Multidrug-Resistant Bacteria Related to Covid-19 Infection: A Minireview

Introduction

Native bacterial resistance to antibiotics is a natural selective pressure event that is currently a problem faced worldwide. Nevertheless, the inappropriate use of these classes of drugs results in treatment failure of infections, hindering therapeutic alternatives and increasing the risk of worsening the condition and spreading diseases. Multidrug-resistant bacteria is considered a pathogen that is not sensitive to three or more classes of antibiotics. The World Health Organization (WHO) recorded antimicrobial multidrug resistance as one of the biggest health risks in 2019. Some of the causes for this acquired resistance are misinformation about the function of antibiotics, leading part of the population to self-medicate to treat common viral infections, inadequate prescription due to inaccurate diagnosis, pollution of the environment with antibiotics and their use in the treatment of animals in areas such as veterinary, and agriculture [1- 4]. SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2) is a single-stranded, positive-sense RNA zoonotic virus identified in late 2019 in Wuhan, China, which spread around the world rapidly, causing a pandemic of the disease called Coronavirus Disease-19 (COVID-19). Immunization of the population is still the only known protective method, in addition to the individual respiratory support that was initiated and recommended as soon as the identification of disease. The world scenario has more than 700 million confirmed cases and more than 6 million deaths reported by WHO, as well as more than 13 billion vaccine doses applied to the population.

The most severe cases of infection require mechanical ventilation and intensive care treatment, with prolonged stays in the hospital environment, which characterizes a very increased risk and predisposition to secondary and opportunistic infections [2,5,6]. In the first phase of the pandemic, from December 2019 to June 2020, antibiotic prescription reached 82.3%, in contrast to 39.7% in the second phase, between June 2020 and March 2021, according to a study by Cong et al. published in 2022 [7]. These data can be explained by the prophylactic use of antibiotics for secondary bacterial infections, which can be corroborate by increased mortality rates from co-infections described after analysis of autopsy data and case studies of the influenza pandemic in 1918. The lack of knowledge of the disease and the initial symptomatic similarity between bacterial and viral infections cannot be ruled out, generating diagnostic misunderstanding, in addition to the high mortality rate 3, [8-10]. As the COVID-19 pandemic gradually accelerated, the investigation of highly effective drug therapies became a major debate in the scientific community. Azithromycin, for example, became one of the antibiotic alternatives raised to combat the syndrome caused by SARS-CoV-2 due to its immunomodulatory properties that were considered promising. Another possibility investigated was hydroxychloroquine, which is used for rheumatoid arthritis, lupus and malaria. Subsequently, studies concluded that both drugs did not have beneficial properties in the treatment of the disease [11-13].

The study by Rawson et al., published in May 2020, describes that 72% of patients were prescribed with antibiotics, although only 8% had confirmed bacterial co-infection after analyzing cases of predominantly Asian infections. It is still difficult to determine whether the conditions of patients infected with COVID-19 who died worsened before or after the bacterial infection acquired during hospitalization in Intensive Care Units (ICU). The interaction of SARS-CoV-2 with other pathogens is still poorly defined in the literature, as well as the impact of acquired resistance in the general public7, [14,15]. The aim of this review is to identify and correlate cases of infection of multidrug-resistant bacteria and the SARS-CoV-2 virus. In particular, addressing the effects of the use of antimicrobials empirically in patients with severe respiratory syndrome infected by antibiotic-resistant pathogens associated with COVID-19. The investigation of diagnosis methods and therapeutic approaches can contribute to a better understanding of the impact on the global scenario of bacterial multidrug resistance.

Materials and Methods

The present work is a narrative review of the literature relating multidrug-resistant bacteria and COVID-19, considering the co-infection scenario, as well as the impact on the development of acquired resistance of these bacteria. The following descriptors were used to carry out this study: “COVID-19”, “bacterial”, “co-infection”, “SARSCoV- 2”, “resistant”, “resistance”, “antibiotic”, “antimicrobial”. At first, the exclusion was made by the titles, resulting in 286 selected and, by eliminating the duplicates, 190 articles were obtained. Of the selected publications, 7 were paid articles. In the end, 81 articles were read partially, 109 in full, and 54 were included in this study.

Results and Discussion

Studies with humans and published in the period from 2020 to 2023 in the PubMed database, obtained 1290 results. Of these, 835 articles were found in the time frame between January 2022 and May 2023 SARS-CoV-2 is responsible for several systemic complications and injuries, differentiating it from other members of the Coronoviridae family, such as: Pulmonary, renal, pancreatic, cardiac, brain, hepatic, and coagulation disorders. Epithelial cells infected by this virus produce inflammatory cytokines such as interleukin-6 (IL-6) and interleukin- 8 (IL-8). The activation of these cytokines is linked to lungs infiltration by inflammatory cells and induction severe lung injury [16]. Co-infection Bacterial infections are related to an increased mortality rate of respiratory viral primary infections. The study by Na and colleagues revealed a rate of more than doubled in-hospital mortality in patients with severe COVID-19 and secondary infections. The damage to the airways caused by the viruses added to the inhibition of the immunology favors the adhesion and bacterial growth in the airways. The inflammation resulting from the cellular damage caused by SARS-CoV-2 infection in lung epithelial cells promotes severe tissue detriment due to bacterial adhesion, invasion, and proliferation. The SARS-CoV-2 spike protein binds to the bacteria’s lipopolysaccharides, increasing anti-inflammatory activity. Acute Respiratory Distress Syndrome (ARDS) is a systemic inflammatory reaction found in severe infections, pneumonia, COVID-19 and activation of the Toll-Like Receptor 4 (TLR4) by lipopolysaccharide stimulation during ARDS causes the pro-inflammatory phase, with high releases of cytokines, acute-phase proteins, and reactive oxygen species [17-20].

Cytokine release syndrome or cytokine storm, defined as the elevated release of pro-inflammatory cytokines, resulting from the activation of dendritic cells, lymphocytes (T), neutrophils, macrophages. Cytokine storm is an excessive response to SARS-CoV-2 and has been recognized as a cause of death in COVID-19. It results from the association of molecules such as interleukins, interferons, chemokines, and TNF-α in patients with COVID-19. A significant elevation of inflammatory markers were observed in patients who received antibiotics, which Have been pointed out as a possible aggravation of the cytokine storm caused by COVID-19 [16,21,22]. Bacteria are able to successfully cause secondary infections in COVID-19 by their virulence factors, such as membrane proteins, secretory systems, adhesins, and glycoconjugates. Bacteria are considered resistant to a specific antimicrobial when the recommended dose is unable to kill it or inhibit its proliferation.
The mechanisms of bacterial resistance can be summarized as follows:
1. Alteration of the site of action of the antibiotic,
2. Drug inactivation,
3. Expulsion of substrates from the interior of the bacterium to the external environment by an efflux system and
4. Alteration in membrane permeability. Acquired resistance is characterized as the alteration of the microbial cell site, antibiotic inactivation, and efflux system.

Gram-positive bacteria mostly adopt the method of decreasing membrane permeability, while gram-negative bacteria can use all four mechanisms. In addition, gram-negative bacteria have a layer of lipopolysaccharides that promotes innate resistance to several groups of antibiotics [20,23]. Bacterial co-infections are often common complications of respiratory infections and are associated with prolonged hospital permanence and increase in ICU admittance and mechanical ventilation. In the study by Patton et al., a high mortality rate of 24% for bacterial co-infections was observed compared to the 5.9% mortality rate for community-acquired pneumonia (CAP) in the pre-pandemic times. Community-acquired bacterial infections confer even greater risks of ICU admission and mechanical ventilation than advanced age and comorbidities, which have been identified as risk factors for COVID-19 mortality. The study by Tiseo et al. conducted in Italy found higher comparative rates of chronic obstructive pulmonary disease (20% vs. 10.2%), chronic kidney disease (24.4% vs. 7%), cerebrovascular disease (22.2% vs. 11.8%), higher sputum frequency (11.1% vs. 2.8%), and PaO2/FiO2 ratio <200 (17.8% vs. 5.4%) between patients with and those without respiratory co-infections, respectively. The rate of co-infections in hospitalized patients with COVID-19 was 8% in this study and was associated with a high risk of progression to severe disease [24-26]. The prevalence rate of co-infection varies in published articles, but it is as high as 50% among non-COVID-19 survivors. Studies show that the risk of acquiring bacterial infection increases according to the severity of COVID-19 and the length of hospital permanence, in which case it is called Healthcare- Associated Infection (HAI). In addition, microbiology differs between HAIs and community-acquired infections [27,28].

Bacterial Species Related to Covid-19

The study by Gu et al. published in 2020 points out that the diversity of the gut microbiota is reduced in patients infected with SARSCoV- 2, with an increase in opportunistic pathogens such as Streptococcus, Rothia, Veillonella, and Actinomyces. In addition, a significant increase in Enterococcus sp. in the gut microbiota, blood infections after ICU admission, especially E. faecalis and E. faecium in studies in Italy [27]. In HAIs, the antibiotic-resistant pathogens found in association with COVID-19 were: carbapenem-resistant Acinetobacter baumannii, Extended-Spectrum ꞵ-Lactamase-Producing Klebsiella Pneumoniae (ESBl), vancomycin-resistant Enterococcus, and other members of the New Delhi-producing Enterobacteriaceae family Metallo-ꞵ-lactamase (NDM) that confers resistance to carbapenem, and Methicillin-Resistant Staphylococcus Aureus (MRSA). S. aureus was associated with both HAIs and community-acquired infections in the 2021 study by Garcia-vidal et al., as was Escherichia coli and Klebsiella pneumoniae in the urinary tract. In this study, the species Streptococcus pneumoniae, Pseudomonas aeruginosa, Enterococcus faecium and Haemophilus influenzae were also identified, with the former and latter being associated only with community-acquired infections [27]. The prevalence of Acinetobacter baumannii and Klebsiella pneumoniae is remarkable and has been reinforced by several studies. The acronym ESKAPE encompasses the pathogens Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and species of the genus Enterobacter spp. which are known for their association with nosocomial infections and mechanisms of multidrug resistance to antibiotics [29]. A. baumannii resistant to carbapenems (CRAB) is on the WHO list of greatest threats to human health. A. baumannii is intrinsically resistant to penicillin and may acquire resistance to all antibiotics used to treat gram-negative drugs, including fluoroquinolones, aminoglycosides, and cephalosporins, as reported in studies in Table 1. Carbapenems are widely used in the treatment of A. baumanii, but some mechanisms of resistance against these antibiotics have been extensively reported and, for that reason polymyxins have been considered the first choice for the treatment of infections caused by CRAB [29]. Infections caused by K. pneumoniae and E. coli are associated with high mortality risks, possibly due to a lack of effective treatments. The main species carrying carbapenemase are K. pneumoniae, E. coli, and Enterobacter spp and have been linked to infections in the COVID-19 pandemic. K. pneumoniae is the most common enterobacterium related to the spread of ESBL and is responsible for the majority of human infections [29]. P. aeruginosa has several virulence factors, in addition to bacterial resistance genes possibly conferred by plasmids, and adaptability to numerous adverse conditions including the immune response. This pathogen has been reported to be significantly elevated in the nasal microbiota of patients infected with SARS-CoV-2. It causes a high risk of mortality, despite its variable resistance in different regions and the high incidence colistin-sensitive diseases caused by P. aeruginosa in hospitals around the world [29]. S. aureus has a marked virulence factor called coagulase, which is used for laboratory identification and is the main agent of blood, cutaneous, and lower respiratory tract infection. Some patients had S. aureus co-infection after hospitalization for COVID-19, some were infected with methicillin- resistant S. aureus (MRSA) and also methicillin-sensitive S. aureus (MSSA). The resistance of S. aureus seems to be due to several mechanisms, such as horizontal gene transfer by plasmids, changes in the target site, and enzyme production [29].

Table 1: Most Frequent Bacterial Species and Multidrug Resistance Rates in COVID-19 Patients.

Note: Legends – MR RATE: Multidrug resistance rate among total bacterial infections; TOTAL: Bacterial infections occurring in COVID-19 patients; NE: Not specified; ERC: Carbapenemase-resistant Enterobacteriaceae; NEC: Coagulase-negative staphylococci; ESBL: Stretched-Spectrum Beta-Lactamase; MRSA: Staphylococcus aureus resistant to meticilina.

Markers for Detection of Secondary Infections

One of the obstacles encountered is determining whether the bacterial infection occurred before or after admission, mainly due to the inability of most patients to produce sputum for culture at the time of arrival at the hospital. In addition, it is difficult to detect secondary infections or co-infections or to easily identify the exact pathogen because the symptoms of COVID-19 are similar to those of bacterial pneumonia [30]. The average time described for detection of secondary infections is 10 days. The most effective method was bronchoalveolar lavage cultures, but it is commonly avoided by the production of aerosols. Thus, sputum cultures and endotracheal aspirates are performed, although studies report that there is no increase in risks to health professionals when performing bronchoscopy [31]. Lymphocytopenia usually occurs within two weeks of the onset of COVID-19 and can result in immune dysregulation and immunodeficiency promoting secondary infections. The study by Chong et al. found a mean value of three to four days for the lowest lymphocyte levels and pointed out that the use of corticosteroids may have contributed to lymphocytopenia. Alnimir et al. found lymphocytopenia in 39 (57.4%) non-survivors. Wu et al. also pointed out that severe immunosuppression marked by low lymphocyte count is associated with mortality risk [28,32-34]. In addition, the administration of medications such as steroids, immunomodulatory agents, and antibiotics can also increase the occurrence of secondary hospital-acquired infections.

