Development, optimization and validation of LC-MS/MS method for the determination of DBS GALT enzyme activity.

Galactosemia is a carbohydrate metabolism disorder often caused by galactose-1-phosphate uridyl transferase (GALT) deficiency. Detecting GALT deficiency involves measuring intra-erythrocyte enzyme activity. We aimed to create a robust liquid chromatography-mass spectrometry (LC-MS/MS) method to assess GALT activity in dried blood spot (DBS) samples. We validated this method and compared it to the fluorometric approach. We investigated the impact of K2EDTA and lithium heparin tubes on enzyme activity to identify the best sample collection tube. We also assessed the reaction-stopping method. The developed approach employed [13C6]-galactose-1-phosphate as a substrate and UDP-N-acetylglycosamine as an internal standard (IS). The mean ± SD value for GALT activity of DBS samples was determined as 6.37 ± 1.96 µmol/gHb/hour. The linear range was 0.4-50 µM (2.4-310% of normal) in the DBS method. The % coefficient of variation (%CV) values were less than 15 for intra-day and inter-day repeatability studies. Over 90% recovery was achieved in recovery studies, and no ion suppression from matrix was detected. DBS samples were quite stable for 31 days under different storage conditions. Enzyme activity results reported as <3.5 U/g Hb by fluorometric method, were quantitatively determined for even very low concentrations by LC-MS/MS method.

Elevating healthcare through artificial intelligence: analyzing the abdominal emergencies data set (TR_ABDOMEN_RAD_EMERGENCY) at TEKNOFEST-2022.

OBJECTIVES: The artificial intelligence competition in healthcare at TEKNOFEST-2022 provided a platform to address the complex multi-class classification challenge of abdominal emergencies using computer vision techniques. This manuscript aimed to comprehensively present the methodologies for data preparation, annotation procedures, and rigorous evaluation metrics. Moreover, it was conducted to introduce a meticulously curated abdominal emergencies data set to the researchers. METHODS: The data set underwent a comprehensive central screening procedure employing diverse algorithms extracted from the e-Nabiz (Pulse) and National Teleradiology System of the Republic of Türkiye, Ministry of Health. Full anonymization of the data set was conducted. Subsequently, the data set was annotated by a group of ten experienced radiologists. The evaluation process was executed by calculating F1 scores, which were derived from the intersection over union values between the predicted bounding boxes and the corresponding ground truth (GT) bounding boxes. The establishment of baseline performance metrics involved computing the average of the highest five F1 scores. RESULTS: Observations indicated a progressive decline in F1 scores as the threshold value increased. Furthermore, it could be deduced that class 6 (abdominal aortic aneurysm/dissection) was relatively straightforward to detect compared to other classes, with class 5 (acute diverticulitis) presenting the most formidable challenge. It is noteworthy, however, that if all achieved outcomes for all classes were considered with a threshold of 0.5, the data set's complexity and associated challenges became pronounced. CONCLUSION: This data set's significance lies in its pioneering provision of labels and GT-boxes for six classes, fostering opportunities for researchers. CLINICAL RELEVANCE STATEMENT: The prompt identification and timely intervention in cases of emergent medical conditions hold paramount significance. The handling of patients' care can be augmented, while the potential for errors is minimized, particularly amidst high caseload scenarios, through the application of AI. KEY POINTS: • The data set used in artificial intelligence competition in healthcare (TEKNOFEST-2022) provides a 6-class data set of abdominal CT images consisting of a great variety of abdominal emergencies. • This data set is compiled from the National Teleradiology System data repository of emergency radiology departments of 459 hospitals. • Radiological data on abdominal emergencies is scarce in literature and this annotated competition data set can be a valuable resource for further studies and new AI models.

Research training program in a Turkish medical school: challenges, barriers and opportunities from the perspectives of the students and faculty members.

