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June 1, 2005
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We review the current knowledge of the pathophysiology of sickle cell disease (SCD), the clinical complications and the state of the art in SCD diagnostics. Today, a flexible laboratory concept allows the fast and economic clarification of the patients' sickle cell hemoglobin (HbS) status, e.g. additional compound heterozygosities. In contrast to a well-investigated pathophysiology of the disease, factors influencing the severity of symptoms as well as some laboratory findings in SCD still lack a final explanation. In this review, we focus on red cell lysis resistance as an additional diagnostic tool in SCD. There is a need for further studies regarding lysis resistance in blood samples from patients with HbS.
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June 1, 2005
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Early epidemiological studies showed that genetic factors contribute to the risk of cardiovascular disease. Genetic epidemiological studies based upon families can be used to investigate familial trait aggregation, to localize genes implicated in cardiovascular diseases in the human genome, and to establish the role of environmental factors. Family studies can be also used to identify the physiological role of candidate genes for cardiovascular diseases, and to characterize shared environmental risk factors and their impact on the expression of genetic predisposition. The present paper reviews the existing family studies with special emphasis on those which have studied healthy populations in relation to cardiovascular disease such as the Framingham Heart Study, the National Heart, Lung, and Blood Institute Family Heart Study, and the STANISLAS cohort.
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June 1, 2005
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Gram-negative bacterial infection, namely Chlamydia pneumoniae has been recently discussed as a risk factor for myocardial infarction. The lipopolysaccharide-binding protein (LBP) and the bactericidal/permeability-increasing protein (BPI) play a role in the processes leading to recognition and neutralisation of the Chlamydia pneumoniae and their endotoxins – lipopolysaccharides (LPS). LPS interact with plasma LBP, and LBP-LPS complex activates monocytes/ macrophages, which can influence the atherosclerotic process. BPI is cytotoxic for Gram-negative bacteria and BPI-LPS complexes do not activate monocytes. We have analysed the polymorphisms in the LBP gene (Gly 98 →Cys; Pro 436 →Leu) and BPI gene (Lys 216 →Glu; PstI polymorphism in intron-5; G 545 →C) in 313 patients after myocardial infarction (MI) and in 302 control individuals. Genotype frequencies in the LBP gene and BPI gene did not differ between MI patients and control individuals. Our findings suggest that LBP and BPI polymorphisms do not influence the risk of MI.
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June 1, 2005
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We investigated the effects of S-(1,2-dicarboxyethyl) glutathione (DCEG) and S-(1,2-dicarboxyethyl) cysteine (DCEC) on the stimulus-induced superoxide generation and tyrosyl phosphorylation of proteins in human neutrophils. When the cells were preincubated with DCEG, the superoxide generation induced by N-formyl-methionyl-leucyl-phenylalanine (fMLP) was enhanced in concentration-dependent manner but DCEC showed no effect. The influence of DCEG and DCEC on phorbol 12-myristate 13-acetate-induced superoxide generation showed no effect. On the other hand, the superoxide generation induced by arachidonic acid was markedly suppressed by DCEG and DCEC in concentration-dependent manner. The suppression of DCEG was more effective than that of DCEC. The superoxide generation induced by fMLP in the DCEG-treated cells was suppressed by genistein. DCEG enhanced tyrosyl phosphorylation of 80.0 kDa, 60.0 kDa, and 45.0 kDa proteins in human neutrophils. The tyrosyl phosphorylation of 80.0 kDa, 60.0 kDa, and 45.0 kDa proteins was suppressed by genistein. The enhancement of tyrosyl phosphorylation of these proteins in the DCEG-treated cells was parallel to the enhancement of the fMLP-induced superoxide generation.
