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Clinica Chimica Acta (v.362, #1-2)

International Society for Enzymology (ISE), Newsletter, August 2005 by David M. Goldberg (pp. s15-s22).
Erich Kaiser; Personal memoir; In memoriam; Birth of ISCE; International Society for Enzymology (ISE) Newsletter August 2005
Editorial Board (pp. iii).

Leptin as a new diagnostic tool in chronic heart failure by P. Christian Schulze; Jrgen Kratzsch (pp. 1-11).
Leptin, the product of the ob-gene, regulates cellular homeostasis and glycemic control. While initially described as an adipocyte-derived protein with expression and secretion restricted to adipose tissue, recent reports have shown local expression of leptin in several tissues including the skeletal muscle, heart, vessels and brain. Leptin acts through the different isoforms of its receptor which are ubiquitously expressed and can be detected in endothelium, vascular smooth muscle and myocardium. In addition to its metabolic effects, leptin has distinct effects in the cardiovascular system leading to increased production of proinflammatory cytokines and oxidative stress, vascular remodeling and neointima formation as well as cardiomyocyte hypertrophy. Notably, recent clinical studies have linked serum levels of leptin to the occurrence of cardiovascular events such as myocardial infarction and stroke suggesting that leptin promotes pro-atherogenic vascular mechanisms. In contrast, less is known about the role and effects of leptin in the setting of chronic heart failure. We here review the current knowledge on cardiovascular effects of leptin and discuss its potential as a new therapeutic tool in chronic heart failure.

Keywords: Leptin; Chronic heart failure; Atherosclerosis; Risk; Biomarker

CSF markers in sleep neurobiology by Jose E. Martnez-Rodrguez; Joan Santamaria (pp. 12-25).
The cerebrospinal fluid has been used in the study of normal and pathological conditions of the central nervous system for more than a century. CSF analysis has also been applied to the study of sleep and its disorders but methodological aspects have often limited the results. The discovery of the hypocretin system (also known as orexin system) and its involvement in the pathophysiology of narcolepsy has opened a new field in the diagnosis of hypersomnia by CSF analysis and has revived the interest on this subject in sleep medicine. Older and new lines of research involving CSF measurement of hypocretin and other neurotransmitters in sleep and its disorders are reviewed.

Keywords: Cerebrospinal fluid; Sleep; Hypocretin; Orexin

Clinical laboratory assessment of acute pancreatitis by Ahmed Z. Al-Bahrani; Basil J. Ammori (pp. 26-48).
Several biochemical markers in blood and urine have been investigated to establish their clinical application in patients with acute pancreatitis (AP). The relevant studies are reviewed and critically appraised.Medline and the World Wide Web were searched and the relevant literature was classified under the following categories: (1) diagnosis of AP and (2) prediction of: a) disease severity, b) pancreatic necrosis and its secondary infection, c) organ failure and death, and d) disease etiology.Serum lipase is a more reliable diagnostic marker of AP than serum amylase. Urinary strip tests for trypsinogen activation peptide (TAP) and trypsinogen-2 provide a reliable early diagnosis of AP. Useful predictors of severity may include serum procalcitonin and urinary TAP and trypsinogen-2 on admission, serum interleukins-6 and -8 and polymorphonuclear elastase at 24 h, and serum C-reactive protein (CRP) at 48 h. Other markers such as amyloid A and carboxypeptidase B activation peptide (CAPAP) need further investigation. Biochemical prediction of pancreatic necrosis requires 72 h to reach reliability and is impractical. However, the daily monitoring of serum procalcitonin provides a non-invasive detection of infected necrosis; the promising role of phospholipase A2 in this regard requires further investigation. Early transient hypertransaminasemia reliably predicts biliary etiology, while serum carbohydrate-deficient transferrin and trypsin may predict an alcoholic etiology.

Keywords: Abbreviations; ALP; alkaline phosphatase; ALT; alanine transaminase; ANP; acute necrotizing pancreatitis; AP; acute pancreatitis; APACHE; Acute Physiology And Chronic Health Evaluation; AST; aspartate transaminase; CAPAP; carboxypeptidase B activation peptide; CDT; carbohydrate-deficient transferrin; cICAM-1; circulating isoforms of ICAM-1; CRP; C-reactive protein; CT; computerized tomography; ERCP; endoscopic retrograde cholangiopancreatography; EUS; endoscopic ultrasound; G-CSF; granulocyte colony stimulating factor; Hct; haematocrit; HLA; human leucocyte antigen; hPASP/PCPB; human pancreas-specific protein/procarboxypeptidase B; ICAM-1; intercellular adhesion molecule-1; IL; interleukins; IU; international unit; LDH; lactate dehydrogenase; MCV; mean corpuscular volume; MRI; magnetic resonance imaging; n; number; NLR; negative likelihood ratio; NPV; negative predictive value; PLA; 2; phospholipase A; 2; PLR; positive likelihood ratio; PMN; polymorphonuclear; PPV; positive predictive value; SAP; severe acute pancreatitis; sICAM-1; soluble intercellular adhesion molecule; sTM; soluble thrombomodulin; TAP; trypsinogen activation peptide; TNF; tumour necrosis factor; U/L; unit/liter; URL; upper reference level; USS; ultrasoundAcute pancreatitis; Markers; Diagnosis; Severity; Pancreatic necrosis; Infection

