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June 1, 2005
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Bacteria can transfer genetic information to provide themselves with protection against most antibiotics. The acquisition of resistance gene arrays involves genetic mobile elements like plasmids and transposons. Another class of genetic structures, termed integrons, have been described and contain one or more gene cassettes located at a specific site. Integrons are defined by an intI gene encoding an integrase, a recombination site attI and a strong promoter. At least six classes of integrons have been determined according to their intI gene. Classes 1, 2 and 3 are the most studied and are largely implicated in the dissemination of antibiotic resistance. A gene cassette includes an open reading frame and, at the 3′-end, a recombination site attC . Integration or excision of cassettes occur by a site-specific recombination mechanism catalyzed by the integrase. However, insertion can occur, albeit rarely, at non-specific sites leading to a stable situation for the cassette. Cassettes are transcribed from the common promoter located in the 5′-conserved segment and expression of distal genes is reduced by the presence of upstream cassettes. Most gene cassettes encode antibiotic resistant determinants but antiseptic resistant genes have also been described. Integrons seem to have a major role in the spread of multidrug resistance in Gram-negative bacteria but integrons in Gram-positive bacteria were described recently. Moreover, the finding of super-integrons with gene-cassettes coding for other determinants (biochemical functions, virulence factors) in Vibrio isolates dating from 1888 suggests the likely implication of this multi-component cassette-integron system in bacterial genome evolution before the antibiotic era and to a greater extent than initially believed.
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June 1, 2005
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We monitored the efficacy of therapy with clodronate, a bisphosphonate drug, in women with postmenopausal osteoporosis, using urinary immunoenzymatic assay of C-telopeptide of collagen type I, an eight amino acid fragment (collagen fragment) of the C-telopeptide of the α 1 -chain of collagen type I (EKAHDGGR). The analysis of the dynamics of collagen fragment concentrations (a marker of bone resorption) during treatment suggests the possibility of early modulation and customization of therapy based on the levels of this marker. This could enable improved control over secondary effects and side effects of clodronate therapy. Pharmacologic inhibition of bone resorption by osteoclasts could be indirectly responsible for the increase in parathyroid hormone found during treatment with clodronate. Increased levels of parathyroid hormone are probably necessary to stimulate residual osteoclast activity and are sufficient for the maintenance of calcium-phosphate homeostasis in a new pharmacologically-induced equilibrium. Outside this context the levels of parathyroid hormone of certain patients would be considered pathologic.
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June 1, 2005
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Instability of β 2 -microglobulin in acidic urine was investigated by identifying an associated protease from normal urine. Degradation was completely blocked by pepstatin, an aspartic protease inhibitor, and the counterpart of the inhibitor was thus sought. The molecular weight of the counterpart was similar to that of the inhibitor, while its cleavage site on β 2 -microglobulin was identical in three products generated in purified β 2 -microglobulin in normal acidified urine (pH 5.0–5.5) and those generated by direct reaction between purified β 2 -microglobulin and cathepsin D in acetic acid (pH 5.0). On Western blotting, the presence of cathepsin D was demonstrated immunochemically in urine, and its urinary concentration correlated well with degree of β 2 -microglobulin degradation. All these findings strongly suggest that cathepsin D is a major urinary acid protease involved in the degradation of β 2 -microglobulin.
