Clinical Biochemistry (v.40, #1-2)

The effect of atorvastatin therapy on lecithin:cholesterol acyltransferase, cholesteryl ester transfer protein and the antioxidant paraoxonase by Andrea Kassai; László Illyés; Hossein Z. Mirdamadi; Ildiko Seres; Tímea Kalmár; Mária Audikovszky; György Paragh (1-5).
The aim of our study was to examine the influence of atorvastatin on lipid parameters, particularly on HDL, and on the activity of LCAT and CETP and how they affect the activity of the HDL-associated antioxidant enzyme paraoxonase.Thirty-three patients with types II.a and II.b primary hyperlipoproteinemia were enrolled into our study. The patients received atorvastatin, 20 mg daily, for 3 months. We measured the serum paraoxonase activity and concentration, oxidized LDL, LCAT and CETP activities.Atorvastatin significantly reduced the levels of cholesterol, triglyceride, LDL-C and apoB, while it did not influence the levels of HDL-C and apo A-I. The increases in serum PON-specific activity, PON/HDL ratio and LCAT activity were significant, while oxLDL and CETP activities were significantly decreased.Atorvastatin may influence the composition and function of HDL, thereby possibly increasing the activity of paraoxonase and preventing atherosclerosis.
Keywords: Atorvastatin; High-density lipoprotein (HDL); Lecithin:cholesterol acyltransferase (LCAT); Cholesteryl ester transfer protein (CETP); Paraoxonase (PON);

Serum soluble Fas levels in patients with autoimmune rheumatic diseases by M. Sahin; O. Aydıntug; S.E. Tunc; H. Tutkak; M. Nazıroğlu (6-10).
The role of the serum soluble Fas (sFAS) system is unclear in diagnosis of several autoimmune rheumatic diseases although there are present contradictory reports on the levels of serum sFas. We therefore assessed levels of sFAS in serum of patients with autoimmune rheumatic diseases.We analyzed sFas levels and their relationship to clinical and laboratory data in patients with systemic lupus erythematosus (SLE, n  = 32), rheumatoid arthritis (RA, n  = 28), Sjögren's syndrome (SS, n  = 20) systemic sclerosis (SSc, n  = 21), polymyositis/dermatomyositis (PM/DM, n  = 15). Patients with osteoarthritis (OA, n  = 20) and healthy volunteers (n  = 20) were used as controls. Serum levels of sFAS were determined by ELISA. sFas levels greater than mean (normals) + 2 SD were considered as elevated.The mean sFas values were found higher in RA, PM/DM and OA than in control although no differences were found in SSc and SS patients. The mean sFas levels in SLE patients were lower than healthy controls. Elevated sFas rates in RA, PM/DM and SS were found to be 21.4%, 60%, 10% higher than in healthy controls, respectively. sFas levels in SLE and SSc did not differ from control values. Mean sFas levels did not show significant difference between active and inactive patients in all disease groups except PM/DM, RA and OA. No correlations of sFas with relevant disease subsets, laboratory findings and treatment modalities were found.The findings indicate that the serum sFas molecule may provide a useful additional marker for presence and assessment of disease in patients with RA and PM/DM.
Keywords: Apoptosis; Autoimmune disease; Polymyositis/Dermatomyositis; Rheumatoid arthritis; Serum soluble Fas;

Life-threatening toxic side-effects following 5-FU exposure have been related to deficiency of dihydropyrimidine dehydrogenase (DPD), the rate-limiting enzyme in its catabolism. We presently report a new DPYD gene SNP in a Spanish woman who died from multivisceral 5-FU-induced toxicity.We looked for 22 known SNPs by pyrosequecing®. Then, we sequenced the whole 23 exons of DPYD, along with adjacent intronic sequences. PCR was carried out to determine whether or not exons were deleted in the DPYD. To determine whether the predicted stop codon indeed resulted in a truncated protein, a bacterial expression vector was employed to generate the predicted protein. 500 patients were genotyped to determine allele frequency.A novel mutation 464 T>A was identified in DPYD gene exon 5, resulting in the replacement of leucine 155 by a stop codon in the protein. We confirmed this mutation by Pyrosequencing® and its involvement by a protein truncation test. We genotyped the patient's family and the allele frequency was 0.2%.The involvement of this variant in 5-FU life-threatening toxicity supports its inclusion in pretherapeutic genetic screening.
Keywords: Dihydropyrimidine dehydrogenase; Single nucleotide polymorphism; 5-Fluorouracil; Toxicity; Pyrosequencing; Toxicity; Colon cancer;

