Toxic potential assessment of hair dye developer 2,4,5,6-tetraaminopyrimidine sulfate exposed under ambient UVB radiation.

Synthetic cosmetics, particularly hair dyes, are becoming increasingly popular among people of all ages and genders. 2,4,5,6-tetraaminopyrimidine sulfate (TAPS) is a key component of oxidative hair dyes and is used as a developer in several hair dyes. TAPS has previously been shown to absorb UVB strongly and degrade in a time-dependent manner, causing phototoxicity in human skin cells. However, the toxic effects of UVB-degraded TAPS are not explored in comparison to parent TAPS. Therefore, this research work aims to assess the toxicity of UVB-degraded TAPS than TAPS on two different test systems, that is, HaCaT (mammalian cell) and Staphylococcus aureus (a bacterial cell). Our result on HaCaT has illustrated that UVB-degraded TAPS is less toxic than parent TAPS. Additionally, UVB-exposed TAPS and parent TAPS were given to S. aureus, and the bacterial growth and their metabolic activity were assessed via CFU and phenotype microarray. The findings demonstrated that parent TAPS reduced bacterial growth via decreased metabolic activity; however, bacteria easily utilized the degraded TAPS. Thus, this study suggests that the products generated after UVB irradiation of TAPS is considered to be safer than their parent TAPS.

Logic-based modeling and drug repurposing for the prediction of novel therapeutic targets and combination regimens against E2F1-driven melanoma progression.

Melanoma presents increasing prevalence and poor outcomes. Progression to aggressive stages is characterized by overexpression of the transcription factor E2F1 and activation of downstream prometastatic gene regulatory networks (GRNs). Appropriate therapeutic manipulation of the E2F1-governed GRNs holds the potential to prevent metastasis however, these networks entail complex feedback and feedforward regulatory motifs among various regulatory layers, which make it difficult to identify druggable components. To this end, computational approaches such as mathematical modeling and virtual screening are important tools to unveil the dynamics of these signaling networks and identify critical components that could be further explored as therapeutic targets. Herein, we integrated a well-established E2F1-mediated epithelial-mesenchymal transition (EMT) map with transcriptomics data from E2F1-expressing melanoma cells to reconstruct a core regulatory network underlying aggressive melanoma. Using logic-based in silico perturbation experiments of a core regulatory network, we identified that simultaneous perturbation of Protein kinase B (AKT1) and oncoprotein murine double minute 2 (MDM2) drastically reduces EMT in melanoma. Using the structures of the two protein signatures, virtual screening strategies were performed with the FDA-approved drug library. Furthermore, by combining drug repurposing and computer-aided drug design techniques, followed by molecular dynamics simulation analysis, we identified two potent drugs (Tadalafil and Finasteride) that can efficiently inhibit AKT1 and MDM2 proteins. We propose that these two drugs could be considered for the development of therapeutic strategies for the management of aggressive melanoma.

Benzo(ghi)perylene (BgP) a black tattoo ingredient induced skin toxicity via direct and indirect mode of DNA damage under UVA irradiation.

