Plasma angiogenesis and oxidative stress markers in patients with diabetic retinopathy.

Background: Neovascularization in the retina and hyperglycaemia-induced oxidative stress are implicated in the pathogenesis of diabetic retinopathy (DR). In this study, we hypothesized that the plasma angiogenic and oxidative stress markers associated with these derangements could aid in the screening of diabetic patients who are at an increased risk of developing retinopathy.Methods: This study included normal (n = 148), type2 diabetes without retinopathy (DNR; n = 148), proliferative DR (PDR; n = 74) and non-PDR (NPDR; n = 148) subjects. Plasma concentrations of vascular endothelial growth factor-A (VEGF-A), hypoxia-inducible factor-1α (HIF-1α), matrix metalloproteinase-9 (MMP-9), pigment epithelium-derived factor (PEDF), nitric oxide (NO), soluble receptors for advanced glycation end products (sRAGE), malondialdehyde (MDA) and protein thiols were estimated.Results: A statistically significant increase was observed in the plasma concentrations of pro-angiogenic factors and markers of oxidative stress in both retinopathy groups. By contrast, the concentrations of anti-angiogenic factors and antioxidants were decreased significantly in these groups. Receiver operating characteristic analysis indicated that the plasma thresholds of HIF-1α and PEDF can be suitable markers in case of NPDR. However, in PDR, HIF-1α, NO, MMP-9 and PEDF showed high sensitivity and specificity.Conclusions: The factors associated with hypoxia, matrix degradation and angiogenic inhibition play a crucial role in predicting DR.

Amino Acid Profile and Lactate Pyruvate Ratio: Potential Adjunct Markers for Differentiating Inborn Errors of Metabolism.

Inborn errors of metabolism (IEM) lead to the physical and mental disability and death of infants, which can be prevented if treated early. Hence it is imperative in diagnosing these disorders at the earliest. The study is planned to differentiate suspected IEM by quantifying the lactate-pyruvate ratio (L/P ratio), Amino acid profiling by HPLC in addition to blood ammonia using the basic methodology in comparison to globally accepted mass spectroscopy technique and specific enzyme activity assays. The objective of this study is not to compete with the existing gold standard methodology rather makes the best of basic diagnostic modalities available. Five cases out of 100 referred cases of IEM suspicion showed the altered pattern of Aminogram correlating with high L/P ratio and Hyperammonemia, which were positive cases, as confirmed by the clinician who referred. Hence study parameters could be used as preliminary protocol to investigate and screen the IEM.

Development and validation of a dried blood spot test for thiamine deficiency among infants by HPLC-fluorimetry.

Thiamine deficiency, if detected early in infancy, can be treated with thiamine supplementation and can prevent seizures, other disabilities and death. The dried blood spot (DBS) sampling technique is an attractive sample collection technique for infants. The present study reports the development and validation of a highly sensitive and precise method for quantification of thiamine diphosphate from DBS. The method utilizes full-spot analysis of a volumetrically deposited 40 µl DBS. The analyte was extracted from the DBS using 50% methanol and then derivatized using potassium ferricyanide to thiochrome. Separation was achieved with the help of an Inertsil ODS C18 column (5.0 µm, 250 × 4.6 mm) using 150 mm phosphate buffer pH 7-acetonitrile (90:10, % v/v) as the mobile phase. The use of a fluorimetric detector gave a good response to the thiochrome derivative offering good sensitivity for the method. The excitation and emission wavelengths were 367 and 435 nm, respectively. The limit of detection and lower limit of quantification were 5 and 10 ng/ml, respectively. Linearity was demonstrated from 10 to 1000 ng/ml, and precision (CV) was <12.08%, at all tested quality control levels. The method accuracy was 89.34-118.89% with recoveries >80%. Bland-Altman analysis of DBS sampling vs. whole blood demonstrated a mean bias of only 1.16 ng/ml, with a majority of the 60 investigated patient samples lying within 7.2% of the corresponding concentration measured in blood, thereby meeting the clinical desirable biological specification criterion and showing that the two methods are comparable.

Cost-Effective HPLC-UV Method for Quantification of Vitamin D2 and D3 in Dried Blood Spot: A Potential Adjunct to Newborn Screening for Prophylaxis of Intractable Paediatric Seizures.