To evaluate the role of non-conventional lipid risk factors like Lipoprotein(a) [Lp(a)], Apolipoprotein A-I (Apo A-I) and Apolipoprotein B-100 (Apo B-100) and other conventional lipid profile parameters in children and adolescents of premature coronary artery disease (CAD) patients in India; and thereby explain the highest occurrence of premature CAD in this population.Forty-five children and adolescents of premature CAD patients (cases, mean age 12.08 ± 3.71 years) and forty-five age and sex matched children and adolescents of healthy parents without any history or clinical evidence suggestive of CAD were studied (controls, mean age 12.14 ± 3.91 years).We found a significant increase in mean levels of Lp(a), Apo B-100, Total cholesterol (TC), Low Density Lipoprotein-Cholesterol(LDL-C) and Triglyceride (TG) in cases than controls. In contrast, Apo A-I and High Density Lipoprotein-Cholesterol (HDL-C) values decreased. Lipid Tetrad Index (LTI) and Atherogenic Index in Indian children and adolescents were also calculated. Kolmogorov D statistic and cumulative probability plot suggest that the new Lipid Pentad Index (LPI) defined by us is able to discriminate case and control populations more precisely than the existing LTI and Atherogenic Index.The new proposed LPI appears to be a better indicator of lipid risk factors in children and adolescents of premature CAD patients from India, than the prior LTI and Atherogenic Index.
Keywords: Apolipoproteins; Lipoprotein(a); Lipid Pentad Index; Atherogenic Index; Lipid Tetrad Index; Premature CAD; Children; Adolescents;

Differential impact of plasma triglycerides on HDL-cholesterol and HDL-apo A-I in a large cohort by André J. Tremblay; Allan D. Sniderman; Claude Gagné; Jean Bergeron; Patrick Couture (25-29).
To examine the relationship between plasma triglycerides (TG) to HDL-cholesterol (HDL-C) or HDL apo A-I.Bivariate and multiple linear regression analyses in a large cohort of 1886 subjects.Higher plasma TG levels were associated with lower concentrations of both HDL-C and HDL-apo A-I. However, the HDL-C/HDL-apo A-I ratio was inversely correlated with plasma TG indicating that the overall composition of the HDL changed as plasma TG changed. Plasma TG levels contributed to 15.9% of the variance of the HDL-C/HDL-apo A-I ratio, whereas gender, HDL-TG, LDL-TG, body mass index and plasma apo B levels represented between 0.15% and 2.21% of this variance.These results indicate that increasing levels of plasma TG result in greater reduction in HDL-C levels than in HDL-apo A-I and this might explain, at least in part, the differences that have been observed in the magnitude of the association of HDL-C versus HDL-apo A-I with the risk of cardiovascular disease.
Keywords: Apolipoprotein; HDL-cholesterol; Triglyceride;

The potential benefits of hyperbaric oxygen therapy (HBOT) have been reported in diabetic patients with foot ulcers. However, the roles of HBOT on wound healing-associated growth factors and inflammatory mediators are not completely understood in diabetes mellitus (DM).The aim of this study was to investigate the effects of HBOT on circulating cytokines, NO, and insulin-like growth factors (IGF) in patients with type 2 DM.Serum samples were collected from patients with type 2 DM (n  = 31) and healthy subjects (n  = 29) before (baseline) and after the first and third exposure.Before HBOT, body mass index (BMI) and serum HbA1c were significantly greater, whereas serum IGF-I was significantly lower in diabetic patients compared to healthy subjects (one-way ANOVA, p  < 0.05). After adjusting for age, gender, and BMI, serum insulin, growth hormone (GH), IGF-II, IGF-binding protein (IGFBP)-1, IGFBP-3, leptin, interleukin (IL)-8, and NO were not significantly altered by HBOT in diabetic patients and healthy subjects (repeated-measures ANOVA). Change in serum insulin (baseline to the third exposure) was a positive predictor of changes in leptin and NO in healthy subjects and diabetic patients, respectively.Our results suggest that short-term HBOT may not alter the circulating insulin, IGF, leptin, IL-8, and NO levels. In addition, healthy subjects and diabetic patients showed differential responses to HBOT in the relationships of leptin, insulin, and NO. Further studies are needed to clarify the mechanism of HBOT-improved wound healing in diabetic patients with foot ulcers.
Keywords: Hyperbaric oxygenation; Type 2 diabetes mellitus; IL-8; Nitric oxide; Leptin; Insulin-like growth factors;

