Showing papers in "Clinical Biochemistry in 2000"

Abstract: Objectives: To evaluate interactions between toxic and essential elements in the mother–fetus relationship and possible predictors of trace element concentrations in placenta and cord blood. Design and methods: A group of 106 Swedish women was investigated for concentrations of cadmium, lead, and several essential elements in placenta as well as cadmium, lead, zinc, and selenium in venous blood collected at gestational week (gw) 36 and umbilical cord blood. Relations between these elements and maternal and child’s characteristics were examined. Results: The concentrations of cadmium in placenta ranged from 10 to 170 nmol/kg, with the median value (Md) being 46 nmol/kg. Cord blood cadmium (Md of 0.19 nmol/L) was only about 10% of that in maternal blood. Smokers had significantly higher cadmium concentrations in blood (p < 0.001) and placenta (p = 0.001) than non-smokers. The median placental concentration of lead was 26 nmol/kg (range 0–630 nmol/kg). The lead levels in cord blood (Md of 54 nmol/L) were almost the same as in maternal blood. Statistically significant negative associations were found between cord blood lead, on one hand, and child’s weight, length, and head circumference, on the other. The placental levels (medians and ranges) of the essential elements (μmol/kg) were 160 (120–280) for zinc, 2.4 (2.0–3.3) for selenium, 15 (10–20) for copper, 0.084 (0.02–0.32) for cobalt, 0.055 (0.03–0.12) for molybdenum, and 1.2 (0.65–5.1) for manganese, respectively. Several of the essential elements in placenta correlated significantly with each other. Multiparous mothers had significantly lower concentrations of zinc (p = 0.002) and selenium (p = 0.049) in serum as well as zinc (p = 0.001) and calcium (p = 0.004) in placenta than nulliparous ones. Also, cord blood zinc decreased with parity. Conclusions: The results showed that lead, but not cadmium crossed easily the placental barrier. There were no negative effects of cadmium on the zinc status. Cord blood lead, on the other hand, was a negative predictor of child’s birth weight, length and head circumference, indicating that lead might have negative influence on growth in children even at very low exposure levels. There was a depletion of maternal stores of essential elements with increasing parity.

Abstract: Objectives: An increasing amount of experimental and epidemiological evidence implicates the involvement of oxygen derived radicals in the pathogenesis of cancer development. Oxygen derived radicals are able to cause damage to membranes, mitochondria, and macromolecules including proteins, lipids and DNA. Accumulation of DNA damages has been suggested to contribute to carcinogenesis. It would, therefore, be advantageous to pinpoint the effects of oxygen derived radicals in cancer development. Design and methods: In the present study, we investigated the relationship between oxidative stress and breast cancer development in tissue level. Breast cancer is the most common malignant disease in Western women. Twenty-one breast cancer patients, who underwent radical mastectomy and diagnosed with infiltrative ductal carcinoma, were used in the study. We determined coenzyme Q10 (Q) concentrations, antioxidant enzyme activities (mitochondrial and total superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase), and malondialdehyde (MDA) levels in tumor and surrounding tumor-free tissues. Results: Q concentrations in tumor tissues significantly decreased as compared to the surrounding normal tissues (p < 0.001). Higher MDA levels were observed in tumor tissues than noncancerous tissues (p < 0.001). The activities of MnSOD, total SOD, GSH-Px and catalase in tumor tissues significantly increased (p < 0.001) compared to the controls. Conclusions: These findings may support that reactive oxygen species increased in malignant cells, and may cause overexpression of antioxidant enzymes and the consumption of coenzyme Q10. Increased antioxidant enzyme activities may be related with the susceptibility of cells to carcinogenic agents and the response of tumor cells to the chemotherapeutic agents. Administration of coenzyme Q10 by nutrition may induce the protective effect of coenzyme Q10 on breast tissue.

Abstract: Background: There is an urgent need for discovery and validation of new serum biomarkers for ovarian carcinoma. Early diagnosis of ovarian cancer with serologic analysis may improve clinical outcomes through administration of effective treatment. Human kallikrein 6 (hK6, encoded by the KLK6 gene) is a serine protease of the kallikrein gene family. Recently, we were able to develop an immunofluorometric procedure for the quantitative measurement of hK6 in biologic fluids, including serum. Methods: We have used an hK6-specific immunofluorometric assay to quantify hK6 protein in a large number of serum samples from normal individuals, as well as from patients with various malignancies. Results: We report for the first time, significant increase of serum hK6 concentration in a large proportion of patients with ovarian carcinoma. The elevations of hK6 appear to be relatively specific for ovarian cancer because other malignancies did not cause any increase in the concentration of this biomarker in serum. Serial hK6 measurements appear to correlate with CA125 levels in patients monitored postsurgery. Conclusions: This is the first report describing significant elevations of hK6 concentration in serum of ovarian cancer patients. These data suggest that hK6 may represent a potential new biomarker for diagnosis and monitoring of ovarian carcinoma.