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June 1, 2005
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Hyperhomocysteinemia has been associated with several pregnancy complications. We have investigated the variation of plasma total homocysteine (tHcys) during the 2 last trimesters of normal pregnancy and related it to blood vitamin B 12 and folate and to the excretion of the degraded intrinsic factor receptor (IFCR) in urine, in a follow-up study of 15 cases. A significant rise in tHcys was observed between the beginning of the second trimester and the third trimester with respective values (median) 6.1, 5.8 and 6.7 μmol/l (p = 0.038). The tHcys/albumin ratio also increased significantly, while no correlation was found between albumin and folate blood concentration. In contrast, a significant decrease in vitamin B 12 was observed (279, 225 and 199 pmol/l, between the 4th and 6th, and the 6th and 9th month, respectively (p = 0.017−0.002)). A significant negative correlation was found between tHcys between the 4th and 9th month of pregnancy and the ratio of vitamin B 12 between the 4th and 9th month of pregnancy (r = 0.55, p = 0.037). The urine excretion of IFCR was increased and was not related to vitamin B 12 and tHcys. In conclusion, we have observed a rise in tHcys between the beginning of the second trimester and the third trimester of pregnancy which was related to the decreased blood level of vitamin B 12 . Subclinical deficiency of vitamin B 12 should be further investigated in pregnant women who remain on inadequate diet.
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June 1, 2005
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Numerous mutations in the galactose-1-phosphate uridyl transferase ( GALT ) gene have been found to impair GALT activity to different extent, causing galactosemia. This disorder exhibits considerable allelic heterogeneity in different populations and ethnic groups. The Q188R mutation accounts for 60–70% of classical galactosemia alleles in the Caucasian population. Individuals homoallelic for Q188R have a severe phenotype with complete loss of enzyme activity. Another form of GALT deficiency is Duarte galactosemia with N314D mutation associated alleles (Duarte-2). Although heterozygotes for classical galactosemia are asymptomatic at birth and Duarte galactosemia appears to be quite benign, there are some indications that these disorders can increase the risk of developing certain diseases later in life. The aim of our study was to analyze a healthy Slovenian population for the frequencies of Q188R and N314D mutations, and for the Duarte-2 indicative intronic variation IVS5-24G>A. DNA samples from 174 healthy subjects were analyzed for all three mutations by polymerase chain reaction and digestion with restriction enzymes. Allele frequencies for Q188R and N314D mutations and IVS5-24G>A intron variation were found to be 0.29%, 8.0% and 5.7%, respectively. These results correlate well with those reported for most other healthy Caucasian populations.
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June 1, 2005
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Leptin, the ob gene product, plays an important role in the regulation of body fat mass and weight. In previous studies, it was demonstrated that leptin is detectable in human fetal cord blood as early as at 18 weeks of gestation and that serum leptin concentrations are significantly reduced in small gestational age newborns. In the present study, we investigated whether umbilical and maternal serum leptin concentrations correlate with intrauterine growth retardation (IUGR). In addition, we aimed to determine the relationships between leptin concentration in the maternal and cord blood. We studied 40 newborn infants (21 female and 19 male; gestational age, 38–42 weeks) and their mothers. Of the infants studied, 10 had IUGR. Serum leptin concentrations were measured by radioimmunoassay. All newborns had detectable leptin concentrations. Leptin concentrations were significantly lower in newborns with IUGR and in their mothers (n=10; 3.53±1.42 ng/ml, 6.75±1.47 ng/ml, respectively) than in infants experiencing normal growth and their mothers (n=30; 5.58±2.98 ng/ml, 9.85±6.50 ng/ml, respectively) (p<0.01 for newborns, p<0.05 for mothers). There was no significant correlation between umbilical leptin concentration and maternal leptin concentration (r=0.229; p=0.155) in all study groups but, significantly, a correlation was observed in the group with IUGR (r=0.736; p=0.015). There were no significant correlations between both umbilical and maternal leptin concentrations and parity, delivery type and gestational age. There was a correlation between umbilical leptin concentration and birth weight of newborns (r=0.383; p=0.015) but no correlation with body mass index (BMI) of the newborns (r=0.034; p=0.834). Maternal leptin concentrations correlated with maternal weight and BMI (r=0.606; p=0.000, r=0.535; p=0.000, respectively). There was no correlation between maternal leptin concentrations and birth weight of the newborns (r=0.179; p=0.269) and with BMI of the newborns (r=0.146; p=0.367). There was no gender difference in leptin concentrations in the newborns (n=21; 5.50±3.37 ng/ml, for females; n=19; 4.58±1.98 ng/ml for males) (p=0.296). In summary, we have shown that IUGR is associated with a decreased leptin concentration in newborns and their mothers. The association between umbilical serum leptin and birth weight in this and other studies suggests a pivotal role of fetal leptin in regulating fetal growth and development.
