[Consequences for clinical biochemists of the modifications of the creatinine-based evaluation of glomerular filtration rate between 2005 and 2008]. / Evolution des modalités d'évaluation de la fonction rénale basée sur la créatinine entre 2005 et 2008: conséquences pour les biologistes.

Since 2005, international guidelines propose a stadification for chronic renal failure based on the glomerular filtration rate (GFR) value. The performance of the creatinine-based equations allowing the estimation of GFR and the bias of the creatinine measurements is, more than ever, a crucial issue. The consequences for the clinical biologists are of importance. First, the Cockcroft-Gault formula must be replaced by the four variable-MDRD equation. Second, the biologists must chose from the "175" and the "186" versions of the MDRD equation. The first one fits the creatinine methods which are traceable to the reference method (liquid or gas chromatography coupled to mass spectrometry). The second equation must be used for creatinine methods, which are not traceable to the reference method. Today, only some enzymatic methods can prove that they are traceable to the reference method. For the colorimetric methods, future is inclear.

Candidate genetic modifiers of individual susceptibility to renal cell carcinoma: a study of polymorphic human xenobiotic-metabolizing enzymes.

The steady increase in sporadic renal cell carcinoma (RCC) observed in industrialized countries supports the notion that certain carcinogens present in the environment (tobacco smoke, drugs, pollutants, and dietary constituents) may affect the occurrence of RCC. Many of the enzymes dealing with such environmental factors are polymorphic and may, therefore, confer variable susceptibility to RCC. This case-control study was designed to test for an association between genetic polymorphism of enzymes involved in xenobiotic metabolism and the risk of sporadic RCC. Genomic DNA was obtained from 173 patients with RCC and 211 controls of Caucasian origin. We used PCR-RFLP to investigate polymorphism for the most common alleles at two cytochrome-P450 mono-oxygenases (CYP1A1 and CYP2D6), one NAD[P]H:quinone oxidoreductase (NQO1), three glutathione S-transferases (GSTM1, GSTT1, and GSTP1), and one N-acetyltransferase (NAT2) loci. The CYP1A1 (m) "variant" genotype, which contains at least one copy of the CYP1A1 variant alleles, was found to be associated with a 2.1-fold [95% confidence interval (CI), 1.1-3.9] increase in the risk of RCC. There was also a higher risk of RCC for subjects with the CYP1A1 (m) variant genotype combined with any of the following genotypes: GSTT1 (+) "active" [odds ratio (OR), 2.3; 95% CI, 1.2-4.5], GSTP1 (m) variant (OR, 2.4; 95% CI, 1.0-5.4), or NAT2 (-) "slow acetylator" (OR, 2.5; 95% CI, 1.1-5.5). A significant association was also found for the GSTM1 (-) "null" and GSTP1 (m) genotypes combined with either NAT2 (-) (OR, 2.6; 95% CI, 1.2-5.8) or CYP1A1 (m) (OR, 3.5; 95% CI, 1.1-11.2). The CYP2D6 (-) "poor metabolizer " and the NQO1 (-) "defective" genotypes were not clearly associated with a higher risk of RCC. Our data demonstrate for the first time a significant association between a group of pharmacogenetic polymorphisms and RCC risk. These positive findings suggest that interindividual variation in the metabolic pathways involved in the functionalization and detoxification of specific xenobiotics is an important susceptibility factor for RCC in Caucasians.

Differential regulation of gamma-glutamyltransferase mRNAs in four human tumour cell lines.

Human gamma-glutamyltransferase (GGT) belongs to a multigenic family and at least three mRNAs are transcribed from the gene that codes for an active enzyme. Four human tumour cell lines (HepG2, LNCap, HeLa and U937) with different GGT levels were used to investigate how GGT activity, total GGT mRNA and each individual GGT mRNA subtype responded to tumour necrosis factor-alpha (TNF-alpha), 12-O-tetradecanoylphorbol 13-acetate (TPA) or sodium butyrate treatment. Butyrate reduced the GGT activity in HepG2 cells, and the level of total GGT mRNA accordingly, whereas TNF-alpha and TPA did not alter these parameters. In LNCap cells, TNF-alpha, TPA, and butyrate reduced the activity as well as the level of GGT total mRNA. In HeLa cells no significant changes were observed either in activity or in mRNA level whereas TPA induced both GGT activity and mRNA levels in U937 cells. The distribution of each GGT mRNA subtype (A, B and C) was found to be cell specific: type B mRNA was the major form in HepG2 cells, while type A was the major form in LNCap and HeLa, type A and type C were expressed almost at the same level in U937 cells. The GGT mRNA subtypes were also differently modulated in these cells after TNF-alpha, TPA or butyrate treatment, suggesting that they are regulated by distinct and cell type specific mechanisms.