The use of corticosteroids to control local inflammation was mentioned and related to consequent immunosuppression in the studies by Gottesman et al. and Wu et al and Ceballos et al. In those studies, authors described that patients who used corticosteroids before admission to the hospital and were exposed to dexamethasone during hospitalization were associated with a higher risk of secondary infections than those who were not exposed in a bivariate analysis [25,34-37]. The Neutrophil/Lymphocyte Ratio (NLR) is a simple and low-cost marker of systemic inflammation, with its desirable values averaging below 2. It is obtained by dividing the absolute values of neutrophils by the absolute count of lymphocytes used in the prognostic evaluation of clinical conditions. NLR increases due to reduced lymphocyte count associated with increased neutrophil count. Levels above 2 suggest a reduction in lymphocyte count or a progressive increase in neutrophil count, which is a marker of worsening of the clinical condition of patients. The likelihood of bacterial co-infection is increased in patients with ≥ 15 NLR within 48 h of hospital admission [24,38-40]. Procalcitonin (PCT) is a peptide or prohormone precursor to calcitonin normally synthesized by the thyroid and, in case of bacterial infection, by other tissues. It is used to screen for bacterial infections and sepsis because it is a biomarker that is generally higher in bacterial infections compared to viral infections, in which the elevation is minimal, and is promising for differentiation.

Normal PCT levels are below 0.1 ng/mL and studies support that an increase above 0.25 ng/mL indicates elevated chances of bacterial infection of the respiratory tract. Increased procalcitonin may indicate bacterial co-infection in patients with COVID-19. The study by Carbonell et al. found that values of up to 0.3 ng/mL of PCT could help rule out bacterial co-infection on admission with a NPV (Negative Predictive Value) of 91.1%. This finding is also supported by the studies of Dolci et al. and May et al. who found the threshold to be up to 0.25 ng/mL with a NPV of 91.7% and 99%, respectively. The study by Alnimr et al. points out that PCT levels were higher at hospital admission among non-survivors compared to survivors 39,43,46. However, approximately 36% of patients with chronic kidney disease have an elevated PCT value of 0.5 ng/mL or above. Some studies also suggest the relationship between elevated PCT and elevated IL-6 release as a result of the cytokine storm caused by SARS-CoV-2, decreasing the discriminatory capacity of PCT. ARDS in patients with COVID-19 can also increase the levels of this marker, as well as clinical conditions of biliary pancretitis, fungal infections, lung carcinoma, among others. However, although PCT is not a reliable marker of the presence of bacterial co-infection, its dosage of up to 0.25 ng/mL is indicative of its absence and is an aid to avoid inappropriate treatment with antibiotics in patients infected with SARS-CoV-2 [38,41-43]. Among the advantages of using procalcitonin as an inflammatory marker, it can be also considered the absence of interference of its levels with the use of corticosteroids, rapid elevation after induction, high sensitivity, and simple dosing through a blood sample [38].

The use of the CURB-65 score to discern between intermediate- risk patients who may need early antibiotic administration to standardize treatment was suggested by Giannella et al. This marker have already been reported to be higher in patients with secondary infections than in those not infected at the time of hospital admission in the study by Na et al. In the study by Giannella et al., leukocyte counts, TSPs, and the Charlson index were also used to guide diagnostic tests and antibiotic use. It is recommended to use antibiotics in patients with CURB-65 >2 [40,44]. C-reactive protein (CRP) is an acute inflammatory protein synthesized mainly in the liver after IL-6 stimulation. It plays an important role in the response to infections and can stimulate the inflammatory process through mechanisms such as activation of complement life, induction of phagocytosis and apoptosis, regulation of nitric oxide synthesis, stimulation of inflammatory cytokine production, and leukocyte chemotaxis and recruitment. It is a nonspecific biochemical marker of the acute phase and has little specificity because it is elevated in several pathologies, and is not very useful for differentiating inflammation from infection. Among the factors that can cause changes in serum CRP levels are age, gender, weight, smoking, dyslipidemia, liver damage, and hypertension. The use of corticosteroids is associated with lower induction of CRP by Carbonell et al. and Schouten et al. while PCT remains unchanged, in addition to the slower elevation of CRP levels compared to PCT.

In healthy patients, the blood concentration of CRP is less than 5 mg/L. Its serum levels are directly associated with the severity of COVID-19 and worse clinical outcome. Patients with elevated CRP levels at the time of admission were associated with a higher risk of disease severity and of developing secondary pulmonary infections [31,38,41,43]. Several studies have demonstrated the correlation between CAP severity and PCT and CRP. In the study by Schouten et al., secondary increases in CRP and PCT were associated with superinfections such as COVID-19 complications, but the use of tocilizumab is associated with a reduction in these markers. On the other hand, a study by Kooistra et al. conducted with patients admitted to the Radboud University Medical Center in the Netherlands demonstrates data from 190 ICU patients who received treatment with immunomodulators such as dexamethasone and tocilizumab had their CRP levels suppressed even after a secondary infection, while these drugs did not affect PCT [41,43,45]. Presepsin serves as a bacterial lipopolysaccharide (LPS) receptor and is the soluble form of CD14, a glycoprotein expressed in monocytes and macrophages. It has been demonstrated as a tool to indicate severity in sepsis diagnoses because it rises early, within the first two hours after infection, surpassing PCT and CRP. Carbonell’s study points to presepsin as a non-specific biomarker for diagnosing pneumonia and its etiology, but several studies have described this marker as reliable for the severity of viral pneumonia and COVID-19 [41,45].

Possible Treatment Alternatives

The development of new therapeutic agents to prevent mortality of patients co-infected with SARS-CoV-2 and bacteria is necessary. Laboratory tests are the tools used during the clinical management of diagnosis and direction of antimicrobial treatment in order to reduce cases of bacterial resistance and prevent mortality 51. Serial PCT measurement provides follow-up of inflammatory dynamics, and can be used to shorten the duration of treatment after antibiotic administration begins. If PCT levels remain low at dosage after 24 and 48 hours from the start of antibiotic treatment in the uncertainty of co-infection, in that case, discontinuation is recommended. If bacterial co-infection has been proven or its presence is very likely, PCT reduced by 80% of the baseline value or below 0.5 μg/L after 48 hours or 72 hours, stopping antibiotic administration is reasonable [38]. Finally, phages are very target-specific, have low toxicity and resistance to them are rare, in addition to reducing the synthesis of reactive oxygen species (ROS), whose levels are elevated in COVID-19 patients [46,47].

Impact of the COVID-19 Pandemic on Bacterial Resistance

The increase in the use of biocidal agents, disorderly use of antibiotics and their empirical use, decreased surveillance of resistance due to the focus on COVID-19, and overcrowding of health systems are factors that may contribute to the increase in antimicrobial resistance. The systematic review study by Langford et al. found no association between COVID-19 and changes in antimicrobial resistance of gram-positive bacteria. However, a possible increase in resistance was found in gram-negative patients, suggesting an important role due to the high use of beta-lactams and third generation cephalosporins in patients with COVID-19 [48,49]. The study by Elton and colleagues at two hospitals in Africa observed an increase in the total number of HAIs caused by multidrug-resistant bacteria in COVID-19 patients. The lack of medical staff and personal protective equipment in some hospitals may have counteracted the possible effect of reducing the transmission of antimicrobial resistance caused by the factors of social distancing, improved hand hygiene, and the use of personal protective equipment. The same was pointed out by Serapide et al., in addition to the overcrowding of patients and failure to quickly isolation of patients colonized by multidrug-resistant bacteria upon admission to hospitals. Protection against COVID-19, such as the quarantine established in early 2020, have been identified as non medicament factors extremely useful in reducing the incidence of infections in the community by the respiratory bacteria S. pneumoniae, Haemophilus influenzae, and Neisseria meningitidis.

Mentions of the incidence of MRSA colonization, reduced by 65.2%, and minimally in resistance rates were related to organized responses to the COVID-19 pandemic, while overloaded environments are directly related to the opposite effect [48,50-52]. A study conducted in Serbia found a statistically relevant difference in carbapenem resistance when COVID-19 patients were compared with patients without COVID-19. Resistance to imipenem was 56.8% vs. 24.5% (p < 0.001), meropenem 61.1% vs. 24.3% (p < 0.001), and ertapenem 26.1% vs. 21.7% (p = 0.03). Another study conducted in Spain reported that patients with COVID-19 revealed an increased risk of nosocomial infection by carbapenemase-producing Enterobacteria, associated with a high risk of mortality and being severe. In Brazil, there was an increase in the incidence of carbapenemase-resistant A. baumannii correlated with COVID-19 both in the ICU and outside. A change in the sequencing of K. pneumoniae and its virulence type (from ST11 to ST15) from 2015 to 2020, which may be related to COVID-19 [48]. The study by Thomas et al. reported an emerging in carbapenemase-resistant Enterobacteriaceae isolates by 42.8% in Brazil, and an increase from 73.6% to 88.4% in the total number of carbapenemase genes detected. The emergence of new genes that have not been identified yet has been reported in Chile, Dominica and Belize, while Peru, Ecuador, Venezuela and Costa Rica have reported the emergence of new combinations of carbapenemase [53-58].

Conclusion

Infections caused by multidrug-resistant bacteria are related to the worsening prognosis and increased mortality in patients with COVID-19. The empirical and prophylactic use of antibiotics has been widely reported in patients with COVID-19 and is one of the main causes for acquired resistance. Studies indicate that PCT levels in SARS-CoV-2 infected patients of up to 0.25 ng/mL are indicative of the absence of bacterial co-infections and are a support to avoid inappropriate antibiotic treatment. In the pandemic scenario characterized by a high use of biocidal agents, empirical and disordered use of antibiotics, and decreased surveillance of resistance worldwide and overcrowding of health systems, lack of medical staff and personal protective equipment in some hospitals were factors that have been pointed out as contributing to the increase in antimicrobial resistance. On the other hand, protective strategies against COVID-19, such as quarantine, improved hand hygiene, and the use of personal protective equipment were identified as factors able to reduce the incidence of infections in the community. In some countries, increasing in the incidence of carbapenem-resistant bacteria and new genes and combinations have been reported, which may be related to the COVID-19 pandemic. More research in the field of therapeutics and alternatives management in cases of bacterial co-infection associated with COVID-19 and its possible markers is needed, as well as investigations of the possible impact of the pandemic on the global scenario of acquired multidrug resistance.

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Antihyperglycemic Effects of Gynura Procumbens: A Review of In Vivo Studies

Introduction

Diabetes mellitus (DM) prevalence is increasing all over the world. Based on data from the World Health Organization (WHO), 8.5% of the world’s population aged 18 years and older were diagnosed with DM in 2014 [1]. Indonesia is ranked as the seventh country with the highest DM prevalence, with a total of 10.7 million cases in 2019 [2]. Uncontrolled DM could lead to various complications, even death. In 2019, DM was attributed to 1.5 million deaths [2]. Pharmacological therapy is often needed to prevent DM complications. While pharmacological therapy remains the primary choice, some medications can cause unwanted adverse effects [3]. Herbal treatments have been recently proposed as an alternative option for DM patients [4]. One of the herbal plants with a potential antidiabetic effect is Gynura procumbens (Lour.) Merr. a herbal plant that grows in tropical Asian countries [5,6]. G. Procumbens has thick leaves and hardened stems with purple tint. G. Procumbens has been used to treat fever, skin rashes, infections from virus and ringworm [7]. G. procumbens has also been studied for its antihypertensive, anticancer, anti-inflammatory properties [8-10]. These properties are attributable by G. procumbens phenolic compounds such as quercetin, kaempferol, astragalin, chlorogenic acid, and rutin [11,12]. Present studies focused on G. Procumbens hypoglycemic effects using in vivo and in silico approaches [13-15]. Despite those promising results, its efficacy and mechanism of action need to be further elucidated to be evaluated in human. A comprehensive and systematic review of the existing literature is still lacking. It is important to evaluate available evidence to establish its efficacy, safety, and potential mechanism of G. procumbens for diabetic therapy. Therefore, this study aims to systematically summarize in vivo studies on the antihyperglycemic effect of G. procumbens and the potential mechanisms involved. 

Methods

Search Strategy

Literature search was conducted on 17 August 2023 from four electronic databases (PubMed, Scopus, ProQuest, and Portal Garuda) with the following terms: (Gynura procumbens) AND ((hyperglycemic) OR (glucose) OR (Type 2 Diabetes Mellitus) OR (T2DM) OR (Diabetes Mellitus) OR (DM)).

Eligibility Criteria

We included in vivo experimental studies using hyperglycemic animal models. There were no restrictions in animal models, method of induction, administration method, dosage, and duration of treatment. We also included full-text studies written in English and Indonesian. Studies without appropriate control or primary outcomes were excluded.

Data Extraction

The following information was extracted: authors, publication year, animal species, types of hyperglycemic-inducing agents, diabetic condition criteria, dosages of G. procumbens, duration of treatment, and measurement outcomes. The primary outcomes were blood glucose levels. Secondary outcomes were HbA1c levels, plasma insulin levels, insulin sensitivity, and glucose tolerance.

Quality Assessment of Studies

The quality of included studies was evaluated using the Collaborative Approach to Meta-Analysis and Review of Animal Data from Experimental Studies (CAMARADES). Three potential judgments for risk of bias were determined for each trial, low risk, unclear risk, and high risk.

