Homocysteine (Hcy) is a thiol containing amino acid related to methionine demethylation. Increased Hcy concentration is considered as a risk factor for cardiovascular diseases. In clinical laboratories, Hcy measurement can be performed using diverse methods such as chromatography, immunonephelemetry, immunochimiluminescence or enzymatic colorimetry. The goal of this work was to correlate two methods: high-pressure liquid chromatography (HPLC) versus immunonephelemetry. We studied 196 plasmatic samples collected in two hospitals, according to established protocols. The statistical analysis showed strong correlation and concordance between the two techniques. Hcy measurements using immunonephelemetry are underestimated by 25% compared to HPLC, leading to normality and cut-off changes for immunonephelemetry to 12 mu M compared to 15 mu M for HPLC. Using this value, sensibitity of the measurement is 82% and specificity is 94.5%. In these conditions, Hcy measurements in immunonephelemetry on BN ProSpec (Siemens (R)) are as dependable as those of HPLC and applicable, as it is currently the case in CHU of Saint-Etienne. (C) 2009 Elsevier Masson SAS. All rights reserved.

Two patients presented massive hypercholesterolemia related to acquired disease, intrahepatic cholangitis with cholestasis in one and nephrotic syndrome in the second. Comparison of the lipoprotein patterns demonstrated distinctive pathophysiological processes different from those operating in primary causes of hypercholesterolemia.

In a previous study we demonstrated that MG-63 cells cultured on Ti-6Al-4V discs covered by alumina ceramic and submitted to intermittent mechanical strain (IMS) presented morphological alteration associated with enhanced differentiation. Here we examine how the mechanical response of osteoblasts can be modulated by the nature of the substrate. MG-63 cells were cultured on four materials: polystyrene and Ti-6Al-4V (average roughness=0.48 μm) as smooth substrates; Ti-6Al-4V (average roughness=5.76 μm) and Ti-6Al-4V covered with alumina (average roughness=5.21 μm) as rough substrates. Mechanical strains were applied for 15 min, three times a day for 1–5 days with a 600 μstrains magnitude and a 0.25 Hz frequency. IMS stimulated alkaline phosphatase activity by 25–35% on all substrates and had no effect on cell growth on either substrate. Fibronectin (FN) was chosen as representative of cell-matrix interaction. FN production was increased by 60% after 1 day of stretching only on alumina-coated discs. FN organization examined on smooth substrates was affected by 5 days of IMS, showing a thickening of the fibres. The same modifications induced by IMS were previously observed on alumina-covered discs. Vinculin expression was not affected by IMS whatever the substrate. Cell–cell interactions were determined by N-cadherin immunoblotting. N-cadherin expression was increased by IMS specifically on rough substrates. Our results suggest that the nature of the surface did not influence the up-regulation of alkaline phosphatase activity induced by IMS, but modulates specifically cell–substrate as well as cell–cell interactions in response to IMS.

A massive release of troponin Ic and CKMB was described in a patient during septic shock. According to experimental animal models previously described, this release of biological markers by myocardial tissue could be due to an inflammatory process of myocardial tissue during septic shock without myocardial infarction in non cardiac critically ill patients.

The following report concerned a 47 year old Caucasian diabetic patient. Routine HPLC of HbA1c (Variant II Biorad Laboratories - hemoglobin A1c program) resulted only in the evidence of HbF (1%) and increase in HbA1c (10%). Considering the presence of HbF a standard agarose gel electrophoresis of patient's hemoglobin was performed and revealed the presence of Hb Athens-Georgia. Consequently the occurrence of HbF during determination of HbA1c by HPLC should lead to perform a standard hemoglobin electrophoresis in order to explore an hidden, unsuspected and clinically silent occurrence of rare Hb variant or additional unsuspected increase in HbA2.

We studied the pattern of acute-phase proteins (orosomucoid, C-reactive protein, and haptoglobin) in hepatocellular deficiency due to chronic alcohol consumption, characterized by a decrease in serum transferrin concentration. We found that their patterns could vary independently of hepatocellular deficiency, but depend on the progression of hepatic disease. The most useful protein for discriminating the stage of inflammatory reaction is orosomucoid. In moderate hepatocellular deficiency, acute-phase proteins are increased independently of the decrease in transferrin, whereas in severe hepatocellular deficiency the acute-phase proteins are also decreased. Thus, it is possible to distinguish the two stages of hepatocellular deficiency by following changes in the concentration of orosomucoid.

