Volume 39, Issue 6

One of the most rapidly developing areas of organelle biology, which has a major involvement in biochemical pharmacology, is the research into the peroxisomal function. There is a large group of compounds that are capable of inducing liver enlargement, proliferation of peroxisomal structures, and induction of peroxisomal and extraperoxisomal fatty acid-oxidizing enzymes in rodent liver, called peroxisome proliferators. This list includes hypolipidemic drugs, analgesics, uricosuric drugs, environmental pollutants, phthalates, etc. Some peroxisome proliferators have also been shown to increase the incidence of liver tumors. This review describes the characteristics of peroxisome proliferation in rodent liver and gives examples of different classes of chemicals that produce this effect. Mechanisms of initiation of peroxisome proliferation in rodent hepatocytes, including peroxisome proliferator-activated receptors, are also described. Rodent peroxisome proliferators are not considered to be genotoxic agents. Proposed mechanisms of liver tumor formation include induction of sustained oxidative stress, enhanced cell replication, promotion of spontaneous preneoplastic lesions, and inhibition of apoptosis. In addition, the absence of effects of peroxisome proliferators on peroxisome proliferator-associated parameters supports the hypothesis that human liver cells are refractory to peroxisome proliferator-induced hepatic carcinogenesis.

Medical biochemistry or clinical chemistry in its roots is an interdisciplinary science between natural sciences and medicine. The largest part of medical biochemistry is natural science (chemistry, biochemistry, biology, physics, mathematics), which is very well integrated in deduction of medical problems. Medical biochemistry throughout the world, including Macedonia, should be a professional field open to both physicians and natural scientists, according to its historical development, theoretical characteristics and applied practice. Physicians and natural scientists follow the same route in clinical chemistry during the postgraduate training of specialization in medical biochemistry/clinical chemistry. However, in Macedonia the specialization in medical biochemistry/clinical chemistry is today regulated by law only for physicians and pharmacists. The study of clinical chemistry in Europe has shown its interdisciplinary character. In most European countries different professions, such as physicians, chemists/biochemists, pharmacists, biologists and others could specialize in clinical chemistry. The question for the next generation of specialists in Macedonia is whether to accept the present conditions or to attempt to change the law to include chemists/biochemists and biologists as well. The latter used to be a practice in Macedonia 20 years ago, and still is in many European countries. Such change in law would also result in changes in the postgraduate educational program in medical biochemistry in Macedonia. The new postgraduate program has to follow the European Syllabus, recommended by EC4. To obtain sufficient knowledge in clinical chemistry, the duration of vocational training (undergraduate and postgraduate) for all trainees (physicians, pharmaceutics, chemists/biochemists and biologists) should be 8 years.

Excretion of urinary proteins (UP) is an important marker for the evaluation of patients with progressive renal disease. In order to analyze quantitative and qualitative variability of UP in relation to physical activity, we used standardized stress tolerance test and SDS-PAGE. Five urine samples were obtained from each patient at rest, during ordinary daily activity and after physical stress. Determination of total proteins was performed using Meulman's classic method with sulfosalicylic acid. UP were separated by ultrathin horizontal gradient SDS-PAGE according to Görg. There were 142 patients; 40 with poststreptococcal glomerulonephritis (PSGN), 11 with diabetes mellitus, 16 with chronic pyelonephritis and 75 who attended for investigation of asymptomatic proteinuria. Functional proteinuria was established in 42 subjects, who displayed maximal UP excretion during stress and the presence of apolipoprotein AI on SDS-PAGE. Children with PSGN showed no significant increase of UP during stress. Some children with diabetes mellitus (27%) and chronic pyelonephritis (47%) displayed microproteinuria or overt proteinuria after stress. Quantitative and qualitative changes in total UP excretion can be detected by stress tolerance test and SDS-PAGE. It remains to be seen whether stress tolerance test can identify children and youths who are at higher risk for disease progression.

