Enzymatic assay of phosphatidylethanolamine in serum using amine oxidase from Arthrobacter sp

BackgroundIn human serum, as for phospholipids not containing choline, phosphatidylethanolamine (PE) exists approximately 5% in a whole phospholipid. PE is well known as one of the main components of biological membranes, and also plays important roles that contribute to apoptosis and cell signaling. However, it could not measure PE with other phospholipids due to a lack of choline in them.MethodsUsing an amine oxidase (EC 1.4.3.6), from Arthrobacter species, a simple and rapid enzymatic assay for measurements of PE in serum was established. That assay used the Hitachi 7170 analyzer to evaluate the analytical performance.ResultsThe average within-run CVs were 0.38–1.27% (n = 20) at 69–160 μmol/l. The correlation between values obtained with the present method (y) and the high-performance liquid chromatography (HPLC) method (x) was: y = 0.944x + 9.441 (r = 0.977, S_y|x = 5.82, n = 34). In addition, the reference interval of healthy subjects was 115 ± 45 μmol/l.ConclusionsThis new enzymatic method shows a high specificity for serum PE and can be easily applied to an automated analyzer. The present method is available as a novel marker of changes in the clinical condition of serum phospholipids.     

The relationship between the levels of gonadotropic hormones and OPG,leptin, TGF-β1 and TGF-β2 in Chinese adult women

BackgroundThe relationship between the levels of gonadotropic hormones and bone metabolism-related cytokines in Chinese women is unclear. We investigated the relationship between FSH and LH and OPG, leptin, TGF-β1, and TGF-β2 in Chinese women.MethodsA cross-sectional study of 694 Chinese women, aged 20 to 82 y was conducted. Levels of serum FSH, LH, OPG, leptin, TGF-β1, and TGF-β2 were determined.ResultsIn premenopausal females, serum follicle-stimulating hormone (FSH) and luteinizing hormone (LH) levels seemly showed no correlation with the cytokine levels. In perimenopausal females, serum FSH and LH levels showed significant positive correlation with osteoprotegerin (OPG) and transforming growth factor-β2 (TGF-β2) levels (r = 0.286 to 0.405, all P = 0.000), whereas they showed negative correlation with TGF-β1 levels (r = ? 0.413 and ? 0.354, all P = 0.000). In postmenopausal females, FSH and LH levels showed positive correlation with OPG levels (r = 0.247 and 0.241, all P = 0.000), negative correlation with leptin and TGF-β1 levels (r = ? 0.234 to ? 0.319, all P = 0.000), and no correlation with TGF-β2 levels. Multiple linear regression stepwise analysis revealed the following results. In premenopausal females, 2.0% and 1.5% of the changes in LH could be explained by OPG and leptin, respectively, while 1.9% of the changes in OPG could be explained by LH. In perimenopausal females, the determinants of OPG and TGF-β1 on FSH were 10.9% and 17.0%, respectively, and the determinants of OPG, TGF-β1 and TGF-β2 on LH were 4.5%, 4.9% and 16.4%, respectively. The determinants of FSH and LH on OPG were 14.5% and 2.5%, respectively. The determinant of FSH on TGF-β1 was 4.5%, while the determinant of LH on TGF-β2 was 16.4%. In postmenopausal females, the determinants of leptin and OPG on FSH were 10.2% and 2.8%, respectively, and the determinants of OPG and TGF-β1 on LH were 5.8% and 2.3%, respectively. The determinant of FSH on OPG, leptin and TGF-β1 were 6.1%, 3.4% and 9.2%.ConclusionsThese results indicate that age-related gonadotropic hormone levels are associated with changes in OPG, TGF-β1, TGF-β2 and leptin, and change with menopausal status.     

Serum tartrate-resistant acid phosphatase 5b (TRACP5b) activity as a biomarker for bone metastasis in non-small cell lung cancer patients