BACKGROUND: Providing medical students with opportunities for research experience is challenging for medical schools in developing countries. The Research Training Program (RTP), which is carried out in Ege University Faculty of Medicine (EUFM) parallel to the core curriculum, aims to improve the scientific competencies of the highly motivated students and to provide them with the opportunity to conduct a research. The purpose of this project is to evaluate RTP through the perspectives of students and faculty members. METHODS: This phenomenological study included two groups; students of RTP and faculty members who contributed to the program. Interviews were conducted with the research group whose selection was determined by maximum variation technique. Interviews with new individuals continued until data saturation was reached. Interpretative data analysis started with close reading of the transcripts and generating a list of codes. Coding by two independently, developing categories and themes were the following steps. RESULTS: Twenty-one RTP students and 14 faculty members were interviewed. The main motivation for students to participate was the desire to learn how to do research. The introduction course providing the students with the basic competencies needs to be improved in terms of practical activities. It was reported that during the project process students needed intensive guidance especially in finding a research topic and a mentor. The students' lack of time, deficit of enough mentoring and the fact that conducting a research does not provide a competitive advantage for residency are important obstacles to the completion of the program. The most frequently mentioned achievement of the students is to learn all the stages of the research as well as getting acquainted with critical thinking. CONCLUSIONS: This research showed that it was realistic to implement research programs for highly motivated students in medical schools with conditions like those in EUFM. The solution of mentor shortage emerged in this study is dependent on the adoption of student research as a national policy. Getting acquainted with the interrogative thinking style, conducting research, and making lifelong learning a core value are more important outcomes of research programs than the number of completed projects.

Development of a New Amperometric Biosensor for Measurement of Plasma Galactose Levels.

Galactosemia is an inherited disease that occurs as a result of insufficient or no synthesis of some enzymes (GALT, GALK, and GALE) in galactose metabolism. Failure to make an early diagnosis, especially in newborns, can lead to severe clinical and even fatal consequences. The aim of this study is to develop a biosensor for measuring free galactose in plasma. The immobilization components of the developed free galactose biosensor are screen printed carbon electrode (SCPE), Prussian blue (PB), chitosan (CHIT), Nafion (NAF), gold nanoparticle (GNP), and galactose oxidase (GaOX). The CHIT/GaOX/NAF-GNP/GaOX/CHIT-GNP/SCPE-PB electrode showed a sensitive amperometric response to detect galactose. While the surface characterization of the biosensor was performed with cyclic voltammetry and scanning electron microscopy, the optimization and performance characterizations were made by applying an amperometry technique. The amperometric operating potential for the free galactose biosensor was determined as -0.05 V. The linear detection range for the free galactose biosensor is between 0.025 and 10 mM. This range includes galactose levels in plasma of both healthy and patients. The percent coefficient of variation values calculated for intraday and interday repeatability of the developed biosensor are below 10%. The practical use of the biosensor, for which optimization and characterization studies were carried out, was tested in 10 healthy 11 patients with galactosemia, and the results were compared with the colorimetric method. In conclusion, the unique analytical properties and effortless preparation of the new galactose biosensor developed in this study make them serious candidates for point-of-care diagnostic testing.

Region-Based Semi-Two-Stream Convolutional Neural Networks for Pressure Ulcer Recognition.

Pressure ulcers are a common, painful, costly, and often preventable complication associated with prolonged immobility in bedridden patients. It is a significant health problem worldwide because it is frequently seen in inpatients and has high treatment costs. For the treatment to be effective and to ensure an international standardization for all patients, it is essential that the diagnosis of pressure ulcers is made in the early stages and correctly. Since invasive methods of obtaining information can be painful for patients, different methods are used to make a correct diagnosis. Image-based diagnosis method is one of them. By using images obtained from patients, it will be possible to obtain successful results by keeping patients away from such painful situations. At this stage, disposable wound rulers are used in clinical practice to measure the length, width, and depth of patients' wounds. The information obtained is then entered into tools such as the Braden Scale, the Norton Scale, and the Waterlow Scale to provide a formal assessment of risk for pressure ulcers. This paper presents a novel benchmark dataset containing pressure ulcer images and a semi-two-stream approach that uses the original images and the cropped wound areas together for diagnosing the stage of pressure ulcers. Various state-of-the-art convolutional neural network (CNN) architectures are evaluated on this dataset. Our experimental results (test accuracy of 93%, the precision of 93%, the recall of 92%, and the F1-score of 93%) show that the proposed semi-two-stream method improves recognition results compared to the base CNN architectures.

Sex-related differences in within-subject biological variation estimates for 22 essential and non-essential amino acids.

