[Evaluation of plasmatic homocysteine determination by gas chromatography-mass spectrometry]. / Evaluation d'un transfert de méthode : dosage de l'homocystéine plasmatique par chromatographie en phase gazeuse couplée à la spectrométrie de masse.

Total plasma homocysteine emerged in the past few years as an independent risk factor for cardiovascular diseases. This test is now currently prescribed for the diagnosis of unexplained thrombosis in young adults or recurrent thrombosis in patients with arteriopathy. This sulphured amino-acid is an important intermediate in transsulfuration and remethylation pathways of methionine metabolism. Within the context of a collaboration between Monastir and Grenoble Universities and because a gas chromatograph mass spectrometer (GC-MS) instrument was available in Monastir, we proposed to transpose a GC-MS method previously developed in Grenoble's hospital for this parameter and to validate it by comparison with the liquid chromatography tandem mass spectrometry (LC-MS-MS) method, used at present. Analytical performances were good: detection limit 0.4 micromol/L and linear range up to 4 mg/L (29.6 micromol/L), and between-run and within-run precision with coefficients of variation < 5% and < 8 %, respectively. The comparison with LC-MS-MS method showed a good correlation (y = 0.9874 x -0.208; r(2) = 0.84). Mean difference from LC-MS-MS was -0.4 micromol/L. Plasma concentrations of homocysteine (mean + SD) determined among Tunisian adults, 29 men, 27 women, of the same age were respectively: 11.6 +/- 2.4 micromol/L and 10.1 +/- 2.7 micromol/L, p = 0.025. This method is now currently used to evaluate tHcy concentration in patients with risk factors for cardiovascular disease.

Hyperhomocysteinaemia is associated with osteoporosis in patients with Crohn's disease.

BACKGROUND: A high prevalence of osteoporosis is observed in Crohn's disease. Recent data have shown that homocysteinaemia is an important risk factor in low-bone mineralization and fracture. AIM: To look for an association between homocysteinaemia and low-bone mineralization in Crohn's disease patients. PATIENTS AND METHODS: Ninety-two consecutive patients (sex ratio M/F 0.87; mean age: 36.6 +/- 13.2 years) were recruited between 2003 and 2005. Bone densitometry was performed on inclusion. The following parameters were analysed: age, sex, Crohn's Disease Activity Index, duration and extent of Crohn's disease, smoking status, corticosteroid treatment, immunosuppressive drugs, plasma homocysteine, folate and vitamin B12 concentration. RESULTS: The prevalence of a high homocysteine level (>15 micromol/L) was 60%. Osteoporosis and low-bone mineralization observed in 26 (28%), and 60 (65%) patients, respectively. On a multivariate analysis, associated factors for osteoporosis and low-bone mineralization were respectively: hyperhomocysteinaemia (OR: 61.4; CI: 95: 23-250; P < 0.001), and ileal Crohn's disease [OR: 13.8; CI: 95: 2.5-150; P = 0.036] for osteoporosis and hyperhomocysteinaemia [OR: 63.7; CI: 95: 8.5-250; P < 0.001] and disease duration of at least 5 years [OR: 11.4; CI: 95: 1.31-99; P = 0.039] for low-bone mineralization. Results were similar whichever site osteoporosis was detected. CONCLUSION: Hyperhomocysteinaemia was observed in 60% of our Crohn's disease patients and was strongly associated with low-bone mineralization and osteoporosis (OR: 61.4).

[Factors associated with hyperhomocysteinemia in inflammatory bowel disease: prospective study in 81 patients]. / Hyperhomocystéinémie et facteurs associés au cours des MICI: étude prospective chez 81 patients.