Patients undergoing hemodialysis are at risk for atherosclerosis and its complications. The aim of this study was to examine the effect of erythropoietin therapy and hemodialysis duration on some of the atherosclerotis risk factors. The patients were divided into four groups: I: patients undergoing hemodialysis for less than 10 years (n=22); II: patients undergoing hemodialysis for more than 10 years (n=17); III: patients on no erythropoietin (n=21); IV: patients on erythropoeitin therapy (n=18). A control group of 20 subjects was also examined. Triglycerides, total cholesterol, low-density lipoprotein and high-density lipoprotein, lipoprotein(a), apolipoprotein-A1, apolipoprotein-B and lipid peroxidation were examined. There was a significant increase in triglycerides, to 2.59±1.2 mmol/l (p<0.001) and in lipid peroxidation in hemodialysis patients, to 5.02±0.9 μmmol/l vs .controls (p<0.001). Significantly elevated triglycerides and lipid peroxidation levels were found in the patients with longer hemodialysis duration. Triglycerides were elevated in group II vs . group I, to 2.90±1.0 mmol/l. (p<0.05). Lipid peroxidation in group II, 5.40±1.0 μmmol/l, showed significant difference compared to group I (p<0.05). Erythropoietin treatment did not affect any of the examined parameters. These results indicate increased risk for atherosclerosis related to hemodialysis duration. Besides the renal disease itself, hemodialysis may also be one of the risk factors for atherosclerosis.

Maternal serum screening identifies women at an increased risk of a pregnancy with Down's syndrome or trisomy 18 or an open neural tube defect. The triple test, consisting of maternal serum α-fetoprotein, unconjugated estriol and human chorionic gonadotropin was carried out by a chemiluminescence immunoassay method in our laboratory. The study consisted of 373 pregnant women. The gestational range for the study group was 14–22 weeks. The mean maternal age for the study group was 28.53 ± 5.46 years (range 17.4 to 43.5 years); 9.1% of the women were considered at high risk for Down's syndrome based on the test results. In our study the detection rate for Down's syndrome by prenatal karyotyping was 66.6%. Maternal serum screening allows reduction of the number of women requiring amniocentesis without a significant decrease in the detection rate.

The aim of this study was to determine the effect of Escherichia coli ( E.coli) -derived lipopolysaccharide on rat plasma low density lipoprotein (LDL), malondialdehyde and 3-nitrotyrosine levels (an indicator of protein nitration). Six hours after intraperitoneal administration of E.coli , plasma LDL was measured electrophoretically and malondialdehyde level was measured by spectrophotometric method. Plasma malondialdehyde was significantly (p<0.001) elevated in E.coli -injected rats (4.97 ± 1.33; n=10) in comparison to control animals (1.83 ± 0.5; n=10). In addition, plasma 3-nitrotyrosine level, determined by reversephase HPLC, was also increased in the infected group (2.84 ± 1.17 to 0.22 ± 0.13; n=10). This increase was statistically significant (p<0.001). An increased level of oxidation of lipids and 3-nitrotyrosine was observed as a result of free radical-mediated damage in plasma. In conclusion, asymptomatic infections may increase the risk of atherosclerosis by inducing free radical formation and a consequent increase in the oxidation of LDL.

The research into non-invasive and invasive prenatal diagnostic techniques developed almost in parallel. On the one hand the need was arising to ensure the birth of normal progeny in all cases, while on the other, it was not possible to eliminate the abortion risks connected with the invasiveness of amniocentesis (risk of abortion 1/200), chorion villi sampling, (risk of abortion 2%) and funicolocentesis (risk of abortion 3–4%). One of the first researchers in the non-invasive field was Adinolfi who published the earliest data (1) in 1974 on the possibility of detecting three types of fetal cells in the maternal circulation using flow cytometry. Adinolfi suggested the possibility of using fetal cells present in the maternal circulation for prenatal diagnosis of chromosome or biochemical anomalies. Our review takes into consideration the latest methodological and technical progress in relation to the study of fetal cells in maternal circulation, without considering cells present in the endocervical canal where from the 8th week of pregnancy it is only possible to obtain trophoblast cells (2). This technique has since been abandoned due to the scarcity of cellular material available, the greater risk of contamination by cells of maternal origin, and also because the recovery of the cells is unpredictable, despite their potential use for the early non-invasive diagnosis of sex (3). The following issues are addressed in this review: the characterization of the fetal cell types present in the maternal circulation, the methods of their separation and enrichment, and the methods of genetic diagnostics applied.

Different methods have been devised to detect point mutations. Some are very sensitive, detecting mutations even in a background of normal tissue, but none provide information about the percentage of cells with mutant DNA. Here we describe an easy, fast and reliable method, melting temperature analysis, which not only detects point mutations but also provides quantitative information on the percentage of cells with mutant DNA. By this method we detected a G-A transition in codon 12 of the K- ras gene in DNA of subjects with colorectal cancer. The K- ras mutation was found in 9/10 bowel cancers and 8/10 normal adjacent samples. It was also detected in 4/7 stool samples from the same patients. In colorectal cancers, the proportion of K- ras mutant cells was variable: in two the mutant/wild-type DNA ratio was 30/70, in three 50/50, and in four 70/30. Melting temperature analysis was sensitive for the detection of point mutations in bowel cancer and also in apparently normal tissue, providing quantitative information about the percentage of cells with mutant DNA.