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June 1, 2005
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We used an atomic absorption spectrophotometric method to determine the concentration of selenium, zinc, iron, copper and calcium in the whole blood of patients with hepatocellular carcinoma. The results demonstrate that these patients have a lower concentration of selenium (0.18±0.02 μg/ml vs. 0.28±0.06 μg/ml) and zinc (11.2±2.75 μg/ml vs. 18.2± 7.33 μg/ml) than healthy controls (p<0.05). On the other hand, the hepatocellular carcinoma patients have higher mean concentrations of iron (651.9±66.2 μg/ml vs. 473.0±88.0 μg/ml; p<0.05), copper (1.43±0.33 μg/ml vs. 0.95±0.19 μg/ml; p<0.05) and calcium (75.0± 13.1 μg/ml vs. 39.9±12.3 μg/ml; p<0.01) than healthy controls. Thus, hepatocellular carcinoma seems to be associated with the changes in the whole blood concentrations of selenium, zinc, iron, copper and calcium.
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June 1, 2005
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Polymorphisms of the gene encoding apolipoprotein E have been implicated in the pathogenesis of peripheral and coronary artery disease and neurodegenerative disorders such as sporadic and late-onset familial forms of Alzheimer's disease. We have developed TaqMan assay systems for the single nucleotide polymorphisms −219G/T, located in the promoter of the apolipoprotein E gene, 113G/C, present in the transcriptional enhancer element of intron 1, 334T/C, determining Cys or Arg as amino acid residue 112 of mature apolipoprotein E, and 472C/T, determining Arg or Cys as residue 158. The accuracy of genotype determination with the TaqMan systems was demonstrated by analyses with restriction endonucleases. We determined the genotypes of the apolipoprotein E polymorphisms in 2349 study subjects. The genotypes were distributed as: −219GG=27.3%, −219GT=49.1%, and −219TT=23.6% (p=0.435); 113GG=41.3%, 113GC=45.2%, and 113CC=13.5% (p=0.343); 334TT=73.4%, 334TC=24.7%, and 334CC=1.9% (p=0.539); 472CC=86.3%, 472CT=12.8%, and 472TT=0.9% (p=0.004) (Hardy-Weinberg equilibrium estimates are given in parentheses). The allele combinations which define the three major isoforms of apolipoprotein E, namely apoE2, apoE3, and apoE4, had the following allele frequencies: 334T/472T ( ∊2 ; 112Cys/158Cys)=7.3%, 334T/472C ( ∊3 ; 112Cys/158Arg)=78.4%, and 334C/472C ( ∊4 ; 112Arg/158Arg)=14.2%, respectively. ApoE genotypes were distributed as: ∊2∊2 =0.9%, ∊2∊3 =11.2%, ∊2∊4 =1.6%, ∊3∊3 =61.3%, ∊3∊4 =23.1%, and ∊4∊4 =1.9% (p=0.014). The TaqMan assays allow for fast and sensitive genotyping and are especially suitable for studies including large numbers of participants.
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June 1, 2005
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Our study was designed to test and compare the levels of enzymatic antioxidants (ARS), endogenous peroxides (POX), non-enzymatic antioxidants (Antiox-cap) and anti-oxidized low-density lipoprotein (LDL) antibody titers (oLAb) in hyperthyroid and hypothyroid patients. We measured POX, ARS, Antiox-cap and oLAb in the plasma from 68 patients (34 patients with hyperthyroidism, thyroid-stimulating hormone (TSH) <0.04; and 34 patients with hypothyroidism, TSH >4.0) and 34 healthy euthyroid controls. POX were highest in hyperthyroid patients, but differences between hyperthyroid and hypothyroid patients and controls were not significant. ARS were significantly higher in patients compared to controls. Antiox-cap were significantly lower in patients compared to controls. oLAb were significantly higher in hypothyroid patients compared to controls and hyperthyroid patients. Our study shows that both hyperthyroidism and hypothyroidism are associated with enhanced oxidative stress involving enzymatic and non-enzymatic antioxidants. Higher POX in hyperthyroid patients might reflect the hypermetabolic state of these patients. oLAb, indicating progression of atherosclerosis, were highest in hypothyroid patients.