Results

Characteristics of the Included Studies

A total of 485 studies were identified from four electronic databases. An additional study was identified through a citation search. Duplicate records and studies that did not meet the inclusion criteria were excluded. The full texts of 30 articles were retrieved and assessed for eligibility. Eight articles were excluded due to different study design. four studies were excluded due to inappropriate control and outcome measures. Three article was retracted by its publisher. As a result, 15 in vivo studies were included in this review. Table summarizes the characteristics of the included studies [12-26] (Table 1). Animal models used in these studies were all rodents. Rats and mice were commonly used in animal studies for the safety and effectiveness of novel compounds [27]. Most studies used male Sprague-Dawley or Wistar rats, while one used C57BL/KsJ-db/db mice [18], two used Swiss albino mice [17,19] and one used Swiss-Webster strained Mus musculus [26]. The Indonesian Food and Drug Authority (BPOM/Indonesian FDA) guidelines for preclinical pharmacodynamic studies required a minimum of five samples in each group [28]. One study did not report the sample size in each group [18]. Except for four studies [14,16,23,25], five or more samples were included in each group. Animal models were either chemically induced or genetically diabetic. One study used C57BL/KsJ-db/db mice as db/db mice is a monogenic model of obesity-induced type 2 DM with mutations in the gene encoding hypothalamic leptin receptors, which causes leptin resistance [18].

Table 1: Characteristics of the included studies.

In most studies, Streptozotocin (STZ) [12,14,15,20–22,24,25] (50-60 mg/kg BW) was used, while newer studies tend to use Alloxan [13,16,17,19,23,26] (100-150 mg/kg BW) as chemical induction for diabetes, as these two agents induced damage to pancreatic islets’ beta cells, resulting in insulin deficiency [29]. The Indonesian Food and Drug Authority (BPOM/Indonesian FDA) mentioned that normal blood glucose levels of rats and mice ranged from 62-175 mg/dL and 50-135 mg/dL, respectively, while blood glucose levels of 200-350 mg/dL were considered diabetic [28]. Except for two study [16,23], the diabetic state criteria used in these studies ranged from ≥200-300 mg/dL. Nath et al, define diabetic criteria as blood glucose increase of 3-4-fold [17]. One study diabetic state criterion is not stated. Almost all of the studies used G. procumbens leaf, except two studies did not mentioned what part of plant that they use. Ethanolic and aqueous extracts of G. procumbens were mostly used with ranging dosages (50-3000 mg/kg BW). Two studies used methanolic extract [15,17]. Nath et al, measure doses using basal diet percentage [17]. Some studies used multiple dosage regimens to assess the dose-dependent effect of extracts [12,14,15,19,20,22,24-26], and the remaining studies used single dosage regimens of extracts. Animal models were treated with G. procumbens extract for a duration ranging from seven hours to six weeks. Two articles studied the acute effects of G. procumbens on blood glucose levels [15, 24].

Glibenclamide (0.025-15 mg/kg BW) and metformin (1.6-500 mg/kg BW) were two antidiabetic medications mostly used as the reference standard to compare the effect of the extract, except for one study that used acarbose [21] and one other used basal diet [17]. Compounds examined in included studies are mostly flavonoids and phenols, while only one study investigating phytol’s [16]. Despite that, only half of the studies mentioned its specific compound. Most compounds in G. procumbens are quercetin, kaempferol, rutin, kaempferol-3-O, stigmasterol, and β-sitosterol [12,15,16,18,20,21,23,24].

Quality of Studies

Figures 1 & 2 show the quality assessment of the included studies using the modified CAMARADES tool. Overall, risk of bias of all 15 studies was low. Only 1 study states their sample calculation method. Quality of assessment of included studies are presented in Figures 1 & 2. Only 1 study states their sample calculation method. One third of included studied displays randomization methods. All of the studies use appropriate animal model. Only four studies were not conducted in controlled environment. Most of studies are done according to animal welfare regulation. All of the included studies were published in peer-reviewed publication. However, none of those studies reported blinded induction of hyperglycemia, and blinded outcomes assessment. Additionally, about half of the included studies did not state their conflict of interest.

Figure 1

Figure 2

Discussion

Of the 15 studies involved, all showed significant results from G. procumbens as an antidiabetic. These studies reported that administration of the leaf extract could reduce blood glucose levels in rats. In addition, reduction in cholesterol, TG, LDL, increased HDL were also reported by some studies. Quercetin is one of the most flavonoids found in G. procumbens. Quercetin works as an antioxidant that protects pancreatic beta cells from oxidative damage by inducing the activity of antioxidant enzymes such as superoxide dismutase (SOD), glutathione peroxidase (GPX), and catalase. In addition, quercetin also works as an antidiabetic by inhibiting the activation of phosphoinositol-3-kinase (PI-3K/Akt), inhibiting the glucose transporter GLUT 2 in the intestine, and reducing lipid peroxidase [29]. Guo et al. also reported the involvement of G. procumbens in the PI3K/Akt signalling pathway in T2DM [18]. Quercetin, other flavonoids, and glycosides of flavonoids also potentially increased glucose uptake in muscle tissue in STZ-induced mice [25]. The study of Algariri et al. stated that flavonoids and phenol are bioactive components that commonly cause pharmacological effects in a herbal plant, especially with their antioxidant properties[24]. The flavonoid content in G. procumbens can also control postprandial glucose levels by influencing the hydrophobic and hydrophilic properties of the alpha-amylase and alpha-glucosidase enzymes [21]. Whereas, phenol can postpone diabetes complication which is glycation [30]. Both carbohydrate metabolism enzymes work to increase the absorption of glucose in the small intestine.

As a result of alpha-amylase and alpha-glucosidase inhibition, carbohydrate absorption is reduced, resulting in decreased postprandial glucose levels. [21,26,31-33] Phytols which is contained in in G. procumbens may also ameliorate insulin resistance and reduced insulin signal transduction caused by TNF-α [16]. Other antidiabetic mechanisms of G. procumbens include increasing the activity of glucokinase, pyruvate dehydrogenase, and phosphorylation of ATP-citrate, which play a role in glucose regulation. Research also found the role of G. procumbens in increasing the activity of fructose-1,6-bisphosphatase, phosphofructokinase, and hepatic hexokinase [34]. Increase of insulin secretion has also been proposed by Tahsin et al as a significant elevation in serum insulin level of rats were seen in treatment group [13]. However, research by Hassan et al. reported that the aqueous extract of G. procumbens did not stimulate insulin secretion [12]. The study concluded that the hypoglycemic effect of G. procumbens stems from increased hepatic or peripheral glucose utilization but not as an insulinotropic [12]. The solvents used for extracting G. procumbens leaves in this study were water, ethanol, and methanol, which are polar solvents. Research conducted by Lee et al. used ethanol and water to extract G. procumbens. This study found that ethanol has a more significant effect than aqueous extract on diabetes parameters, one of which is fasting blood glucose. This study also concluded that the ethanolic extract of the leaves had an antidiabetic effect equivalent to that of metformin [25].

This finding probably occurs due to the low solubility of flavonoids in water [24]. From these findings, the different solvents used in the studies could affect the antidiabetic properties of G. procumbens. Three studies by Zhang et al., Algariri et al., and Tahsin et al., observed the toxicity evaluation of the G. procumbens leaf extract were reported to be safe. In the study by Zhang et al., acute toxicity was evaluated in two groups of BALB/c mice, where one group was given the extract at a dose of 1 g/kg, and the other group was given 5 g/kg. Both groups showed no signs of toxicity, such as restlessness, respiratory distress, seizures, or coma, and remained alive for seven days. There was also no alteration of P450 enzymes; hence it is unlikely to have pharmacokinetic interaction with other medicines [14]. The study of Algariri et al. assessed the acute and sub-chronic toxicity of the ethanolic extract of the leaves of G. procumbens in female rats. At a maximum dose of 2,000 mg/kg, no signs of toxicity were found after 14 days of observation. Extracts can be declared safe based on these tests because the LD50 exceeds 2000 mg/kg. There were also no signs of illness in the mice involved in the subacute test for 28 days, and all mice lived until the end of the observation period. The assessments were animal growth rate, liver function examination, kidney profile, and hematological analysis [22]. Tahsin et al. reported, 50 times greater than that of the medium dose (750 mg/kg) of G. procumbens did not cause lethality whereas all of rodents dies from 50-fold dose of metformin [13].

This systematic review reinforces the initiation of clinical trials. The included studies used a similar animal model, and almost all studies conform to the DM criteria set by BPOM. Moreover, this paper only involves studies with defined control which ensures the validity and reliability. However, this systematic review has several limitations. First, different range of doses, solvents, and duration of the study, which could affect the significance of the results. In addition, more results could be obtained from other Asian countries as our search strategy only included articles with English and Indonesian language.

Conclusion

In conclusion, based on our systematic review, G. procumbens leaf extract has sufficient evidence of efficacy and safety as an antihyperglycemic agent in animal research by increasing hepatic and peripheral glucose regulation and promoting insulin release. Further clinical trials on human subjects are needed to determine its efficacy, safety, and dosage of G. procumbens extract as an antidiabetic.

Acknowledgement

This article was presented at the 7th International Conference and Exhibition on Indonesian Medical Education and Research Institute (7th ICE on IMERI), Faculty of Medicine, Universitas Indonesia. We appreciate the exceptional support of the 7th ICE on the IMERI committee during the manuscript preparation and peer-review process. We also thank the Basic Herbal Medicine module held by the Department of Medical Pharmacy, Faculty of Medicine Universitas Indonesia, for the opportunity to conduct the project.

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Alterationss Serum Sodium Levels on Intravena Administrations of 20% Manitol and Hypertonic Sodium Lactate in Male Wistar Rats

Introduction

Mannitol, hypertonic saline and hypertonic sodium lactate has been in clinical for intracranial hypertension. Hyperosmoler theraphy has been use in neurosurgical patients to maintain circulation, prevent hypovolemia, hypervolemia, hypoosmoler condition, and hyperglycemia. Maintain the systemic pressure is very important in traumatic brain injury patients [1]. Mannitol is the most an osmotic diuretic recommendation as a first-line agent to reduce intracranial pressure for decades [2]. However, side effects of mannitol are electrolyte imbalances induced by slow elimination and accumulation of mannitol in serum [3]. Repeated administration of mannitol induced water loss through osmotic diuresis is associated with an increase in sodium levels. The retrospective study on 2006, the use of mannitol as a therapy for intracranial hypertension affected the incidence of hypernatremia (24.3%) [4]. The other study in 2010, using mannitol for patients with intracranial hypertension induced hypernatremia in 10% of the total patients on the first day and 10-21% on mannitol administration up to the seventh day [5].

In traumatic brain injury (TBI), hypernatremia is associated with length of Intensive Care Unit (ICU) stay and an increased risk of death. The previous study in 2008, mortality in TBI with hypernatremia reached 65% [6]. The higher level of sodium in patients with traumatic brain injury significantly affected in mortality [7]. The incidence of hypernatremia mostly showed in the first week after trauma and is associated with the use of hyperosmoleric fluids. as management of increasing ICP [8]. Lactate is known as a key intercellular or interorgan metabolite between glycolysis and oxidative phosphorylation that can be produced and used by the brain as an energy source under pathological conditions [9]. An experimental data on rat hippocampus shows that lactate is a better substrate of glucose and is able to significantly improve cognitive function deficits compared to 3% NaCl at 24 hours and 30 days after surgery on orientation, registration, attention, remembering and language values. High sodium content stimulate vagal respon to secreting Atrial Natriuretic Peptide (ANP) and affected to increase natriuresis to maintain blood pressure and decrease sodium or water levels [10,11]. In a study in mice in 2007, the use high concentrations sodium solution is associated with the activation of Transient Receptor Potential Vanilloid type 1 (TRPV1) which results in an increase in the process of natriuresis and diuresis [12].

The use of hypertonic sodium lactateassosiated with increase the Strong Ion Difference (SID) or pH (alkalosis) and prevent acidosis hyperchloremia condition [13]. Based on the high incidence intracranial hypertension, increase in sodium levels is associated with high mortality and morbidity because using mannitol and hypertonic sodium lactate appears as a new alternative therapy to reduce ICP. Lack of this solution is the absence of long-term studies to evaluate the alterations in sodium levels. Therefore, researchers try to compare alterations in sodium levels between mannitol and hypertonic sodium lactate in experimental animals as a first step. The experimental animals used in this study were male iistar rats.

Methods

Animal Groups and Protocols

This study was an experimental laboratory study with a simple randomized design. All experiments were approved by the Institutional Animal Care and Use Committee of Gajah Mada University (KE/FK/0958/EC/2019). 36 male Wistar rats weighing above 250g, were housed in the animal facility for 1 week before used in the experiments. During the adaptation period the mice are in adequate cages suitable for food, bedding, and equipment related to maintenance. Air exchange, temperature, humidity, noise, light intensity and the light cycle are maintained within limits according to the health and welfare of animals. Feeding uses the manufacturer’s standard feed (AD II) which meets the nutritional requirements of rat maintenance both macro and micronutrients.

Experimental Procedure

On the day of treatment the rats were divided into 2 groups (group A as treatment using mannitol, group B with hypertonic sodium lactate treatment). Rats were anesthetized with intraperitoneally ketamine 1mg/100grBB before IV line insertion with abocath number 27G in the rat tail vein then fixed with silkam 2.0. A rat given mannitol through intravenous infusion through the caudal vein as much as 0.5 mL/100gr. Rat B was given hypertonic sodium lactate as much as 0.15ml/100gr. Rats left back to the cage after the protocol. During the trial period, rats feeding was adjusted to the nutritional requirements of mouse rearing. There was no limit on intake volume in mice during the study. After 6 hours of fluid administration, blood drawn using venous blood through the sinus orbital in the eye as much as 0.5cc. Total blood sampling in rats was 2.5cc during the trial period. This is adjusted to the maximum blood loss limit of rats with a weight of 250gr is 15% of the total blood volume of the rat.

Measurement

Blood is sent to the UGM Clinical Pathology Laboratory for examination of sodium levels. This research was conducted on all mice for 5 days.

