Supplemental Digital Content is available in the text To investigate the risk factors for postoperative venous thromboembolism (VTE) in patients undergoing spinal surgery.Literature published in PubMed, Embase, the Cochrane Library, and Web of Science was systematically reviewed to assess risk factors for VTE following spinal surgery. The data analysis was conducted with STATA 12.0. Data were pooled using fixed-effects or random-effects models according to the heterogeneity among the included studies.P < .001), female sex , diabetes , chronic kidney disease , nonambulatory preoperative activity status , D-dimer level , long duration of operation , spine fusion , and blood transfusion , and the differences were statistically significant. However, there were no significant differences in body mass index, obesity, hypertension, coronary heart disease, spondylolisthesis, intraoperative blood loss, surgical procedures , or surgical site (lumbar vs thoracic) .Twenty-six studies involving 3,216,187 patients were included in this meta-analysis, and the total incidence of VTE after spinal surgery was 0.35% (0.15–29.38%). The pooled analysis suggested that the incidence of VTE after spinal surgery was higher in such aspects as increasing age (weighted mean difference [WMD] 0.55 years, 95% confidence interval [CI] 0.33–0.78, Based on our meta-analysis, we identified several important factors that increased the risk of VTE after spinal surgery. We hope our study provides assistance to spine surgeons so that they can adequately analyze and assess risk factors in patients and then develop preventive measures to reduce the incidence of VTE. Protocols for the use of drug prophylaxis are well established for some surgeries, most notably hip and knee arthroplasty. The recommendations for chemoprophylaxis in spinal surgery are less clear, and clear evidence-based guidelines are lacking. Given the relative paucity of consensus concerning the efficacy and safety of chemoprophylaxis in spinal surgery, management varies widely and tends to be carried out according to surgeons’ personal experiences. Therefore, it is very necessary for spine surgeons to be aware of the incidence of VTE and its risk factors.Venous thromboembolism (VTE), including pulmonary embolism (PE), and deep venous thrombosis (DVT), are well-known and feared complications of major orthopedic surgery.–8 Therefore, spine surgeons should be more cautious when making decisions regarding anticoagulant therapy. They have to face the dilemma of how to balance the risk of morbidity and mortality from VTE and the risk of permanent neurological injury from epidural hematomas. Numerous studies have focused on the risk factors for VTE in patients undergoing spine surgery.–18 The incidence varies on account of these relatively small studies, which are conducted in heterogeneous populations using various detection methods. Collectively, these studies indicate that patients with an older age undergoing long periods of bedrest for paralysis and pain are reported to have a higher incidence of VTE than other patients.,10,12–14 While the incidence of VTE after spine surgery is influenced by other factors, such as D-dimer level, duration of operation, intraoperative blood loss, and surgical procedures, the resulting incidence of VTE after spine surgery is not inconsistent or ever contradictory.Spinal surgery is unique because its bleeding complications can lead to an epidural hematoma, which is a devastating but comparatively rare complication with an approximated incidence of <1%.Therefore, we carried out this study to systematically review and meta-analyze the existing literature to more precisely describe the incidence of VTEs and the risk factors associated with VTEs for patients who underwent spine surgery; we aimed to summarize the evidence-based proof for this issue, with the goal of supporting surgeons to make wise clinical decisions.22.1There was no need to seek informed consent from patients as this meta-analysis was based on published data and there was no potential harm to patients; this was approved by Ethics Committee of Qilu Hospital of Shandong University.2.2A PRISMA -compliant search was performed in the PubMed, Embase, Cochrane Library, and Web of Science databases by using combinations of the following keywords: venous thromboembolic, pulmonary embolism, deep venous thrombosis, spine surgery, and spinal surgery. References cited in the relevant articles were also reviewed. All studies were carefully reviewed to identify repeated data.2.3Studies were included if they conformed to the following criteria: original articles; randomized or nonrandomized controlled studies; studies on spinal surgery; studies on VTE after spine surgery; and studies on patients with spine degeneration diseases. Studies that were duplicate reports of an earlier trial, reviews, letters, and case reports were excluded.2.4Two investigators independently reviewed all subjects, abstracts, and the full text of the selected literature. Then, the eligible studies were selected according to the inclusion criteria. Any controversy between the investigators was resolved by discussion and consensus. When consensus could not be reached, a 3rd author was consulted to resolve the disagreement.2.5Two independent authors extracted the data from the eligible studies, discussed discrepancies, and reached a consensus for all items. The essential information extracted from all primary studies included the titles, author names, year of publication, study location, study design, sample size, number of patients with VTE, demographic characteristics , the level of D-dimer, preoperative walking disability, spine fusion vs nonfusion, anterior lumbar interbody fusion (ALIF) vs posterior intervertebral fusion (PLIF)/translaminar lumbar interbody fusion (TLIF), surgical duration, blood loss, and blood transfusion. We used the modified Newcastle–Ottawa scale (NOS) to evaluate the quality of the studies. This method consists of 3 items: selection, comparability, and outcomes. Scores ranged from 0 to 9, and higher scores indicated better quality. Any study was considered to be of high quality if the NOS score was >5 points.2.6P < .05 was considered statistically significant. Heterogeneity was tested using I2 statistic. If I2 >50%, heterogeneity was implied, and a random-effects model (DerSimonian–Laird method) was used; if I2 < 50%, a fixed-effects model (Mantel–Haenszel method) was used. When significant heterogeneity was identified, a sensitivity analysis was performed to explore the potential source of heterogeneity. Publication bias was evaluated with Begg and Egger test.The STATA 12.0  was used to analyze the data. Dichotomous variables are expressed as odds ratios (ORs) with 95% confidence intervals (CIs), and continuous variables are expressed as weighted mean differences (WMDs) with 95% CIs. A probability of 33.1–18,24–39 was designed as a prospective study, and the other 25 are retrospective studies. The total sample size was 3,216,187, of which 1196 were complicated with VTE, and the total incidence of VTE after spinal surgery was 0.35% . The incidence of VTE was 8.43% in Asian patients and 0.33% in occidental patients, with a statistically significant difference (P < .0001).The article screening and selection process for inclusion in this study is shown in Figure 3.2Assessment of the study-specific quality scores from the NOS is shown in Table 3.33.3.1,14,16,37 including 6218 patients investigated the relationship between increased age and the incidence of VTE. The pooled results suggested that spinal surgery patients with VTE were older than those without VTE .Seven studies3.3.3,12,14,16 including 5572 patients reported the relationship between BMI and the incidence of VTE. Pooled estimates indicated that patients with high BMI values had a similar risk of VTE compared to those with normal BMI . There was significant heterogeneity ; thus, we conducted subgroup analyses. When we excluded a Chinese study, the pooled data for the remaining Japanese studies,12,14,16 did not change substantially , but there was still heterogeneity present .Five studies3.3.4,28,31 including 8777 patients reported the relationship between obesity and the incidence of VTE. The pooled results suggested that obesity patients had a similar incidence of VTE compared to those of normal weight . There was no significant heterogeneity among the studies .Three studies3.3.5–36,38,39 including 2,866,773 patients assessed the relationship between diabetes and the incidence of VTE. The pooled data suggested that diabetes patients had a 1.34-fold increased risk of VTE compared to those without diabetes .Nine studies3.3.6,11,13,14 including 47,172 patients investigated the relationship between HT and the incidence of VTE. The pooled estimate suggested that HT patients had a similar incidence of VTE compared to that of normal patients . There was significant heterogeneity among the studies . Thus, we conducted sensitivity analyses. When we excluded a study with a relatively small sample (n = 80), the pooled data for the remaining studies changed dramatically , but there was still heterogeneity present .Four studies3.3.7,13 including 3725 patients reported a relationship between coronary HD and the incidence of VTE. The pooled results indicated that patients with coronary HD had a similar incidence of VTE compared to normal patients . There was no significant heterogeneity among the studies .Two studies3.3.8,32 including 328,240 patients reported a relationship between CKD and the incidence of VTE. The results showed that patients with CKD had an 8.31-fold increased risk of VTE compared to those without CKD . Heterogeneity was significant . Thus, a subgroup analysis was performed . There was no significant heterogeneity among the studies .Two studies3.3.10,11,12,14 including 44,510 patients investigated the relationship between a nonambulatory preoperative activity status and the incidence of VTE. The pooled results suggested that a preoperative walking disability notably increased the incidence of VTE .Four studies3.3.11,14,16 including 2249 patients investigated the relationship between a high level of serum D-dimer and the incidence of VTE. The results revealed that a high level of D-dimer prominently increased the incidence of VTE .Three studies3.3.12,14,16,37 including 46,891 patients reported the relationship between the duration of the operation and the incidence of VTE. The results revealed that long surgical times dramatically increased the incidence of VTE .Six studies3.3.13,14,16,37 including 3114 patients observed a relationship between intraoperative