Determination Of Plasma Levels Research Articles

A quantitiative LC-MS/MS method for the measurement of arachidonic acid, prostanoids, endocannabinoids, N-acylethanolamines and steroids in human plasma.

Free arachidonic acid is functionally interlinked with different lipid signaling networks including those involving prostanoid pathways, the endocannabinoid system, N-acylethanolamines, as well as steroids. A sensitive and specific LC-MS/MS method for the quantification of arachidonic acid, prostaglandin E2, thromboxane B2, anandamide, 2-arachidonoylglycerol, noladin ether, lineoyl ethanolamide, oleoyl ethanolamide, palmitoyl ethanolamide, steroyl ethanolamide, aldosterone, cortisol, dehydroepiandrosterone, progesterone, and testosterone in human plasma was developed and validated. Analytes were extracted using acetonitrile precipitation followed by solid phase extraction. Separations were performed by UFLC using a C18 column and analyzed on a triple quadrupole MS with electron spray ionization. Analytes were run first in negative mode and, subsequently, in positive mode in two independent LC-MS/MS runs. For each analyte, two MRM transitions were collected in order to confirm identity. All analytes showed good linearity over the investigated concentration range (r>0.98). Validated LLOQs ranged from 0.1 to 190ng/mL and LODs ranged from 0.04 to 12.3ng/mL. Our data show that this LC-MS/MS method is suitable for the quantification of a diverse set of bioactive lipids in plasma from human donors (n=32). The determined plasma levels are in agreement with the literature, thus providing a versatile method to explore pathophysiological processes in which changes of these lipids are implicated.

Determination of plasma levels of interleukin-17 in patients with vitiligo

Objective To investigate the relationship between interleukin-17 (IL-17) and vitiligo.Methods Totally,32 vitiligo patients and 30 healthy human controls were enrolled in this study.Blood samples were collected from all the subjects,and enzyme-linked immunosorbent assay (ELISA) was performed to determine the plasma levels of IL-17.The relationship of plasma IL-17 levels with disease stage,clinical course and lesion area was assessed.Results The plasma levels of IL-17 were significantly higher in the patients with progressive and stable vitiligo than in the healthy controls (both P ＜ 0.05),and higher in the patients with progressive vitiligo than in those with stable vitiligo (P ＜ 0.05).Moreover,the plasma levels of IL-17 were positively correlated with the area of vitiligo lesions (r =0.456,P ＜ 0.05),but unrelated to the clinical course of vitiligo (r =0.239,P ＞ 0.05).Conclusion IL-17 may play a certain role in the occurrence and development of vitiligo. Key words: Vitiligo; Interleukin-17; Immunity

PO-0018 Plasma Homocysteine In Overweight And Obese School Children

High values of plasma homocysteine (Hcy) level were highlighted and in the child obesity, elevated levels can be an independent risk factor for cardiovascular disease. Objective. Research of plasma Hcy values in overweight and obese school children compared with a control group of non obese children and without cardiovascular diseases and correlations with blood pressure values and blood levels of total cholesterol and tryglycerides. Methods. The authors researced in ambulatory 36 children (8–18 years) of which 26 overweight and obese and 10 non-obese healthy children, without cardiovascular diseases. All children were investigated by clinical exam, body mass index, determination of plasma levels of homocysteine in the morning after 12-hour fasting, total cholesterol, tryglyceride, bloog glucose. Results. Plasma levels of homocysteine were elevated in children with obesity, 42,18% of cases, compared to the control values (the mean 8,98 ± 1,73 μmol/ vs 6,1 ± 2,4 μmol/l). Increased levels of homocysteine in obese children was correlated with body mass index and more important to the in obese children with hypertension (5 cases). Elevated Hcy were often associated with high total cholesterol ± triglycerides in obese children compared with control values, normal values of blood glucose. Conclusions. Obesity in school children and especially association and the presence of high blood pressure werw associated with an increase in plasma homocysteine values often correlated with elevated total cholesterol ± triglyceride levels. Research and systematic monitoring of values of plasma homocysteine correlated with cholesterol and triglyceride levels allows an objective estimation of cardiovascular risk in children with obesity and an indication for cardiovascular prevention effective measures.