Statistical Analysis

Statistical processing and analysis was performed by comparing alterations sodium levels in the two groups. Normality test with Shapiro-Wilk for numerical scale data. Independent T-test is used to analyze data with normal distribution and Mann Whitney if not normally distributed. Researchers used General linear models (GLM) to assess the effect of giving both hyperosmolar solution in sodium levels alterations from day to day conducted for 5 days.

Results

There were no significant differences in the age of the rats and variations in sodium levels between the two groups with respect to the age of the experimental animals (Table 1). There were significant differences between the two groups on body weight before the study (p <0.05), but after analysis (Table 2) there was no significant effect of weight differences between the two groups in sodium levels alterations (p = 0.486). After administration of hyperosmolar solution in both groups, serum sodium levels variation in the mannitol group from the first day to the fifth day is 141-152 mEq/L, whereas in the hypertonic sodium lactate group is 139-156 mEq/L. Sodium levels alterations (∆) between days can be seen in Figure 1. The highest sodium levels alterations in the mannitol group occurred on the first day, where the level of serum sodium increase to 5.67±2.96 mEq/L. This situation was similar in the sodium lactate group (5.06±4.25 mEq/L). In this analysis (Table 2) also no significant difference was found between administration of hypertonic sodium lactate to sodium levels alterations compared with administration of mannitol (p> 0.05).

Table 1: Demographic data.

Note: *p<0,05 (Significant)

Table 2: Analysis of the relationship between the use of hyperosmoleric fluids (mannitol and hypertonic sodium lactate) and body weight on alterations in sodium levels.

Note: GLM test by Wilks’ Lambda value, where

1. There is no significant difference between body weight and alterations in sodium levels;

2. There was no significant difference between the mannitol and hypertonic sodium lactate groups on alterations in sodium levels from day to day; Δ = alterations sodium levels

Figure 1

The highest serum sodium level in mannitol was highest on the first day (148.17 ± 2.96), whereas hypertonic sodium lactate occurred on the fifth day (147.83 ± 3.35). The difference in mean serum sodium between mannitol and hypertonic sodium lactate varies considerably from the first day to the fifth day (Figure 2). Serum sodium levels in the hypertonic sodium lactate group were lower in the first 3 days, whereas mannitol was lower on the fifth day. From the statistical analysis using the GLM (Table 3) it was stated that the treatment (hyperosmolar solutions) in both groups would significantly increase the sodium level when compared with before giving (p <0.05). The significance only occurred when compared to before administration, but in an analysis there was no significant difference between the use of mannitol and hypertonic sodium lactate after treatment (first day to fifth day) (p> 0.05). In this analysis also found no significant difference between administration of hypertonic sodium lactate on sodium level compared with administration of mannitol (p>0.05).

Figure 2

Table 3: Analysis of the relationship between time and use of hyperosmolar solutions (mannitol and hypertonic- sodium lactate) on average sodium levels.

Note: GLM test by Wilks’ Lambda value, where

1. There was no significant difference between the mannitol and hypertonic sodium lactate groups on alterationss in sodium levels from day to day;

2. There is a significant difference between the days before and after administration of both hyperosmolar solutions (mannitol and hypertonic sodium lactate); *p<0,05 (significant)

Discussion

The results of this study found a significant difference between body weight in mannitol mice and hypertonic sodium lactate mice (p <0.05). This condition cannot be controlled by researchers because there is no difference in treatment between the two groups of mice. The administration of fluid doses in both groups was adjusted to the body weight of mice in grams. In the analysis using GLM significant weight differences in the two groups did not affect the results of the study. No significant difference was found between the effect of body weight on alterationss in serum sodium levels in the two study groups (p = 0.486), as a whole or when viewed from changes in sodium levels from day to day until the fifth day (p> 0.05). Evaluation of sodium levels in both groups before administration of hyperosmolar fluid showed the same mean sodium level. Both groups had a mean initial sodium level of 142.5 mEq / L. Looking at the data from this study (gender, age, and average sodium level), no significant differences in the characteristics of the sample between the mannitol and hypertonic sodium lactate groups. no significant effect of weight difference was found on alterationss in sodium levels after statistical analysis. From these characteristics it can be concluded that this study is worth comparing. The primary outcome in this study was the alterationss in sodium levels in the administration of hypertonic mannitol and sodium lactate in male wistar rats. From this study, there is no difference between administration of hypertonic sodium lactate and mannitol to changes in sodium levels for 5 days. At the beginning of administration of the two hyperosmolar solutions, both of them will cause an increase in sodium levels, but after that the sodium levels in both groups are relatively fixed. In this study the hypothesis of hypertonic sodium lactate administration has a lower effect on increasing sodium levels in wistar male rats compared with mannitol administration was not proven.

Be observed from the average serum sodium levels, the use of hypertonic sodium lactate and mannitol both increased the mean sodium in rats when compared to before the study. The results of this study showed a significant effect on mannitol administration (142.5±2.25 vs 148.17±2.96) on the first day and hypertonic sodium lactate administration (142.5±2.25 vs 147.56±4.25) compared to the day before treatment (p <0.05). This is consistent with the previous theory, where an increase in sodium levels is one of them influenced by the administration of hyperosmolar solutions (mannitol and hypertonic sodium lactate). From the first day to the fifth day, sodium levels varied in both groups (> 145mEq/L). hypertonic sodium lactate on the first day to the third day had a lower mean sodium concentration than mannitol, while on the fifth day the mannitol group was lower. This variation was considered not significant in statistical analysis. There was no difference between administration of hypertonic sodium lactate and mannitol to changes in mean sodium from the first day to the fifth day (p> 0.05). The other factors that influence the discrepancy of research results and hypotheses are factors that cannot be controlled and are not assessed by researchers so that this becomes a limitation in this study. Increased serum sodium levels in the hypertonic sodium lactate group can be caused by ANP secretion, regulation of osmolarity changes and stimulation of TRPV1.

ANP Secretion

In this study, there may be a disorder that inhibits ANP secretion so that it affects natriuresis and diuresis. In this study no volume status measurement was carried out. Measurement of volume status can be seen by assessing fluid intake and urine output. The situation was not assessed because of the difficulty of measuring the intake of volume and shape of the cage that was inadequate to maintain the condition of the urine does not evaporate. The effect of hemodynamic changes that can affect ANP secretion cannot also be assessed in this study due to the limitations of measuring devices for measuring hemodynamics in laboratory animals.

Osmolarity

Increased osmolarity will cause increased thirst response, ADH secretion and ANP secretion by carotid baroreceptors. Not achieving osmolarity in the use of hypertonic sodium lactate will cause no increased thirst response in mice and reduced ADH secretion so that water retention does not occur. The loss of water can cause disruption of sodium regulation. In future studies, consideration should be given to the assessment of changes in osmolarity.

Activation of TRPV1

Activation of TRPV1 is influenced by the use of sodium-based liquids with high concentrations, pathological conditions such as decreased pH, body temperature, and increased metabolic lipid concentrations. In this study these factors have not been excluded because they cannot be included in the study variables. If you view the results, both hypertonic sodium lactate and mannitol will significantly increase sodium levels compared before administration. Manifestations that can arise in elevated levels of sodium are restlessness, lethargic, hyperflexia and can lead to seizures, coma to death. Manifestations in hypernatremia did not appear in this study. So it can be stated that it is safe to be given to experimental animals for 5 days.

Conclusion and Suggestion

Conclusion

In this study, administration of hypertonic sodium lactate and mannitol significantly increase sodium levels compared before administration, but not followed by significant changes on the first day to the fifth day. There was no significant difference between hypertonic sodium lactate and mannitol to alterations in sodium levels over 5 days.

Suggestion

In this study, we have a significant weight difference between the mannitol group and hypertonic sodium lactate as a confounding factor in the study, therefore in subsequent studies an effort was needed to maintain homogeneity before the study was conducted. In the next research, it is necessary to evaluate other variables that affect the research, such as hemodynamics, nutrient intake and nutritional status, urine output and volume status, sampling place, rat weight evaluation after treatment or before treatment the next day to minimize confounding factors in the study. This research is quite safe when given to experimental animals, so it is recommended to continue the research on humans for more than 48 hours. This is because there is no significant difference between the use of hypertonic sodium lactate and mannitol to changes in sodium levels for 5 days in experimental animals.

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Bioactivities and Animal Clinical Studies of Fat-Soluble Carbon-60: A Minireview

Mini Review

Carbon, with the chemical symbol C and atomic number 6, is classified within group 14 of the periodic table [1]. Carbon atoms exhibit diverse bonding capabilities, resulting in the formation of various carbon allotropes. Well-known allotropes include graphite, diamond, amorphous carbon, and fullerenes. Carbon-60 (C-60), also called fullerene, comprises 60 carbon atoms arranged in a football-shaped structure, with a molecular weight of 720 (https://en.wikipedia.org/ wiki/Buckminsterfullerene). In 1996, three scientists were awarded the Nobel Prize in Chemistry for their pioneering discovery of C-60 [2-4](https://www.sesres.com/unlocking-the-mysteries-of-carbon- 60-discovery-and-implications/). C-60 is an inorganic form of carbon. However, inorganic forms of carbon exhibit limited biological activity and minimal clinical applications in humans. Research has shown the therapeutic potential of liposoluble C-60 in addressing inflammatory skin diseases, cancers, and intestinal diseases owing to its robust antioxidant properties [5]. C-60 holds promise for applications in the fields of cosmetics, medical devices, and medicine [6,7]. In 1992, the Journal of the American Chemical Society highlighted the potent free radical scavenging abilities of C-60 fullerene and first introduced the concept of the “free radical sponge.” Studies published in the journal Recent Patents on Biotechnology have demonstrated the robust scavenging ability of fullerene against reactive oxygen species and its remarkable potential as a biological antioxidant, advocating its utilization as an active ingredient in the preparation of cosmetic formulations [8].

Therefore, given its vital effects on skin health, hair follicle growth, and anti-aging, markets in Japan, the United States, and China have incorporated C-60 into cosmetic formulations to treat, prevent, and protect against skin damage [9-14]. Our pioneering patented research showed the efficacy of fat-soluble C-60 oil in alleviating itching caused by mosquito bites [15,16], suggesting its ability to effectively penetrate human skin. Studies by Russian researchers also suggest C-60 as a viable alternative for the treatment of allergic and inflammatory conditions [17]. Our previous study demonstrated the efficacy of orally administered fat-soluble C-60 oil in treating colitis in mice [18]. Relevant patents and additional studies have further supported the role of fat-soluble C-60 oil in colitis treatment in mice [19-22]. Fat-soluble C-60 is insoluble in water but soluble in edible oil at concentrations ranging from 1–4 g/L. Various studies have underscored the antioxidant and anti-inflammatory properties of fat-soluble C-60 [23-28]. Investigating the transmission pathways of fat-soluble C-60 in aqueous solutions or human bodily fluids constitutes an intriguing research avenue [29-36]. Our previous study elucidated that fat-soluble C-60 oil disperses in aqueous solutions, producing a potent antioxidant effect [5]. Fat-soluble C-60 oil exerts a more robust antioxidant effect compared to vitamin C in aqueous environments [5]. Similarly, even a small volume of fat-soluble C-60 oil effectively inhibits the proinflammatory effects of human neutrophils in cell culture media [5]. Conversely, fat-soluble vitamin E fails to produce antioxidant effects in aqueous solutions. These studies were the first to propose the hypothesis that fat-soluble C-60 could disperse in aqueous solutions or human bodily fluids [5].

Researchers at the Chinese Academy of Sciences discovered the efficacy of fat-soluble C-60 in treating central nervous system diseases [37], suggesting its direct entry into the central nervous system [5] and subsequent anti-inflammatory effects [37]. French scientists conducted related research, wherein they orally administered C-60 oil to mice once a week, thereby almost doubling their lifespan [38]. Our previous research yielded notable findings: oral administration of fat-soluble C-60 oil significantly reduces the levels of the inflammatory marker C-reactive protein in the blood of beagle dogs [5], suggesting entry into tissues and organs, thereby producing anti-inflammatory effects. C-reactive protein in the blood serves as a marker for cardiovascular and cerebrovascular inflammation and may promote such conditions [39]. These findings collectively suggest that oral administration of fat-soluble C-60 oil may offer therapeutic or preventive benefits for cardiovascular and cerebrovascular inflammatory diseases [5,39-41]. In summary, orally administered fat-soluble C-60 enters tissues and cells in the body, thereby lowering the inflammatory marker C-reactive protein levels and treating inflammatory diseases in humans. It is expected to serve as a therapeutic agent for skin inflammation, colitis, cardiovascular and cerebrovascular inflammation, as well as central nervous system inflammation. C-60 demonstrates potential as a versatile anti-inflammatory drug for managing systemic inflammatory diseases.

I focus my analysis on the offense of storage of illicit information, in which infringement is conditional upon on the offender intentionally storing the information, that is, the information is under the control of the offender.

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Health Care Tourism

Definition

Medical tourism is the process of traveling to a distant location to obtain general medical, dental, or cosmetic surgery at a higher value (quality divided by cost) than is commonly available in one’s own local system.

Types

Medical tourism can be sub-divided into domestic and international.

1. Domestic medical tourism refers to patients traveling within their own country to receive care at a ‘center of excellence’ (COE) – e.g., institutions with large patient volumes and documented quality outcomes (e.g., University of Pittsburgh Medical Center for transplantation), or those that enjoy this status via common public recognition (e.g., Mayo Clinic, Cleveland Clinic).

2. International medical tourism refers to traveling outside of one’s country (sometimes to an international COE) to obtain care at significantly reduced cost or (for those traveling to more modern countries) increased quality.