blood loss and the incidence of VTE. The results indicated that intraoperative blood loss did not affect the incidence of VTE .Five studies3.3.14,15,17,18 including 49,278 patients researched the relationship between spine fusion and the incidence of VTE. The pooled results showed that spine fusion significantly increased the incidence of VTE .Five studies3.3.15,24,37 including 179,844 patients investigated the difference in VTE risk between ALIF patients and PLIF/TLIF patients. The results suggested that ALIF patients had a similar risk of VTE compared to PLIF/TLIF patients .Three studies3.3.16,27,29,33 including 72,493 patients assessed the relationship between a blood transfusion and the incidence of VTE. As 1 study included the patients undergoing lumbar and thoracic fusion surgery, another study included patients undergoing cervical fusion surgery, subgroup analysis was performed according to the surgery site. The pooled data revealed that a blood transfusion remarkably increased the incidence of VTE . No significant heterogeneity was observed . Thus, a subgroup analysis was performed according to the surgical site .Two studies3.3.18The assessment of publication bias using Begg and Egger tests showed that there was no potential publication bias for any of the included studies Table .4The incidence of VTE in the field of orthopedics varies with surgical site or surgical procedure. Compared with the incidence of VTE after lower limb surgery, such as hip or knee arthroplasty, the incidence of VTE after spinal surgery is relatively low. Nonetheless, if not promptly treated, thromboembolic complications, such as lower extremity DVT, PE, myocardial infarction, and cerebral infarction after spinal operation, could lead to severe malfunction of the extremities, heart, and brain, and even death. VTE after spine surgery has drawn increasing attention from researchers; however, the incidence of and risk factors for VTE following spine surgery remain unclear. This meta-analysis summarized and analyzed the risk factors for VTE after spine surgery. The difference in the incidence of VTE between Asians and occidentals was also investigated. The results indicated that Asian ethnicity, older age, being female, preoperative walking disability, high levels of serum D-dimer, longer surgical times, spine fusion surgery, and blood transfusion, combined with diabetes or CKD, were risk factors for VTE after spine surgery. In addition, it was found that the incidence of VTE in Asian patients was significantly higher than that in occidental patients, which indicated that Asian patients were more likely to suffer from VTE after spine surgery; the reason for this remains to be investigated by further research. Our study indicated that elderly patients were more likely to develop VTE, which was in line with previous studies.,10,12 In a retrospective study of 2861 patients that investigated risk factors for DVT in patients undergoing posterior lumbar interbody fusion, the authors suggested that age was an independent risk factor for DVT. The mean age in the DVT and non-DVT groups was 61.3 and 52.6 years (P = .002), respectively, suggesting that elderly age increased the risk of DVT. In some of the studies included in this meta-analysis, although the mean age of the VTE group was older than that of the non-VTE group, no significant difference was shown. Tominaga et al conducted a retrospective clinical study and performed a logistic regression analysis of 80 patients who had undergone spine surgery. Among these patients, 20 experienced postoperative VTE, and 60 had no VTE. The median ages for the patients with VTE and those without VTE were 75.0 and 70.5 years, respectively, and they were not statistically significant (P = .075). Likewise, Takahashi et al undertook a review of a case series to identify the incidence of VTE after spinal surgery. The results showed that the mean age did not differ significantly between the PE and non-PE groups . We think that the respectively small sample size and selection bias are influencing factors of the results. To obtain more objective conclusions, we pooled all the data from the included studies together in a meta-analysis to weaken the bias of a single study. As is commonly known, aging patients tend to have a high incidence of diabetes and cardiovascular and cerebrovascular diseases and have a long postoperative bedrest period after surgery. Therefore, it is reasonable that elder patients are at higher risk for VTE. Increasing age is one of the risk categories in the Autar DVT scale. Attention must be paid to elderly patients after spinal surgery, and steps must be taken to prevent the incidence of VTE.A total of 3,216,187 patients from 26 studies were included in this study, including 11,196 patients with VTE after spine surgery. The overall incidence of VTE was 0.35%, which was much lower than in a previous meta-analysis.,12 Yoshioka et al and Ikeda et al found that female sex was one of the risk factors in multivariate analyses. However, in the other 5 studies,,15,31,37 a negative relationship was found between female sex and VTE. The pooled data indicated that females had a significantly higher incidence of VTE than males, and there was no significant heterogeneity among the studies. We consider that female patients recover more slowly and stay in bed longer after spine surgery than male patients, thereby increasing the risk of VTE.Consistent with prior studies, female sex was significantly associated with VTE after spine surgery.,34,35 Guzman et al performed a retrospective database analysis. They enrolled a total of 403,629 controlled diabetic patients, 19,421 uncontrolled diabetic patients, and 2,145,944 nondiabetic patients who underwent degenerative lumbar spine surgery. They found that relative to nondiabetic patients, uncontrolled diabetic patients had significantly increased odds of having DVT (P < .001); moreover, controlled diabetic patients also had an increased risk of DVT when compared with nondiabetic patients (P < .001). However, some other studies had the opposite idea that a history of diabetes would not increase the incidence of VTE.,14,38 Our results suggested that a history of diabetes markedly increased the incidence of VTE. It is known that hyperglycemia and endocrine metabolic disturbances may lead to hormone imbalances in the whole body.,41 In addition, patients who have diabetes are more prone to vascular stenosis, which affects hemodynamics, thereby increasing the risk of VTE.Diabetes was another risk factor for VTE following spine surgery, and this result was comparable to previous studies. analyzed 164,097 patients who underwent treatment for degenerative spine disease. Among these, 1047 had CKD, and 270 had ESRD . After multiple logistic regression analyses, the authors found that patients with CKD and ESRD were dramatically more likely to experience DVT and PE when compared with patients without kidney disease. Another study had the opposite idea, that is, that the incidence of PE after lumbar decompression and fusion was similar between the CKD group and the no-CKD group. Our meta-analysis revealed that a history of CKD was a significant risk factor for VTE in patients who had undergone spinal surgery. This may be related to the imbalance of the internal environment and anemia in patients with CKD. Due to the significant heterogeneity , we performed a subgroup analysis. However, the subgroup analysis data suggested that the incidences of both PE and DVT were similar between CKD patients and non-CKD patients. Therefore, further studies are needed to determine whether CKD is a risk factor for VTE.There are few studies on CKD and VTE. De la Garza Ramos et al,27,29,33 Three studies,27,33 observed a relationship between postoperative transfusion and the incidence of VTE in patients who had undergone lumbar spinal surgery. Aoude et al retrospectively analyzed the American College of Surgeons National Surgical Quality Improvement Program (ACS-NSQIP) database to identify patients who underwent lumbar or thoracic fusion. They enrolled 13,695 patients, of whom 13,170 had lumbar fusion and 525 had thoracic fusion. In 2016, they reported that patients receiving transfusions who underwent lumbar fusion were more likely to develop VTE, which was in line with the other 2 studies,,27 while there was no increased risk of VTE in patients receiving transfusions who underwent thoracic fusion. Again, Aoude et al identified 11,588 patients who had cervical fusion from the ACS-NSQIP database. Their results suggested that all transfused patients had an increased risk of VTE. It was assumed that the postoperative transfusion increased the viscosity of the blood, which may affect hemodynamics and the blood coagulation mechanism and that this may be correlated with an increased risk of VTE postoperatively.In the present study, we found that blood transfusion was a remarkable risk factor for VTE in patients who had undergone spinal surgery. These results were in line with previous studies.4.1Some limitations of this study should be recognized. First, the sample size was huge, so the chance of internal errors, significant heterogeneity among the studies and samples, and bias was also high. Consequently, in some results, observable heterogeneity was identified among the included studies. Despite sensitivity analyses and subgroup analyses being performed to detect potential sources of heterogeneity, no valuable information was observed. Second, clinical heterogeneity might be caused by the different indications for surgery, different postoperative bedrest times and the postoperative functional rehabilitation exercises used at different treatment centers. Moreover, all of the studies were performed with a retrospective design, and their results might be biased by inherent shortcomings. This may have had a potential impact on our pooled estimates.5Our study indicates that Asian ethnicity, advanced age, female sex, diabetes, CKD, high levels of D-dimer, preoperative walking disability, long duration of operation, spine fusion, and blood transfusion are independent risk factors for VTE following spine surgery, whereas BMI, obesity, HT, coronary HD, spondylolisthesis, intraoperative blood loss surgical procedures, and surgical site are not. Knowing these risk factors, surgeons can fully analyze and assess risk factors in patients and then formulate preventive measures to reduce the incidence of VTE.Data curation: Yunzhen Chen.Formal analysis: Hongxin Cao.Investigation: Hongxin Cao.Software: Hongxin Cao.Supervision: Yunzhen Chen.Writing – original draft: Lu Zhang.Writing – review & editing: Jiao Guangjun.