Decreased dickkopf-1 levels in chronic lymphocytic leukemia and increased osteopontin levels in non-Hodgkin's lymphoma at initial diagnosis: Could they be playing roles in pathogenesis?

AimsWe determined plasma levels of dickkopf-1 (DKK-1) and osteopontin (OPN) which have roles in the Wnt pathway in chronic lymphocytic leukemia (CLL) and non-Hodgkin lymphoma (NHL) patients and in healthy controls. We also tested whether DKK-1 and OPN levels could be of clinical or prognostic significance in CLL and NHL.MethodsWe included 36 CLL, 24 NHL patients, and 21 healthy controls. Patients’ clinical and demographic features, treatment modalities, and response to treatment were recorded. DKK-1 and OPN levels in plasma obtained at initial diagnosis were determined with enzyme-linked immunosorbent assay.ResultsCLL patients had significantly lower DKK-1 levels than NHL and control groups (P levels, respectively, 0.048 and 0.017). OPN level was significantly higher in NHL group than in CLL and control groups (P values, 0.017 and <0.001). CLL patients with early and late Rai stages of disease had similar DKK-1 and OPN levels. After a median follow-up of 48 months, 13 CLL patients died. Univariate analysis showed that advanced Rai stages and older age were significantly poor prognostic factors. DKK-1 level in CLL patients who have died was significantly lower than those who were alive (P = 0.035). NHL patients with extranodal involvement had significantly higher OPN levels than those with no involvement (P = 0.04).ConclusionsOur results demonstrated that the Wnt pathway inhibitor DKK-1 was decreased in CLL. OPN was increased in NHL and associated with extranodal involvement. In order to reveal the pathogenic and clinical roles of DKK-1 and OPN in CLL and NHL, larger studies need to be conducted.

A Specific and Sensitive HPLC–MS/MS Micromethod for Milrinone Plasma Levels Determination After Inhalation in Cardiac Patients

Milrinone administered through inhalation is an emerging method aimed at specifically reducing pulmonary hypertension without affecting systemic pressures. Its administration has been shown to be useful both in patients undergoing cardiac surgery and for persistent pulmonary hypertension of the newborn. These populations are prone to receive many concomitant medications and/or blood sampling may require a low volume quantification method. To address these issues in view of pharmacokinetic studies, this article aims to develop and validate a specific and sensitive analytical assay using high performance liquid chromatography (HPLC) and tandem mass spectrometry (MS/MS) detection for the quantification of milrinone plasma concentrations after inhalation in patients undergoing cardiac surgery. Plasma samples (50 μL) were extracted using ethyl acetate. Milrinone was separated on a C18 analytical column at 50°C. The mobile phase consisted of methanol and 10 mM ammonium acetate (45:55 vol/vol). The electrospray was operated in the negative ionization mode and monitored the following mass transitions: m/z 212.1 → 140.0 at 36 eV for milrinone and m/z 252.1 → 156.1 at 32 eV for olprinone. Calibration curves followed a quadratic regression in the concentration range of 0.3125-640 ng/mL. The lower limit of quantification is 0.3125 ng/mL and is based on a low plasma volume of 50 μL. Mean drug recovery and accuracy were ≥72.3% and 96.0%, respectively. Intraday and interday precision coefficient of variation (%) was ≤7.4% and ≤11.5%, respectively. The specificity allowed milrinone quantification in the multidrug administration conditions of cardiopulmonary bypass. This validated micromethod proved to be highly sensitive and specific while using a low volume of plasma. Its low volume and its lower limit of quantification indicate that this approach is suitable for further characterization of milrinone pharmacokinetics in both adults (inhalation) and neonates.

Efficient removal of colistin A and B in critically ill patients undergoing CVVHDF and sorbent technologies.