Lipid profile, oxidant–antioxidant status and glycoprotein components in hyperlipidemic patients with/without diabetes by K. Kaviarasan; M.M. Arjunan; K.V. Pugalendi (pp. 49-56).
Plasma lipoproteins are protected against oxidative modification by the antioxidant defense system. An imbalance in the antioxidant defense system seems to result from the accumulation of low density lipoprotein (or) very low density lipoprotein in the course of hyperlipidemia.The lipid profile, glycoprotein components, glucose, total proteins, albumin, lipid peroxidation and antioxidant status of plasma and erythrocytes were investigated in hyperlipidemic patients and hyperlipidemic patients with diabetes on treatment with glibenclamide (or) glipizide along with normal subjects and compared.A significant increase was observed in the levels of total cholesterol (TC), VLDL-C, triglycerides (TG), lipid peroxidation, ceruloplasmin (CP), glycoprotein components and glucose in the hyperlipidemic patients with/without diabetes and the increase was more pronounced (except TC) in patients with diabetes. The activities of erythrocyte antioxidant enzymes such as superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase (CAT) and plasma concentrations of vitamins C and E, and reduced glutathione (GSH) decreased in hyperlipidemic patients with/without diabetes. GPx, CAT, vitamin C and GSH levels exhibited a further decrease in hyperlipidemic patients with diabetes.An enhanced levels of VLDL-C, TC/HDL-C ratio, TG, lipid peroxidation, glycoprotein components, and decreased concentrations of total proteins (TPs) and albumin were observed in hyperlipidemic patients with diabetes while the decrease was more marked in GSH, vitamin C, CAT and GPx among antioxidants.

Keywords: Hyperlipidemia; Diabetic hyperlipidemia; Lipids; Lipid peroxidation; Glycoprotein components

Caffeic acid phenyl ester in propolis is a strong inhibitor of matrix metalloproteinase-9 and invasion inhibitor: Isolation and identification by Un-Ho Jin; Tae-Wook Chung; Sung-Koo Kang; Seok-Jong Suh; June-Ki Kim; Kang-Hyun Chung; Yeun-Hwa Gu; Ikukatsu Suzuki; Cheorl-Ho Kim (pp. 57-64).
Propolis has been used as a folk medicine and has several proven biological activities. Herbal remedies recommended for cancer therapies in Korea.Matrix metalloproteinase (MMP)-9-inhibitory activity of propolis has been assessed. CAPE as an acting compound was isolated and molecular structure was determined. Anti-invasion activity of CAPE was assayed using hepatocarcinoma cells.Propolis ethanol extracts showed a strong inhibitory effect of MMP-9 activity, which is known to be involved in tumor cell invasion and metastasis in a concentration-dependent manner on zymography. Assay guided fractionation led to the isolation of a caffeic acid phenyl ester (CAPE) as the compound responsible for the anti-MMP-9 activity. CAPE was obtained by reversed-phase HPLC, and its structure was elucidated by fast atom bombardment mass spectrometry and tandem mass spectrometry. The purified CAPE inhibited MMP-9 activity with the IC50 of 1.0–2.0 nmol/l.CAPE possesses selective antiproliferative activity toward hepatocaricoma cell line Hep3B, but not primary cultured mouse hepatocytes.

Keywords: Zymography; Matrix metalloproteinase-9; Caffeic acid phenyl ester; Propolis

Diagnostic potential of phosphorylated cardiac troponin I as a sensitive, cardiac-specific marker for early acute coronary syndrome: Preliminary report by David Bar-Or; Gregory W. Thomas; Raphael Bar-Or; Leonard Rael; James V. Winkler (pp. 65-70).
Cardiac troponin I (cTnI) has low sensitivity in the early hours of acute coronary syndrome (ACS). For patients with early ACS symptoms, we determined the diagnostic potential of an immunoassay for phosphorylated cTnI (PO4-cTnI).In a prospective study of 61 emergency department patients with suspected ACS, we compared a novel plasma immunoassay for PO4-cTnI to cTnI overall and in a subgroup with symptoms ≤4 h duration ( n=31). Admission PO4-cTnI and cTnI assays (thresholds determined by ROC curve) were analyzed in a blinded fashion against the clinical, ECG and coronary angiographic diagnosis of ACS.Overall, PO4-cTnI sensitivity was significantly higher than cTnI (82% vs. 50%, respectively, P<0.05) and PO4-cTnI specificity was 81% ( n=61). Addition of PO4-cTnI to cTnI improved sensitivity to 91% vs. 50% for cTnI alone ( P<0.001). In the ≤4 h subgroup ( n=31), PO4-cTnI sensitivity was significantly higher than cTnI (79% vs. 26%, respectively, P<0.01) and PO4-cTnI specificity was 75%. In the same ≤4 h subgroup, addition of PO4-cTnI to cTnI improved sensitivity to 84% vs. 26% for cTnI alone ( P=0.001).The results suggest that PO4-cTnI, alone or in combination with cTnI, warrants further investigation as a sensitive, cardiac-specific diagnostic tool for early ACS.

