Clinical Biochemistry

Published by Elsevier
Print ISSN: 0009-9120
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Considerable variation in serum sodium (Na) and chloride (Cl) is found in patients at hospital admission. Our goal was to quantify the respective impact of drugs, comorbidities, demographic factors and weather conditions on serum Na and Cl. For 13 277 consecutive patients without terminal kidney disease admitted to the department of internal medicine of the Kantonsspital St. Gallen drug history on admission, age, sex, body weight, ICD-10 diagnoses, and laboratory data were extracted from electronic medical records. Weather parameters prior to hospital admission were also integrated in a multivariate regression analysis. Both serum Na and Cl showed an asymmetric left-tailed distribution. Median (interquartile range) Na was 138 (136/140) and Cl 104 (101/106). The distribution of sodium in patients with one or more risk factors for SIADH was best explained by the presence of two populations: one population with a similar distribution as the unexposed patients and a smaller population (about 25%) shifted to lower sodium levels. Lower weight, lower blood pressure, kidney dysfunction, fever, and diabetes were associated with both lower Na and Cl. Higher ambient temperature and higher air humidity preceding admission were associated with both higher Na and Cl values. Na and Cl at hospital admission are highly influenced by ambient weather conditions, comorbidities and medication. The bimodal distribution of Na and Cl in persons exposed to risk factors for SIADH suggests that SIADH may only affect a genetically distinct vulnerable subpopulation.
 
To compare automated platforms with the routinely used methods in our clinical laboratory for the quantitation of 25-hydroxyvitamin D [25(OH)D] and 1,25-dihydroxyvitamin D [1,25(OH)2D]. The NEXgen Four and Triturus ELISA platforms, utilizing the IDS enzyme immunoassay (EIA) kit for 25(OH)D, and the DiaSorin Liaison 25(OH)D methods were compared with the DiaSorin radio immunoassay (RIA) kit. The NEXgen Four and the Triturus, utilizing IDS EIA for 1,25(OH)2D, were compared with the thymus radioreceptor assay (RRA) for measurement of 1,25(OH)2D. NEXgen correlated best with DiaSorin RIA (r(2)=0.652). NEXgen correlated best with the thymus RRA method (r(2)=0.541). Imprecision CV values for NEXgen 1,25(OH)2D were 2.8-9.4% within-run and 10.2-13.9% between-run compared with a between-run precision of 14.0-16.9% with the thymus RRA method. NEXgen correlated best with DiaSorin RIA for measurement of 25(OH)D. NEXgen correlated best and demonstrated better precision than thymus RRA for quantitation of 1,25(OH)2D.
 
To investigate whether there is a relationship between serum 1,25 dihydroxy vitamin D3 [1,25(OH)2D3], which is an inhibitor of angiogenesis, concentrations and severity of diabetic retinopathy (DR). Serum 1,25(OH)2D3, 25 hydroxy vitamin D [25(OH)D] and parathormone (PTH) concentrations were measured in diabetic patients (n = 66) and nondiabetic healthy subjects (n = 20). The mean serum 1,25(OH)2D3 concentration in diabetic patients was lower than that in nondiabetics (57.3+/-21.44 vs. 89.4+/-18.01 pmol/L, p<0.001); mean 1,25(OH)2D3 concentrations fell with increasing severity of DR [being 63.4+/-17.26 pmol/L for background DR (BDR), 47.7+/-13.27 pmol/L for preproliferative DR (pre-PDR), and 43.1+/-19.45 pmol/L for proliferative DR (PDR)]. Compared with the control group, serum 25(OH)D concentrations were found to be decreased in diabetic patients (p<0.001). There were negative correlations between 1,25(OH)2D3 and age (r = -0.331, p<0.01) and duration of diabetes (r = -0.255, p<0.05). From these findings, it was found that there was an inverse relationship between the severity of the retinopathy, i.e., neovascularization, and serum 1,25(OH)2D3 concentrations, being the lowest in PDR and the highest in diabetic patients without retinopathy (NDR) patients. The measurement of serum 1,25(OH)2D3 concentrations might be helpful to predict severity of DR in patients with diabetes mellitus.
 
The analytical and clinical validation of the DiaSorin 1,25 dihydroxyvitamin D RIA is described. The analytical parameters assessed included analytical sensitivity, dilution linearity, intra- and inter-assay precision, recovery, specificity, and interference studies. Where appropriate, assessments were performed according to NCCLS guidelines. The clinical validation assessed normal individuals and end-stage renal disease patients. The analytical sensitivity of the assay is < 2.0 pg/mL or < 4.8 pM. The assay is specific for both 1,25 dihydroxyvitamin D2 and D3. Recovery ranged from 97% to 108% for spiked samples. Intra-assay precision, as %CV, ranged from 7% to 11%, while inter-assay precision was 12% to 15%. No interference was observed from bilirubin, cholesterol, hemoglobin, or triglycerides. Clinical validation demonstrated complete discrimination between normal and ESRD populations. These data demonstrate that the DiaSorin 1,25 (OH)(2) vitamin D RIA is a robust, accurate, and precise tool for the assessment of 1,25 (OH)(2) vitamin D.
 
We investigated clinical relevance of serum 1,5-anhydroglucitol (1,5-AG) levels in fulminant type 1 diabetes mellitus (FT1DM) patients, because 1,5-AG is known to reflect short term glycemic control. Subjects comprised 7 patients with FT1DM and 32 patients with type 2 diabetes mellitus (T2DM) with HbA1c <8.5%. All of them have never been treated for diabetes. HbA(1C) showed no significant difference between both groups. On the other hand, serum 1,5-AG levels were significantly lower in the FT1DM patients than in the T2DM patients. Serum 1,5-AG levels were < 5.0 μg/ml in 6 of 7 (86%) FT1DM patients, compared with only 1 of 32 (3%) T2DM patients. Serum 1,5-AG levels were lower in the FT1DM patients than in the T2DM patients. Serum 1,5-AG, but not HbA(1C), reflects short-term exacerbation of glycemia in patients with FT1DM.
 