Abstract: Objectives: We have identified an acyl glucuronide (M-2) of the immunosuppressant mycophenolic acid (MPA) Acyl glucuronides have toxic potential and may contribute to drug toxicity Whether acyl glucuronides are able to induce release of proinflammatory cytokines is unknown Gastrointestinal disturbances have been observed during MPA therapy and may involve an inflammatory reaction This study investigated whether M-2 can induce IL-6 and TNF-α release as well as gene expression of these cytokines in leukocytes Design and methods: M-2 was produced by incubation of MPA with human liver microsomes Human mononuclear leukocytes were incubated in the presence of M-2 Concentrations of IL-6 and TNF-α were measured by ELISA Expression of mRNA was determined by quantitative RT-PCR Results: Incubation of 3 × 10 6 cells with M-2 resulted in a time and dose dependent release of cytokines, whereas MPA or its phenolic glucuronide MPAG were without effect Cytokine liberation depended on mRNA induction Response to M-2 showed much inter individual variability (30-fold for IL-6, 3-fold for TNF-α) Conclusions: If M-2 promotes release of cytokines in vivo, these may mediate some of the toxic actions of MPA

Abstract: Background: The human kallikrein gene family has contributed the best prostatic biomarkers currently available, including prostate-specific antigen (PSA) and human glandular kallikrein 2 (hK2). Recently, new members of the human kallikrein gene family have been identified. One new member is the KLK6 gene, encoding for human kallikrein 6 (hK6), which is also known as zyme/protease M/neurosin. In this paper, we describe development of antibodies and a sensitive immunofluorometric procedure for hK6 protein. Methods: Recombinant hK6 protein was used as immunogen to develop polyclonal antibodies in rabbits and mice. These antibodies were used to develop a sandwich-type time-resolved immunofluorometric procedure for hK6. Results: The newly developed hK6 immunofluorometric assay has a detection limit of 0.5 μg/L and upper concentration range of 200 μg/L. The assay is highly specific (no detectable cross-reactivity from PSA and hK2) and was used to quantify hK6 protein in various biologic fluids. Highest concentrations of hK6 were found in milk of lactating women, cerebral spinal fluid, nipple aspirate fluid, and breast cyst fluid. hK6 was also detected in male and female serum, in the majority of seminal plasmas and in a small fraction of amniotic fluids and breast tumor cytosols. hK6 was not detectable in urine. Chromatographic studies indicated that hK6 is present in these biologic fluids in its free, 30-kDa form. Conclusions: This is the first reported sensitive immunofluorometric procedure for quantifying hK6 protein. hK6 is a secreted proteolytic enzyme that is found at high levels in cerebrospinal fluid and all breast secretions. This assay will facilitate further studies to examine the possible application of hK6 in diagnostics, including cancer and neurodegenerative disorders.

Abstract: Objectives: In determining the plasma malondialdehyde MDA levels in some Taiwanese college students, we found rather different results by using different thiobarbituric acid TBA tests, even by the high-performance liquid chromatography HPLC-based methods. Here, we re-evaluated four commonly used TBA tests and improved the HPLC-based test. Design and Methods: We used the blood plasma of 16 college volunteers to determine plasma MDA by using four methods: a spectrophotometric measurement of thiobarbituric acid-reactive substances (TBARS) in the TCA-supernatant of plasma (Method A); a fluorescence measurement of plasma lipid peroxides (Method B); and two different HPLC-based measurements of MDA with either 532-nm measurement (Method C, HPLC/532 nm) or fluorescence measurement (Method D, HPLC/fluor.). Results: The levels of MDA or TBA reactive substances obtained from the four methods differed substantially (0.39 ± 0.15; 2.14 ± 0.73; 0.75 ± 0.22; and 0.38 ± 0.15 μM for Methods A, B, C, and D, respectively). The results were positively correlated between Methods A and B (r = 0.740, p < 0.02) and between Methods C and D (r = 0.516, p < 0.05). However, results were negatively correlated between Methods B and D (r = −0.548, p < 0.05). Because most plasma MDA is bound to proteins, we modified the HPLC-based methods (C and D) by adding an alkaline hydrolysis step, and the plasma TBA-MDA adduct detected by HPLC/532 nm was referred to as total MDA. Results show that alkaline hydrolysis was a critical step for measurement of total MDA in plasma because this treatment led to release of MDA from plasma proteins. We also adapted the potassium iodide (KI) treatment of plasma from Method D to reduce lipid hydroperoxides. Our modified method gave a total MDA level in the 16 volunteers of ∼1.5 μM, which was equal to protein-bound MDA plus free MDA. This total MDA level was positively (p < 0.05) correlated with the level of TBA reactive substances obtained from Methods C (r = 0.63, p < 0.05) and D (r = 0.48, p < 0.07), but was not correlated with those from Methods A and B. The recovery (84∼105%), precision (within-assay coefficient of variation: 2.4%, between-assay coefficient of variation: 4∼8%) and sensitivity of the modified procedure were comparable to other HPLC-based methods. Conclusion: By using a validated modification of HPLC-based TBA method, the total plasma MDA in 16 Taiwanese college students was found to be 1.54 μM, which was relatively high compared to those obtained by other HPLC-based method, primarily due to the release of protein-bound MDA by alkaline hydrolysis. This level equaled the sum of protein-bound MDA and free MDA in plasma, confirming that this level represents total plasma MDA.

