The Greek way to the Register: the establishment and operation of the Register for Clinical Chemists-Clinical Biochemists in Greece.

In Greece, there is no officially organized training in clinical chemistry for scientists. The Greek Society of Clinical Chemistry-Clinical Biochemistry (GSCC-CB), following the encouragement of the EC4/RC decided to organize a voluntary Register for specialists in clinical chemistry. The following criteria for registration were defined: 1) University degree in Chemistry, Biochemistry, Biology, Medicine, Pharmacy or other relevant subject. 2) A total of 9 years of university studies and postgraduate specialization in clinical chemistry-clinical biochemistry. 3) A minimum of 4 years of postgraduate specialization in clinical chemistry-clinical biochemistry on the job. 4) The candidate must be practicing clinical chemistry-clinical biochemistry in a laboratory in a medical environment in Greece. The postgraduate specialization in clinical chemistry-clinical biochemistry includes the laboratory training and the theoretical education. The laboratory training is organized by the GSCC-CB according to the Professional Training Dossier. The theoretical education was organized in a series of 18 "Seminars" which was the content of the "Educational program" of the GSCC-CB. Successful completion of the Educational program leads to a Certificate of Competence. The Greek Register has gained equivalence with the EC4 Register and it has 218 members, more than 80 of whom are European clinical chemists.

Evaluation of methods for the measurement of low-density lipoprotein cholesterol.

A high concentration of low-density lipoprotein cholesterol (LDL-C) in plasma is one of the strongest risk factors for atherosclerotic cardiovascular disease and mortality. The most common approach to determining LDL-C in the clinical laboratory is the Friedewald calculation. There is an increased interest to improve the accuracy of LDL-C estimated by this equation. The expert panel convened by National Cholesterol Education Program has recommended the development of accurate direct methods to measure LDL-C. Several homogeneous and fully automated methods have been introduced in recent years that show improved precision and accuracy over earlier methods, especially the Friedewald calculation. Each of the atherogenic particles in plasma--very-low, intermediate-, and low- density lipoprotein--as well as lipoprotein (a), contain one molecule of apolipoprotein B (apoB) and thus, plasma total concentration of apoB reflects the number of atherogenic particles. Several studies suggested that the measurement of apoB could improve the prediction of risk of coronary artery disease. Thus, in addition to the newly developed direct assays, alternative calculation procedures have been proposed that also take into consideration total serum apoB concentration for the estimation of LDL-C and the presence of small, dense LDL particles. The new generation of homogenous methods for the measurement of LDL-C and the use of serum apoB concentration for the estimation of LDL-C can contribute to the accurate LDL-C determination.

Hypouricemia in individuals admitted to an inpatient hospital-based facility.

BACKGROUND: Decreased serum uric acid levels resulting from renal urate wasting occasionally are reported in hospitalized patients because of isolated or generalized proximal tubular damage. There are limited recent findings with regard to the incidence and cause of hypouricemia in patients admitted to an internal medicine clinic. The aim of this study is to examine the prevalence of hypouricemia in individuals admitted to our inpatient hospital-based facility and identify underlying causes and pathogenetic mechanisms and any association of hypouricemia and uricosuria with other tubular defects. METHODS: A total of 7,250 serum urate measurements were available on patients' admission. Hypouricemia is defined as a serum urate level less than 2.5 mg/dL (149 micromo/L). In all hypouricemic cases, a detailed clinical and laboratory investigation was performed. RESULTS: Hypouricemia was found in 90 patients (1.24%). In all except one patient, hypouricemia was associated with inappropriate uricosuria (urate fractional excretion [FE] > 10%; range, 10.8% to 94%). There was an inverse correlation between serum uric acid level and its FE (r = -0.73; P < 0.0001). The most common causes of hypouricemia were obstructive jaundice of any cause (n = 18), solid or hematologic neoplasias (n = 17), diabetes mellitus (n = 12), drugs affecting urate homeostasis (n = 10), and intracranial diseases (n = 8). Seventeen patients with hypouricemia showed one or more other manifestations of proximal tubular damage, such as glucosuria, inappropriate phosphaturia leading to hypophosphatemia, and kaliuria resulting in hypokalemia. CONCLUSION: Hypouricemia caused by inappropriate uricosuria is not rare in patients admitted to an internal medicine clinic, is related to underlying diseases, and may be associated with other abnormalities of proximal tubular function.

Evaluation of alternative calculation methods for determining low-density lipoprotein cholesterol in hemodialysis patients.