Objectives: The aim of this study was to examine the relationship between serum levels of uric acid (UA) and 1,5-anhydroglucitol (1,5-AG) in elderly subjects (60 years or older; mean age, 73.0±7.2 years) with and without type 2 diabetes mellitus (DM). Methods: Subjects with DM (n=97) and without DM (n=360) were recruited from among our outpatients (estimated glomerular filtration rate≥45 mL min⁻¹ 1.73 m⁻², and urine protein equivalent to <1.0 g/L), and a cross-sectional study was performed with simple linear regression and stepwise multiple linear regression analyses. Results: The mean serum UA levels of men were significantly higher than those of women in both groups. The mean serum 1,5-AG levels of men were significantly higher than those of women in the non-DM group. There were positive correlations (indicated by Pearson's correlation coefficients) between serum UA levels and 1,5-anhydroglucitol levels in all patients and in both men and women. Simple linear regression and multiple linear regression analyses showed that the serum 1,5-AG levels were significantly and positively correlated with the serum UA level in both the non-DM group and the DM group. In the non-DM group, HbA1c levels, as well as 1,5-AG levels, were positively correlated with serum UA levels. Furthermore, the correlation between 1,5-AG and UA levels was stronger in subjects with DM than in subjects without DM. Conclusions: These results suggest that the serum 1,5-AG level is an independent factor associated with serum UA levels in the nondiabetic state, as in DM.
 
1,5-anhydroglucitol (AG) is a nonmetabolizable glucose analogue found in plasma due to ingestion. The normal steady-state concentration can be dramatically decreased by inhibition of tubular reabsorption during periods of hyperglycemia. For this reason, monitoring of AG has been plausibly advocated for detection of periodic glucosuric hyperglycemia. In this review, we examine the influence of variation in factors affecting both steady-state and transient changes in plasma AG. Among normals, the lower and upper limits of the plasma AG reference range vary by a factor of 5. Using a simplified mass balance model (a single compartment model with 3-6x larger-than-plasma volume of distribution), reasonable inter-individual variations of ingestion rate, glomerular filtration rate and fractional post-filtration reabsorption are each able to account for the wide range of normal, steady-state AG concentrations. In monitoring of changes in AG, inter-individual variations in the threshold for glucose excretion, volume of distribution and glomerular filtration rate are all likely to significantly affect correspondence of integral changes in AG to integral glucosuria/hyperglycemia. This combination of variables, affecting both steady-state and transient changes, is significantly confounding with respect to interpretation of serial plasma AG concentrations. Resolution of information content of AG monitoring is thus largely that of crossing simple characterization of deltas [+,0,-] for changes in AG concentration against the information content of hemoglobin A1c monitoring. Despite this limitation, AG monitoring can in principle provide information about glycemic control in the short term that is not apparent through monitoring of hemoglobin A1c alone. However, whether AG monitoring can lead to improved outcomes in diabetes management remains to be established.
 
The study aim was to establish conditions for stabilization the activity of fructose-1,6-bisphosphatase (FBP-1) in stored urine. The FBP-1 was determined by the method Kepka et. al in a collected fraction of purified urine. EDTA and mercaptoethanol stabilized FBP-1 activity in stored urine. At optimal conditions urine may be stored up to 7 days at a temperature of 4 degrees C.
 
The incubation of whole blood with fructose-1,6-diphosphate (FDP) entails a statistically significant increase of intraerythrocytic FDP together with a decrease of blood glucose. The increase is not significant when equimolar amounts of fructose plus twice molar phosphate are used. The effect of FDP is decreased in the presence of an excess of oxygen. FDP added to the whole blood is removed from plasma by the activity of plasma enzymes and by the presence of blood cells as well. No specific interaction of FDP with plasma proteins seems to occur and the effects of FDP addition last longer than is compatible with the presence of FDP in the plasma.
 
To evaluate analytical performance of Sysmex UF-1000i for peritoneal fluid analysis. Functional sensitivity, imprecision, linearity and comparison studies were performed on peritoneal fluids. Total imprecision was 1.6-4.7%, functional sensitivity 27/μL for white blood cell (WBC) and 32/μL for total nucleated cell (TNC) count. Linearity was excellent up to 983cell/μL, carry-over <0.2%, correlation with manual microscopy always greater than 0.992. The instrument exhibited optimal performance at the conventional WBC diagnostic thresholds.
 
This study was designed to apply the rapid Elecsys S100 immunoassay for real-time measurement of S100 protein serum levels indicating acute brain damage in patients undergoing carotid artery stenting (CAS) or endarterectomy (CEA). Data of 14 CAS patients were compared to those of 43 CEA and 14 control patients undergoing coronary angiography (CA). S100 serum levels were measured by the full-automatic Elecsys S100 immunoassay and compared to those obtained by the well-established LIA-mat S100 system. In contrast to CAS and CA patients, median S100 serum levels of CEA patients significantly increased to 0.24 ng/mL before declamping, but subsequently returned to baseline. Three CEA patients with neurological deficits showed sustained elevated S100 levels 6 h after extubation. Absolute S100 values were not significantly different between the two methods. Bland-Altman plot analyses displayed a good agreement, mostly indicating slightly smaller values applying the Elecsys S100 system. The Elecsys S100 system appears to be suitable for rapid real-time detection of neurological deficits in patients undergoing CAS and CEA. Persistent elevations of Elecsys S100 levels during CEA were associated with prolonged neurological disorders, whereas transient increases seem to represent impaired blood-brain barrier integrity without neurological deficits.
 
To investigate the occurrence of subclinical neurologic involvement in patients with essential hypertension employing serum biochemical markers. Fifty patients with essential hypertension and 42 controls with no clinical evidence of neurological disease were recruited. Serum S100B protein and neuron specific enolase (NSE) were determined by employing immunoassay kits from CanAg Diagnostics AB (Sweden). Brain MRI and fundoscopic exploration were conducted. S-100B and NSE levels were significantly higher in hypertensive patients than in controls. In hypertensive patients, multivariate analysis revealed that NSE was independently associated with two variables expressing severity of hypertension: diastolic blood pressure and grade of retinopathy. Brain MRI studies demonstrated higher NSE levels in patients with more severe white matter lesions. Raised NSE levels are associated with a higher severity of hypertension and of white matter lesions, providing preliminary evidence that suggests the presence of silent brain damage in a subset of hypertensive patients.
 
Cardiac arrest often represents the first expression of an underlying cardiac disease. Despite advances in neurocritical care, the neurological assessment of cardiac arrest patients relies on clinical, instrumental and biochemical parameters. The clinical significance of S-100 calcium binding protein B (S-100B) has substantially increased throughout several areas of clinical neuroscience, but reliable evidences attest it can be used as a reliable and early predictor of poor physiological and cognitive neurological outcomes after cardiac arrest.
 