25-Hydroxyvitamin D (25-(OH)D) deficiency is recently been described as one of the multiple factors responsible for pediatric seizures. 25-Hydroxyvitamin D3 and 25-Hydroxyvitamin D2 are the well-known markers to determine Vitamin D status. In this work we report the development of a sensitive and cost effective HPLC technique for the quantification of the vitamin D metabolites from dried blood spot samples (DBS). The metabolites were extracted using acetonitrile-methanol-0.1% formic acid (60 : 20 : 20 (v/v)) and analyzed on an Acclaim C18 column (150 × 4.6 mm i.d., 3 µm) at a flow rate of 1 mL/min. The method was linear in the range of 10-80 ng/mL. Limit of detection and limit of quantification (LOQ) of the method were 5 and 10 ng/mL respectively. Extensive stability studies demonstrated the analytes to be stable in stock and matrix with a percent change within the acceptable range of ±15%. Comparison of the newly developed HPLC-DBS method with the reported LC-MS-DBS and electrochemiluminescence immunoassay (ECLIA) methods followed by Bland-Altman analysis demonstrated a bias of 0.08 and -0.14, respectively proving the methods are comparable. Application of the developed method to a pediatric seizure cohort depicted 46.6% of cases as deficient and 26.6% as insufficient for 25-(OH)D. Among deficient cases 8 samples were below 10 ng/mL and exact amount was not calculated since these were below the LOQ levels. The mean ± standard deviation (S.D.) in the remaining 6 deficient cases was 13.22 ± 2.80 ng/mL. The levels in healthy infants were 33.9 ± 6.11 ng/mL. The method can be used routinely for assessing 25-(OH)D deficiency in newborn.

Epidemiological study of burn patients hospitalised at a burns centre, Manipal.

Burn injuries constitute a major public health problem and account for a significant cause of mortality and morbidity amongst the Indian population. This 2-year prospective study was undertaken to find out the exact epidemiological determinants of 737 patients admitted with burn injuries and, thus, to try and formulate effective preventive strategies. This study was conducted in the department of plastic surgery and burns from September 2011 to June 2013. All consecutive patients with major burns admitted for in-hospital treatment during the study period were included in the study. The data collected included age, gender, cause and mode of burns, total burn surface area (TBSA), microbiological profile and outcome. More than 55% of the patients were female (58.61%). The mean age was 31.3 years ranging from 8 months to 89 years. A higher number of females (97.5%) sustained burn injuries at home compared with 36.11% males sustaining injuries outdoors. (P = 0.000). Almost one third of injuries (40.36%) occurred between 4 pm and 8 pm, followed by 28% between 7 am and 12 noon. Synthetic garments were worn by 70% of females at the time of injury, whereas 40% of males had worn mixed clothing (P = 0.000). Flame injuries contributed to 80.1% of burns in females (P = 0.006). The rate of electrical injuries (9.8%) was significantly higher in males (P = 0.005). In almost 40% of males, TBSA was <19%, whereas in 40% of females, TBSA was >68%. (P = 0.004). Microbial profile showed that pseudomonas aeruginosa (n = 260; 35.3%), Klebsiella pneumoniae (n = 209; 28.5%), and Escherichia coli (n = 145; 22.6%) were the most frequent types of Staphylococci bacterial growths. The cause for burn injury was ignition of clothes in 68.74% females, and in 35.48% males, it was because of an attempt to save other burn injury victims (P = 0.013) The case fatality rate was 29.3%. A majority of the males (60.7%) recovered, whereas 45.89% of females succumbed because of their burn injury. (P = 0.001). As the aetiological factors of burn injuries vary considerably in different communities, careful study of the pattern in every community is needed before a sound prevention programme can be planned and implemented. Hence, this study was conducted to assess epidemiological, modes, causes, and risk factors and the microbial profile of burn injuries and to study the outcomes.

A comparative study to evaluate the effect of limited access dressing (LAD) on burn wound healing.