Keywords: Abbreviations; cTnI; cardiac troponin I; PO; 4; -cTnI; phosphorylated cardiac troponin I; CV; coefficient of variation; Sens; sensitivity; Spec; specificity; STEMI; ST elevation myocardial infarction; Non-STEMI; non-ST elevation myocardial infarction; UA; unstable anginaPhosphorylation; Inflammation; Troponin; Cardiac markers; Acute coronary syndrome; Myocardial infarction

Molecular analysis of homocystinuria in Brazilian patients by Marianna P.R. Porto; Luciano C. Galdieri; Vanessa G. Pereira; Naja Vergani; Jos Cludio C. da Rocha; Ceclia Micheletti; Ana Maria Martins; Ana Beatriz A. Perez; Vnia D`Almeida (pp. 71-78).
Cystathionine β-synthase (CBS) deficiency is the most common cause of homocystinuria. However, no data are available concerning the molecular basis of this disease in Brazilian populations.We studied 14 Brazilian patients from 11 unrelated families using a combined screening approach, involving restriction analysis, single-strand conformational polymorphism (SSCP) scanning, and sequencing.All patients presented homocysteine levels higher than 200 μmol/l before the beginning of treatment. The most common CBS gene mutations, p.G307S (c.919G>A) and p.I278T (c.833T>C), were evaluated and the allele c.919A was not found. One allele with the c.844 ins68 (4.5%) in the CBS gene was found. Three families (6 patients) presented the allele c.833 C (13.6%), without the insertion in the heterozygous state. SSCP scanning and sequencing showed 3 alleles p.T191M (13.64%) in 2 families. One allele with a novel mutation was found in exon 4 (c.168T>A) of the CBS gene (4.5%). We also analyzed c.677C>T and c.1298A>C polymorphisms in the methylenetetrahydrofolate reductase ( MTHFR) gene and the 2756A>G polymorphism in the methionine synthase ( MTR) gene. The frequencies of mutated alleles were: 50% c.677T and 18.2% c.1298C for MTHFR, and 27.3% c.2756G for MTR.In spite of the high level of racial mixing in the country, Brazilian homocystinuric patients did not present a high prevalence of the most common mutations described in the literature.

Keywords: Abbreviations; CBS; Cystathionine β-synthase; SSCP; single-strand conformational polymorphism; MTHFR; methylenetetrahydrofolate reductase; MTR; methionine synthase; FVL; Factor V Leiden; WHO; World Health OrganizationHomocystinuria; Cystathionine β-synthase; Methylenetetrahydrofolate reductase; Methionine synthase; Mutation analysis; Factor V Leiden; Prothrombin

Rapid and highly sensitive determination of trace proteins using dibromomethyl carboxyazo–Pb(II) complex as a spectroscopic reagent by Qin Wei; Yuan-Yuan Li; Yan Li; Bin Du; Cai-Hong Duan (pp. 79-84).
The concentration of protein in body fluids is an important measure in disease diagnosis and in analytical biochemistry. Searching for a sensitive and rapid method for proteins is an ongoing subject of investigation. We describe the reaction of dibromomethyl carboxyazo–Pb(II) with proteins found in urine.In a buffer containing of HCl and KCl at pH 1.80, dibromomethyl carboxyazo–Pb(II) complex combined with proteins to form a colored compound. The reaction was completed in 2 min and remained stable for 80 min. Under optimum conditions, the product of reaction between protein and dibromomethyl carboxyazo–Pb(II) absorbed at 530 nm and was linear in the range of 0–50 μg/ml of BSA. Moreover, the maximum binding number was defined to express the binding ability of dibromomethyl carboxyazo–Pb(II) to protein under a given set of conditions.The method was applied to the determination of urine proteins. The recovery of protein from human urine was 97.6–103% and the precision was <4.8%. Results compared favorably to the Biuret and Bradford protein methods on 50 urine samples.This method had high sensitivity and specificity and may be applicable to clinical analysis.

Keywords: Protein; Spectrophotometry; Dibromomethyl carboxyazo–Pb(II) complex

Comparison between three molecular methods for detection of blood melanoma tyrosinase mRNA. Correlation with melanoma stages and S100B, LDH, NSE biochemical markers by Concetta Santonocito; Paola Concolino; Maria Michela Lavieri; Franco Ameglio; Stefano Gentileschi; Rodolfo Capizzi; Sandro Rocchetti; Pierluigi Amerio; Massimo Castagnola; Cecilia Zuppi; Ettore Capoluongo (pp. 85-93).
The molecular monitoring of circulating tumor cells by reverse transcriptase-PCR (RT-PCR) for patients with melanoma, is still under debate. It may be affected by: a) pre-analytical variability, b) frequency of melanoma-associated gene transcripts and c) the reliability of the methods employed. Few commercial methods are available for the detection of tyrosinase mRNA in blood.Comparison between two RT-PCR-nested methods with a third one based on real-time methodology, for detection and quantitation of tyrosinase transcripts, respectively.Sixty-two melanoma patients with different AJCC stages and 20 healthy subjects were enrolled. All blood samples were extracted in duplicate with two different methods. Two nested-PCR methods (one commercial and one in house) plus a real time commercial kit were employed.The two nested PCR methods employed were overimposable, specific and sensitive, at least in the stage III, where there was a concordance between sentinel lymph nodes detection and blood tyrosinase positivity. The different extraction methods did not affect the quality of results, while the commercial real-time kit cannot be used.Tyrosinase mRNA detection may be therefore employed to monitor the melanoma patients over time in function of response to therapy.