History of Medical Tourism

Medical tourism dates back thousands of years to when Greek pilgrims traveled from all over the Mediterranean to the small territory in the Saronic Gulf called Epidaurus. Epidaurus became the original travel destination for medical tourism. Spa towns and sanitarium were the form of early medical tourism. People traveled to these destinations for medical benefits. From the 18th century wealthy Europeans travelled to spas from Germany to the Nile. Early the 16th century, Europe became a destination for medical tourism, due to roman baths or spa. In the 1900, USA and Europe became medical centers of major interest, but only for rich persons who had the possibility to travel in order to take care of their health [1-7]. In the 80’s –90’s travels for aesthetic surgical procedures and dentistry appeared. These tourists were attracted by medical tourism especially due to low costs, taking into consideration that in their countries these services were not covered by insurance policies.

Why do Patients Go Abroad?

1. Affordable is probably the major reason and this is particularly true for patients from the well-off, developed countries like America and UK, where private health care is expensive, and some surgeries are not covered by their insurance.

2. Available is often because the medical treatment they need is not available in their local areas or not trusted by the patients, as is often the case with Omani patients.

3. Accessible applies more particularly to patients from countries where the waiting list is long, particularly to national health service patients in the UK and in Canada.

4. Acceptable applies to services, which may be affordable, available, and accessible, but they are not acceptable in the patient’s own country for religious, political reasons or other social reasons.

5. Additional refers to the availability of better care, perhaps better technology, or a better specialist, or simply better service and personalized care abroad compared to care in the home country.

6. Cost savings Using US costs across a variety of specialties and procedures as a benchmark, average range of savings for the most-traveled destinations:

India: 65-90%

Thailand: 50-75%

Mexico: 40-65%

Singapore: 25-40%

South Korea: 30-45%

Global Top Destinations for Medical Tourism

1. India

2. Thailand

3. Mexico

4. Israel

5. Malaysia

6. Singapore

7. South Korea

Top Specialties for Medical Travelers

1. Cosmetic surgery

2. Dentistry (general, restorative, cosmetic)

3. Cardiovascular (angioplasty, CABG, transplants)

4. Orthopedics (joint and spine; sports medicine)

5. Cancer (often high-acuity or last resort)

6. Reproductive (fertility, IVF, women’s health)

7. Weight loss (LAP-BAND, gastric bypass)

8. Scans, tests, health screenings and second opinions.

Risk Exposure to Medical Tourists

Three Broad Categories have Been Identified

1. Risk of travel

2. Risk post-operative procedure

3. Risk that might affect the health of patients during the procedure.

Risks of Travel

1. Psychologically and emotionally, being away from the closest people such as family, spouse, and parents may lead to acquired stress to medical tourist. Such health risk related to travel is due to the mental burden and particularly during the healing period abroad.

2. Infectious Diseases like COVID 19 which spread across borders.

3. More susceptible to deep vein thrombosis.

4. Furthermore, there might be a possibility of pulmonary embolism.

Risks-During the Procedure

1. Quality of care

2. Infection and cross-border spread of antimicrobial resistance and dangerous pathogens

Risks -Post Operative

1. Follow-up care: Many medical procedures require follow-up care after surgery. However, patients who have traveled abroad for medical procedures may have problems finding US physicians who are willing to provide follow up care after their return.

2. Malpractice and liability: In addition to quality of care and follow-up care, many patients are concerned about malpractice.

India as Top Destination

1. Low Cost

2. Availability Of Medical Experts

3. Availability Of Advanced Equipment’s

4. No Waiting Time

5. Specialty Treatment

SWOT Analysis of Medical Tourism in India

Strengths

1. Indian doctors are recognized as amongst the best at international levels; skillful, qualified, share information with patients and are readily available, whenever required.

2. High quality treatment in low cost.

3. Medical technology, equipment, facilities and infrastructure are at par with international standards.

4. Doctors and staff are good at English which makes it comfortable for tourists from English speaking countries.

5. Because of absence of racial discrimination, customers, especially from Africa, are comfortable in India

6. Education system provides 30,000 doctors and nurses each year which support the growing medical sector in India.

7. Foreigners are also attracted to Indian Systems of Medicine like Ayurveda, Yoga.

Weakness

1. Though the Cost of treatment is less in India, other costs like accommodation may prove to be inhibitive, especially for customers from low income economies.

2. Maximum medical tourist are from non-English speaking parts of the world which highlights the need for training of linguists for example specialists of Arabic.

3. Accreditation

Opportunities

1. Cost of medical treatment in developed western world remaining high, provides Indian medical tourism sector with a unique opportunity.

2. Patients from third world countries, where comparable quality medical care is not available, seek treatment outside their home countries. They compare western service providers with Indian service providers and find Indian medical care cost effective.

3. Employers in US are looking for ways to decrease their employes medical expenses providing appropriate health coverage concurrently. Employers will look for low cost care in India and other Asian countries.

4. Insurance companies in western countries are offering full cover and care in home country at a higher premium payment. Insurance companies are offering packages where customers can choose a lower premium but will have to get them treated at hospitals with comparable quality outside the country, with which they have tie-ups. Indian accredited hospitals can choose to compete for a share of this segment.

5. The medical care facilities in other South Asian countries are also not up to the mark. Patients from these countries find good quality care in neighborhood, where travel time as well as the cultural divide is less.

Threats

1. Cost to the local population-non-availability of services.

2. High competition from other Asian countries like Singapore, Thailand, Malaysia etc.

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Questions for Proponents of Bekesy’s Theory

Questions About the Traveling Wave Theory

1. How the resonance of a longitudinal sound wave in the fluid of the atrial canal, acting in the frontal plane with transverse waves of the basilar membrane in the sagittal plane, works? Is it possible to transmit 100% of energy when a 100 Hz sound wave has a wavelength of 14,500 mm and the length of the basilar membrane is 35 mm in humans, but in small mammals and birds – 2-5 mm [1]? Moreover, a pigeon can hear loud sounds from 3 Hz upwards, and normal sounds from 5 Hz [2]. How does the resonance of wave with the basilar membrane come about? Another problem is that some mammals perceive frequencies up to 100 kHz [2]. There is a lack of compatibility with own vibrations of the basilar membrane. If excited wave is suppressed, the amplitude of the excited wave is smaller. High frequencies are attenuated to a greater extent. The vibrations of the basilar membrane are strongly attenuated by the massive organ of Corti, which is intimately attached to the basilar membrane. Even if a resonance of a longitudinal wave with a transverse wave occurs, which is doubtful, there is no possibility of precise transmission.

2. It is assumed that own vibrations of the basilar membrane in humans range from 16 Hz to 20,000 Hz. Vibrations were determined for a very thin strip of connective tissue of the basilar membrane. Not included is the organ of Corti, which lies on the basilar membrane, that cannot vibrate on its own, without the organ of Corti, which has a much larger mass. The reception of frequencies up to 100 kHz by mammals having the same hearing organ is not considered. Moreover, the 2-5 mm long basilar membranes of small mammals and birds also receive all frequencies. It is difficult to imagine a wave traveling on basilar membrane of an owl hearing sounds at a level of -10 dB [2], when the input wave amplitude is 0.001 nm and in the cochlea the wave amplitude decreases by about 100-200 times [3]. A wave 10 times smaller than the diameter of a hydrogen atom can’t produce a traveling wave on the basilar membrane. Such a sound wave fading in the cochlear canal is not able to move the cochlear fluid by acting through the organ of Corti located on the basilar membrane. A 0.001nm wave fading about 100 times on its way to the cap can’t tilt or bend hairs of hair cells, 4000 nm long and 200 nm in diameter. Besides, studies on the natural vibrations of human tissues have shown that the results range between 2 Hz and 100 Hz [4].

3. Bekesy’s theory relates frequency resolution to the resonance of the sound wave with the basilar membrane. When the frequency of the sound wave and own vibrations of the basilar membrane match, the resulting wave peak transmits wave energy encoding auditory information [5]. Is it possible that the natural vibrations of the basilar membranes in mammals reach 100 kHz? Where on the basilar membrane does the peak of the wave form when the wavelength is many times greater than the length of the basilar membrane?[6] How does the wave on the basilar membrane originate and travel in the case of polytones? The frequency is recognized with cochlear implantation due to partial deafness, when the basilar membrane is immobilized. Frequency resolution is related to tonotopy.

4. The vibrations of the basilar membrane are supposed to move the fluid of the cochlear duct according to the frequency of the sound wave [7]. The wave energy is conducted through the elastic structure of the organ of Corti, which has damping properties. The fluid has mass, is in constant motion, has positive and negative acceleration in accordance with the wave frequency. A non-inertial system is formed with the possibility of inertia action. As the frequency increases, the acceleration in wave motion increases rapidly. The attenuation of the sound wave increases.

5. The energy of the sound wave is quantized, which makes precise transmission possible [8]. Does the tilting or bending of the hairs of the hair cells take place in a staggered manner? Or continuously, which interferes with the transmission of information. A similar problem relates to cadherin filaments connecting adjacent hairs of hair cells. Their tension and release of tension, which determines the regulation of the gating mechanism of mechano-dependent potassium channels, does not occur in steps.

6. The contraction of the OHC after depolarization of the OHC is supposed to be responsible for the amplification of quiet sounds by 40 dB [9]. Quiet sounds below the auditory threshold cannot be amplified because they are unable to excite the OHC – so there is no OHC contraction. Sounds above the threshold are received and the signal is transmitted via the afferent pathway to the CNS. Outer hair cells have afferent innervation and the signal can be transmitted to the brain. Traveling wave theory holds that OHCs serve solely as amplifier of quiet tones. The following question arises: For what purpose do OHCs possess afferent innervation if they do not use it? If we assume that the contraction of the OHC amplifies the quiet tones by OHC contraction, then the question arises: What waves does it amplify? After the 0.2 ms needed to amplify wave, there is already another wave on the basilar membrane. In addition, in the case of polytones, when we hear loud and quiet tones at the same time, the loud tones are received and information is sent to the brain. In contrast, quiet tones are separated and routed through the time-consuming signal amplification pathway of OHC contractions. But those waves that have been separated from the loud ones can’t be amplified, because the pull of the basilar membrane disrupts the new traveling wave existing on the basilar membrane at that time. Such splitting of a polytone wave is unacceptable. There is an intracellular signal amplification at the molecular level, not interfering with the reception of polytones.

7. Traveling wave theory assumes that the auditory signal pathway leads from the middle ear through cochlear fluids, basilar membrane, then through cochlear fluid, tilting or bending of hairs of hair cells, and tip-links mechanism [10]. The transmission of energy is in accordance with the frequency and amplitude of the sound wave. Pulling cadherin “strings” acts on the mechanism responsible for gating mechano-dependent potassium ion channels. Myosins, which do not have the ability to operate the channel opening and closing to 200 kHz, are supposed to be involved in the regulation of the closing of ion channels. This described pathway is energy-consuming, time-consuming, and highly prone to distortion of the conducted information.

Final Question

If there are so many theoretical and practical problems with the accurate transmission of information via the route adopted by the traveling wave theory, the possibility of a pathway that is fast, simple, energy-saving and ensures precision in the transmission of auditory information should be considered. This path leads from the middle ear through the bony casing of the cochlea directly to the hearing receptor. Sound waves are transmitted from the eardrum, incus and plate of the stapes to the bony casing of the cochlea. Bone conducts sound waves at a speed of approximately 4000 m/s. It is very close from the crista spinalis to the hair cells. The sound wave carries the quantized mechanical energy to the receptor of the auditory cell. The difference in the portions of energy contained in a sound wave must be a multiple of 1 quantum of energy.

New Theory

The new, sub molecular theory of hearing differs significantly from the traveling wave theory in several points. The basis of the new theory of hearing is the reception and transmission of quantized energy encoded in a sound wave to the centers of the brain while maintaining proportions, regardless of molecular changes at the atomic and electronic levels along the way. The adequate stimulus for the hearing organ is the mechanical energy of the sound wave, previously properly formed, which reaches a specific receptor in the form of molecules sensitive to the energy of the sound wave, called sound-sensitive molecules. These molecules have their own basic energy, which depends on several variable factors [11]. The length of their atomic bonds is constantly lengthened and shortened due to the normal atom vibrations. Atoms reach their maximum deflections at the same time. They perform constant oscillatory and rotational vibrations and vibrations around their equilibrium positions. The vibration frequency is between 1013 Hz and 1014 Hz. The vibration period is on average 1012 s. The oscillation deflections are 10% of the bond length. For the hydrogen molecule H-2, it is about 0.1 Å. Each molecule has its own basic energy. The mechanical energy of the sound wave causes a change in the potential energy of the receptor, rotational excitation, a change in bond length, a change in covalent and torsion angles, a change in the total energy of the molecule, and finally a change in the conformation of the receptor molecule, which transfers energy to the next molecule [8].

The kinetic energy of the molecule is associated with motion. In addition to kinetic energy, the molecule has potential energy, which is represented by chemical bonds and forces of electrostatic attraction and electromagnetic interactions. The sum of these energies creates the internal energy of the molecule’s body. The internal energy still depends on the temperature and on the mass of the molecule. The transmission of the sound wave external energy to the receptor molecule causes the internal energy of the receptor to increase at a given moment. Particles transfer energy to the next particle through the energy of molecular collisions and the merging of the electron clouds of these particles. Each atom has electrons that form an electron cloud around the nucleus of the atom. The size of this cloud depends on the number of orbitals in which the electrons are distributed. If an atom in a molecule receives energy from another atom, molecule or sound wave, the electron jumps to an orbital closer to the nucleus – its internal energy increases – in a quantized jump. The so-called excited state of the atom is formed, which, unlike the ground state, is unstable. Such state is unstable and there is an instantaneous attempt to return to the ground state by emitting 1 photon of energy — when 1 orbital transition of an atom is concerned. If, in the transfer of information to the next molecule, we have an innumerable number of such transitions, or at least 2 orbital transitions, there are 1020 possibilities of transmitting different types of quantized energy.