Curcumin-induced cell death in two leukemia cell lines: K562 and Jurkat.

Curcumin presents strong antioxidant and anticancer properties. However, molecular mechanisms leading to curcumin-induced cell death are poorly understood. The effect of curcumin was compared in two different leukemia cell lines: K562 and Jurkat. Cell death was induced in both cell lines, and apoptosis pathways were investigated by Western blot analysis. Decreases in pro-caspase 8 and 9 levels were observed. BH(3) interacting domain death agonist (Bid) was also cleaved. Jurkat cells appeared to be more sensitive to curcumin, and apoptosis takes place earlier.

Lipid and lipoprotein genetic variability: an important contribution from the French health examination centers.

OBJECTIVE: To assess lipid and lipoprotein genetic variability in the French Population. METHODS: Many health examination centers are covering a great part of France (700,000 individuals are examined in 54 centers every year). Each citizen has the right to have a personal examination every 5 years. This unique system was modified in 1992, and we are presenting our 25-year experience focusing on the results we have more recently obtained in the lipid and lipoprotein field. RESULTS: First of all, the 600 items of information collected on every patient coming to our Center give us the data and facilitate the development of the theory of reference values. Having mastered the analytical variations, we studied the biological variations. Age, sex, overweight, tobacco, alcohol, and drugs are the main factors we should control for obtaining reference values. More recently, the genetic part in the production of reference values has become of greater importance, particularly for apolipoproteins. Apolipoprotein E is an important lipoprotein for which the two frequent mutations influence the level of circulating Apolipoprotein E. The risks linked to these alleles are also very different in cardiovascular disease and extremely important (for epsilon 4) in Alzheimer's Disease. Apolipoproteins B and A-IV also have interesting polymorphisms, but currently have no systematic applications in clinical chemistry. The familial recruitment we have in Nancy also permitted us to constitute a large tool "La Cohorte Stanislas." CONCLUSION: The thousand families recruited will be followed for 10 years. It is an open tool for collaboration.

Dextromethorphan O-demethylase activity in rat brain microsomes.

CYP2D, a genetically variable isoform of cytochrome P450, has been characterized mainly in the liver and the brain of mammals by measurement of debrisoquine hydroxylase activity. Moreover, 'poor debrisoquine metabolizer' phenotype is significantly increased in Parkinson's disease patients. We present here the first demonstration that the activity of the CYP2D isoform can be characterized in rat brain microsomes by the measurement of dextromethorphan O-demethylase capacity. The cerebral formation of dextrorphan, an antagonist of the N-methyl-D-aspartate receptor, was inhibited by the presence of quinidine and N-methyl-4-phenylpyridinium (MPP+), a dopaminergic neurotoxin inducing a chemical parkinsonism in humans.

Continuous in vivo measurement of creatine kinase variations in man during an exercise.

In order to obtain information concerning the CPK turnover during exercise, we have elaborated a technique to measure its activity continuously in vivo. A colorimetric method has been adapted to total blood. CPK was measured continuously in 5 physically fit athletes exercised on an ergometric bicycle for 30 min. There was practically no increase in enzymatic activity. The starting level was very high, corresponding perhaps to a "permanent state of enzyme release"; the exercise was not hard enough because of the fitness of the athletes or the maximal increase did not occur immediately after the exercise.

Enzyme induction by drugs and toxins.

Enzyme induction by drugs mostly concerns those enzymes involved in drug metabolism: cytochromes P-450, UDP-glucuronosyltransferases, glutathione S-transferases, gamma-glutamyltransferases and epoxide hydrolases. A large variety of molecular forms exists, but not all of them are inducible (e.g. the inducible cytochromes P-450 in man are members of family IA, IIA, IIC, IIE, IIIA). Induction is most common in the liver, but also occurs in other organs (lung, placenta, lymphocytes). Over the past 20 years a relatively small number of drugs and environmental chemicals have been identified as enzyme inducers, perhaps fewer than early studies suggested. Information on inducing properties must be obtained as early as possible during the development of a new drug and made available to clinicians and clinical chemists when the drug is marketed. The main consequences of enzyme induction are changes in pharmacokinetics of the drug itself or of an associated drug. Much progress has been made in methods to identify these inducers.