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June 1, 2005
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Although there is increasing evidence for anti-oxidized low-densitiy lipoprotein (LDL) autoantibodies in human sera, their diagnostic utility remains controversial. We examined the difference in autoantibody titers between patients with Achilles tendon xanthoma and control subjects. Fifteen hyperlipidemic patients with Achilles tendon xanthoma (group A+) and 94 hyperlipidemic patients without Achilles tendon xanthoma (group A−) were studied. Quantification of anti-oxidized LDL and anti-native LDL autoantibodies was performed using an ELISA method. To calculate antibody titers, we used the ratio between the spectrophotometric reading of anti-oxidized LDL and anti-native LDL wells. Using oxidized LDL that was purified by gel-permeation chromatography as antigen, immunoglobulin G level differed significantly between groups A+ and A− (p ＜ 0.01). In contrast, using native and oxidized LDL as antigens without chromatographical purification revealed no significant difference between the two groups. Furthermore, immunoglobulin autoantibody titer did not correlate with age, body mass index, total cholesterol, high-density lipoprotein cholesterol, LDL cholesterol, or triglyceride in the entire group of subjects. Thus, immunoglobulin G autoantibody values appear to correlate with Achilles tendon xanthoma.
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June 1, 2005
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The aim of the present study was to analyze the hepatic endothelin system and its regulation in liver cirrhosis due to bile duct obstruction. Wistar rats were subjected for 6 weeks to: 1) sham operation; 2) bile duct obstruction; 3) bile duct obstruction and the selective oral endothelin A receptor antagonist LU 135252; 4) bile duct obstruction and oral silymarin, a hepatoprotective and antifibrotic compound. We determined tissue concentrations of endothelin-1 and big-endothelin-1 by ELISA and the density of both endothelin receptor subtypes in plasma membrane fractions by Scatchard analysis. The hepatic endothelin system in liver cirrhosis due to chronic bile duct obstruction is characterized by a simultaneous up-regulation of both endothelin-1 tissue concentration (7.2 fold compared to sham operation; p<0.001) as well as the density of both endothelin receptor subtypes (ET A 7.4-fold, ET B 4.9-fold, p<0.001, respectively) suggesting a synergistic activation of the hepatic endothelin system in this rat model of non-inflammatory cirrhosis. Treatment with proven antifibrotic agents such as silymarin or a selective endothelin-A-receptor blocker (LU 135252) did not reduce the activity of the hepatic endothelin system, suggesting that the hepatic endothelin system is not activated by the fibrotic process itself.
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June 1, 2005
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The determination of plasma methoxyamines is the most informative parameter for the diagnosis of pheochromocytoma. Very good sensitivity and specificity are necessary for this. The measurement of deconjugated (free plus sulfate—conjugated) metanephrines is more useful. Concentrations are 10—fold higher than free metanephrine concentrations. The methodology generally uses a column purification and high performance liquid chromatography with electrochemical detection. We have tested six commercial columns used for the purification step. We chose a rapid and reliable method with a mixture of strong cation-exchange and strong anion-exchange groups bonded onto a silica column. This protocol has been validated with samples of plasma from normal subjects, healthy elderly people, renal failure patients and patients with pheochromocytoma.
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June 1, 2005
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Brain natriuretic peptide is proposed as a biochemical marker which could provide a useful screening test to select patients for further cardiac investigations in heart failure. The applicability of such a biochemical test in clinics, hospital wards, and clinical laboratories is dependent on its ease of use and on the complexity of sample handling. The present study was undertaken to evaluate the stability of brain natriuretic peptide under a number of different handling conditions (sample collection, storage temperatures, freezing temperatures) assayed with a commercially available kit. The results clearly demonstrate that brain natriuretic peptide is stable at room temperature for 24 hours, or in up to 30°C for 12 hours in the presence and absence of aprotinin, on the condition that brain natriuretic peptide is assayed within one month (frozen at −20°C) after blood collection. The presence of aprotinin prevents brain natriuretic peptide degradation in samples preserved for more than 1 month at −20°C before assay.