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June 1, 2005
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This is a secondary analysis of data from a cross-sectional study to evaluate the diagnostic efficiency of cystatin C as a marker of the glomerular filtration rate in the elderly. Thirty patients (15 male, 15 female, mean age 75.4±7.1 years) attending a geriatric ward were enrolled. Exclusion criteria were previously diagnosed renal disease, dementia and heart failure (NYHA III or IV). Cystatin C in serum was determined by a particle-enhanced turbidimetric assay. Inulin clearance was assessed using a single-shot method. Also, Cockcroft-Gault formula was calculated. Twelve patients had a reduced glomerular filtration rate (<70 ml/min/1.73m 2 ). The mean values were 88.4 μmol/l (±27.7) for serum creatinine, 1.57 mg/l (±0.34) for cystatin C and 88.7 ml/min/1.73 m 2 (±34.6) for inulin clearance. Maximum efficiency was 0.73 for serum creatinine (cut-off limit 82 μmol/l), 0.67 for cystatin C (cut-off limit 1.63 mg/l) and 0.8 for Cockcroft and Gault estimation (cut-off limit 54 ml/min/1.73 m 2 ). A receiver operating characteristics (ROC) analysis did not show any differences between the various methods. Therefore, cystatin C in serum may not improve the diagnostic efficiency in detecting a reduced glomerular filtration rate in the elderly. Furthermore, normal ranges for serum creatinine in the elderly might need to be adjusted.
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June 1, 2005
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Slightly elevated values of homocysteine are commonly associated with thromboembolic diseases, while high values can be found in patients with congenital metabolic defects or nutritional problems. The clinical use of homocysteine as an independent marker of cardiovascular disease was limited in the past by technical problems with its measurement, the instrumentation (HPLC, radioenzymatic assays, gas chromatography-mass spectrometry, etc. ) and the necessary skills required. Commercially available immunoassays now permit a simpler and more rapid measurement of homocysteine, that is more suitable for routine clinical laboratories; in this paper we analyze the results obtained by using three fully automated methods for homocysteine determination (Abbott IMx immunoassay, Abbott AxSYM immunoassay and Immulite 2000 homocysteine immunoassay) and their correlation with the widely used HPLC method. The results clearly indicate that all three automated immunochemical methods correlate well with the HPLC method (slope 0.97–1.03; intercept 0.95–1.91 with a recovery above 95% for all three methods).
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June 1, 2005
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Total protein, albumin, α 1 -microglobulin, and immunoglobulin G (IgG) were analyzed in 1622 urine samples without Bence-Jones proteinuria or gross hematuria. There was correlation with the histological picture obtained on renal biopsy in 61 patients. We established 24-h reference intervals for α 1 -microglobulin and IgG on 659 urine samples with total protein and albumin excretion rates below 100 mg/24 h and 30 mg/24 h, respectively, and creatinine clearance above 80 ml/min. The central 95% reference interval was found to be between 4 and 17 mg/24 h for α 1 -microglobulin and between 3 and 8.5 mg/24 h for IgG. In 80 urine samples with albumin excretion rate above 30 mg/24 h and α 1 -microglobulin and IgG within their reference intervals, we analyzed the 95% central interval of the distribution of the IgG/albumin ratios, and it was found to be within 0.01 and 0.20 (0.90 confidence interval: 0.17–0.24). Proteinuria was considered to be of the selective glomerular type if the albumin excretion rate was abnormal and the IgG/albumin ratio was under 0.20, even when the IgG excretion was within a pathological range. For the classification of proteinuria as predominantly tubular, we estimated the α 1 -microglobulin/albumin ratio in 173 urine samples with normal excretion rates of albumin and IgG and pathological excretion of α 1 -microglobulin. The discriminating value of 0.91 (0.90 confidence interval: 0.78–1.08) was accepted in order to define proteinuria of a tubular origin in the presence of a pathological albumin excretion rate. The association between albumin and IgG excretion rates and tubular reabsorption of the α 1 -microglobulin normally filtered by the glomerulus was studied in 33 urine samples from patients with no histologically significant tubulo-interstitial or vascular disease and a serum creatinine concentration below 141 μmol/l. The optimal curve-fitting function between albumin plus IgG and α 1 -microglobulin excretion rates was of the quadratic type (r=0.927). Mixed proteinuria was considered when both, albumin and α 1 -microglobulin excretion rates were pathological and could not be included in the previously described groups.
