Possible Effect of Chelation Treatment on Metabolomic and Lipidomic Analysis in Lead Exposure.

OBJECTIVE: This study aimed to examine patients with lead poisoning in terms of metabolomic profiles and bioactive lipids (oxysterols and sphingosine 1-phosphate [S1P]) before and after chelation therapy. METHODS: Consent was obtained from 42 individuals diagnosed with lead poisoning and blood and urine samples were collected before and after chelation therapy. The levels of 7-ketocholesterol (7-KC), cholestan-3b,5a,6b-triol (Ctriol), and S1P were measured via LC-MS/MS. Metabolomic analysis was performed via GC-MS. RESULTS: 7-KC and C-triol levels were detected higher before chelation therapy compared with after therapy (P < 0.001 for both). S1P levels were measured higher before the therapy. The results also showed that sphingolipid metabolism-related pathways were affected by lead toxicity as well as other related pathways. CONCLUSION: This preliminary study showed that lipid metabolism is affected in lead exposure and chelation therapy is effective in reversing possible damage.

Does bisphenol A bioaccumulate on zebrafish? Determination of tissue bisphenol A level.

Bisphenol A (BPA) is a high-production-volume industrial chemical mainly used in the production of polycarbonates and epoxy resins utilized in the manufacture of containers, bottles, toys, and medical devices. It has systemic effects as an endocrine disruptor even at low doses. To analyze its quantity in biological materials, sensitive and reproducible methods have to be used. Different doses and duration (90 and 900 µg/L, 24 and 120 h, and 21 days) of BPA exposure to whole body zebrafish were analyzed after specific homogenization of tissue, and then a modified method HPLC was used. The mobile phase was acetonitrile and water using a gradient method of reversed-phase C18 column, and excitation = 227 nm/emission = 313 nm. The calibration curve for BPA using HPLC-fluorescence detection method was between a concentration range of 1 and 1000 ng/mL and linear, and r2 = 0.999. The mean and standard error of mean values were 4.29 ± 1.05, 2.50 ± 0.92, and 2.53 ± 0.68 for control; 10.43 ± 2.61, 11.46 ± 3.24, and 8.55 ± 3.11 for BPA-90 µg/L; and 17.78 ± 4.39, 21.55 ± 4.37, and 25.32 ± 3.25 for BPA-900 µg/L (24 h, 120 h, and 21 days, respectively). Although some statistical significance among dose/time was observed between two different dose-treated groups, statistical significance was not found in dose/time results within the group. However, the positive result of BPA in the control group can be explained by low-dose, chronic exposure or prevalence of endocrine-disrupting chemicals.

The evaluation of serum tryptophan and kynurenine levels in patients with obstructive sleep apnea syndrome.

PURPOSE: The kynurenine (Kyn) pathway may play a role in certain physiological functions such as behavior, sleep, thermoregulation, and pregnancy. Tryptophan (Trp) is oxidized with tryptophan 2,3-dioxygenase and indolamine 2,3-dioxygenase (IDO). Under normal conditions, hepatic kynurenine is a transcription factor and IDO expression in healthy tissues is very low. The ratio of Kyn to Trp can be used as an indicator to assess IDO activity. This study aimed to determine the relationship between Kyn/Trp ratio and obstructive sleep apnea syndrome (OSAS) disease activity. METHODS: Study participants were categorized in 3 groups: Group 1 included patients with mild OSAS, Group 2, patients with moderate to severe OSAS, and Group 3, individuals considered normal to serve as controls. The demographic characteristics of the patients were recorded. Apnea-hypopnea index (AHI) and oxygen desaturation index (ODI) measurements were performed by diagnostic polysomnography (PSG). Trp and Kyn levels were determined by HPLC-UV method. RESULTS: Group 1 included 30 patients (18 men) with mild OSAS; Group 2 included 42 patients (31 men) with moderate to severe OSAS; and Group 3 included 25 controls (13 men). While there was no statistically significant difference between the levels of tryptophan and kynurenine in the groups, a significant difference was found between the Kyn/Trp ratios. A significant correlation was observed in individuals with a body mass index less than 25 with the Kyn/Trp ratio. In individuals with mild OSAS, a significant correlation was observed between ODI and BMI. In individuals with moderate to severe OSAS, there was a significant correlation between ODI, AHI, and BMI. CONCLUSION: In this study, there was no relationship between OSAS disease severity and IDO activity as assessed by immunoreactivity via the Kyn/Trp pathway.

Evaluation of oxysterol levels of patients with silicosis by LC-MS/MS method.