Serum neural protein S-100B concentration is considered as a marker of CNS lesions. Phenylketonuria (PKU) is a metabolic disorder characterized by high phenylalanine (Phe) levels in blood and foci of myelin absence in the CNS of untreated patients. To evaluate S-100B blood levels in PKU patients. Twenty-five (N = 25) PKU patients of comparable age, who were diagnosed by neonatal screening and "followed up" in our Inborn Error of Metabolism Department, were divided into two groups: group A (N = 13) with almost normal Phe levels and group B (N = 12) "off diet" with high Phe concentrations. Their MRI examinations were normal 12-14 months before the beginning of the study. Twenty-three (N = 23) healthy children were the controls. Serum S-100B levels, measured with an immunoluminometric assay, were greatly elevated in the group B (0.48 +/- 0.6 microg/l) as compared to those of group A (0.16 +/- 0.4 microg/l, P < 0.001) and controls (0.10 +/- 0.02, P < 0.001). Positive correlation was found between S-100B and Phe blood concentration (r = 0.46, P < 0.01). Foci of myelin absence in MRIs were observed in 1/13 of group A and in 10/12 of group B at the end of this study. (a) Serum S-100B protein level, for the first time evaluated in PKU, was positively correlated with Phe blood level in PKU patients. (b) S-100B blood estimation could be a useful peripheral marker of CNS lesions in patients with demyelinated disease such as PKU.
 
Isoelectric focusing in cylindrical polyacrylamide gels has been used to demonstrate the formation of asymmetric hybrids between an abnormal hemoglobin, Hb Alberta (alpha 2 beta 2 101 Glu replaced by Gly), and Hb A (alpha 2 beta 2), Hb A2 (alpha 2 delta 2) or Hb F1 (alpha 2 gamma 2 Acetyl). There was no discernible difference in the stabilities of the three hybrids (alpha 2 beta A beta Alberta, alpha 2 delta beta Alberta and alpha 2 gamma Acetyl beta Alberta) in the oxy, carboxy or cyanmet hemoglobin forms. Two mixed liganded hybrids, (alpha beta A)oxy (alpha beta Alberta)cyanmet and (alpha beta A)cyanmet (alpha beta Alberta)oxy were also demonstrated and found to have stabilities similar to that of the Hb Alberta hybrids.
 
To compare the accuracy of BiliCheck™ (Respironics, Marietta, GA) and Konica-Minolta Air Shield JM-103 (Drager Medical Inc, Telford, PA) to evaluate total serum bilirubin (TSB). Prospective blinded study comparing two diagnostic devices in 630 neonates requiring TSB measurement. Linear regression analysis showed a good correlation between BiliCheck™ and TSB (r=0.8212) as well as between JM-103 and TSB (r=0.8686). BiliCheck shows a tendency to underestimate TSB. The mean difference in TSB-TcB was -1.4 mg/dL for BC (-4.7/+1.8 mg/dL) and 0.3 mg/dL for JM-103 (-2.6/+3.2mg/dL). ROC analysis for TSB≥ 12 mg/dL showed area under the curve for BiliCheck™ significantly lower than those for JM-103 (p<0.0001). JM-103 resulted less time expensive than BiliCheck. In spite of similar diagnostic accuracy JM-103 could be preferred for some practical advantages, but its suitability in performing universal screening for severe hyperbilirubinemia deserves further investigations.
 
Background and objectives: Increased plasma low-density lipoprotein-cholesterol (LDL-C) levels in hypercholesterolemic subjects are associated with enhanced LDL oxidation that represents an additional risk for atherosclerotic disease. Human serum paraoxonase (PON1), a high-density lipoprotein (HDL) associated enzyme, has been shown to protect LDL from oxidation, thus playing an important role in reducing the risk of atherosclerosis. PON1 gene polymorphisms have been found to be associated with the variations in serum PON1 levels and activities, and with the risk for coronary artery disease (CAD). This study was performed to evaluate the contribution of the PON1 promoter (-107)T>C and the coding region Gln 192 Arg (Q192R) and Leu 55 Met (L55M) polymorphisms to the presence of carotid atherosclerosis in 208 Sicilian subjects with primary hypercholesterolemia. Methods: Carotid artery intima-media wall thickness (IMT) was measured as an indicator of early atherosclerotic disease. The subjects were classified according to whether they have a normal (<or=1 mm) or an abnormal (>1 mm) IMT. Subjects were also investigated for physical and biochemical parameters, including PON1 activity. Results: No significant differences were detected among the PON1 genotypes with respect to age, sex, BMI, plasma lipids, systolic blood pressure in both groups of patients. There were significant differences between PON1 genotypes with respect to PON1 activity. The 192QQ, 55MM and (-107)TT genotypes showed lower PON1 activity compared to the RR, LL and CC genotypes. The PON1 (-107)T>C genotype distribution in both IMT groups showed no significant differences in percentage of TT, CT and CC genotypes. Similar results were obtained analyzing the Q192R and L55M genotype frequencies. Stepwise forward logistic regression analysis confirmed the lack of association between PON1 genotypes and carotid abnormalities. Conclusions: In conclusion, our data provided no evidence of a significant association between either PON1 promoter (-107)T>C or coding region, Q192R and L55M, polymorphisms and early carotid atherosclerosis in Sicilian hypercholesterolemic subjects.
 
B-type natriuretic peptide (BNP) and amino-terminal proBNP (Nt-proBNP) are derived from a common precursor, the proBNP(1-108) (proBNP), synthesized by cardiomyocytes. We determined proBNP concentrations in patients admitted to ED and suspected of CHF. One hundred fifty six consecutive patients admitted to ED were included. ProBNP, BNP and Nt-proBNP levels were determined at admission. In this ED population, assays for proBNP, BNP and Nt-proBNP were positively and significantly correlated. Circulating levels of proBNP were higher in patients admitted to ED for CHF than in patients admitted to ED other reasons. Applying receiver operating characteristic curve (ROC) analysis for the diagnosis of CHF, the area under the curve (AUC) was 0.92 for proBNP. Our study demonstrated that proBNP testing, the precursor of BNP and Nt-proBNP, appears as a relevant tool to assist the diagnosis of CHF in patients admitted to ED.
 