Serum prolidase activity and oxidative status in Helicobacter pylori infection by Mehmet Aslan; Yasar Nazligul; Mehmet Horoz; Cengiz Bolukbas; Fusun F. Bolukbas; Nurten Aksoy; Hakim Celik; Ozcan Erel (37-40).
During the course of Helicobacter pylori infection, increased oxidative stress plays an important role in the pathogenesis of gastroduodenal mucosal inflammation, which can cause gastric mucosal atrophy that characterized by the replacement of the gastric mucosal glands by collagen fibers. In the present study, we aimed to determine serum prolidase activity and oxidative status, and to find out if there is any association between serum prolidase activity and oxidative status in H. pylori infection.Forty H. pylori-positive and 32 H. pylori-negative subjects were enrolled. Serum prolidase activity was measured spectrophotometrically. Oxidative status was determined using total antioxidant capacity and total oxidant status measurement and calculation of oxidative stress index.Total antioxidant capacity level was lower in H. pylori-positive group than H. pylori-negative group (p  < 0.001), whereas total oxidant status, oxidative stress index and prolidase activity were higher (all p  < 0.05). Significant correlation was observed between serum prolidase activity, and total antioxidant capacity, total oxidant status and oxidative stress index (p  < 0.01, r  = − 0.367; p  < 0.05, r  = 0.283; p  < 0.01, r  = 0.379; respectively) in H. pylori-positive subjects. H. pylori infection may be associated with increased oxidative stress and increased serum prolidase activity. Increased oxidative stress seems to be associated with increased serum prolidase activity and this association may help to provide a better understanding about the pathogenesis of H. pylori infection.
Keywords: Helicobacter pylori; Prolidase activity; Total antioxidant capacity; Total oxidant status; Oxidative stress index;

Hyperhomocysteinemia is associated with deep venous thrombosis of the lower extremities in Tunisian patients by Souheil Omar; Imed Ben Ghorbel; Habib Feki; Malek Souissi; Moncef Feki; Habib Houman; Neziha Kaabachi (41-45).
To test the association between hyperhomocysteinemia (HHC) and deep venous thrombosis (DVT) of lower extremities in Tunisians.This case–control study included 90 patients with DVT of the lower extremities and 160 healthy controls. Plasma homocysteine, vitamin B12 and folate were determined using immunoenzymatic methods. Logistic regression models were performed to test whether the association between HHC and DVT is independent and to precise determinants of HHC in DVT patients.Plasma total homocysteine concentrations were significantly higher in patients with DVT (17.4 ± 11.5 μmol/L) and in patients with idiopathic DVT (15.2 ± 6.4 μmol/L) as compared to controls (11.5 ± 3.3 μmol/L). HHC was significantly associated (p  < 0.001) with all DVT (OR, 8.82; 95% CI, 3.96–19.6) as well as idiopathic DVT (OR, 7.40; 95% CI, 3.01–10.8). These associations persisted after adjustment for several thrombosis risk factors. In patients with DVT, HHC was related to folate and vitamin B12 concentrations, but neither to the type of occurrence nor to the recurrence of DVT.HHC is independently associated with first DVT of lower extremities in Tunisians. Homocysteine should be assessed in patients with DVT and the effect of vitamin B supplementation should be tested among them.
Keywords: Folic acid; Deep venous thrombosis; Homocysteine; Hyperhomocysteinemia; Venous thromboembolism; Recurrent deep venous thrombosis; Vitamin B12;

Muscle biopsies from chronic steroid (glucocorticoid) myopathy, non-steroid histochemical type-2 fiber atrophy, and muscle denervation patients were studied to determine if their glycogen contents, or enzymes involved in glycogenolysis and glycolysis might be related to their fiber atrophy.Fast frozen muscle biopsies from the above patients and from patients later judged by histochemistry to be normal were assayed enzymatically for glycogen content, for enzymes involved in glycogenolysis, and for 6 of the enzymes involved in glycolysis.All three groups of patients had glycogen content, but only the chronic steroid myopathy muscle had statistically less glycogen content than did normal human muscle. All 3 groups had statistically low mean values compared to normal muscles for glycogen phosphorylase activity. This suggests that the biosynthesis and phosphorolysis of glycogen are not involved in muscle fiber atrophy, and glucocorticoid administration does not activate muscle glycogen biosynthesis. Histochemical type-2 fiber atrophy muscles were low compared to normal muscles in three glycogenolysis enzyme activities plus four glycolysis enzyme activities. Muscles from denervation patients were low compared to normal muscles in three glycogenolysis enzyme activities plus five glycolysis enzyme activities. This suggests that muscle denervation may lower the rate of glycolysis enough to fail to provide sufficient pyruvate for mitochondrial ATP biosynthesis, resulting in insufficient protein biosynthesis in both fiber types.
Keywords: Human muscle; Steroid myopathy; Denervation; Muscle type-2 fiber atrophy; Glycogen; Glycolysis;