Colistin pharmacokinetics data are scarce regarding patients undergoing renal replacement therapy (RRT), or even absent as in patients treated with sorbent technologies potentially capable of removing colistin by extensive absorption on many polymeric materials. Twelve septic shock patients with acute kidney injury (AKI) undergoing RRT [continuous venovenous hemodiafiltration (CVVHDF) n = 7, coupled-plasma filtration adsorption-HF (CPFA-HF) n = 4, hemoperfusion n = 1] treated with colistin methanesulfonate at a dose of 4.5 × 10(6) U bid were studied. Colistin A (Col-A) and colistin B (Col-B) concentrations on plasma and effluent at time 0, 0.2, 1, 3, 6, 12, 24 and 48 h were determined by the liquid chromatography-tandem mass spectrometry method. With CVVHDF the sieving coefficient was lower for Col-A, peaked early (0.40 for Col-A at 10 min, and 0.59 for Col-B at 3 h) and declined after 48 h (0.22 and 0.30 for Col-A and Col-B, respectively). Colistin's filter clearance showed a similar pattern, with the highest clearance value of 18.7 ml/min for Col-B at 1 h. With CPFA-HF after the cartridge the Col-A and Col-B levels were negligible (<0.2 mg/l) or not detectable. The sum of the effluent and cartridge clearances reached values of 30 and 40 ml/min for Col-A and Col-B, respectively. With hemoperfusion the postcartridge concentrations for Col-A and Col-B were about 30 % lower than those determined precartridge. During CPFA-HF and CVVHDF, the extent of colistin removal is high, and patients should receive an unreduced dosage. However, due to risk of accumulation in long-term administration colistin plasma levels determination is recommended.

Association of PS gene polymorphism and soluble P-selectin levels in atrial fibrillation thromboembolism population in Xinjiang

To investigate the association between the polymorphism of P choose element (p. selectin, PS) and soluble P-selectin levels in atrial fibrillation (AF) thromboembolism in Han and Uigur population of Xinjiang. Method: Using ELISA method determination of plasma level of sPs. The frequency distributions of SNP sP-selectin gene promoter (−2123C/G) and SNP in exon region (Thr715Pro) were investigated by polymerase chain reaction (PCR)-restriction fragment length polymorphism and direct DNA sequence analysis among 302 Xinjiang Uigur and 340 age- and sex-matched Han people. Results: Cases sPs exist significant difference serum level and the control group. The frequencies of the −2123C/G allele among the Uigur population had no significant differences from those of the Han population. Thr715Pro did not show any polymorphism in the two populations. Conclusions: The sP-selectin gene polymorphisms are associated with serum sP-selectin levels or thromboembolic events, suggesting that the patients with nonvalvular AF and thromboembolic events may have genetic susceptibility.

IL-8 Gene Variants are Associated with Lung Function Decline and Multidimensional BODE Index in COPD Patients But Not with Disease Susceptibility: A Validation Study

Background and objective: COPD is a leading cause of dead worldwide and tobacco smoking is its major risk factor. IL8 is a proinflammatory chemokine mainly involved in the acute inflammatory reaction. The aim of this study was to test the association of IL-8, CXCR1 and CXCR2 gene variants and COPD susceptibility as part of a replication study and explore the effect of these variations in disease progression. Methods: 9 tagSNPs were genotyped in 728 Caucasian individuals (196 COPD patients, 80 smokers and 452 non-smoking controls). Pulmonary compromise was evaluated using spirometry and clinical parameters at baseline and annually over a 2 years period. We also determined plasma levels of TNF-α, IL-6, IL-8 and IL-16 in COPD patients. Results: There was a lack of association between gene variants or haplotypes with predisposition to COPD. No correlation was observed between the polymorphisms and cytokines levels. Interestingly, significant associations were found between carriers of the rs4073A (OR = 3.53, CI 1.34-9.35, p = 0.01), rs2227306C (OR = 5.65, CI 1.75–18.88, p = 0.004) and rs2227307T (OR = 4.52, CI = 1.49–12.82, p = 0.007) alleles in the IL-8 gene and patients who scored higher in the BODE index and showed an important decrease in their FEV1 and FVC during the 2 years follow-up period (p < 0.05). Conclusions: Despite no association was found between the studied genes and COPD susceptibility, three polymorphisms in the IL-8 gene appear to be involved in a worse progression of the disease, with an affectation beyond the pulmonary function and importantly, a reduction in lung function along the follow-up years.

