Dibyajyoti Banerjee

Urinary hydrogen peroxide: a probable marker of oxidative stress in malignancy

BACKGROUND Urinary hydrogen peroxide was postulated to be a biomarker of oxidative stress. We estimated urinary hydrogen peroxide along with other established parameters of oxidative stress in malignancies where oxidative stress is well documented. METHODS The oxidative stress markers tested were concentrations of erythrocyte glutathione, erythrocyte malonaldehyde (MDA) and plasma hydroperoxide, and activities of plasma glutathione-S-transferase (GST) and erythrocyte catalase. Urinary hydrogen peroxide was measured by a modified ferrous ion oxidation xylenol orange version-2 (FOX-2) method on a spot random sample of urine. RESULTS In healthy controls (n=10), erythrocyte glutathione concentration was 4.41+/-0.057mg/g of hemoglobin, plasma hydroperoxide was 2.5+/-0.07 micromol/l, erythrocyte MDA was 0.9+/-0.15 nmol/ml of packed cell suspension and erythrocyte catalase and plasma GST were 74.66+/-9.2/s/ml of packed cell suspension and 6.12+/-0.84 IU/l, respectively. In cancer patients (n=25), erythrocyte glutathione, plasma hydroperoxide and erythrocyte MDA were 9.32+/-0.42 mg/g of hemoglobin, 6.2+/-0.13 micromol/l and 2.3+/-0.27 nmol/ml of packed cell suspension, respectively; and activities of erythrocyte catalase and plasma GST were 151.04+/-6.5/s/ml of packed cell suspension and 10.9+/-0.36 IU/l, respectively. Urinary hydrogen peroxide concentration was 15+/-9.8 micromol/l in the healthy controls and 56.3+/-3.9 micromol/l in cancer patients. CONCLUSION Urinary hydrogen peroxide may be a marker of oxidative stress in malignancies.

Vitamin D regulates the production of vascular endothelial growth factor: A triggering cause in the pathogenesis of rheumatic heart disease?

Rheumatic heart disease (RHD) remains a major cause of cardiac related mortality and morbidity in the developing countries due to poor diagnosis and lack of proper therapeutics. The definite reason of heart valve injury during RHD is poorly understood. Valvular endothelial cells play an important role in pathogenesis of different cardiovascular diseases. Besides, the regulation of vitamin D (calciferol) and VEGF (vascular endothelial growth factor) results in the functional changes in endothelial cells. However, the crosstalk between vitamin D and VEGF in the pathogenesis of RHD is not yet unfurled. Evidences in the concerned fields are documented by searching through Google Scholar and Pubmed. Literature based survey has revealed that vascular endothelium, especially endothelial cells play important roles in valvular remodelling during cardiovascular diseases. Endothelial cell dysfunction leads to heart valve remodelling, which furthermore initiates the pathogenesis of valvular heart disease. Vitamin D has the potential to maintain the concentration of VEGF in the circulation and induce the function of endothelial cells. Hence, we hypothesize that vitamin D and VEGF homeostasis can alter the function of endothelial cells, which may subsequently trigger the valvular remodelling or even damage of heart valves during the progression of RHD pathogenesis. Our hypothesis shed light on the evidence based knowledge translation of plausible cellular phenomena due to vitamin D/VEGF homeostasis during valvular vandalism in RHD.

Identification of orcinol reactive substance in pleural fluid cell lysate—A new parameter for classification of pleural effusion

BACKGROUND Cell-free DNA is observed to be more in exudative pleural effusions. Based on this fact development of a clinical chemistry test for classification of pleural effusion will require DNA extraction followed by PCR amplification and electrophoresis. These procedures may not be cost effective for the purpose for classification of pleural effusion as already established parameters are popular for the purpose which can be estimated by comparatively low cost colorimetric procedures. Therefore development of a simple colorimetric test for the classification of pleural fluid based on nucleic acid identification test can be attempted. The aim of this work is to develop such colorimetric test for classification of pleural effusion using only pleural fluid sample. METHODS Cell pellet is obtained from 5 ml pleural fluid which is lysed and subjected to DNA extraction, followed by identification under UV-transilluminator after electrophoresis and orcinol and diphenylamine reaction. RESULT Exudates show extractable DNA from 5 ml biofluid (n=52) which are not observed from transudate (n=32). Orcinol reaction is significantly positive in exudates (n=52) compared to the transudates (n=32). Diphenylamine test cannot differentiate exudate from transudate. CONCLUSION Orcinol reaction of cell lysate obtained from pleural fluid can classify pleural fluid sample into exudate or transudate.