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June 1, 2005
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In order to assess the short- and long-term stability of apolipoprotein (apo) E concentration in serum, we compared the apo E concentrations measured in fresh human serum samples with those determined after storage at +4°C, −20°C or −80°C. The serum apo E concentration was measured by immunoturbidimetry using an anti-human apo E polyclonal antibody from goats. One week storage at +4°C did not significantly affect the serum apo E concentration. At −20°C or −80°C no significant change in apo E concentration occurred during up to three months of storage. Moreover, the concentration of apo E was not modified after long-term storage of serum samples kept at −196°C in liquid nitrogen for up to four years. In addition, 15 freeze-thaw cycles, over a 3-week period, did not affect the apo E concentration in serum. A similar freezethaw procedure applied to purified human recombinant apo E showed that apo E2 isoform was the most stable in comparison with the apo E3 and apo E4 isoforms.
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June 1, 2005
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We compared several “new” risk factors (autoantibodies to oxidatively modified low density lipoprotein (LDL), sialic acid content of LDL, bilirubin and C-reactive protein) with “conventional” risk factors (apolipoprotein (apo) AI, AII and B, lipoprotein(a), triglycerides, and total, LDL and high density lipoprotein (HDL) cholesterol) for the presence and the extent of coronary or carotid atherosclerosis. Forty male patients with angiographically proven coronary atherosclerosis and 31 male patients with ultrasound-proven extracranial carotid atherosclerosis were compared to 40 age matched (53 ± 5 years) healthy males as control subjects, with negative parental history of atherosclerosis, no clinical signs of systemic or organ-related ischemic disease and normal extracranial carotid arteries. The apo B/apo AII ratio most powerfully indicated the presence and the extent of coronary or carotid atherosclerosis. Elevated lipoprotein(a) contributed significant additional information in the assessment of the atherosclerotic risk. Increase in Creactive protein indicated the presence (but not the extent) of coronary or carotid atherosclerosis with a similar power as lipoprotein(a). Decreased values of total bilirubin indicated the presence of atherosclerosis only in smokers. Autoantibodies to oxidatively modified LDL additionally described the atherosclerotic process, but were less important than apolipoproteins, lipoprotein(a), C-reactive protein or bilirubin. Sialic acid content of LDL added no information to the parameters discussed above. We demonstrated that in male patients apolipoproteins, especially the apo B/apo AII ratio, were better indicators of the presence and the extent of coronary or carotid atherosclerosis than C-reactive protein, bilirubin, autoantibodies to oxidatively modified LDL or sialic acid content of LDL.
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June 1, 2005
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To date, no data have been available on relationship between apolipoprotein E polymorphism and lipid levels in Serbian populations. Blood samples were obtained from 591 healthy normal individuals (193 women and 398 men). A 244 bp sequence of the apolipoprotein E gene including the two polymorphic sites was amplified by polymerase chain reaction. After digestion with Hha l, DNA fragments were visualized by microplate array diagonal gel electrophoresis. In men, levels of both total and low-density lipoprotein cholesterol among the three apolipoprotein E genotype groups differed significantly (p <0.05). The ε2 allele was associated with lower concentrations of both total and low-density lipoprotein cholesterol, where the ε4 allele had the opposite effects. No significant effects of apolipoprotein E polymorphism on serum lipid levels were observed in women. The presented data could be taken into consideration in any future disease risk evaluation in this population.
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June 1, 2005
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A model for characterization of measurements on the ordinal scale is presented. It is based on transformation of the calculated fractions (fractiles) of positives from measurements on samples with known concentrations to a probit-natural log (probit-ln) scale. Such measurements could be made by other methods on ratio or difference scales but, for convenience (for example for speed or low cost), are measured on the ordinal scale by “simple” methods. The model is examined, and verified, using three examples from published data (haemoglobin, glucose, and leukocytes) and an external quality assessment survey on measurements of streptococcus. We show that it is possible to obtain reliable analytical quality specifications and to establish design of control systems for measurements on the ordinal scale. It is concluded that the presented probit-ln model for the ordinal scale is a tool which can improve and facilitate (i) characterizing methods with measurements on the ordinal scale, (ii) defining analytical quality specifications, (iii) designing external assessment as well as internal control schemes, (iv) validation of methods with measurements on the ordinal scale according to the analytical quality specifications, and further, (v) reduction of the number of samples required for method validation and the number of replicate measurements needed.
