patients with unobstructed coronaries, and 53 patients with abnormal coronary dilatation (aneurysm and ectasia). A total of 1105 coronary segments were analysed using QAngio XA 3D . The combined volume of the major coronary arteries was significantly different between each group (1240 ± 476 mm Qualitative coronary artery dilatation is evident in up to 10% of all diagnostic coronary angiograms3. However, defining true aneurysm and ectasia is challenging with a lack of clearly quantifiable and reproducible cut-offs. An aneurysm is simple to define when discrete, arising in mid-vessel, and bordered by normal calibre proximal and distal segments. However, aneurysms frequently associate with atheromatous or diffusely ectatic segments and consequently defining the abnormalities is more challenging. The difficulties in effective quantification are reinforced by the myriad definitions for aneurysm and ectasia found within the literature4.Aneurysmal vascular disease was first reported post-mortem over 200 years ago3. In considering strategies to improve diagnosis it was noted that paediatric cardiologists have developed a robust method for coronary artery calibre measurement, however, paediatric and adult coronary assessments pose different challenges. In paediatric cardiology, an echocardiography-based assessment of coronary dimensions is commonly used. This assessment, usually based on visual internal diameter provides a z-score  and is used to guide further management5. Assessment for aneurysm and ectasia in adult coronary arteries is not possible by echocardiography and is complicated by the potential for concomitant development of stenotic atherosclerotic disease.We have previously expressed a major concern regarding the accuracy of reporting and recognition of aneurysm/ectasia in adult coronary angiographic assessment6. However, 2D QCA has fundamental limitations as coronary angiography only provides a 2D image of the 3-dimensional (3D) structure of a coronary artery7. Advances in imaging software have facilitated 3D QCA, with generation of a 3D reconstruction of the coronary artery from two angiographic images, acquired at different angles8. This allows evaluation of vessel segment length, diameter and volume. We propose that plotting coronary volumes against a population-based normal range, with z-scoring, equivalent to the paediatric echocardiographic method, would assist in the diagnosis of aneurysm and ectasia, where the angiographic appearances are ambiguous.Conventional 2-dimensional (2D) quantitative coronary angiography (QCA) is a highly reproducible computer-assisted technique, and is widely used to evaluate CAD in research settingsAs proof of principle, we have tested this new method of vessel assessment in 3 distinct populations of patient assessed by coronary angiography. In this study, we report the 3D QCA characteristics of the coronary arteries of patients with diabetes without evidence of significant coronary artery disease (CAD), in non-diabetic patients without evidence of significant CAD and in patients with angiographic evidence of aneurysmal or ectatic coronary arteries. Furthermore, we tested inter-observer variability of this novel analysis to ensure that the technique could be deployed clinically.p < 0.01). Differences in risk factor profile were observed between groups, with the greatest number of risk factors in the diabetic cohort (3.4 ± 1.1) and least in the normal group . The majority of patients (74.8%) had right dominant coronary anatomy. No difference in height was observed between groups (172.8 ± 10.1 cm) but variation in weight was observed , resulting in a significant difference in BMI & BSA between groups. All baseline characteristics are shown in Table From a total of 1437 patients, 131 individuals were included from three groups: 42 , 36 (diabetic group), and 53 . The average age of the 131 patients was 43.2 ± 5.3 years, 76.3% were male, with a near-exclusive population of males in the abnormal group , or anatomical absence of the LMCA (5 patients). Data collected by the 3D QCA software included lumen volume, proximal and distal diameters, and segment length.3 diabetic group, 1646 ± 391mm3 normal group, and 2072 ± 687 mm3 abnormal group—see Fig. p < 0.001—see Fig. p < 0.001).The total coronary volume was defined as the combined volume of the major coronary arteries which was the summation of volumes from the RCA , LAD (segments 6 and 7), and LCX (segments 11 and 13), and LMCA (segment 5). Quantification of total coronary volume was possible in 118 (90.1%) of 131 patients, including 40 (95.2%) in the normal group, 36 (100%) in the diabetic group, and 42 (79.2%) in the abnormal group. The total coronary volume was significantly different between each group , see Table The total length of the LAD and RCA did not differ, however differences in the total length of the LCX and LMCA were observed between groups. The diabetic group had a shorter LCX length, due to a significantly shorter mid segment (CASS segment 13). Contrary to this, the diabetic group had a significantly longer LMCA compared with the normal group , and demonstrated a consistent difference between all 3 patient groups , see Table Consistent with the observation that the abnormal group had greater proximal segment volumes, the segment averaged and proximal reference diameters for all measurements in the abnormal group were larger in comparison to both other groups. Segmental analysis of the diabetic group coronary lumen diameters consistently demonstrated the smallest measurements across all three groups, see Table p < 0.001)  . Furthermore, the intraclass correlation coefficient values for the measurements of LAD, LCX, and RCA were 0.963, 0.978, and 0.999, respectively, demonstrating excellent inter-observer measurement consistency  abnormal group individuals. There was a very strong and significant correlation regarding the volume measurements between the two observers  Fig. A. This r01) Fig. B. The abTo our knowledge, this work constitutes the first demonstration of a quantitative comparison of 3D coronary artery characteristics by coronary volume reconstruction. This evaluation was undertaken to test the feasibility and reproducibility of 3D QCA derived coronary volume analysis, in the first instance to assist with the challenging angiographic diagnosis of ectasia/aneurysm. Confirmation of a difference in coronary volume between a normal population and selected cohort of patients with angiographic ectasia or aneurysm is reassuring but not surprising. However, our segmental analysis has confirmed that despite increased vessel dimensions throughout all major epicardial coronary arterial segments, a significant increase in coronary volume is predominantly observed in the proximal segments. Importantly, low inter-observer variability was observed, however, automation of this enhanced QCA would further enhance the operator’s interpretation of the coronary angiogram and improve the diagnostic yield/identification of important non-stenotic coronary abnormalities.10. Previous studies have demonstrated that thrombosis and embolisation of the involved segments are the leading cause of acute MI in patients with coronary artery dilation12. In our previous report focusing on the abnormal cohort, despite low prevalence (3.4%) in patients under 50 years of age, 71.4% of all patients with coronary artery dilation presented with an acute ST-elevation myocardial infarction3. Furthermore, angiographic signs of turbulent and stagnant flow, have been demonstrated in patients with coronary artery dilation by using doppler velocity and thrombolysis in myocardial infarction (TIMI) frame count method, an index of coronary flow velocity14. However, at present, there is no data regarding an association between the coronary volume and coronary flow velocity. Our results are consistent with 3D coronary artery volume reconstructions of dilated coronary arteries being significantly larger than the volume of equivalent normal and diabetic arteries. In the future, use of 3D QCA may enable a better understanding of the association between coronary volume and flow dynamics in patients with dilated coronary arteries.Coronary artery aneurysm and ectasia are associated with a poor prognosis16. Furthermore, increasing blood glucose may also have an effect on vasoconstriction17. As a consequence of this hyperglycaemic phenomenon, diabetes may be associated with CAD leading to MI and angina18. Indeed, Mosseri et al. showed that coronary arteries of diabetic patients had a smaller diameter than those of normal subjects19. In the present study, we observed that this is particularly apparent in the LAD (segment 6 and 7), and LCX (segment 11 and 13) with a trend towards smaller diameters in the RCA  have been used for classifying coronary artery dilation in young patients with Kawasaki disease22. However, as the paediatric Z-score is an ultrasound-based measurement and is not possible