Biochemical and histopathological analyses are commonly used objective parameters in research and clinical fields to assess the healing status of burn wounds. In this study, the effect of newer intermittent negative-pressure wound therapy in combination with moist environment [limited access dressing (LAD)] on burn wound healing is studied. Various biochemical parameters like hydroxyproline, hexosamine and total protein, and antioxidants like reduced glutathione (GSH), glutathione peroxidase (GPx) and oxidative biomarker malondialdhyde (MDA) were measured in the granulation tissue. Histopathologically, necrotic tissue, amount of inflammatory infiltrate, angiogenesis and extracellular matrix deposition (ECM) were studied to determine wound healing. A total of 55 patients were divided into two groups as follows: LAD group (n = 28) and conventional dressing group (n = 27). Patients treated with LAD have shown significant increase in the mean levels of (±SD) hydroxyproline (75·2 ± 26·30 versus 27·8 ± 15·5; P = 0·010), hexosamine (9·0 ± 1·99 versus 8·0 ± 1·18; P = 0·038), total protein (15·6 ± 8·23 versus 10·26 ± 4·94; P = 0·003), GSH (7·40 ± 1·91 versus 5·1 ± 1·28; P = 0·037), GPx (112·6 ± 46·4 versus 92 ± 32·4; P = 0·016), and decrease in MDA (6·5 ± 2·24 versus 1 0·6 ± 3·8; P = 0·002). Histopathologically, between LAD and conventional dressing groups, there was a significant difference after 10 days of treatment (mean±SE) in necrotic tissue of (LAD versus conventional dressing groups = 10 ± 1·8 versus 11·9 ± 2·6; P = 0·033), inflammatory cells (8·4 ± 1·9 versus 13 ± 3·46; P = 0·021), new blood vessels (12·5 ± 2·87 versus 9·4 ± 1·7; P = 0·047), ECM deposit (12·9 ± 2·41 versus 9·68 ± 1·3; P = 0·018) and showed comparatively fewer inflammatory cells, increased and well-organised extracellular matrix deposit, more angiogenesis in LAD group as compared with that in conventional dressing group. To conclude, LAD exerts its beneficial effects on wound healing by reducing oxidative stress, decreasing necrotic tissue and amount of inflammatory infiltrate, and increasing ECM deposition and angiogenesis.

Pathophysiology and diagnosis of Guillain-Barré syndrome - challenges and needs.

Guillain-Barré syndrome (GBS) is an autoimmune polyneuropathy which presents with acute onset and rapid progression of flaccid, hyporeflexi quadriparesis. Both sensory and autonomic nerve involvement is seen. GBS has various subtypes that vary in their pathophysiology. The pathogenesis involves an immune response triggered by a preceding event which may be an infection, immunisation or surgical procedure. Clinical diagnosis has been largely the primary diagnosing criterion for GBS along with electrodiagnosis, which has several pitfalls and is supported by ancillary testing of cerebrospinal fluid (CSF) analysis and Nerve Conduction Studies. Measurement of anti-ganglioside antibodies is also an effective tool in its diagnosis. Further understanding of pathophysiology and better diagnostic methods are required for better management of GBS.

Effects of limited access dressing in chronic wounds: A biochemical and histological study.

BACKGROUND: Negative pressure wound therapy has emerged as an attractive treatment modality for the management and healing of chronic ulcers. Though numerous clinical studies are available, there is a lack of biochemical and histological studies evaluating the healing of chronic wounds. MATERIALS AND METHODS: In the present study, a total 60 patients were divided into two groups: Limited access dressing (LAD) group (n = 30) and conventional dressing group (n = 30). Various biochemical parameters such as hydroxyproline, total protein and antioxidants such as reduced glutathione (GSH), glutathione peroxidase (GPx), catalase (CAT) and oxidative biomarker malondialdhyde (MDA) are measured in the granulation tissue. Histologically amount of inflammatory infiltrate, angiogenesis, and collagen deposition are studied to assess wound healing. RESULTS: Patients treated with LAD have shown significant increase in the mean (±standard deviation) hydroxyproline (77.3 ± 30.1 vs. 32.3 ± 16.18; P = 0.026), total protein (13.89 ± 9.0 vs. 8.9 ± 4.59; P = 0.004), GSH (7.4 ± 1.91 vs. 5.1 ± 1.28; P = 0.039), GPx (122.3 ± 59.3 vs. 88.7 ± 34.11; P = 0.030), CAT (1.80 ± 1.14 vs. 0.9 ± 0.71; P = 0.002) and decrease in MDA (13.4 ± 5.5 vs. 8.6 ± 3.8; P = 0.004). Histological study showed comparatively fewer inflammatory cells, increased and well organised collagen bundles, and more angiogenesis in the LAD group when compared with that with conventional dressing after 10 days of treatment. CONCLUSION: In the present study, we have found beneficial effect of newer intermittent negative pressure therapy in combination with moist environment (LAD) on chronic wound healing by increasing collagen deposition and angiogenesis; and reducing oxidative stress and inflammatory infiltrate.