BACKGROUND: Measurement of serum amino acid (AA) concentrations is important in particular for the diagnosis and monitoring of inborn errors of AA metabolism. To ensure optimal clinical interpretation of AAs, reliable biological variation (BV) data are essential. In the present study, we derived BV data for 22 non-essential, conditionally essential, and essential AAs and assessed differences in BV of AAs related to sex. METHODS: Morning blood samples were drawn from 66 subjects (31 males and 35 females) once a week for 10 consecutive weeks. All samples were analyzed in duplicate using liquid chromatography-tandem mass-spectrometry. The data were assessed for outliers, trends, normality and variance homogeneity analysis prior to estimating within-subject (CVI) and between-subject (CVG) BV. RESULTS: CVI estimates ranged from 9.0 % for histidine (male) to 33.0 % for taurine (male). CVI estimates in males and females were significantly different for all AAs except for aspartic acid, citrulline and phenylalanine, in most cases higher in females than in males. Apart from for arginine, CVG estimates in males and females were similar. CONCLUSIONS: In this highly powered BV study, we provide updated BV estimates for 22 AAs and demonstrate that for most AAs, CVI estimates differ between males and females, with implications for interpretation and use of AAs in clinical practice.

Impact of the empirical therapy timing on the clinical progress of septic shock patients.

AIM: To evaluate the effect of timing of antimicrobial therapy on clinical progress of patients with septic shock. MATERIALS AND METHOD: We included 204 adult patients diagnosed with septic shock according to Sepsis-3 criteria between March 2016 and April 2021. One-month survival was evaluated using univariate and logistic regression analysis. RESULTS: Antibiotic treatment was initiated within 1 h of the vasopressors in 26.4 % of patients. One-month mortality did not differ significantly between patients with and without empirical therapy coverage on etiological agents. Univariate factors that significantly affected one-month survival were starting antibiotics at the first hour, the unit where the case was diagnosed with septic shock, SOFA scores, qSOFA scores, and lactate level. In multivariate analysis, diagnosis of septic shock in the Emergency Service, SOFA score ≥11, qSOFA score of three and lactate level ≥4 were significantly associated with one-month mortality. CONCLUSION: Training programs should be designed to increase the awareness of septic shock diagnosis and treatment in the Emergency Service and other hospital units. Additionally, electronic patient files should have warning systems for earlier diagnosis and consultation.

Erythrocyte deformability and oxidative stress in inflammatory bowel disease.

BACKGROUND: Oxidative stress and reduced microvascular flow are important factors in the pathogenesis of inflammatory bowel disease (IBD). The increased oxidative stress reduces the erythrocyte deformability. However, in IBD, there are no studies in the literature which evaluate erythrocyte deformability. AIMS: In our study, we investigated the effect of oxidative stress and erythrocyte deformability in IBD. METHODS: Forty-three patients with active IBD, 48 patients with inactive IBD and 45 healthy controls were included. The erytrocyte deformability, malonyldialdehyde levels, glutation peroxidase and sulfhydryl levels were measured in peripheral venous blood samples. RESULTS: Erytrocyte malonyldialdehyde levels in both active and inactive IBD were significantly increased compared with control groups. Plasma glutation peroxidase levels did not show statistically significant difference between all groups. The decreased plasma sulfhydryl levels in active IBD were statistically significant compared with both the inactive IBD and the control group, but plasma sulfhydryl levels in inactive IBD group did not show statistically significant differences when compared with the control group. Elongation index values in both active and inactive IBD increased significantly compared with the control group. Statistically significant correlations were not found between the elongation index and glutation peroxidase, malonyldialdehyde, sulfhydryl levels in all groups. CONCLUSIONS: Our study is the first to evaluate the erythrocyte deformability in IBD. In our study, increased erytrocyte malonyldialdehyde levels and decreased plasma sulfhydryl levels manifested the role of oxidative stress in the pathogenesis of the disease. It is thought that the increased erythrocyte malonyldialdehyde values cause the reduction in erythrocyte deformability.

Artificial Intelligence in Healthcare Competition (TEKNOFEST-2021): Stroke Data Set.