Abstract: Point-of-care testing (POCT) has evolved from the demand for analytical information more rapidly than is available from central laboratories. By bringing the analysis closer to the patient several process steps have been eliminated, facilitating a shorter time to result and faster management response with improved outcomes. Thus benefits include better therapeutic turnaround times, decreased blood loss as a result of reduced phlebotomy secondary to clinical improvement, and diminished resource utilization. These advantages depend on acceptable analytical performance in comparison with central laboratory methods and in relation to clinical criteria. Generally these requirements are met but there are problems particularly with atypical specimens. Outcomes and cost-benefit analyses have been difficult to perform and evaluate. Given the multitude of participants, quality assurance and program management are recognized as resource intensive. However, recognition of problem areas is driving continuous improvement and we envisage expansion of this paradigm.

Abstract: Objective: To investigate whether the preincubation of brain homogenates with L -phenylalanine (Phe) could reverse the free radical effects on brain acetylcholinesterase (AChE) activity, since it has been reported that Phe binds hydroxyl radicals (•OH). Design and methods: Two well established systems were used for production of free radicals: (a) FeSO4 (84 μM) plus ascorbic acid (400 μM), and (b) FeSO4, ascorbic acid and H2O2 (1 mM) at 37 °C in homogenates of adult rat whole brain. Changes in brain AChE activity were studied in the presence of each system separately. Results: AChE was inhibited (18–28%) by both systems of free radicals. This inhibition was reversed when the brain homogenate was preincubated with Phe 1.8 mM. Conclusions: In accordance with our previous reports, Phe could protect against the direct action of •OH radicals on brain AChE and in this way it might be useful in the prevention of certain cholinergic neural dysfunctions.

Abstract: Background: Alzheimer’s disease (AD) is a major cause of dementia in the elderly. It is generally difficult to diagnose accurately early AD. A few biomarkers, including τ protein and amyloid β-42, are now used as aids for diagnosis and monitoring of AD. Our aim was to examine the possible use of cerebrospinal fluid, blood and tissue, and human kallikrein 6 (hK6) concentration as a marker of AD. Methods: We have used a highly sensitive and specific immunofluorometric procedure for measuring hK6. We measured hK6 in tissue extracts from AD brain or normal individuals, in cerebrospinal fluids of AD patients or normals and in whole blood of AD patients and normals and compared the findings. We have used ten pairs of AD/normal controls in all cases. Results: We found that hK6 concentration is tissue extracts from AD brain were approximately twofold lower than extracts from normal controls. Further, we found that cerebrospinal fluid hK6 concentration is approximately a threefold increase, in comparison to cerebrospinal fluid controls ( p = 0.001). We have also found that the whole blood hK6 concentration in AD patients is about ten times higher than hK6 concentration in normal controls ( p = 0.002). We have immunohistochemically localized the expression of hK6 in epithelial cells of the chorioid plexus. Conclusions: This is the first report describing significant elevations of cerebrospinal fluid and plasma and whole blood hK6 concentration in AD patients, in comparison to controls. These data suggest that hK6 may constitute a new biomarker for diagnosis and monitoring of AD.

Abstract: Objectives: 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). Design and methods: 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). Results: 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). Conclusion: 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.

Abstract: Atherosclerosis remains the leading cause of morbidity and mortality in Western countries. Recent evidence has demonstrated that atherosclerosis is not simply a disease of lipid deposition. Inflammation plays a major role in the initiation, progression, and destabilization of atheromas. High-sensitivity C-reactive protein (hs-CRP) is a circulating acute-phase reactant that reflects active systemic inflammation. Large prospective trials have shown hs-CRP to be a strong predictor of future cardiovascular events. Increased hs-CRP concentration is in fact associated with higher cardiovascular events in individuals with and without clinical evidence of atherosclerotic disease. The relative risk associated with hs-CRP is independent of other cardiovascular disease risk factors. Assays for hs-CRP measurement are currently available but must be standardized because patients' results will be interpreted by using population-based cutpoints. A risk-stratifying algorithm incorporating hs-CRP and total cholesterol to high-density lipoprotein cholesterol ratio has been proposed. Further research into the mechanisms and pharmacological treatment of vascular disease will provide novel management strategies in the very near future.