in adults, there is no diagnostic equivalent for adults with coronary aneurysm or ectasia. Beyond the choice of imaging modality, there are other important differences between the paediatric and adult populations. The adult population has a stable height but fluctuant body weight that may lead to a less consistent correlation between coronary volume and BSA, consequently indexing against other anthropomorphic parameters may prove more useful. Assessment of total coronary volume and height in our population provided a similarly moderate correlation (R = 0.42), which was partially enhanced when restricted to the normal group (R = 0.49). Correction of measurements to myocardial mass may provide the most accurate method of indexing, however, this would require multi-modality imaging. It is anticipated that extending our analysis to a larger cohort will further enhance our correlations and provide the potential for producing a Z-score equivalent using total coronary artery volume to differentiate pathological coronary artery dilation from healthy individuals.Despite the pilot nature of our data, we have demonstrated a moderate correlation between total coronary volume and BSA. It has previously been demonstrated that there is a linear correlation between coronary artery diameter and BSA in children25. In the recent study by Medrano-Garcia et al., diameter and lengths of normal coronary artery were evaluated using computed tomography angiograms26. It is useful to compare our angiographically derived measurements against CT-derived measurements, albeit from separate populations. Our population had larger vessel diameters across all proximal segments, however, the derived LAD and LCX proximal segment lengths were shorter . Whereas, LMCA and RCA lengths appeared remarkably consistent . The variance in this comparison is likely to reflect the dependence of our 3D QCA analysis on optimally selected angiographic projections to generate an accurate reconstruction. Ultimately it would be useful to undertake a comparison of 3D QCA by multiple modalities to validate the coronary volume measurements.Analysis of luminal diameters of normal coronary arteries are well established although differences in study methodology can make comparison challengingThere are several limitations that should be acknowledged. Firstly, this study was designed as a ‘proof-of-concept’ evaluation and consequently was not adequately powered to assess for significant differences between individual QCA parameters, or to provide sufficient data to generate a nomogram or thresholds for identification of coronary volume abnormalities. For this reason, angiographically evident coronary disease was excluded, however, we acknowledge that clinical application of this 3D QCA analysis will necessitate inclusion of a full spectrum of coronary disease. We anticipate that segmental-level analysis would facilitate comparative assessment in the presence of significant CAD. Secondly, as indicated by the comparison with CT-derived coronary dimensions, the retrospective nature of our analysis prevented optimisation of angiographic acquisition for each coronary segment. Of the initial population of 131 patients, 13 were excluded due to an inability to generate 3D reconstructions of all 3 coronary arteries. The majority of these patients had significant ectasia or aneurysm that significantly distorted image reconstruction. However, it is important to recognise that retrospective foreshortening with underestimation of coronary segment lengths is likely to contribute to the differences observed between modalities. Our intention is to repeat the analysis using a validation cohort from a collaborating centre and the ultimate goal is to undertake a prospective study utilising a dedicated acquisition/analysis protocol. Particular care will be taken to optimise image acquisition and minimisation of distortion of the 3D reconstruction of the coronaries, before considering our novel method as a clinical application for diagnosis of non-atherosclerotic coronary artery abnormalities.Evaluation of coronary angiographic images tends to be qualitative in clinical practice, and consequently non-stenotic disease can be challenging to detect. We have demonstrated the feasibility of a 3D QCA methodology to quantify coronary volume in three distinct patient populations. This quantitative analysis correlates with BSA, has good reproducibility, and therefore offers potential as a tool for measuring coronary artery volume and identification of non-atherosclerotic coronary abnormalities. Larger scale, prospective, studies will be needed to generate adult Z-scoring for accurate identification and monitoring of abnormal coronary artery dilation, including aneurysm and ectasia.3. We have extended the analysis to include two further populations from this cohort: patients found to have angiographically normal arteries, with or without a history of diabetes mellitus.We have previously reported the prevalence of aneurysmal and ectatic disease within an unselected population of 1437 patients, under 50 years-of-age, that underwent coronary angiography between 2011 and 2015, at the Bristol Heart Institute, Bristol, UKPatients were excluded if they had poor angiographic images or if there was only one angiographic view per artery, as neither are suitable for 3D QCA.This study falls outside the scope of the UK policy framework for health and social care research and was registered with University Hospitals Bristol and Weston NHS Foundation Trust as a service evaluation. It is an analysis of routinely collected anonymized data, and followed the national “Guidance on the use of patient images obtained as part of standard care for teaching, training and research” issued by the Royal College of Radiologists, UK.There were 344 patients with atypical chest pain who underwent coronary angiography to investigate for CAD, without evidence of any luminal stenosis/coronary atheroma. We excluded patients who had a history of myocardial infarction (MI), renal failure, diabetes, and > 2 risk factors for CAD, including hypertension, dyslipidaemia, smoking, and family history of CAD in first degree relatives less than 60 years of age, as these factors could affect coronary volume.There were a total of 150 patients with insulin dependent diabetes investigated by coronary angiography for stable anginal symptoms. Insulin treatment was identified as as a surrogate for extended hypo-glycaemic treatment and thereby significant exposure to the potential vasculopathic effects of diabetes. Patients with evidence of luminal stenosis secondary to overt atherosclerosis were excluded from the analysis.27.All coronary angiograms demonstrating any discrepancy of vessel calibre were reviewed, independently, by two interventional cardiologists. Coronary aneurysm was defined by quantitative dilatation of the coronary artery exceeding 50% of an adjacent segment diameter and ectasia was dichotomized by evidence of extension across 2 or more coronary segments3D angiographic coronary artery reconstruction was performed using QAngio XA 3D . The 3D reconstruction procedure consisted of the following steps: (1) from the routine coronary angiography acquisitions, two image sequences acquired from two angiographic projections at least 25° apart were highlighted; (2) end-diastolic still frames with complete vessel contrast-filling were selected . If the AHA classification was not suitable for evaluating a segment, the segment would be defined using the modified AHA classification29. Volumetric analysis was restricted to the proximal and mid-vessel segments of all three major epicardial arterial territories. According to the segment classification, the right coronary artery (RCA) analysis included segments 1, 2, and 3. Regarding the left coronary system, the left main coronary artery (LMCA) segment 5, the left anterior descending artery (LAD) segments 6 and 7, and the left circumflex artery (LCX) segments 11 and 13 were included in the analysis.Two interventional cardiologists determined each coronary segment according to the American Heart Association (AHA) classification2 test. Correlations were assessed using the Pearson’s correlation coefficient, and the intraclass correlation coefficient was used to evaluate consistency of inter-observer agreement of coronary volume measurements. All variables were considered significant when the p value was < 0.05. Statistical analysis was performed using SPSS 22.0 for Windows  and R version 3.6.0 (2019-04-26—R Core Team (2019). R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. URL https://www.R-project.org/.)Continuous variables were expressed as mean with standard deviation (SD) and differences were investigated by use of one-way analysis of variance (ANOVA). Post-hoc analysis was conducted using Tukey Honest Significant Differences. Categorical variables were reported as numbers with percentage and compared with χSupplementary Tables.