BACKGROUND: A high prevalence (52%) of hyperhomocysteinemia is observed in Crohn disease (CD), however it is not well documented in ulcerative colitis (UC). Furthermore, in the different works studying hyperhomocysteinemia the associated factors are different. AIM: Prospective evaluation of hyperhomocysteinemia in inflammatory bowel disease (IBD) patients, of the risk factors and the determination of a potential risk of colorectal carcinoma in case of hyperhomocysteinemia. PATIENTS AND METHODS: IBD patients followed in our department were prospectively recruited between November 2003-September 2004. To be included patients should have passed a coloscopy in the two years. Patients with kidney failure or drugs supposed, to interfere with homocystéine metabolism (folates, vitamin B12, methotrexate) were excluded from the study. The following parameters were analysed: age, sex, clinical activity indexes (CDAI for Crohn disease and CAI for ulcerative colitis), length-extent and type of the disease (CD or UC), smoking, plasma homocystein concentration, folates and vitamin B12. RESULTS: Eighty-one patients (60 CD, 21 UC, mean age 43.8 +/- 17.3) were included, 30 had an active disease at inclusion and 16 were smokers. The prevalence of high homocystein concentration was 55.6%. In univariate analysis a low rate of folates was the only risk factor for a high homocystein concentration (74 vs. 52.8%; P = 0.018). Smoking was almost an associated factor. In multivariate analysis, a low rate of folate was the only risk factor of hyperhomocysteinemia, OR = 3.59 [1.27-10.17]. Five endoscopic lesions considered as precancerous were described; these patients had all a hyperhomocysteinemia. CONCLUSION: The prevalence of hyperhomocysteinemia is high in UC and in CD. A low folate rate is the only risk factor observed in our study. There is a possible link between colorectal cancer and hyperhomocysteinemia. A high Plasma homocystein concentration must be search in inflammatory bowel disease patients and a substitutive treatment of folates and vitamin B12 is necessary in case of hyperhomocysteinemia.

[Severe hyperhomocysteinemia revealing homocystinuria in two young adults with mild phenotype]. / Hyperhomocystéinémie sévère révélant une homocystinurie chez deux jeunes adultes présentant un phénotype peu marqué

INTRODUCTION: To the request of total plasma homocysteine determination in the investigation of vascular disease, diagnosis of homocystinuria in young adult patients with mild phenotype is not so rare. EXEGESIS: A 26-year-old man developed embolic cerebral infarction and a 22-year-old woman presented a right renal venous thrombosis one week after delivery. In each case, high concentration of total plasma homocysteine was first found and plasma and urinary amino acids analysis later on directed the diagnosis towards homocystinuria. Finally, reduced skin fibroblast cystathionine beta-synthase activity confirmed the diagnosis of homocystinuria. CONCLUSION: Total plasma homocysteine determination must be determined for screening for hyperhomocysteinemia in young adults with venous thromboembolism without characteristic phenotypic features of homocystinuria.

[Belated decompensation of an Imerslund-Grasbeck disease]. / Décompensation tardive d'une maladie d'Imerslund-Grasbëck.

Imerslund-Gräsbeck disease is an autosomic recessive disease characterised by a megaloblastic anemia due to a vitamin B12 deficiency and by a moderate proteinuria without kidney failure. It is caused by the malabsorption of Cobalamin-intrinsic factor complex bringing into play cubulin and other proteins (megaline, amnioless), some mutations of which are described at present. We report herein the observation of a child whose diagnosis was made belatedly during an acute decompensation with biological hemophagocytic syndrome. Its evolution was marked by the appearance of neurological disorders at the beginning of the vitamin B12 substitution treatment. These disorder regressed as the dosage was increase. The purpose of this observation is to recapitulate the main characteristics of this disease and to review the current data.

Lack of cell death enhancement after irradiation with monochromatic synchrotron X rays at the K-shell edge of platinum incorporated in living SQ20B human cells as cis-diamminedichloroplatinum (II).

In this paper we describe the results of experiments using synchrotron radiation to trigger the Auger effect in living human cancer cells treated with a widely used chemotherapy drug: cis-diamminedichloroplatinum (II) (cisplatin). The experiments were carried out at the ID17 beamline of the European Synchrotron Radiation Facility, which produces a high-fluence monochromatic beam that is adjustable from 20 to 80 keV. Cisplatin was chosen as the carrier of platinum atoms in the cells because of its alkylating-like activity and the irradiation was done with monochromatic beams above and below the platinum K-shell edge (78.39 keV). Cell survival curves were comparable with those obtained for the same cells under conventional irradiation conditions. At a low dose of cisplatin (0.1 microM, 48 h), no difference was seen in survival when the cells were irradiated above and below the K-shell edge of platinum. Higher cisplatin concentrations were investigated to enhance the cellular platinum content. The results with 1 microM cisplatin for 12 h showed no difference when the cells were irradiated with beams above or below the platinum K-shell edge with the exception of the higher cell death resulting from drug toxicity. The intracellular content of platinum was significant, as measured macroscopically by inductively coupled plasma mass spectrometry. Its subcellular localization and particularly its presence in the cell nucleus were verified by microscopic synchrotron X-ray fluorescence. This was the first known attempt at K-shell edge photon activation of stable platinum in living cells with a platinum complex used for chemotherapy. Its evident toxicity in these cells leads us to put forth the hypothesis that cisplatin toxicity can mask the enhancement of cell death induced by the irradiation above the K-shell edge. However, K-shell edge photon activation of stable elements provides a powerful technique for the understanding of the biological effects of Auger processes. Further avenues of development are discussed.