The need for early and accurate prediction of outcome in Hypoxic-Ischaemic Encephalopathy (HIE) remains critical. We have previously demonstrated that Interleukin 16 (IL-16) is raised in the umbilical cord blood (UCB) of infants with moderate and severe HIE and has the potential to be developed as a predictive biomarker. Normal reference ranges for IL-16 in UCB have not been previously described. The aim of this study was to determine normative levels of IL-16 in full term neonates using UCB following uncomplicated deliveries and to examine the effect of labour on cord IL-16 values. Full term infants were recruited as part of an ongoing birth cohort study, the Cork BASELINE Birth Cohort Study. All had UCB drawn and bio-banked at -80°C, within 3 hours of birth. Samples for this experiment were chosen from this population based cohort study to represent uncomplicated pre-labour caesarean sections and spontaneous vaginal deliveries. Analysis was performed on plasma EDTA, using ELISA Quantikine® (R&D Systems, Europe). Samples were analysed from 48 infants with two modes of delivery; spontaneous vaginal delivery (n=12 male, n=12 female) and elective caesarean section (n=12 male, n=12 female). The range of all samples was normally distributed between 87.0 and 114.6 pg/ml. Overall mean (SD) for IL-16 was 102.9 (21.5) pg/ml. Levels were not affected by spontaneous vaginal delivery or gender. For the first time we have described the expected range of cord plasma IL-16 levels in healthy term infants following pre-labour and post-labour delivery.
 
To determine the levels of serum and synovial fluid (SF) interleukin (IL)-11 in patients with various arthritides and estimate the contribution of IL-11 to acute phase response (APR). Serum and SF IL-11 were measured by ELISA in patients with rheumatoid arthritis (RA, n = 31), seronegative spondyloarthritis (SSA, n = 23), gout (GT, n = 14) and osteoarthritis (OA, n = 20) and were correlated with ESR and acute phase proteins as well as with cytokines IL-1 alpha, IL-1 beta, IL-6, and TNF alpha. IL-11 was detected in both serum and SF in each group, with IL-11 being statistically higher in SF than serum in all groups, suggesting reduced catabolism or increased synthesis of IL-11 intra-articularly. Median SF IL-11 levels were higher in OA patients than in other groups and in the treated than in the untreated RA subgroup. Moreover, serum and SF IL-11 were correlated significantly with each other, and moderately with the other cytokines examined in RA, SSA, and GT, but not in OA patients, while a significant negative correlation was found with a few of the inflammatory markers examined in each group. Our findings provide evidence of extensive intra-articular expression of IL-11 in arthritides, especially in OA and treated RA patients, suggesting a protective role for IL-11 in joints, probably through the induction of tissue inhibitor of metalloproteinases.
 
Human kallikrein 11 (hK11) is a secreted serine protease, highly expressed in hormonally regulated tissues, including the prostate and the ovary. Our preliminary studies indicate that hK11 may represent a diagnostic and prognostic biomarker for ovarian cancer. The aim of the present study was to examine the prognostic value of hK11 expression in ovarian tumors. Using our established immunofluorometric assay, hK11 levels were quantified (ng per mg of total protein) in 134 ovarian tumor extracts and correlated with various clinicopathological variables and outcome [progression-free survival (PFS), overall survival (OS)], over a median follow-up period of 42 months. hK11 concentration in ovarian tumor cytosols ranged from 0 to 155 ng/mg of total protein, with a median of 1.45 ng/mg. An optimal cutoff value of 6.3 ng/mg was selected to categorize tumors as hK11-positive or negative. hK11-positive tumors were most often of early stage (Stage I/II) and grade (G1/G2) (P < 0.05). Univariate analysis revealed that patients with hK11-positive tumors had a significantly longer PFS (HR of 0.39, P = 0.005) and OS (HR of 0.44, P = 0.033). Cox multivariate analysis indicated that hK11 was an independent prognostic indicator of PFS (HR of 0.47, P = 0.042). Kaplan-Meier survival curves further confirmed that women with hK11-positive tumors have longer PFS and OS (P = 0.003 and P = 0.028, respectively). Also, a weak positive correlation was found between the expression levels of tissue hK11 and tissue CA125 (rs = 0.508; P < 0.001). These results further validate our initial findings that hK11 is an independent marker of favorable prognosis in ovarian cancer patients.
 
Human kallikrein 11 gene (KLK11) encodes a secreted serine protease. In view of its diagnostic and prognostic strength in many malignancies, we investigated the mRNA expression levels of KLK11 in laryngeal tissues in order to unveil its clinical usefulness in laryngeal cancer. KLK11 expression was quantified in 163 tissue samples from 105 laryngeal cancer patients with the development of a highly sensitive real-time PCR methodology, using SYBR Green® chemistry. KLK11 expression in laryngeal cancer specimens of primary or recurrent nature was significantly inferior compared with their non-malignant counterparts (P<0.001 and P=0.026, respectively), a finding of immense diagnostic value as illustrated in the ROC curve analyses (P<0.001). Survival analysis showed that patients harboring KLK11-positive tumors had a significantly decreased risk of death (HR=0.26, P=0.042). Our data recommend KLK11 mRNA expression as a novel and independent biomarker in laryngeal cancer for diagnostic and prognostic purposes.
 