Elevated hair levels of cadmium and lead in school children exposed to smoking and in highways near schools by Tülin Ayşe Özden; Gülbin Gökçay; Hayriye Vehid Ertem; Özlem Durmaz Süoğlu; Ayşe Kılıç; Semra Sökücü; Günay Saner (52-56).
Heavy metal pollution has become a serious health concern in recent years. Cadmium (Cd) and Lead (Pb) are toxic heavy metals. This study was aimed to determine the risk factors for high cadmium and lead levels in school children.The scalp hair samples were obtained from 760 children at 13 schools in Istanbul. A questionnaire was prepared to gather information about demographic and socioeconomic characteristics of the children. Hair cadmium and lead concentrations were determined by atomic absorption spectrophotometer.Household exposure to smoking and attending a school near to Main Streets were found to be the most important risk factors for the high hair cadmium and lead levels in our study.These findings support the public health recommendations that children should not have household exposure to smoking, schools should not be near to the main streets and unleaded gasoline use should be promoted.
Keywords: Toxic metals; Cadmium; Lead; Environmental exposure; School children; Hair analysis; Smoking;

Effects of treatment with dehydroepiandrosterone (DHEA) on oxidative energy metabolism in rat liver and brain mitochondria were examined.Young adult rats were administered DHEA (0.1, 0.2, 1.0 or 2.0 mg/kg body weight) by subcutaneous route for 7 consecutive days.DHEA treatment resulted in general, in stimulation of state 3 respiration rates without having any uncoupling effect on ADP/O ratios. The stimulation of state 3 respiration rate for a given substrate was dose dependent in a tissue-specific manner. Parallel increases in the contents of cytochromes aa3 and b were also noted. DHEA treatment stimulated the glutamate dehydrogenase (GDH) and succinate DCIP reductase (SDR) activities. Under the treatment conditions, mitochondrial ATPase activity was also stimulated.Treatment with DHEA significantly stimulated oxidative energy metabolism in liver and brain mitochondria.
Keywords: Dehydroepiandrosterone (DHEA); Oxidative energy metabolism; ATPase activity; Cytochromes; Dehydrogenases;

Asymmetric dimethylarginine (ADMA) and hyperhomocysteinemia in patients with acute myocardial infarction by Claudia Korandji; Marianne Zeller; Jean-Claude Guilland; Catherine Vergely; Pierre Sicard; Laurence Duvillard; Philippe Gambert; Daniel Moreau; Yves Cottin; Luc Rochette (66-72).
We sought to investigate the association between increased levels of asymmetric dimethylarginine (ADMA), an endogenous nitric oxide synthase inhibitor, and total plasma homocysteinemia (tHcy) in patients with acute myocardial infarction (AMI).In 138 patients hospitalized for AMI < 24 h on admission, serum levels of ADMA, its symmetric stereoisomer (SDMA) and tHcy were measured.ADMA was positively associated with SDMA (p  < 0.001) and tHcy (p  = 0.03) but not with estimated glomerular filtration rates (eGFR, p  = 0.96), while tHcy strongly correlated with eGFR (p  = 0.002) and SDMA (p  < 0.001). By multiple linear regression, SDMA but not ADMA was independently associated with tHcy (p  = 0.005).Our findings suggest that, in AMI patients, hyperhomocysteinemia is indirectly related to ADMA levels via renal function. Moreover, ADMA level was independent of traditional cardiovascular risk factors in AMI patients. Interestingly, our findings suggest that SDMA could be a good risk indicator for cardiovascular disease in AMI patients.
Keywords: Asymmetric dimethylarginine; Symmetric dimethylarginine; Homocysteine; Estimated glomerular filtration rate; Acute myocardial infarction;

Clinical, biochemical and molecular diagnosis of a compound homozygote for the 254 bp deletion–8 bp insertion of the APRT gene suffering from severe renal failure by Valentina Di Pietro; Italia Perruzza; Angela Maria Amorini; Alessandro Balducci; Lia Ceccarelli; Giuseppe Lazzarino; Paola Barsotti; Bruno Giardina; Barbara Tavazzi (73-80).
To determine the type of mutation in a patient with clinical diagnosis of suspected APRT deficiency.A 51-year-old male patient, with a clinical history of two prior episodes of renal colic with urinary stone excretion (reported as uric acid stones in the first episode and as calcium oxalate stones in the second), was admitted to the hospital with severe non-oliguric renal failure (1.06 mmol/L serum creatinine), severe hyponatremia (114 mmol/L Na+), metabolic acidosis (14 mmol/L HCO3 ) and uricemia in the normal range. Abnormalities at renal scan and persistency of severe renal failure required to start haemodialysis. Results of renal biopsy prompted us to undertake a biochemical and molecular biological evaluation of the patient for suspected adenine phosphoribosyltransferase (APRT) deficiency.HPLC analysis of serum and urine, for determining purine derivative profile, showed the pathological presence of adenine in both biological fluids (3.57 μmol/L and 7.11 μmol/mmol creatinine in serum and urine, respectively; not detectable in both fluids in healthy controls). APRT assay in a sample of patient hemolysate showed no detectable activity of the enzyme (25.56 ± 9.55 U/L red blood cells in control healthy subjects). Molecular biological analysis of the amplified APRT gene revealed that the patient harboured in exon 3 a homozygous 254 bp deletion–8 bp insertion, previously described only once in a compound heterozygote. Analysis of the patient family showed that heterozygotes for this APRT gene mutation, in spite of a 69% lower APRT enzymatic activity than that of healthy subjects, had no detectable adenine concentrations in both serum and urine.Results of the first patient harbouring the homozygous 254 bp deletion–8 bp insertion of the APRT gene strongly indicated that definitive diagnosis of APRT deficiency (often under or misdiagnosed) would require a combined clinical, biochemical and molecular biological evaluation.
Keywords: APRT deficiency; Renal failure; HPLC; Serum purine analysis; Urine purine analysis; Urolithiasis; PCR; Molecular biological diagnosis;