Intussusception is defined as the invagination or telescoping of a proximal portion of the intestine into the distal portion of the intestine. Intussusception can occur at any age but is more common among children. Most cases of intussusception in adults have a pathological lead point. Inflammatory fibroid polyp (IFP) is a rare benign tumor-like lesion arising from the submucosa of the gastrointestinal tract that can cause intussusception in adults. Here, we report a case of adult intussusception due to IFP. We also present a literature review of 31 reports including 34 cases between 2012 and December 2019, which shows a mean age of 45.4 ± 14.2 years and female dominance (23/34) of intussusception due to IFP.A 47-year-old man presented with a half-day history of epigastric abdominal pain. Physical examination revealed distension and tenderness of the upper abdomen. Computed tomography (CT) of the abdomen and pelvis demonstrated intussusception of the jejunum along with a suspicious jejunal mass associated with mesenteric lymphadenopathies.Intussusception of the jejunum along with a suspicious jejunal mass, and histopathological examination of the resected specimen showed IFP.The patient underwent emergency laparotomy. The intussusception was resected without attempts for reduction.The postoperative period was uneventful, and the patient was discharged on the fourth postoperative day.Intussusception in adults is rare, especially that secondary to IFP. The most commonly used diagnostic tool for adult intussusception is abdominal CT, and the optimal management is resection of the involved bowel segment without reduction if malignancy cannot be ruled out. IFP is a rare benign tumor-like lesion of the gastrointestinal tract, which can lead to small bowel intussusception. In this article, we present a case of jejuno-jejunal bowel intussusception caused by IFP in a 47-year-old male patient. A literature review of 34 cases between 2012 and December 2019 is also presented.Intussusception is rare among adults. Diagnosis of intussusception can be difficult in adults because of non-specific symptoms. Abdominal CT is the most sensitive tool for diagnosis of adult intussusception, which demonstrates concentric rings in the axial view referred to as the “target sign”. Surgical intervention is ideal. However, a consensus has not been reached on whether to perform reduction before resection.2A 47-year-old man presented to the emergency department with a half-day history of epigastric abdominal pain. The abdominal pain was not relieved even after morphine and nalbuphine injections.He had a past history of gastric ulcer and was under medication. He had no history of prior abdominal surgery. He was a non-smoker, a non-drinker, and had no relevant family history.9/L, and hemoglobin level 9.4 g/dL. Other serum parameters  were all within the normal range. Chest radiograph showed no abnormalities. CT of the abdomen and pelvis, performed 6 hours after his presentation to the emergency department, demonstrated intussusception of the jejunum along with a suspicious jejunal mass ; 23 were women  and 11, men , including two adolescent boys aged 10 and 16 years. The size of IFPs was 5 ± 2.9 cm, and the five, 28, and one IFPs were located in the jejunum, ileum, and colon, respectively. Reduction was performed before resection in five of the 34 cases, with one case suspected to be lipoma through CT. Laparoscopic reduction was performed without resection; however, intussusception recurred after 2 weeks; thereafter, segmental resection was performed. Length of the resected bowel mentioned in six cases was 22.8 ± 5.2 cm. Among the 34 cases, two underwent laparoscopy with mini-laparotomy approach, four, laparoscopy approach, 17, laparotomy approach, while the rest of the cases did not mention having undergone any procedure. Among 34 cases, 29 were diagnosed using computed tomography, including six that initially used ultrasonography and one that initially used barium. Of the remaining five cases, three were diagnosed using ultrasonography alone and two were diagnosed using colonoscopy alone. The clinical characteristics of the 34 patients are summarized in Table  Most IFPs usually measured between 2 and 5 cm in diameter. The size of IFPs in our review was 5 ± 2.9 cm, and the previous review was 4.1 ± 1.8 cm. However, giant IFP up to 18 cm in diameter was also reported in our review. Age of onset was lesser, and the females were more was predominant in our review compared to the previous review. Furthermore, the use of CT as the diagnostic tool had increased significantly, and the use of barium had decreased markedly.Compared with the previously reviewed article of 85 cases from 1976 to 2011, the size and location of IFPs were similar. The IFPs as a lead point causing intussusception were more commonly located in the ileum (28 of 34 cases in our review and 63 of 85 in previous) and lesser in jejunum.4Intussusception is defined as the invagination or telescoping of a proximal portion of the intestine (intussusceptum) into the distal portion of the intestine (intussuscipiens). Intussusception can occur at any age but is more common among children. Intussusception in adults differs from that in children in various aspects, including etiology, clinical manifestations, and prognosis.,4Approximately 90% of intussusception in children is idiopathic, while about 90% of that in adults has a lead point, which is a well-defined pathological abnormality. In adults, the lead points in the small-bowel intussusceptions are benign lesions (60%) and malignant (30%) lesions, while the rest (10%) are idiopathic. Further, up to 66% of large bowel intussusceptions are caused by malignancy. The most common presentation in adult is abdominal pain. Patient may also present with vomiting, nausea, diarrhea, hematochezia, abdominal distension, and palpable mass. Intussusception in adults requires surgical intervention due to the high incidence of well-definable pathological abnormality and the possibility of malignancy. The clinical condition of the patient and the surgeon's experience determine whether laparoscopy or laparotomy must be performed.Pediatric intussusception is often acute with sudden intermittent colicky pain, vomiting, bloody mucoid stools, and presence of a palpable mass. Conversely, adults may present with acute, subacute, or chronic nonspecific symptoms.,4–6 To avoid potential dissemination of malignant cells, resection without reduction is performed in most cases of colonic intussusception owing to higher incidence of malignancy. However, reduction of small bowel intussusception could be performed if malignancy is not suspected.,4Some controversy still exists whether to reduce the intussusception before resection. Reduction of intussusception can reduce the extent of the intestinal resection. Conversely, inappropriate manual reduction may lead to dissemination of the malignant cells, bowel perforation, and increased risk of anastomotic complications. The diagnosis of IFP is confirmed by histopathologic analysis. IFPs are usually treated by surgical resection. However, only a single case of polyp recurrence is found in the literature. According to the location and size of the lesion, patients present with different clinical manifestations. IFPs of the small bowel can lead to intussusception. Furthermore, recurrent intussusception secondary to IFPs of the small intestine may cause bowel obstruction, ischemia, necrosis, or perforation.IFPs are rare benign tumor-like lesions of the gastrointestinal tract. The most common site is the gastric antrum, followed by the small bowel and colorectal region.In conclusion, intussusception in adults is rare, especially that secondary to IFP. The most commonly used diagnostic tool for adult intussusception is abdominal CT, and the optimal management is resection of the involved bowel segment without reduction if malignancy cannot be ruled out.Conceptualization: Yi-Kai Kao, Jian-Han Chen.Resources: Jian-Han Chen.Software: Jian-Han Chen.Visualization: Jian-Han Chen.Writing – original draft: Yi-Kai Kao.Writing – review & editing: Jian-Han Chen.


We carried outtests of reliability, construct and criterion validity and a comparison ofmeans based on the diagnosis and socio-demographic variables. Thereliability and validity analyses showed adequate psychometric propertiesfor this scale, with better results obtained for a single dimension aftereliminating 3 items.adolescents with type 1 diabetes and girls were found to have an increasedthreat perception of their illness. Anxiety/depression was positivelyassociated with the perception of illness.this questionnaire is a useful and practical tool for evaluating adjustmentto illness in pediatric patients. Those who perceive their illness as more threatening, appear topresent more anxiety, depression and a poorer quality of life(-).The evaluation of the perception of illness has aroused great interest as it m factors-6. Th of life-3.(-).The processing of emotional and cognitive responses to illness helps patients toregulate themselves emotionally in order to better adapt to their condition, whichcan in turn change their cognitive or emotional representation. The term “perceptionof illness” therefore refers to a patient’s mental representations of their illness,in relation to its identity (name and symptoms), causes, consequences, course andcontrol-8..All this seems particularly relevant in the case of chronic diseases (CD), sincethese are health problems that persist over time and requires continuous managementand lifestyle changes over the years, and may affect the possible future evolutionof the disease..However, not all CD appear to have the same effects. These effects seem to be moreserious in the case of endocrine diseases, such as Diabetes Mellitus Type 1(DM1),than in respiratory diseases, such as asthma..Despite its importance, few studies have addressed this concept in adolescence, aparticularly complicated period with lower rates of adherence to treatment, agreater presence of risk behaviors and more psychopathological problems,11.(-), the prevailing model in the literature for some years has been theCommon Sense Self- Regulation Model.Although there are different models and instruments for the study of adaptiveresponse to disease-13, ton Model. was designed, and is probably the most widely used instrument atinternational level for assessing this construct. It initially had 38 items, and an extended revised version (IPQ-R) wassubsequently designed, consisting of 70 items, including cognitive and emotionalresponses. In recent years, in order to simplify and facilitate its application,the short IPQ (IPQ-R) was developed. This is composed of 9 items providing a quick and simple evaluation of this construct.Based on this model, the Illness Perception Questionnaire (IPQ) was desonstruct,15. Iesponses. In rec 9 items providionstruct. among patients aged 8 to 80 years old. The linguistic adaptation ofIPQ-R to Spanish was carried out with adults with different CD, including endocrinesand respiratory. However, despite its widespread use, few studies analyze itspsychometric properties, and these are even more scarce in the Spanish context. Likewise, there do not appear to be any studies analyzing thepsychometric properties of IPQ-R pediatric patients with chronic conditions.The IPQ-R has been used in 36 countries and translated into 26 languages, and hasbeen used in numerous diseases-,-19 ampiratory. Howeveerties,-22, a context. Likewi, others postulate the existence of two (cognition andemotion) or three dimensions (adding understanding of the disease to the twoprevious dimensions). Similarly, there seems to be no consensus on the effect that sex, age,or type of disease have on threat perception. Another limitation of the instrument refers to the non-existence ofinterpretative scales that facilitate its interpretation depending on the type ofdisease or chronic condition.One aspect in relation to this instrument that continues to be a source of debaterefers to its dimensionality. While some studies suggest using each item as asubscale, othersemotion) or threensions). Similaeption,-24. AThe main objective of the study will therefore be to analyze the psychometricproperties of BIP-Q in a sample of chronically ill adolescents, while studying thedimensionality of the scale and the effect of age, sex and medical diagnosis, and