A simple, specific, accurate, precise and sensitive radioimmunoassay procedure developed for plasma 11-deoxycortisol is described. 1. The assay employs an anti-11-deoxycortisol serum generated against 11-deoxycortisol-3-(0-carboxymethyl) oxime coupled to bovine serum albumin, crystalline 11-deoxycortisol as standard, and [3H] 11-deoxycortisol as the radioactive ligand. 2. Cross-reactivity studies performed with structurally related steroids indicated cross reactivities with 17 alpha-hydroxyprogesterone, deoxycorticosterone and progesterone of 2.0%, 1.3% and 0.4% respectively; cortisone, corticosterone, cortisol, testosterone, less than 0.1%; and estrone, 17-beta-estradiol, estriol, and metyrapone less than 0.001%. Due to the high specificity of the anti-11-deoxycortisol serum, the method is simplified by the lack of need for chromatographic purification of the organic solvent extract of the plasma prior to the radioimmunoassay. The procedure was validated by comparing values for plasma 11-deoxycortisol with and without preliminary purification by chromatography on Sephadex LH-20 columns (y = 0.99 R/-x + 4.0, r = .98). Pretreatment of the plasma with n-hexane was found to eliminate interferences from high concentrations of 17 alpha-hydroxyprogesterone or progesterone. 3. Parallel dose-response curves were demonstrated between dilutions of plasma with elevated 11-deoxycortisol concentrations and the standard reference preparation. A non-specific binding less than 4% of the total [3H] 11-deoxycortisol was routinely observed. The detection limit of the assay was approximately 10 pg of 11-deoxycortisol which corresponds to a plasma concentration of approximately 0.7 micrograms/L 4. The analytical recovery of 11-deoxycortisol added to human plasma varied from 88 to 108%, with a mean recovery of 100%. The inter-assay variation was determined by assaying (n = 30) three different quality control pools. The following data were obtained: x 1 = 3.8 +/- 0.6 micrograms/L (CV = 15.8%); x 2 = 18.5 +/- 2.0 micrograms/L (CV = 10.8%); x 3 = 43.0 +/- 3.7 micrograms/dl (CV = 8.6%).
 
The aim of the study was to assess the association between visceral and subcutaneous fat with glucose intolerance, adipocytokines, inflammatory markers and carotid IMT in Asian Indians. Subjects with NGT (n=85), IGT (n=49) and T2DM (n=93) were randomly selected from CURES. Total abdominal, visceral and subcutaneous fat were measured using Helical CT scan. Adiponectin, hs-CRP, TNF-alpha, oxidized LDL, visfatin and leptin and IMT and insulin resistance were assessed. Total abdominal fat (p=0.041) and the visceral fat (p=0.039) but not subcutaneous fat progressively increased from NGT, IGT and T2DM subjects. With increasing quartiles of visceral fat, there was a significant increase in insulin resistance (p=0.040); significant decrease in adiponectin (p=0.043) and increase in TNF-alpha (p=0.028), hs-CRP (p=0.043), OX-LDL (p=0.034) and visfatin (p=0.040), and carotid IMT (p=0.047) was observed. Visceral fat levels increased with increasing glucose intolerance and are associated with decreased levels of adiponectin and increased levels of hs-CRP, TNF-alpha, oxidized LDL, visfatin, HOMA-IR and IMT.
 
The aim was to evaluate the relationships of the T-1131C (rs662799) polymorphism variants of apolipoprotein A5 (Apo A5) gene and variants of apolipoprotein E (Apo E) gene common polymorphism (rs429358, rs7412) to signs of metabolic syndrome (MetS). We examined 590 asymptomatic dyslipidemic patients divided into MetS+(n=146) and MetS- (n=444) groups according to criteria of NCEP ATPIII Panel. We evaluated genotype frequencies and differences in MetS features between individual groups. Logistic regression analysis was used for evaluation of Apo A5/Apo E variants as possible risk factors for MetS. We found no statistical differences between genotype and allele frequencies for both Apo A5 and Apo E polymorphisms between MetS+and MetS- groups. In all subjects and MetS- group, we confirmed well-known association of the -1131C Apo A5 minor allele with elevated triglycerides (TG, p<0.001). The Apo E gene E2 and E4 variants were associated with higher levels of TG (p<0.01) in comparison to E33 common variant. However, no statistical differences were observed in MetS+subjects, regardless of significantly higher TG levels in this group. Apo A5/Apo E variant analysis in all dyslipidemic patients revealed significant increase of TG levels in all subgroups in comparison to common -1131T/E3 variant carriers, the most in -1131C/E4 variant subgroup. Logistic regression analysis models showed no association of Apo A5, Apo E and all Apo A5/Apo E variants with metabolic syndrome, even after adjustment for age and sex. Our study refined the role of Apo A5 and Apo E genetic variants in the group of adult dyslipidemic patients. We demonstrate that except of TG, Apo A5 T-1131C (rs662799) and Apo E (rs429358, rs7412) polymorphisms have no remarkable effect on MetS characteristics.
 
Citrullinemia type I (CTLN1) is an urea cycle defect caused by mutations in the argininosuccinate synthetase gene. We report the first identification in Argentina of patients with CTLN1 in a limited geographic area. Molecular analysis in patient/relatives included PCR, sequencing and restriction enzyme assay. The studied families showed the same mutation: ASS~p.G390R, associated with the early-onset/severe phenotype. We postulate a possible population cluster. A program to know the carrier frequency in that population is in progress.
 
There has been growing interest in the analysis of certain polyphenols in wine, especially flavonoids, trihydroxystilbenes and phenolic acids, stimulated by intense research into their potential benefits to human health. One of their main properties in this regard is their antioxidant activity, which enables them to attenuate the development of atherosclerosis, inflammatory diseases, and cancer. A two stage CD-1 mouse skin cancer model using 9,10-dimethyl-1,2-benzanthracene (DMBA) as initiator and phorbol 12-myristate 13-acetate (TPA) as promoter was employed to compare the antitumorigenic activities of one polyphenol from each of four different classes: flavanols [(+)-catechin], stilbenes (trans-resveratrol), flavonols (quercetin) and hydroxybenzoic acids (gallic acid). Animals were treated with specific polyphenols at doses ranging from 0 to 25 micromoles (dissolved in 200 microL acetone), twice a week for eighteen weeks. The solution was applied topically to the shaved dorsal region of each animal. The relative potencies of the polyphenols were compared by evaluating the percentage inhibition of tumor formation in individual mice and the number of mice developing one or more tumors with the different dose schedules. Probit analysis revealed that quercetin was the most (ED(50)<1 micromole) and gallic acid the least effective (ED(50) 5-10 micromoles). (+)-Catechin and trans-resveratrol were intermediate, with ED(50) values of 5 and 6 micromoles, respectively. We have shown recently that trans-resveratrol is absorbed much more efficiently than (+)-catechin and quercetin in humans after oral consumption. Taking this and the relative concentrations in red wine into account, together with the present results, we conclude that trans-resveratrol may be the most effective anticancer polyphenol present in red wine as consumed po by healthy human subjects.
 