OBJECTIVES: To evaluate alternative equations for the estimation of low-density lipoprotein cholesterol (LDL-C) than the Friedewald equation in hemodialysis patients. DESIGN AND METHODS: The equations LDL-C = 0.41TC - 0.14TG + 0.66ApoB - 10.43 and LDL-C = 0.94TC - 0.94HDL-C - 0.19TG were evaluated in 86 patients and compared with the Friedewald equation and the ultracentrifugation procedure. RESULTS: The alternative equations yield significantly lower bias than the Friedewald equation and are less affected by increased triglycerides (TG) levels. CONCLUSION: The alternative equations for LDL-C yield slightly better results than the Friedewald equation especially in hypertriglyceridemia.

Content of the HMG-17 chromosomal protein in porcine tissues.

HMG-17 proteins are nucleosomal proteins, implicated in control of chromatin structure and transcription, in a way that has not yet been fully understood. In this report, quantification of HMG-17 in porcine tissues was performed, by ELISA, using previously produced and characterized specific rabbit anti-HMG-17 antibodies. Our results showed high levels of HMG-17 compared to the DNA and H1 content of the tissues (on the contrary to previous reports), and more specifically that there were 1.7 molecules of HMG-17 per molecule of histone H1.

The organization of an educational program for specialists in clinical chemistry by the Greek Society of Clinical Chemistry-Clinical Biochemistry.

In Greece, there is no officially organized training in clinical chemistry for scientists. The Greek Society of Clinical Chemistry-Clinical Biochemistry decided to organize an intensive educational program of 18 seminars on clinical chemistry content as it is described in the EC4 Syllabus. The duration of each seminar was about 6 hours and consisted of 6 to 9 lectures. At the end of each seminar there was a voluntary written examination, comprised of 24 multiple choice questions. Successful completion of the Educational program was leading to a Certificate of Competence. Two cycles of the 18 seminars were performed: 1st cycle from October 2003 to December 2005 and 2nd cycle from March 2005 to October 2007. One hundred eighty nine colleagues was the mean attendance per seminar for the seminars of the 1st cycle and 38 colleagues for the seminars of the 2nd cycle. The mean participation to the examination for each seminar was almost 80% for the 1st cycle and 68% for the 2nd cycle. More than 80% of the participants performed Good or Very good in the examination in both cycles. It is estimated that more than 40% of the scientists who practice Clinical Chemistry in Greece, participated to this educational activity. This program is now provided as an e-learning application, and it is open for all scientists who want to follow the discipline of clinical chemistry.

Promoter methylation status of hMLH1, MGMT, and CDKN2A/p16 in colorectal adenomas.

AIM: To investigate aberrant DNA methylation of CpG islands and subsequent low- or high-level DNA microsatellite instability (MSI) which is assumed to drive colon carcinogenesis. METHODS: DNA of healthy individuals, adenoma (tubular or villous/tubulovillous) patients, and colorectal carcinoma patients who underwent colonoscopy was used for assessing the prevalence of aberrant DNA methylation of human DNA mismatch repair gene mutator L homologue 1 (hMLH1), Cyclin-dependent kinase inhibitor 2A (CDKN2A/p16), and O-6-methylguanine DNA methyltransferase (MGMT), as well as their relation to MSI. RESULTS: The frequency of promoter methylation for each locus increased in the sequence healthy tissue/adenoma/carcinoma. MGMT showed the highest frequency in each group. MGMT and CDKN2A/p16 presented a statistically significant increase in promoter methylation between the less and more tumorigenic forms of colorectal adenomas (tubular vs tubullovillous and villous adenomas). All patients with tubulovillous/villous adenomas, as well as all colorectal cancer patients, showed promoter methylation in at least one of the examined loci. These findings suggest a potentially crucial role for methylation in the polyp/adenoma to cancer progression in colorectal carcinogenesis. MSI and methylation seem to be interdependent, as simultaneous hMLH1, CDKN2A/p16, and MGMT promoter methylation was present in 8/9 colorectal cancer patients showing the MSI phenotype. CONCLUSION: Methylation analysis of hMLH1, CDKN2A/p16, and MGMT revealed specific methylation profiles for tubular adenomas, tubulovillous/villous adenomas, and colorectal cancers, supporting the use of these alterations in assessment of colorectal tumorigenesis.

Analysis of the composition of plasma lipoproteins in patients with extensive coronary heart disease using 1H NMR spectroscopy.