BackgroundDiagnosis and follow-up of bone metastasis (BMet) in non-small cell lung cancer (NSCLC) patients usually rely on symptoms and image studies. A serum marker of bone resorption may improve the quality of treatment in such patients. Tartrate-resistant acid phosphatase 5b (TRACP5b) is a specific marker for osteoclasts and we proposed it can be used as a marker of BMet in NSCLC patients.MethodsIn November 2002 till August 2008 serum samples were obtained from 141 newly diagnosed stage IIIA, IIIB or IV NSCLC patients and 41 normal subjects. All patients received baseline bone scintinography examination and evaluation of clinical symptoms as a standard of BMet diagnosis. Patients were divided into 2 groups by having BMet (Group I, n = 72) or not (Group II, n = 69). An in-house immunoassay using a TRACP-specific monoclonal antibody, 14G6, was used to measure the serum TRACP5b activity at pH 6.1.ResultsThe mean serum TRACP5b activities of Group I, Group II and normal subjects were 3.50 ± 2.23 U/l, 2.09 ± 0.72 U/l and 2.33 ± 0.52 U/l, respectively. After adjusting for age, stage, gender, and histology in a generalized linear model, Group I has significantly higher TRACP5b activity than Group II (p < 0.001). The receiver operating characteristic analysis established a cutoff value of 2.551 U/l to identify BMet in NSCLC patients with a sensitivity of 63.9% and a specificity of 76.8%. TRACP5b activity declined in patients who responded to treatment (p = 0.047), and elevated in patients who developed new BMet (p = 0.05).ConclusionsSerum TRACP5b activity test is a potentially useful adjunct in diagnosing and monitoring BMet in NSCLC. Further study is warranted to establish its real value in diagnosis and monitoring of BMet in NSCLC patients.     

Anserine inhibits carnosine degradation but in human serum carnosinase (CN1) is not correlated with histidine dipeptide concentration

BackgroundWe reported an association of a particular allele of the carnosinase (CNDP1 Mannheim) gene with reduced serum carnosinase (CN1) activity and absence of nephropathy in diabetic patients. Carnosine protects against the adverse effects of high glucose levels but serum carnosine concentration was generally low.MethodsWe measured the concentration of two further histidine dipeptides, anserine and homocarnosine, via HPLC. CN1 activity was measured fluorometically and for concentration we developed a capture ELISA.ResultsWe found an association between the CNDP1 Mannheim allele and reduced serum CN1 activity for all three dipeptides but no correlation to serum concentrations although anserine and homocarnosine inhibited carnosinase activity. Patients with liver cirrhosis have low CN1 activity (0.24 ± 0.17 μmol/ml/h, n = 7 males; normal range: 3.2 ± 1.1, n = 104; p < 0.05) and CN1 concentrations (2.3 ± 1.5 μg/ml; normal range: 24.9 ± 8.9, p < 0.05) but surprisingly, histidine dipeptide concentrations in serum are not increased compared to controls.ConclusionsSerum histidine dipeptide concentrations are not correlated to CN1 activity. The protective effect of low CN1 activity might be related either to turnover of CN1 substrates or a protective function of dipeptides might be localized in other tissues.     

Enzyme-linked immunosorbent assay (ELISAs) for metalloproteinase derived type II collagen neoepitope, CIIM--increased serum CIIM in subjects with severe radiographic osteoarthritis

ObjectivesIn joint degenerative diseases, the collagens are degraded by matrix metalloproteinases and protein fragments are released to serum as potential biomarkers.MethodsA collagen type II specific neoepitope, CIIM, was identified (…RDGAAG¹⁰⁵³) by mass spectrometry. Two ELISAs against the neoepitope were developed. CIIM was measured in cartilage explants in the presence or absence of protease inhibitors. CIIM was measured in OA synovial fluid (n = 51) and serum (n = 156). Knee OA was graded by standard Kellgren–Lawrence (KL) score.ResultsThe ELISAs showed good technical performance; CV%, < 13%. CIIM release from cartilage explants was blocked by the MMP inhibitor. CIIM was detected in synovial fluid. Furthermore, serum CIIM levels were significantly higher (P < 0.05) in those individuals with mild or severe OA than in those with no OA.ConclusionWe developed a new biomarker for joint degenerative diseases, which we demonstrated was derived from MMP-degraded type II collagen.     