[Comparison of plasma total homocysteine determinations in 9 French hospital laboratories by using 6 different methods]. / Comparaison du dosage de l'homocystéine plasmatique totale dans neuf laboratoires par six techniques différentes.

A lot of methods are now available for total plasma homocysteine (tHcy) determination. Commercial kits using immunoassay, easier to use, begin to supplant in-house laboratory methods. Our aim is to evaluate the interchangeability of tHcy measurements in 9 French hospital laboratories. Six different method types were used: 2 gas chromatography-mass spectrometry (GC-MS), 2 HPLC with fluorescence detection subdivided in one in-house method and one commercial kit (Bio-Rad ), 3 fluorescence polarization immunoassays (FPIA), 1 enzyme immunoassay, 1 amino acid analyser, 1 capillary electrophoresis coupled with laser-induced fluorescence detection (EC-LIF). Each laboratory analysed 41 patient's plasma samples in which 8 samples contained added homocystine. Results were analysed for imprecision, recovery, and methodological differences. The mean among-laboratory imprecision (CV) ranged from 12.5 to 18% in function of plasma sample type and was identical to the mean among-method variation. In terms of recovery, we obtained underestimated results with immunoassays. The bias relative to the GC-MS method was less than 12.5% except for two laboratories, one using FPIA assay and the other EC-LIF. In conclusion, the interchangeability of tHcy results between laboratories is not satisfactory and does not allow us to evaluate cardiovascular risk linked to moderate increases of tHcy.

Effects of selenium supplementation on malignant lymphoproliferative pathologies associated with OF1 mouse ageing.

Low plasma selenium (Se) levels have been shown to correlate with increased cancer incidence in humans and in mice. This study was undertaken to investigate the ability of Se to decrease mortality rate and tumor production in ageing mice. Se (2.5 ppm) given as sodium selenite in drinking water to 8 months old OF1 mice, for 4 consecutive months, reduced significantly the mortality of mice with 6% and 50% mortality rate for Se and control groups, respectively. In addition 80% of control deaths resulted from a lymphoid cell neoplasma, while no one of Se supplemented mice produced tumor. Evaluation of parameters of free radical metabolism showed highly significant reduction of the antioxidant defence system in the liver of cancer mice, with a 78% decrease in GSH-Px activity, a 65% decrease in superoxide dismutase (SOD) activity, a 75% decrease in the GSH/GSSG ratio and a 62% decrease of plasma Se level, as compared to healthy old mice. Nevertheless in the conditions of our experiment, Se didn't really improve the endogenous antioxidant status of ageing mice.

Identification of amylase crystalloids in cystic lesions of the parotid gland.

OBJECTIVE: To identify alpha-amylase crystalloid formations in parotid specimens obtained by fine needle aspiration. STUDY DESIGN: The study concerned three cases of sialadenitis with crystalloid formation observed between 1993 and 1998. In one of these cases, transmission electron microscopy, mass spectrometry and measurement of amylase activity were used to characterize the nature of the crystalloids. RESULTS: Light microscopy revealed the same crystalloid structure in all three cases. In one case, where the material was saved, a biochemical method made it possible to reveal high amylase activity, while protein electrophoresis and mass spectrometry were used to identify salivary alpha-amylase. CONCLUSION: Crystalloids of salivary alpha-amylase can be identified by May-Grünwald-Giemsa and Papanicolaou stain and can be rapidly confirmed through determination of amylase activity.

Gas chromatographic-mass spectrometric determination of total homocysteine in human plasma by stable isotope dilution: method and clinical applications.

The detection and quantitation of slight increases of plasma homocysteine levels is of growing interest. This has prompted us to develop a highly sensitive and accurate capillary gas chromatography-mass spectrometry (GC-MS) method. The method proved to be highly sensitive (DL=0.17 micromol/l) with between- and within-run precision less than 6% and 7%, respectively. Reference values of plasma total homocysteine have been determined for men (n=39) and women (n=36), showing a significant difference (P=0.003) between gender. Preliminary results in cerebrovascular accidents and in venous thrombosis are presented.

Urinary 8-oxo-7,8-dihydro-2'-deoxyguanosine and 5-(hydroxymethyl) uracil in smokers.