Differential solubilization of rabbit liver plasma membrane gamma-glutamyltransferase by proteases and detergents: effect of phenobarbital treatment.

In an attempt to understand the mechanism of gamma-glutamyltransferase transfer from the liver to the plasma, potassium chloride, sodium dodecyl sulfate and proteases (papain and bromelain) were used to solubilize rabbit liver plasma membrane gamma-glutamyltransferase. Potassium chloride solutions solubilized 10-30% of membrane proteins but only 1-3% of membrane gamma-glutamyltransferase activity. However, when sodium dodecyl sulfate is used, even at low concentration (0.1-0.2%, w/v) greater than 90% of membrane gamma-glutamyltransferase activity and about 80% of membrane proteins can be solubilized. Furthermore, we showed that unlike the effect of bile salts on the membrane gamma-glutamyltransferase of phenobarbital-treated animals, the same treatment seems to have no influence on membrane gamma-glutamyltransferase solubilization by proteases. Indeed, the ratios of gamma-glutamyltransferase solubilization by papain or bromelain were the same for liver membranes obtained from control and phenobarbital-treated animals.

Malondialdehyde adducts to, and fragmentation of, apolipoprotein B from human plasma.

Many recent in vitro experiments support the hypothesis that oxidatively modified low density lipoproteins (LDLs) could participate in atherogenesis. Oxidation of LDLs, especially derivatization by aldehydes originating from peroxidation of fatty acids and fragmentation of apolipoprotein (apo) B-100 which is their major apolipoprotein, probably occurs extravascularly and the presence of oxidized LDLs in the circulation is not well documented. Using electrophoresis and immunodetection techniques, we studied the structure of apo B and the presence of adducts of malondialdehyde (MDA) to this protein in LDLs from plasma of a limited population of five healthy subjects and nine patients with severe atherosclerosis. In the patient-derived LDLs, apo B appeared extensively fragmented, much more so than in those from the healthy subjects, although LDLs were isolated in all cases in the presence of antioxidants, protease inhibitors and antibiotics. Additionally, in all healthy subjects, we found a minor fragment of apo B-100, apo B-74, whereas the complementary peptide, apo B-26, was not detected; thus the presence of this minor form cannot be related to cleavage of apo B-100, either by proteolysis or by oxidation. We also present evidence that MDA adducts are present in circulating apo B and most of its fragments not only in atheromatous patients, but also in healthy subjects. Our results are consistent with the existence of oxidized LDLs in the human circulation. However, the role of non-oxidative phenomena in the structural modifications affecting apo B which are reported here cannot be excluded.

Drug metabolizing enzymes related to laboratory medicine: cytochromes P-450 and UDP-glucuronosyltransferases.

Many studies on drug metabolism have been carried out during the last decades using protein purification, molecular cloning techniques and analysis of polymorphisms at phenotype and genotype levels. These researchers led to a better understanding of the role of drug metabolizing enzymes in the biotransformation of drugs, pollutants or foreign compounds and of their use in laboratory medicine. The metabolic processes commonly involved in the biotransformation of xenobiotics have been classified into functionalization reaction (phase I reactions), which implicate lipophilic compounds. These molecules are modified via monooxygenation, dealkylation, reduction, aromatization, hydrolysis and can be substrates for the phase II reactions, often called conjugation reactions as they conjugate a functional group with a polar, endogenous compound. This review, devoted to cytochromes P-450 (CYP) and UDP-glucuronosyltransferases (UGT), describes essentially the genetic polymorphisms found in humans, their clinical consequences and the methods to assess the phenotypes or genotypes, with a view to studying the interindividual differences in drug monooxygenation and drug glucuronidation. Variations in drug glucuronidation reported here focused essentially on variations due to physiological factors, induction, drug interactions and genetic factors in disorders such as Gilbert's Syndrome and Crigler-Najjar type I and II diseases.