Silicosis is one of the prolonged and irreversible occupational diseases. Crystalline silica dust, which has been linked with silicosis, occurs in different industrial areas such as constructions, ceramic, quarry, and pottery. There are significant numbers of newly diagnosed cases every year in Turkey. Patients with silicosis suffer from inflammatory respiratory disorders and silicosis-related complications such as rheumatoid arthritis, systemic sclerosis, and vasculitis. Oxysterols are defined as 27-carbon intermediates or end products of cholesterol. They are also implicated in the etiology of disease states such as atherosclerosis, neurodegenerative, and inflammatory diseases. The aim of the study is to evaluate cholesterol oxidation products in the patients with silicosis and determination of sphingosine-1-phosphate (S1P) levels which is a sphingolipid metabolite. In addition to these parameters, it is aimed to determine the possible lipid peroxidation by different parameters. For this purpose, blood samples and urine were collected from 47 patients and 30 healthy individual with their consents. In order to evaluate oxysterols, 7-ketocholesterol and cholestan 3ß,5α,6ß-triol levels were measured by LC-MS/MS method. The measured levels of 7-KC were 0.101 ± 0.005 µmol/l in patient and 0.050 ± 0.003 µmol/l in control plasma samples. Triol levels were measured as 0.038 ± 0.005 µmol/l in patient group and 0.033 ± 0.004 µmol/l in control group (p < 0.001). In addition, lipid peroxidation products were measured by human-8-isoprostane, human-4-hydroxynonenal (4-HNE), and human malondialdehyde (MDA) ELISA kits. The measured levels of HNE in the patient and control groups were 735.14 ± 288.80 pg/ml and 595.72 ± 108.62 pg/ml in plasma and 606.02 + 118.23 pg/ml and 531.84 + 107.18 pg/ml in urine, respectively (p < 0.05). F2-iP results of patients and controls were 450.0 + 101.40 pg/dl and 386.9 + 112.7 pg/ml for urine and 432.7 ± 188,8 pg/dl and 321.9 ± 69.4 pg/dl for plasma, respectively (p < 0.05). MDA levels of plasma were measured as 44.1 ± 14.6 nmol/ml in the patient and 31.9 ± 10.5 nmol/ml in the control (p < 0.05). Levels of MDA for urine samples were 30.15 + 5.06 nmol/ml and 25.15 + 6.07 nmol/ml in patients and controls, respectively (p < 0.05). S1P levels were decreased in patients compared to control group (49.05 ± 10.87 and 67.57 ± 16.25, p < 0.001). The results not only indicate a correlation between cholesterol oxidation, lipid peroxidation, and silicosis, but also provide better understanding of the role of the lipids in the mechanism of this inflammatory disease.

Determination of Tryptophan and Kynurenine by LC-MS/MS by Using Amlodipine as an Internal Standard.

Tryptophan is an essential amino acid that plays an important role in cell metabolism, and kynurenine is its main metabolic pathway. By using ultra-high-performance liquid chromatography coupled to electrospray ionization triple-quadrupole mass spectrometry, tryptophan and kynurenine were determined using amlodipine as an internal standard. The analysis was carried out on an ACE-C18 (4.6 mm × 50 mm, 5 µm) reversed-phase analytical column using the gradient elution mode. For quantitative determination, amlodipine was used as an internal standard. Detection was performed using multiple reaction monitoring in electrospray ionization mode at m/z 205.1 → 117.7 and 187.9 for tryptophan, m/z 209.1 → 146 and 93.9 for kynurenine, and m/z 409.2 → 294.1 for the internal standard. Good linearity of the analyte to internal standard peak area ratios was seen in the concentration range 1.25-4000 ng/mL for tryptophan and 0.5-1600 ng/mL for kynurenine. The method showed excellent linearity with regression coefficients of 0.99 for kynurenine and 0.996 for tryptophan. The limits of quantification were 0.55 ng/mL for tryptophan and 0.47 ng/mL for kynurenine. The % RSD for all analytes ranged from 0.3 to 3.4% for intraday and 0.4 to 8.9% for interday experiments. A simple LC-MS/MS method has been developed and validated for measuring Kyn and Trp by using an affordable and more easily available internal standard, which is amlodipine.

Does Pepsin Play a Role in Etiology of Laryngeal Nodules?

OBJECTIVES: Vocal fold nodules (VFNs) are benign disorders affecting the superficial lamina propria of the true vocal folds. The etiology of VFNs still remains unclear but laryngeal trauma caused by vocal abuse, tobacco, alcohol, and laryngopharyngeal reflux (LPR) plays a crucial role on the pathogenesis. The aim of this study was to assess the presence of pepsin in formalin-fixed, paraffin-embedded (FEPE) specimens of VFNs to evaluate the role of LPR as a risk factor for VFNs. MATERIALS AND METHODS: A total of 28 pathology specimens of patients suffering from VFNs who had undergone laser microsurgery under general anesthesia were evaluated. The specimens were maintained in paraffin blocks in the pathology department. Western blot (WB) and enzyme-linked immunosorbent assay (ELISA) analyses were used to measure pepsin enzyme levels in the VFNs tissue specimens. Signs of LPR were assessed according to the reflux finding score. RESULTS: The mean reflux finding score of the patients was 13.6 ± 2.89 (8-21). According to WB and ELISA analyses, pepsin was detected with both the WB the ELISA tests in positive controls, but there was no pepsin enzyme in any of the 28 laryngeal FEPE VFNs specimens. CONCLUSION: The pepsin enzyme was not detected in any of the FEPE VFNs specimens, and it is concluded that further studies are needed to reveal the role of pepsin in the etiology of VFNs.