Circulating osteoprotegerin is increased in the metabolic syndrome and associates with subclinical atherosclerosis and coronary arterial calcification

ContextThe relationship between osteoprotegerin (OPG) a glycoprotein related to bone metabolism and the metabolic syndrome (MS) has not been established.ObjectiveThe aim of this study is to evaluate OPG concentration in patients with MS and its association with subclinical atherosclerosis and coronary arterial calcification (CAC).Materials/methodsThe study included 238 asymptomatic patients. MS was diagnosed according to the NCEP/ATPIII guidelines. OPG was measured by ELISA. All subjects underwent ultrasonography of the common carotid arteries to measure intima-media thickness (IMT) and evaluate the presence of atheroma plaques. In a subgroup (n = 39) CAC was quantified by ECG-triggered cardiac computed tomography. Adipose tissue was excised from 25 patients and OPG expression by RT-PCR and immunohistochemistry was studied.ResultsPatients with the MS (n = 60) had higher OPG than patients without (n = 178) (p < 0.05). OPG correlated with IMT (r = 0.2, p = 0.005) and patients with atheroma plaques had higher OPG (p = 0.008) and also those with coronary artery calcification (p < 0.05).OPG expression was confirmed in adipose tissue (n = 12) and the expression was significantly higher in patients with MS than in those without (p = 0.003).ConclusionsThis study shows that OPG may potentially be a biomarker for cardiovascular risk/damage in the MS and identifies adipose tissue as a potential source of OPG.     

ADAMTS13, FVIII, von Willebrand factor, ABO blood group assessment in preeclampsia

IntroductionPreeclampsia (PE) is a multifactorial disease characterized by high blood pressure and proteinuria after the 20th week of pregnancy. PE is associated with fibrin deposition in placental microcirculation and intrauterine fetal growth retardation. We evaluated FVIII activity, VWF and ADAMTS13 plasma levels, according to O and “non O” blood groups, in women with severe PE (sPE).MethodsThis case-control study included 140 women; 55 pregnant with sPE, 35 normotensive pregnant and 50 non-pregnant women. VWF and ADAMTS13 antigen levels were assessed by ELISA (American Diagnostica). FVIII activity was measured by automated coagulometric method (Dade Behring) and ABO blood groups phenotyping was performed by indirect technique.ResultsFVIII activity and VWF levels were significantly higher comparing either sPE to normotensive pregnant (P = 0.01; P = 0.05) and to non-pregnant women (P = 0.00 in both cases) or normotensive pregnant and non-pregnant women (P = 0.00 in both cases). A significant decrease in ADAMTS13 levels was observed comparing either sPE to normotensive pregnant (P = 0.02) and non-pregnant women (P = 0.00) or normotensive pregnant and non-pregnant women (P = 0.00). FVIII activity and VWF levels were associated to O and “non O” blood groups only in non-pregnant women.ConclusionsThe increase of FVIII activity and VWF levels and the decrease of ADAMTS13 in sPE are not associated to O and “non O” blood groups. These alterations in hemostatic markers in sPE largely surpass those physiologically determined by ABO blood groups influence and may have masked the effect of O and “non O” groups in this disease. A concomitant analysis of VWF levels and ADAMTS13 activity and antigenic levels will be important to clarify the imbalance between these parameters found in sPE in the present study.     

Polymorphisms in the CC-chemokine receptor-2 (CCR2) and -5 (CCR5) genes and risk of myocardial infarction among Tunisian male patients

ObjectivesThe aim of the present study was to investigate the association between CCR2-Val64Ile and CCR5-Δ32 variants and the estimation of haplotypes with MI in a sample of the Tunisian population.Design and methodsA total of 290 unrelated MI patients and 282 healthy controls were studied. The CCR2-Val64Ile and CCR5-Δ32 variants were analyzed by PCR-RFLP.ResultsSubjects carrying at least one copy of the CCR5-deletion allele were significantly more common in the control group, suggesting an atheroprotective effect (adjusted OR = 0.44, 95% CI = 0.28–0.72, p = 0.001). Haplotype analysis showed that MI patients had significantly less 64Val-Del haplotype (9.9% vs. 21.3%, OR = 0.30, 95% CI = 0.21–0.43, p < 0.001) and 64Ile-Ins haplotype (12.3% vs. 16.7%, OR = 0.58, 95% CI = 0.42–0.80, p < 0.001).ConclusionA protective effect of the CCR5-Δ32 polymorphism against MI in the Tunisian population was found.     