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June 1, 2005
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The aim of this study was to establish reference ranges for children (neonates to young adults), for serum lutropin (LH), follitropin (FSH), estradiol (E2), progesterone, prolactin, sex hormone-binding globulin (SHBG), dehydroepiandrosterone sulfate (DHEAS), cortisol and ferritin, using the nonisotopic, automated chemiluminescence immunoassay system, Immulite ® (DPC). Serum samples from 762 children (369 female; age 1 day to 19 years) were examined. Of these, 381 were classified as pubertal. Due to non-normal distribution, the 2.5th, 50th and 97.5th percentiles (central 95% interval) were calculated for each group. Statistical differences between the reference ranges were analyzed with respect to age, sex and the stage of sexual maturation. The median concentrations of E2, prolactin, progesterone, DHEAS, cortisol and ferritin were higher during the first 2 weeks post-partum than thereafter. The largest difference was seen with prolactin, which showed up to 27-fold higher values during this period. In contrast, before the onset of puberty, hardly any sex difference was observed and all analyte concentrations remained relatively constant, apart from SHBG which increased steadily after the neonatal period. The increase of gonadal activity in females with the onset of sexual maturation included an increase in LH and FSH, which was accompanied by a strong increase in E2, progesterone and prolactin. Cortisol increased to a lesser extent during puberty. In males, the increase in the median concentrations of the hormones was smaller, except for DHEAS. The concentration of ferritin was high in the neonatal period but did not change during sexual maturation. Our findings agree with earlier studies. The calculated reference intervals can be used to assess the development of children, particularly for measurements performed by the Immulite and Immulite 2000 chemiluminescence assay systems.
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June 1, 2005
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The goal of this work was to check the validity of a model previously described for the study of kinetics of the processes taking place during the immuno-analytical measurement of insulin. The antibody was immobilized on the suface wall of the reaction tube. Tracer and insulin concentration, temperature, viscosity and ionic strength in the reaction medium were taken as independent variables. Biexponential kinetics depending on the concentration was observed. The results of the viscosity study show a clear negative influence of this parameter on the direct reaction velocity. Ionic strength had a slight effect, which suggests that the observed variation due to the addition of glycerol is not induced by the influence of the dielectric constant of the solution used. The effect of the temperature shows activation parameters similar to water flow-viscosity energy, which suggests a diffusion control for the reaction. The proposed model correctly interprets the influence of the studied variables.
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June 1, 2005
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Counting of cells in cerebrospinal fluid is currently performed manually. Because of the inherent analytical and economical disadvantages, we attempted to introduce a fully automated method. Therefore, we validated the Abbott CellDyn-4000 haematology analyser for counting cells in cerebrospinal fluid. The analyser was used in its standard configuration with the simple precaution of a preceding blank sample. As for leukocyte counting the analyser yielded high precision (CV ~5% above the upper reference limit), good linearity, low limit of detection (2/μl) and excellent correlation (r > 0.99) with the counting chamber method. The differential leukocyte count was equally accurate and precise, even in the low concentration range. Performance of the erythrocyte count was impaired by its high limit of detection (6/nl) and it appeared satisfactory only for detecting blood admixture due to traumatic puncture. The specificity of the analyser is excellent, since it correctly classified non-viable leukocytes and excluded yeast cells from the leukocyte count in a patient with cryptococcal meningitis. We conclude that the CellDyn-4000 is well suited for quickly and reliably counting leukocytes in cerebrospinal fluid. Developing some software modifications might make the analyser useful also for performing erythrocyte counting in cerebrospinal fluid.
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June 1, 2005
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June 1, 2005
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July 27, 2005