The aim of this study was to evaluate the concentration of penicillin G in bone affected by antiresorptive agent-related osteonecrosis of the jaw (ARONJ) following a single preoperative dose of 10 million international units (6000 mg). ARONJ is a major concern in patients administered antiresorptive agents for conditions associated with pathologically increased bone resorption. Antibiotic therapy is a key component of most treatment approaches for ARONJ and penicillin based regimens, providing a cost effective therapy option with a favorable side effect profile, are administered most frequently. In this study, high performance liquid chromatography with tandem mass spectrometry (HPLC-MS/MS) was applied to evaluate penicillin G concentration in serum and bone samples of 19 patients suffering from ARONJ and undergoing surgical treatment under perioperative intravenous (IV) antibiotic therapy. Penicillin G bone concentrations were above the limit of detection (0.1 μg/g bone tissue) in 16 out of 19 samples, with a median concentration of 2.7 μg/g (range 0.1–8.8 μg/g). Penicillin G concentrations in intraoperative serum samples were above the limit of detection in all serum samples, with a median concentration of 116 μg/mL (range 1–232 μg/mL). Thus, considering bacteria frequently found in ARONJ lesions, penicillin G at levels providing adequate antimicrobial activity was detected in the serum and 16 out of 19 osteonecrotic lesions of patients suffering from ARONJ. Actinomyces, Fusobacteria, Streptococcus, Staphylococcus, and Treponema species are reported as a frequent feature of ARONJ [Antiresorptive agents like bisphosphonates or denosumab are a major component of the medical treatment of conditions associated with pathologically increased bone resorption . Antiresorptive agent-related osteonecrosis of the jaw (ARONJ) is a major concern in these patients . Despiteof ARONJ ,4,5.As of now, there is no consensus on the treatment of ARONJ, with various treatment strategies having been proposed that can generally be grouped into conservative/non-surgical and surgical approaches . DespiteConservative approaches generally aim at eliminating pain, controlling infection, and minimizing the progression of bone necrosis . In thisAs with the treatment approaches for ARONJ in general, heterogeneity can be found in the literature regarding the antibiotic regimens administered. However, a recent literature review of perioperative antibiotic administration in surgery for MRONJ/ARONJ found that penicillin-based antibiotics  were the agents administered most frequently . Treatment of bone infections is challenging as the efficacy of antibiotic treatment depends significantly on the penetration capacity of the administered antibiotic to the infection site . In boneIn the past, studies have been conducted to analyze the antibiotic concentration in  jawbone after intravenous administration of penicillin G , piperacTo the authors’ knowledge, to date, there are no data available on the concentration of antibiotics achievable in bone affected by ARONJ. Thus, the aim of this study was to obtain qualitative and quantitative data on penicillin G concentration in bone affected by ARONJ following intravenous (IV) administration.The patients included in this study had a median age of 72.5 years (range 51–81 years) and a median BMI of 23.4; four of the patients were smokers. Fifteen patients were administered antiresorptive therapy for malignant diseases and four patients were administered antiresorptive therapy for osteoporosis. Twelve patients were administered bisphosphonates , six patients were administered denosumab, and one patient had been administered bisphosphonates and denosumab. Twelve patients presented with ARONJ in the mandible, six patients presented with ARONJ in the maxilla, and one patient presented with ARONJ in both jaws. The bone samples had a median weight of 76 mg (range 16–550 mg). The median interval between the start of infusion and the resection of bone was 43 min (range 29–88 min). Concentrations of penicillin G were above the limit of detection in 16 out of 19 bone samples; the median penicillin G concentration in bone was 2.7 μg/g (range 0.1–8.8 μg/g).The median duration of penicillin G infusion was 11 min (range 6–22 min). Penicillin G concentrations were above the limit of detection in all intraoperative serum samples; the median concentration of penicillin G in the intraoperative serum samples was 116 μg/mL (range 1–232 μg/mL). No penicillin G was detected in the preoperative serum samples. There was a ratio of 1:43 between the median bone and serum concentration of penicillin G. The aim of this study was to quantify the level of penicillin G concentration achievable in jawbone affected by antiresorptive-agent related osteonecrosis by measuring penicillin G jawbone concentrations following a single preoperative dose of 10 million IU (6000 mg) administered to patients with a baseline penicillin G serum concentration < LOD.Penicillin G was detected in all bone samples except for three, in which concentrations were below the limit of detection of 0.1 μg/g . ConsideIn our study, the median penicillin concentration in jawbone affected by ARONJ was 2.7 μg/g (2.3% of the serum concentration) in bone specimens obtained at 18 to 72 min after completion of infusion of 10 million IU (6000 mg) of penicillin G. As expected, this value was lower than the one reported for healthy jawbone: In an investigation on penicillin G concentration in patients undergoing wisdom teeth extraction, Otten et al.  reportedEvaluating the concentration of ampicillin in irradiated jawbone, Heibel et al.  found a In their study on penicillin G concentration in healthy jawbone, Otten et al. reported bone concentrations to be highest within the first 40 min after the end of infusion  and a coStreptococcus mutans, Streptococcus anginosus, Fusobacterium nucleatum, Parvimonas micra, and Eubacterium spp. were found in 16 samples. Penicillin G concentrations above the MIC90 for Streptococcus sanguinis, Lactobacillus spp., and Actinomyces spp. were found in 14 samples and penicillin G concentrations above the MIC90 for Prevotella intermedia were found for six samples. Penicillin G at bacteriostatic or even bactericidal concentrations regarding the pathogens listed in To provide a basis for clinical conclusions from the bone and serum concentrations of penicillin G determined in our study, the literature was searched for bacteria frequently found in bone affected by ARONJ ,4,5 and Prevotella intermedia, even high dose intravenous administration of penicillin G may not be sufficient to achieve an adequate antimicrobial effect in bone affected by ARONJ. Thus, in conservative treatment approaches, a beneficial effect of the (long-term) administration of antibiotic therapy seems questionable. Whereas invasive surgical approaches aiming at complete elimination of necrotic bone yielded high success rates, these were much lower in approaches performing  debridement only ,24. RegaIn general, the results of our study can be considered to support the need for surgical resection in ARONJ treatment approaches with a curative intention. In ARONJ therapy approaches aiming at complete surgical resection of necrotic bone, perioperative administration of antibiotics has been reported as an important factor for successful treatment ,26,27,28It can be seen as a shortcoming of this study that we did not consider patient specific parameters like smoking status, BMI, or the type of antiresorptive agent administered. These factors might have had an impact on the penicillin G concentrations found in our study; however, evaluating the pharmacodynamic impact of these parameters would require a larger sample size. Moreover, while only bone tissue determined to be necrotic by visual evaluation  was analyzed, it cannot be fully ruled out that small amounts of healthy bone were unintentionally included in some of the analyzed samples. Prevotella intermedia) have relatively high MIC90 values for penicillin G. Thus, it might be advisable to apply a combined regimen with penicillin and metronidazole as we do at our center.As penicillin-based regimens are the ones administered most frequently in the treatment of ARONJ , there iWhile the literature provides some information on the concentrations of antibiotics achievable in jawbone following IV administration, with this study adding data on concentrations achievable in jawbone affected by ARONJ, there is a lack of information on the concentration of antibiotics in jawbone following oral administration. As this is a route of administration commonly applied in clinical practice, a similar study might be carried out focusing on antibiotics administered orally.This non-randomized, non-blinded, prospective clinical investigation was performed at the Department of Oral and Maxillofacial Surgery, University Medical Center Freiburg, Germany after obtaining approval by the University of Freiburg Ethics Committee (Reference 287/13). All patients included in this study provided written informed consent.Patients were included, if they(i.)presented with a clinically and radiographically confirmed diagnosis of ARONJ;(ii.)had an indication for surgical resection of necrotic bone with subsequent primary closure and preoperative/perioperative administration of IV antibiotic therapy (penicillin G 10 million IU and metronidazole 500 mg); and (iii.)provided informed written consent for this study.Patients were excluded if they(i.)presented with osteonecrosis of the jaw due to other medical circumstances ;(ii.)presented with an allergy to penicillin and/or metronidazole;(iii.)were pregnant or breastfeeding; and(iv.)were minor or incapacitated to provide informed consent.A bone sample as well as pre- and intraoperative blood samples were obtained from a total of 19 patients meeting all inclusion criteria. Control samples were obtained from two patients that underwent procedures with the resection of alveolar bone and without an indication for the administration of antibiotics. As part of this study, the point in time of pre- and intraoperative blood draw, the point in time and duration of administration of preoperative IV antibiotic therapy and the point in time of bone resection were documented.Venous blood was drawn prior to the IV administration of antibiotic therapy (penicillin G 10 million IU and metronidazole 500 mg) and directly after surgical resection of the necrotic bone using serum tubes . Immediately after blood draw, the serum tubes were taken to the laboratory, where the samples where centrifuged at 5300 rpm for 5 min. The serum obtained this way was pipetted into cryotubes and deep-frozen at −80 °C. For further processing, the serum samples were thawed, diluted in methanol in a 1:2 proportion, and subsequently centrifuged at 5300 rpm for 5 min. The supernatant was pipetted into screw thread vials and deep-frozen at −80 °C until HPLC-MS/MS analysis.Bone samples were obtained from the bone affected by ARONJ directly after surgical removal. Subsequently, any adherent tissue was removed from the samples using sterile surgical instruments and all samples were cleaned using a moisturized sterile swab until they were macroscopically free of blood, as has been reported in the literature ,18. RinsCalibration standards were prepared from blank (penicillin G free) blood and bone of the control group, processed the same way as described above. Standard antibiotic solutions  were prepared using the following amounts of penicillin G: 10 mL, 5 mL, 1 mL, 500 μL, 100 μL, 50 μL, 25 μL, and 0 μL. To prepare the bone standards, 0.5 g of the antibiotic-free bone sample was mixed with 5 mL of each of the standard antibiotic solutions. Serum standards were prepared in the same manner.Concentrations of penicillin G in bone and serum were determined by the University Medical Center Freiburg Pharmacy by applying high performance liquid chromatography (Dionex) with quadrupole time-of-flight (q-TOF) mass spectrometry . Defined sample volumes of 2 μL (serum) and 20 μL (bone extract) were applied to the liquid chromatography (LC) column . The calibration standards were used to obtain a calibration row as the basis for calculating the penicillin G concentrations. In this study, resulting from the method applied, the limit of detection (LOD) for penicillin G was 0.1 μg/g bone tissue. In this study, penicillin G at levels providing adequate antimicrobial activity/prophylaxis was found in the serum and 16 out of 19 osteonecrotic lesions of patients suffering from ARONJ. Thus, systemic (IV) administration of penicillin based antibiotic regimens can potentially serve as a useful adjuvant measure in surgical ARONJ treatment regimens aiming at complete elimination of necrotic bone.