Keywords: Abbreviations; ABL; Abelson; AJCC; American Joint Committee on Cancer; EDTA; Ethylenediaminetetraacetic acid; EORTC; European Organisation for the Research and Treatment of Cancer; FAM; realtime probe; GAPDH; Glyceraldehyde 3-phosphate dehydrogenase; Gp100; Glycoprotein 100; HMB-45; monoclonal antibody HMB-45; LDH; Lactate dehydrogenase; Mart-1; Melanoma-associated antigen recognized by T cells; MelanA/Mart-1; Melanocyte-associated monoclonal antibody; NSE; Neuron-specific enolase; PCR; Polymerase chain reaction; RT-PCR; Reverse transcriptase polymerase chain reaction; S-100B; S-100 protein, beta chain; VIC-MGB; realtime probeTyrosinase mRNA; Nested-PCR; Real-time; Melanoma

Experimental spinal cord injury induced an increase of extracellular ascorbic acid concentration in anesthetized rats: A microdialysis study by Pi-Ju Tsai; Wen-Ying Chen; Shun-Fen Tzeng; Wei-Ming Liang; Chung-Shi Yang (pp. 94-100).
Ascorbic acid plays important roles in mammalian central nervous system. We employed an on-line analytical system to monitor the extracellular ascorbic acid concentrations in anesthetized rat spinal cord before and after the experimental injury. A microdialysis probe (216 μm od, 200 μm id, 3 mm in length) was implanted into an anesthetized rat spinal cord (Thoratic-12). Microdialysis perfusate (2 μl/min) was collected in the sample loop (20 μl) of an on-line injector for direct injection onto a High Performance Liquid Chromatography (HPLC) system equipped with an electrochemical detector. Normal ascorbic acid concentrations in the spinal cord extracellular fluids ranged from 1.8 μM to 10.8 μM (mean±S.D. 5.6±2.4 μM, n=8). The experimental spinal cord injury, induced by a lesion at T-10, gradually yet significantly increased the extracellular ascorbic acid levels. The effect of exogenous glutamate perfusion (0.2 mM, 2 mM, and 20 mM) through the microdialysis probe also increased the extracellular ascorbic acid concentrations in a dose dependent manner. These results suggested that the injury-induced ascorbic acid accumulation may result from elevated extracellular glutamate levels that are commonly observed in spinal cord injury. This on-line, continuous and automatic monitoring system can be applied to future investigations on the roles of ascorbic acid in spinal cord injuries.

Keywords: Microdialysis; Spinal cord; Lesion injury; Anesthetized rats; On-line analysis; High performance liquid chromatography (HPLC)

Biochemical properties of β-glucosidase in leukocytes from patients and obligated heterozygotes for Gaucher disease carriers by Kristiane Michelin; Alessandro Wajner; Hugo Bock; ngela Fachel; Roberto Rosenberg; Ricardo Flores Pires; Maria Luiza Saraiva Pereira; Roberto Giugliani; Janice Carneiro Coelho (pp. 101-109).
Gaucher's disease (GD) is a disorder caused by the deficiency of lysosomal β-glucosidase, an enzyme that participates in the degradation of glycosphingolipids. Deficiency of this enzyme results in the storage of glucocerebrosides in lysosomes of macrophage. No studies are available in the literature comparing biochemical and kinetic behavior of this enzyme in leukocytes and fibroblasts from normal individuals, obligate heterozygotes and patients with GD.The behavior of β-glu in terms of optimum pH, heat stability, Km and Vmax in leukocytes from patients with GD and obligated heterozygotes with different genotypes and normal individuals were characterized.Optimum pH was similar in all groups analyzed. In terms of Km and Vmax, several differences among heterozygotes and homozygotes groups and among these groups and normal enzyme were observed. Enzyme from all groups were inactivated when preincubated at 60 °C, but some enzymes were more stable than other. Results showed a different behavior of the enzyme in the 3 groups under analysis. Such behavior varied according to individual mutation.The catalytic gradient presented by β-glu allowed the correlation of N370S mutation–which presented more stable biochemical properties–with the non-neurological clinical condition of the disease and the catalytically less stable mutation (D409H), with the neurological clinical condition of GD. This study contributes to a better understanding of the repercussion of the different mutations on the protein function, thus allowing to predict the severity of such complex metabolic disorder and to anticipate the most appropriate intervention for each case specifically.

Keywords: Gaucher's disease; β-Glucosidase; Lysosomal storage diseases; Sphingolipidosis

Contemporaneous carrier-state of two or three “proatherosclerotic? variants of APOE, ICAM1, PPARA and PAI-1 genes differentiate CAD patients from healthy individuals by Iwona Żak; Anna Balcerzyk; Beata Sarecka; Paweł Niemiec; Zbigniew Ciemniewski; Stanisław Dyląg (pp. 110-118).
Atherosclerosis is the most important cause of coronary artery disease (CAD). Genetic predisposition to CAD is related to polymorphisms of genes encoding products functionally involved in pathogenesis of atherosclerosis. Polymorphisms of genes participating in monocyte adhesion and diapedesis, lipid metabolism and fibrinolysis regulation may be partially responsible for this process. The aim of our study was to assess the polymorphic variants frequencies of ICAM1, APOE, PPARA and PAI-1 genes in CAD patients and healthy blood donors and to find specific arrangement of polymorphic variants which would differentiate both groups.We studied 146 CAD patients and 121 healthy blood donors. Polymorphisms in analyzed genes were examined using PCR-RFLP analysis.We found significantly higher frequency of 5G allele of PAI-1 gene in patients than in control subjects ( p=0.038, OR=1.44). We observed also a considerably higher frequency of contemporaneous carriers of two or three “proatherosclerotic? variants: 1) PPARA and PAI-1, 2) APOE and ICAM1 and 3) PPARA, ICAM1 and PAI-1 in CAD group comparing to control subjects. The number of “proatherosclerotic? variants carriers differentiate studied groups also independently of specific genotype arrangement.In conclusion, contemporaneous carrier-state of two or three polymorphic variants within analyzed genes is associated with CAD.