Tattooing is a very common fashion trend across all the ages and gender of the society worldwide. Although skin inflammatory diseases are very frequent among tattoo users because of the active chemical ingredients used in tattoo ink, yet no ingredient-specific toxicity study has been performed. Benzo(ghi)perylene (BgP) is one of the PAHs and an important ingredient of black tattoo ink that shows strong absorption in UVA and UVB radiation of sunlight. Therefore, understanding the hazardous potential of BgP especially under UVA exposure is important for the safety of skin of tattoo users by considering the fact that penetration of UVA is in the dermis region where tattoo ingredients reside. To evaluate the hazardous potential of BgP on human skin under UVA exposure, different experimental tools i.e., in-chemico, in-silico and in-vitro were utilized. Our results illustrated that BgP photosensitized under UVA (1.5 mW/cm2) irradiation shows a degradation pattern till 4 h exposure. Photosensitized BgP reduced significant cell viability (%) at 1 µg/ml concentration. However, the pretreatment of singlet and hydroxyl radical quenchers, restoration of cell viability observed, confirmed the role of type-I and type-II photodynamic reactions in phototoxicity of BgP. Further, intracellular uptake of BgP in HaCaT cells was estimated and confirmed by UHPLC analysis. Molecular docking of BgP with DNA and formation of γ-H2AX foci demonstrated the DNA intercalation and double-stranded DNA damaging potential of BgP. Furthermore, acridine orange and ethidium bromide (AO/EB) dual staining showed apoptotic cell death via photosensitized BgP under UVA irradiation. The above findings suggest that BgP reached the human skin cell and induced dermal toxicity via direct and indirect mode of DNA damage under UVA exposure finally promoting the skin cell death. Thus, BgP-containing tattoo ink may be hazardous and may induce skin damage and diseases, especially in presence of UVA radiation of sunlight. To minimize the risk of skin diseases from synthetic ingredients in tattoo ink, the study highlights the importance of developing eco-friendly and skin-friendly tattoo ingredients by companies.

Involvement of type-1 pathway in phototoxicity of benzo[ghi]perylenean ingredient of tattoo ink at ambient exposure of UVR and sunlight.

Tattooing on different parts of the body is a very common fashion trend in all sections of society globally. Skin allergies and other related skin diseases are very common among tattoo users. Benzo[ghi]perylene (BP) is a PAH and an important component of tattoo ink that showed prominent absorption under ultraviolet radiation (UVR) region. Therefore, to provide safety to the skin, a thorough safety study of BP exposed under UVR and Sunlight is very essential to understand their hazardous impact on the skin. BP showed a strong absorption of UVA and UVB radiation of sunlight. It is photolabile and degraded under UVA, UVB, and Sunlight in progressing order of time (1-4 h) without generating any novel photoproducts. Further, BP showed a specific generation of O2.- and OH radicals via activation of type I photodynamic reaction under exposure to UVA, UVB and Sunlight. Photocytotoxicity results illustrated concentration-dependent cell viability reduction in all exposure conditions of UVA, UVB, and Sunlight, respectively. Fluorescent probes (2',7'-dichlorofluorescein diacetate and dihydroethidium) for intracellular reactive oxygen species (ROS) generation supported the involvement of ROS in the phototoxicity of BP in the HaCaT cell line. Hoechst staining showed significant genomic insult induced by BP under UVA and UVB. Photoexcited BP promoted cell cycle arrest in the G1 phase and induced apoptosis confirmed via acridine orange/ethidium bromide staining. The findings of gene expression also supported apoptotic cell death in photoexcited BP via an increase in the level of pro-apoptotic gene (Bax) and a decrease in the level of anti-apoptotic gene (Bcl-2). The aforementioned finding indicates that tattoo users should avoid using BP since it can cause skin damage/diseases if they are exposed to UVR or Sunlight while tattooing on the body.

Tattoo inks are toxicological risks to human health: A systematic review of their ingredients, fate inside skin, toxicity due to polycyclic aromatic hydrocarbons, primary aromatic amines, metals, and overview of regulatory frameworks.