Abstract: Objectives: We have investigated the AAG and its genetic variants concentrations in plasma samples of 61 patients suffering from different types of cancers. Design and methods: The patients were shared out in three groups, breast, lung, and ovary cancers groups. AAG concentration was measured by an immunonephelometric method and the phenotype was determined, after desialylation of plasma by analytical isoelectric focusing. Detection of AAG variants was made by immunoblotting and their proportions were determined by laser densitometry analysis. A population of 74 healthy individuals served as controls. Results: The plasma concentrations of AAG in the breast and lung cancer groups were 2.5 times increased, while in the ovary cancer group, the concentrations were 1.6 times increased. AAG concentrations in the cancer population ranged between 0.45 and 2.85 g/L (mean value 1.12 ± 0.51 g/L). The proportions of the ORM1 and ORM2 variants were similar to those in the healthy population. In breast and lung cancer groups, the relative concentrations of genetic variants were increased more than 2.5 fold, whereas a 1.6-fold increase was observed in the ovary cancer group. Conclusions: These results show that AAG plasma concentrations are increased in these types of cancers and that changes in the expression of the genetic variants of AAG could also occur according to the type of cancer.

Abstract: Objectives: The plasma apolipoprotein B (apo B) concentrations have been considered to be a more accurate representation of atherogenic particles and it has been proposed that the formula LDL-C (mmol/L) = 0.41TC − 0.32TG + 1.70apo B − 0.27 is reliable for the estimation of LDL-C ( Clin Chem 1997; 43: 808–15). We undertook the present study to investigate the reliability of this formula in a large number of hyperlipidemic patients. Design and methods: 1) The Friedewald formula (LDL-F) and the apo B-based formula (LDL-B) were compared with the β-quantification reference procedure in 130 individuals with a wide range of total cholesterol (TC) and triglyceride (TG) levels, and 2) the LDL-C levels obtained by the Friedewald formula were compared with those calculated by the apo B-based formula in 1010 individuals attending our outpatient lipid clinic. Results: The LDL-F and the LDL-B formulae for LDL-C estimation were found to be in good agreement with the β-quantification ( r = 0.96 and 0.97, respectively). The bias of each method plotted as a function of TG (up to 4.52 mmol/L) was found positive for the LDL-F, whereas the LDL-B was independent of the concentrations of TG. When a large number of individuals were examined, a good correlation between the two equations was found ( n = 1010, r = 0.98). The difference between the two methods was not correlated with serum TG levels. However, it was correlated to serum TC, and apo B levels. Conclusions: The LDL-B formula is a more reliable and accurate method than the LDL-F formula, especially at TG levels >2.26 mmol/L, although it underestimates LDL-C concentrations. Furthermore, this equation can be used in hypertriglyceridemic patients (TG >4.52 mmol/L) in whom the Friedewald equation is inaccurate.

Abstract: Background: Plasma homocysteine has been reported to be useful in the evaluation of patients with suspected vitamin B12 or folate deficiency. In November 1998, Canada began its mandatory fortification of all flour, and some corn and rice products, with folic acid. We evaluated the status of folate and vitamin B12 in Ontario since this fortification program began, and also studied the role of plasma homocysteine in the assessment of vitamin B12 deficiency since that time. Methods: A retrospective cross-sectional study design was performed using a community database of all Ontario samples analyzed by MDS Laboratories, a major provider of diagnostic laboratory services in Canada. All consecutive single-patient fasting samples for plasma homocysteine collected between January 1 and September 30, 1999 were included, as well as corresponding red cell folate and serum B12 concentrations. Data for serum folate were included when available. Descriptive statistics included the arithmetic and geometric means for each measure, as well as the lower and upper centile values. After excluding cases with a concomitant serum creatinine > 120 μmol/L or red cell folate Results: The mean age of all subjects was 58.4 years (95% CI 57.4 to 59.4). Plasma homocysteine samples were obtained from 403 males (56.7%) and 308 females. The geometric mean homocysteine concentration for the entire population was 8.3 μmol/L, and was significantly higher among males (9.3 μmol/L) than females (8.3 μmol/L) (unpaired t-test: 2-p 15 μmol/L did not discriminate between cobalamin concentrations below versus above 120 pmol/L (positive and negative predictive values 7.4% and 97.2%, respectively), nor did it discriminate “indeterminate” B12 levels between 120 and 150 pmol/L (positive and negative predictive values 6.3% and 94.0%, respectively). Conclusion: In a large select group of Ontarians, serum and red cell folate concentrations appear to be higher than expected, possibly due to a recent national folate fortification programme; cobalamin levels are no higher than expected. Given our inability to detect mild B12 deficiency using such indicators as plasma homocysteine, and considering the substantial growth in the elderly segment of the Canadian population, occult cobalamin deficiency could become a common disorder. Accordingly, we recommend either consideration of the addition of vitamin B12 to the current folate fortification programme, and/or the development of better methods for the detection of cobalamin deficiency.