The present study was undertaken to clarify the role of the impaired renal function and the dialysis therapy on plasma levels of proatherogenic cytokines and Cu/Zn superoxide dismutase (Cu/Zn SOD)—as a marker of oxidative stress (SOX) in uraemia.We have measured the levels of Cu/Zn SOD, monocyte chemoattractant protein-1 (MCP-1) macrophage inflammatory proteins (MIP-1α, MIP-1β) and vascular endothelial growth factor (VEGF) in the plasma of predialysis (CRF) (n  = 42), on maintenance hemodialysis (HD) (n  = 25) or peritoneal dialysis (PD) (n  = 45) patients and in the healthy volunteers (n  = 20).The increase in Cu/Zn SOD levels was in PD and HD patients compared to controls (215.56 ± 125.18 and 356.28 ± 122.57 versus 53.53 ± 23.65 ng/ml, respectively). In plasma of the CRF, PD and HD subjects we have also observed the significant increase in the levels of MIP-1β: [31.5 (2–149), 33.0 (1–203) and 76.0 (9–345), respectively]; MCP-1 (616.50 ± 240.15, 943.64 ± 348.99 and 968.50 ± 355.85, respectively) and VEGF (387.93 ± 184.63, 371.56 ± 125.18 and 645.56 ± 136.30, respectively) compared to healthy people. In the predialysis group, creatinine clearance correlated with Cu/Zn SOD and cytokine levels. Moreover, the cytokine levels were also associated with age. In dialysis patients, the correlations were between duration of dialysis treatment and both Cu/Zn SOD and cytokine levels. There was also a direct relationship between Cu/Zn SOD and both MIP-1β and VEGF levels.This study has shown that impaired renal function, age and duration of dialysis treatment are associated with increased oxidative stress and proatherogenic cytokine levels in uremic patients.
Keywords: End-stage renal disease; Cytokines; Oxidative stress; Hemodialysis; Peritoneal dialysis;

Immunoassay of serine-phosphorylated isoform of insulin-like growth factor (IGF) binding protein (IGFBP)-1 by Javad Khosravi; Radha G. Krishna; Umesh Bodani; Anastasia Diamandi; Najmuddin Khaja; Bhanu Kalra; Ajay Kumar (86-93).
Development of an ELISA for phosphorylated isoform of IGFBP-1. Serine phosphorylation is an important regulator of IGFBP-1 bioactivity, but specific immunoassays for its measurement are currently lacking.Assay design was based on a novel approach of first capturing the phosphorylated and non-phosphorylated IGFBP-1 by an anti-IGFBP-1 antibody and then selectively detecting the phosphorylated form by an anti-phosphoserine antibody. Method development involved pair-wise evaluation of the candidate antibodies and determinations of analytical performance and specificity. Specificity was monitored by reactivity with dephosphorylated IGFBP-1, with antibodies against other phosphorylated residues that are not expressed, and by comparative analysis of sample containing different IGFBP-1 phosphorylation profile.Analytical evaluation demonstrated acceptable performance; detection limit 0.3 μg/L, dynamic range 1.56–100 μg/L; intra- and inter-assay CVs 2.1–8.6%; mean recovery (± SD) 97.8 ± 9.2%, and mean recovery of sample dilution 93.4 ± 6.0%. The phosphorylated and total IGFBP-1 medians in non-pregnant adult serum, which mostly contain the highly phosphorylated isoform, were 11.9 and 18.6 μg/L, respectively, and the sample values were tightly correlated (r  = 0.99). As expected, the corresponding medians in 1st trimester (17.4 and 63.0 μg/L) and 2nd trimester (30.9 and 75.8) samples with altered IGFBP-1 phosphorylation were significantly different (p  < 0.001). Similarly, a fraction (1.29%) of total IGFBP-1 (13.3 mg/L) in amniotic fluids was found to be phosphorylated (0.172 mg/L). There was no reactivity with dephosphorylated IGFBP-1.The present ELISA is highly specific for the phosphorylated isoform of IGFBP-1 and its availability should help expedite further investigations of IGFBP-1 phosphorylation.
Keywords: Growth hormone; Insulin-like growth factors; Phosphorylation; IGFBP;