The prognostic value of D‐dimer in lung cancer

Activation of coagulation and fibrinolysis is frequently encountered among cancer patients. Such tumors are supposed to be associated with higher risk of invasion, metastases and eventually worse outcome. The aim of this study is to explore the prognostic value of blood coagulation tests for lung cancer patients. Between 2009 and 2012, 72 newly diagnosed patients with lung cancer and 40 healthy subjects as control group were included in this prospective study. Patients were staged according to the seventh edition of the tumor, node, metastasis (TNM) classification. The treatment responses of patients were evaluated according to the World Health Organization (WHO) criteria. We measured plasma D-dimer level, activated partial thromboplastin time (APTT), prothrombin time (PT), international normalized ratio (INR), lactate dehhydrogenase (LDH), hemoglobin (Hb), platelet (Plt), white blood cells (WBC) count before, during and after chemotherapy. We investigated association of the results with stage and histologic type of the disease, as well as with response to therapy and survival in lung cancer patients. The median D-dimer, PT and INR levels of the patients with lung cancer were significantly higher than in the control group (P = 0,000). D-dimer, APTT, PT, INR, LDH levels after four cycles of treatment were significantly lower in responders than in nonresponders (P = 0,000). Plasma D-dimer levels were evaluated according to histopathological type and stage of diseases; D-dimer level was found significantly higher in metastatic disease (P < 0,5) and significantly lower in small cell lung cancer (SCLC) (P < 0,05). The mean follow-up was 574,14 ± 463,48 days. The mean survival was 750,866 ± 74,857 days (95% CI: 604,147 - 897,586). After second and fourth cycles of treatment, the plasma D-dimer, APTT, and LDH levels were higher in mortality group than in survival group (P = 0,000). After four cycles of treatment, the mean survival of the patients with serum D-dimer level above and below 1900 ng/mL was found to be significantly different (P = 0,000). The results suggest that determination of D-dimer plasma levels that is an inexpensive, easy and non invasive method may be useful in predicting clinical outcome, survival and treatment response of patients with lung cancer.

Pharmacokinetics of cefovecin (Convenia) in white bamboo sharks (Chiloscyllium plagiosum) and Atlantic horseshoe crabs (Limulus polyphemus).

Cefovecin was administered to six healthy adult white bamboo sharks (Chiloscyllium plagiosum) and six healthy adult Atlantic horseshoe crabs (Limulus polyphemus) to determine its pharmacokinetics in these species. A single dose of cefovecin at 8 mg/kg was administered subcutaneously in the epaxial region of the bamboo sharks and in the proximal articulation of the lateral leg of the horseshoe crabs. Blood and hemolymph samples were collected at various time points from bamboo sharks and Atlantic horseshoe crabs. High performance liquid chromatography was performed to determine plasma levels of cefovecin. The terminal halflife of cefovecin in Atlantic horseshoe crabs was 37.70 +/- 9.04 hr and in white bamboo sharks was 2.02 +/- 4.62 hr. Cefovecin concentrations were detected for 4 days in white bamboo sharks and for 14 days in Atlantic horseshoe crabs. No adverse effects associated with cefovecin administration were seen in either species.

Características Clínico-Epidemiológicas de la Oftalmopatía de Graves: Significado Etiopatogénico

OBJETIVO: Determinar si las características clínico-epidemiológicas y de laboratorio que presentan a su ingreso los pacientes hipertiroideos por Enfermedad de Graves, influyen en la ocurrencia de la oftalmopatía de Graves (OG). MATERIALES Y MÉTODOS: Estudio analítico, observacional, de corte transversal de 97 pacientes hipertiroideos, 69 cumplieron los parámetros del protocolo. RESULTADOS: La OG es más frecuente en mujeres en proporción de 1, 1: 1 y en la 51 década de la vida, aunque ninguno de estos factores mostró significancia. El inicio paralelo, dentro de los 6 primeros meses de manifestaciones sistémicas, sí es estadísticamente significativo. El tabaco demostró significancia estadística en el desarrollo de OG. Los grados severos son más frecuentes en varones. El exoftalmos está presente en 27% de pacientes y predominan los signos clínicos dependientes de partes blandas. CONCLUSIONES: Los primeros 6 meses de iniciadas las manifestaciones sistémicas y el antecedente de tabaquismo deben constituir motivo de alerta para detectar la aparición de OG. El exoftalmos es detectado en un tercio de pacientes, siendo la Exoftalmometría de Haertel un método 100% específico para su determinación. El dosaje sérico de Ig no es útil en la valoración del paciente en riesgo.