tooffer interpretative scales.This will all enable the development of intervention and prevention programmes thatimprove the quality of life of these patients.The participants (n=510) were adolescents with chronic diseases between 9 and 16years of age . After obtaining the informed consent of the tutors,the data were collected between June 2016 and January 2018, the Endocrinology orPediatric Pneumology Units of hospitals in the Valencian Community. This researchhas been endorsed by the Ethics Committees of the University of Valencia and thevarious participating hospitals.The information was collected through an interview using an instrument composed of anad hoc record with sociodemographic variables and two standardized instruments(BIP-Q and HADS) by the same professional in all cases. For the ad hoc record, thevariables analyzed were age, sex of patient and type of pediatric chronic disease,and the instruments used to analyze the variables were the the Brief IllnessPerception Questionnaire (IPQ-R) and the Hospital Anxiety and Depression Scale(HADS). consists of 9 items. The first 8 items  are answered on a Likert scale from 0 to 10, dependingon the degree of agreement. The last item, causes, is in open response format, andassessed by citing the 3 most important responses believed to have caused thedisease. The overall score is obtained by inverting items 3, 4 and 7 and adding themto the score for items 1, 2, 5, 6 and 8. The higher the total score, the greater theperception of the illness as a threat. The BIP-Q has been demonstrated to haveadequate psychometric properties in previous studies not performed in adolescentpopulations. The questionnaire showed adequate internal consistency indices rangingfrom α=.67-.89, depending on the study and the type of sample.The Spanish version of the Brief Illness Perception Questionnaire (IPQ:R)15)   and depression (HADS-D). Both subscales are composed of 7 itemsinterspersed throughout the questionnaire (Anxiety is the odd-numbered items andDepression is the even-numbered items). The response format ranges from 0 to 3, with0 being the minimum score or no symptomatology, and 3 being the maximum score orpresence of symptomatology. The higher the score, the higher the level ofanxiety-depressive symptomatology. Previous studies have found adequate psychometricproperties in relation to its reliability or internal consistency: α=.68-.93 (Mα=.83) for the anxiety scale and for the depression scalebetween .67 and .90 (Mα=.82)(-) generally obtaining higher scores on the anxiety scale. In this sample,α= .63 for anxiety and α=.56 for depression was obtained.The version of the Hospital Anxiety and Depression Scale (HADS) adapted Spanish is a Lisistency: α=.68-(Mα=.82)-28 ge using the unweighted least squares (ULS) method, parallel analysis anddirect Oblimin rotation. The CFA were carried out by means of robust estimation ofmaximum likelihood (ML), with the objective of correcting the absence ofmultivariate normality. The suitability of the CFA was tested using the chi-squaresignificance and robust Satorra-Bentler adjustments (S-B χ²)(-), the ratio between χ² and its degrees of freedom (χ²/df), as well asthe S-B X² and its degrees of freedom (proper setting≤5), the comparative setting index (CFI), the incremental setting (IFI)(proper setting≥.90) and the mean square error approach (RMSEA) (propersetting≤.08).For data analysis: first, the reliability of the scale  and mean extracted variance index (MEVI)) was analyzed, and itsvalidity , confirmatory factorial analysis(CFA) and predictive validity of the instrument) was then analyzed. The EFA wasperformed using the recommended process using t(S-B χ²)-31, ttting≤5), the coing≥.90) and theing≤.08). software and EQSv6.3.The relationship between BIP-Q and HADS was then analyzed using multiple hierarchicallinear regressions, followed by differences in means according to the diagnosis(single factor ANOVA), sex and the two age groups of preadolescents (9-12 years) andadolescents (12-16 years) (t-test). They were recoded into two groups by age.Finally, scales were calculated based on centile scores according to diagnosis andsex. Statistical analyses were performed using SPSSv24, FACTOR softwarThe participants (n=510) were minors with chronic diseases between 9 and 16 years old, 42.4% (n=216) were preadolescents (9-12 years old) and 55.6%(n=294) were adolescents (12-16 years old). 54.5% of them were children (n=278). Thesample was distributed as follows: 51.1% (n=262) presented chronic respiratoryproblems (PRC), 22.4% (n=113) had DM1 and 26.5% (n=135) had short stature (SS).By analyzing the internal consistency of the BIP-Q scale, we found the initialreliability of the original structure for the unifactorial and bifactorial solutionshowed inadequate indices (α≤.70). In the final version of the questionnaire aftereliminating three items, the data showed an improvement in the reliability for theunifactorial solution, complying with the acceptable indices (α=.76) but thisimprovement did not occur for the bifactorial solution . Similar2=393.6, gl=28; p≤.001)obtained adequate values, so EFA and CFA were performed. Then, as suggested by theliterature(-), the sample was segmented into two groups, controlling for age, sex anddiagnosis: group A (n=255) was used for EFA, group B (n=255) for CFA.Prior to EFA and CFA, we determined the adequacy of the data using theKaiser-Meyer-Olkin analysis (KMO) and Bartlett’s sphericity test. KMO analysis(KMO=.75) and Bartlett’s sphericity test , since its factorial load wasless than .40 . After this elimination, the factorialsolution presented good adjustment indices , the variance wasexplained by the first factor of 23.66% and by the second factor of 11.46%. For theunifactorial dimension, the data suggested eliminating items 3, 4 and 7 becausetheir factorial loads were below .40 , and the total variance was explained as 21.33%.Various CFAs were then carried out. The goodness-of-fit indicators for the twosolutions  were inappropriate, so it was necessary tore-specify the model by eliminating items  . After t, we then analyzed the effect of age and the perception of threat of theillness (BIP-Q) on anxiety and depression, using two two-step hierarchical linearregressions using age (step 1) and the perception of threat of the illness (step 2)as predictor variables, and anxiety and depression measured by HADS as criterionvariables. In the first step, age explained 3%  of anxietyand 2%  of depression, inclusion of the perception ofthreat of the illness increased 3%  of the varianceexplained for anxiety and 4%  for depression. Bothvariables positively predicted 6% of anxiety and depression (R2adj=.06)with perception of threat of the illness being slightly more explanatory  than age .As suggested by the literature ,-24, wDifferences in the perception of the disease according to sociodemographic variableswere then analysed. Significant differences were found between all diagnostic groups, with moderate to large effect sizes, with diabetics showinggreater threat perception  than those with SS  and respiratory disease , and a greater threatperception among respiratory patients with SS (d=0.49), Likewise, no differenceswere found between preadolescents and adolescents, but differences were observeddepending on gender . Girls showed a greater perception ofillness threat  than boys  with a smalleffect size (d=0.21).Finally, in order to facilitate the interpretation of the scores, the main centileswere calculated according to diagnosis and sex, ignoring age groups, given the lackof differences between these groups ., which may influence the course and resolution of thedisease. It is therefore necessary to have instruments for itsmeasurement(-), and the BIP-Q is the most widely used,The presence of CD in adolescence, whether endocrinological or pneumological, may beperceived as a threat to health and survival, which disease,8. Itsurement-13, ay used,,20,However, to date, psychometric properties have not been analysed in adolescents withCD, or in the specific case of Spain. Our study therefore aims to analyze thepsychometric properties of BIP-Q in a sample of 510 adolescents with CD., although both the unifactorial and the bifactorial models seemadequate, and the unifactorial model generally seems better. The unifactorialsolution has been chosen because after analysing the overall results obtained inboth the reliability analysis and the validity analysis (exploitative andconfirmatory factor), the reliability improves by eliminating the items loaded below.40 ranging from .56 with 8 items, and .76 with 5 items, which is consideredacceptable in the unifactorial model. However, scores below .70 are obtained for thebifactorial solution in both the original version and the 5-item version proposed inthis study.After analysing the psychometric properties, the final number of items was reduced to5, thereby obtaining the reduced version presented in this study that presentsadequate psychometric properties. Although the scale itself was already reduced, itwas necessary to eliminate items because when analyzing the psychometric propertiesin our study sample, the adjustment indices were not adequate, and as such thereliability and validity of the questionnaire improved significantly after items 3,4 and 7 were eliminated. As regards the dimensionality of the scale,,20, a(-), the main predictor of anxiety- depressive symptomatology is the levelof perception of illness threat, followed by age.As for criteria validity, as suggested by the literature-3, th, there are differences depending on the type of disease, as adolescentswith DM1 show higher levels of threat than adolescents with respiratory problems.Likewise, there are no differences according to age, as indicated in theliterature, but there are differences according to sex, as in the specializedliterature. Finally, in order to facilitate diagnosis, interpretation scales arepresented that can help health professionals to assess the threat perception oftheir CD patients quickly and easily.Regarding the analysis of the influence of sociodemographic variables with respect tothe perception of illness threat, as suggested by previous research, there terature, but thterature. FinallDespite the contributions of this research, it is not without limitations,particularly the sampling procedures for the sample studied are neitherprobabilistic nor representative of all CD. It contains a higher proportion ofpatients with respiratory disease, making it difficult to generalize the resultsfound. Finally, although the use of self-reports is a common tool, it can introducebiases due to social desirability.Future research should use probability sampling, extend the sample to other settingsand even include other paediatric diseases while combining other objective orphysiological measures. but not in adolescent populations. The fact that the questionnaire isnot specific to a chronic disease as such, but that it allows substitution of termssuch as asthma or diabetes, medical treatment by inhalers or insulin when it iscompleted facilitates its use. It is therefore useful for any pediatric CD, as wellas being easy to administer and correct. The BIP-Q questionnaire is therefore auseful and practical tool with which to evaluate the adjustment to disease.Nevertheless, the study is of particular interest in view of the lack of studies inthe paediatric context and the sample obtained. The use of this instrument inpaediatrics is new, since this questionnaire in the Spanish context has only beenused in adult populations but notThe results of this study show that the reduced Spanish version of the DiseasePerception Questionnaire (BIP-Q) is a reliable, valid instrument with solidpsychometric properties, which works effectively in the evaluation of the threatperception of pediatric chronic diseases