Keywords: Abbreviations; ACE; angiotensin converting enzyme; APOE; apolipoprotein E; BMI; body mass index; CAD; coronary artery disease; HDL; high density lipoprotein; ICAM1; intercellular adhesion molecule-1; LRP; LDL-related receptor; ox-LDL; oxidized low density lipoprotein; PAI-1; plasminogen activator inhibitor 1; PCI; percuteneous coronary intervention; PCR-RFLP; polymerase chain reaction–restriction fragments length polymorphism; PPARA; peroxisome proliferator-activated receptor alpha; VLDL; very low density lipoproteinPolymorphisms; Plasminogen activator inhibitor-1 gene (; PAI-1; ); Coronary artery disease (CAD); Peroxisome proliferator-activated receptor alpha gene (; PPARA; ); Apolipoprotein E gene (; APOE; ); Intercellular adhesion molecule-1 gene (; ICAM1; )

Oxidative susceptibility of apolipoprotein AI in serum by Takanari Nakano; Atsuo Nagata (pp. 119-124).
We compared the oxidative susceptibility of apolipoprotein AI (apoAI), the major protein component of high-density lipoprotein, in serum with that of low-density lipoprotein (LDL) and of thiobarbituric acid reactive substance (TBARS).Serum samples were oxidized by adding AAPH (2,2′-azobis (2-amidinopropane) dihydrochloride), and exposure to fluorescent light or ultraviolet (365 nm) light for 4 days at 23 °C. Then the levels of oxidized apoAI, total apoAI, oxidized LDL and TBARS were measured.The apoAI in serum samples was readily oxidized on exposure to light, the amount of oxidized apoAI increasing 18.4-fold under fluorescent light and 58.8-fold under UV light compared to that in the untreated serum. On the other hand, the level of TBARS increased only 1.9- and 4.9-fold with the respective exposure to light. The amount of oxidized LDL remained unchanged with the treatment of AAPH and fluorescent light, and decreased under ultraviolet light. The oxidation of apoAI on exposure to the lights was inhibited by quercetin, an antioxidant.ApoAI was more susceptible to oxidation than LDL and TBARS. Thus, oxidized apoAI can be used as an early marker for oxidative stress in humans.

Keywords: Abbreviations; AAPH; 2,2′-Azobis (2-amidinopropane) dihydrochloride; apoAI; apolipoprotein AI; BHT; 2,6-di-; tert; -butyl-4-methylphenol; ELISA; enzyme-linked immunosorbent assay; HDL; high-density lipoprotein; HRP; horseradish peroxidase; LDL; low-density lipoprotein; mAb; monoclonal antibody; TBARS; thiobarbituric acid reactive substance; UV light; ultraviolet lightApolipoprotein AI; Oxidation; Quercetin; Ultraviolet light; Fluorescent light

Detection of autoantibodies to survivin and livin in sera from patients with breast cancer by Atsuhito Yagihashi; Tohsei Ohmura; Koichi Asanuma; Daisuke Kobayashi; Naoki Tsuji; Toshihiko Torigoe; Noriyuki Sato; Koichi Hirata; Naoki Watanabe (pp. 125-130).
Survivin and livin are highly expressed in cancer cells and transformed cells, but show little or no expression in normal differentiated tissues. Human antibody responses to tumor-associated antigens have been detected, but little is known about the response to survivin and livin in breast cancer patients.We examined the prevalence of anti-survivin and livin antibodies in breast cancer patients with a specific enzyme-linked immunosorbent assay (ELISA) using recombinant protein.Using a cutoff value for positivity determined as the mean absorbance +2SD for healthy control samples, sera from 11 of 46 breast cancer patients (23.9%) were positive by the ELISA using recombinant survivin protein. Of 46 samples from the same breast cancer patients, 15 (32.6%) were positive for anti-livin antibodies. In addition, 24 (52.2%) were positive for 1 or both ELISAs using the respective proteins. Intensity of anti-livin antibody responses did not correlate with intensity of anti-survivin responses.Anti-livin antibodies were detected in sera from breast cancer patients by an anti-livin ELISA using full-length recombinant livin protein. Like survivin, livin may act as a major cancer antigen in breast cancer patients.