This provides an endless amount and variety of transmitted auditory information. The molecule that received the information from the sound wave has an increased total energy. According to the law of entropy, there is a tendency to return to the lowest free energy state possible the obtained energy, which is in excess, is transferred to the adjacent molecule, which changes its shape through its conformational changes. These physical changes of the molecule are the driving force for the mechanism regulating the openness of the mechano- dependent potassium channel of the hair cell membrane. The openness of the potassium channel from 0.3-1.0 nm at the frequency of the sound wave allows for the flow of 6 million/s of potassium ions from the endolymph to the interior of the hair cell. The influx of positive ions into the negatively charged cell interior initiates the depolarization of the hair cell. A change in the cell membrane potential of approximately 5-10 mV activates voltage-gated calcium and sodium ion channels. Cell depolarization increases rapidly. Once the equilibrium for the sodium ions is achieved, the sodium channels close and the repolarization of the hair cell begins. The influx of calcium during depolarization causes the release of calcium from intracellular stores – the endoplasmic reticulum of mitochondria and the nucleus. Calcium binds with proteins whose activity is dependent on calcium.

A typical protein that increases its activity many times over is Calmodulin, which, together with other mechanisms, plays an important role in the intracellular amplification of the signal received, but too weak to reach the brain. In the hair cell, processes related to cell life rapidly accelerate. But in the hair cell, the most important are the processes associated with the reception and processing of auditory information. The so-called second relays, molecular motors, are produced, a transmitter is produced, transported from the Golgi apparatus to the cell membrane in the vicinity of the synapses and secreted into the synapse. At the synapse, a postsynaptic excitatory potential is created and conducted to the spiral ganglion nerve cells, where, after integration with other dendrites, the signal is encoded and transmitted via the auditory nerve as an action potential to the brain [12].

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Methodological Approaches to Contrasting Sections for Electron Microscopy

Mini Review

Tissue sections, even fixed in solutions of O₃O₄ or KMnO₄, i.e. in solutions that simultaneously serve as both fixative and contrasting agent, are usually insufficiently contrasted. Greater contrast can be achieved by selectively increasing the density of the structures. High molecular weight contrast agents, preferably heavy metals and their compounds, are suitable for this purpose. However, organic compounds are also suitable if they are bound by the tissue in sufficiently large amounts per unit volume (e.g. the azo dye hexazonium-pararararosaniline). When contrasting biological material, the increase in electron density is determined by the total number of atoms or molecules of the contrast agent captured by the tissue. Thus, the final electron density depends on the mass of molecules (atoms) of the contrasting substance, on the number of binding groups in the tissue and on the number of molecules (atoms) of the substance captured by the tissue. It follows, in particular, that the contrast intensity is inversely dependent on the thickness of the slice [1,2].In the process of contrasting there is often an extraction of various tissue components, which may be accompanied by both contrast enhancement and, on the contrary, its partial decrease [3].Elements used for contrast include: lead, uranium, tungsten, osmium, manganese, as well as iodine, iron, mercury, barium, vanadium, chromium, gold, silver, nickel, lanthanum, ruthenium, platinum, thallium, indium, bismuth, strontium, and thorium. In this manual, we will focus on only a few commonly used contrast agents that can be used in conjunction with numerous histochemical procedures. In addition, methods for selective contrasting of specific structures also deserve mention. Heavy metal salts with selective contrasting action will be discussed below in the relevant section.

Lead Contrasting

The remarkable qualities of lead salts as a contrasting agent have long been recognized. At present, according to Watson, lead salts are used mainly in alkaline solutions. The use of lead salts as a contrast agent is complicated by the formation of insoluble lead carbonate and other undesirable precipitates [4]. To overcome this disadvantage, a number of modifications of the method have been proposed, among which the Reynolds and Karnovsky modifications are the most popular. Although both methods give a fairly stable solution of lead salt, attention should still be paid to the purity of the reagents used: they should be fresh and free of CO₂ admixture (NaOH granules without film, fresh distilled water). The contrasting effect depends on the choice of the preceding fixation. The different methods of contrasting in alkaline medium seem to be based on the same mechanism, as they all give similar results. According to Reynolds, divalent lead salts in an alkaline medium (pH=12) form so called “basic” salts:

The connection of divalent cations (with two lead atoms) with phosphate, carboxyl and sulfhydryl groups, as well as with negative charges of osmium oxide, is apparently carried out through ionic bonds or through hydrogen bridges, as is probably the case with glycogen [5]. Lead is poisonous! Inhalation of its vapours and ingestion through the mouth and skin should be avoided!

Uranyl Acetate Contrasting

The uranyl cation binds to phosphate and carboxyl groups. Uranil acetate UO₂(CH3COO)₂ (uranyl nitrate or double salts are used less frequently) is used in aqueous or alkaline solutions after fixation, during dehydration or when contrasting sections. In 20 min uranyl cation from aqueous solution is able to penetrate into the tissue filled in the eponge to a depth greater than the thickness of the ultrathin slice. In solutions of lead salts the diffusion rate is even higher [2,6]. Zobel and Veer found that at pH=3.5, 7 times more uranyl acetate binds to DNA than to bovine serum albumin [7]. At pH=5, the ability of both of these substances to bind to uranyl acetate increases and the difference in contrastability decreases. The effect of differential contrasting can also be observed at different concentrations of the contrasting agent. Thus, DNA is contrasted already at uranyl acetate concentration equal to 10-5M, while myosin is contrasted only at concentrations above 10-3M. The contrastability of lyophilised tissue with worse structure preservation was much better.

Double Contrasting

Significant contrast enhancement can be achieved by double contrasting ultrathin sections first with uranyl acetate and then with lead according to Reynolds or Karnovsky. Double contrasting using solutions of lead salts on slices or simultaneously on tissue pieces also gives good results [8]. In tissues fixed in aldehyde, contrastability with uranyl salts and/or lead hydroxide can be improved by treating the material with alcoholic solution of dinitrofluorobenzene at pH=8. The contrastability of the fabric with potassium permanganate or phosphorus-tungstic acid does not change.

Phosphorus-Tungstic Acid (PTA) Contrasting

General contrasting by phosphorus-tungstic acid H₃P(W₃O₁₀)₄ (mol. weight 2880) is carried out due to its interaction with proteins, the electron density of which can almost double. Proteolysis impairs the contrastability of phosphorus-tungstic acid. Like uranyl acetate, phosphorus-tungstic acid is used in aqueous solutions as well as in alcohol or acetone solutions to contrast both tissue pieces and slices. Because phosphoric tungstic acid strongly extracts some tissue components, it is best used mainly for contrasting slices, and its effect on tissue should not be prolonged [9]. The contrast results depend on the pretreatment of the tissue and on the pH of the phosphorus-tungstic acid solution. After fixation in osmium tetra oxide, the affinity of tissue to phosphorus-tungstic acid is partially masked, but it can be brought back to the initial level by exposure to oxidants (2% H₂0₂, 30-60 min) or longer treatment in phosphorus-tungstic acid. Material fixed in KMnO₄ is not suitable at all [10]. Phosphorus-molybdenum acid is also suitable for contrasting, but it is weaker in this respect than phosphorus-tungstic acid.

Potassium Permanganate Contrasting

With KMnO₄ it is possible to obtain a strong contrast in slices, which can be further enhanced by subsequent contrasting with lead. Contrasting in slices is easier, but it involves, as in the case of fixation in KMnO₄, a marked extraction of substances from the tissue and may lead to the disappearance of ribosomes and rupture of membranes [1]. In material treated with an aqueous solution of strontium permanganate, contrast is observed only in cut structures into which the contrasting agent can diffuse from the cut surface. Apparently, even a thin layer of resin makes it difficult to contrast with permanganate.

OTO-Contrasting

Zeligman et al. described a new imaging principle with general application for both light and electron microscopy. According to this method, metallophilic reagents are introduced into the tissue, the localisation of which is then revealed on electron micrographs due to the subsequent capture of metal ions during the contrasting process. Reagents with two or more functional groups are suitable for simple contrasting. They react primarily with osmium, which is bound in the process of fixation, but, in addition, retain the ability to form additional bonds with the same or with another metal. The most active ligands were thiocarbohydrazide H₂NNHCSNHNH₂ and carbohydrazide. The first of them is recommended for general contrasting by the O_SO₄– thio carbohydrazide – O_SO₄ method (OTO-method) [4]. In such contrasting, a pigment, the so-called “osmium black”, is formed, which is considered as a copolymer. In model experiments after fixation in O_SO₄, lipids are most intensively blackened; proteins and nucleic acids react much weaker, and starch does not react at all. Unlike other methods, the intensity of contrasting by the OTO-method corresponds to the true osmiophilia of the tissue. Membranes are sharply delineated and appear thinner, apparently due to the strong contrast of the inner lipid layer. If contrast is found to be insufficient, the procedure can be repeated. This procedure, carried out according to the scheme: metal-thio carbohydrazide- O_SO₄ or metal-thiocarbohydrazide-metal (MTO- or MTM-reaction), can also be used to detect other cations. Pd, Os, U, Pb, Hg, Fe, Cr, Cu, Ca, Zn, Sn give positive reactions [2]. Magnesium and aluminium salts give a negative reaction. Similarly, the metals in the histochemical reaction product can be used to enhance contrast.

Prolonged Osmosis

After conventional fixation in OSO4, intensive impregnation of individual structures can be achieved by longer treatment in an unbuffered solution of osmium tetraoxide at elevated temperature. With the help of such osmation it was possible to demonstrate the directionality of chemical synthesis processes in the Golgi apparatus. In cells of the cortical layer of the adrenal gland (rat), substances in the endoplasmic network and in mitochondria are contrasted. In addition, the outer segments of retinal bacilli of amphibians and gastropods and the outer segments of photosensitive cells of the Rana pineal organ are osmised, apparently due to photopigment. Fat droplets are not contrasted [11]. Osmation of material fixed in glutaraldehyde or in glutaraldehyde with acrolein gives the same results as fixation in 0S04. Blocking the aldehydes with dimedone has no effect on the reaction results. Pretreatment with mercuric (II) chloride releases additional reactive groups [12]. The chemical affinities of the substances that can undergo osmation have not yet been identified. However, some lipids are thought to be involved in the reaction, although cholesterol and neutral fats and possibly aldehydes do not seem to play a role. Perhaps the reason should be sought in the peculiarities of solubility of lamellae of the outer segments of the rod (but not cones) of the frog retina, as well as in the data obtained with the 0_S0₄-zinc iodide reaction on sensitive cells of the rat retina [8].

Reaction 0_s0₄ – Zinc Iodide

Incubation of fresh tissue in medium with 0_S0₄ and ZnI₂ gives intensive contrasting of different cell components: Golgi apparatus, lysosomes, contents of perinuclear space, granular endoplasmic network, mitochondrial matrix, cytoplasm in nerve and glial cells, synaptic vesicles, granules in cells of islets of Langerhans, keratohyalin granules in epidermis, as well as granules in eosinophilic leukocytes, sensitive cells of retina, cells of the brain layer of adrenal gland, etc [13]. A positive reaction is only observed in certain cells, but even in these cells identical structures can react differently. Little is yet known about the substances involved in this reaction. On the grounds that a negative reaction was obtained after lipid extraction, the lipids or lipoproteins which appear to acquire the ability to react with 0_S0₄ as a result of exposure to zinc iodide are thought to be responsible for the development of the colouring. At one time it was assumed that this staining was selective for cholinergic nerve endings, but later it turned out that synaptic vesicles in adrenergic fibres can also stain. On the basis of the available data, it can be assumed that this method is based on the involvement in the reaction of a certain grouping in lipids or in lipid-containing compounds. Any histochemical conclusions based on this reaction are not yet possible [6,14]. However, for contrasting certain structures on a purely empirical basis, the 0_S0₄-zinc iodide reaction may prove useful.The method of contrasting with bismuth-iodine complex is similar to this method.

Silver Impregnation

Numerous attempts have been made to modify the various silvering methods used in light microscopy: after all, the presence of metallic silver deposits at the reaction site is a good prerequisite for electron microscopic studies. Quite good impregnation is achieved by treating ultrathin sections of tissue fixed in 0_S0₄ with a solution of methenamine-silver or borax-AgNO₃. In such treatment, cell nuclei, mitochondria, cell membranes, as well as mucus, glycogen, collagen fibres and other structures are exposed to silvering. When treated with the same silver solutions of tissues fixed in acrolein or glutaraldehyde, only chromatin and ribosomes, as well as granules of eosinophilic leukocytes (especially their inner part) and granules of enterochromaffin cells are intensively impregnated; reticular and collagen fibres are impregnated somewhat weaker; mitochondria and nuclei in this case are usually free of silver deposits [15-18]. After fixation in formalin the impregnation is much weaker; a marked contrast is observed only in structures having a pronounced reducing power. There is a method of selective detection of nuclei. When treated with silver nitrate solution in vivo, intensive silver deposition is observed in connective tissue, although not in the cells themselves.The available data indicate that the results depend significantly on the pretreatment of the tissue and the choice of silvering method. Therefore, the specificity of the silver impregnation method is low [9]. The reduction of silver to free metal is mainly due to aldehydes, sulfhydryl groups, histones (i.e. arginine and histidine-rich proteins), and unsaturated lipids that bind lower oxides of osmium. Interpretation of the results is often difficult.