Today, tattooing has become very popular among people all over the world. Tattooists, with the help of tiny needles, place tattoo ink inside the skin surface and unintentionally introduce a large number of unknown ingredients. These ingredients include polycyclic aromatic hydrocarbons (PAHs), heavy metals, and primary aromatic amines (PAAs), which are either unintentionally introduced along with the ink or produced inside the skin by different types of processes for example cleavage, metabolism and photodecomposition. These could pose toxicological risks to human health, if present beyond permissible limits. PAH such as Benzo(a)pyrene is present in carbon black ink. PAAs could be formed inside the skin as a result of reductive cleavage of organic azo dyes. They are reported to be highly carcinogenic by environmental protection agencies. Heavy metals, namely, cadmium, lead, mercury, antimony, beryllium, and arsenic are responsible for cancer, neurodegenerative diseases, cardiovascular, gastrointestinal, lungs, kidneys, liver, endocrine, and bone diseases. Mercury, cobalt sulphate, other soluble cobalt salts, and carbon black are in Group 2B, which means they may cause cancer in humans. Cadmium and compounds of cadmium, on the other hand, are in Group 1 (carcinogenic to humans). The present article addresses the various ingredients of tattoo inks, their metabolic fate inside human skin and unintentionally added impurities that could pose toxicological risk to human health. Public awareness and regulations that are warranted to be implemented globally for improving the safety of tattooing.

Superoxide anion radical induced phototoxicity of 2,4,5,6-Tetraminopyrimidine sulfate via mitochondrial-mediated apoptosis in human skin keratinocytes at ambient UVR exposure.

2,4,5,6-Tetraaminopyrimidine sulfate (TAPS) is worldwide the most commonly used developer in hair dyes. As skin is the major organ, which is directly exposed to these permanent hair dyes, a comprehensive dermal safety assessment is needed. Hereto, we studied the photosensitization potential and mechanism involved in dermal phototoxicity of TAPS exposed to the dark and UVA/UVB/Sunlight by using different in-chemico and mammalian (HaCaT) cells, as test systems. Our experimental outcomes illustrate that TAPS get photodegraded (LC-MS/MS) and specifically generated superoxide anion radical (O2•-) under UVA and UVB via type-I photodynamic reaction. The phototoxic potential of TAPS is measured through MTT, NRU, and LDH assays that depicted a significant cell viability reduction at 25 µg/ml concentration and higher. Different cellular stainings (PI uptake, AO/EB, JC-1, NR uptake) suggested the role of mitochondrial-mediated apoptosis. Further, the transcriptomics study revealed upregulation of Apaf-1, Bax, Cytochrome c, Caspase 3, Caspase 9 and downregulation of Catalase and Bcl-2 by TAPS treated cells that strengthen our findings. Thus, the above findings suggest that chronic application of TAPS may be hazardous for human skin and promote various skin diseases.

Impact of glutathione S transferases P1 (Ile105Val) variants on the risk of GSTp, phosphorylated c-Jun kinase, and P53 phenotypic expression and their implications on overall survival outcomes in non-small cell lung cancer patients treated with chemotherapy.

AIM: This study focused on GST-M1, T1 null, and P1 Ile105Val variant genotypes associated with the risk of altered expression of GSTp, pJNK, and P53 in NSCLC patients. These markers and overall survival (OS) were correlated with a key set of clinicopathological characteristics. METHODS: Genotyping of GST- M1, T1 (+/-), and P1 (Ile105Val) was performed using PCR-RFLP.The expression of GSTp, pJNK, and P53 phenotypes was assessed by immunohistochemistry. The Spearman test was used to examine the correlation between GSTp, pJNK, and P53. Kaplan-Meier test was used for OS analysis. RESULTS: GSTP1 Val/Val and Ile/Val genotypes notably increased GSTp expression by 1.8 and 1.7 fold, respectively (p = 0.04,p = 0.06). GSTP1 Val/Val and Ile/Val genotypes considerably reduced P53 expression by 0.61 and 0.57 fold, respectively (p = 0.03& p = 0.05), respectively. GSTp, pJNK, and P53 were significantly co-expressed (p < 0.001). GSTp and pJNK expression showed a moderate negative correlation (ρ = -0.32, p = 0.046). In contrast, GSTp and P53 expression exhibited a strong negative correlation (ρ = -0.53, p < 0.0001). There was no correlation between P53 and pJNK expression(ρ = 0.07, p = 0.54). The patient's median OS was 8.9 months, and it was significantly related to pack-years, stage, metastasis, and GSTM1(-/-) genotypes (p > 0.05). SQCLC showed poor OS than ADC (5.7 months vs.9.1 months, p = 0.2). Stage IV and metastasis significantly reduced the OS (p = 0.001). The tumour size and lymph nodes reflected poor OS (p = 0.07&p = 0.06). Gemcitabine+Cisplatin and Gefitinib showed a slightly higher rate of survival (9.3 months and 8.1 months) than Pemtrexe+Cisplatin treatment (7.0 months,p = 0.8). Multivariate analysis revealed that pack-years and GSTp were independent predictors for OS (p = 0.03). CONCLUSION: GSTp, pJNK, and P53 showed interconnected cascading. Age, pack-year, stage, and GSTp were found to be significant predictive factors for OS.Pack-years, GSTp independent OS predictor.