During thin layer or macrocolumn gel filtration with Sephadex G 200, a 7S macroamylase dissociated completely to normal-sized amylase when the eluant was phosphate-benzoic acid buffer. In contrast a 11S macroamylase was not affected by the buffer and amylase activity was associated with 11S proteins. Since phosphate-benzoic acid buffer is used in a thin layer screening method described by Peeters et al. (9), some macroamylases may go undetected. After treatment of the 7S macroamylase with phosphate-benzoic acid buffer, the amylase-free 7S proteins obtained formed macroamylase with added pancreatic amylase. This indicates that the buffer did not inhibit the activity of the macroamylase, but caused a dissociation of the macroamylase during gel filtration. The difference in the effect of phosphate-benzoic acid buffer on the two types of macroamylases may be attributed to the difference in affinity of binding proteins for the amylase molecule.
 
Although the population prevalence and mutation spectrum of β-thalassemia in most areas of south China have been characterized, the mutations have not been elucidated in Guizhou Province. The aim of this study was to investigate the spectrum of β-thalassemia in this province. We detected and analyzed β-globin gene mutations in 407 β-thalassemia patients and carriers by PCR-based reverse dot blot (RBD) and direct sequencing methods. Twelve types of β-globin gene mutations were detected. Among the 12 different mutations, six mutations are common, accounting for 97% of mutated alleles. The most prevalent mutation is codon 17(A→T) with an allele frequency of 40.7%. In addition, codon 121 (GAA>TAA), a rare dominant mutation, was detected in a patient with β-thalassemia intermedia for the first time in China. The results of this study will be useful in genetic counseling and prenatal diagnostic service of β-thalassemia in Guizhou Province.
 
MicroRNA miR-124 has been suggested as a tumor suppressor for its role in inhibiting cell growth, inducing differentiation and promoting apoptosis. The present study was aimed to investigate the expression status of miR-124-1 and its clinical relevance in patients with acute myeloid leukemia (AML). Real-time quantitative PCR was performed to detect the expression level of miR-124-1 in AML patients. The clinical significance of miR-124-1 expression in AML was investigated. miR-124-1 underexpression was identified in 30 (36%) of 83 AML patients. No significant difference could be observed in sex, age and blood parameters between the patients with and without miR-124-1 underexpression. The frequency of miR-124-1 underexpression was higher in the patients with t(15;17) than in others (62% versus 30%, P=0.040). The status of miR-124-1 expression was not correlated with the mutations of nine genes (FLT3-ITD, NPM1, C-KIT, IDH1/IDH2, DNMT3A, N/K-RAS and C/EBPA). The patients with miR-124-1 underexpression had borderline longer overall survival and relapse-free survival than those without miR-124-1 underexpression (P=0.052 and 0.045, respectively). These findings suggest that miR-124-1 underexpression is a common event and might have a favorable impact on prognosis in AML.
 
We examined analytical characteristics of new CA 15-3, CA 19-9, CA 125 II, Carcinoembryonic Antigen (CEA), and Alpha-Fetoprotein (AFP) assays on the Dimension Vista® System. Imprecision studies used CLSI-EP5-A2, Limit of Blank and Limit of Detection used CLSI-EP17 and measurement ranges were determined. Method comparisons were evaluated with Passing-Bablok, least-squares regression and residual plots. Reference intervals were determined and valid specimen types, lot-to-lot variability and sample storage stability were defined. Clinical monitoring patterns for each tumor marker in patients were examined. Reproducibility for each method was <6.5%. Limits of Blank and Detection were low. Comparisons between methods showed slopes ranging from 0.89 to 1.32 with low y-intercepts and scatter. Minimal lot-to-lot variability was documented; serum/plasma specimens provide valid results; sample stability at -70°C was >9months. Clinical monitoring patterns correlated with established methods in >89% of cases. Measurement of CA 15-3, CA 19-9, CA 125 II, CEA and AFP on the Dimension Vista® System is an attractive alternative.
 
To evaluate the analytical performance of a radioimmunoassay for measurement of erythropoietin. The INCSTAR Epo-trac 125I radioimmunoassay was examined for applications requiring sensitivity and precision within the normal range. Sensitivity was improved by increasing sample volume to 300 microL. Minimal detectable concentration was determined at 5.3 U/L, %CV ranged from 4.8-5.8 and 13.3-6.4 for intra- and inter-assay imprecision, respectively. Accuracy was maximized by controlling for lipemic and hemolyzed samples and ensuring serum was separated from the clot within 1 h of collection. Values demonstrated good correlation to the Diagnostic Systems Laboratory RIA-kit. With sample volume increased to 300 microL and control of sample preparation, the INCSTAR Epo-trac 125/RIA showed improved precision. Assay sensitivity at lower values allows resolution of changes in erythropoietin within the normal reference range. Age was not found to influence erythropoietin concentration. Within 10 days sojourn at moderate altitude an increase in circulating erythropoietin and reticulocytosis was observed in swimmers.
 
The present study was undertaken to characterize the antigenic determinant of the CA 125 macromolecules recognized by newly developed monoclonal antibodies (M 11, 130-22, 145-9, 602-1, and 602-6), examine their relationship among the epitopes recognized by these antibodies, and develop a series of enzyme-linked immunosorbent assays (ELISAs) in which various combinations of antibodies are used in a double-determinant sandwich mode. The antigenic determinants of CA 125 were characterized by several methods including competitive ELISA and immunoblotting. These antibodies, as well as OC 125, reacted with ovarian cancer (HOC-I) cell extract in a dose-dependent manner. Purified CA 125 antigen with a molecular mass of less than 200 kDa (CA 125 < 200 kDa) had a significant inhibitory effect on the reaction between the cancer cell extract and all these antibodies. The reactivity of M 11, 130-22, 145-9, 602-1, and 602-6 to the cancer cell extract was not significantly inhibited by purified CA 125 > or = 200 kDa, whereas the reactivity of OC 125 was completely inhibited by CA 125 > or = 200 kDa. The antigenic determinants of M 11, 145-9, 602-6, 130-22, and 602-1 were closely related to each other, in this order; whereas OC 125 recognized a different epitope on a structurally identical molecule. The use of these monoclonal antibodies in combination with each other may result in the development of a more specific and sensitive assay for CA 125.
 