OBJECTIVE: The artificial intelligence competition in healthcare was organized for the first time at the annual aviation, space, and technology festival (TEKNOFEST), Istanbul/Türkiye, in September 2021. In this article, the data set preparation and competition processes were explained in detail; the anonymized and annotated data set is also provided via official website for further research. MATERIALS AND METHODS: Data set recorded over the period covering 2019 and 2020 were centrally screened from the e-Pulse and Teleradiology System of the Republic of Türkiye, Ministry of Health using various codes and filtering criteria. The data set was anonymized. The data set was prepared, pooled, curated, and annotated by 7 radiologists. The training data set was shared with the teams via a dedicated file transfer protocol server, which could be accessed using private usernames and passwords given to the teams under a nondisclosure agreement signed by the representative of each team. RESULTS: The competition consisted of 2 stages. In the first stage, teams were given 192 digital imaging and communications in medicine images that belong to 1 of 3 possible categories namely, hemorrhage, ischemic, or non-stroke. Teams were asked to classify each image as either stroke present or absent. In the second stage of the competition, qualifying 36 teams were given 97 digital imaging and communications in medicine images that contained hemorrhage, ischemia, or both lesions. Among the employed methods, Unet and DeepLabv3 were the most frequently observed ones. CONCLUSION: Artificial intelligence competitions in healthcare offer good opportunities to collect data reflecting various cases and problems. Especially, annotated data set by domain experts is more valuable.

Utilization of biological variation data in the interpretation of laboratory test results - survey about clinicians' opinion and knowledge.

INTRODUCTION: To interpret test results correctly, understanding of the variations that affect test results is essential. The aim of this study is: 1) to evaluate the clinicians' knowledge and opinion concerning biological variation (BV), and 2) to investigate if clinicians use BV in the interpretation of test results. MATERIALS AND METHODS: This study uses a questionnaire comprising open-ended and close-ended questions. Questions were selected from the real-life numerical examples of interpretation of test results, the knowledge about main sources of variations in laboratories and the opinion of clinicians on BV. A total of 399 clinicians were interviewed, and the answers were evaluated using a scoring system ranked from A (clinician has the highest level of knowledge and the ability of using BV data) to D (clinician has no knowledge about variations in laboratory). The results were presented as number (N) and percentage (%). RESULTS: Altogether, 60.4% of clinicians have knowledge of pre-analytical and analytical variations; but only 3.5% of them have knowledge related to BV. The number of clinicians using BV data or reference change value (RCV) to interpret measurements results was zero, while 79.4% of clinicians accepted that the difference between two measurements results located within the reference interval may be significant. CONCLUSIONS: Clinicians do not use BV data or tools derived from BV such as RCV to interpret test results. It is recommended that BV should be included in the medical school curriculum, and clinicians should be encouraged to use BV data for safe and valid interpretation of test results.

LC-MS/MS measurement of leukocyte cystine; effect of preanalytic factors.

Cystinosis is an autosomal recessive disorder characterized by the accumulation of cystine in lysosomes, causing irreversible damage to organs, especially the kidneys. Intracellular leukocyte cystine concentrations are used to diagnose cystinosis and to monitor cysteamine treatment. The aim of this study was to develop and validate a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method without derivatization capable of measuring leukocyte intracellular cystine concentrations. During development, the effects of using three different protein precipitation agents were evaluated in terms of sensitivity and the matrix effect, with 12% trichloroacetic acid providing the highest sensitivity. The effects of different blood collection tubes were also assessed in terms of recovery, matrix effect, and protein content. Compared to other methods, our method was quicker (run time of 3 min), was linear over the range 0.078-100 µM, and had lower limits of detection (0.0192 µM) and quantification (0.0582 µM). The intra-day and inter-day reproducibility %CVs were ≤10%. and the method had excellent recovery rates (94%-106%). Other parameters including matrix selectivity, injection carryover, leukocyte lysate stability were also validated and met the acceptance criterias of European Medicines Agency (EMA) Guideline. The assay was successfully applied to quantify cystine leukocyte concentration in healthy and cystinosis patients.

The decision-making levels of urine tetrasaccharide for the diagnosis of Pompe disease in the Turkish population.