Independent and Interactive Effect of CYPs and GSTs Genetic Variants and Tobacco Smoking on the Risk of Non-Small Cell Lung Carcinoma.

BACKGROUND: CYP and GST gene families detoxify tobacco carcinogens and have been linked to the risk of non-small cell lung carcinoma (NSCLC). AIM: Independent and combined effects of CYP and GST genetic variations and smoking on the risk of non-small cell lung carcinoma (NSCLC) and its sub-histological types. METHODS: We modelled an epistatic interaction via the effects of particular genotypes in two genes as OR (odds ratio), OR1, and OR2, a combination of both genotypes were characterized as ORcombine. In contrast, the two ORs' epistatic interaction for the individual genotypes has been represented as ORinteraction = ORcombine/(OR1 × OR2). RESULTS: The variant genotypes of CYP2A6 (OR:4.2, p <0.001), GSTT1 (OR:3.9, p <0.001), and GSTM1 (OR: 4.5, p <0.001) were showed a significant risk with NSCLC. GSTM1 (del.)/CYP2A6 (variant) genotype was associated with a higher risk of NSCLC (OR:12.5, p <0.001). GSTM1 (del.)/CYP2A6 (Ser/Pro+Pro/Pro) and GSTM1 (del.)/CYP2A13 (CT+TT) interacted redundantly (ORintraction = 0.66 and 0.64). A co-suppressive interaction was observed between GSTT1 (del.)/CYP2A6 (Ser/Pro+Pro/Pro) (ORintraction = 0.41). Simultaneously, both GSTT1/GSTM1 del. genotype was associated with a significantly higher risk to NSCLC. In contrast, GSTT1 del./GSTM1 del. genotype interaction displayed a co-suppressive effect (ORintraction = 0.15). CYP1A1(TC+CC)/CYP2A13(CT+TT)mutually interacted synergistically (ORintraction = 1.27).CYP1A1 (TC+CC)/GSTP1 (Val/Val+Ile/Val) genotype demonstrated an additive (ORintraction = 1) effect. GSTP1(Val/Val+Ile/Val) interacts with GSTT1 (del.) genotype exerted a suppressive effect (ORintraction = 0.69). CYP2A6 in smokers increased risk by 4.2 (p = 0.001) to 5.6 fold (p <0.001), while GSTM1 and GSTT1 were independent of smoking. CONCLUSION: Epistatic interactions revealed that CYPs/GSTs might follow a web of the interactions to modify the risk of NSCLC.

Ibuprofen Protects from Cypermethrin-Induced Changes in the Striatal Dendritic Length and Spine Density.