SFLT1/PIGF angiogenic relationship in the experimental model of preeclampsia induced by sympathetic activation in the mouse (679.5)

It has been reported that angiogenic factors involved during pregnancy, are altered during the pathogenesis of preeclampsia. Despite some conflicting results, the majority of studies indicate an increase of soluble factor‐like tyrosine kinase 1 (sFlt‐1) with reduced placental growth factor (PIGF). However, many doubts still prevail about the angiogenic response and its relation to this disease. The purpose was to determine plasma levels of sFlt‐1 and PIGF in healthy pregnant CD1 mice (n = 7 / duplicate) and a group of pregnant mice subjected to chronic cold stress (Walking on cold platform 0°C/20 min/day) for 11 days (from day 7 to 17), a validated model for experimental preeclampsia in rats. Results: the group subjected to cold stress showed decreased levels of sFlt‐1 in respect to control group (ct 7137±487 &lt; st 9742±846 pg/mL; p&lt;0.001 Student t). PIGF levels increased significantly with stress (ct 3.88±1.5 &lt; st 40.20±4.2 pg/mL; p&lt;0.001 Student t). Besides we quantified plasma glucose levels, which increased in a non significant in the stress group (163±44 ct &lt; st 174.6 ± 54 mg/dL). Moreover, stressed mice showed a non significant decrease in weight at 17 day of gestation (53.1 ± 4.4 ct &gt; st 51.9±8.3 g). These data may indicate that the results are polarized to a sympathetic nervous system response. In our work, the angiogenic factors behaved contrary to several reports. This could be due to release of Neuropeptide Y by sympathetic activation. This compound has been related to cause pro‐angiogenic effects. These may indicate that the diagnostic criteria of preeclampsia, not necessarily are associated with endothelial and / or angiogenic dysfunction.

Reduced Levels of Circulating 7α-Hydroxy-Dehydroepiandrosterone in Treated Adolescent Obese Patients

Elevated levels of glucocorticoids lead to the development of obesity and metabolic syndrome. Local glucocorticoid levels are regulated through the enzyme 11beta-hydroxysteroid dehydrogenase 1 (11beta-HSD 1), an enzyme that regenerates active cortisol from inert cortisone. Increased expression of 11beta-HSD 1 in adipose tissue promotes higher body mass index (BMI), insulin resistance, hypertension, and dyslipidemia. Human 11beta-HSD 1 is also responsible for inter-conversion of 7-hydroxylate metabolites of dehydroepiandrosterone (7-OH-DHEA) to their 7-oxo-form. To better understanding the mechanism of the action, we focused on 7-OH- and 7-oxo-DHEA, and their circulating levels during the reductive treatment in adolescent obese patients. We determined plasma levels of 7alpha-OH-DHEA, 7beta-OH-DHEA, and 7-oxo-DHEA in 55 adolescent patients aged 13.04-15.67 years, BMI greater than 90th percentile. Samples were collected before and after one month of reductive therapy. Circulating levels of 7alpha-OH-DHEA decreased during the reductive therapy from 1.727 (1.614; 1.854, transformed mean with 95 % confidence interval) to 1.530 nmol/l (1.435; 1.637, p<0.05) in girls and from 1.704 (1.583; 1.842) to 1.540 nmol/l (1.435; 1.659, p<0.05) in boys. With regard to the level of 7-oxo-DHEA, a significant reduction from 1.132 (1.044; 1.231) to 0.918 nmol/l (0.844; 1.000, p<0.05) was found after the treatment, but only in boys. No significant difference in 7beta-OH-DHEA levels was observed. In conclusions, diminished levels of 7alpha-OH-DHEA indicate its possible effect on activity of 11beta-HSD 1. Further studies are necessary to clarify whether competitive substrates for 11beta-HSD 1 such as 7alpha-OH-DHEA could inhibit production of glucocorticoids and may be involved in metabolic processes leading to reduction of obesity.