Keywords: Survivin; Livin; Autoantibody; Breast cancer

Urate oxidation during percutaneous transluminal coronary angioplasty and thrombolysis in patients with coronary artery disease by Sevgi Yardım-Akaydın; Mehmet Kesımer; Ersin Imren; Aylin Sepici; Bolkan Şimşek; Meral Torun (pp. 131-137).
Thrombolysis and percutaneous transluminal coronary angioplasty (PTCA) are kinds of procedures that can be used to restore the blood flow of previously ischemic myocardium that can be the result of excessive production of reactive oxygen and nitrogen species, such as superoxide and hydroxyl radical, hypochlorous acid and peroxynitrite. Reaction of urate with some of these potent oxidants results in allantoin production. In this study, we measured the serum allantoin levels, an oxidation product of urate, and “in vivo? marker of free radical generation in reperfusion of ischemic myocardium.After an overnight fasting state, blood samples were collected from 35 patients with coronary occlusive diseases (7 women and 28 men) and 31 healthy subjects (8 women and 23 men). Serum allantoin and urate levels were measured by a GC-MS method.Serum allantoin levels of patients on PTCA therapy (mean±SD, 27.4±15.2 μmol/l) and thrombolytic therapy (24.6±8.6 μmol/l) were significantly higher than those of the patients without therapy (15.8±6.2 μmol/l, p<0.05 with PTCA and p<0.006 with thrombolysis) and healthy controls (12.6±6.3 μmol/l, p<0.002 with PTCA and p<0.0001 with thrombolysis). Although serum urate levels in PTCA (380.1±72.6 μmol/l) and thrombolysis (359.5±60.0 μmol/l) were higher than those in the non-therapy patients (336.6±53.8 μmol/l) and controls (318.3±81.0 μmol/l), there were no significant differences among groups ( p>0.05). The results of the study are consistent with others which have demonstrated, higher urate levels are associated with coronary occlusive diseases. Our data support the hypothesis that generation of ROS occurs during myocardial reperfusion. Increased allantoin levels may be used as an index of increased oxidative stress during reperfusion.

Keywords: Allantoin; Urate; Oxidative stress; Coronary occlusive disease; Reperfusion injury

Endothelial nitric oxide synthase and fractalkine chemokine receptor polymorphisms on angiographically assessed coronary atherosclerosis by Domingos L.S. Rios; Sdia M. Callegari-Jacques; Mara H. Hutz (pp. 138-146).
The CX3CR1 is a fractalkine chemokine receptor expressed by leukocytes attracting them to the arterial wall inflammation. The endothelial nitric oxide synthase ( eNOS) produces nitric oxide that acts on the vascular wall and circulating blood cells, lessening the inflammatory atherogenic damage. We determined if − 786T> C and E298D eNOS and 745G> A CX3CR1 variants were associated with CAD risk and/or severity in Southern Brazilians of European descent.We investigated these polymorphisms in 358 patients who had undergone coronary angiography and 129 non-symptomatic controls by PCR followed by restriction analyses.The 745 G> A CX3CR1 variant was not associated with CAD in this sample. Patients with significant CAD (coronary stenosis≥75%) presented higher frequencies of the eNOS786C, but not of 298D allele than those observed among patients in whom significant CAD was ruled out by angiography (control group 1, p=0.022) and non-symptomatic controls (control group 2, p<0.001). The eNOS haplotypes derived from these 2 sites revealed that the frequency of haplotypes carrying the − 786C allele (− 786C/298D and − 786C/298E) was increased and of the wild haplotype (− 786T/298E) was decreased in patients with significant CAD ( p=0.003). After controlling for other classical risk factors carriers of haplotypes containing the − 786C allele were at increased CAD risk (− 786C/298D, OR=2.95, p=0.007; and − 786C/298E, OR=2.41, p=0.030).The − 786T> C was the polymorphism associated with severe CAD in this study. Haplotype analyses can be extremely helpful in unraveling the influence of different markers within a gene.

Keywords: CAD; eNOS; CX3CR1; DNA polymorphism

Effects of the CYP3A5 genetic polymorphism on the pharmacokinetics of diltiazem by Takehito Yamamoto; Takahiro Kubota; Takeshi Ozeki; Mika Sawada; Shin-ichi Yokota; Yasuhiko Yamada; Yuji Kumagai; Tatsuji Iga (pp. 147-154).
The cytochrome P450 (CYP) 3A subfamily plays an important role in the metabolism of various endogenous and exogenous compounds. Among CYP3A subfamily members, CYP3A5 is polymorphically expressed and the CYP3A5*3 and CYP3A5*6 alleles are known not to express functional CYP3A5. Thus, these mutant alleles are thought to be responsible for interindividual variability of CYP3A activity.Subjects possessing CYP3A5*1/*1, *1/*3 or *3/*3 received oral administration of diltiazem hydrochloride (60 mg), and plasma and urinary concentrations of diltiazem and its metabolite N-desmethyldiltiazem were measured. Before drug intake, cortisol metabolic clearance in each subject was measured to estimate in vivo CYP3A4 activity.The mean values of total oral diltiazem clearance of subjects with *1/*1, *1/*3 and *3/*3 were 183.439.4, 197.949.6 and 293.6141.1 (l/h), respectively, and were not significantly different among the 3 genotype groups. The cortisol metabolic clearance was not significantly different among the three genotype groups, indicating that the CYP3A4 activity is not significantly different.The results suggest that CYP3A5*3 has only a minor effect on the pharmacokinetics and metabolism of diltiazem. Although our results did not indicate significance of CYP3A5, the effects of CYP3A5*3 on the metabolism of other CYP3A substrates remain to be investigated.