Vitamin K (VK) and vitamin D (VD) deficiency/insufficiency is a common feature of chronic kidney disease (CKD), leading to impaired bone quality and a higher risk of fractures. CKD patients, with disturbances in VK and VD metabolism, do not have sufficient levels of these vitamins for maintaining normal bone formation and mineralization. So far, there has been no consensus on what serum VK and VD levels can be considered sufficient in this particular population. Moreover, there are no clear guidelines how supplementation of these vitamins should be carried out in the course of CKD. Based on the existing results of preclinical studies and clinical evidence, this review intends to discuss the effect of VK and VD on bone remodeling in CKD. Although the mechanisms of action and the effects of these vitamins on bone are distinct, we try to find evidence for synergy between them in relation to bone metabolism, to answer the question of whether combined supplementation of VK and VD will be more beneficial for bone health in the CKD population than administering each of these vitamins separately. Chronic kidney disease (CKD) represents a global health issue involving about 13% of the general population, of which about 11% are patients in the 3–5 stage of CKD . AbnormaCKD-MBD impacts bone remodeling —the dynaMany clinical studies reported on VK and vitamin D (VD) deficiency in patients with CKD or undergoing dialysis ,10,11,12Nondietary determinants of VK status in CKD include taking drugs prescribed to patients with CKD, such as warfarin, statins, proton-pump inhibitors, phosphate binders, steroids, or antihypertensives drugs ,19,20. TRegarding the general population, a recent report recommended that serum 25D concentrations should be maintained at 20–50 ng/mL, and values >30 ng/mL should be considered normal . HoweverEstablishment of the reference value of VK in patients with CKD is a challenge, because there is no gold standard for the measurement of VK levels and there is a lack of standardization. Instead, a functional deficiency of VK is commonly used as a surrogate of VK status in these patients. Measurements of uncarboxylated prothrombin (known as protein induced by VK absence/antagonism II (PIVKA-II), uncarboxylated OC (ucOC), and desphospho-uncarboxylated matrix Gla protein (dp-ucMGP) MGP are indicative of VK deficiency ,11,23.This review focuses on the contribution of VD and VK to skeletal health in CKD, discussing their effects on bone remodeling, derived from in vitro, in vivo, and clinical studies. In particular, we tried to find a functional synergy between these vitamins in relation to bone health in CKD and answer the question of whether simultaneous supplementation with VD and VK may be more beneficial in counteracting the effects of CKD-MBD than supplementing the deficiency of a particular vitamin.Lactococcus or Bacteroides) and fermented food [VK constitutes a group of fat-soluble chemical compounds, whose common property is a structure containing 2-methyl-1,4-naphthoquinone. Naturally, VK occurs in two forms—VK1 (phylloquinone) and VK2 . VK1 is the main source of dietary VK and is mainly found in green leafy vegetables and plant oils. MKs are derived from intestinal bacteria (ted food . The mosted food . The rected food . The maiThe main physiologic role of VK is to act as cofactor for the γ-glutamyl carboxylase (GGCX) enzyme in the gamma-carboxylation reaction that add carboxyl groups to glutamic acid (Glu) residues in proteins. GGCX oxidizes VK into VK epoxide and then adds CO2. The newly carboxylated residues in such proteins are referred to as gamma-carboxyglutamic Gla domains. This process transforms inactive (uncarboxylated) proteins into active carboxylated VKDPs, enabling them to bind to calcium. Adequate calcium binding is a critical physiological step in blood coagulation, bone mineralization, and vascular calcification. The most acknowledged extrahepatic VKDPs are MGP, osteocalcin (OC), growth arrest specific protein 6 (Gas6), and Gla-rich protein (GRP) .In addition to protein modification, a novel mechanism was uncovered in the signaling that regulates the transcription of target genes by VK through the activation of a nuclear receptor, the steroid and xenobiotic receptor  and pregnane X receptor (PXR), which is the mouse and rat ortholog of SXR) . VK2 wasThe determination of VK levels is difficult because of its physicochemical properties and low levels of VK in circulation. Measurements can be done using direct and indirect methods. One of the most popular direct methods is the determination of VK using high-performance liquid chromatography (HPLC). A disadvantage of this method is the possibility of interaction between an HPLC column and lipoproteins transporting VK, which may affect the results . The mosThe ucOC level, the total OC level, and the ratio between the two (%ucOC) are frequently used to reflect VK status linked to bone health . The diaMany observational and interventional studies reported that patients with CKD undergoing conservative treatment, peritoneal dialysis (PD), or hemodialysis (HD) suffer from subclinical VK deficiency ,20,23,37There are a few studies showing that PD patients have a comparable degree of VK deficiency to HD patients. Stankowiak-Kulpa et al.  demonstrIt is widely known that VK is crucial for the activation of OC, which is involved in bone metabolism. OC  is one of the main noncollagenous proteins that is synthetized by OBs during bone formation. Modification by VK-dependent carboxylation converts ucOC to an active carboxylate form (cOC). The cOC binds calcium ions and incorporates them into hydroxyapatite crystals in the bone matrix to promote bone formation . TranscrVD is a prohormone that acts in a variety of paracrine and autocrine systems. VD exerts a pleiotropic effect in the body, plays an important role in calcium–phosphate homeostasis and the regulation of PTH, bone metabolism, immune system, and cardiovascular disease ,52.2) and cholecalciferol (VD3) [2 are vegetables and “fortified” food, whereas VD3 is derived from animal-based foods but is mainly synthetized in the skin [3 is produced through the action of ultraviolet (UV) sunlight in the skin by photolytic conversion of 7-dehydrocholesterol (pro-VD3) to pre-VD3 ; then, it is subsequently is changed to VD3 [2 and VD3) are biologically inactive, they need further metabolism to be activated. In the next step, they are transported by VDBP in the liver, where they are subjected to the hydroxylation process by 25-hydroxylase (CYP2R1) to create 25D  [VD is a fat-soluble vitamin that exists in two distinct forms, ergocalciferol (VDol (VD3) . The southe skin ,13. VD3 d to VD3 ,54. Due lcidiol) . The finlcidiol) . The levlcidiol) ,58. The 2+ absorption in small intestines, increases PTH-dependent Ca2+ reabsorption in kidney, and mediates PTH-stimulated calcium release from bone [In a healthy individual, the kidneys are the main place of 1,25D synthesis, but under specific conditions , other cell types can also release it into circulation . Interesrom bone . Thus, Prom bone .3 [Serum 25D concentration is the most reliable biomarker for assessing VD status. To date, there is a lack of standardized methods for quantifying the level of 25D . Althoug3 .Low levels of 25D are common in CKD as well as in the general population, but the prevalence of 25D deficiency is much greater in the CKD population ,71. VD dBoth the Kidney Disease Outcomes Quality Initiative (KDOQI) and KDIGO guidelines recommend checking and supplementing low serum 25D levels in CKD and dialysis patients . AdditioVK (in particular K2) improves the function of OBs by inducing their proliferation, differentiation ,79, and κB) expression in OBs as well as in the OC precursors, explaining its dual pro-anabolic and anti-catabolic activities. Interestingly, the combined use of VK2 and 1,25D enhanced calcium deposition and OC expression in the OBs of obese diabetic mice [α (IL1α), and 1,25D [Yamagushi et al.  observedtic mice , suggesttic mice ). VK dectic mice ,84. Morend 1,25D . The stuIn the available literature, there are few data on the in vitro effect of 1,25D on OBs from patients with CKD. Over the course of the last decades, 1,25D has been studied extensively for its pleiotropic actions promoting bone remodeling in the general population, and numerous in vitro studies have implicated 1,25D in the regulation of both osteoblastic and osteoclastic activity ,95. Both1,25D has been shown to stimulate bone formation and mineralization in all studies using hOBs, and it induced osteogenic differentiation from hMSCs. Ten nM of 1,25D stimulated the differentiation of hMSCs to OBs, and osteoblastogenesis was stimulated to a greater degree by 1,25D in hMSCs that were obtained from subjects with inadequate or deficient 25D levels than the people who were VD sufficient . hMSCs, 1,25D has been shown to increase RUNX2, small mother against decapentaplegic (SMAD) 1–3,5, osterix (OSX), ALP, and BGLAP expression in hOBs ,106,107.The Wingless-type, Wnt-β-catenin pathway is an important regulator of OBs differentiation and function. Cytosolic β-catenin is translocated into the nucleus to stimulate osteoblastogenic gene transcription. The levels of β-catenin expression represent the functional status of the Wnt/β-catenin signaling pathway in OBs . The impAutophagy was recently recognized as an important regulator of OB survival and function. During autophagy, the toxic cytoplasmic components are removed, while nutrients are recycled to maintain cell functions and to protect against apoptosis. Impaired autophagy causes cellular dysfunction and cell death. Therefore, modulating the functional autophagy in bone cells is of therapeutic interest ,114. RecVascular endothelial growth factor (VEGF) is one of the most important pro-angiogenic factors involved in the regulation of new bone blood vessel formation. Human osteoblastic cells produce VEGF, and receptors for VEGF have been identified on these cells, allowing VEGF to directly regulate survival, chemotactic migration, and OB activity . 1,25D tIn vitro experiments with primary bone cells isolated from humans demonstrate that treatment with 1,25D inhibited OB proliferation and enhanced OB maturation and mineral deposition. The expression of many genes key to OB maturation and mineral deposition are modulated by 1,25D, as has been above described ,88. The In addition to the stimulation of bone formation and mineralization, 1,25D has a certain protective potential to avoid pathological over-mineralization. It may induce activin A and osteopontin (SPP1) gene expression—the recognized inhibitors of mineralization ,121. MorCurrent evidence suggests that endogenous 1,25D synthesis and the response to this vitamin in human bone is linked with coordinated functions in both the osteoclastic and osteoblastic cells, controlling bone remodeling ,124.The stimulation of osteoclastogenesis by 1,25D via the OB is one of better established effects of this vitamin on OC activation. With respect to gene regulation in OBs, the 1,25D–VDR complex induces the expression of RANKL that activates RANK on OCs and their hematopoietic precursors, stimulating bone resorption through osteoclastogenesis. OPG, the soluble decoy receptor for RANKL, is repressed by 1,25D in OBs, so that the biological effect of RANKL is reinforced ,125. TheIn vitro studies on the effect of 1,25D on osteoclastogenesis and hOC function are conflicting, showing both stimulatory as well as inhibitory effects of this vitamin on OC differentiation and resorptive activity ,136,137.The study of Zarei et al.  showed tKim et al.  examinedWnt ligand 10b (Wnt-10b) is a key pathway for bone formation through increases in the number of OBs and the rate of mineral apposition . A recenAutophagy has been reported to increase the number and function of OCs ,137. TheTaken together, the in vitro studies revealed that 1,25D may function to optimize osteoclastogenesis, but on the other hand, it can mitigate hyperactive osteoclastic resorptive activity. The main effects of VK and VD on bone cells derived from in vitro studies are summarized in According to our best knowledge, there is only one report examining the influence of VK2 on cortical bone mass and bone strength in rats with renal insufficiency. This study demonstrated that the administration of VK2 increased cortical bone strength without changing bone mineral density (BMD) in nephrectomized rats , suggest3 showed an additional protective effect on osteoporosis versus VK2 treatment alone [However, several animal models of osteoporosis have been used to study the effects of VK on bone metabolism. nt alone . In addint alone  did not nt alone  showed tIn general, evidence from in vitro and animal studies supported the role of VK2 in bone health, suggesting a potential benefit for its use in patients with osteoporosis.Despite widespread clinical use, there are limited studies on animal models of CKD that examine the bone tissue