Evidence for the presence of somatic mitochondrial DNA mutations in right atrial appendage tissues of coronary artery disease patients.

Coronary artery disease (CAD) is a multifactorial disease with the underlying involvement of environment, life style and nuclear genetics. However, the role of extranuclear genetic material in terms of somatically acquired mutations in mitochondrial tRNA and protein coding genes in the initiation or progression of CAD is not well defined. Hence, in the present study, right atrial appendage tissues and matched blood samples of 150 CAD patients were screened for mutations in nucleotide regions encompassing the Cytochrome c oxidase subunit II (MT-CO2), tRNA lysine (MT-TK), ATP synthase F0 subunit 8 (MT-ATP8) and Cytochrome b (MT-CYB) genes of mitochondrial DNA. We have found 9 different somatic mutations in 6 % of the CAD patients. Out of these mutations, 4 each were localized in MT-TK gene (T8324A, A8326G, A8331G and A8344G) and MT-CYB genes (T15062C, C15238A, T15378G and C15491G) in addition to one mutation in non-coding region 7 (A8270T) of mitochondrial genome. In addition, we noticed that majority (85.3 %) of CAD patients showed double repeats of germ-line "CCCCCTCTA" intergenic sequence between MT-CO2 and MT-TK genes. Our in-silico investigations of missense mutations revealed that they may alter the free energy and stability of polypeptide chains of MT-CYB protein of complex III of mitochondrial respiratory chain. Based on our study findings, we hypothesize that the somatically acquired variations in MT-TK and MT-CYB genes may negatively impact the energy metabolism of cardiomyocytes in right atrial appendage tissues and contribute in the cardiac dysfunction among CAD patients. In conclusion, our findings may be likely to have potential implications in understanding the disease pathophysiology, diagnosis as well as for the better therapeutic management of CAD patients.

Correlation of serum homocysteine levels with the severity of coronary artery disease.

Coronary artery disease (CAD) has become the most common cause of mortality in the entire world. Homocysteine is implicated as an early atherosclerotic promoter. We studied the relationship between levels of serum homocysteine with severity of coronary artery disease. Total of 70 subjects who scheduled for coronary angiogram consented to participate in this study. In all the patients Gensini scoring system was used to assess the severity of CAD. Venous samples were taken from the patients in fasting state before angiography. Homocysteine levels in patients were measured by enzyme linked immunosorbant method and were compared with respective Genseni scores of participants. Fasting serum homocysteine levels in CAD patients were significantly higher than patients without coronary artery disease (p < 0.001). Also Homocyseine levels correlated significantly with increasing severity of CAD (p < 0.001). Serum homocysteine levels correlated well with the severity of CAD.

A Case of Sanfillippo's Disease Correlating Clinical and Biochemical Findings.

The mucopolysaccharidoses (MPSs) are a heterogeneous group of rare inherited disorders caused by deficiency of lysosomal enzyme necessary to break down mucopolysaccharides or glycosaminoglycans (GAGs). We had combined clinical findings and ophthalmological features. Biochemical test for urine glycosaminoglycans was done for confirmation of diagnosis in the patient. The case of Sanfillippo's disease was characterized by delayed development, hyperactivity with aggressive behaviour. Coarse facial feature, hirsutism and sleep disorder. Urine GAG tests for MPS was positive in the case. Based on clinical findings and biochemical tests for MPS, this case was diagnosed as a type III mucopolysaccharidoses. Urinary GAG's electrophoresis is an important screening test for MPS suspected cases.

Effect of limited access dressing on hydroxyproline and enzymatic antioxidant status in nonhealing chronic ulcers.