A drop of hydrogen peroxide can differentiate exudative pleural effusion from transudate — development of a bedside screening test

BACKGROUND There is no bedside test to classify pleural fluid as exudate or transudate. The aim of the present study is to develop such a test. METHODS We analyzed the Lights criteria parameters from bloodless pleural fluid and classified the biofluid as exudate or transudate and also estimated some parameters of oxidative stress in the biofluid by established spectrophotometric procedure. Two hundred microliters of sample was taken and added with 10 microl of 30% hydrogen peroxide followed by inspection of the sample for appearance of bubbles. RESULT All exudative fluids (n=52) have shown appearance of profuse bubbles within 1 min of addition of hydrogen peroxide along with significantly more catalase activity compared to transudate. All transudative fluids (n=32) have not shown bubble formation within 1 min after addition of hydrogen peroxide. The exudate does not show bubble formation if supplemented with catalase inhibitors. Blood mixed transudate have shown profuse bubble formation after addition of hydrogen peroxide. CONCLUSION In the case of blood uncontaminated pleural fluid, this newly developed protocols sensitivity and specificity will be equivalent to Lights criteria probably with more advantage as by this procedure transport of the sample to the clinical laboratory is not required due to its inherent simplicity.

High dose targeted delivery on cancer sites and the importance of short-chain fatty acids for metformin's action: Two crucial aspects of the wonder drug

Metformin is a popular anti-diabetic drug currently being explored for its role in cancer and gut microbiome amongst other areas. Recently, Adak T et al. explicatively reviewed metformins effects as an anti-cancer drug and a gut microbiome modulator. We feel that the authors have not adequately addressed some of the key concerns around metformin in their report and in this correspondence, we seek to add some of the issues that need to be addressed by researchers.

Pseudoesterase activity of albumin: A probable determinant of cholesterol biosynthesis

The association between hypoalbuminemia and coronary artery disease is known from some time. However, the reason as to how such phenomenon is correlated remains unknown. We have observed from published scientific literature that HSA has the potential to control cholesterol biosynthesis due to its pseudoesterase activity. In-silico observations have supported our view since acetyl coA, the precursor molecule of cholesterol biosynthesis is shown to bind with Tyr 411 of HSA. Incidentally, Tyr411 is a critical moiety for pseudoesterase activity of albumin. With this frame of reference in mind we hypothesize that pseudoesterase activity of HSA is an important determinant of lipid metabolism including cholesterol biosynthesis. Therefore, albumin has the potential to influence the atherosclerotic state important for pathogenesis of coronary artery diseases.

The lesson to be learned from Broccoli and turmeric

Broccoli, a salad vegetable, and turmeric, herbs are known to humankind from times immemorial [1,2]. There are several observations documented in the literature regarding the beneficial effects of the compounds present in these two food gradients [3-5]. Now it is the high time for translating ingredients from kitchen to clinic because of translational and nutrigenomics research. In that context, broccoli and turmeric are two ideal foodstuffs. There are phytochemicals and antioxidant compounds in these two herbs that have profound health benefit ranging from preventive to the curative phase of diseases [6-8]. Antioxidants are believed to be beneficial, but it can act both as friend or foe varying upon the microenvironment of the milieu interior in the scenario of the molecular pathogenesis of a disease [9,10]. Clinical Trials with antioxidants have yet not produced many encouraging results [11,12]. Instead, other than ascorbic acid (vitamin C) the antioxidants including Vitamin E are proved to increase mortality in all causes [13,14]. Therefore, use of antioxidants must consider seriously, and from the point of the molecular pathogenesis of the diseases, their use should be justified. It is found that oxidative stress is associated with most of the diseases [15]. The major noncommunicable chronic diseases of the recent times like diabetes mellitus, obesity, atherosclerosis etc. are related to oxidative stress [16]. In such conditions, supplementations of the antioxidant vitamins are not expected to improve the levels of the antioxidant enzymes [17]. It is particularly noteworthy that the activity of antioxidant enzymes is observed to be diminished in these chronic illnesses. In this context, there is a ray of hope from broccoli and curcumin.