Perturbation of laboratory test results by analytical interferences and drug effects.

An account is given of ways in which drug treatment can influence the results of laboratory tests for certain species of clinical importance, and of methods of detecting such interferences or effects.

Drug effects on clinical laboratory tests.

Drugs and xenobiotics can affect clinical laboratory test results either by interfering with the analytical systems themselves, or by influencing endogenous constituents. National and international bodies have brought widespread recognition to this problem and have proposed protocols for its thorough scientific study. In this survey the authors discuss studies in their laboratories concerning the effects of drugs on thousands of patients undergoing a routine clinical screen. A database is described for storing both patient information and a detailed analysis of the published literature on drug effects. Analytical interferences in clinical tests must be examined in validating the procedure. However, highly specific analytical techniques are increasingly helping to reduce such interferences. Biological effects can be classified as physiological, pharmacological or toxicological. In some cases, biological effects can be used to advantage in monitoring treatment by potentially hazardous drugs, such as the cardiac glycosides. The requirement for a well-defined reference population for each drug and for access to all clinical and medical data for each patient is discussed. The need for greater awareness of the influence of drugs on clinical laboratory results is considered, together with the suggestion that the health professions should try to exploit such effects in monitoring possible toxicity problems, in defining genetic constitution and in designing medication programmes.

Hydroxylation of debrisoquine using perfused liver isolated from Sprague Dawley and DA rats: comparison with in-vivo results.

The hydroxylation of debrisoquine was investigated in Sprague-Dawley (SD) and Dark-Agouti (DA) rats. Female and male rats were phenotyped in-vivo with debrisoquine six times during their growth. The ratios debrisoquine/4-hydroxydebrisoquine of the female DA rats increased until the 15th week and then decreased; but the values of the metabolic ratios never exceeded 2. Female DA rats cannot be considered as genetically deficient for hydroxylation of debrisoquine in regard to the metabolic ratio, but the percentage of debrisoquine excretion is up to ten fold higher than that in the other strains. Therefore SD and DA rat livers were perfused for 2 h when the clearance of debrisoquine was significantly lower in the female DA group than in the other groups. 4-Hydroxydebrisoquine in the perfusate increased with time, but the amount after 120 min was 12 fold lower in the female DA rat group than in the female SD rat group. The results of the male DA group fell between. This study confirms that female DA rats present a lower debrisoquine 4-hydroxylating capacity than other rats but shows that urinary metabolic ratio is not sufficient to assess the deficiency of debrisoquine hydroxylation.

[Changes in blood cholesterol levels over a 10-year period; value of a single sample for predicting future blood cholesterol levels]. / Evolution de la cholestérolémie sur 10 ans; intérêt d'un dosage ponctuel pour prédire la cholestérolémie future.

The aim of this study was to report the serum cholesterol changes over a 10 year period in 12,238 subjects aged 4 to 64 years (mean 28 +/- 14 years) based on 3 health check-ups at an average of 5.5 yearly intervals between 1973 and 1989, and to determine the value of a single sample for predicting the serum cholesterol level at 5 and 10 years, and the influence of blood pressure and Quetelet index on this predictability. After identification of the influencing factors, the different variables were adjusted using a step-by-step regression analysis. The correlation coefficients calculated between the adjusted cholesterol level at the first examination and that measured at 5 and 10 years, were all significant (0.38 to 0.59) and varied with age at the time of the first examination and gender. The positive predictive value of having a cholesterol level higher than the 90th centile at 5 and 10 years when it was already higher than this value at the first examination varied from 26 to 46% respectively with respect to the subgroups. The sensitivity of the test was 25 to 48%. The negative predictive value and specificity were 93 to 95%. The lowering of this threshold to the 80th centile increased the positive predictive value from 35 to 45% and decreased the specificity from 94 to 87% for the whole population. When the first two sampling results, five years apart, were taken into consideration simultaneously, the predictive value of having a raised cholesterol level at 10 years increased from 35 to 61%.(ABSTRACT TRUNCATED AT 250 WORDS)

[Changes of plasma and tissue gamma-glutamyltransferase under the influence of drugs]. / Interprétation des variations de la gamma-glutamyltransférase plasmatique et tissulaire sous l'influence des médicaments.