The practical application of elevated carbohydrate antigen 125 (CA125) to predict clinical outcome in chronic heart failure (CHF) is under debate. The mechanism for this CA125 elevation remains unknown. We hypothesize that mechanical stress on mesothelial cells initiates CA125 synthesis. Design and Methods Total 191 patients suffering from edema and/or dyspnea were enrolled. 109 patients were diagnosed as CHF, and 82 patients without CHF were assigned as control group. Echocardiography, CA125, N-terminal pro-brain natriuretic peptide (NT-proBNP), and other biochemical parameters were measured. All enrolled patients underwent heart function classification. Patients with serous cavity effusion (SCE) demonstrated higher serum CA125 than patients without SCE (82.91 (61.90-103.92) vs. 44.98 (29.66-60.30) U/ml, P<0.001). In the absence of SCE, CA125 levels in CHF patients were slightly higher than non-CHF patients (52.37 (34.85-69.90) vs. 35.15 (23.81-46.49) U/ml, P=0.017). Additionally, compared with non-CHF patients, CHF patients had higher levels of high-sensitivity C-reactive protein (hsCRP) and lower superoxide dismutase (SOD). In all enrolled patients, CA125 levels were negatively correlated with SOD concentrations (r=-0.567, P<0.001), and positively correlated with hsCRP levels (r=0.608, P<0.001). Receiver operating characteristic curve analysis showed that CA125 was better in predicting SCE than NT-proBNP, while NT-proBNP was more suitable for predicting CHF than CA125. The in vitro study demonstrated that MUC16, the CA125 coding gene, was up-regulated by mechanical stretch on human mesothelial cell line (MeT-5A). CA125 elevation in CHF was associated with SCE. Mechanical extension of mesothelial cells from SCE plays an important role in CA125 increase.
 
CA 125 is a tumour marker usually used to monitor the clinical course of the patients with ovary cancer. The frequently used reference value of this marker is 35 U/mL. However, some arguments to allow us to question us the validity of the classical reference value: i) a second generation of immunoassays, ii) diverse studies related to the factors that influence in the CA 125 serum concentrations and iii) the new applications of CA 125 in pathologies different to the ovary cancer. 1) To propose a reference value of CA 125 in men; 2) To evaluate the CA 125 serum concentration according to different variables, some of which can be altered in pathologies where CA 125 level can be monitored and/ or altered. 65 healthy men were included (age: 40.21+/-10.60 years). A survey containing different parameters and an analytic that contained a hemogram, hepatic, renal, pancreatic profile, ionogram, thyroid function, tumour markers and NT-Pro-BNP was carried out to exclude the presence of a pathological situation. The percentile 95 (P(95)) was calculated to obtain the reference value. Correlations among the CA 125 and the different variables were analyzed by the Spearman test. The median [ranges] and the P(95) were: 7.50 [3.00-25.00] and 20.17 U/mL, respectively. 78% of the values of CA 125 were < or =10 U/mL, 94% were < or =15 U/mL and 95% were < or =20 U/mL. Furthermore, the studied variables don't seem to influence in the concentrations of this marker. The proposed reference value obtained in healthy male subjects is significantly lower than the one used in the clinical practice. This value should be kept in mind when extending the use of this marker to other pathologies which was not used up to now.
 
Objectives: To investigate the expression profile of miR-1258 and heparanase (HPSE) in breast cancer and to assess their clinicopathological significance. Design and methods: The expression levels of miR-1258 and HPSE were analyzed in normal, benign and malignant breast tissues. Their serum levels were evaluated in healthy women and in patients with benign and malignant breast disease. We studied the correlation between the expression of miR-1258 and HPSE and the clinical features presented by the patients. Results: MiR-1258 was down-regulated and HPSE was up-regulated in breast cancer, with a significant inverse correlation. A reduced miR-1258 expression and an elevated HPSE expression were associated with the lymph node status, late clinical stages, a short overall survival and a short relapse-free survival. In frozen fresh tissue samples, the miR-1258 levels in breast cancer with lymph node metastasis were significantly lower than that of breast cancer without lymph node metastasis and benign disease (BD). In contrast, the HPSE levels in breast cancer with lymph node metastasis were the highest. In serum samples, the miR-1258 levels in metastatic breast cancer (M1) were lower than that of primary breast cancer (M0) and BD. However, serum HPSE levels of M1 patients were significantly higher than that of M0 patients and BD patients. Conclusions: MiR-1258 may play an important role in breast cancer development and progression by regulating the expression of HPSE, and they might be potential prognostic biomarkers for breast cancer.
 
We developed a sensitive radioimmunoassay (CYCLO-Trac SP) that specifically measures cyclosporine A in serum, plasma and whole blood of transplant patients. The specific monoclonal antibody was from Sandoz and the tracer was an 125I derivative of cyclosporine C. The assay is performed at room temperature for 1 h followed by a 20 min centrifugation. The sensitivities of the assays are 2.6 ng/mL and 8.7 ng/mL for the serum/plasma assay and the whole blood assay, respectively. Within-run and between-run CVs for both types of assays using cyclosporine concentrations of 80 and 58 ng/mL (serum) and 186 and 199 ng/mL (whole blood) were less than 5% and 9%, respectively. Averaged recovery of serum/plasma and whole blood assays at various levels ranged from 93% to 115%. Interferences by bilirubin, triglyceride, cholesterol, hemoglobin, OKT-3, azathioprine, methylprednisolone and 20 other drugs were insignificant. Multicenter proficiency studies showed an excellent correlation between the CYCLO-Trac SP and the specific 3H-Sandimmune assay from Sandoz: whole blood assay (r = 0.998) and serum assay (r = 0.997).
 
To compare a new radioimmunoassay (RIA), measuring vitamin D metabolites, against an established, competitive-protein binding assay (CPBA) that uses rat vitamin D binding protein. For the CPBA, we first isolated 25-hydroxyvitamin D (25(OH)D) by eluting sample through silica cartridges. For the RIA, serum was added to acetonitrile and centrifuged, followed by RIA using 125I-25(OH)D3. The within-run CV was 1.5% to 4.1% by RIA, compared to 8.1% to 15.0% by CPBA. For both methods, analytical recovery was not significantly different from 100%, and both methods were appropriately linear with sample dilution. We compared RIA values (y axis) from 90 subjects with 25(OH)D ranging from 11 to 232 nmol/L by CPBA (x axis). The slope was 0.9992 (not significantly different from 1). However, the RIA exhibited a bias of 11.15 nmol/L (p < 0.05 vs 0), and a correlation coefficient of r = 0.923. When the comparison was confined to the 37 samples in the lower half of our reference range, the RIA did not compare well: slope, 0.764 (p < 0.05 vs 1.0); intercept 18.7 nmol/L (p < 0.05 vs 0); r = 0.517. For the samples below 36 nmol/L by CPBA, there was no discrimination by RIA between the 11 values below, and the 11 above the decision level (25 nmol/L by CPBA) for vitamin D deficiency. We conclude that the RIA is not optimized to detect vitamin D deficiency, and for this purpose it is not a valid substitute for conventional 25(OH)D methods.
 