Patient selection has a strong impact on cystatin C and Modification of Diet in Renal Disease (MDRD) estimated glomerular filtration rate

ObjectiveEstimation of the glomerular filtration rate (GFR) is essential for the evaluation of patients with kidney disease, and for correct dosage of drugs that are eliminated from the circulation by the kidneys. In most cases GFR is estimated based on serum creatinine and the Modification of Diet in Renal Disease (MDRD) formula. As both cystatin C and creatinine are used for the determination of GFR it is important to investigate if estimated GFR by the two methods differ in various patient groups.Design and methodsWe have compared cystatin C and MDRD estimated GFR calculated from the same request from primary care units (n = 488), a cardiology ward (n = 826), the cardiointensive care unit (n = 1026), two oncology wards (n = 919 and 1021), and the neurosurgical intensive care unit (n = 1515) in an observational cross-sectional study.ResultsWe found better agreement between the two GFR estimates in samples from primary care patients and patients in the cardiology wards, than in samples from oncology wards or the neurosurgical intensive care unit. In the latter settings there was a pronounced difference between the two GFR estimates.ConclusionThe comparisons show that differences in patient selections have a strong impact on the agreement between cystatin C and MDRD estimated glomerular filtration rate.     

Specific increase in serum autotaxin activity in patients with pancreatic cancer

ObjectivesTo evaluate the potential clinical significance of serum autotaxin (ATX) level in patients with cancers of the digestive system.Design and methodsSerum ATX activity was measured as the lysophospholipase D activity in patients with cancer of the esophagus (n = 8), stomach (n = 18), colorectum (n = 21), biliary tract (n = 19), or pancreas (n = 103) and in patients with benign pancreatic diseases (n = 73).ResultsAmong patients with various cancers of digestive system, increased serum ATX activity was predominantly observed among pancreatic cancer patients. Serum ATX activity was not increased in patients with chronic pancreatitis or pancreatic cysts. In the diagnosis of pancreatic cancer, the area under the receiver operating curve for serum ATX activity was 0.541 (95% CI, 0.435–0.648) for men and 0.772 (95% CI, 0.659–0.885) for women. No significant correlation was observed between serum ATX activity and CEA, CA19-9 or Dupan2 levels.ConclusionSerum ATX activity may be useful for identifying pancreatic cancer when used together with other serum markers of pancreatic cancer.     

Lipoprotein(a)-cholesterol: a significant component of serum cholesterol

BackgroundLipoprotein(a) [Lp(a)] is known to be a cholesterol-rich lipoprotein, however, the contribution of Lp(a)-cholesterol [Lp(a)-C] to the serum cholesterol and LDL-C levels has not yet been fully evaluated.MethodsWe determined the serum Lp(a)-C in 55 subjects with serum Lp(a) concentrations ranging from 9 to 129 mg/dl. To measure the serum Lp(a)-C concentrations, we developed an immunoaffinity gel assay; serum was incubated with Sepharose 4B gel coupled with immunoglobulin G (IgG) prepared from a polyclonal anti-Lp(a) goat antiserum. After separating Lp(a) from other lipoproteins, we determined the serum Lp(a)-C concentrations. Validation of the assay showed satisfactory results in terms of the specificity and reproducibility.ResultsThe mean cholesterol content of Lp(a), determined as Lp(a)-C/Lp(a), was 29.5 ± 10.4%. The serum Lp(a)-C values were found to be highly correlated with the serum Lp(a) mass (r = 0.923, p < 0.001). At serum Lp(a) levels of over 50 mg/dl, the contribution of Lp(a)-C to the serum total cholesterol was 10.2%. Further, the Friedewald formula overestimated the serum LDL-C by 20.4%.ConclusionsLp(a) contains approximately 30% cholesterol in each molecule. In subjects with markedly elevated serum Lp(a) concentrations, the Lp(a)-C values should be taken into account when evaluating the serum LDL-C.     

Circulating Nampt and RBP4 levels in patients with carotid stenosis undergoing carotid endarterectomy (CEA)

BackgroundObesity is a risk factor for atherosclerotic vascular disease. Altered adipokine secretion, including increased production of nicotinamide phosphoribosyltransferase (Nampt) and retinol binding protein 4 (RBP4) may link adipose tissue dysfunction to cardiovascular complications.MethodsWe determined Nampt and RBP4 serum concentrations in 193 consecutive patients with carotid stenosis prior to carotid endarterectomy (CEA) in relation to recently experienced ischemic events, markers of atherosclerosis and obesity, as well as anthropometric and clinical characteristics.ResultsNampt but not RBP4 was significantly higher in symptomatic patients who experienced an ischemic event within 6 months before surgery compared to asymptomatic patients (p = 0.001). In multivariate regression analysis Nampt was the only independent predictor of symptomatic carotid stenosis. Nampt correlated with peripheral leukocyte blood count (p < 0.0001) and with the number of macrophages/foam cells within carotid plaques (p = 0.042). However, Nampt and RBP4 serum concentrations did not correlate with the maximum percentage of carotid stenosis.ConclusionOur data suggest circulating Nampt as an independent predictor of recently experienced ischemic events in patients with carotid stenosis despite the lack of an association between Nampt and carotid atherosclerosis severity.     