Abstract: Objectives: The pathogenesis of hepatic fibrosis is accompanied with several biochemical and metabolic abnormalities. To obtain more information about the alteration of biochemical and metabolic parameters during alcoholic liver fibrosis, we have monitored the changes of certain important biochemical compounds in experimentally induced hepatic fibrosis. Design and methods: The liver injury was induced in adult male albino rats by using dimethylnitrosamine (DMN) in doses of 1 mg/100 g body weight. Total collagen, total protein, cholesterol, lipid peroxides, glucose, urea, and inorganic phosphorus were monitored in liver and blood/serum samples on Days 0, 7, 14, and 21 after the start of DMN administration. Serum insulin levels were assayed by radioimmunoassay. The serum and urinary levels of hydroxyproline, uric acid, and creatinine were also monitored. Results: The total collagen content in the liver was increased about 4-fold by Day 21 after the start of DMN administration. A significant increase was observed in lipid peroxide levels in both liver and blood samples. Although inorganic phosphorus level decreased in both serum and liver tissue, cholesterol was lowered only in the serum. Increased serum insulin level with impaired glucose tolerance was observed after 21 days. Serum hydroxyproline level increased throughout after the start of DMN administration. The urinary excretion of hydroxyproline was also significantly increased with a striking elevation on Day 7. Elevated uric acid levels were recorded in serum and urine samples during the latter periods of DMN treatment. No alteration was observed in blood urea and creatinine levels. Conclusions: The results of the present investigation demonstrated important alterations in metabolic parameters and biochemical abnormalities during experimentally induced liver damage. All alterations are compatible with the deterioration of liver functions during the pathogenesis of hepatic fibrosis. Copyright © 2000 The Canadian Society of Clinical Chemists

Abstract: Objectives: Develop fully automated assay of antioxidant catalatic activity of catalase. Design and methods: The assay is based on standard, clinical chemistry automated analyzer methods for measuring hydrogen peroxide by using the Trinder reagent. Catalase competes with 324 U/L horseradish peroxidase (type XII) and Trinder reagent for hydrogen peroxide produced by 46 U/L uricase action on urate. Unit activity is defined as 50% inhibition of maximal color development. Results: Within-run coefficients of variation (cv) were 2% for standards and samples, whereas between-run cv was 3.1% for standards and 7.3% for samples. Dilutional parallelism and linearity were demonstrated for 8-fold dilutions of samples over the range 0.1 to 1.1 U/mL. Recovery of added catalase was complete. Samples are stable to freezing and storage for 1 week at −80 °C. Activities (units/mL) ranged from 0.29 to 0.41 in human and canine plasma, and for erythrocytes from 48 to 70 in man, 17 to 19 in dogs, and 60 to 89 in rats. Rat liver activity (units/g wet weight) was age-dependent and ranged from 17 to 24 at 2 months, and from 19 to 37 at 6 months. Conclusion: The first, fully automated assay for the measurement of catalatic activity of catalase in plasma, erythrocytes, and liver is demonstrated for multiple species. The assay is simple, precise, relatively inexpensive, and rapid.

Abstract: Objectives: To develop a simple and practical enzyme immunoassay (EIA) for oxidatively modified low density lipoprotein (Ox-LDL) in human blood, a biological marker of atherogenesis. Design and methods: A sandwich EIA suitable for the measurement of human Ox-LDL was developed using the mouse monoclonal antibody FOH1a/DLH3. This antibody, specific for oxidized phosphatidylcholine, was used as the capture antibody, and a horseradish peroxidase (HRP)-labeled goat anti-human apolipoprotein-B (Apo-B) IgG was used for detection. Copper-oxidized human LDL, prepared under controlled conditions, was used as a standard and the results of the EIA were expressed in arbitrary units (U/mL). Results: This EIA meets all the requirements for use in routine clinical assays in terms of sensitivity (detection limit: 1 U/mL), reproducibility (total CV: 2.3–7.7%), accuracy (recovery: 90.6–103.8%), simplicity and rapidity ( Conclusions: We have developed a new assay, suitable for the measurement of Ox-LDL in human blood, which meets the requirements for routine clinical assay.

Abstract: Objectives: Evaluation of glutathione (GSH) system in different tumors to reveal its potential usefulness in a clinical setting. Design and methods: In addition to 10 normal controls, blood and tissue samples (85 benign and 109 malignant) from patients with breast, ovarian, prostatic, and liver neoplasms were investigated. The GSH concentration, glutathione S-transferase, glutathione peroxidase, and glutathione reductase activities were biochemically measured. Results: Whereas all the components of the GSH system increased in patients with breast tumors, few components were significantly changed in patients with malignant ovarian, prostatic as well as metastatic liver diseases. GSH had the highest Z scores in ovarian and breast tumors. It was correlated (p < 0.05) with both glutathione S-transferase, glutathione peroxidase in breast cancer and with glutathione S-transferase only in prostate cancer. No correlation could be found in the expression of the GSH system in the blood and tissues of the same group of patients. Conclusion: This work revealed that measurement of some and/or all components of the GSH system might be of clinical value in some malignant cases.