material and structural properties after VD  treatment, and the obtained results are inconclusive. In the study by Jokihaara et al. , 5/6 nepThe effect of daily or intermittent calcitriol administration in enhancing bone growth in CKD was studied by Sanchez et al. . The aniNewman et al.  used a rIn contrast to the above data, some studies reported that 1,25D therapy can lead to bone turnover alteration and a reduction of cortical thickness in CKD rats. Male subtotally nephrectomized Sprague–Dawley rats were treated with 0.25 μg/kg/day of calcitriol starting 2 weeks after subtotal nephrectomy and continued for the next 14 weeks. In rats treated with 1,25D, a dramatically increased bone formation rate, an irregular osteoid deposition, and chaotic mineralization were observed. The dynamic bone histomorphometric parameters could not be measured in these animals due to the chaotic tetracycline incorporation. An excessive amount of osteoid, in combination with reduced bone resorption, led to a high bone area, which was improperly mineralized in rats treated with 1,25D. Moreover, kidney function was significantly more impaired, whereas aortic calcification was increased in rats treated with calcitriol compared to the CKD group .Bisson et al.  treated Summarizing, the recently performed studies showed that the treatment of CKD animals with 1,25D may not improve bone quality , or evenSuch divergent data obtained during the treatment of renal osteodystrophy with 1,25D may result from the use of different animal species, doses, and treatment regimens, a differential degree of PTH reduction, and possibly distinct effects of this vitamin in different skeletal sites. Nevertheless, data obtained from animal models indicated that 1,25D may have a direct effect on bone, independent of its effect on PTH suppression.Studies regarding the association between poor VK status and bone metabolism, BMD, and the risk of fracture in CKD patients are limited. The main results obtained using VK supplementation and its association with bone health are summarized in Kohlmeier et al.  were theStudies on the effect of clinical VK supplementation on BMD are also scarce. Sasaki at al.  showed tTo date, very little is known about the VK insufficiency and bone remodeling in CKD. Holden et al.  performeSo far, VK supplementation and bone fractures in CKD patients have not been studied in a controlled, randomized clinical trial. Currently, there is one randomized controlled double-blind trial, RenaKvit, being performed in Denmark to address the effect on VK2 (MK7) on cardiovascular and bone disease in CKD patients. This study is evaluating the impact of VK2 supplementation on arterial stiffness and bone mineral density in HD patients. The RenaKvit trial is evaluating the impact of 360 μg of VK2 during a period of 2 × 12 months . IncreasTogether with the declining kidney function, many abnormalities concerning 1,25D, FGF-23, and PTH levels were observed. A disruption of the delicate balance between 1,25D, calcium, phosphorus, and PTH lead to secondary hyperparathyroidism and increased risk of bone disease. Several studies showed an inverse association between VD deficiency/insufficiency and PTH levels ,181,182.Studies showing an association between VD and BMD or bone fractures in the CKD population are limited. A summary of the key findings is presented in 3 for VD repletion, but it confirmed that some patients with CKD may require a more aggressive therapeutic strategy [The optimal management of CKD-MBD is a daily challenge for nephrologists. VD supplementation is required for CKD patients to suppress PTH increases as well as to correct abnormalities of bone and mineral metabolism. In daily practice, VD  can be used in daily, weekly, or monthly doses. On the other hand, when VD supplementation is ineffective, therapy with VDRA  can be initiated . Neverthstrategy . Howeverstrategy . Accordistrategy . The excstrategy , hyperphVitamin D analogs (VDRAs), which are less calcemic and phosphatemic than the active form of vitamin D, are becoming the standard for the treatment of secondary hyperparathyroidism. The experimental models and clinical studies suggest that VDRAs can promote VC probably only at high doses if they induce or exacerbate hyperphosphatemia, while the use of these agents in more physiological doses (just enough to correct secondary hyperparathyroidism) might even be protective against VC ,196,197.3 supplementation for six months in adults with diabetes mellitus and CKD. Summarizing, all the above-mentioned randomized studies demonstrated that a correction of VD deficiency with cholecalciferol supplementation led to the efficient achievement of a sufficient level of 25D in CKD patients.In a study by Oksa et al. , a 12-moIn another study, Moe et al.  comparedStudies regarding VD supplementation, using ergocalciferol in CKD patients with VD insufficiency, have shown effective correction of 25D ,209,210.A growing body of evidence from in vitro  and in vOC is produced by OBs during bone formation. It is one of the most abundant proteins in bone and is necessary for bone mineralization. The synthesis of fully functional OC and its expression are controlled by both VK and VD . 1,25D iVD can exert an anabolic effect in bone through increasing OB activity and reducing OC activity . KoshihaOn the other hand, there are suggestions that excessive amounts of VD can increase VK requirements, inducing a relative VK deficiency by direct stimulation of the synthesis of VK-dependent proteins ,227.Another field of VD and VK cooperation is inflammation, which is casually implicated in osteoporosis . VK is rVK and VD also overlap metabolically at the cellular level. The VK cycle is a source of electron transfer for antioxidant power in hOBs, and 1,25D can enhance the reductive recycling of MK4 . SXR, whOn the other hand, the activation of SXR in the liver can lead to the down-regulation of CYP2D25, which is an enzyme involved in 1,25D biosynthesis  that may3, and VK2 on the inhibition of OVX-induced bone loss in rats. The combination of these three elements increased cortical and trabecular bone quality as well as improved biochemical and densitometrical parameters. Meanwhile, Iwamoto et al. [In vivo evidence also reported that combined VD and VK2 supplementation prevented bone loss by stimulating OC production in ovariectomized (OVX) rats ,237,238,o et al.  found noThe above presented data suggest that combined VD and VK supplementation may be beneficial for bone health in the course of CKD. Unfortunately, there have been no randomized, controlled trials that examined the effects of such a combination in this population. Many studies have investigated the combined effect of VK and VD supplementation on skeletal integrity in the general population, especially in postmenopausal women with osteoporosis ,217,241.Taken together, despite the growing body of evidence from in vitro and in vivo studies, as well as clinical trials, the synergy between VK and VD in relation to bone quality and quantity remains not fully elucidated. Therefore, further studies are needed to explain the exact mechanisms of the combined effects of these vitamins on bone health, especially in the CKD population.The current understanding is that patients with CKD are a clinical group at high risk for VD and VK deficiency. Therefore, these patients are prone to suffering from many consequences of VK and VD deficiency, such as poor bone health and a higher risk of fractures. Therefore, finding new solutions for the prevention/treatment of osteoporosis in this population is a particular challenge. The majority of randomized-controlled studies performed on osteoporotic patients without CKD suggest that combined VK and VD supplementation may be more beneficial for the prevention and treatment of bone loss. Moreover, supplementation with these vitamins is easily accessible, safe to use, and relatively inexpensive. However, there is still not enough evidence to recommend VK and VD supplementation. Based on studies in the general population, we can suspect that VD and VK supplementation in CKD patients may be a possible therapeutic target for improving bone health. Due to the lack of adequate clinical studies in this population, the question arises whether CKD patients might benefit from simultaneous VK and VD supplementation. This creates a need for further research, in which an investigation of the potential synergistic effect of combined supplementation of VD and VK on bone health in this population should receive more attention.


Non-alcoholic steatohepatitis (NASH) is characterized by an abnormal hepatic lipid accumulation accompanied by a necro-inflammatory process and a fibrotic response. It comprises from 10% to 30% of cases of patients with non-alcoholic liver disease, which is a global health problem affecting around a quarter of the worldwide population. Nevertheless, the development of NASH is often surrounded by a pathological context with other comorbidities, such as cardiovascular diseases, obesity, insulin resistance or type 2 diabetes mellitus. Dietary imbalances are increasingly recognized as the root cause of these NASH-related comorbidities. In this context, a growing concern exists about whether magnesium consumption in the general population is sufficient. Hypomagnesemia is a hallmark of the aforementioned NASH comorbidities, and deficiencies in magnesium are also widely related to the triggering of complications that aggravate NASH or derived pathologies. Moreover, the supplementation of this cation has proved to reduce mortality from hepatic complications. In the present review, the role of magnesium in NASH and related comorbidities has been characterized, unraveling the relevance of maintaining the homeostasis of this cation for the correct functioning of the organism. Non-alcoholic steatohepatitis, or NASH, is a term used to define a pathophysiological stage of the liver characterized by an abnormal lipid accumulation (steatosis), inflammation, hepatocellular damage and fibrosis development . NASH isThe liver plays a key role in the metabolism of all biomolecules, but perturbations in lipid balance lead to the development of steatosis. In NAFLD, the genetic background or nutritional imbalances lead to a downregulation of the pathways involved in hepatic lipid clearance: (i) very-low-density lipoprotein (VLDL) secretion and (ii) fatty acid oxidation (FAO) or an upregulation in those that promote hepatic lipid content as (iii) de novo lipogenesis (DNL) and (iv) fatty acid (FA) uptake . AlthougUnhealthy nutritional habits and dietary imbalances are beginning to be recognized as the root cause of many diseases. Particularly, it was previously mentioned that NASH has metabolic perturbations that are the most common cause of development . High-fa2+ in its free form, is a micronutrient widely distributed in the food supply, both in plant and animal foods. Most green vegetables, legumes, peas, beans and nuts are rich in magnesium, as are some shellfish and spices. The daily recommended intake (DRI) for elemental magnesium is age-dependent, and lower for women  than men . Magnesium DRI increases for pregnant women and lactation periods. The cation is absorbed in the duodenum and ileum by both active and passive processes, and this process is affected by different nutrients, such as fiber content [Magnesium, or Mg content . Under h content .2+ is the most predominant divalent cation in the cell, with concentrations ranging from 5–20 mM, and extracellular Mg2+ accounts for only 1% of the total content in the organism [2+ homeostasis have been reported, not only in liver pathologies but also in concomitant systemic complications [Mgorganism ,20. Magnorganism . The maiorganism . These aications ,23,24.2+ homeostasis and the development of systemic complications accompanying NASH. In the present review we aim to highlight the relevance of Mg2+ in the development of NASH has not been explored in depth, a clinical study showed a protective effect of Mg2+ intake in patients with liver diseases. Remarkably, an increased intake of 100 mg (25–33% increase of the daily recommended intake) showed a 49% reduction of liver-derived mortality [2+ has also been characterized, as hypomagnesemia leads to a deficient PKCε translocation and subsequent fibrinogen and collagen deposition [2+ content, an over-activation of leukocytes and