Impact of sleep-disordered breathing on myocardial damage and metabolism in patients with chronic heart failure

Sleep-disordered breathing (SDB) has a critical association with mortality and morbidity of patients with chronic heart failure (CHF). Troponin T is a marker of ongoing myocardial damage and predicts adverse clinical outcomes in patients with CHF. Carnitine plays an important role in the utilization of fatty acids in the myocardium. It has been reported that myocardial carnitine levels decrease in the failing heart. We hypothesized that plasma troponin T and carnitine are increased due to the leakage from damaged cardiomyocytes or the alteration of myocardial metabolism in CHF patients with SDB. We examined the relation of plasma troponin T and carnitine levels with severity of SDB in CHF. We used portable sleep monitor and measured the apnea-hypopnea index (AHI), plasma levels of high-sensitive troponin T and carnitine in 131 CHF patients. These patients were divided into three groups based on AHI: group A (None-mild SDB AHI<15/h, n=45), group B (Moderate SDB 15≤AHI<30/h, n=32) and group C (Severe SDB AHI≥30/h, n=54). Levels of high-sensitive troponin T and plasm total carnitine were significantly higher in group C than in groups A and B [high-sensitive troponin T; group A 0.009 (0.005-0.016), group B 0.012 (0.006-0.021), group C 0.021 (0.011-0.039) ng/ml, total carnitine; group A 61.0±15.1, group B 65.0±13.5, group C 73.3±17.5μmol/l, P<0.01 vs. group A and P<0.05 vs. group B, respectively]. Furthermore, in the multiple regression analysis, the independent factors to determine plasma levels of log (high-sensitive troponin T) were high-sensitive C-reactive protein and AHI, and the independent factors to determine plasma levels of carnitine were glomerular filtration rate and AHI. The present study suggests that SDB is associated with latent myocardial damage and alteration of myocardial carnitine metabolism in patients with CHF, presented by higher circulating troponin T and carnitine levels.

Determination of pergolide in horse plasma by UPLC–MS/MS for pharmacokinetic applications

Pergolide, an ergot-derived dopamine D2 receptor agonist, is used extensively as an orally administered treatment for pituitary pars intermedia dysfunction (PPID) in horses. One of the barriers associated with pergolide determinations in plasma for pharmacokinetic applications has been the technically demanding requirement for sensitivity. The objective of our work was to develop a simple assay for the determination of pergolide in plasma and demonstrate its potential application in the study of pergolide pharmacokinetics (PK) in horses. A UPLC–MS/MS assay was developed with a simple sample preparation involving methanol protein precipitation and injection of supernatant. The assay was applied to samples from a horse dosed with 10mg pergolide (as the mesylate salt) by nasogastric intubation. Plasma samples were collected over a 48h period. The assay demonstrated performance sufficient to enable application to low level PK studies. Within-batch precision and accuracy were within acceptance criteria; precision was less than 10% RSD (n=5) and accuracy was −7.3% at 0.014ng/mL, the lower limit of quantification was 0.006ng/mL and the method detection limit was 0.002ng/mL. In the treated horse, Cmax was 0.40ng/mL and the assay easily allowed determination of plasma levels in the elimination phase to 48h. In conclusion, this assay using UPLC–MS/MS and methanol protein precipitation easily meets the challenging demands of pergolide analyses in plasma.

Association of plasma GDF-9 or GDF-15 levels with bone parameters in polycystic ovary syndrome

We aimed to determine plasma levels of growth and differentiation factor (GDF)-9 and GDF-15, and their possible association with bone turnover parameters and bone mineral density (BMD), in patients with polycystic ovary syndrome (PCOS). Forty-two obese PCOS women aged 25-35years, 23 women with idiopathic hirsutism (IH) and 20 healthy controls matched for age and body mass index were enrolled. Anthropometric, metabolic and hormonal patterns, plasma GDF-9 and GDF-15 concentrations, bone turnover markers and BMD were measured. No significant differences were observed in bone turnover markers, BMD measurements, plasma GDF-9 and GDF-15 levels in subjects with PCOS compared with the other two groups. In the combined population of all three groups, GDF-15 concentrations were negatively correlated with osteocalcin (r=-0.317, p<0.01). Analysis of PCOS patients showed a significant correlation of GDF-15 concentrations with age and homeostasis model assessment index (r=0.319, p<0.05, and r=0.312, p<0.05, respectively). In addition, GDF-15 concentrations were negatively correlated with osteocalcin (r=-0.395, p<0.01) and positively correlated with urine deoxypyridinoline (r=0.353, p<0.05). GDF-9 did not correlate with bone markers and BMD measurements. In conclusion, plasma GDF-9 and GDF-15 levels as well as bone turnover markers and BMD measurements in subjects with PCOS (25-35years of age) were comparable with those either in subjects with IH or in healthy controls with similar anthropometric and metabolic profiles. GDF-15 might be a marker of a crossregulation between bone and energy metabolism.