Platelet activation and vascular endothelial growth factor 165 release in hepatocellular cancer

BackgroundVascular endothelial growth factor (VEGF₁₆₅) is stored, transported and released by platelets. Platelet functional abnormalities have been described in patients with hepatocellular carcinoma (HCC). Thus, this study was designed to investigate the behavior of VEGF₁₆₅ with respect to platelet activation in HCC.MethodsPlasma and serum VEGF₁₆₅ and plasma sP-selectin levels were analyzed in patients with HCC (n = 70) or cirrhosis (n = 45) and control subjects (n = 70). Given the thrombocytopenia that characterizes both HCC and cirrhotic patients, plasma VEGF₁₆₅ and sP-selectin as well as serum VEGF (plt-VEGF₁₆₅-load) levels were normalized by platelet counts.ResultsMedian concentrations of plasma VEGF₁₆₅/platelet (p = 0.002) and sP-selectin/platelet (p < 0.0001) were higher in HCC or cirrhotic patients compared to controls. Moreover, sP-selectin/platelet was the only independent variable predictive of plasma VEGF₁₆₅/platelet at multivariate analysis (p < 0.0001). Conversely, plt-VEGF₁₆₅-load correlated with tumor diameter (p < 0.05) but not with sP-selectin/platelet and was an independent predictor for 5 year overall survival (p = 0.012).ConclusionsThe results obtained are suggestive for VEGF₁₆₅ release by tumor in HCC. It is plt-VEGF₁₆₅-load, but not plasma VEGF₁₆₅ or serum VEGF₁₆₅ that is an independent predictor for overall survival of HCC patients.     

Variation of serum C-reactive protein (CRP) over time in pediatric cancer patients with febrile illness and its relevance to identified pathogen

ObjectiveTo evaluate the correlation of serum CRP with clinical and laboratory parameters proven to be related to the cause of infection in pediatric cancer patients.MethodsWe studied prospectively for a 12-month period, 37 pediatric cancer patients, who presented with 70 episodes of febrile illness (38 bacterial and 13 viral infections).At fever's onset and 48 h later, infection indices, such as CRP, WBC, ANC were measured in the peripheral blood. Moreover we calculated the change rate of CRP over 48 h [CRP/t = (CRP48h ? initial CRP) / t (t = 2 days)]. Cultures of biological fluids, PCR and antibody detection of infectious agents were also obtained.ResultsWhen comparing patients with viral vs. bacterial infections, mean CRP levels on admission (11.0 vs. 33.1 mg/L, p = 0.005) and at 48 h (13.4 vs. 71.9 mg/L, p = 0.0007), and CRP/t (0.9 vs. 18.8 mg/L/day, p = 0.030) were significantly lower in the group with viral infection.At 48 h - follow-up, patients with positive culture had higher CRP levels (57.3 vs. 43.3 mg/L, p = 0.048) and higher CRP/t (15.9 vs. 7.7 mg/L/day, p = 0.025), compared to those without proven infection. CRP/t at 48 h was correlated with both the fever duration (r = 0.27, p = 0.027) and maximum temperature (Tmax) during the febrile episode (r = 0.30, p = 0.013).ConclusionsSingle CRP values on fever initiation can differentiate between viral and bacterial infections in febrile pediatric cancer patients. Moreover the change rate of CRP over time (CRP/t) is offered as a prognostic index of bacterial infection and a marker of the total duration of fever and Tmax.     

Association between serum retinol-binding protein 4 and small dense low-density lipoprotein cholesterol levels in young adult women