Dual action of H2O2 on placental hCG secretion: Implications for oxidative stress in preeclampsia by Aziz Kharfi Aris; Samuel Leblanc; Annie Ouellet; Jean-Marie Moutquin (94-97).
Our previously published findings showed that circulating levels of H2O2 were increased and correlated with high levels of hCG in women with preeclampsia, suggesting that oxidative stress modulates placental hormone synthesis. The aim of this study was to investigate in vitro the effects of H2O2 on placental secretion of hCG. In vitro trophoblasts were stimulated with increasing concentrations of H2O2 and the de novo hCG secretion was assayed.Stimulation with low concentrations of H2O2 (1–50 μM) enhances cytotrophoblastic hCG secretion, whereas concentrations of H2O2 > 50 μM reduce hCG secretion in a dose-dependent manner.Our findings emphasize that: (1) H2O2 may have dual action on placental activity and acts not only as a cytotoxic mediator, but also as a signaling molecule able to induce hCG secretion; (2) hCG may be a protective antioxidant released by the placenta to counter low oxidative stress challenge.
Keywords: hCG; H2O2; Placenta; Trophoblasts; Oxidative stress; Preeclampsia;

Development of a novel ELISA for human insulin using monoclonal antibodies produced in serum-free cell culture medium by Megha S. Even; Chad B. Sandusky; Neal D. Barnard; Jehangir Mistry; Madhur K. Sinha (98-103).
Development of an ELISA for human insulin that utilizes monoclonal antibodies (mAbs) produced in serum-free medium.Insulin mAbs were produced in vitro by hybridomas in serum-free medium. A two-step ELISA was developed to replace bovine insulin (standard) and bovine serum albumin (assay buffer) with non-animal reagents.The sensitivity of the insulin ELISA was 0.73 uU/mL with a dynamic range of 2–200 uU/mL. No cross-reactivity with either human C-peptide or human proinsulin was observed. The intra- and inter-assay CVs were < 7%. The mean recovery of insulin added to plasma samples was between 102.2% and 105.7%. The mean linearity of dilution was between 93% and 110% of undiluted plasma samples. The animal serum-free (ASF) insulin ELISA showed no marked degradation of any kit component when stored at 37°C for up to 7 days. Significantly higher fasting insulin levels were observed in overweight or obese subjects (n  = 12) compared to lean subjects (n  = 10, p  < 0.05). Feeding markedly increased fasting insulin levels in both lean (p  < 0.02) and overweight or obese (p  < 0.005) subjects. Excellent correlation was observed between insulin levels measured by ASF insulin ELISA and another CE marked insulin ELISA (y  = 1.06x  − 0.44, r  = 1.00, n  = 44).This novel insulin ELISA provides precision and reliability equal to methods currently used in clinical research and serves as a guide for the development of other serum-free immunoassays.
Keywords: Animal use alternatives; Serum-free hybridomas; Immunoassay; Insulin ELISA; Insulin monoclonal antibodies; Diabetes clinical research;

Development of an immunofluorometric assay for human kallikrein 15 (KLK15) and identification of KLK15 in tissues and biological fluids by Julie L.V. Shaw; Linda Grass; Georgia Sotiropoulou; Eleftherios P. Diamandis (104-110).
Human kallikrein 15 (KLK15) may have some utility as a prostate, ovarian, and breast cancer biomarker, based on previous studies, which examined mRNA levels of KLK15. The aim of this study was to develop analytical technology for human kallikrein 15, including recombinant protein, specific antibodies, and a sensitive and specific ELISA immunoassay. The assay was then used to examine levels of KLK15 in tissues and biological fluids.We produced human, recombinant pro-KLK15 in HEK 293 cells. Recombinant KLK15 was purified with various chromatographic steps and used to immunize rabbits and mice for production of KLK15 polyclonal antibodies. We used these antibodies to develop a highly sensitive and specific KLK15 immunoassay and to study KLK15 expression in various tissues and biological fluids.Large amounts of pure, recombinant KLK15 have been produced and characterized. KLK15 mouse and rabbit polyclonal antibodies have been employed for development of a KLK15 immunoassay. This assay has a lower detection limit of 0.05 μg/L, and no cross-reactivity with any of the other fourteen kallikreins. Using this assay, KLK15 was detected in prostate, colon, and thyroid tissues, as well as in breast milk and seminal plasma.The KLK15 reagents developed here will allow for analysis of KLK15 protein expression levels in tissues and biological fluids, both normal and cancerous. This will expand upon previously characterized tissue KLK15 mRNA expression studies which suggested that KLK15 might be useful as a biomarker for breast, ovarian, and prostate cancer. KLK15 is another serine protease that is produced in prostate and other tissues and is secreted in seminal plasma and other fluids. Its physiological function needs to be further elucidated.
Keywords: Kallikrein; KLK15; Immunoassay; Tissue expression; Serine proteases;