Microgliosis and inflammation are major wrongdoers in cypermethrin-induced Parkinsonism along with oxidative stress, mitochondrial dysfunction and α-synuclein aggregation. Dopamine depletion could alter dendritic morphology, length and spine number in the striatum. Present study investigated the effect of ibuprofen on the dendritic morphology, length and spine density in cypermethrin PD model. Male pups were treated intraperitoneally with cypermethrin during postnatal days followed by adulthood to induce Parkinsonism using standard procedure along with controls. Subsets of animals were pre-treated with ibuprofen 2 h prior to cypermethrin treatment during adulthood. Standard methods were used to confirm Parkinsonism/neuroprotection. Striatal dendritic morphology, length, spine number and expression of synaptophysin and postsynaptic density protein-95 (PSD-95) along with the nigrostriatal pro-inflammatory and apoptotic proteins were measured. Cypermethrin induced Parkinsonian traits and attenuated the dendritic length, spine number and expression of synaptophysin and PSD-95. While cypermethrin increased the expression of interleukin-1ß, interleukin-4, interferon-γ, inducible nitric oxide synthase, caspase-3, caspase-9 and B-cell lymphoma (Bcl)-xl proteins, it attenuated Bcl-2 expression. Ibuprofen normalized the changes in dendritic morphology, length, spine number and expression of synaptophysin, PSD-95, and pro-inflammatory and apoptotic proteins. Results demonstrate that cypermethrin induces inflammation and alters dendritic morphology, length and spine number, which are encountered by ibuprofen.

Early prognostic markers for fatal fulminant hepatic failure cases with viral hepatitis: proton nuclear magnetic resonance spectroscopic studies of serum.

BACKGROUND: Fulminant hepatic failure is associated with liver metabolic derangements which could have fatal consequences. The aim of the present study is to identify serum markers for early prediction of the outcome. METHODS: Proton nuclear magnetic resonance spectroscopic studies of serum of fulminant hepatic failure patients due to viral hepatitis with grade II/III of encephalopathy (twenty-four: ten prospective and fourteen retrospective) and twenty-five controls were undertaken. Of the twenty-four patients, fifteen survived with medical management alone while nine had fatal outcome. RESULTS: The results demonstrated significantly elevated indices of amino acids (alanine, lysine, glutamine, histidine, tyrosine, phenylalanine and 1,2-propanediol) in fatal cases compared to survivors and controls. Principal component analysis showed clear separation of fatal and surviving cases. Liver function parameters were significantly deranged in patients but they failed to provide early significant differences between surviving and fatal cases. Compared to model for end-stage liver disease scores, principal component analysis appear to be better as an early prognostic indicator. Biochemical mapping of pathways suggested interruptions in amino acid metabolism and urea cycle. CONCLUSIONS: Proton nuclear magnetic resonance studies of serum have the potential of rapidly identifying patients with irreversible fulminant hepatic failure requiring liver transplantation as life saving option.

Restoration of hepatocytes function following decompression therapy in extrahepatic biliary obstructed patients: metabolite profiling of bile by NMR.

Prolonged biliary obstruction and infection cause retention of biliary constituents in liver followed by hepatocyte dysfunction. Decompression therapy is important for both management and prognostic reasons and restores hepatocyte function. Quantitative metabolite profiling of bile using NMR spectroscopy at the time of obstruction and serially following decompression therapy using percutaneous transhepatic biliary drainage (PTBD) from nineteen patients with extrahepatic malignant biliary obstruction are presented. Based on detailed history, clinical condition, total leucocyte counts (TLC) and microbiological cultures of bile, patients were classified in two groups depending upon absence or presence of cholangitis. Statistical analysis was performed for comparison within each group using Wilcoxan sign square rank test. TLC and liver function tests indicated a trend towards recovery following decompression by one week. While on day 0 biliary constituents were undetectable in both the groups of patients, they increased significantly following one week of drainage with better recovery in patients with cholangitis compared to without. Free amino acids' signals were detected in all specimens starting from day 1 after decompression. This indicates disruption of blood-bile barrier during cholestasis and slow restoration of tight junction of hepatocytes following decompression leading to the appearance of biliary constituents in bile. Decompression therapy tends to restore biliary constituents with a prompt recovery in patients with cholangitis further supports such therapy for clinical management and outcome. To our knowledge this is the first report on the detection of amino acids in bile taken from common bile duct though they have been reported in hepatic bile.