Background Recently, urinary excretion of the tetrasaccharide 6-α-D-glucopyranosyl-maltotriose (Glc4) has been proposed as a marker for the diagnosis and monitoring of Pompe disease (PD). We aimed to determine the reference intervals and reliable decision-making levels of urine tetrasaccharide concentrations for the diagnosis of infantile- and late-onset Pompe patients in the Turkish population. Methods In this study, nine patients with PD (five of them with late-onset PD [LOPD]) and 226 healthy individuals (aged 0-64 years) were included. Urine Glc4 concentrations were determined using the ultra-high-performance liquid chromatography (UHPLC) tandem mass spectrometry (MS/MS) method. Results Our data showed that the urine tetrasaccharide levels decreased with age in healthy individuals (p < 0.001, r = -0.256). It was higher especially during the first year of life compared to that in the elder subjects. The tetrasaccharide level of Pompe patients was higher compared to that of healthy controls of the same age: 99 ± 68 mmol/mol creatinine for infantile onset vs. 4.0 ± 3.0 mmol/mol creatinine for healthy controls of the same age group and 12.1 ± 17.4 mmol/mol creatinine for late onset vs. 1.7±1.2 mmol/mol creatinine for healthy controls of the same age group. Conclusions The results of this study showed that the reference intervals of tetrasaccharide in urine changed over time; therefore, it is critically important to define age-based decision levels for the diagnosis of LOPD.

Non-invasive cortisol detection in saliva by using molecularly cortisol imprinted fullerene-acrylamide modified screen printed electrodes.

In this study, we developed an impedimetric sensor system by using molecularly cortisol imprinted acrylamide polymers on fullerene modified carbon electrode to detect cortisol in real saliva samples. The polymer layer was formed on fullerene modified screen printed carbon electrodes with the ratio of 1:2 (cortisol:acrylamide) and ammonium persulfate (APS) was used as the initiator of polymerization. The sensor surface was investigated by cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS) and Scanning Electron Microscopy (SEM). Performances of the electrode were determined as follows; calibration curve was calculated between 0.5 nM and 64 nM, R2 = 0.9939, LOD and LOQ were 0.14 nM and 0.44 nM, respectively. Real human saliva samples were obtained from 15 participants between 25 and 32 ages regardless of gender. The samples, which were collected at least 30 min after waking up and the new sensor method analysis were compared tandem mass spectroscopy coupled liquid chromatography system (LC-MS/MS). The sensor analysis showed a significant correlation with LC-MS/MS results (R2 = 0.9727).

Clinical Decision Support Systems: From the Perspective of Small and Imbalanced Data Set.

Clinical decision support systems are data analysis software that supports health professionals' decision - making the process to reach their ultimate outcome, taking into account patient information. However, the need for decision support systems cannot be denied because of most activities in the field of health care within the decision-making process. Decision support systems used for diagnosis are designed based on disease due to the complexity of diseases, symptoms, and disease-symptoms relationships. In the design and implementation of clinical decision support systems, mathematical modeling, pattern recognition and statistical analysis techniques of large databases and data mining techniques such as classification are also widely used. Classification of data is difficult in case of the small and/or imbalanced data set and this problem directly affects the classification performance. Small and/or imbalance dataset has become a major problem in data mining because classification algorithms are developed based on the assumption that the data sets are balanced and large enough. Most of the algorithms ignore or misclassify examples of the minority class, focus on the majority class. Most health data are small and imbalanced by nature. Learning from imbalanced and small data sets is an important and unsettled problem. Within the scope of the study, the publicly accessible data set, hepatitis was oversampled by distance-based data generation methods. The oversampled data sets were classified by using four different machine learning algorithms. Considering the classification scores of four different machine learning algorithms (Artificial Neural Networks, Support Vector Machines, Naive Bayes and Decision Tree), optimal synthetic data generation rate is recommended.

Assessing Anticancer Potential of Blueberry Flavonoids, Quercetin, Kaempferol, and Gentisic Acid, Through Oxidative Stress and Apoptosis Parameters on HCT-116 Cells.