Novel homogenous time-resolved fluorometric RT-PCR assays for quantification of PSA and hK2 mRNAs in blood by Maria Rissanen; Pauliina Helo; Riina-Minna Väänänen; Veikko Wahlroos; Hans Lilja; Martti Nurmi; Kim Pettersson; Jussi Nurmi (111-118).
The purpose of this study was to design, validate, and optimize internally standardized real-time quantitative RT-PCR assays and to identify and avoid problems with assay reliability and examine the impact of an exogenous internal standard.The model system consisted of internally standardized quantitative real-time RT-PCR assays specific for PSA and hK2 mRNA based on time-resolved fluorometric detection of lanthanide chelates.Reproducibility was best when large copy numbers (> 5000 per milliliter blood) were analyzed. Addition of an exogenous target-mimicking internal standard had no significant effect on the reproducibility of the method, but increased the calculated copy numbers by an average of 2-fold.We developed an internally standardized, specific and reproducible real-time RT-PCR analysis method for PSA and hK2 mRNA in circulating cells in the bloodstream. Both PSA and hK2 assays are sufficiently sensitive to detect two LNCaP cells per milliliter whole blood.
Keywords: Reverse transcriptase polymerase chain reaction; Tissue kallikreins; Reproducibility of results; Neoplasm circulating cells; Sensitivity and specificity;

Rapid separation of serum does not avoid artificially higher matrix metalloproteinase (MMP)-9 levels in serum versus plasma by Raquel F. Gerlach; Caroline Demacq; Klaus Jung; Jose E. Tanus-Santos (119-123).
To examine whether the time between blood drawing and centrifugation (TBDC) affects the levels of matrix metalloproteinase (MMP)-9 and MMP-2 levels in serum and in plasma samples, and to assess whether there is correlation between MMP-9 and MMP-2 levels in serum and plasma samples.Serum and plasma samples (N  = 8) were separated from venous blood collected into citrate, heparin, and EDTA tubes, which were either centrifuged immediately or after 5, 10, 20, or 30 min after blood drawing. We assessed the correlation between MMP-9/MMP-2 in serum and citrate, heparin, and plasma samples (N  = 20), which were assayed for gelatine zymography of MMP-2 and MMP-9.MMPs are released by platelets or leukocytes during platelet activation or sampling process, thus leading to artificially higher MMP-9 levels in serum compared with citrate, heparin, or EDTA plasma samples, independently of TBDC. Citrate and heparin plasma samples had the lowest Pro-MMP-9 and MMP-9 levels, which correlated with each other. Pro-MMP-9 levels in serum correlated with Pro-MMP-9 levels in EDTA or citrate plasma, but not with heparin plasma. While no significant correlations were found between MMP-9 levels in serum and those found in plasma samples, the total MMP-9 levels (Pro-MMP-9 + MMP-9) in serum and in plasma samples correlated with each other. No significant differences were found in pro-MMP-2 levels.These results suggest that the circulating levels of MMP-9 should be assessed in citrate or heparin plasma samples, but not in serum samples because of artificially higher MMP-9 levels in serum, independently of TBDC, and because they do not correlate with the MMP-9 levels in plasma samples.
Keywords: Metalloproteinases; Methods; Zymography;

Compare GFR estimates (eGFR) calculated by formulas (CG, CGI, MDRD) and creatinine clearance (CrCl), in subgroups based on sex, age, BMI and serum creatinine (Cr).eGFR is calculated with Cockgroft–Gault (CG), CG corrected for ideal or lean weight (CGI), and Modification of Diet in Renal Disease (MDRD) formulas using a compensated kinetic Jaffé method. Comparison of means and correlations relative to CrCl is done with 666 male and female adult subjects in different subgroups.Comparatively to CrCl, most subgroups are best correlated with MDRD, especially with high Cr. However, in men < 65 years, a factor of 222, instead of 186 in MDRD formula, is better.eGFR from MDRD formula, modified for men < 65 years, is better correlated to CrCl.
Keywords: Creatinine; Creatinine clearance; Cockgroft–Gault and MDRD formula;