Circulating Angiogenesis Factors in Patients with Non-Hodgkin’s Lymphoma

Aims: Non-Hodgkin’s lymphoma (NHL) is a heterogeneous group of hematological malignancies. Several angiogenic factors are important in NHL. The objective of this study was to determine plasma levels of various proangiogenic [vascular endothelial growth factor (VEGF), platelet derived growth factor (PDGF), tumor necrosis factor (TNFα), transforming growth factor (TGF-β), interleukin (IL-6), IL-8] and antiangiogenic [IL-4, IL-12, interferon gamma (IFN-)] factors in NHL patients and implication of CHOP (cyclophosphamide, hydroxydaunorubicin, oncovin, prednisone) therapy on these mediators. Study Design: This study was conducted on 100NHL patients recruited from the Oncology Hospital, Menofia University, Egypt. Fifty patients had different doses of CHOP chemotherapy and 50 patients were without treatment. Another 119 healthy blood donors were served as healthy controls. Methodology: Enzyme-linked immunosorbent assay (ELISA) was used to detect the concentrations of these mediators in plasma of NHL patients and normal controls. Results: Several proangiogenic (VEGF, PDGF, IL-8, and TNF-α) (P<.001, P<.01, P<.01, Original Research Article British Journal of Medicine & Medical Research, 4(17): 3366-3383, 2014 3367 and P<.001) and antiangiogenic (IL-4,IL-12) (P=.05,P<.01) mediators were decreased in NHL patients. In contrast, IL-6, and IFN-) were increased (P<.001). After CHOP treatment, VEGF, and PDGF were significantly increased (P<.001) as compared to NHL without therapy Fig.1. Reduction in IL-8 (P<.01) and TNF-α (P<.001) in untreated NHL patients was continued after CHOP treatment. IL-6 was the only elevated cytokine in NHL patients with (P<.001) or without (P<.001) treatment compared to healthy individuals. Concerning the antiangiogenic mediators, treatment resulted in reduction in the secretion level of IFN-) (P<.001) and IL-12 (P<.001). Although CHOP treatment required more than 8 doses to be able to down regulate proangiogenic (VEGF,PDGF, IL-8,TNF-α and IL-6) and elevate antiangiogenic (IL-4,IL-12,IFN-) mediators, changes were statistically insignificant. Conclusion: Collectively, our study stressed on the importance of having an angiogenic profile of NHL patients under treatment which could be used to monitor the efficacy of cancer therapy particularly in therapy with antiangiogenic drugs. Further studies are required to confirm these results.

Homocysteine: validation and comparison of two methods using samples from patients with pulmonary hypertension

Introduction and objective: The determination of homocysteine plasma levels has been reported as a risk marker of interest in severe diseases involving endothelial injury and associated with the development or progression of atherosclerotic lesions and thrombus formation. The aims of this study were to validate method for quantification of plasma homocysteine by high performance liquid chromatography (HPLC) with fluorimetric detection, and to compare the results obtained from patients with pulmonary hypertension by HPLC with those obtained by spectrophotometric enzymatic cycling (S-Ec) method. Materials and methods: The validation parameters, such as linearity, matrix effect, precision, accuracy, detection and quantitation limits, and robustness of the method were evaluated aiming to demonstrate that it is suitable for the intended use. The data obtained in the quantification of homocysteine using the validated method (HPLC) and the spectrophotometric enzymatic cycling (S-Ec) method, were compared. Results: The method was precise, accurate, and robust; it also had good recovery and showed no matrix effect. The linearity covered a range of 5.0-85.0 µmol/l and the limits of detection and quantification were 1.0 µmol/l and 3.4 µmol/l, respectively. The results obtained for homocysteine determination by HPLC and S-Ec methods were comparable. Conclusion: The validated HPLC method showed good performance for quantification of plasma homocysteine levels, while S-Ec method provided results for homocysteine comparable with those obtained by the validated method; therefore, this methodology is a potential alternative of automated method for clinical laboratories.