In recent years, natural products gained popularity with their anti-inflammatory and antioxidant effects mediated by chemical compounds within their composition. Study results offering them as palliative therapy options in cancer or as anticancer agents with high levels of cytotoxicity brought a new approach to combine cancer treatment protocols with these products. From a different perspective, edible types of these products are suggested in daily diets due to their potential cancer preventive effects. Our preliminary work was on blueberry extracts (Vaccinium myrtillus) as a main representative of these natural products, and the contents of the extracts were analyzed with liquid chromatography tandem mass spectrometry (LC MS/MS) to reveal the composition and distribution of polyphenolic compounds within. The most abundant polyphenols detected in V. myrtillus extracts were quercetin, kaempferol, and a phenolic acid, gentisic acid (GA). The compounds were further evaluated on treated HCT-116 cells for their potential anticancer effects by measuring total antioxidant status, total oxidant status, and 8-hydroxydeoxyguanosine levels for evaluation of oxidative stress and through protein array analysis and flow cytometric analysis for evaluation of apoptosis. In analysis of oxidative stress parameters, reduced total oxidant levels and reduced oxidative stress index levels were found in cells treated with the compounds in comparison with untreated cells. In apoptosis-related protein profiles, at least twofold reduction in various apoptotic proteins was observed after quercetin and kaempferol treatment, whereas a different profile was observed for GA. Overall, results of this study showed that quercetin and kaempferol have strong cytotoxic, antioxidant, and apoptotic effects, although GA is mostly effective as an antioxidant polyphenol on HCT-116 cells.

Neuroprotective effects of melatonin upon the offspring cerebellar cortex in the rat model of BCNU-induced cortical dysplasia.

Cortical dysplasia is a malformation characterized by defects in proliferation, migration and maturation. This study was designed to evaluate the alterations in offspring rat cerebellum induced by maternal exposure to carmustine-[1,3-bis (2-chloroethyl)-1-nitrosoure] (BCNU) and to investigate the effects of exogenous melatonin upon cerebellar BCNU-induced cortical dysplasia, using histological and biochemical analyses. Pregnant Wistar rats were assigned to five groups: intact-control, saline-control, melatonin-treated, BCNU-exposed and BCNU-exposed plus melatonin. Rats were exposed to BCNU on embryonic day 15 and melatonin was given until delivery. Immuno/histochemistry and electron microscopy were carried out on the offspring cerebellum, and levels of malondialdehyde and superoxide dismutase were determined. Histopathologically, typical findings were observed in the cerebella from the control groups, but the findings consistent with early embryonic development were noted in BCNU-exposed cortical dysplasia group. There was a marked increase in the number of TUNEL positive cells and nestin positive cells in BCNU-exposed group, but a decreased immunoreactivity to glial fibrillary acidic protein, synaptophysin and transforming growth factor beta1 was observed, indicating a delayed maturation, and melatonin significantly reversed these changes. Malondialdehyde level in BCNU-exposed group was higher than those in control groups and melatonin decreased malondialdehyde levels in BCNU group (P<0.01), while there were no significant differences in the superoxide dismutase levels between these groups. These data suggest that exposure of animals to BCNU during pregnancy leads to delayed maturation of offspring cerebellum and melatonin protects the cerebellum against the effects of BCNU.

Pomegranate wine has greater protection capacity than red wine on low-density lipoprotein oxidation.

Although there is a large body of evidence on the main role of red wine in protection of low-density lipoprotein (LDL) against oxidation, there are few data on the role of pomegranate juice, which has high phenolic content. We conducted this study considering the possible importance of pomegranate wine as an antioxidant and in order to make a comparison between red and pomegranate wines. The phenol levels of pomegranate and red wines (4,850 mg/L gallic acid equivalents and 815 mg/L gallic acid equivalents, respectively) were in accordance with their total antioxidant activity (39.5% and 33.7%, respectively). Both wines decreased LDL-diene levels following a 30-minute incubation period compared with controls (145 +/- 3.2 micromol/mg of LDL protein). However, pure pomegranate wine demonstrated a greater antioxidant effect (P < .01) on diene level (110 +/- 4.6 micromol/mg of LDL protein) than pure red wine (124 +/- 3.2 micromol/mg of LDL protein). In conclusion, we suggest that pomegranate wine has potential protective effects toward LDL oxidation, and it may be a dietary choice for people who prefer fruit wines.

Methods for Determination of α-Glycosidase, ß-Glycosidase, and α-Galactosidase Activities in Dried Blood Spot Samples.