Metabolomic analysis of serum by (1) H NMR spectroscopy in amyotrophic lateral sclerosis.

BACKGROUND: Amyotrophic lateral sclerosis (ALS), an invariably fatal neurological disorder shows complicated pathogenesis that poses challenges with respect to diagnosis as well as monitoring of disease progression. METHODS: We investigated metabolite profiles in the serum of 30 patients with ALS, 10 patients of Hirayama disease, which served as a neurological disease control and 25 healthy controls by using (1) H NMR spectroscopy. RESULTS: Compared to healthy controls, the ALS patients had higher quantities of glutamate (P<0.001), beta-hydroxybutyrate (P<0.001), acetate (P<0.01), acetone (P<0.05), and formate (P<0.001), and lower concentrations of glutamine (P<0.02), histidine (P<0.001) and N-acetyl derivatives. On the other hand, Hirayama disease patients had significantly higher median concentrations of pyruvate (P<0.05), glutamate (P<0.001), formate (P<0.05) and lower median concentrations of N-acetyl derivatives. Furthermore, we also found that serum glutamate showed a positive correlation (P<0.001, r=0.6487) whereas, histidine showed a negative correlation (P<0.001, r=-0.5641) with the duration of the disease in ALS. CONCLUSIONS: Such (1) H NMR study of serum may reveal abnormal metabolite patterns, which could have the potential to serve as surrogate markers for monitoring ALS disease progression.

1H NMR spectroscopic study of blood serum for the assessment of liver function in liver transplant patients.

BACKGROUND AND AIMS: Plausible reasons for the failure of liver graft in liver transplantation are explored. 1H-NMR spectroscopy of serum is employed for assessment of liver graft function. Differences in concentrations of specific metabolites between patients with successful and unsuccessful liver grafts following transplantation were used as possible markers to assess the graft quality. METHODS: Blood samples from the patients undergoing liver transplantation were obtained preoperatively, immediately after transplant followed by every 24 hrs of post-transplantation until patients were discharged or expired. 1H-NMR spectroscopic studies of serum were performed at each time point and concentrations of various metabolites measured. Conventional biological tests were also performed at each time point. RESULTS: Elevation of concentrations of the nine metabolites (lactate, alanine, lysine, glutamine, methionine, asparagine, tyrosine, histidine and phenylalanine) in non-survivors using NMR was attributed to the graft dysfunction. The information on the graft dysfunction using conventional biological tests was obtained much later. However, elevation in aminotransferases and bilirubin levels was indicated after about one week and 3 days respectively in non-survivors. Hepatic failure causes alteration in the concentrations of amino acids due to impairment of amino acid metabolism and urea cycle. 1H-NMR spectroscopy provides the information of all the metabolites in a single step without involving any chemical pretreatment implying better accuracy since each step involved can introduce its own experimental error. CONCLUSION: Distinct metabolic profile in non-survivors compared to survivors following transplantation promises potential of 1H-NMR studies in the assessment of liver graft function.

(1)H and (31)P NMR studies indicate reduced bile constituents in patients with biliary obstruction and infection.

Patients with extrahepatic biliary obstruction present with impairment of the normal bile flow, with jaundice and cholangitis as common complications. (1)H and (31)P NMR quantitative analysis of bile specimens from patients with extrahepatic biliary obstruction (n = 80) (with/without jaundice and cholangitis separately and together) was carried out for the chief biliary constituents to determine the relationship between biliary constituents and jaundice (serum bilirubin concentration >or=1.0 mg/dL) and cholangitis (total leucocyte count >11,000 cells/mm(3) and/or fever >38.5 degrees C with/without bile culture positivity). Compared with controls (patients without jaundice and cholangitis), median indices of the chief biliary constituents (total bile acids, cholesterol, phosphatidylcholine and inorganic phosphate) were significantly suppressed in patients with cholangitis and/or jaundice. Quantities of total bile acids, cholesterol and phosphatidylcholine correlated negatively with the quantity of bilirubin and with cholangitis, i.e. total leucocyte count. Suppression of biliary constituents correlated significantly with the severity of jaundice and cholangitis. The decrease in biliary constituents in the presence of jaundice and cholangitis is possibly the result of downregulation of the function of transporters located at the canalicular side of hepatocytes, leading to their suppressed indices in bile. This information may have implications in the examination of bile for clinical studies.