Abstract: Objectives: The aim of the present study was to investigate the effect of dietary treatment on serum and erythrocyte lipid peroxidation and erythrocyte antioxidative enzyme activity of patients with Type 2 diabetes. Design and methods: A total of 30 patients with newly diagnosed as Type 2 diabetes were enrolled to the study. A total of 30 healthy subjects served as controls. Diabetic patients were given standard dietary treatment that was composed of 50% to 55% carbohydrate and 30% fat for 2 months. No diet was applied for controls. For both groups serum and erythrocyte lipid peroxidation and erythrocyte superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) were obtained at first and at the end of 2 months. Results: Diabetic patients had higher serum and erythrocyte lipid peroxidation than those of controls before dietary treatment( p p > 0.05). At the end of 2 months of dietary treatment, while diabetics had still higher glucose and erythrocyte lipid peroxidation than controls ( p p > 0.05). In diabetic patients, after 2 months of dietary treatment, whereas serum and erythrocyte lipid peroxidation decreased, erythrocyte SOD and GSH-Px activities showed significant increase ( p Conclusions: Our results showed significant alteration in serum and erythrocyte lipid peroxidation and erythrocyte antioxidant enzyme status of patients with Type 2 diabetes by dietary treatment. However, whether such alterations have clinical importance for diabetic patients needs further investigation.

Abstract: Objectives: To validate the novel lymphocyte toxicity assay (LTA) based on the mitochondrial succinate dehydrogenase (SDH) activity vs. the LTA by using trypan blue exclusion and to determine the utility of the assay to confirm drug hypersensitivity syndrome (DHS) to sulphonamides (SMX) and aromatic anticonvulsants. Methods: Incubation of patient lymphocytes, with or without murine hepatic microsomes with anticonvulsants or SMX. The viability of lymphocytes was based on SDH activity that can be measured spectrophotometrically. The percentage of cells displaying cytotoxicity compared to controls (cells treated only with drug) was calculated. Seventy-two immunocompetent and 16 immunocompromised (HIV) patients with DHS to SMX were sampled. The results were validated vs. 26 controls that had not experienced DHS to SMX. Sixty-two patients who had DHS to anticonvulsants were compared with 24 controls that did not have any DHS to the same anticonvulsants. Results: The results showed a very good percentage of sensitivity 98 and specificity 96 with a κ-score of 0.96. LTA higher than 13.5% was considered positive for the immunocompetent population and LTA higher than 22% was positive for the immunocompromised population. In two of the 26 controls, LTA was positive. Conclusion: The high quantitative κ-value 0.96 emphasizes that the novel LTA is at least as good as the trypan blue assay. The latter is labor intensive, time consuming, and prone to human error. The new assay is objective, faster, and has been reproducible in assessing cytotoxicity.

Abstract: Objectives: To validate a specific enzyme-linked immunosorbent assay for the rapid-acting human insulin analogue, insulin aspart, in human serum, human plasma, and porcine plasma. Design and methods: For the enzyme-linked immunosorbent assay, two murine monoclonal antibodies were developed that bind to two different epitopes on the insulin aspart molecule. Key parameters for validation were imprecision, accuracy, matrix effects, dilution-linearity, and cross-reactivity. Results: No cross-reactivity was found with human and porcine insulin, human proinsulin, or human C-peptide. The assay is sensitive (limit of quantification = 11.5 pmol/L), accurate (95–107% recovery with human serum, human plasma, and porcine plasma in the range 16–800 pmol/L), and has a 14.7% total imprecision within the entire analytical range. Dilution of samples gave linear results with human serum as the diluent. Conclusions: The insulin aspart-specific enzyme-linked immunosorbent assay described in this study is well suited to study the bioavailability, bioequivalence, and pharmacokinetics of this insulin analogue.