The lysosomal storage diseases (LDSs) are a heterogeneous group of inherited genetic disorders caused by defects of lysosomal proteins. The accumulation of undigested substrates from different catabolic pathways leads to cellular dysfunction. LSDs generally presents during early childhood and have a devastating impact on the families and on public health. Over the years, approaches for treatment of some LSDs have been developed with different strategies. Increasing availability of treatments of these diseases has accelerated the development of new methods and techniques for rapid diagnosis in patients with clinical indication.The use of dried blood spot (DBS) test has been proposed as a first tier test to identify patients with Gaucher, Pompe, and Fabry diseases. DBS usage is advantageous for the purpose of screening as it is non-invasive, sensitive, has low-cost and fast turnaround time compared to measurements in leucocyte and/or fibroblast culture. This chapter focuses on the activity measurement of three lysosomal enzymes (α-glucosidase, ß-glucosidase, and α galactosidase) in DBS samples by using fluorescent substrates and by the LC-MS/MS (liquid chromatography-mass spectrometry) method. All steps of the methods, from preparation of the solutions to calculation of the enzyme activity, will be explained in detail.

Infertility and the presence of insulin resistance are associated with increased oxidative stress in young, non-obese Turkish women with polycystic ovary syndrome.

STUDY OBJECTIVE: To investigate the relationship between both insulin resistance and fertility and the oxidant/antioxidant system in young, non-obese patients diagnosed with polycystic ovary syndrome (PCOS). DESIGN: Case-control study. SETTING: Department of Obstetrics and Gynecology, Ege University, Izmir, Turkey. PARTICIPANTS: PCOS patients without insulin resistance (IR-) (n = 33), PCOS patients with insulin resistance (IR+) (n = 27), and healthy controls (n = 30). Patients with PCOS and regular sexual intercourse were further divided into infertile (n = 14) and fertile (n = 15) groups. MAIN OUTCOME MEASURES: The malondialdehyde (MDA) and thiol levels as well as the catalase (CAT) and superoxide dismutase (SOD) enzyme activities. RESULTS: Both IR+ and IR- PCOS patients had higher MDA levels and lower thiol levels when compared to the controls (each P < .001). However, only IR- patients had significantly higher SOD (3700.81 ± 410.13 vs 2614.19 ± 611.80 U/g Hb; P < .001) and CAT (7565.06 ± 628.27 vs 6819.61 ± 539.2 U/g Hb; P < .001) activities when compared to the controls. Infertile PCOS patients had significantly higher MDA levels (347.5 ± 22.8 vs 278.6 ± 42.6 nmol/g Hb, P < .001) and lower thiol levels (498.5 ± 56.2 vs 568.5 ± 38.6 µmol/l, P = .001) when compared to fertile patients. CONCLUSIONS: The results of this study demonstrated an imbalance in the oxidative-antioxidative system of PCOS patients. This imbalance was worse in IR+ and infertile PCOS patients.

The effects of estrogen and raloxifene treatment on the antioxidant enzymes and nitrite-nitrate levels in brain cortex of ovariectomized rats.

Number of studies indicate that the female gonadal hormone estrogen protects women against several neurodegenerative diseases and cerebral ischemia via various mechanisms. The possible protective effects of estrogen are mediated mainly by three ways; the activation of steroid receptors and/or modulation of a neurotransmitter and/or direct antioxidative action. Therefore we aimed to investigate the effects of estradiol and raloxifene on levels of nitric oxide (NO) and antioxidant enzymes in brain cortex of ovariectomized female rats. Ten Sprague-Dawley rats were used as naive controls while 32 rats were ovariectomized at 120-140 days of age. Twelve weeks after ovariectomy: (1). Ovariectomized Placebo group (n=11), was given physiologic saline. (2). Estrogen group (n=10) was given Ethynyl estradiol, 0.1 mg/kg sc. (3). Raloxifene group (n=10) was given raloxifene, 1 mg/kg sc. At the end of the treatment period (8 weeks), rats were decapitated and cortex samples were dissected. Results showed that ovariectomy caused a decrease in total nitrite-nitrate levels. The NO levels of both the estrogen and the raloxifene group were higher than the placebo group. Catalase activities did not show any significant difference between the groups, while superoxide dismutase (SOD) activities were elevated via ovariectomy. Estradiol and Raloxifene treatment had no statistically significant effect on SOD activity.