Nevertheless, if certain conditions are met, silver impregnation often allows important histochemical conclusions to be drawn.For general contrasting purposes, silvering is unlikely to be suitable due to the fact that silver deposits precipitate as granules. However, for the detection of morphological details or specific cells and to establish their spatial location, this method may be useful. Examples include the silvering of connective tissue fibres, mitochondria and various structures in nervous tissue by the Nauta or Golgi method, and the silvering of the cell wall in endothelium. These are all entirely empirical methods, and little is known for certain about their mechanisms. Histochemical interpretation of these data is therefore very difficult. Valverde believes that the Golgi method is based on the formation of a lipoprotein-chromium-silver compound by the catalytic action of 0_S0₄. When the tissue is simultaneously exposed to osmium tetraoxide and some other oxidising agent (e.g. potassium bichromate), the mechanism of action of the former is reversed; the reduced osmium is immediately reoxidised to 0_S0₄ [12]. In addition, those numerous compounds that 0_S0₄ under normal conditions oxidises, in a mixture of osmium tetraoxide with the second oxidant do not react. In Golgi staining 0_S0₄ reacts with ethylene groups of lipids. Under the action of bichromate, however, these bonds are broken again. Then bichromate oxidises also the released two OH-groups of ketone bodies, whose compounds with chromium react with silver nitrate, which leads to blackening of the tissue.

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SAR Reduction Method of PIFA Antenna with Metal Frame

Introduction

The description of SAR, the radiation problem of mobile phone has attracted more and more attention. In order to analyze the influence of electromagnetic radiation on human body more effectively, people put forward the concept of specific absorption rate (SAR) to measure the radiation intensity of electromagnetic wave [1], which is defined as the electromagnetic power absorbed or consumed per unit mass of organism, in W/kg:

We normally use the SAR simulation Software, Microwave studio CST as following Figure 1. About the SAR reduction methods of mobile antenna, there is many studies in recent decays, such as in 2003, Minseok lung, Bomson Lee mentioned use EM absorption material to reduce SAR [2,3]. In 2012, Sungtek Kahng; Kyungseok Kahng presented a metamaterial-inspired handset antenna with the SAR reduction [4]. In 2016, M. Haridim presented to Use of Rod Reflflectors to reduce the SAR [5]. In 2021 , Vidya R. Keshwani mentioned textile wearable antenna used in various applications need novel designs to achieve objectives of compact antenna with lowest possible Specific Absorption Rate (SAR).One method used to reduce SAR using EBG structure are briefly described. ISM 5.8 GHz band antenna is designed and modeled in Ansys High Frequency Structure Simulator (HFSS). SAR values with this antenna are computed in human body model. The effectiveness of EBG array structure to reduce SAR to acceptable values is demonstrated by simulations [6,7]. In 2022, Harri Varheenmaa;Pasi Ylä-Oijala presented SAR Reduction with Antenna Cluster Technique [8].

Figure 1

In this work, A new mobile antenna SAR reduction method mentioned in this paper by Ground metal frame resonator. In theory the display frame decreases SAR values when it is grounded, due to the opposite current phases in PWB and in the display frame. To achieve this, the length and the height of display to proportion to length of the PWB and the wavelength are the critical variables. In this case the purpose was to decrease the SAR values of the lower band. Variables and the characteristics were naturally chosen for lower band. The used head model was simplified to ball, due to the decrease in the simulation time. The diameter of the phantom head was 18cm, including skull with thickness 6mm and inside of skull the “head part”. Conductivity, permittivity and values were same than defined on the standard, density value was found from IEEE article.

Simulation Section

Simulated Construction

The structure is simplified version of the real model. If the structure is complicated it can cause problems in the simulation and increase the simulation time. So we use the simulated Structure as Figure 2. The metal parts were modeled as a 0.15mm thick copper. The pins of the antenna are made from the same metal part as the radiator. The frame and chassis has modeled as low loss plastic with a permittivity of 2.5 and loss tangent 0.005. The antenna radiator pattern is close to the pattern. The size of the ground plane is 40mm x 99mmx0.85mm. The size of the antenna was approximately 36mm x 16mm. Simulated phone structure is Figure 3.

Figure 2

Figure 3

Display Frame Modifications

In simulation the effect length of the display frame to SAR value was first investigated. Second the position and the length of the ground pins. Third the best results of these two simulations were simulated together. Because of the tight schedule it was impossible to do all the simulation f.e for length of display with 0.5 steps. Simulation was tried to do also by respecting the mechanical issues. The Results, which were found to decrease SAR value remarkably, are also included for additional information. In the initial case the dimensions of the display were the same as presented in Figure 4.

Figure 4

Length of the Display

The length of the display frame was decreased and increased first with 0.5 steps, then with 1mm steps as Figure 5.

Figure 5

Length of the Ground Pin

In the second case, as presented in Figures 6-8, only the length of the ground pins was changed. The height of the pin and also height of the display frame was constant. Also, the length of the display was constant, 30.5mm. Length 4.5mm for ground pin was chosen concerning mechanical issues.

Figure 6

Figure 7

Figure 8

Position of the Ground Pin

In the third case, as presented in Figures 9-14, only position of the ground pins was changed. The length of the ground pin was 10mm and length of the display frame 30.5mm. The height of the pin and also height of the display frame was constant.

Figure 9

Figure 10

Figure 11

Figure 12

Figure 13

Figure 14

Simulation Results

Results for the Simulations of Length of the Display Frame in Figures 15-17 [9-11]

Results for the Simulations of Length of the Ground Pin in Figures 18-19

Results for the Simulations of Position of the Ground Pin in Figures 20-21

Simulation Results of the Best Possible Combination in Figures 22-23 [12-14]

In the end, the best possible cases were combined. The simulation was Done by placing 4.5mm long ground pin on the top corner of the 34mm long Display frame. The result was good only 1.96W/Kg, which 1W/kg less than in the beginning.

Figure 15

Figure 16

Figure 17

Figure 18

Figure 19

Figure 20

Figure 21

Figure 22

Figure 23

Conclusion

The simulation results show that the length of the display and position and length of the ground pins is critical and sensitive value. Those should be chosen carefully considering the dimensions of the PWB. Lowest SAR value was achieved when length of the display frame was 34mm adding the height of the frame, the total length is 37.85 which is really close to l/8 wavelength of the GSM. When combining the two best results pins with 4.5 mm length and display frame with 34mm length even lower SAR value was achieved. Correlation between measured and simulated results is expected to be good. In engineering, the size corresponding to the smaller SAR value is found through parameter scanning. It needs to be explained from the physical principle that the redistribution of current leads to the redistribution of the near-field, making the near-field amplitude behind the antenna smaller, thus reducing SAR.

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Hepatotoxicity of Aqueous Extract of Dialium Guineense Stem Bark in Rats

Introduction

Toxicology deals with the deleterious impact of chemical, physical or biological agents on living organisms and the environment [1]. It encompasses toxicity and the underlying principles. The amount of a substance may account for its toxicity: dose is what distinguishes a poison from a remedy. Toxicity is the extent to which a substance can damage an organism. It could impact negatively on whole organism, as well as organelles [1,2]. The major principle of toxicology is that “dose and effect are directly proportional to each other”; seemingly harmless substance like water can intoxicate if consumed in a very high dose, while a toxic substance such as snake venom may not produce any toxicity if present in very low concentration. It has been established that toxicity is species-specific [3,4]. Toxicity measurement is carried out via meticulous observation of the effect of a substance on whole organism, organ, tissue or cell. The assessment is usually population-based [1]. Toxic effects are classified based on the site of effect. When the effect is observed at a single site, it is known as “specific target organ effect”, but, when it happens at several sites, it is called “systemic toxicity”. Acute, sub-chronic, and chronic toxicities are examples of systemic toxicity [1]. Acute toxicity occurs almost immediately after an exposure, while continuous exposure for weeks or months results in sub-chronic toxicity. As a common human exposure pattern sub-chronic toxicity assessment examines non-lethal impacts of toxicants on behavior, growth and reproduction, caused by biochemical, haematological, physiological and histopathological disturbances [5].

As the principal organ for the detoxification and distribution of drugs the liver is closely monitored to confirm the relative safety of a substance [6]. The liver is an indispensable organ involved in metabolism, detoxication and excretion. Some medicinal substances, chemicals and herbal preparations may cause liver injury [7]. Hepatotoxicity refers to injury to liver cells (hepatocytes) or liver function impairment. It means liver damage driven by chemicals. Hepatocellular damage may be caused by drugs, food additives, alcohol, chlorinated solvents, peroxidized fatty acids, fungal toxins, radioactive isotopes, environmental toxicants, and certain herbs [8,9]. Playing a key role in the transformation and clearance of chemicals the liver is highly vulnerable [10]. The organ may be injured by some medicinal substances, when taken in overdoses (for example, paracetamol) or within therapeutic ranges (for example, halothane). Hepatotoxins refer to agents that cause liver injury [11]. The aim of this study was to investigate the hepatotoxicity of aqueous extract of D. guineense stem bark in Wistar rats.

Materials and Methods

Chemicals and Reagents

Analytical grade reagents were used in this study. Kits used to perform liver integrity tests were products of Randox Laboratories Limited (United Kingdom). The other chemicals/reagents were bought from Pyrex Scientific Limited (United Kingdom), Merck (Germany), British Drug House (BDH) (England), and Sigma-Aldrich Ltd. (USA).

Plant Material and Authentication

Freshly harvested stem barks of D. guineense were collected from Auchi, Etsako West, Edo State, Nigeria. Their identification and authentication took place at the University of Benin herbarium in the Department of Plant Biology and Biotechnology, Faculty of Life Sciences (No. UBHD330).

Plant Extraction

The ground plant material was extracted via maceration for 72 h [12]. Exactly 500 g of the pulverized stem bark was steeped in 5 L distilled water. The aqueous extract produced after filtration was freeze-dried via lyophilization.

Animals

Thirty-five (35) male Wistar rats (mean weight = 170.00 ± 10.00 g) were bought from the Animal House of the Department of Anatomy, University of Benin, Benin City, Nigeria. The rats were kept in metal cages under standard laboratory conditions. They had unrestricted access to feed and water. The rats were acclimatized to the laboratory environment for seven days, prior to commencement of the study. Standard experimental procedure was adopted for this study.

Subchronic Toxicity Study

The rats were randomly divided into 7 groups (5 rats/group): “Group I served as control, while others received graded doses of extract (200 – 5000 mg/kg bwt) for 28 days”. Basal samples were collected before the commencement of treatment. At the expiration of the 28-day treatment the rats were euthanized after an overnight fast. The collected blood was centrifuged at 3000 rpm for 10 min to obtain clear plasma.

Organ Function Tests

Liver function tests (LFTs) such as ALT, AST, GGT, total protein, albumin, globulins and bilirubin were carried out in rat plasma [13-17].

Determination of Lipid Peroxidation in Liver

Malondialdehyde (MDA) concentration was measured in 20 % liver homogenate [18].

Statistical Analysis

Data obtained in this study are expressed as mean ± standard error of mean (SEM, n = 5). One-Way Analysis of Variance (One-Way ANOVA) was performed with SPSS (version 20). Groups were compared using Duncan multiple range test. Values of p < 0.05 were considered statistically significant.

Results

Percentage Weight Increase and Relative Organ Weight of Rats

Percentage increases in body weights of rats treated with aqueous extract of D. guineense stem bark were significantly reduced, when compared with control group (p < 0.05). However, there were no significant differences in the corresponding relative organ weights among the groups (p > 0.05; (Table 1). Data are percentage weight increase and relative liver weight, and are expressed as mean ± SEM (n = 3). ^ap < 0.05, when compared with control group; ^bp < 0.05, when compared with the other treatment groups. Liver Function of Rats Treated with Varied Doses of Plant Extract “There were no significant differences in the activities of plasma ALT and AST in rats exposed to doses ranging from 200 – 1000 mg/kg bwt aqueous extract, relative to control group (p > 0.05). However, the corresponding activities of GGT were significantly reduced, when compared with control group (p < 0.05). While the activities of ALT in groups VI and VII, and AST in groups V, VI and VII were significantly increased (p < 0.05), the corresponding activities of GGT were not significantly different from that of control group (p > 0.05). There were no significant differences in AST/ALT among the groups (p > 0.05; (Table 2). There were also no significant increases in the concentrations of albumin, total protein, globulins, bilirubin and plasma MDA in treated rats, relative to the control group (p > 0.05; (Tables 3-5). Furthermore, the basal activities and concentrations of the measured indices of liver function were not significantly different from those after treatment (p > 0.05; (Tables 2-4)”.

Table 1: Percentage Body Weight Increase and Relative Liver Weight of Rats.

Table 2: Liver Function Parameters in Rats Treated with Aqueous Extract of D. guineense Stem Bark.

Table 3: Concentrations of Liver Proteins in Rats Treated with Aqueous Extract of D. guineense Stem Bark.

Table 4: Concentrations of Bilirubin in Rats Treated with Aqueous Extract of D. guineense Stem Bark.

Table 5: Concentration of MDA in Rat Liver.

Data are indices of liver function, and are expressed as mean ± SEM (n = 5). Data are indices of liver function, and are expressed as mean ± SEM (n = 5). Data are indices of liver function, and are expressed as mean ± SEM (n = 5). Data are indices of liver function, and are expressed as mean ± SEM (n = 5). T. Bilirubin = total bilirubin; D. Bilirubin = direct bilirubin; and Ind. Bilirubin = indirect bilirubin. Data are concentrations of liver MDA and are expressed as mean ± SEM (n = 5).