Abstract: Objectives: To describe the biochemical and clinical features in hereditary coproporphyria (HCP). Design and method: Within the last 20 years, we investigated 53 patients (male:female = 1:2.5; age = 8–86 years) suffering from HCP. We describe the characteristic levels of urine, and fecal porphyrins and their precursors in hereditary coproporphyria and present the clinical features. Especially, we measured the coproporphyrin isomers I and III. Results and conclusion: The group of hereditary coproporphyria patients exhibited a significantly higher (p < 0.0001) excretion of urinary porphyrin precursors, δ-aminolevulinic acid (median = 84 μmol/24 h) and porphobilinogen (median = 39 μmol/24 h), as compared to controls (δ-aminolevulinic acid: 22 μmol/24 h, porphobilinogen: 3 μmol/24 h; median, n = 20). The median of coproporphyrin in urine (1315 nmol/24 h) and feces (1855 nmol/g) were enhanced 12- and 168-fold, as compared to healthy subjects (urinary coproporphyrin: 106 nmol/24 h, fecal coproporphyrin: 11 nmol/g; median, n = 20). During therapy on one female patient, with IV application of heme arginate, a considerable decline of porphyrin precursors and porphyrin excretion was observed. The examination of urinary and fecal coproporphyrin isomers I and III revealed an excessive elevation of the coproporphyrin isomer III of 87% in urine and 94% in feces, respectively (normal: urinary isomer III = 69–83% and fecal isomer III = 25–40%). In feces the increase of isomer III caused an inversion of the physiologic coproporphyrin isomer III:I ratio that could be recognized in all various stages in hereditary coproporphyria and in children. Acute attacks of hereditary coproporphyria are accompanied by an acute polysymptomatic clinical syndrome, and this is associated with high levels of urinary porphyrin precursors. On review of our patients, the highest percentage had abdominal pain (89%), followed by neurologic (33%), psychiatric (28%), cardiovascular (25%), and skin symptoms (14%).

Abstract: Objective: The purpose of the study is to identify biochemical tests that are good predictors for the diagnosis of pheochromocytoma in patients at hypertension. Setting: Review of data from of 3826 patients studied over a 5-year period, between 1994 and 1998, at the University Hospital “Virgen de la Arrixaca” Murcia, Spain. Design and Methods: A retrospective study for the diagnosis of pheochromocytoma of 24-h urinary free catecholamines (norepinephrine, epinephrine, and dopamine) measured by high-performance liquid chromatography (HPLC)-electrochemical detector (ECD), total metanephrines (MNt), and vanillylmandelic acid measured by spectrophotometric methods. Results: During this period, 57 patients were found to have pheochromocytoma, being 47 sporadic, 9 with multiple endocrine neoplasia type 2A, and 1 with neurofibromatosis. In all patients multiple endocrine neoplasia type 2A the tumor were bilateral but only in four of the sporadic tumor group (p Conclusion: Urinary 24-h MNt excretion level is the best single biochemical test for screening and, in combination with norephinephrine, is diagnostic of the presence of pheochromocytoma.

Abstract: Objectives: To evaluate a simple assay for macroprolactin for use with the Bayer Immuno 1 analyzer, and to compare the reactivity of macroprolactin in commonly used automated prolactin assays. Methods: Macroprolactin in serum was precipitated in a buffer containing 13.3% polyethylene glycol (PEG) 8000, redissolved, and assayed on the Bayer Immuno 1 for PRL. Presence of macroprolactin was confirmed in some sera by FPLC using a Pharmacia Superose 12 column, followed by prolactin assay of the fractions on the Immuno 1. Sera with and without macroprolactin were then also assayed on the Abbott AxSYM, Bayer Centaur, Beckman Access, and Roche Elecsys. Results: The PEG precipitation assay is simple and reproducible (CVs Bayer Immuno 1 > Abbott AxSYM > Bayer Centaur > Beckman Access, with the Centaur showing more variability than other assays. Conclusion: Macroprolactin can be easily quantitated using the Immuno 1 PRL assay after PEG precipitation. It cross-reacts to different degrees with common prolactin assays, and is a major source of variability between them.

Abstract: Objectives: This study investigates the sensitivity of various standard clinical techniques in the detection of albumin fragments. The significance of this work is in the detection of urinary proteins, such as albumin, which has recently been discovered to be excreted as mainly peptide fragments as a result of filtered albumin undergoing degradation during renal passage. All filtered proteins undergo a similar degradation process. Design and Methods: Albumin digested with trypsin was used as a model urine solution. The solution was assayed for albumin concentration by various methods including the biuret assay that is known to detect urinary albumin fragments. The digest solution was also analyzed by various clinically used chromagen assays, electrophoretic and chromatographic methods to determine whether they are able to detect the fragmented protein. Results: The benzethonium chloride, Coomassie blue, and pyrogallol red assays for urine protein, the immunoassay for human albumin and sodium dodecyl sulfate polyacrylamide gel electrophoresis with Coomassie blue staining were unable to detect the albumin fragments. Capillary electrophoresis was sensitive to the fragments but with low resolution. High-performance liquid chromatography gave the best results. Conclusions: Many techniques utilized to assay patient urine samples are unable to detect fragmented albumin and, hence, will severely underestimate albumin and protein excretion.