BACKGROUND: Healing ability of nonhealing chronic ulcers can be assessed by estimating hydroxyproline, total protein and enzymatic antioxidants such as glutathione peroxidase (GPx), glutathione S-transferase (GST) in the granulation tissue. MATERIALS AND METHOD: A total of 34 patients were analysed from two groups: Limited access dressing (LAD) group (n = 17) and conventional dressing group (n = 17). RESULTS: Patients treated with LAD that exerts combination of intermittent negative pressure and moist wound-healing had shown a significant increase in the hydroxyproline (P = 0.026), total protein (P = 0.004), GPx level (P = 0.030) and GST level (P = 0.045). CONCLUSION: Patients treated with LAD indicated significantly better anabolic effect on wound-healing compared to that of patients treated with conventional dressing.

Importance of Serum Copper and Vascular Endothelial Growth Factor (VEGF-A) Levels in Postmenopausal Bleeding.

The main aim of this study is association of serum copper and vascular endothelial growth factor (VEGF-A) in postmenopausal bleeding (PMB) patients. Blood samples were collected from female patients suffering with postmenopausal bleeding (n = 50) as well as healthy females as controls (n = 50). Serum copper levels were estimated by spectrophotometric method and serum VEGF-A by ELISA technique and compared with ultrasonographic measurement of endometrial thickness in both patients and controls. A significant increase in serum copper levels and an almost twofold increase in serum VEGF-A was observed in DUB patients when compared with controls. Correlation (r) between serum VEGF-A levels and endometrial thickness was 0.96. Odds ratio for copper, VEGF-A and combination of copper and VEGF-A was 0.0426, 0.0947 and 0.0313 respectively, in all these cases odds ratio was <1. The abnormal angiogenesis in PMB could be due to increased serum copper levels,which in turn stimulates factors like VEGF-A, thereby causing an increase in endometrial growth.

Serum Inflammatory Markers in Patients with Guillain Barre Syndrome.

Background: Guillain Barre syndrome (GBS) is an immune-mediated peripheral neuropathy characterized by the demyelination and axonal damage of the peripheral neurons. The pathogenesis of GBS involves the breakdown of the blood-brain barrier after which pro inflammatory cytokines attack the neurons in the peripheral nervous system. Aims: This study aims to evaluate five markers, namely matrix metalloproteinase (MMP)-2 and MMP-9, vascular endothelial growth factor (VEGF)-A, basic fibroblast growth factor (bFGF), and SFLT-1, which could have a role in the inflammatory response in patients with GBS and healthy controls. Settings and Design: In this prospective study, patients diagnosed with GBS at the department of neurology, Kasturba Medical College, Manipal, Karnataka were enrolled. Methods and Material: The markers selected for this study were analyzed using the ELISA method and expressed as given in the kit provided by the company. Ethical clearance was obtained from the Institutional Ethical Committee. Statistical Analysis: Results were evaluated using SPSS version 17.0 and expressed as mean ± SD. Error bars for each were drawn. Results: The levels of all five parameters showed a significant increase in patients as compared to controls. Conclusions: Disruption of the basement membrane of endoneurium by MMP-2 and MMP-9, recruitment and migration of macrophages and other cytokines by VEGF-A, bFGF, and soluble fms-like tyrosine kinase-1 (SFLT-1) are plausible, which leads to inflammation process and thus neuronal damage leading to the development of GBS.

Need and Viability of Newborn Screening Programme in India: Report from a Pilot Study.

India, a country with the second largest population in the world, does not have a national newborn screening programme as part of its health policy. With funding support from the Grand Challenges Canada, a pilot newborn screening programme was implemented for the Udupi district of South India to study the need and viability of a national programme in India. Six disorders were selected for the study based on the availability of funding and recommendation from pediatricians in the district. Here, we report the observed incidence during the study. A cost-effectiveness analysis of implementing newborn screening in India was performed. It is evident from our analysis that the financial loss for the nation due to these preventable diseases is much higher than the overall expenditure for screening, diagnosis, and treatment. This cost-effectiveness analysis justifies the need for a national newborn screening programme in India.

Micro-Raman spectroscopy study of optically trapped erythrocytes in malaria, dengue and leptospirosis infections.