(1)H NMR spectroscopy of ascitic fluid: discrimination between malignant and benign ascites and comparison of the results with conventional methods.

It is often difficult to distinguish benign ascites from malignant ascites by conventional examination of ascitic fluid. Therefore, (1)H NMR spectroscopy of ascitic fluid specimens was explored as a one-shot experiment to identify potentially interesting metabolic indices that might help to differentiate between the two. Seventy ascitic fluid specimens (15 cytologically positive for malignant cells, eight cytologically negative for malignant cells but remaining suspicious for malignant ascites, and 47 due to liver cirrhosis) were subjected to (1)H NMR spectroscopy for quantitative estimation of 14 metabolites. Mean concentrations of the metabolites were compared with the Mann-Whitney U test. Multivariate discriminant function analysis was performed to determine important descriptors in the discrimination process. The sensitivity and specificity of the proposed model were compared with conventional methods using ascitic fluid protein and serum ascitic albumin gradient. Then, probable predictions for the doubtful cases were made using the proposed model. Patients with malignant ascites had significantly higher mean concentrations (microM) of beta-hydroxybutyrate (594 vs 61), lactate (5384 vs 2104), acetone (136 vs 69), and acetoacetate (122 vs 48) than patients with cirrhotic ascites, and significantly lower concentrations of glutamine (359 vs 615), citrate (62 vs 118), glucose (4933 vs 8411), tyrosine (44 vs 124), and phenylalanine (51 vs 93) (P < 0.05 for all). In the discriminant function analysis model, the best discrimination (P < 0.001) was achieved when beta-hydroxybutyrate, lactate, citrate and tyrosine were considered together as markers. Sensitivity and specificity of the proposed model, ascitic fluid protein and serum ascitic albumin gradient were found to be 100% and 97.9%, 53.3% and 76.6%, and 60% and 87.2%, respectively. The proposed model put five of the eight doubtful cases in the malignant group. This is encouraging and may provide useful information for clinical purposes.

Malabsorption syndrome with and without small intestinal bacterial overgrowth: a study on upper-gut aspirate using 1H NMR spectroscopy.

Biochemicals in the upper-gut aspirate in 31 patients with malabsorption syndrome (MAS) with and without small intestinal bacterial overgrowth (SIBO), and 10 disease-free controls were analyzed using high-resolution (1)H-NMR spectroscopy, and were correlated with the degree of SIBO and severity of MAS. Compared to controls, the patients had higher quantities (micromol/L: median [range]) of total bile acids/cholesterol (2000 [0-12000] vs. 300 [0-600]), lactate (700 [0-5200] vs. nil [0-30]), acetate (200 [0-6500] vs. 20 [0-200]), and formate (80 [0-900] vs. nil [0-50]) (P < 0.01, Mann-Whitney U-test). However, amino acids and glucose were comparable in both. Quantities (micromol/L: median [range]) of acetate (1330 [220-6500] vs. 100 [0-1430]), lactate (1430 [670-3300] vs. 300 [0-5200]), formate (360 [0-600] vs. 25 [0-800]), and unconjugated bile acids (500 [40-600] vs. 10 [0-300]) were higher in MAS patients with SIBO than those without SIBO (P < 0.01, Mann-Whitney U-test, for all). In patients with MAS the quantity of acetate positively correlated with the degree of SIBO, and unconjugated bile acids correlated with the degree of steatorrhoea (Spearman's rank correlation coefficient, two-tailed, P < 0.05: 0.46 and 0.52, respectively). This study demonstrates the bacterial production of metabolites and deconjugation of bile acids in patients with MAS.