INTRODUCTION: Alterations in the lipid composition and overall structure of plasma lipoproteins have been correlated with pathological situations such as dyslipidaemia, coronary heart disease (CHD), hypertension, and renal disease. In the present study 1H NMR spectroscopy was used to analyse the lipid composition of HDL and nonHDL lipoproteins in patients with triple vessel CHD and in patients with normal coronary arteries. METHODS: Serum samples were collected from 50 patients with triple vessel CHD and 41 patients with normal coronary vessels, both documented angiographically. The classical risk factors for CHD were recorded and each patient's standard lipid profile was determined. HDL and nonHDL lipoprotein particles were separated by precipitation with Dextran Sulphate/MgCl2. HDL and nonHDL lipid fractions were extracted with chloroform:methanol (1:2, v/v). 1H NMR spectra were recorded on a Bruker DRX-600 spectrometer. RESULTS: In the HDL fraction of patients with triple vessel disease the percentage of triglycerides was significantly higher than in those with normal coronary arteries, whereas the percentages of cholesterol esters, phosphatidylcholine and sphingomyelin, as well as polyunsaturated fatty acids, such as linoleic, arachidonic, and eicosapentaenoic, were significantly lower. In the nonHDL fraction significantly higher levels of triglycerides and lower levels of polyunsaturated fatty acids were observed. CONCLUSIONS: Patients with established CHD show significant alterations in the composition of plasma lipoproteins compared to those with normal coronary arteries. Further study of plasma lipoprotein composition might be able to identify components as indexes for the existence of CHD.

Gender-related and age-related urinalysis of healthy subjects by NMR-based metabonomics.

NMR-based metabonomic analysis is a well-established approach to characterizing healthy and diseased states. The aim of this study was to investigate inter-individual variability in the metabolic urinary profile of a healthy Greek population, not subjected to strict dietary limitations, by NMR-based metabonomics. The overall metabonomic urinalysis showed a homogeneous distribution among the population. The metabolic profile was examined in relation to gender and age, and reference intervals of major metabolites were determined. Multivariate data analysis led to the construction of two robust models that were able to predict the class membership of the subjects studied according to their gender and age. The most influential low molecular weight metabolites responsible for the differences in gender groups were citrate, creatinine, trimethylamine N-oxide, glycine, creatine and taurine, and for the differences in age groups they were citrate, creatinine, trimethylamine N-oxide and an unidentified metabolite (delta 3.78).

Evaluation of tubulointerstitial lesions' severity in patients with glomerulonephritides: an NMR-based metabonomic study.

An 1H NMR-based metabonomic approach was used to investigate the correlation of histopathologically assessed tubulointerstitial lesions with the urinary metabolite profile in 77 patients with glomerulonephritides submitted to renal biopsy. The presence of renal damage was predicted with a sensitivity of 96% and a specificity of 99%. Patients with mild, moderate, and severe tubulointerstitial lesions were progressively differentiated from the healthy individuals in the Orthogonal Signal Correction Partial Least-Squares-Discriminant Analysis (OSC/PLS-DA) models with a statistically significant separation between those with mild and with severe lesions. The onset of the tubulointerstitial lesions is characterized by decreased excretion of citrate, hippurate, glycine, and creatinine, whereas further deterioration is followed by glycosuria, selective aminoaciduria, total depletion of citrate and hippurate, and gradual increase in the excretion of lactate, acetate, and trimethylamine-N-oxide. NMR-based metabonomic urinalysis could contribute to the early evaluation of the severity of the renal damage and possibly to the monitoring of kidney function.

Antirestenotic effects of a novel polymer-coated d-24851 eluting stent. Experimental data in a rabbit iliac artery model.