macrophages has been characterized, together with the recruitment of more inflammatory cells to the liver [The spread of unhealthy lifestyle habits, together with inadequate nutritional behavior, are making NAFLD and the pathologies comprised in its spectrum the leading cause of chronic liver disease worldwide. Although, to date, the role of Mgortality ,27. The ortality . The rolposition . There ihe liver . Moreovehe liver .2+ plays a key role in DNA synthesis. Therefore, hypomagnesemia leads to dysfunctions in DNA damage repair mechanisms, modulation of cell cycle progression, cell proliferation and differentiation, and apoptosis with the subsequent tumor growth promotion and metastasis of the tumor [As aforementioned, the development of NASH and other pathologies ranging in NAFLD may increase the risk of developing HCC. Related to this, the biological functions of the cation have been characterized, as Mghe tumor ,33. Althhe tumor .2+-deficient livers show an increased KC activation through the toll-like receptor type 4 (TLR4), promoting ROS generation [2+ supplementation in patients with alcoholic steatohepatitis prevents the progression of the disease, reducing the transaminases levels in serum and decreasing liver-related morbidity [Finally, a continuous magnesium loss has been related to alcohol consumption , leadingneration  and pro-neration . Remarkaorbidity .In summary, even though several studies suggest the relationship between hypomagnesemia and the development of liver diseases , to dateOverweight and obesity are complex and multifactorial diseases that affect over 1/3 of the world population. Similar to the epidemiology of NASH and NAFLD, these conditions are expected to even increase to a 38% prevalence of overweight and a 20% prevalence of obesity by 2030 . Obesity2+ perturbations may have in the development of alterations in obesity, hypomagnesemia has been identified in serum from obese patients [2+ consumption prevents the induction of obesity in in vivo models, suggesting a pivotal role of the cation in maintaining energy homeostasis [Related to the possible role that Mgpatients . Remarkapatients . Relatedpatients . Other spatients . Additioeostasis . Howevereostasis . Nevertheostasis .Finally, other studies have focused on the impact of magnesium modulation in the adipocyte. It has been known for a long time that the adipocyte is not only a simple storage depot for body energy, but in fact an endocrine organ playing a very relevant role in obesity . Interes2+ cation appears to prevent and reduce obesity prevalence by targeting inflammation and adipocyte proliferation  and type 2 Diabetes mellitus (T2DM) constitute together with obesity the main worldwide global epidemics, termed “diabesity”. Whereas the prevalence rates of IR syndrome have been reported to be 3–16%, with a higher incidence in white populations, the global prevalence of T2DM has been estimated to be 6.3% of the world’s population, being higher in elder patients . IR is p2+ reabsorption [2+ deficiencies have been reported to alter the pancreatic insulin recreation by disrupting the normal activity of β-cells [2+ directly affects the GK activity [+ (KATP) channels that depolarize the β-cell membrane for the insulin release, leading to an uncontrolled insulin secretion [2+ perturbations over insulin resistance development has been reported to be reversible, so that the recovery of normal levels of the cation could solve the pathology [2+ supplementation shows a significant improvement in plasma glucose, lipoprotein and triglycerides profile and BP in diabetic patients [Hypomagnesemia in both IR and T2DM patients has been described, as they often show not only a reduced intake of the cation but they also present an augmented urinary loss ,53. As msorption . T2DM pasorption . For inssorption . Relatedsorption . The devsorption . Relatedsorption . The tra β-cells . In β-ceactivity . Thus, hecretion . Furtherecretion  and pro-ecretion . Remarkaathology . In thispatients .Therefore, as summarized in Hypertension is a growing health concern that over a decade ago was already estimated to affect around 31.1% of the global population (1.39 billion patients) . This co2+ deficiencies are related to a reduction in endothelial functionality, a risk factor for atherosclerosis [2+ plays a role in the proliferation and migration of endothelial and vascular muscle cells, the modulation of neuronal excitation and intracardiac conduction, as well as the myocardial contraction [2+ plays a role in the depolarization–repolarization processes, in that Mg2+ depletion alters electrocardiogram activity [2+ in skeletal muscle are related to aortic distensibility [2+ levels show alterations in echocardiographic indices and increased heart volume [2+ in patients with CVD has also been reported to exert a protective role [2+ acts as a calcium channel blocker, increasing the production of nitric oxide (NO), improving endothelial dysfunction and inducing vasodilatation [2+ deficiencies and an elevated risk of stroke [Regarding the involvement of magnesium perturbations in the development of hypertension and cardiovascular diseases, there are several works than point out a relationship between the two conditions. Similar to the other conditions previously mentioned, the supplementation of the cation reduces BP in case of IR, pre-T2DM and other non-communicable chronic diseases . Remarkaclerosis ,72, whilclerosis . The catclerosis . Investitraction . During activity . Low seractivity . Correlasibility  and the sibility . The catsibility . Patientt volume . Similarive role . Houstonlatation . Additiof stroke . Regardif stroke , togethef stroke Overall, magnesium appears to play a role in the development of CVDs as deficiencies in the cation are related to atherogenesis and perturbations in skeletal muscle functionality .2+ deficiencies has been widely characterized.Between the systemic complications that normally accompany NASH development, obesity, IR and TD2M, hypertension and CVDs are the most common and relevant . Indeed,2+ could contribute to the transition from IR to T2DM [2+ in the prevention of oxidative and inflammatory stress could be a common mechanism. Moreover, considering the impact of mitochondrial dysfunction in the NASH-related comorbidities, other potential underlying mechanisms include the role of magnesium in the regulation of mitochondrial function. Finally, as the Mg2+ content can influence Ca2+ levels, and, for example, it has been shown that T2DM may cause endothelial dysfunction by remodeling the intracellular Ca2+ toolkit [2+ in IR, T2DM, CVD, and other NASH-related comorbidities.Although NASH and related pathologies have their own specific complications, studies about the role of magnesium point in the same direction. Low magnesium concentrations have been related to many liver pathologies as cirrhosis, liver cancer or alcoholic liver disease. Moreover, hypomagnesemia is related to the development of a higher BMI—characteristic of obesity development—a higher risk of developing T2DM, and the development of atherogenesis and alterations in the cardiac muscle that lead to CVDs. Indeed, in the case of the pancreas, the cation has been characterized to play a role in insulin secretion by β-cells so that the loss of Mg to T2DM  and, in  to T2DM . Cardiom to T2DM ,79. Anot to T2DM  so a pos toolkit , this me2+, the encouragement of the global population by health authorities to increase their nutritional intake of Mg2+ may reduce the prevalence of the pathologies mentioned in the present work. This can be achieved either by supplementation or by promoting the consumption of magnesium-rich food, such as green vegetables, nuts, seeds and unprocessed cereals [On the one hand, considering the preventive role of the supplementation of Mg cereals . Conside cereals . The pot2+ transporters that allow its flux across cell membranes participating in the processes of intake, excretion and reabsorption [2+ homeostasis in the development of several diseases in which immune responses or essential biological processes are altered, the role of these proteins might be a research topic of interest for therapy development against NASH or related morbidities.On the other hand, the physicochemical properties of the cation make essential the activity of Mgsorption  In this sorption ,85,86, tsorption , whereassorption . Otherwisorption  and SLC4sorption . Related


The aim of our study was to describe the balance between health benefits and risks associated with the consumption of crawfish and nine fish species from lake Trasimeno. We thus determined both fatty acid profiles  and docosahexaenoic (DHA) acids) and chemical pollutants  in fish muscle tissues. The contents of all fatty acids varied significantly among species. Sand smelt, carp, and tench, which have a high fat content, contained considerable amounts of EPA and DHA; lean fish, like perch, pike, and largemouth bass, which have relatively high percentages of the predominant n-3 fatty acids EPA and DHA, showed lower amounts of these fatty acids because of their low lipid contents. Some species contributed strongly to the Dietary Reference Intake (RDI) of EPA and DHA. The contribution of lean fish to the RDI of EPA and DHA was more limited. The concentrations of all contaminants in fish muscle tissues were lower than the regulatory limits, demonstrating the safety of the environmental conditions of the lake. The contribution to health-based reference values and benefit–risk quotients indicated that the health benefits of consumption of fish from lake Trasimeno outweigh the potential risks. The nutritional benefits of fish consumption are attributable to the high-value proteins and unsaturated fatty acids, as well as certain minerals and vitamins, contained in fish. Particularly, fish are considered a very important source of n-3 polyunsaturated fatty acids (PUFA), especially eicosapentaenoic EPA, C 20:5n-3) and docosahexaenoic  ,2. Sever0:5n-3 anNevertheless, Li et al.  found thIn the present work, in order to define the nutritional quality and safety of nine economically important species of freshwater fish and crawfish caught in lake Trasimeno , the fatty acid composition, as well as the concentrations of certain chemical pollutants , were defined. Furthermore, aiming to describe the balance between health benefits and risks due to fish and crawfish consumption, the intake of n-3 PUFA and the estimated daily intake (EDI) of the mentioned contaminants were assessed.2), it is considered quite shallow . This Umbrian lake is characterized by a remarkable naturalistic importance, as it is, indeed, a Site of Community Interest, Special Protection Zone and Regional Park  is the largest laminar lake in Italy and, more generally, is the fourth among the largest Italian lakes; furthermore, in spite of its large extension  [Cyclops, whereas in summer, Daphnia galeata, a small species of crustaceans belonging to the Order Cladocera, is the main zooplankton representative [Since the 20th century, the composition of the lake Trasimeno fauna has changed considerably due to several factors, such as eutrophication, pollution, significant hydrologic fluctuations, and the introduction of allochthonous species. Despite the above-mentioned factors, the Umbrian lake is considered a high-value ecosystem due to its wide biodiversity: its fish fauna, in fact, comprises 19 species dominated by those belonging to the Cyprinidae Family . The repnoficee) . Similarentative .Cyprinus carpio, L.), golden fish , tench , pike , eel , sand smelt , perch , black bullhead , largemouth bass , and crawfish .Nowadays, small-scale traditional fishing is still one of the main commercial activities of the lacustrine area, promoted by both governmental guidelines encouraging the production and consumption of local food and consumer preference for Km 0 foodstuff. Small-scale traditional fishing, as well as the consumption of fish in the area of lake Trasimeno, can be considered sustainable. In fact, the freshwater fish industry of this area is expanding into the market by exploiting the great potential of abundant undervalued species consumed as ingredients in processed food and by the adoption of long-term preservation through a cold chain system to compensate the seasonality of fish . In thisFish and crawfish collected for the present study were caught from lake Trasimeno. Fishes and