The mean SLO levels were the highest in the ferrous treatment, followed by nZVI and mZVI treatments; the differences in the mean SLO levels were significant (p < 0.05). The findings indicate that oral exposure to stabilized ZVI nanoparticles may induce sensory properties different from that of ferrous salt, likely predictive of diminished detection of metallic flavor by humans.Zerovalent iron nanotechnologies are widely used for groundwater remediation and increasingly considered for advance oxidation treatment in drinking water applications. Iron nanoparticles have been detected in drinking water systems and considered for food fortification; therefore, the potential for human exposure through ingestion can be a concern. This study aimed to assess whether ingestion of iron nanoparticles from drinking water could be detected through flavor perception using In Vitro salivary lipid oxidation as an indicator for metallic flavor perception. Ten female subjects, aged 29–59 years, donated saliva samples for use in the In Vitro experiments. Test samples consisted of 1:1 mixture of saliva and bottled drinking water (control) and three treatment solutions, spiked with ferrous sulfate, stabilized zerovalent iron nanoparticles (nZVI), and an aggregated/microsized suspension of mixed zerovalent iron and microsized suspension of iron and iron oxide metal powder, (mZVI). Upon mixing, samples were subjected to 15 min incubation at 37 °C to resemble oral conditions. Salivary lipid oxidation (SLO) was measured in all samples as micromoles of thiobarbituric acid reactive substances (TBARS)/mg Fe. Exposure to iron in all three forms induced significant amount of SLO in all treatment samples as compared to the control ( Metallic iron in its zerovalent form has been widely used in the treatment of contaminated groundwater since the early 1990s, when it was first discovered that chlorinated hydrocarbons, such as trichlorethene (TCE), could be dehalogenated in the presence of iron metal . Since tEnvironmental nanoparticles (NPs), sized 1 to 100 nm, are classified as naturally occurring or engineered. Naturally occurring NPs are ubiquitous in nature and exist in all environmental media: air, water, and the subsurface . On the While elemental iron is a vital nutrient for the maintenance of body functions in all organisms, its toxicity potential is widely recognized due to its ability to cause oxidative stress in living cells by damaging membrane lipids and DNA. Oxidative stress is defined as an imbalance between production of free radicals and reactive oxygen species and their destruction by the protective actions of antioxidants and enzymes within the body . AssociaThe ability of iron to induce oxidative stress in biological tissues and fluids has been quantified by measuring lipid oxidation products using the method of thiobarbituric acid reactive substances (TBARS). Lipid oxidation has been extensively studied using the TBARS assay, which involves derivatization of malondialdehyde (MDA) with thiobarbituric acid to produce a pink product that is quantified in a UV–VIS spectrophotometer . IntriguThe aims of this manuscript are to communicate findings of this study, which utilized salivary lipid oxidation (SLO) as an indirect measure of metallic flavor intensity to compare iron-induced SLO between soluble ferrous iron salt and nanoparticles of stabilized zerovalent iron products in micro- and nanoscale suspensions. The study aimed to explore whether the use of SLO can be a useful screening tool for predicting sensory properties and oxidative stress potentials from oral exposure to iron nanoparticles.This study was approved by the Institutional Review Board (IRB) at Virginia Tech (IRB Project No. 06-395). Human subjects were recruited from the community, students, faculty, and staff of Virginia Tech and Blacksburg, Virginia, by means of paper and email flyers. Subjects were required to have no chronic oral or general health problems, be nonsmokers, and not pregnant. All subjects read and signed an informed consent form in accordance with the approved IRB protocols. Ten female subjects, aged 29–59 years, donated saliva for use in this study.® bottled water and wait 1 min to stabilize the conditions in their mouth. Then, subjects’ oral pH was measured using a pH indicator strip  by placing the pH strip on their tongue until it became moist with their saliva; then, using the color scale, the pH was read and recorded after 1 min and while the strip was still moist. Subjects were asked to expectorate approximately 4 mL of saliva into 50 mL propylene tubes during each session for a total volume of 12 mL collected over the three daily sessions. Between daily collections, saliva samples were kept stored in a freezer; at the end of final collection, saliva samples were frozen immediately and stored at −50 °C for up to one month until analysis. For long-term storage, temperature ranges of −20 to −80 °C have been shown to preserve stability of salivary constituent for up to 3 months [Subjects were asked to refrain from eating, drinking, and smoking for at least one hour prior to collecting their saliva sample. Three daily saliva collection sessions were conducted within the same week and time frame of 10 a.m. to 12 noon. For each daily session, subjects were first asked to thoroughly rinse their mouth with Aquafina3 months .®) to provide for 10 mg/L of total iron concentration in the resulting solution. For the NANOFER 25S, the suspension as provided by the manufacturer contained approximately 15% Fe(0), and it was diluted accordingly to the targeted concentration of 10 mg/L total Fe. The NANOFER STAR solution was prepared by mixing 1 part (10 g) of the powder with 4 parts (40 mL) of the bottled drinking water using a blender to provide for approximately 15% Fe(0) suspension; then, the mixture was diluted to the targeted concentration of 10 mg/L total Fe. As pH level is important in iron chemistry as well as stability of nanoparticles in suspension, pH was measured in the stock solutions of the prepared samples using pH indicator strips (Cen-Med/Fisher M95883). The total iron concentration of each stock solution was quantified using inductively coupled plasma mass spectroscopy , following Standard Method 3120B from the American Public Health Association Standard Methods for Examination of Water and Wastewater [Three different stock solutions of iron were prepared immediately before use in the experiments. The stock solutions included iron (II) sulfate  and two aqueous dispersions of manufactured nZVI products, NANOFER 25S and NANOFER STAR . The iron solutions were prepared by mixing the iron salt or the nanoiron product with bottled drinking water (Aquafinastewater .3O4 (6–2%), carbon (0–1%), and a surfactant (3%) as an organic stabilizer. The NANOFER STAR is described as an air-stable highly reactive powder consisting of Fe(0) surface-stabilized nanoparticles coated by a thin organic surface layer to protect against air oxidation and allow for ease of transport and long-term storage. The powder mixture was composed of ≥80% iron and iron oxide. For both products, the average particle size was reported as <50 nm; the specific surface area was >25 m2/g with spherical morphology; the dispersion density was 1210 kg/cm3. As indicated by the product manufacturer, the NANOFER STAR is expected to behave like a micro-ZVI particle if the powder is applied directly to water without dispersing. To verify the particle size distribution and zeta potentials for freshly prepared suspensions, the iron nanoparticles were analyzed by dynamic light scattering . For reliable particle size and zeta potential determinations, the suspensions had to be diluted 100 times  solutions) and were filtered (1 µm) as necessary for particle size determination.Characteristics of the iron nanoparticle products were provided by the manufacturer . The NANOFER 25S is described as a stabilized water dispersion of nanoscale zerovalent iron consisting of Fe (14–18%), Fe®). The choice of Aquafina® as the control and dilution water was based on its low level of total dissolved solids (<10 mg/L) as used in a previous metallic flavor research study with human subjects [g. The absorbance of the supernatant from each sample and standard was measured with a spectrophotometer at 532 nm. The concentration of TBARS was obtained from the standard curve and absorbance values. The standard curve was developed by running the TBARS method on known MDA standards at 0.03 to 10 μM concentrations. TBARS analysis was run in duplicates for each sample. As equal volumes of saliva and control or treatment solutions were mixed for In Vitro experiments, a 50% dilution factor was applied in the calculation to obtain the actual concentration of TBARS in each sample.In Vitro experiments were performed using individual saliva samples from each of the 10 subjects as well as a single pooled saliva sample from multiple subjects. The pooled saliva sample consisted of a mixture of equal volumes of saliva from all 10 subjects. Saliva pooling was done to account for potential differences in experimental outcomes due to variation in salivary fluid between subjects. In Vitro test sample preparations followed procedures utilized in previous In Vivo  experiments for determination of ferrous iron sensory threshold and oral lipid oxidation measures in human subjects ,31. Thussubjects . The tresubjects  was modisubjects . Using tStatistical software, JMP 9.0 , was used for all data analyses. One-way analysis of variance (ANOVA) and comparison of the means using Tukey’s honestly significant difference (HSD) or Wilcoxon/Kruskal–Wallis rank sum test were performed on the mean oxidative stress responses as measured by salivary lipid oxidation in the control and test samples. The SLO was also reported as delta SLO as the arithmetic difference between the measured salivary TBARS in the control and the iron-containing test samples. To normalize the measured iron-induced SLO levels, the delta SLO values in micromoles of TBARS were divided by the concentration of total iron in milligrams. All statistical analyses were performed at alpha level of 0.05, and results are presented as means ± standard error (SEM).The particle size distribution of the stabilized suspension of the iron nanoparticle product, NANOFER 25S, ranged from 18 to 110 nm with an average size of 52 nm; the zeta potential was measured at −60 mV a. The prThe pH levels in saliva ranged from 6.0 to 7.0 . The pH was 5.0 in the three iron stock solutions, while the measured pH was 7.0 in the control and all treatment samples.F  = 15.60; p < 0.0001) as well as the pooled saliva