1H NMR spectroscopic method for diagnosis of malabsorption syndrome: a pilot study.

Despite its well-documented limitations, colorimetry has been commonly used for the d-xylose test in the diagnosis of malabsorption syndrome (MAS). With a possibility of overcoming its limitations, the use of (1)H NMR spectroscopy for D-xylose test is explored herein. Urine samples from 35 adults with suspected MAS were obtained before and after oral ingestion of D-xylose. The diagnosis of MAS was based on fecal fat (72 h excretion using Van de Kamer's technique, normal < 7 g/24 h and/or Sudan III stain of spot stool specimen, normalor=1 g/5 g/5 h). In vitro experiments on the standard specimens of D-xylose were also performed independently using both methods. Colorimetry showed a lower value for the quantity of D-xylose excreted in urine than NMR [median 0.73 (0.17-1.89 g) vs 1.37 (0.17-3.23 g), respectively; p<0.0001, Wilcoxon's signed ranks test]. Colorimetry and NMR correctly diagnosed 11/12 and 10/12 (p=N.S.) patients with MAS and 14/23 and 20/23 (p<0.05) without MAS, respectively. Sensitivity and specificity of colorimetry and NMR were 91.6 and 60.7% vs 83.3 and 86.9%, respectively. In in vitro experiments, the values obtained for standard xylose using NMR showed a maximum error of 7%, whereas the colorimetric method showed 20%. The NMR method is simple and may be more accurate for the D-xylose absorption test. Colorimetry was found to be inferior as compared with NMR due to its low specificity.

Enhancement of human T cell response to a peptide epitope of 38 kDa antigen of Mycobacterium tuberculosis by liposomes.

Diagnosis of tuberculosis a problem, specially in the regions harboring an abundance of both pathogenic and non-pathogenic mycobacteria. This study was undertaken to assess in such a situation the predictive value of proliferative T cell response to a peptide epitope ('38G') of the 38 kDa membrane protein of Mycobacterium tuberculosis. 3[H]-thymidine incorporation assays were done with peripheral blood mononuclear cells of tuberculoid leprosy and pulmonary tuberculosis patients. The donors were also classified as PPD responders (Stimulation Index, SI> 3) or non-responders (SI < or = 3) on the basis of their T cell response to the 'Purified Protein Derivative (PPD)' of M. tuberculosis. 38G peptide was used in either free or liposome-associated form prepared by the technique of 'Dehydration-rehydration Vesicles' (Kirby and Gregoriadis, 1984). While free peptide failed to induce a positive response in study subjects, its liposomal form was T cell stimulatory and distinguished, to certain extent, between PPD responders (corresponding SI > 3 in 54% subjects) and non-responders (SI > 3 in 29% subjects). However, it did not differentiate between leprosy and tuberculosis. The study supports use of liposomes as adjuvant vehicles for antigenic peptides designed to activate human T cells.

Enhancement of human T cell response to a peptide epitope of 38kDa antigen of Mycobacterium tuberculosis by liposomes

Diagnosis of tuberculosis a problem, specially in the regions harboring an abundance of both pathogenic and non-pathogenic mycobacteria. This study was undertaken to assess in such a situation the predictive value of proliferative T cell response to a peptide epitope ('38G') of the 38 kDa membrane protein of Mycobacterium tuberculosis. 3[H]-thymidine incorporation assays were done with peripheral blood mononuclear cells of tuberculoid leprosy and pulmonary tuberculosis patients. The donors were also classified as PPD responders (Stimulation Index, SI> 3) or non-responders (SI 3 in 54 per cent subjects) and non-responders (SI > 3 in 29 per cent subjects). However, it did not differentiate between leprosy and tuberculosis. The study supports use of liposomes as adjuvant vehicles for antigenic peptides designed to activate human T cells.