β-Naphthyl Acetate in Acetone Produces a Dark Background for Staining of Esterase Activity on Gel

Staining of gels for esterase activity is a routinely performed procedure in any biological laboratory. The substrates popularly known for such purpose are a-naphthyl acetate or b-naphthyl acetate (BNA). After the electrophoresis run the gels are incubated with the ester substrates, and azo dye like Fast Red B. The separated protein having esterase function that cleaves the ester bond of the substrate concerned and forms either a-naphthol or bnaphthol which is subsequently stained by the azo dye. By the above procedure esterase activity can be easily visualised on gel [1]. As on date, there is no consensus about the preparation of the ester substrate. Earlier workers have dissolved the substrates in acetone or ethanol and detected the enzyme activity [2–5]. While working in this line, we have observed that if BNA is dissolved in acetone, it produces a dark background compared to the situation when the substrate is prepared by dissolving in ethanol. Enzyme activity bands are visible in both the cases [Fig. 1A, B for agarose and native polyacrylamide gel electrophoresis (PAGE) respectively; for methods, please refer the supplementary]. It is needless to emphasise that low background staining is a prerequisite for electrophoresis experiment. It is interesting to note that destaining is sometimes necessary when the ester substrates are prepared in acetone [5]. This supports our observation that acetone as a dissolving medium of the ester substrate produces a dark background in the gel. With ethanol using BNA we have observed no (or very light) background staining. So if ethanol is used as a dissolving medium of BNA for esterase staining of gels, no destaining will be necessary.

Anti-endothelial cell antibody rich sera from rheumatic heart disease patients induces proinflammatory phenotype and methylation alteration in endothelial cells

Rheumatic heart disease (RHD) is a major cause of cardiovascular morbidity and mortality in developing nations like India. RHD commonly affects the mitral valve which is lined by a single layer of endothelial cells (ECs). The role of ECs in mitral valve damage during RHD is not well elucidated. In here, anti-endothelial cell antibody from RHD patients has been used to stimulate the ECs (HUVECs and HMVECs). ECs proinflammatory phenotype with increased expression of TNFα, IL-6, IL-8, IFNγ, IL-1β, ICAM1, VCAM1, E-selectin, laminin B, and vimentin was documented in both ECs. The promoter hypomethylation of various key inflammatory cytokines (TNFα, IL-6, and IL-8), integrin (ICAM1) associated with leukocyte transendothelial migration, and extracellular matrix genes (vimentin, and laminin) were also observed. Further, the in-vitro data was in accordance with ex-vivo observations which correlated significantly with the etiological factors such as smoking, socioeconomic status, and housing. Thus, the study sheds light on the role of ECs in RHD which is a step forward in the elucidation of disease pathogenesis.

Inhalational supplementation of metformin butyrate: A strategy for prevention and cure of various pulmonary disorders

The management of chronic lung diseases such as cancer, asthma, COPD and pulmonary hypertension remains unsatisfactory till date, and several strategies are being tried to control the same. Metformin, a popular anti-diabetic drug has shown promising effects in pre-clinical studies and has been subject to several trials in patients with debilitating pulmonary diseases. However, the clinical evidence for the use of metformin in these conditions is disappointing. Recent observations suggest that metformin use in diabetic patients is associated with an increase in butyrate-producing bacteria in the gut microbiome. Butyrate, similar to metformin, shows beneficial effects in pathological conditions found in pulmonary diseases. Further, the pharmacokinetic data of metformin suggests that metformin is predominantly concentrated in the gut, even after absorption. Butyrate, on the other hand, has a short half-life and thus oral supplementation of butyrate and metformin is unlikely to result in high concentrations of these drugs in the lung. In this paper, we review the pre-clinical studies of metformin and butyrate pertaining to pathologies commonly encountered in chronic lung diseases and underscore the need to administer these drugs directly to the lung via the inhalational route.