Experimental and clinical data suggest that stents eluting antiproliferative agents can be used for the prevention of in-stent restenosis. Here we investigate in vitro the antiproliferative and apoptotic effect of D-24851 and evaluate the safety and efficacy of D-24851-eluting polymer-coated stents in a rabbit restenosis model (n = 53). Uncoated stents (n = 6), poly (DL: -lactide-co-glycolide) (PLGA)-coated stents (n = 7), and PLGA-coated stents loaded with 0.08 +/- 0.0025 microM (31 +/- 1 mug; low dose; n = 7), 0.55 +/- 0.02 microM (216 +/- 8 mug; high dose; n = 6), and 4.55 +/- 0.1 microM (1774 +/- 39 mug; extreme dose; n = 5) of D-24851 were randomly implanted in New Zealand rabbit right iliac arteries and the animals were sacrificed after 28 days for histomorphometric analysis. For the assessment of endothelial regrowth in 90 days, 12 rabbits were subjected to PLGA-coated (n = 3), low-dose (n = 3), high-dose (n = 3), and extreme-dose (n = 3) stent implantation. In vitro studies revealed that D-24851 exerts its growth inhibitory effects via inhibition of proliferation and induction of apoptosis without increasing the expression of heat shock protein-70, a cytoprotective and antiapoptotic protein. Treatment with low-dose D-24851 stents was associated with a significant reduction in neointimal area and percentage stenosis only compared with bare metal stents (38% [P = 0.029] and 35% [P = 0.003] reduction, respectively). Suboptimal healing, however, was observed in all groups of D-24851-loaded stents in 90 days in comparison with PLGA-coated stents. We conclude that low-dose D-24851-eluting polymer-coated stents significantly inhibit neointimal hyperplasia at 28 days through inhibition of proliferation and enhancement of apoptosis. In view of the suboptimal re-endothelialization, longer-term studies are needed in order to establish whether the inhibition of intimal growth is maintained.

Rabbit anti-HMG-17 antibodies recognize similar epitopes on the HMG-17 molecule as lupus autoantibodies. Relation with histone H1 defined epitopes.

HMG-17 is a nucleosomal protein which is an immune target of autoantibodies in systemic lupus erythematosus (SLE) and other autoimmune diseases. Autoantibody production in SLE is believed to result from autoantigen specific immune stimulation and subsequently, it is expected that antigenic determinants recognized by SLE autoantibodies and induced antibodies by immunization are quite similar. To examine this issue, rabbits were immunized with purified HMG-17. The produced antiserum showed cross reactivity on blots and in inhibition ELISA with histone H1, even after its affinity purification with immobilized HMG-17. Finally, purification of the antiserum over H1 absorbed on nitrocellulose membrane produced specific anti-HMG-17 antibodies in the supernatant and anti-HMG-17/H1 antibodies that were bound to H1. SLE sera positive for HMG-17 had also cross reactivity with H1, and following the same procedure as before we received HMG-17 specific SLE autoantibodies and anti-HMG-17/H1 autoantibodies. Using the multipin epitope mapping technology, 19 overlapping 15-mer HMG-17 peptides and six 15-peptides, corresponding to known epitopes of histone H1, were synthesized. Four major epitopes were identified on the HMG-17 molecule, reactive with induced anti-HMG-17 antibodies, and these were the same as major autoepitopes In SLE. The sequence 25-51 of HMG-17, part of its DNA-binding domain, was recognized by the anti-HMG-17/H1 antibodies that were bound to H1. These antibodies recognized also defined epitopes of H1. Our results show that SLE autoantibodies can be directed against the same or similar epitopes as do IgGs evoked during the active immunization of animals, and provide additional evidence that autosensitization with an autoantigen might be operative. The possibility that the same or similar epitopes are found on different molecules (in this study HMG-17 and H1) supports the fact that there are rules by which nature selects the most dominant immunodeterminant to a given protein, which often represents functional or structural sites in the autoantigen.

The recombinant subdomain IIIB of human serum albumin displays activity of gonadotrophin surge-attenuating factor.

BACKGROUND: Gonadotrophin surge-attenuating factor (GnSAF) is an as yet unidentified ovarian factor that acts on the pituitary to attenuate the pre-ovulatory LH surge. In a previous study, GnSAF bioactivity was proposed to derive, at least in part, from a C-terminal domain (95peptide) of human serum albumin (HSA). METHODS AND RESULTS: We employ here the expression-secretion system of Pichia pastoris to produce and assay selected recombinant polypeptides of HSA for GnSAF activity. We show that the C-terminal 95peptide of HSA (residues 490-585; subdomain IIIB) can be expressed from P.pastoris in secreted form and supernatants from clones expressing this polypeptide reduce the GnRH-induced LH secretion of primary rat pituitary cultures by 50-82%. When expressed in the same system, HSA domain III (residues 381-585) or full-length HSA (residues 1-585) are inactive. The bioactive subdomain IIIB is also separable from either domain III or full-length HSA on Blue Sepharose chromatography. CONCLUSIONS: Taken together, the findings highlight the putative importance of HSA subdomain IIIB as a GnSAF-bioactive entity and introduce a unique experimental tool to engineer this molecule for structure-function analysis.