We employed the analysis of single-strand conformation polymorphisms to identify mutations in exon 4 of the low density lipoprotein receptor gene causing familial hypercholesterolemia. Three familial hypercholesterolemia heterozygotes had abnormal singlestrand conformation polymorphism patterns. DNA sequencing revealed that the abnormal pattern of exon 4A was due to heterozygosity (T/C) at nucleotide 442. Nucleotide 442 is the first base of codon 127, and the T→C mutation (C127R) changes this codon from Cys TGT to Arg CGT . Abnormal patterns of exon 4B were due to heterozygosity (A/G) at nucleotide 662: nucleotide 662 is the second base of codon 200, and the A→G mutation (D200G) changes this codon from Asp GAC to Gly GGC . Mutation D200G was previously described as FH Padova, but mutation C127R (FH Zagreb) has not been reported previously. This novel mutation was confirmed by restriction endonuclease analysis with Dsa I. The screening of 420 familial hypercholesterolemia heterozygotes suggests that C127R and D200G account for about 0.7% of mutations causing familial hypercholesterolemia in Croatia.

The plasma levels of endothelin-1 and big endothelin-1 were evaluated in blood of rats in the superior and inferior vena cava , in normal posture (synchronous controls), and after 12 days head-down suspension and 1 day recovery in normal posture. In synchronous controls, the mean plasma concentration of endothelin-1 in inferior vena cava or superior vena cava was almost the same (5.89±0.63 pmol/l and 5.67±0.64 pmol/l, respectively), whereas the mean plasma concentration of big endothelin-1 was higher (p<0.05) in superior vena cava compared to inferior vena cava (5.49±0.75 pmol/l and 1.39±0.15 pmol/l, respectively). In samples from superior vena cava of head-down suspended rats big endothelin-1 levels were significantly lower (p<0.05) up to day 9 of suspension, compared to non-suspended synchronous controls, whereas endothelin-1 values were higher (p<0.05). Big endothelin-1 concentration was higher (p<0.05) in inferior vena cava compared to non-suspended synchronous controls. The behaviour of endothelin-1 was more complex, endothelin-1 levels were lower (p<0.05) on day 1 of head-down suspension and higher (p<0.05) in samples taken on days 9 and 12. After 1 day recovery endothelin-1 and big endothelin-1 concentrations returned to normal in both superior vena cava and inferior vena cava . These data indicate that the endothelial system involvement for the two venous beds is different and suggest that local rather than systemic evaluation could better explain endothelial involvement and the contribution of different anatomic sites to the biosynthesis, conversion and clearance of the various involved molecules.

We present the establishment of a sensitive immunoassay for the determination of α 1 -acid glycoprotein (orosomucoid, AGP) in rat serum. The assay is based upon antigen capture by surface-immobilized specific polyclonal rabbit anti-AGP antibodies with biotinylated rat AGP (rAGP) as the tracer, and formatted as competitive enzyme immunoassay. Signaling is performed by streptavidin-conjugated horseradish peroxidase. Enzyme activity is quantified by an enhanced chemiluminometric method, allowing the sensitive detection of rAGP serum levels in small sample volumes.

The aim of this study was to explore anti- Candida albicans systemic and mucosal humoral responses against Candida virulence antigens such as somatic antigen and secreted aspartic proteases (Saps) in HIV-infected patients with oral candidiasis. Twenty-eight subjects were included in the study: 11 HIV-positive patients without oral candidiasis (group A), 6 HIV-positive patients with oral candidiasis (group B) and 11 HIV-negative healthy controls (group C). Total IgA, IgG and IgM concentrations and antibodies to C . albicans (somatic antigen, Sap1, Sap6) were measured in serum and saliva. We developed a time-resolved immunofluorometric assay with biotin and europium-labeled streptavidin for this purpose. Salivary total IgA, IgG and IgM concentrations were higher in group B. IgA, IgG and IgM anti- C . albicans antibodies (against somatic antigen, Sap1, Sap6) were higher in saliva and serum from patients from group B compared with patients from group A and controls. Our results suggest that, in oral candidiasis, HIV-infected patients have a high mucosal response, specifically directed against C . albicans virulence antigens, such as somatic antigen, Sap1 and Sap6.