sample  = 164.32; p < 0.0001)  .F  = 3.72, p = 0.035). Follow-up comparison of the mean delta SLO responses using Wilcoxon’s test indicated a significance difference only between the Fe(II) and nZVI treatments (p = 0.038) and Fe(II) and mZVI (p = 0.044) but no significant differences between nZVI and mZVI (p = 0.47)  indicated a significant difference between at least one pair of the mean responses ( = 0.47) .F  = 34.75, p = 0.0005). Follow-up comparison of the mean delta SLO responses using Tukey‘s HSD test indicated a significant difference between the treatment pairs Fe(II) and nZVI (p = 0.0045) and Fe(II) and mZVI (p = 0.0004) but no significant difference between nZVI and mZVI (p = 0.058)  .2O3) induced lower toxicity with no considerable difference between the particle sizes in terms of induced oxidative stress [The findings from this study indicate that ferrous iron salt, being the most soluble compared to the two nanoiron products, induced the highest level of oxidative stress response in human salivary fluid as measured by lipid oxidation. After ferrous iron, the stabilized suspension of nZVI product, NANOFER 25S, produced the next highest level of iron-induced oxidative stress, with the stabilized powder, NANOFER STAR, showing the least oxidative stress response. Based on these observations, it is evident that, in the case of stabilized zerovalent iron composed of an outer layer of iron oxide, the potential for iron-induced salivary oxidative stress is higher when particle size becomes smaller, whereas the oxidative stress declines when particles get larger by size characteristics and/or the aggregation effect. Previous research studies exploring the impact of particle size on nanometals toxicity have produced varying results. In one study, nanometer or micrometer particle size toxicity varied by the type of metal; for example, copper oxide in its nanoform induced higher oxidative stress in human lung cells when compared to microsized particles. In the case of iron, iron oxide (Fee stress . Other se stress ,37,38.In aqueous systems, the role of pH in influencing zeta potential is critical as it indirectly influences particle charge and stability by changing the zeta potential in aqueous suspensions . NanosizIn human saliva, the pH remains relatively neutral, with levels ranging from 5.5 to 7.5 ,44. The In human sensory studies, the unpleasant flavor of iron in water has been described as “metallic” and “rusty nail”, leaving an aftertaste in the mouth of tasters . UtilizeExposure of humans to iron and other essential, trace, or toxic metals and their associated nanoparticles are likely scenarios as they are widely present in the environment through natural and engineered systems and products ,55. TherWith increasing and innovative use of iron nanoparticles in a variety of applications, concerns for potentially toxic exposure to humans as well as other organisms are well founded. Equally important are considerations for the positive benefits of using iron nanoparticles for enhancing food quality and nutrition ,10. ThisFor improved detection ability, In Vitro lipid oxidation studies using human saliva as a biomarker of exposure to metals should be conducted with pooled saliva in order to reduce variability among human subjects. As indicated by this research, coefficient of variation for SLO response could range from 45% to 60% when using individual saliva samples, whereas it was reduced to 3% to 16% with the pooled saliva sample, thus enhancing the ability to detect significant differences between the mean SLO responses among different treatment groups for exposure studies. Reducing variability is especially important for using the test as a screening method.Lastly, with human salivary fluid enriched with numerous proteins, including those with metal-binding capacities, fatty acids and lipids, electrolytes, and antimicrobial agents, human saliva, in real or simulated formulation, can be more widely used as an innovative and simple means by which interactions and toxicity effects of stabilized nZVI and other metal nanoparticles in biological fluids can be studied and compared.


Bis (2-Ethylhexyl) phthalate (DEHP) was the major pollutant  in all types of indoor dust with a concentration up to 3,901,500 ng g−1. While dibutyl phthalate (DBP) and DEHP together contributed >70% in PM10 (1900 ng m−3), which indicate PM10 as a significant source of exposure for DBP and DEHP in different Saudi indoor settings. Tris (1-chloro-2-propyl) phosphate (TCPP) was the major OPE in PM10 with a concentration of up to 185 ng m−3 and the occurrence of OPEs in indoor dust varied in studied indoor settings. The estimated daily intake (EDI) of studied chemicals via dust ingestion and inhalation of PM10 was below the reference dose (RfD) of individual chemicals. However, estimated incremental lifetime cancer risk (ILCR) with moderate risk (1.5 × 10−5) for Saudi adults and calculated hazardous index (HI) of >1 for Saudi children from DEHP showed a cause of concern to the local public health.In this study, we measured the occurrence of organophosphate esters (OPEs) and phthalates in the settled dust (floor and air conditioner filter dust) and in suspended particulate matter (PM10) from different microenvironments (households ( Organophosphate esters (OPEs) and phthalates are used in various consumer products to add elasticity and retard the onset of unwanted fire to minimize fire hazards ,2,3. TheRecent studies have reported these chemicals’ presence in indoor dust and other environmental samples ,14,15,16Saudi Arabia is going through fast industrialization for the past few decades Saudi population’s lifestyle. As a result, the lifestyle of the Saudi population has also changed dramatically. Still, studies are lacking to understand the impact of changing lifestyles and changing indoor environments on their health. Due to these scenarios, the current study reported these chemicals’ incidence into the suspended particulate material (PM10) of the Saudi indoor environment for the first time in Saudi Arabia. This primary study objective is to explore the profiling of selected OPEs and phthalates in PM10 and dust of selected Saudi indoors and estimate the exposed population’s exposure via dust ingestion, dermal intake and air inhalation.Analytical standards of phthalates namely bis(2-ethylhexyl) phthalate (DEHP), benzyl butyl phthalate (BzBP), bis(2-ethylhexyl) adipate (DEHA), dibutyl phthalate (DBP), diethyl phthalate (DEP), dimethyl phthalate (DMP) and OPEs namely tris  phosphate (TCPP), tris (2-chloroethyl) phosphate (TCEP), tris  phosphate (TDCPP) and triphenyl phosphate (TPhP) were purchased from AccuStandards and Sigma Aldrich. Corresponding deuterated (d4-labelled) phthalates were used as internal standards (ISs) for phthalates while TCEP-d12 was used for OPEs. All stock solutions for the analytical standards were prepared in iso-octane and toluene. Acetone, dichloromethane (DCM), n-hexane (n-Hex) and iso-octane were of analytical grade obtained from Sigma Aldrich.n = 20), hotels (n = 10) and university offices (n = 10) of Jeddah, Saudi Arabia. The households sampled in this study were from Jeddah’s town, and they were all apartments that are the major part of housing in the city of Jeddah. This type of apartment/housing is typical and shared throughout the country. The sampled hotels were of 3- and 4-star categories that are usually used for most visiting pilgrims. Floor dust was obtained using the vacuum cleaner method previously reported by Ali et al. [TM, MSP Corporation, was used in the selected households (kitchen and living rooms), hotel rooms and university offices to collect PM10 samples. The sampler was installed for 24 h in each selected indoors, and the PM10 sample was collected on 47 mm quartz fiber filter paper using the flow of 10 L min−1. Before sampling filter papers were oven-baked at 400 °C for 6 h and kept in the desiccators until use. This precondition of the filter paper helped in getting rid of any moisture and prior contamination. A microbalance was used to measure PM10 levels, and after that, sampled filter paper stored at −20 °C until analysis.For this study, paired particulate material (PM10) and indoor dust samples were collected simultaneously from households ) was added, and samples were extracted using ultrasonication (20 min) followed by centrifugation (3000 rpm for 10 min). The supernatant was collected in a clean tube, the same extraction procedure was repeated twice with the leftover samples. The extracts were pooled and brought to incipient dryness using a gentle stream of nitrogen. After drying, samples were resolubilized again in 1 mL the solvent mixture (hexane and toluene) and cleaned further using silica BondElut  and 10 mL solvent mixture (hexane/dichloromethane) and after with 4 mL of ethyl acetate for quantitative analysis. After elution, the obtained fraction was concentrated to incipient dryness under a gentle stream of nitrogen. It then was resolubilized in 100 µL of iso-octane for gas chromatography–mass spectrometry (GC–MS) analysis. The same extraction procedure used for the extraction of OPEs and phthalates from PM10.A detailed description of sample preparation is provided by Ali et al. . Briefly−1. The oven temperature was raised from 90 to 300 °C using a ramp of 15 °C min−1 and held for 1 min.The detailed description of the used instrument analysis is provided elsewhere ,19. Brie2SO4 (dust replica) spiked with a known concentration of standards were used to evaluate the procedure accuracy. The analytes’ levels found in procedural blanks corrected from the concentrations of the analysts in the samples. The values of OPEs and phthalates in SRM 2585 were similar (RSD < 25%) with other reported values [2SO4 ranged between 70 and 130%. To have good QA/QC during the experimental procedure, all the glass-wares used were baked at 400 °C overnight and kept at 100 °C until use. Standard reference material (SRM) 2585 from the National Institute of Standards and Technology (NIST), procedural blanks (1 for every 8 samples), washed Nad values  and otheIn the present study, health risk assessment for the local population was calculated by per day exposure, incremental lifetime cancer risk (ILCR), hazard quotient (HQ) and hazardous index (HI). For this purpose, different equations were used as reported earlier ,22. SincThe following Equations (1)–(3) were used to calculate non-carcinogenic chronic daily intake via dust ingestion, inhalation and dermal contact. For HQ calculation of each exposure route Equation (4) was used and HI was calculated by combining the HQ of different exposure routes (Equation (5)).n represents the chemicals’ concentration, and for these calculations, we used the 90th percentile of the concentrations. Concentrations in dust were considered for ingestion and dermal contact while levels of these chemicals in PM10 were considered for the inhalation route. Since the AC filter dust is trapped in the filter and humans are not directly exposed to this dust. However, humans are directly and indirectly getting exposed to floor dust. Though, a part of AC filter dust, which escaped the AC filter, also settled down on the floor dust. Therefore, for these calculations, we only considered floor dust values relevant to ingestion and dermal exposure ro utes. The parameters of the Equations (1)–(3) are explained in In the above equations, CWe estimated carcinogenic risk exposure via different exposure routes using Equations (6)–(8) and the total carcinogenic risk was calculated combining all calculated exposure routes and cancer slope (SF) for children and for adults using the below Equation (9) .