Measurement of gamma-glutamyltransferase (GGT) activity in plasma is widely used in clinical biology (in order to detect hepatic diseases or to monitor treatment for alcoholism), and also in pharmacology (since this test is the only plasmatic marker for hepatic induction in human). However, the correct interpretation of a high plasmatic activity should take into account the various analytical factors which can affect results, as well as the physiological parameters known to modify this activity. It also requires its comparison to defined reference values. Several mechanisms may be involved in the increase of plasmatic activity as an index of hepatic induction, such as an increase in the protein synthesis, a release of the enzyme from the membrane or a modification in the biliary flux.

[Human plasma lactic dehydrogenase activity: individual variations and reference intervals]. / Activité déhydrogénase lactique humaine plasmatique: variations interindividuelles et intervalles de référence.

Out of a population of 8 7600 people of all origins, we drew up frequent values for the activity of total lactate dehydrogenase. From these values, we defined a reference interval on a selected population. Variations between individuals were determined in relation to the following factors: age (8 to 70 years), sex, height, weight, socio-prefessional categories, any excess weight, exercise and drugs. There exist definite variations in relation to age, especially in children. On the other hand, this enzyme shows little variation in relation to the morphology of the individual adult. Socio-professional groups have more influence on the activity of total lactate dehydrogenase.

[Viral theory of atherosclerosis. Role of cytomegalovirus]. / Théorie virale de l'athérosclérose. Place du cytomégalovirus.

The multifactorial aetiology of atherosclerosis is nowadays well established. In parallel with confirmation of the lipidic hypothesis, a tumoral theory for this pathology was built up during the past decade. This theory considers atheroma a benign tumor. Among agents that can induce cell proliferation, oncogenic viruses seem to be the most efficient. In this review, we present several works suggesting viral involvement in atherosclerosis. The viral theory is based not only on clinical and epidemiologic data, but also on cell and molecular biology research. These three complementary approaches have established a relationship between herpes viruses and the atherogenic process. Cytomegalovirus (CMV) seems to be the suspect in human atherosclerosis. Several studies of the effects of CMV on cells involved in atheroma, especially smooth muscle cells and endothelial cells, are consistent with a possible role of this virus in atherosclerosis. This paper presents not only recent research developments in this increasingly explored field, but also questions that remain to be elucidated and the consequences of the viral theory of atherosclerosis.

[Biological tests as indirect indicators of the activity of drug metabolizing enzymes. Use of glucaric acid and gamma-glutamyltransferase]. / Les examens biologiques témoins indirects de lactivité des enzymes du métabolisme des médicaments. Place de l'acide glucarique et de la gamma-glutamyltransférase.

Laboratory tests are increasingly important in the surveillance of drug treatments. People in charge of clinical laboratory science must now become aware of a new development, the use of as indicators of the activity of drug-metabolizing enzymes, especially of the induction of such enzymes. Ideally, the activity of such enzymes should be measured in the tissues. In man, however, easier indirect methods of application are now most often used : measurement of antipyrine half-life, urinary and plasma drug metabolites, protein and specific enzyme levels in palsma, changes in endogenous substrates and endogenous metabolites, and enzymes in circulating blood cells. Of all these, two examinations are now widely performed, those for urinary glucaric acid and gamma-glutamyltrasferase. The value and limitations of these two tests are discussed.

[Application of bidimensional electrophoresis in the determination of genetic variants]. / Application de l'électrophorèse bidimensionnelle à la détermination des variants génétiques.

Study of the polymorphism of human plasma proteins presents a major advantage in clinical biology to discover abnormal or rare forms, associated with risks or involved with well-specified pathological conditions. If this polymorphism corresponds to structural differences causing a variation of the isoelectric point (Ip) or the molecular weight, it is easily visualized with bidimensional electrophoresis, starting with a few microliters of plasma. On a single mapping per patient, we were able to establish the frequency of various isoforms of Gc globulin, transferrin, haptoglobin, alpha-1-antitrypsin and apolipoproteins E as well as A-IV, in a Lorraine population consulting for a health check-up. This method has already enabled us to demonstrate the effect of polymorphic apolipoproteins on the cholesterol and triglycerides level: patients carrying the epsilon 2 allele of the Apo E, have a mean cholesterol level which is lower than that of the carriers of allele epsilon 3. Patients carrying allele epsilon 4 have the highest mean cholesterol level.