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June 1, 2005
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Data transformations enable expression of original data in a new scale, more suitable for data analysis. In computer-aided interactive analysis of biochemical and clinical data an exploratory data analysis often finds that the sample distribution is systematically skewed or does not accept a sample homogeneity. Under such circumstances the original data should be transformed. The power transformation and the Box-Cox transformation improve sample symmetry and also stabilize variance. Both the Hines-Hines selection graph and the plot of logarithm of a maximum likelihood function allow selection of an optimum transformation parameter. The proposed procedure of data transformation in univariate data analysis is illustrated on a determination of 17-hydroxypregnenolone in umbilical blood of a population of newborns. Lower levels of free 5-ene steroids in umbilical blood and elevated levels of 5-ene steroid sulfates indicate a congenital sex-specific placental sulfatase insufficiency. After examination of statistical assumptions by diagnostic plots of an exploratory data analysis the best estimate of a mean value of 17-hydroxypregnenolone is derived.
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June 1, 2005
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Numerous methods are proposed to quantify antithyroid peroxidase autoantibodies. No standardization exists but most assays use the standard MRC 66/387 with a calibration factor. Costs of the tests vary between the different kits. We evaluated the concordance of eight peroxidase autoantibodies assay kits in two centres, using a panel of sera from 269 subjects: controls (n=100), patients with autoimmune thyroid disease (n=77; Graves' disease, Hashimoto's thyroiditis), patients with non-autoimmune thyroid disease (n=69; nodular goiter, differentiated thyroid carcinoma) and individual sera with thyroglobulin antibodies only (n=23). The concordance between the eight methods was high, ranging from 88.3% to 98.8% with the total panel of sera. The majority of assays demonstrated high diagnostic performance. We encountered some false-positive results at borderline positive levels, and the nonrecognition of some sera by competitive assays.
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June 1, 2005
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We present the results of a multicenter evaluation of a new point-of-care system (Cardiac Reader) for the quantitative determination of cardiac troponin T (CARDIAC T Quantitative test) and myoglobin (CARDIAC M test) in whole blood samples. The Cardiac Reader is a CCD camera that optically reads the immunochemical test strips. The measuring range is 0.1 to 3 μg/l for CARDIAC T Quantitative and 30 to 700 μg/l for CARDIAC M. Both tests are calibrated by the manufacturer. The reaction times of the tests are 12 or 8 minutes, respectively. Method comparisons were performed with 281 heparinized blood samples from patients with suspected acute coronary syndromes. The results obtained with CARDIAC T Quantitative showed a good agreement compared with cardiac troponin T ELISA (r = 0.89; y = 0.93x + 0.02). The method comparison between CARDIAC M and Tina-quant Myoglobin also showed a good agreement between both assays (r = 0.98; y = 0.92x + 1.6). Test lot-to-lot comparisons yielded differences of 2% and 6% for CARDIACT Quantitative and of 0 to 11% for CARDIACM. The within-run imprecision with blood samples and control materials was acceptable for CARDIAC T Quantitative (CV 10 to 15%) and good for CARDIAC M (CV 5 to 10%). The between-instrument CV was below 7% for CARDIACT Quantitative and below 5% for CARDIACM. The cross-reactivity of CARDIAC T Quantitative with skeletal troponin T was approximately 0.003%. No significant analytical interference was detected for any of the assays in investigations with biotin (up to 100 μg/l), hemoglobin (up to 0.125 mmol/l), hematocrit (26 to 52%), bilirubin (up to 340 μmol/l), triglycerides (up to 5.0 mmol/l), and 18 standard drugs. With the Cardiac Reader reliable quantitative results can be easily obtained for both cardiac markers. The system is, therefore, particularly suitable for use in emergency rooms, coronary care units and small hospitals.
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July 27, 2005
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