Discussion

The liver in vertebrates and other animals’ function in detoxification, protein synthesis, and synthesis of other important biomolecules. Since there is no way to compensate for its absence in the long-term the liver is necessary for survival [6]. The organ is involved in metabolism: glycogen storage, hemolysis, synthesis of plasma proteins/hormones, and detoxification. Located below the diaphragm of the abdomen the liver secretes bile, an alkaline substance that facilitates digestion (that is, lipids emulsification) [6]. The specialized tissues of the organ control a number of biochemical/physiological reactions [19]. Karat parenchymal and non-parenchymal are the two principal cells found in liver lobes. Commonly referred to as hepatocytes, parenchymal cells constitute approximately 80 % of liver volume, while non-parenchymal cells makeup 40 % of the total number of liver cells, but only 6.5 % of its volume. Sinusoidal hepatic endothelial, Kupffer and hepatic stellate cells are some of the non-parenchymal cells that line liver sinusoid [20]. Liver function is assessed in blood. Liver Function Tests (LFTs) can reveal the degree of liver injury [21]. These tests are employed to screen for liver disease, speculate the possible cause, determine the severity, evaluate prognosis and monitor effectiveness of therapy [22]. Aminotransferases, Alkaline Phosphatase (ALP), GGT, 5’-nucleotidase, and leucine aminopeptidase are LFTs that detect injury to hepatocytes. Serum and urine bilirubin are tests of the liver’s capacity to metabolize drugs and transport organic anions. The hepatocyte’s ability to carry out synthetic function is determined with albumin [22].

During cellular necrosis and increased plasma membrane permeability, liver enzymes released into systemic circulation are used as diagnostic tool to evaluate liver damage. Most proteins in plasma are synthesized by cells of the liver and released into systemic circulation. A sharp decrease in plasma albumin level below normal range suggests a marked decline in the liver’s protein synthetic function. Liver injury or damage results in elevated bilirubin (breakdown product of haemoglobin) level. Total bilirubin elevation caused by reduced uptake and conjugation of bilirubin is as a result of hepatocyte dysfunction, while elevated levels of direct/conjugated bilirubin is due to declined secretion from liver or obstruction of bile ducts. Elevated albumin, bilirubin and total protein levels due to hemolysis or liver disease or both can result in jaundice of the skin or brain kernicterus [23,24]. In this study, treatment of Wistar rats with graded doses of aqueous extract of the medicinal plant did not produce any observable elevation in liver enzymes, proteins and metabolites to subject hepatotoxicity. Even in few cases were ALT and AST were elevated, they were not significantly different from the basal values. The hepatoprotective capacity of extracts of D. guineense stem bark has been reported [24-26]. The results of this study are in agreement with those of previous reports [27-48]. Studies have demonstrated important biological effect of extracts of the medicinal plant [49-58].

Conclusion

The results of this study indicate that the aqueous extract of D. guineense stem bark may not be deleterious to the liver at dose not exceeding 5000 mg/kg bwt. This study has provided first-time evidence as to the relative safety of the plant extract.

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The Relationship Between Erectile Dysfunction and Monocyte/HDL Ratio

Introduction

Penile erection is a complex phenomenon in which neurological, endocrine, and vascular structures work in a sensitive and balanced manner [1]. During penile erection, dilation of the arteries supplying the penis, relaxation of the penile trabecular smooth muscles and activation of the corporeal veno-occlusive mechanism are required [1]. Erectile Dysfunction (ED) is defined as the inability to achieve and/or maintain a penile erection sufficient for satisfactory. sexual intercourse [2]. ED occurs in more than half of the male population aged 40-70 years [3]. Among the organic pathologies that cause ED, the most common cause is impaired arterial blood flow to the erectile tissues [4]. Vasculogenic risk factors that may cause ED include diabetes, dyslipidemia, Cardiovascular Diseases (CVD), Hypertension (HT), obesity, metabolic syndrome, sedentary lifestyle and smoking [4]. The common pathophysiological pathway underlying these risk factors is inflammation, atherosclerosis and endothelial dysfunction resulting in decreased blood flow, arterial insufficiency, or arterial stenosis [4,5]. Macrophages and monocytes have important roles in the secretion of proinflammatory and prooxidant cytokines in inflammation areas [6,7]. High-Density Lipoprotein Cholesterol (HDL- C) has been shown to protect endothelial cells against the adverse effects of Low-Density Lipoprotein Cholesterol (LDL-C) and inhibit the oxidation of LDL molecules [7,8]. The Monocyte/HDL Ratio (MHR), recognized as a new marker of inflammation, is the ratio of inflammatory markers (monocytes) to Anti-Inflammatory Markers (HDL-C) [9]. Some studies have shown that MHR is associated with metabolic syndrome, CAD and diabetic microangiopathy [10-12]. In this study, considering that CVD and ED have many common risk factors and pathophysiology, we have aimed to determine whether MHR is associated with ED.

Material and Methods

Participants were included in the study after obtaining the approval of the clinical research ethics committee and in accordance with the principles of the Declaration of Helsinki. Patients who applied to the urology outpatient clinic of our hospital between November 2021 and January 2024 due to andrological complaints were retrospectively reviewed. The erectile function of the patients was evaluated using the 5-question form of the International Index of Sexual Function (IIEF-5). Patients with an IIEF-5 score of 22 and above were considered healthy/normal in terms of erection, while those with a score below 22 were considered ED. Exclusion criteria; patients with any endocrinological disease other than type 2 DM such as hyperprolactinemia, hypogonadism and hypo/hyperthyroidism, neurological disease, hematological disorders, accompanying malignancies, psychiatric disease, ED-related drug and addictive substance use, collagen tissue disease, history of previous penile or pelvic surgery/ trauma/radiotherapy, history of spinal cord trauma, presence of penile curvature/Peyronie’s disease, chronic liver failure, chronic renal failure. In addition, patients with missing data were excluded from the study. IIEF-5 score, anamnesis and physical examination findings of the patients were obtained from archive records. Hemogram, FBG, HbA1c, total cholesterol, HDL, LDL and total testosterone results were recorded from peripheral blood samples. MHR value was calculated by dividing serum monocyte level by HDL cholesterol level.

As a control group, patients who were examined for an andrological reason other than ED (Peyronie’s disease, ejaculation disorders, infertility, etc.) and whose IIEF-5 score was 22 and above were included in the study. The patients were divided into two groups as Group 1 (ED) and Group 2 (control), and comparisons were made. Statistical analysis was performed using SPSS 21.0 (IBM SPSS for Windows). Data were expressed as number, percentage, mean and standard deviation. Comparison between two independent groups was performed by Student’s t-test. ROC analysis was performed for the cut-off value of MHR in predicting ED. Statistical significance was accepted as p<0.05.

Results

A total of 133 patients with a mean age of 42.49 ±12.49 years were included in the study. There were 84 (63.15%) patients in Group 1 and 49 (36.85%) patients in Group 2. The mean age of Group 1 was 47.14 ±11.70 years and the mean age of Group 2 was 34.51 ±9.45 years, which was significantly higher in Group 1 (p=0.001). The mean BMI was 27.88 ± 3.49 kg/m2 in Group 1 and 26.85±2.09 kg/m2 in Group 2 and was significantly higher in Group 1 (p=0.036). The mean FBG was 116.21±64.79 mg/dL in Group 1 and 87.59±20 mg/dL in Group 2 and the difference was significant and higher in Group 1 (p=0.003). The mean HbA1c was 6.15±1.68 % in Group 1 and 5.39±0.43 % in Group 2, which was significantly higher in Group 1 (p=0.002). The mean HDL cholesterol level of Group 1 was 41.56±9.63 mg/dL, while the mean HDL cholesterol level of Group 2 was 46.99±9.90 mg/dL, which was significantly higher in Group 1 (p=0.002). The mean monocyte/ HDL ratio was 0.0131±0.005 in Group 1 and 0.0106 ± 0.004 in Group 2, which was significantly higher in Group 1 (p=0.009). The data of all participants are presented in Table 1 and the comparison between Group 1 and Group 2 is presented in (Table 2). MHR was found to be associated with ED in ROC analysis, with 0.66 of AUC, 0.536-0.730 of 95% CI and p=0.011. According to ROC analysis, MHR cut-off value above 0.0124 seems to be associated with ED with 47% sensitivity and 76% specificity (Figure 1).

Table 1: Data of all participants.

Table 2: Comparison of data between the two groups.

Figure 1

Discussion

The main aim of this retrospective study was to investigate whether MHR was associated with ED. As a result of our analyses, we found that MHR had a relationship with ED and MHR was significantly higher in the ED group. In addition, other main findings we obtained were that age, BMI, FBG and HbA1c were significantly higher in ED patients. HDL cholesterol was found to be significantly lower in the ED group. Inflammation and oxidative stress are well recognized mechanisms in the development and progression of atherosclerosis [7]. Monocytes play a critical role in this process. Activated monocytes interact with the endothelium, causing overexpression of proinflammatory cytokines. Monocytes then differentiate into macrophages that digest oxidized LDL cholesterol and form dangerous foam cells [13]. On the contrary, HDL molecules inhibit the migration of macro-phages and stimulate the outflow of oxidized cholesterol from these cells [13]. Monocytes exert proinflammatory and prooxidant effects, whereas HDL-C acts as an anti-inflammatory and antioxidant factor that reverses these processes [7,12]. Canpolat et al. showed that higher MHR levels were significantly and independently associated with the presence of SCF in Coronary Slow Flow Phenomenon (SCF) associated with inflammation, oxidative stress, and endothelial dysfunction [7]. Kanbay et al. reported that MHR increased during decline in the glomerular filtration rate and was associated with a worse cardiovascular profile and was an independent predictor of major cardiovascular events during follow-up in their study with patients with chronic renal failure.

Çetin et al. emphasized that MHR was an independent predictor of the severity of coronary artery disease and future cardiovascular events in patients with ACS in their study, which included 2661 patients with Acute Coronary Syndrome (ACS) and a mean follow-up period of 31.6 months [11]. Bolayır et al. reported in their study including 466 Acute Ischemic Stroke (AIS) patients that MHR was an independent predictor of 30-day mortality in AIS [14]. In our study, the relationship between MHR and ED was investigated and a significantly higher MHR level was found in ED patients compared to the control group. When ROC analysis was performed for MHR, patients with MHR cut-off value above 0.0124 were found to be associated with ED with 47% sensitivity and 76% specificity. The relationship between age and ED, which is one of the important risk factors in the development of ED, has been demonstrated in many studies. In the European Male Ageing Study (EMAS) conducted in 2010 in eight European Union countries with 3369 male participants with an average age of 60±11 years; while the prevalence of moderate or severe ED was reported in 30% of all participants, this rate was reported as 64% in men aged 70 years and over [15]. In a study conducted by Braun et al. in Germany with participants aged 30-80 years, the overall prevalence of ED was 19.2% and increased with age; 2.3% in the 30- 39 age range and 53.4% in the 70-80 age range [16]. In our study, the mean age was found to be significantly higher in the ED group in accordance with the literature.

When the literature is analyzed, it is understood that obesity is among the common causes of ED. Kratzik et al. reported in their study that each 1 kg/m2 increase in BMI, IIEF-5 decrease by 0.141 (p=0.005) regardless of age and that high BMI rates strongly contributed to the development of ED [17]. In a meta-analysis aiming to demonstrate the relationship between BMI and ED, it was found that ED was significantly associated with high BMI ratios [18]. In this context, the present study is consistent with the literature and BMI was found to be significantly higher in the ED group. DM causes sexual dysfunction in both men and women, and ED is the most important dysfunction in men with DM [19]. Epidemiological studies show that the age of onset of ED in diabetic men is on average 10-15 years earlier than in non-diabetic men, and that the duration of DM and ED are closely related [20]. According to a meta-analysis of 145 studies, the overall ED prevalence rate in men with DM was reported to be 52.5% (95% CI, 48.8- 56.2), whereas the prevalence rates in patients with Type 1 and Type 2 DM were 37.5% and 66.3%, respectively [21]. Today, the concept of prediabetes, which is considered as a metabolic state between normoglycemia and diabetes, has emerged [22]. According to the World Health Organization, the definition of prediabetes is defined as a FBG of 110-125 mg/dL and an HbA1c level of 5.7-6.4% [23]. According to the meta-analysis published by Jin et al., compared with normoglycemic men, prediabetic men were reported to have a higher prevalence of ED (OR 1.62; 95% CI 1.28-2.07; p<0.001) [22].

The results reported in various studies support this situation [24,25]. In the present study, in accordance with the literature, mean FBG and HbA1c levels were significantly higher in the ED group compared to the control group. The role of HDL cholesterol in mediating atherosclerotic heart disease is unclear, but results from epidemiological studies suggest that low HDL cholesterol levels are an independent risk factor for atherosclerotic heart disease [26,27]. The MMAS study reported an inverse association between the likelihood of having an ED and HDL cholesterol level [28]. However, high serum total cholesterol and low HDL cholesterol levels have been shown to be associated with an increased risk of ED [29]. In the present study, HDL cholesterol was found to be significantly lower in the ED group compared to the control group and this result supports the studies reporting that HDL cholesterol is protective in terms of ED. Our study has some limitations. Firstly, its design was retrospective, single-centre, cross- sectionally, and the participants did not have specific follow- up periods. In addition, the habits of the participants included in the study, such as smoking/alcohol/nutrition, which may have effect on ED, could not be recorded and analyzed due to lack of data.

Conclusion

In our study aiming to reveal the relationship between ED and MHR, which has a common pathophysiology with atherosclerosis- based vascular diseases, MHR level was found to be significantly higher in ED patients. To the best of our knowledge, this is the first study to demonstrate the relationship between ED and MHR and we think that our study is a valuable contribution to the literature. However, prospective studies with larger participation are needed to support our findings.

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