Determination of glucuronidated 5-hydroxytryptophol (GTOL), a marker of recent alcohol intake, by ELISA technique by Jutta Dierkes; Manfred Wolfersdorf; Katrin Borucki; Wolfgang Weinmann; Gerhard Wiesbeck; Olof Beck; Stefan Borg; Friedrich M. Wurst (128-131).
To compare markers of alcohol consumption.Measurement of urinary ethyl glucuronide, 5-hydroxytryptophol, 5-hydroxytryptophol glucuronide, 5-hydroxyindole acetic acid in 10 patients during alcohol withdrawal.5-Hydroxytryptophol glucuronide was measured by ELISA with good analytical precision, its diagnostic specificity and sensitivity was better than that of 5-hydroxytryptophol and its correlation was closer to ethyl glucuronide than to 5-hydroxytryptophol.Determination of 5-hydroxytryptophol glucuronide by ELISA offers promising results in detection of previous alcohol consumption.
Keywords: 5-Hydroxytryptophol glucuronide; ELISA; Alcohol withdrawal;

Everolimus (Certican®) is a new immunosuppressant derived from sirolimus (Rapamune®) with a 2-hydroxyethyl chain at position 40 of the macrolide ring. The aim of our study was to evaluate the possible determination of everolimus in whole blood using a commercialized microparticle enzyme immunoassay for sirolimus determination.Everolimus concentrations were determined in blood samples from 11 kidney transplant patients (n  = 51) and different control materials (n  = 35) using the Seradyn Innofluor® Certican® fluorescence polarization immunoassay (FPIA) and the Abbott IMx® sirolimus microparticle enzyme immunoassay (MEIA).The MEIA gave a concentration-dependent cross-reactivity with the everolimus, with a linear regression between the assigned values (y) for the Innofluor® Certican® calibrators and those obtained (x) using this immunoassay: y  = 0.96x  + 0.67 (ma68 = 0.21 μg/L, r  = 0.974, p  < 0.001). The within- and between-run coefficients of variation using the MEIA were ≤ 7.3%. In analyzing the different blood samples from patients and control materials using the MEIA and FPIA, a linear regression was found: MEIA = 0.99FPIA − 0.46 (ma68 = 0.32 μg/L, r  = 0.967, p  < 0.001). Correcting the MEIA results by means of the linear regression equation found between the assigned values for the Innofluor® Certican® calibrators and those obtained using this immunoassay led to a reduction in the deviation with respect to the FPIA values. The possible effect of the hematocrit on the results is analogous for both immunoassays.The Abbott IMx® sirolimus MEIA permits the simple and precise determination of everolimus in whole blood, making it a valid alternative for the therapeutic monitoring of this immunosuppressant agent.
Keywords: Everolimus; Fluorescence polarization immunoassay; Microparticle enzyme immunoassay;

Rapid molecular characterization of Hb Queens and Hb Siam: Two variants easily misidentified as sickle Hb by Supan Fucharoen; Sanita Singsanan; Abdulloh Hama; Goonnapa Fucharoen; Kanokwan Sanchaisuriya (137-140).
To establish a rapid differential diagnosis of hemoglobin (Hb) Queens and Hb Siam from other clinically relevant variants.Molecular and hematological features associated with two pregnant Thai women who were mistaken for Hb S were investigated. A simultaneous DNA diagnosis based on multiplex allele specific PCR approach was developed and tested with other common variants.Apart from mild anemia, the two subjects were generally healthy. DNA analysis identified that they were respectively carriers of Hb Siam [α15(A13)Gly–Arg] and Hb Queens [α34(B15)Leu–Arg]. A successful application of the multiplex allele specific PCR for differential diagnosis was demonstrated.Diagnosis of these clinically relevant hemoglobinopathies is problematic in the routine setting, and the method developed should prove useful in complementing routine Hb analysis for providing accurate diagnosis.
Keywords: Multiplex PCR; Hb Queens; Hb Siam; Hb S;

Regular exercise attenuated lipid peroxidation in adolescents with Down's syndrome by Francisco Javier Ordonez; Manuel Rosety-Rodriguez (141-142).
This study was designed to ascertain whether regular exercise may attenuate lipid peroxidation in adolescents with Down's syndrome.Thirty-one male adolescents with Down's syndrome performed a 12-week training program, 3 days/week at a work intensity of 60–75% of peak heart rate (HRmax = 194.5–[0.56 age]). Plasmatic malondialdehyde (MDA) was determined by high performance liquid chromatography (HPLC).When compared to baseline, MDA content was decreased significantly.Regular exercise significantly decreased lipoperoxidation in Down's syndrome.
Keywords: Down's syndrome; Exercise; Lipid peroxidation; Malondialdehyde;

by Gary Green (143).

by Hassan M.E. Azzazy; Mai M.H. Mansour; Robert H. Christenson (144-145).