Malaria, dengue and leptospirosis are three tropical infectious diseases that present with severe hematological derangement causing significant morbidity and mortality, especially during the seasonal monsoons. During the course of these infectious diseases, circulating red blood cells are imperiled to the direct ill-effects of the infectious pathogen in the body as well as to the pro-inflammatory cytokines generated as a consequence of the infection. RBCs when exposed to such inflammatory and/or pathogenic milieu are susceptible to injuries such as RBC programmed eryptosis or RBC programmed necrosis. This research aimed to explore the Raman spectra of live red cells that were extracted from patients infected with malaria, dengue, and leptospirosis. Red cells were optically trapped and micro-Raman probed using a 785 nm Diode laser. RBCs from samples of all three diseases displayed Raman signatures that were significantly altered from the normal/healthy. Distinct spectral markers that were common across all the four groups were obtained from various standardized multivariate analytical methods. Following comprehensive examination of multiple studies, we propose these spectral wavenumbers as "Raman markers of RBC injury." Findings in our study display that anemia-triggering infections can inflict variations in the healthy status of red cells, easily identifiable by selectively analyzing specific Raman markers. Additionally, this study also highlights relevant statistical tools that can be utilized to study Raman spectral data from biological samples which could help identify the very significant Raman peaks from the spectral band. This approach of RBC analysis can foster a better understanding of red cell behavior and their alterations exhibited in health and disease.

Biomarker profiling of vitamin responsive seizures: a potential tool to detect pediatric seizures of unknown aetiology.

Aim: Certain rare inborn errors of metabolism clinically present with intractable seizures that readily respond to vitamin therapy. If identified early, brain damage due to seizures can be prevented. Methodology: A LC-MS method was developed and validated for the simultaneous quantification of the biomarkers in selected vitamin responsive pediatric seizures from dried blood spots. Results: Application of the validated method to a seizure cohort of 46 patients indicated strong agreement of the method for clinical validity. Reference intervals for these biomarkers in dried blood spots were also determined for the population, after screening 956 neonates. Conclusion: The developed method was seen to be sensitive, linear, accurate and precise for testing vitamin responsive pediatric seizures.

Cadaver as a first teacher: A module to learn the ethics and values of cadaveric dissection.

OBJECTIVES: The undergraduate medical students must be made aware of the ethical and humanistic values of cadaveric dissection. This study therefore designed, implemented, and evaluated the impact of the module 'Cadaver as a Teacher' (CrAFT) that examines the ethical values of cadaveric dissection. METHODS: This prospective, multimethod study involved 447 first-year undergraduate medical students who had participated in all three sessions of the CrAFT module. Activities included interactive lectures, individual assignments, and a poster-making competition. Students offered a silent tribute and wrote words of gratitude down on a tribute wall. They also expressed their thoughts in the form of essays, poems, and collages. These reflections were qualitatively analysed to generate themes. At the end of the module, an online quiz was conducted to assess the knowledge gained by the students. Their scores were correspondingly recorded and calculated. RESULTS: The major themes identified were: cadaver as a teacher, acknowledgement and thanksgiving, bonding, and empathy. Out of all the test takers, 316 students (94.32%) scored more than a five out of ten. The students strongly felt that the module effectively sensitised them towards the ethical and humanitarian aspects of handling cadavers. CONCLUSIONS: The implementation of an educational module about cadavers is a novel approach towards sensitising medical students. The students believed that sensitising them early on would have helped them establish a practice grounded in professionalism, human values, and empathy.

Micro-Raman Spectroscopy Analysis of Optically Trapped Erythrocytes in Jaundice.

Derangements in bilirubin metabolism and/or dysfunctions in the hepato-biliary system lead to the unhealthy buildup of bilirubin in blood, resulting in jaundice. During the course of this disorder, circulating red cells are invariably subjected to toxic effects of serum bilirubin and an array of inflammatory compounds. This study aimed to investigate the vibrational spectroscopy of live red cells in jaundice using micro-Raman spectroscopy combined with optical-trap. Red cells from blood samples of healthy volunteers and patients with jaundice were optically immobilized and micro-Raman probed using a 785 nm diode laser. Raman signatures from red cells in jaundice exhibited significant variations from the normal and the spectral-markers were obtained from multivariate analytical methods. This research gives insightful views on how different pathologies can act as "stress-milieus" for red cells in circulation, possibly impeding their normal functions and also exasperating anemia. Raman spectroscopy, an emerging bio-analytical technique, is sensitive in detecting molecular-conformations in situ, at cellular-levels and in real-time. This study could pave way in understanding fundamental red cell behavior in different diseases by analyzing Raman markers.