BackgroundSerum retinol-binding protein 4 (RBP4) and small dense low-density lipoprotein (sdLDL) have been suggested to be associated with insulin resistance, but no information is available on the relationship between RBP4 and sdLDL.MethodsWe determined serum RBP4, sdLDL-cholesterol, and other metabolic variables on 38 young women, aged 19–29 years. The homeostatic model assessment of insulin resistance (HOMA-IR) was used for the estimation of insulin resistance.ResultsIn simple regression analyses, RBP4 levels had significant correlations with total cholesterol (r = 0.354, P = 0.029), LDL-cholesterol (r = 0.396, P = 0.014), and sdLDL-cholesterol (r = 0.510, P = 0.001) levels. The sdLDL-cholesterol levels also correlated significantly with total cholesterol (r = 0.402, P = 0.012), LDL-cholesterol (r = 0.627, P < 0.001) and triglycerides (r = 0.449, P = 0.005). Stepwise multiple regression analyses showed only sdLDL-cholesterol (β coefficient (ß) = 0.510, P = 0.001) level was a significant independent predictor of RBP4 levels (adjusted R² = 0.240), whereas RBP4 (ß = 0.289, P = 0.026) level was one of major factors affecting sdLDL-cholesterol levels (adjusted R² = 0.519). There was no significant association of HOMA-IR with RBP4 or sdLDL levels.ConclusionsWe showed an independent linkage between serum RBP4 and sdLDL-cholesterol levels in young adult women. These findings may contribute to understanding of lipoprotein metabolisms involved in diabetes and cardiovascular disease.     

Vital and functional outcomes of the first-ever hemispheric stroke, epidemiological comparative study between Kunming (China) and Limoges (France)

BackgroundClinical outcomes and socioeconomic consequences after a stroke may differ between regions.MethodsOne cohort was established prospectively in Kunming (China) to compare with a cohort of 156 stroke patients included in Limoges (France). During 1 year, patients hospitalized within 48 hours for a first-ever hemispheric stroke were included. Demographic data and neurocardiovascular risk factors were registered. Hemiplegia was evaluated. Functional outcome was assessed using the Barthel Index (BI) after 3 months.ResultsOne hundred and eighteen patients were included in Kunming. Patients of Kunming were younger (61.4 ± 13.4 vs 72.3 ± 14.6 years in Limoges, P < 0.0001), more involved in professional activity (36.4% vs 12.8%, P < 0.0001). Survival analysis indicated that mortality did not differ between cohorts, but independently predicted by coma at the 2nd day (HR = 9.33, 95% CI [4.39, 19.78]) and age > 70 years (HR = 6.29, 95% CI [2.36, 16.59]). Despite a better baseline BI for patients of Kunming (50.0 ± 34.9 vs 37.4 ± 34.2, P = 0.0031), after adjustment for confusing, patients in Limoges had a 2.11 OR 95% CI [1.03, 4.31]) to reach a BI > 80 at 3 months.ConclusionsFunctional recovery for patients of Kunming was not as good as expected. The socioeconomic consequences of stroke in Kunming are significant as they involved younger subjects who were still in work.     

Specific, rapid, and sensitive enzymatic measurement of sphingomyelin, phosphatidylcholine and lysophosphatidylcholine in serum and lipid extracts

ObjectiveHuman serum sphingomyelin (SM) and phosphatidylcholine (PC) play important roles in the development of atherosclerosis. However, there are no rapid and sensitive methods for SM and PC measurement. The present report describes a novel enzymatic method for measuring SM, PC and lysophosphatidylcholine (lyso-PC) levels in plasma and lipid extracts.Design and methodsThe total choline-containing phospholipids (total PL), SM and PC were measured using a two-reagent system involving specific enzymes for choline-based phospholipids. The procedure was performed using either microplate or automatic analyzer technology. The concentration of lyso-PC was calculated by subtracting the concentration of SM plus PC from the total PL concentration.ResultsAssay results showed linear correlations between sample concentration and absorbance. The within-run and between-run coefficients of variation for PC, SM, and lyso-PC concentrations were 2.0–4.4% for the microplate analyzer and 0.9–2.9% for the automatic analyzer. Analysis of normal human serum showed that the total PL concentration strongly correlated with the SM plus PC concentration (r = 0.9850). There were moderate correlations between serum PC and SM levels (r = 0.6228) and between serum PC and lyso-PC levels (r = 0.7806). SM, PC, and lyso-PC levels in normal human serum (n = 50) were 0.54 ± 0.07, 1.99 ± 0.22 and 0.60 ± 0.15 mmol/L, respectively.ConclusionThe present enzymatic method allowed for rapid, simple, and accurate measurement of SM, PC, and lyso-PC levels in lipid extracts and in serum. The method is suitable for both microplate and automatic analyzer assays.     