Cigarette smoke is known to generate free radicals by various mechanisms. In this study involving 30 non-smokers and 30 smokers, we show that urinary excretion of 5-(hydroxymethyl) uracil (HMUra) was not different in the two groups (6.54+/-2.07 vs. 6.70+/-1.68 nmol/mmol creatinine). In contrast, 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dGuo) excretion increased by 16% (1.16+/-0.35 vs. 1.35+/-0.50 nmol/mmol creatinine, p = 0.039). Results concerning 8-oxo-dGuo are in agreement with those of previous studies. We observed significant multiple correlations between HMUra and creatinine (r(p) = 0.44), BMI (r(p) = -0.27) and nicotine derivatives (r(p) = 0.26). Multiple correlation analysis showed relations between 8-oxo-dGuo on the one hand, and: creatinine (r(p) = 0.36), nicotine derivatives (r(p) = 0.29), BMI (r(p) = -0.24) on the other.

Foreign body histiocytosis reaction after hip replacement with concomitant metastatic adenocarcinoma in the same lymph node.

Infiltration of regional lymph nodes by macrophages has been shown after total joint arthroplasty. These pelvic lymph nodes were obtained most often from patients during staging procedures for carcinoma and may be a diagnostic pitfall in the frozen section diagnosis of nodal metastasis. We report an unusual case of association in the same lymph node between histiocytosis and prostatic carcinoma metastasis. Histiocytosis was caused by wear debris from two different prostheses. Inductively coupled plasma mass spectrometry verified this diagnosis.

Analyzed dietary intakes, plasma concentrations of zinc, copper, and selenium, and related antioxidant enzyme activities in hospitalized elderly women.

OBJECTIVE: The purpose of the study was to assess the actual dietary intakes of zinc (Zn), copper (Cu) and selenium (Se) intakes in relation with some indicators of trace element status in a selected group of hospitalized elderly patients. SUBJECTS: 24 elderly women aged 76-99 years were recruited in the Geriatric Department of the Grenoble University Hospital. MEASURES OF OUTCOME: Zn, Cu, and Se dietary intakes were estimated by duplicate portion analysis. Plasma trace element concentrations, Cu-Zn superoxide dismutase (Cu-Zn SOD) and Se glutathione peroxidase (Se GSH-Px) activities were determined in parallel. RESULTS: Mean daily intakes of Zn (5.6 mg), Cu (0.67 mg), and Se (23 micrograms) were low, in relation with poor energy intake and nutrient densities. Zn and Se levels in plasma were lower and plasma Cu increased compared to reference values obtained from healthy younger subjects. Thirty-eight percent of the elderly patients had plasma Zn concentrations < 10.7 mumol/l, but Cu status appeared adequate as suggested by the lack of decline in Cu-Zn SOD activity. A high proportion of plasma Se concentrations < 0.76 mumol/l and the parallel decrease in erythrocyte and plasma GSH-Px activities suggest a Se deficiency in this population. CONCLUSION: Our findings indicate that French hospitalized elderly patients may be at risk of Zn and Se marginal status and present altered antioxidant defenses in relation with low dietary intakes. It underlines the interest of supplementation studies in this population.

5-Hydroxymethyluracil excretion, plasma TBARS and plasma antioxidant vitamins in adriamycin-treated patients.

The thymine oxidative lesion-5-hydroxymethyluracil (HMUra)-was measured in urine collected from cancer patients. These patients all received chemotherapy using Adriamycin. Adriamycin (ADR) intercalates DNA coils and interferes with normal cell metabolism through diverse biochemical mechanisms that may explain its different actions. The anticancer action of ADR could derive from its interaction with topoisomerase II, resulting in DNA nicking followed by DNA fragmentation and apoptosis. Side effects of ADR-mainly its cardiotoxicity-may derive from the fact that ADR generates superoxide and hydroxyl radicals in two ways: redox-cycling and a Haber-Weiss type reaction due to Fe-ADR complexes. The oxygen free radicals, particularly .OH, are thought to be produced by ADR directly in genomic material and attack all its components. 5-Hydroxymethyluracil is a thymine lesion provoked by these attacks, and it has been proposed as a marker of DNA alterations. In this article, we report the results of a study involving 14 cancer patients treated with ADR. We found that urine HMUra is significantly increased by the anticancer therapy (HMUra (nmol/24 h): 74.4 9.46 vs. 96.3 8.74; p < .01), this increase reveals a higher risk of mutagenesis. Our study is the first to show an in vivo alteration of DNA by ADR. Results also show that thiobarbituric acid reactants increase significantly, and that the vitamin levels for retinol and alpha-tocopherol, which are antioxidant vitamins, are lower at the end of chemotherapy. We suggest to supplement these patients with vitamins A and E, and selenium to reduce the side effects of ADR.