Keywords: CYP3A5; Genetic polymorphism; Diltiazem; Cortisol; CYP3A activity

Ischemia-modified albumin levels in cord blood: A case-control study in uncomplicated and complicated deliveries by Alejandro Gugliucci; Ricardo Hermo; Carolina Monroy; Masahide Numaguchi; Satoshi Kimura (pp. 155-160).
In the past few years ischemia modified albumin (IMA) has emerged as a new biomarker of ischemia in the area of monitoring acute coronary syndromes. We hypothesized that reduced blood flow, such as that resulting from vascular compression in complicated labors or placental ischemia, may increase IMA. IMA level in cord blood could then serve as an indicator of fetal hypoxia and fetal tissue ischemia and serve as a biomarker of the severity of these conditions.We performed a case-control study with 26 newborns (12 normal term deliveries, Apgar 8–9; and 14 complicated labors or pre-term deliveries, Apgar 5–8). Complications were: prematurity (3), fetal distress (6), premature rupture of membranes (6), intrauterine growth retardation (3), pre-eclampsia (1). We also studied 30 healthy adults. IMA was measured in serum from cord blood (or venous blood for adults) by the decrease in cobalt 2+ binding.IMA levels in neonates from non-complicated deliveries are significantly higher (45%, p<0.005) than those of an adult control population, suggesting that IMA may increase as a consequence of labor. This increased IMA in neonates could not be accounted for by the changes in albumin concentration. It is conceivable that a transient increase in IMA reflects, in part, transient localized tissue ischemia due to the external forces exerted on the fetus during the mechanism of labor. IMA levels in cord blood from neonates from complicated deliveries are 50% higher than in neonates from uneventful deliveries ( p<0.05) while their albumin values are not significantly different (32±3 vs. 33±2 g/l). Moreover, IMA seems to be responsive to hypoxic fetal distress, showing values more than 300% higher in cases of severe fetal hypoxia (Apgar 5 n=2: 2.19±0.01 AU vs. 0.64±0.24 for controls). IMA values did not correlate significantly with either lipoperoxides or CRP levels.This is the initial reporting of IMA levels in cord blood from normal deliveries compared to healthy adult ranges and neonates from complicated deliveries. Cord blood IMA levels may be an indicator of fetal ischemia and/or hypoxia. This test could become an additional biomarker to be used in conjunction with other markers and/or clinical scores aimed at determining risk of neurological complications of fetal distress.

Keywords: Abbreviations; IMA; ischemia modified albumin; CRIB; clinical risk index for babies; CRP; C-reactive protein; DTT; dithiothreithol; SNAP; score for neonatal acute physiologyFetal distress; Oxidative stress; Hypoxia

The effects of a novel synthetic retinoid, seletinoid G, on the expression of extracellular matrix proteins in aged human skin in vivo by Mi-Sun Kim; SeRah Lee; Ho Sik Rho; Duck Hee Kim; Ih Seop Chang; Jin Ho Chung (pp. 161-169).
Although retinoids have potential efficacy in aged skin, their side effect (skin irritation) remains a clinical problem. We designed a novel synthetic retinoid, seletinoid G, by using computer-aided molecular modeling, and investigated its effects on the expression of extracellular matrix proteins in human skin in vivo.Twenty-three subjects were tested on the buttocks using 4-day occlusive application of seletinoid G and all- trans retinoic acid ( tRA). Skin irritation after topical application was quantified by the degree of erythema and cutaneous blood flow. The expression of extracellular matrix proteins and interstitial collagenase (MMP-1) in skin biopsies was investigated by immunohistochemical staining and Western blotting.The topical application of seletinoid G under occlusion induced no skin irritation in contrast to tRA, which caused severe erythema. The topical treatment with seletinoid G increased the expressions of type I procollagen, tropoelastin, and fibrillin-1, and reduced MMP-1 in old skin in vivo. Seletinoid G was found to inhibit not only the UV-induced decrease of type I procollagen but the UV-induced increase of MMP-1 and c-Jun protein in young skin in vivo.Seletinoid G is a novel synthetic retinoid, which has little the side effect of skin irritation after topical application. Seletinoid G can repair altered connective tissue in old skin and inhibit UV-induced collagen deficiency in young skin.

Keywords: Abbreviations; t; RA; all-; trans; retinoic acid; RAR; retinoic acid receptor; AP-1; activator protein-1; ECM; extracellular matrix; MMP-1; matrix metalloproteinase-1; UV; ultraviolet; MED; minimal erythema dose; EI; erythema index; LDF; laser Doppler flowmetryRetinoid; Skin irritation; Extracellular matrix; Matrix metalloproteinase; Aging

Formation of 8-nitroguanine in blood of patients with inflammatory gouty arthritis by Horng-Rong Chang; Chih-Chieh Lai; Jong-Da Lian; Chiu-Chu Lin; Chau-Jong Wang (pp. 170-175).
NOx causes DNA damage due to an inflammatory effect of gouty arthritis. We investigated the concentration of 8-nitroguanine (8-NO2-G) in the blood of patients with arthritis.Subjects were divided into 3 groups: (1) high inflammatory (HI) group ( n=21) with hyperuricemia (mean, 8.9 mg/dl) and leukocytosis, (2) low inflammatory (LI) group ( n=14) with mild hyperuricemia (mean, 7.6 mg/dl) but normal leukocyte count, (3) non-inflammatory (NI) healthy control ( n=19) with mean serum uric acid concentration 5.3 mg/dl and normal leukocyte count. Serum C-reactive protein (CRP) concentrations were measured by a visual agglutination method. The blood concentrations of 8-NO2-G were determined by high performance liquid chromatography-electrochemical detection and were compared between groups.There was significant difference in percentage of positive CRP (NI: 55.6%, LI: 64.3%, HI: 100%, p=0.003) between the 3 groups. The leukocyte count (mean±S.E., NI: 7400±528, LI: 7686±433, HI: 10952±691/mm3, p<0.001), uric acid (NI: 5.3±0.24, LI: 7.6±0.4, HI: 8.9±0.36 mg/dl, p<0.001), NO2 (NI: 6.5±1.2, LI: 11.1±2.9, HI: 35.6±5.1 μg/ml, p<0.001) and the 8-NO2-G (NI: 0.08±0.03; LI: 0.34±0.13; HI: 0.59±0.09 ng/μg DNA, p=0.002) were significantly increased by inflammation.Gouty inflammation induces DNA damage by increasing 8-NO2-G through endogenous NO and ROS formation.