A detailed method for the determination of iodothyronine deiodinase type 1 (DI-1) activity is described. The objective of the present method development was to consolidate the effective procedures of previous methods and produce an efficient assay that can be easily reproduced. This method uses a 5',-125I labelled rT3 as substrate and ion-exchange chromatography to separate released ionic iodine. Released 125I- collected in the eluate is counted, and the results used to calculate DI-1 activity. Results were found to be linear for tissue homogenates containing 3-11 mg protein.mL-1. Day-to-day coefficient of variation of liver homogenate was determined to be 13%. This method was found to be reliable, reproducible, and sample sizes as small as 10 microL could be readily assayed. The use of centrifuge filter units to contain the ion-exchange medium decreased handling of the material, and potential sources of error.
 
Aim of this study was to evaluate the accuracy and precision of the detection of individual miRNA as clinical biomarkers in the serum. miRNA-126 was quantified in serum using endogenous and exogenous controls for normalization and the accuracy and precision of the method evaluated. The diagnostic value of serum miRNA-126 was evaluated in malignant mesothelioma (MM) and non-small-cell lung cancer (NSCLC) patients using both relative and absolute qRT-PCR methods. The use of endogenous invariant and exogenous synthetic controls as well sample dilution markedly improves the accuracy and precision of the assay. The inter- and intra-assay analyses revealed that relative qRT-PCR is a more reliable method. Circulating miR-126 detected in the serum by relative qRT-PCRs was found low-expressed in both malignancies, significantly differentiated MM patients from healthy controls and NSCLC from MM, but do not discriminate NSCLC patients from control subjects. Kaplan-Meier analysis revealed that low level of circulating miR-126 in MM patients was strongly associated with worse prognosis. We propose that this approach can be adopted for accurate analysis of other suitable circulating miRNA markers of different types of cancer.
 
To test for possible association of hsp70-2 (+1267A/G), hsp70-hom (+2437T/C), HMOX-1 (number of GT repeats) and TNF-α (+489G/A) polymorphisms with chronic obstructive pulmonary disease (COPD) in Croatian population. Genotyping of DNA isolated from whole blood of 130 COPD patients (as defined by spirometry) and 95 healthy controls was performed. Fragment size analysis upon restriction enzyme digestion and/or sequencing was used for genotype/allele definition. Significance of findings was tested using χ(2) test. hsp70-2 (+1267A/G) polymorphism was significantly associated with COPD. Results of genotyping analysis indicated that a genotype carrying G allele was preferentially associated with COPD; odds ratio (OR)=1.50, 95% confidence interval (CI)=1.00-2.24 and P=0.061. OR for the GG genotype was 3.47 with CI=1.26-9.56 and P=0.04. No association for hsp70-hom (+2437T/C), TNF-α (+489G/A) and HMOX-1 (number of GT repeats) polymorphisms were found. In addition, comparison of genotype frequencies among different stages of disease severity (GOLD II-IV) revealed no discrimination for any of the tested polymorphisms. This study is supporting the association of hsp70-2 (+1267A/G) polymorphism and COPD. Higher frequency of G allele and GG genotype in Croatian COPD patients was observed. There was no evidence for the association of hsp70-hom (+2437T/C), TNF-α (+489G/A) SNPs and HMOX-1 (number of GT repeats) polymorphism with COPD. Allele and genotype frequencies for all of the tested polymorphisms show no association with disease severity (GOLD II-IV).
 
To compare ion exchange and boronate affinity chromatography for HbA(1c) estimation in patients with type I and II diabetes having hemoglobin D. Systems based on ion exchange and boronate affinity chromatography were evaluated and compared for their performance for HbA(1c) estimation in patients with homozygous and heterozygous D disease. Boronate affinity chromatography shows least interference by HbD in heterozygous as well as homozygous diabetic patients for HbA(1c) estimation. The use of boronate affinity chromatography was found to be helpful in evaluating glycemic control in diabetic subjects with HbD.
 
To evaluate 13C-NMR spectroscopy as a method for fat quantitation in human feces without time consuming or unpleasant preparation steps. Stool samples of seven healthy subjects were collected for 18 days before and during oral intake of the inhibitor of gastrointestinal lipases Orlistat. Fecal lipid content was determined first using 13C-NMR, then by conventional gravimetry after homogenization and Bligh & Dyer lipid extraction. The correlation between gravimetry and 13C-NMR was excellent (R2 = 0.91). In repeated measurements, the mean percentage error was 2.8%. On average, 13C-NMR yielded 1.27 g less fat than gravimetry. Orlistat efficacy for fat excretion assessed by 13C-NMR and by gravimetry was 34.3% and 33.9%, respectively. With a total measurement time of three minutes, 13C-NMR spectroscopy of unprocessed whole stool provides an accurate alternative to gravimetry for assessing total fecal fat excretion. 13C-NMR is superior with regard to practicability and speed.
 
The MTG-BT estimates the hydrolysis of triacyl-glycerols by pancreatic lipase, and appears attractive for monitoring exogenous lipase requirements in patients with exocrine pancreatic insufficiency. To assess the test's discrimination capacity and repeatability, 9 CF patients with PERT and 10 healthy children underwent the (13)C-MTG-BT twice, at a 2- to 4-week interval. The test distinguished well between patients with severe exocrine pancreatic insufficiency (SEPI) and healthy subjects. However, within-subject variability for postprandial per thousand(13)C-enrichment and postprandial % dose recovery (PDR) was high in both groups. Therefore, the (13)C-MTG-BT seems useful to distinguish between SEPI and normal exocrine pancreatic function, but requires further development to improve its repeatability.
 
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Ozcan Erel
  • Ankara Yildirim Beyazit University & Ankara City Hospital
Jose Mates
  • University of Malaga
Steven Soldin
  • National Institutes of Health
Chukuka S Enwemeka
  • San Diego State University
Kesava Reddy