(6)CDII−1 d−1) was available for only TCEP, DEHA and DEHP for oral and inhalation routes [Cancer slope factor (SF)  and PM10 (ng m−3), IR is the dust ingestion rate (100 mg d−1 for adults and 200 mg d−1 for young children) and inhalation rate  for working people) and for people working in hotels . With the lack of knowledge on these chemicals’ bioaccessibility, we assumed 100% bioaccessibility for the EDI. Bodyweight of 70 kg for adults and 15 kg for young children was considered for the calculations.Estimated daily intake (EDI) was calculated via dust ingestion and air inhalation using the following Equation (10):t-test was applied using GraphPad to study the difference of PM10 in different microenvironments, and the significance level was p <0.05. Since the data did not show normal distribution, therefore data was log-transformed, and outliers were removed before using two-sample t-tests.For descriptive analysis, Microsoft Excel 2007 was used. A two-sample −3 (−3), followed by those from household living rooms (median: 68 ng m−3), hotel rooms (median: 62 ng m−3) and offices (median: 20 ng m−3). Levels of ∑OPEs were significantly (p < 0.05) lower in the PM10 collected from university offices than those of other studied indoor microenvironments. To the best of our knowledge, this is the first study reporting OPEs in PM10 from various microenvironments of this region. Among studied OPEs, TCPP was present at the highest concentrations in PM10 of all microenvironments, while TDCPP contributed the lowest proportions in the PM10 samples. Although OPEs in PM10 collected from the kitchen and living rooms of the households varied but were not significantly different (p > 0.05). Similarly, the profile of these chemicals was also similar in both microenvironments (p < 0.05) in PM 10 samples from household than those found in the hotel and office, while levels of TCPP were significantly (p < 0.05) lower in the office PM10 than other indoor microenvironments. These significant differences indicate that the household air was more contaminated with OPEs than hotel and university offices. This showed there were more sources of emission inside the households than other studied microenvironments. Contrary to homes, most of the sampled hotel rooms and all the university offices had a centralized cooling and ventilation system, which might be another reason for the low concentrations of OPEs in these indoor air. The levels of OPEs in present indoor air samples were similar to more deficient than those reported in indoor air from other countries [The total concentrations of OPEs (∑OPEs) in PM10 ranged between 9 and 295 ng m−3 . Among vronments . This miountries ,28,29,30−1) was the major OPE in AC filter dust, while TCPP (median: 2650 ng g−1) and TDCPP (median: 1530 ng g−1) were the major OPEs in the hotel and household floor dust, respectively. The high levels of TDCPP in the households were due to the high concentration of this chemical in a couple of floor dust samples. By removing outliers, TCPP (median: 1300 ng g−1) was the major OPE in household floor dust as well. The levels of TCEP were significantly higher in AC filter dust than household and hotel floor dust, which might indicate material used in the air conditioning as a source of their emission. TDCPP was also significantly high (p < 0.05) in AC filter dust than household and hotel floor dust. Except for TCEP, the other three OPEs were detected in all the dust samples from studied microenvironments. The levels of OPEs in indoor dust were different from those reported from other countries , electronic types of equipment, PVC, glues, nail polishes, etc.) and a plasticizer in many products. After the phasing-out of Penta-BDE, the Firemaster® 550 (Chemtura) was used in polyurethane foam and other applications, resulting in its widespread occurrence in indoor environments found in this study too.The consistent occurrence trends for the high levels of OPEs in studied samples might indicate their widespread use as FRs and plasticizers in different consumer products. These chemicals reported to be used in flexible and rigid polyurethane foam, furniture foam, acrylic latexes for the back coating and binding of non-woven textiles products, leather, electronics and building construction laminates, furniture and baby products such as nursing pillows, portable mattresses, car seats, seat positioners and changing table pads ,32. Halo−3 (−3), followed by those collected from household living rooms (median: 830 ng m−3), hotel rooms (median: 650 ng m−3) and offices (median: 315 ng m−3). Levels of ∑phthalates were significantly lower in office PM10 (p < 0.05) than other studied indoor microenvironments. As discussed in the previous section (p > 0.05). DEP levels were significantly lower in homes (living room and kitchen) PM10 than those found in hotels and offices. DEP was used to improve the performance and durability of several products. As a plasticizer, it was added to plastic polymers to help maintain flexibility. It has been used in various products, including plastic films, rubber, tape, toothbrushes, automotive components, tool handles and toys. In addition to plastics, DEP is present in a wide range of personal care products , industrial materials  and medical products  [p > 0.05) in PM10 samples from offices than other sampled microenvironments. Although less volatile nature of DEHP, the high levels of its presence in PM10 than some of the other analyzed phthalates can be justified due to their increased fact in indoor dust, indicating extensive use in indoor consumer products. Number of studies have reported the concentrations of phthalates in the indoor environment [The total concentrations of  in PM10 ranged between 245 and 1900 ng m−3 . To the  section  centrali section , which iterials) . These eironment ,14, and −1), hotel floor  and household floor dust . DBP was the 2nd significant phthalate in all indoor microenvironment dust with a median concentration of 18, 850 ng g−1 in household floor dust, 41,450 ng g−1 in hotel floor dust and 26,700 ng g−1 in AC filter dust. The high DEHP levels were not surprising considering its extensive application as a plasticizer in polymer products, which are extensively utilized in furniture material, cosmetics and personal care products [rd dominant phthalate in dust samples from all three microenvironments with median concentrations of 3550, 7810 and 1620 ng g−1 of dust in AC filter, hotel and household floor dust, respectively. DEHA were significantly higher (p < 0.05) in hotel floor dust than the AC filter dust, indicating AC is not a primary source of DEHA in indoors. Still, other indoor inventories are mainly responsible for their indoor presence. The major application of DEHA is in hydraulic fluid and PVC-based plastic food wrap but is not chemically bound to the polymer, thus migrates into the surrounding environment [−1. DMP contributed the least with the highest median concentration of 410 ng g−1 of dust in the household floor dust, and this is understandable because of the volatile nature of DMP.The total concentrations of ∑phthalates in indoor dust samples from different microenvironments are presented in products . DBP is Phthalates contamination has been reported into indoor dust from different countries . The varSeveral studies have reported that exposure to OPEs and phthalates is associated with various health issues such as DEHP, an endocrine disruptor and have carcinogenic properties . Rowdhwa−4 and 1.00 × 10−6 [−5, this indicates a moderate carcinogenic risk for the public health according to Health Canada Guidelines [As discussed above, many of the studied chemicals reported to be carcinogenic; therefore, the main interest in calculating the ILCR was to look at the potential long-term cancer risk via dust exposure to the Saudi young and adult people from the indoors. The probabilistic ILCR assessment was highest via dust ingestion, followed by dermal contact and inhalation . The USE0 × 10−6 . Therefoidelines . In a reidelines . Limitedidelines  and its This is the first study reporting OPEs and phthalates in the indoor PM10 of Saudi Arabia. The ∑phthalates and ∑OPEs concentrations in PM10 from the kitchen were highest, which highlighted the risk for people working in the kitchen for a longer time. Dust from the AC filter was more contaminated with these chemicals than dust from other microenvironments. The air conditioning system (AC window and split AC) helps in indoor air circulation, but at the same time, it might circulate more chemicals in the indoor air, which otherwise settle down to the ground. However, large scale studies are needed to establish the overall contribution of different air conditioning systems to indoor pollution. DEHP was the major pollutant among studied chemicals in both PM10 and indoor dust from all microenvironments, which indicates its high use in consumer products in the local market. The EDI and ILCR calculations showed a cause of concern to the Saudi public health from exposure to DEHP via contaminated indoor dust and PM10. Dust ingestion and dermal contact were the primary exposure routes for both adults and young children. The probabilistic cancer risk assessment from ∑phthalates and DEHP, total ILCR, was high up to 1.5E-5 for Saudi adults, which might indicate a moderate risk to the exposed Saudi population and a cause of concern public health. Limited studies are available on this topic from this region. Therefore, extensive scale studies will be needed to understand the dynamics of organic pollution in Saudi indoor environments and their impact on public health.