crawfish sampling was conducted in three different months: December 2018 and January and February 2019. After collection, all fishes were transported the same day under refrigeration to the Department of Veterinary Medicine, University of Perugia. Upon arrival, each individual was beheaded and dressed (for fish) or decarapaced (for crawfish), and the muscle tissue (edible muscle) was filleted and stored at −80 °C until analysis. Sand smelt, due to their small size (adult maximum length around 7 cm), were left whole. Four individuals of each freshwater fish species and six individuals for crawfish and sand smelt, each month, were used for the analysis.The chemical composition and fatty acid profiles of each individual were determined according to the procedure of Branciari et al. ,18.2SO4-acidified Extrelut NT-3 column connected on top to an SPE silica column (1 g/6 mL), and the analytes were eluted with 13 mL of n-hexane. The purified extract was re-dissolved in 0.25 mL of a 13C12-PCB-155 iso-octane solution at 10 ng/mL and submitted to GC–MS/MS analysis . The injection was carried out in pulsed splitless mode, and the chromatographic separation was achieved in temperature-programmed mode  on an SGE-HT8 PCB capillary column , using helium at 1 mL/min as the carrier gas. The transfer line was held at 280 °C, the source at 230 °C, and the quadrupoles at 150 °C. The method was submitted to multi-level validation in intra-laboratory reproducibility conditions, following the prescription of Commission Regulation (EU) 2017/644. The procedure enabled to measure 0.10 ng/g fresh weight for each of the six PCB congeners (limit of quantification = LOQ); moreover, the analyses were conducted in isotopic dilution for all the six PCBs, and acceptable internal standards recoveries were between 60% and 120%, as requested by Regulation (EU) 2017/644 [Samples for contaminant determination were collected by local official competent authorities from fishermen and sent to the official local laboratory located at Istituto Zooprofilattico Sperimentale dell’Umbria e delle Marche. The number of samples collected was defined according to the integrated regional control plan, and sampling was carried out randomly. Fish muscles were analyzed to assess PCB levels . A sample amount yielding roughly 1 g of fat was weighted, spiked with 2.5 ng of the six 13C12-labelled indicator PCBs, and freeze-dried (6 h). The sample powder obtained was mixed with diatomaceous earth and extracted with ASE 200 , using a mixture of hexane/acetone (1:1 v/v). The extracts, after solvent evaporation, were purified on an H2017/644 .Pb, Cd, Hg, Ni, Cr, and As were analyzed in 1 g of sample after microwave digestion with 6 mL HNO3 , 2 mL of H2O2 , and 100 µL HF  (Milestone-Ethos1-HPR1000). The appropriately diluted solutions were analyzed by inductively coupled plasma mass spectrometry  in standard mode using specific mass-to-charge ratios (m/z) for each element . We used 103Rh as the internal standard, and quantification was matrix-matched. The analytical methods were fully validated in intra-laboratory reproducibility conditions, following Commission Regulation (EC) No 333/2007 [4, 4 g; NaCl, 1 g; tri-sodium citrate dehydrate, 1 g; sodium hydrogen citrate sesquihydrate, 0.5 g) was added, and the tubes were vortexed for 1 min and centrifuged for 5 min at 4000 rpm. Eight milliliters of ACN were then transferred to a clean tube and stored in a freezer overnight. Six milliliters of cold ACN were purified by d-SPE (150 mg PSA + 900 mg Mg2SO4) and centrifuged for 8 min at 3500 rpm. Finally, 1 mL of purified extract was dried in a rotary evaporator  and resuspended in 0.2 mL of isooctane. The sample solution was injected in splitless mode, and gas chromatography was carried out using an Agilent EI GC-MS/MS system equipped with a 7890A GC system, 7000B GC/MS triple QUAD, and a 7963 autosampler. Chromatographic separation was carried out through a Rtx-Pesticides2 column in temperature-programmed mode. The method was validated according to the European Commission Guidance Document SANTE\11945\2015 on Method Validation and Quality Control Procedures for Pesticide Residue Analysis in Food and Feed. Performance parameters of the method in terms of repeatability, reproducibility under intra-laboratory conditions, linearity, recovery, specificity, and sensitivity were established by validation in a single laboratory. Within-laboratory reproducibility (RSDWLR) combined with data from EURL-AO proficiency tests were used for the estimation of measurement uncertainty according to Document SANTE\11945\2015. The LOQ was fixed at 0.002 mg/kg for all organochlorine pesticides and dinitroaniline, and at 0.005 mg/kg for organophosphorus and pyrethroids.Pesticide analysis was conducted by the QuEChERS method, as reported. In brief, extraction was performed on homogenized samples (5.00 ± 0.05 g) with 10 mL acetonitrile (ACN). δ-lindane, triphenilphoshate, and PCB 198 were added as internal standards (IS) for organochlorine, organophosphorus (P), and pyrethroids (Py) pesticides, respectively. The extract was shaken on an orbital shaker for 20 min, and afterwards a salt mix  is the recommended daily intake of EPA + DHA. In this study, the RDI of 250 mg/d for a healthy adult [FA (mg/g) represents the concentration of EPA + DHA in fish muscles.The benefit–risk quotient (BRQ) was employed to evaluate the risk–benefit of the simultaneous ingestion of PUFA and contaminants through freshwater fish consumption, according to the following equation :(1)BRQ=Qhy adult  was applT) can be define according to the following equation:The maximum allowable fish consumption related to toxic effects , and an analysis of variance (ANOVA) model was defined using the GLM procedure in SAS version 2001 . The differences between means were considered to be significant if The proximate and fatty acid composition and cholesterol content of muscle tissues of freshwater fish and crustaceans collected in this study are presented in The lipid content varied markedly among the fish and crustacean species (0.41%–22.35%). According to the lipid content, the fish species studied were categorized into different groups as lean, low-fat, medium-fat, and high-fat, as reported by other authors . Perch, A total of 23 major fatty acids, including eight saturated fatty acids (SFA), six monounsaturated fatty acids (MUFA), and nine PUFA, were identified and quantified. All fatty acids varied significantly among species. Total SFA ranged from 25.29% in the crustacean red swamp crawfish to 36.99% in golden fish, MUFA ranged from 19.55% in perch to 49.67 in eel, and PUFA from 19.59% in eel to 51.26% in pike. The most abundant SFA in all fish studied was palmitic acid (16:0), which varied from 16.2% in crawfish to 22.87% in tench. The second was myristic acid (14:0), which varied from 0.9% in crawfish to 7.7 in sand smelt, and the third was stearic acid (18:0), which varied from 3.6% in sand smelt to 7.5% in golden fish. These values are in agreement with previous studies on freshwater fish from other Italian lakes . The mosPUFA contents in the studied species have been reported to be in a very wide range. The major fatty acids identified as PUFA were EPA and DHA in all fish species and crawfish. Among PUFA, total n-3 fatty acids ranged from 9.6% in eel to 39.8% in pike. DHA and EPA varied greatly among species. DHA ranged from 3.4% in eel to 29.2% in pike, whereas EPA ranged from 1.7% in eel to 11.6% in crawfish. Perch and pike presented the highest percentages of EPA and DHA, followed by largemouth bass. The lowest percentage of the sum of EPA and DHA was found in eel, followed by black bullhead. The percentages of the EPA and DHA of pike and perch were similar to those reported by other authors ,29 and cLinoleic acid 18:2n-6) and arachidonic acid (20:4n-6) were the predominant n-6 PUFA in all freshwater fish. Similar results have also been shown in many previous studies of freshwater fish n-6 and a. An incrTo evaluate the effective quantitative level of EPA and DHA introduced by diet, the amounts of EPA and DHA were calculated in fish flesh and expressed as milligrams of fatty acid per 100 g of fish meat .Eel, sand smelt, carp, and tench, which have a higher fat content than the other fish, contained higher amounts of EPA and DHA . In geneThe PCBs analyzed represent the sum of six congener  considered as suitable indicators of all PCBs due to their predominance in biotic and non-biotic environments and listed as priority food contaminants monitored by the European Food Safety Authority . FurtherThe residual levels of these chemicals varied among the fish groups considered, ranging from 28 ng/g for the HFF group (represented only by eel) to 1.9 ng/g for LFF. The total PCB concentration in HFF was approximately 15 times higher than that in other fishes, confirming the higher capacity of eel to accumulate PCB congeners. This result is consistent with those reported by other researchers, who found higher levels of PCBs in eel muscle with respect to other species, attributable mainly to the high level of tissue fat peculiar to this species . For thiIn contrast to this study, data reported for other countries  show that the EU limit set for PCB was exceed in eel, which thereby represents a risk to human health. Low levels were found in Polish water bodies; indeed, only 9 of 125 eels exceeded the maximum level. Although PCBs were banned in most developed countries in the 1970s, the extensive and careless use of these compounds for almost 50 years and their persistence have resulted in widespread contamination of the environment and chronic pollution of terrestrial and aquatic ecosystems in Europe ,48,49.Regarding pesticides, According to the results of the questionnaire distributed to the target population, freshwater fish accounted for a modest proportion of the consumers’ diets (33 g/d); however, as reported in the literature, it may represent one of the main sources of human exposure to EPA and DHA and, eventually, environmental contaminants ,24. The In order to assess the potential risks associated with the consumption of fish from lake Trasimeno, the mean EDI and the contribution to ADI/TDI were calculated, considering a standard Italian adult weight of 70 kg and the above-mentioned consumption values. The EDIs for PCBs were 1.1, 0.9, 1.4, and 13.2 ng/kg bw/day for LM, LFF, MFF, and HFF fish groups, reflecting a contribution to ADI/TDI below 10% with the exception of the HFF category, which accounted for 66% of the reference value due to the high lipid content of this species. For the two pesticides molecules considered , the EDI was negligible in all fish groups examined, as was their contributions to the reference value were, in all cases, below 0.000 mg/kg bw/day and below 1%, respectively . SimilarRecently, monitoring programs have been implemented by several countries aiming to assess the presence of chemical pollutants in foods as well as to define human health risks resulting from dietary exposure to these contaminants. In the present study, the toxic effects of the targeted contaminants in muscle tissues of lake Trasimeno freshwater fish were evaluated using BRQ. The BRQ for all of the groups of fish analyzed were <1, ranging between 0.00 and 0.59 . This stThe contents of n-3 long-chain PUFAs (EPA and DHA) were higher in species such as eel, sand smelt, carp, and tench and were associated with the lipid contents of the fish, strongly contributing to the RDI of EPA and DHA. In lean fish that showed lower amounts of these fatty acids due to their low lipid contents, the contribution of EPA and DHA to the RDI was more limited than in fatty fish. The concentrations of all of the contaminants that were analyzed in fish muscle tissues were very low, below the limits when present , demonstrating the safety of the environmental conditions of the lake. Together, the contribution to reference values and BRQs indicated that the health benefits of consumption of fish from lake Trasimeno outweigh the potential risks; therefore, these species can be consumed regularly by the general population, without posing significant health risks related to the presence of contaminants. The population should be encouraged to consume higher amounts of freshwater fish from lake Trasimeno, as they can be considered a healthy choice and can allow consumers to meet the recommended levels of EPA and DHA.