Plasma Activity of Endothelial Lipase Impacts High-Density Lipoprotein Metabolism and Coronary Risk Factors in Humans

Endothelial lipase (EL) is a determinant of plasma levels of high-density lipoprotein cholesterol (HDL-C). However, little is known about the impact of EL activity on plasma lipid profile. We aimed to establish a new method to evaluate EL-specific phospholipase activity in humans. Plasma samples were obtained from 115 patients with coronary artery disease (CAD) and 154 patients without CAD. Plasma EL protein was immunoprecipitated using an anti-EL monoclonal antibody after plasma non-specific immunoglobulins were removed by incubation with ProteinA. The phospholipase activity of the immunoprecipitated samples was measured using a fluorogenic phospholipase substrate, Bis-BODIPY FL C11-PC. The EL-specific phospholipase assay revealed that plasma EL activity was inversely correlated with HDL-C levels (R = -0.3088, p＜0.0001). In addition, the EL activity was associated with cigarette smoking. Furthermore, EL activity in CAD patients was significantly higher than that in nonCAD patients. Concomitantly, the HDL-C level in CAD patients were significantly lower than that in non-CAD patients. We have established a method for human plasma EL-specific phospholipase activity by combination of EL immunoprecipitation and a fluorogenic phospholipid substrate. Plasma EL activity was associated with not only plasma HDL-C levels but also the risks for CAD.

Decreased Mitogen Inducible Gene 6 (MIG-6) Associated with Symptom Severity in Children with Autism.

BACKGROUNDIndividuals with autism spectrum disorders (ASDs) demonstrate impairment in social interactions and problems in verbal and nonverbal communication. Autism spectrum disorders are thought to affect 1 in 88 children in the US. Recent research has shown that epidermal growth factor receptor (EGFR) activation is associated with nerve cell development and repair. Mitogen inducible gene 6 (MIG-6) is a 58-kDa non-kinase scaffolding adaptor protein consisting of 462 amino-acids, which has been shown to be a negative feedback regulator of EGFR and Met receptor tyrosine kinase (RTK) signaling.SUBJECTS AND METHODSIn this study, we determined plasma levels of MIG-6, which suppresses the EGFR RTK pathway in autistic children, and compared MIG-6 levels with the EGFR ligand, epidermal growth factor (EGF), and the cMET ligand, hepatocyte growth factor (HGF). MIG-6 levels were also compared to the symptom severity of 19 different autistic behaviors.Plasma MIG-6 concentration was measured in 40 autistic children and 39 neurotypical, age, and gender similar controls using an enzyme linked immunosorbent assay (ELISA). Plasma MIG-6 levels were compared to putative biomarkers known to be associated with EGFR and cMET and severity levels of 19 autism related symptoms [awareness, expressive language, receptive language, (conversational) pragmatic language, focus/attention, hyperactivity, impulsivity, perseveration, fine motor skills, gross motor skills, hypotonia (low muscle tone), tip toeing, rocking/pacing, stimming, obsessions/fixations, eye contact, sound sensitivity, light sensitivity, and tactile sensitivity].RESULTSIn this study, we found that plasma MIG-6 levels in autistic children (182.41 ± 24.3 pg/ml) were significantly lower than neurotypical controls (1779.76 ± 352.5; P = 1.76E − 5). Decreased MIG-6 levels correlated with serotonin, dopamine, Tumor necrosis factor alpha (TNF-alpha), and urokinase receptor (uPAR) concentration, but not with other tested putative biomarkers. MIG-6 levels also correlated significantly with severity of expressive language, receptive language, tip toeing, rocking/pacing, and hand flapping/stimming.CONCLUSIONSThese results suggest a relationship between decreased plasma MIG-6 levels, biomarkers associated with the EGFR pathway, and symptom severity in autism. A strong correlation between plasma MIG-6 and dopamine and serotonin levels suggest that decreased MIG-6 levels may be associated with abnormal neurotransmitter synthesis and/or action. A strong correlation between MIG-6 and uPAR and the inflammatory marker TNF-alpha suggests that low MIG-6 levels may be associated with the HGF/Met signaling pathway, as well as inflammation in autistic children.