Background and purpose: Ischemia–reperfusion injury is encountered in numerous processes such as cardiovascular diseases or kidney transplantation; however, the latter involves cold ischemia, different from the warm ischemia found in vascular surgery by arterial clamping. The nature and the intensity of the processes induced by ischemia types are different, hence the therapeutic strategy should be adapted. Herein, we investigated the protective role of tannic acid, a natural polyphenol in a rat model reproducing both renal warm ischemia and kidney allotransplantation. The follow-up was done after 1 week. Experimental approach: To characterize the effect of tannic acid, an in vitro model of endothelial cells subjected to hypoxia–reoxygenation was used. Key results: Tannic acid statistically improved recovery after warm ischemia but not after cold ischemia. In kidneys biopsies, 3 h after warm ischemia–reperfusion, oxidative stress development was limited by tannic acid and the production of reactive oxygen species was inhibited, potentially through Nuclear Factor erythroid-2-Related factor 2 (NRF2) activation. In vitro, tannic acid and its derivatives limited cytotoxicity and the generation of reactive oxygen species. Molecular dynamics simulations showed that tannic acid efficiently interacts with biological membranes, allowing efficient lipid oxidation inhibition. Tannic acid also promoted endothelial cell migration and proliferation during hypoxia. Conclusions: Tannic acid was able to improve renal recovery after renal warm ischemia with an antioxidant effect putatively extended by the production of its derivatives in the body and promoted cell regeneration during hypoxia. This suggests that the mechanisms induced by warm and cold ischemia are different and require specific therapeutic strategies. Ischemia is the cessation of blood flow to an organ and reperfusion is its restoration. The combination of both processes induces a range of lesions at the cellular and organ level, leading to increased cell death and immunogenicity, assembled under the term Ischemia–Reperfusion Injury (IRI). This pathology is widely encountered in medicine, particularly during vessel and heart surgery and is unavoidable in organ transplantation. Indeed, all organs commonly require transport from the donor to the recipient, cold flushing with a preservation solution, and maintenance of hypothermia (approx. 4 °C) during transport from 2 to 36 h). IRI induces several pathways, such as inflammation, cellular death by necrosis or apoptosis and oxidative stress, particularly during the reperfusion stage ,2. Oxida to 36 h.O-galloyl-beta-D-glucose (PGG) nucleus and several gallic acid moieties. It has shown several beneficial effects, such as reducing serum cholesterol and triglyceride levels, as well as antioxidant activities [Tannic acid is a plant-derived polyphenol from the family of hydrolysable tannins. It is composed of a penta-tivities ,4,5. Thetivities   is a transcription factor sensitive to ROS and a key player in oxidative metabolism . InteresIn the present study, we hypothesized that tannic acid, metabolized to gallic acid and PGG, will provide lasting protection against IRI. We also hypothesized that such protection could differ between warm and cold ischemia.®, column C18X Terra MS, Waters) associated to spectrophotometry, according to the method previously described with some modifications [2 solution (Merck), 0.1 mL of 0.6 M CaCl2 solution, and 500 UI of β-glucuronidase . The samples were incubated for 45 min at 37 °C. Enzymatic hydrolysis was stopped by the addition of 0.5 mL of 1N sulfuric acid and component were extracted twice with 4 mL of ethyl acetate. After evaporation, the residue was dissolved in 0.2 mL of ethyl acetate and evaporated again and dissolved in 0.2 mL of mobile phase . The chromatogram analysis conditions are flow rate 0.7 mL/min (isocratic), run time: 10 min.To determine the bioavailability of tannic acid after injection and to identify the kinetics of its metabolite formation, gallic acid concentrations were determined in rat blood. After intraperitoneal injection of tannic acid (50 mg/kg), blood was collected in the tail vein at 0.5, 1, 2, 4 and 24 h. Gallic acid determination was performed by HPLC .Sixty-four rats were randomized into eight groups of eight animals. The 7 d groups designate animals with 7 d of reperfusion after ischemia (sacrificed on day seven after IR) and the 3 h groups designate animals with 3 h of reperfusion (sacrificed 3 h after IR). The IR groups designate rats which underwent bilateral renal ischemia–reperfusion and injection of a vehicle solution according to the protocol described below. The groups IR + tan designate the rats which underwent bilateral renal ischemia–reperfusion and injection of a tannic acid solution. The groups Sham 3 h, Sham 7 d, Sham + tan 3 h, Sham + tan 7 d designate rats with the same surgery, but without a renal ischemia–reperfusion sequence with the injection of vehicle solution or tannic acid .Male Sprague–Dawley rats , from 265 to 350 g, were used. They were maintained in a climate-controlled room at 22 ± 2 °C and a humidity of 60 ± 5%, on a light/dark cycle, and provided a standard commercial diet and water ad libitum. After an adaptation period of three days, rats were anesthetized by isoflurane inhalation (induction 4% and maintenance 1.5%) associated with ketamine intraperitoneal injection (12.5 mg/kg). Rats were then randomly injected intraperitoneally with tannic acid (50 mg/kg) or the vehicle (NaCl 0.9%). Bilateral renal ischemia was performed 30 min after injection, by right and left renal pedicles clamping using appropriate vascular clamps . After 60 min, clamps were removed. Good vascularization of the kidneys was visually checked by the coloration change. Blood collection was performed by the tail vein. At the time of euthanasia, the kidneys were collected and biopsies performed for tissue analysis .Thirty-five rats were randomized into four groups:n = 12): kidney transplantation with a bilateral nephrectomy and vehicle solution injection;-KT (Kidney Transplantation) (n = 11): kidney transplantation with a bilateral nephrectomy and tannic acid solution injection;-KT + tan (n = 6), and Sham + tan. (n = 6): rats with a dissection of the left renal pedicle and ureter and right nephrectomy with vehicle solution or tannic acid.-Sham , from 250 to 350 g, were used with similar housing conditions as above. After an adaptation period of three days, rats were anesthetized by isoflurane inhalation (induction 4% and maintenance 1.5%). In the donor rat, after dissection of the left renal artery and vein, the ureter was sectioned and the kidney dissected. Then, ligation of the infrarenal aorta and the suprarenal aorta were performed and the renal vein was ligated and cut. An arteriotomy was performed and a 25-gauge needle was inserted into the aorta. The kidney was flushed with 10 mL of SCOT 15 at 4 °C and the renal artery was sectioned. The kidney graft was placed in 30 mL of SCOT 15 at 4 °C for 6 h. The recipient rats were then randomly injected intraperitoneally with tannic acid (50 mg/kg) or vehicle solution (NaCl 0.9%). Thirty minutes after injection, kidney transplantation began. The surgical time, including bilateral nephrectomy and anastomosis, did not exceed 60 min. After dissection of the left renal artery and vein in the recipient, the ureter was sectioned and the kidney dissected. Then, clamping of the renal vessels was performed. The artery and vein were cut as close as possible to the hilum. The renal graft was set in place of the recipient left kidney and end-to-end vascular anastomoses were performed. The donor ureter was anastomosed with the ureter of the recipient with an end-to-end anastomosis. Right nephrectomy was then performed. Sham procedure consisted in a dissection of the renal pedicle and the ureter with a mobilisation of the kidney and a right nephrectomy. Blood collection was performed by the tail vein. At the time of euthanasia, the kidneys were collected and biopsies performed for tissue analysis .Plasma creatinine concentrations were measured on the Cobas C701 automatic analyzer  by the compensated Jaffé technique.Total glutathione (GSH) and superoxide dismutase (SOD) activity within kidney biopsies were measured, respectively, with the glutathione assay kit  and the SOD activity kit (Bertin Pharma), following the manufacturer’s instructions. Protein levels in homogenate were determined by the Bicinchoninic acid protein assay kit .Briefly, frozen sections on renal cortex samples were used for Cell ROX assay and NRF-2 staining  detected by immunofluorescence. Protein levels were determined through western blotting according to standard protocols [Interleukin 6 (IL-6), Interleukin 10 (IL-10) and tumor necrosis factor alpha (TNFa) levels were determined in blood with Enzyme-Linked ImmunoSorbent Assay (ELISA) kit assays  following the manufacturer’s instructions.To mimic the ischemia–reperfusion found in vivo, we used a standard protocol of hypoxia–reoxygenation in human aortic endothelial cells , cultured as recommended by the manufacturer . Cells w-). The deprotonation was at the carboxyl group, as the pKa of the COOH group of gallic acid is around 4.3, while the phenolic OH groups have pKa ranging from nine to 13 [To obtain the necessary parametrisation for the molecular dynamics (MD) simulations, tannic acid was split into two parts, the PGG  moiety and the gallic acid (phenolic) tails, both with an acetyl cap. Both moieties were optimised with the B3LYP/6-31G* method with the Gaussian software. The optimised structures were used for charge calculation with the HF/6-31G* method. The partial charges were obtained using the Amber RESP procedure performed according to the official Amber manual. The structures of the sugar moiety and the gallic acid tail were then aligned in a pairwise manner in the PyMol software, using the carbonyl carbon, carbonyl oxygen and ether oxygen. The caps were removed, and the structures were connected between sugar carbonyl carbon and the corresponding aromatic ring carbon of the tail. The gaff2 forcefield parameters were used for the tail part of the molecule and the GLYCAM forcefield was used for the sugar moiety. To check the parametrisation and stability of the molecule, a 50 ns run was performed in water. The same starting structure of the molecule was then inserted into a simulation box containing an equilibrated solvated 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) bilayer solvated in water, and the whole system was minimised. The simulations were conducted with three different replicas, for 1µs each. Gallic acid was modelled in the POPC bilayer in a similar way to tannic acid. Gallic acid was inserted in the POPC membrane box and the whole system was simulated for 500 ns, which allowed sufficient sampling of insertion events and diffusion of gallic acid in the lipid bilayer. To evaluate the impact of the protonation state, gallic acid was simulated in its two major protonation states, namely the fully protonated one  and the deprotonated one . The values shown are means ± SD. We used Newman–Keuls or Kruskal–Wallis tests for multiple comparisons or a Mann–Whitney test for comparisons between two groups. Statistical significance was considered for A high level of gallic acid (around 19 mg/L) was detected in the plasma 1h after injection and remained detectable at 24 h , indicatIntraperitoneal injection of tannic acid 30 min before surgery improved renal function recovery after 60 min of renal ischemia: at 24 h post ischemia, plasma creatinine levels in the group without tannic acid injection increased up to 300 µmol/L, while treated animals showed a mild increase (approximately 100 µmol/L). After 7 d, the IR + tan group showed a full recovery of renal function compared to the untreated group .According to the literature, 6 h of cold ischemia was chosen  to inducKidney biopsies at 3 h after warm ischemia-reperfusion were collected and analyzed. Tannic acid limited the expressions of major pro-oxidant enzymes such as xanthine oxidase and p67 phox, a subunit of NADPH oxidase involved in superoxide anion production A,B. In aThe mechanism of tannic acid protection was characterized using a cellular model of endothelial cell subjected to warm hypoxia–reoxygenation. Tannic acid was added to the culture medium during hypoxia and reoxygenation steps, mimicking the rat treatment. A dose-dependent reduction of cytotoxicity was observed with the different tannic acid derivatives A. These The capacity of tannic acid to be inserted into membranes, and then to act as an antioxidant to protect against lipid oxidation, was evaluated by MD simulations performed on in silico models of membranes solvated in water. The model was made of POPC lipids, being one of the most representative phospholipids present in the organism. Tannic acid was primarily positioned in the bulk water, where MD simulations revealed a large structural variability. Several stable tannic acid conformations were identified, mainly driven by π–π stacking interactions . Within Both the protonated and deprotonated forms of gallic acid interact with the membrane, albeit in slightly different manners. The protonated  form moves quickly at the surface of the membrane and is positioned in close contact with the glycerol moiety and the upper part of the lipid tails; during the simulation, it occasionally reaches the middle of the lipid tails. The deprotonated (anionic) form quickly reaches the surface of the membrane; however, it is positioned further from the membrane centre, fluctuating from the water phase to the glycerol moiety, with only little contact with the lipid tails .The potential of plant-derived medicine in the fields of aging, cancer, neurodegenerative disorders and cardiovascular diseases has attracted increasing interest in past decades. However, the beneficial effect is linked to an appropriate concentration in the body in the long term. Tannic acid is a polyphenol found frequently in plants. It is known for its pharmacologic properties, such as antiseptic, veinotonic, bactericides and antioxidant ,17,18, tIn this study, we used a rat model of renal warm ischemia by renal pedicle clamping . We firsAfter renal warm ischemia–reperfusion, animals were followed for 1 week. Renal recovery was significantly improved by tannic acid: the increase in plasma creatinine at day one was reduced and returned to basal levels at day seven. Plasma creatinine was not affected by tannic acid in the sham group, supporting a lack of nephrotoxicity.In order to investigate the efficiency of tannic acid in transplantation, a rat model of allo-transplantation was used. However, the injection of tannic acid was ineffective in improving renal recovery. These results could be explained by the intensity of injury induced by transplantation, higher than that of warm ischemia, even with conditions adapted to promote animal survival at day seven. Allo response could be also involved in this context with the lack of immunosuppressive therapy. In this regard, Lewis are known as particularly syngeneic and are able to minimize the effects of immune rejection related to transplantation. On the other hand, in warm ischemic protocol, we choose Sprague–Dawley rats because of their sensitivity to renal warm ischemia, allowing 7 d survival ,26,27,28In order to investigate the mechanism of action of tannic acid regarding antioxidant activity, we explored the redox status 3 h after reperfusion. This time was chosen because oxidative stress is currently described at the beginning of reperfusion although some studies have described it during ischemia . InteresNRF2 is a transcription factor activated by ischemia–reperfusion but also by polyphenolic compounds . After rIn order to decipher if tannic acid or its derivatives were efficient against IRI, we used an in vitro model of endothelial cells subjected to hypoxia–reoxygenation. Endothelial cells are the first cells affected by ischemia–reperfusion injury and are also involved in inflammatory and regeneration processes ,30. As sAs with many other polyphenols, tannic and gallic acids are efficient free radical scavengers due to the presence of the galloyl moiety, allowing efficient H-atom transfer to the free radicals . HoweverIn conclusion, our results show that the antioxidant tannic acid exhibits protective effects on renal recovery after warm ischemia, as found during vascular surgery. This is particularly interesting considering the fact that tannic acid is a natural compound, easily accessible in nature, and is soluble in aqueous solution, which is rare for polyphenols. However, its beneficial role was not observed in kidney transplantation, potentially explained by the intensity of injury or by the additional alloimmune response. The beneficial activity after warm ischemia–reperfusion could be linked to the formation of active derivatives in the body, which offers an extended period of protection against ROS production up to 24 h, limiting cytotoxicity and promoting regeneration to accelerate renal recovery. In addition to the ROS scavenging activity of polyphenols, we suggested a stimulation of endothelial cell proliferation associated to NRF2 process induction. This study underlined that the protection by tannic acid has to be applied during ischemia and also during reperfusion, and that the mechanisms induced by warm and cold ischemia could be different and require adapted therapeutic strategies.