The carbohydrate antigen 19–9 (CA 19–9), a determinant (sialylated lacto-N-fucopentaose 119) of a circulating oligosaccharide antigen, is a frequently used tumor marker. Echinococcus spp. infects humans throughout the world and may be able to synthesize closely related molecules which could interfere with the measurement and interpretation of CA 19–9 concentration. The main objective of the present study was to determine the range of CA 19–9 levels in the sera of patients infected by E. granulosus (cystic hydatide disease; CYSHD) or E. multilocularis (alveolar hydatide disease; ALVHD). Serum samples were collected from patients (aged 10–85 years) over a period of 5 years: from 19 patients with CYSHD and from 20 patients with ALVHD. Infection was confirmed by positive Echinococcus serology and clinical evidence provided by imaging and/or histopathological findings. CA 19–9 was detectable in 13 patients with CYSHD (13.5 ± 8.5 kU/l) and 13 patients with ALVHD (30.0 ± 21 kU/l; p < 0.05). Thus ALVHD patients exhibited a significantly higher plasma level of CA 19–9 than CYSHD patients. The serum level of CA 19–9 assessed with an increased cut-off value (> 22 kU/l) was elevated in nine (45%) of 20 ALVHD patients compared to two (11%) of 19 CYSHD patients (p < 0.05). Sera from patients with Echinococcus multilocularis infection contain substances which cross-react with CA 19–9. These substances originate either from the parasite or are synthesized by the host in response to the infection, and possibly bear the Lewis-a antigen or closely related structures which are recognized by anti-CA 19–9 antibodies. Our findings are relevant to the investigation of patients presenting with cystic lesions for which the differential diagnosis includes an infectious or neoplastic origin.

The main objective with cancer antigen 125 (CA 125), carcinoembryonic antigen (CEA), and tissue polypeptide antigen (TPA) monitoring of ovarian cancer patients is to detect an early change of disease activity with high reliability. We hypothesized that a monitoring scheme for ovarian cancer patients with serological tumor markers should take into account the stochastic variation, i.e. the probability that observed increases and decreases may solely be due to analytical imprecision and normal intra-individual biological variation. The aim of this study was to provide a detailed characteristic of the within-subject mean steady state concentrations and the associated variability in healthy individuals with an age distribution representative for ovarian cancer patients. Thirty-one healthy women with a median age of 55 years comprised the study population. Sixteen blood samples were collected from each subject in four series, with four samples per series, over a period of approximately 1 year. We found that, i) natural logarithmic-transformed concentrations were more homogeneously distributed between individuals than the original concentrations, ii) the within-subject mean steady state levels, the standard deviations, and the coefficients of variation differed among subjects, and iii) the steady state variability differed among the markers. In conclusion, our data indicate that the assessment of sequential CA 125, CEA, and TPA concentrations is more complex than hitherto recognized. We suggest that it is necessary to adjust the assessment criteria to the type of marker, and that assessment may be facilitated if based on natural logarithmic transformed concentrations.

COBAS INTEGRA 400 is a random-access analytical system consolidating assays for clinical chemistry analytes, electrolytes, serum proteins, drugs of abuse and therapeutic drugs. Analytical performance and practicability of the instrument were evaluated in seven laboratories over a 2-year period in parallel with system development. Good within-run and total imprecision for all assays was observed with a few exceptions for specimen pools with low concentration or activity. The coefficients of variation for total imprecision were well below 3.0% for clinical chemistry analytes and electrolytes, and below 5.0% for serum proteins and therapeutic drugs. Method comparisons demonstrated a good agreement with the various systems used for comparison, with slopes varying typically from 0.94 to 1.05, and Spearman correlation coefficient generally > 0.975. Accuracy was verified by recovery of controls and certified reference materials within 90 to 110% of target values. Assay ranges were linear within ± 5%. No carry-over on reagent or sample pipetting systems was observed. Manufacturer-specified interference limits and onboard stabilities of reagents were confirmed. A time study for calculating direct personnel times and total processing time was carried out in three laboratories under different conditions including consolidated, STAT and dedicated use. On a scenario-independent basis, the total working time was shorter on the COBAS INTEGRA 400 than on routine systems in all three laboratories. Personnel time, in particular, was significantly reduced when compared to routine instruments. In general, system practicability was judged very positively in all laboratories. Owing to its versatility, the instrument is best placed as a consolidated workstation in small- to medium-sized laboratories or as an instrument for special determinations such as serum proteins, drugs, urinalysis or emergency analyses in large laboratories.