[Systemic granulomatous reaction in hip prosthesis. Apropos of 2 anatomoclinical cases]. / Réactions granulomateuses systémiques et prothèse de hanche. Deux observations anatomocliniques.

PURPOSE OF THE STUDY: The production of particulate wear debris is a recognised complication of joint arthroplasty. Focus was made on local tissue reactions. Systemic distribution of wear debris are less know. We report two new cases of distant granulomatous reaction. MATERIAL: The first case concerned a 71-year-old man with lymph node histiocytosis incidentally discovered during the staging of a prostatic carcinoma. The second case concerned a 61-year-old man with a visceral granulomatosis reaction (liver, spleen and lymph node) associated to hepatic ans splenic enlargement. METHODS: We realised an histological analysis of several tissue specimens for these two patients and for one case an inductively coupled plasma-mass spectrometry technique (ICPMS). RESULTS: In all specimens we identified a foreign body granulomatous reaction characterized by the presence of particles coming from hip arthroplasty. The ICPMS identified titanium in spleen. DISCUSSION: Distant granulomatous reaction are generally localised in regional lymph nodes and discovered by accident. A systemic granulomatous reaction attributable to wear particles raises the question of long term biocompatibility of prosthetic materials. CONCLUSION: Besides local reaction, it is of utmost importance that physicians involved in prosthetic field take into consideration the possible systemic adverse effect of wear particles from arthroplasty.

Effect of double-blind crossover selenium supplementation on biological indices of selenium status in cystic fibrosis patients.

Twenty-seven cystic fibrosis patients received selenium supplementation (2.8 micrograms of sodium selenite per kilogram of body weight per day) or a placebo. This 5-month trial was conducted as a double-blind, placebo-controlled study. After an interval of 2 months, treatments of the two groups were interchanged (crossed over) for another 5-month period. A group of healthy subjects, living in the same area, was investigated simultaneously. No selenium deficiency was found either in plasma or in erythrocytes before the supplementation. This result was inconsistent with a previous study performed in 1988 in our laboratory. This change in selenium status can be explained by progress in the nutritional nursing care of children and by the addition of selenium to the diet. During the study, selenium concentrations in plasma decreased when patients received placebo treatment and increased during selenium intake. In one of the two groups a similar variation was found for glutathione peroxidase activities in plasma and erythrocytes, whereas erythrocyte selenium was normal and did not change in any group. Nowadays, in the Grenoble area, the selenium status of cystic fibrosis patients is close to normal. Nevertheless, this study indicates a fragile equilibrium, given that selenium concentrations cn be lowered by placebo or mildly increased by supplementation.

Gas chromatographic-mass spectrometric determination of 5-hydroxymethyluracil in human urine by stable isotope dilution.

A method for the determination of 5-hydroxymethyluracil in urine is described. 5-Hydroxymethyluracil was extracted by reversed-phase chromatography and quantified by gas chromatography-mass spectrometry as tert.-butyldimethylsilyl derivative. Since natural 5-hydroxymethyluracil contained ca. 22% of M + 2 species, an internal standard consisting of [1,3-15N2,5-2H2]hydroxymethyluracil was used to correct losses during extraction, evaporation and derivatization. Between-run precision of this method was 7.79%, and concentrations as low as 1.87 nM could be measured. This sensitivity and precision could not be obtained with trimethylsilyl derivatives.

Measurement of oxidative base damage to DNA by using HPLC-32P-postlabelling and GC/MS-selective ion monitoring assays.

A 32P-postlabelling assay has been developed for singling out specific oxidized base lesions. Emphasis was placed on the quantitative aspect and the accuracy of the assay, which require the use of calibration curves and microreactions, respectively. The method was successfully applied to the detection and the measurement of adenine N1-oxide and 5-hydroxymethyluracil in cells exposed to agents inducing oxidative stress including H2O2 and UV-A radiation. The sensitivity of the assay allows the detection of one lesion in 10(6) normal bases in 1 microgram of DNA. The GC/MS method when coupled to the selective ion monitoring (SIM) technique is about twenty times less sensitive, even for suitable substrates such as 5-hydroxymethyluracil and 5-hydroxyuracil, than the 32P-postlabelling assay. However, the former assay is much easier to apply, even though a derivatization step is necessary, and provides unambiguous structural information on the compound to be measured. Accurate quantitative measurements can be obtained when stable, isotopically labelled standards are available.