Keywords: Abbreviations; 8-NO; 2; -G; 8-nitroguanine; 8-OH-dG; 8-hydroxy-2′-deoxyguanosine; CRP; C-reactive protein; HI; high inflammatory; HPLC-ECD; high performance liquid chromatography-electrochemical detection; LI; low inflammatory; NI; non-inflammatory; ROS; reactive oxygen speciesGout; 8-nitroguanine

Angiotensinogen M235T polymorphism is associated with coronary artery disease severity by Jos R. Lanz; Alexandre C. Pereira; Pedro A. Lemos; Eulogio Martinez; Jos E. Krieger (pp. 176-181).
Previous reports relating coronary artery disease and functional variants of the renin-angiotensin system have been contradictory in establishing the role of these polymorphisms in coronary artery disease (CAD) development. The aim of the present study is to determine if there is an association between the M235T variant of the angiotensinogen gene and severity of coronary artery disease in patients with clinically suspected disease undergoing cineangiogram.The angiotensinogen M235T variant was analyzed in 871 consecutive patients with clinically suspected coronary artery disease submitted to coronary angiography study. Three different angiographic scores were used to determine severity of the disease analyzing 20 coronary segments. All patients were evaluated considering classical risk factors for coronary artery disease throughout a medical oriented questionnaire, anthropometric measures, and blood glucose and lipid profile determination.Presence of the 235T allele was associated with higher angiographic extension scores in univariate analysis ( p<0.05). The 235T allele was also associated with an increased risk of presenting 3-vessel disease ( p<0.05). In addition, the T allele was significantly associated with higher Gensini's scores both in univariate ( p=0.05) and multivariate analyses ( p<0.05). Finally, a 1.86 fold increase in the risk of multivessel disease (95% CI: 1.174–2.947) was associated with the TT genotype independently of other cardiovascular risk factors associated with disease extension.The data hereby provide further support for the association between angiotensinogen M235T polymorphism and CAD severity independently of other cardiovascular risk factors.

Keywords: Coronary artery disease; Extension; Angiotensinogen polimorphism

Pharmacogenetic study of apolipoprotein E, cholesteryl ester transfer protein and hepatic lipase genes and simvastatin therapy in Brazilian subjects by Marilu Fiegenbaum; Fabiano R. da Silveira; Czar R. Van der Sand; Luiz Carlos Van der Sand; Maria E.W. Ferreira; Renan C. Pires; Mara H. Hutz (pp. 182-188).
In recent years, one of the focuses of genetic investigation in cardiology has been to identify the genetic factors associated with variable response to statin treatment. Polymorphisms in apolipoprotein E (APOE), cholesteryl ester transfer protein (CETP) and hepatic lipase (LIPC), proteins with major roles in lipid metabolism and homeostasis have been shown associated with lipid-lowering drugs response.One hundred forty-six hypercholesterolemic patients of European descent were prospectively enrolled and treated with simvastatin 20 mg per day for over 6 months. Ninety-nine subjects completed the 6-month follow-up. Plasma lipids and lipoproteins were measured before and throughout the study. APOE ( E*2, E*3 and E*4), LIPC−250A>G and CETP TaqIB genotypes were determined by PCR and restriction mapping.After a 6-month follow-up, no differences among genotypes in the percentage variation in lipid and lipoprotein concentrations for APOE and LIPC SNPs were observed. After adjustment for covariates, CETP B2B2 homozygotes showed a greater HDL-cholesterol increase compared to B1B2 and B1B1 subjects (14.1% vs. 1.7% and 1.3%, P<0.05, respectively).Our study demonstrates that individual plasma HDL-cholesterol response to simvastatin is mediated, in part, by the CETP gene locus, with the B2 homozygotes having more benefit in HDL-C improvement than carriers of B1 allele.

Keywords: APOE; CETP; HDL-C; LIPC; Pharmacogenetic; Simvastatin

Publications in clinical chemistry journals in the European Union by X. Fuentes-Arderiu; M.J. Castieiras Lacambra (pp. 189-191).
Thiobarbituric acid reactive substances, seric superoxide dismutase and catalase activities in healthy subjects by Ana Cristina Andreazza; Diana Lilian Bordin; Mirian Salvador (pp. 192-194).
Does plasma nitrite determination by the Griess reaction reflect nitric oxide synthesis? by Luci Maria Sant Ana Dusse; Rvia Mara Morais e Silva; Lauro Mello Vieira; Maria das Graas Carvalho (pp. 195-197).
Calendar (pp. 198-198).
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