Abstract: Objective: To review some of the recently proposed improvements and the corresponding apparent issues in the field of biochemical markers of cardiac damage. Conclusions: The continuous development of new analytical tools for the biochemical evaluation of patients with suspected myocardial injury brings without doubt new challenges of careful technological evaluation, implementation, and standardization but it may also provide a unique opportunity to markedly enhance our diagnostic performance in the clinical setting of acute coronary syndrome.

Abstract: Objectives: To set up an optimized multiplex polymerase chain reaction for real-time genotyping of the prothrombotic risk factors methylenetetrahydrofolate reductase C677T and factor V Leiden on the LightCycler. Design and methods: Novel primer and probe sets were designed on the basis of thermodynamic double-strand DNA stability calculations. Detection probes were labeled with LC-Red640 or Cy5.5 dye. Results: The polymerase chain reaction efficiency was reduced in multiplex polymerase chain reaction but this could be overcome by the design of novel amplification primers. The selection of detection probes with a lower melting temperature ( T m ) and high Δ T m improved the discrimination of heterozygous samples. Color compensation was not compromised by either the use of the Cy5.5 dye or different fluorescein linker chemistries. Conclusions: Probes with a Δ T m of 5 °C or more between the matched and mismatched state are desirably for genotyping. Such probes can be selected by using a priori calculations based on the thermodynamic nearest neighbor model.

Abstract: Objective: To implement a quality control program for the standardization and harmonization of lipid and lipoprotein analyses as performed at two core laboratories (St. Paul’s Hospital, UBC [Vancouver], and NPHI [Helsinki]) for the Diabetes Atherosclerosis Intervention Study (DAIS). Design and methods: A DAISSOFT computer program was designed to minimize the occurrence of data and sample management errors during the course of the study. Fresh human serum was used for the provision of an accuracy based external quality control program that monitored the analytical performance of lipid testing at these two laboratories. A separate program was designed for monitoring hemoglobin A 1c (HbA 1c ). At the outset of the study, allowable total error goals were established for each analyte. Ongoing performance was monitored using bimonthly blinded challenges of fresh human serum. The two EQA programs routinely monitored the analysis of total cholesterol, calculated LDL-cholesterol, HDL-cholesterol, net triglycerides, apoprotein A-1, apoprotein B, and HbA 1c . Results: The EQA precision and accuracy data for the measurement of total cholesterol at the two core laboratories over the last 5 years indicated both laboratories operated with good precision, approximately 1% CV over the time period. The accuracy at both laboratories was similar initially. Part way through the study, the accuracy of the cholesterol method at NHPI tended to drift upward with an operating positive bias (+3%) relative to the Abell Kendall reference method. Triglyceride measurements were the most problematic for the study. By EQA cycle 8, the accuracy of the method at UBC had stabilized and was meeting the accuracy goals of the study. NPHI’s method was negatively biased relative to the accuracy base of the DAIS study. In spite of recalibrating their method, NPHI found it difficult to maintain consistent accuracy for the measurement of triglycerides during the study. Both laboratories operated their HDL methods with excellent precision. Accuracy at NHPI was well maintained over the course of the study whereas the accuracy of HDL measurements at UBC was more problematic. There was an inconsistent variation in the accuracy of apoprotein A-1 measurements at both laboratories. In most cases, the bias would be corrected by the time of the next EQA challenge. In the case of apo B, one laboratory was standardized to the CDC while the other laboratory was standardized to IFCC/WHO. The discrepancy between these two accuracy bases was > 20%. Recalibration to a common accuracy base rectified the problem. Only minor problems were encountered with the precision and accuracy of the DIAMAT assay for hemoglobin A-1c. The two DAIS core laboratories consistently operated within the 9% total error goals of the study for HbA 1c . Conclusions: Through the use of this program, the two DAIS core laboratories were able to maintain their lipid analyses within the limits of allowable total error that had been established for the study.

Abstract: Objectives: Autoantibodies against the p53 tumor suppressor protein have been detected in the serum of a proportion of patients with various cancers. The generation of such antibodies has been proposed to be due to either tumor p53 protein accumulation or to the type of p53 gene mutation. These hypotheses are examined in the present study. Design and methods: Using immunofluorometric assays, we studied 195 patients with primary breast cancer for the presence of p53 antibodies in serum and p53 protein accumulation in the corresponding tumor. Seventeen patients (9%) were p53 antibodypositive and 77 (40%) overexpressed p53. Ten of the 17 p53 antibody-positive patients had tumor p53 accumulation and 7 were negative for p53. Statistical analysis revealed a weak association between the presence of p53 antibodies and p53 protein accumulation (p 5 0.05). Direct DNA sequencing of exons 1‐11 of the p53 gene was performed for 16 p53 antibody-positive and 16 p53 antibody-negative patients. Results: Five of the seropositive and eight of the seronegative patients had a p53 gene mutation. Four of the five mutations in the p53 antibody-positive patients affected a Tyr residue, whereas none of the gene abnormalities in the seronegative patients had such an