Influence of pre-analytical and analytical factors on osteoprotegerin measurements

IntroductionOsteoprotegerin (OPG), an osteoclastogenesis inhibitor implicated in bone remodelling, has emerged as a potential biomarker for cardiovascular disease. In order to implement OPG determination in the clinical laboratory, it is crucial to identify the most appropriate specimen type, preparation and measurement conditions. The present study focuses on identifying the pre-analytical variables that may influence OPG measurements.MethodsSerum and plasma (in EDTA, heparin and citrate) were collected from 45 healthy volunteers (men (n = 21, 46.7%), women (n = 24, 53.3%)). OPG was analysed by ELISA. The influence of the centrifugation speed, the number of freeze–thaw cycles, delay in sample processing, thermo-stability and endogenous interfering agents (haemolysis, triglycerides, bilirubin, cholesterol and RANKL) were studied.ResultsOPG concentrations were significantly lower (p < 0.0001) in serum (1015 ± 357 pg/mL) than in all plasma samples (1314 ± 448 pg/mL in EDTA, 1209 ± 417 pg/mL in heparin and 1260 ± 498 pg/mL in citrate).Increasing centrifugation speed (200 g to 3000 g) did not change serum OPG concentration (p = 0.88). However, OPG concentration significantly increased when centrifuged serum samples were stored at 48 h at room temperature (p < 0.0001). Repeated freeze–thaw cycles did not modify OPG levels until 4 cycles (p < 0.0001). Increasing time before processing the samples (2 h and 6 h) raised OPG concentrations both at room temperature (p < 0.0001) or 4 °C (p < 0.001).Positive concentration-dependent interference of triglycerides was found in the analysed pooled samples; however, OPG concentrations were falsely diminished with haemoglobin interference. Bilirubin, cholesterol and RANKL did not interfere with OPG measurements.     

Increased inflammatory response and imbalance in blood and urinary oxidant-antioxidant status in South Indian women with gestational hypertension and preeclampsia

ObjectiveThe objective of this study is to assess the association of blood and urinary oxidative stress parameters and inflammatory markers in women with gestational hypertension and preeclampsia.Design and methodsMalondialdehyde, protein bound sialic acid and C-reactive protein were estimated in serum and urine of pregnant women diagnosed with preeclampsia (n = 30) and gestational hypertension (n = 30) and the results were compared with 30 normal pregnant women.ResultsWhole blood glutathione level was reduced, and malondialdehyde and C-reactive protein levels were significantly higher and correlated with each other in preeclampsia (p < 0.05). Urinary malondialdehyde significantly correlated with urinary protein bound sialic acid in preeclampsia (r = 0.412; p = 0.02). Receiver operating curve analysis of serum protein bound sialic acid and serum malondialdehyde showed reasonable cutoff values for the differential diagnosis of preeclampsia.ConclusionsOxidative stress and inflammatory response are greater in women with preeclampsia in comparison to pregnant women with gestational hypertension and there is an association between oxidative stress and inflammatory response.     

Serum vascular adhesion protein-1 is higher in subjects with early stages of chronic kidney disease

ObjectivesAn increased level of serum vascular adhesion protein-1 (VAP-1) has been found in patients with diabetes mellitus and vascular disorders. This study examined whether serum VAP-1 levels are associated with chronic kidney disease (CKD).Design and methodsWe included 262 subjects aged 30 and above with fasting plasma glucose level < 7 mmol/L checked within 1 year. First morning urine specimens were collected. Microalbuminuria was defined if urinary albumin-to-creatinine ratio ≥ 30 μg/mg creatinine. The glomerular filtration rate (GFR) was estimated. CKD stages were defined according to the suggestions of the National Kidney Foundation. Serum VAP-1 levels were analyzed by immunofluorometric assay.ResultsSerum VAP-1 levels were positively associated with the urinary albumin-to-creatinine ratio ( r = 0.29, p < 0.0001) and negatively associated with estimated GFR (r = ?0.24, p =  0.0001). Subjects with CKD stage 2 (N =  51) and stage 3 (N =  91) had significantly higher levels of serum VAP-1 than those without CKD (p =  0.0003 and p =  0.035, adjusted for age and gender, respectively). A high serum VAP-1 level was associated with the presence of CKD (OR 1.63 for 1 SD increase of VAP-1, p =  0.018), adjusting for age, sex, and smoking. Ordered logit models revealed that high serum VAP-1 levels correlated with advanced stages of CKD.ConclusionsSerum levels of VAP-1 are associated with the severity of kidney damage or stages of kidney disease. The true mechanism which links the serum VAP-1 and CKD remains to be elucidated in further studies.