The female inbred strains of C3H/HeJ (C3H) and C57BL/6J mice (B6), having high and low femoral peak bone mass, respectively, were proposed as models for studying the genetic regulation of bone mass. Here, we compared the known bone phenotype, in 4.5-month-old C3H versus B6 mice, in both genders. Femoral bone mineral content, trabecular bone mass, and thickness at the distal metaphysis were higher in C3H mice. In the long bones, deoxypyridinoline content was lower and pyridinoline/deoxypyridinoline ratios were greater in C3H. Intrafibrillar collagen packing is different not only within strains but also within sexes. Bone resorption activity, evaluated by urinary pyridinium crosslinks and active resorption surfaces in the femoral metaphysis, was lower in C3H. Bone formation activity, evaluated by serum osteocalcin and alkaline phosphatase (ALP) levels, as well as histomorphometric indices of bone formation in the femoral metaphysis and the cortical tibia, was lower in C3H. Conversely, the ALP- and Von Kossa-positive colony-forming units were more numerous in bone marrow cell cultures originating from male C3H. In both strains, resorption and formation activities were lower in males than in females. In C3H, males had lower bone mass than females whereas the opposite was seen in B6. In conclusion, we found that the lower cellular activities in C3H were associated with high cancellous bone mass and pyridinium crosslink levels, which might account for the more mineralized bone in C3H mice compared to that in B6 mice.

The course of glycation of calf skin fibrous type I collagen was monitored in vitro under physiological conditions during an 8-week incubation period in order to take into account the long half-life of this protein. The formation of glycated compounds was measured by determining fructosamine, pentosidine, and carboxymethyllysine content. The incubation conditions were as physiological as possible in sterile saline phosphate buffer, except glucose concentration. With incubation medium containing 200 mmol glucose, fibrous collagen underwent solubilization; in addition an increase in fructosamine, pentosidine, and carboxymethyllysine content in both solubilized and remaining insoluble collagen was noticed. There was a spontaneous, restricted, and time-dependent native glycated state of collagen; high concentration glucose enhanced the formation of glycated compounds and induced changes in solubility and glycoxidated products. The production of pentosidine during incubation without glucose should be considered as an event resulting from the initial fructosamine. Whereas the production of carboxymethyllysine during long-term incubation with glucose provided indirect proof of an additional oxidative process after early glycated product formation. These experimental observations provide insight into the in vivo context of advanced glycation end product formation in chronic hyperglycemia and aging.

The deleterious effects of glycoxidation are dependent on the half-life of proteins. Collagen, the main component of extracellular matrices, is a long live protein and thus may be sensitive to the glycoxidation process. We incubated calf skin fibrous type I collagen in PBS at 37°C with glucose. The fibrous type I collagen was solubilized and an increase in the amount of advanced glycation end products of the solubilized fraction was observed. As there was no bacterial contamination and no proteolytic activities in the incubation medium, the solubilization of fibrous type I collagen is probably due to the speculative production of the free radicals in our experimental conditions. To test this hypothesis, fibrous type I collagen was incubated in PBS with AAPH (2,2′azo-bis 2-aminodinopropane) a free radicals generator. AAPH induced a dramatic and dose dependent solubilization of fibrous type I collagen.

We have developed an in vitro mechanical stretching model of osteoblastic cells cultured on metallic biomaterials in order to study the effects of mechanical strain on osteointegration of orthopaedic implants. Titanium alloy discs coated with alumina or hydroxyapatite were used as substrates. Three Dynacell® devices were especially designed to apply cyclic strains on rigid biomaterials. The regimen (600με strains, 0.25Hz) was defined on the basis of physiological data and estimated deformation on hip stem prostheses. The performances of these apparatus were reproducible and provided controlled deformations. Human osteosarcoma cell line MG-63, human osteoblasts obtained from primary cultures and ROS 17/2.8 rat osteosarcoma cells were used as cell models. Cell behaviour was assessed in terms of growth and alkaline phosphatase (ALP) activity by in situ assays for two regimens: 15-min deformations repeated three times a day to mimic rehabilitation exercises and 24-h continuous deformations. We demonstrated that continuous deformation did not affect the growth and ALP activity of MG-63 cells, in contrast with sequential deformations which had no effect on cell number, but which stimulated ALP activity after 5 days of stretching. This sequential regimen can also modify the behaviour of human bone-derived cells resulting in increased proliferation after 5 days and stimulation of ALP activity after 15 days. ROS 17/2.8 rat osteosarcoma cells submitted to sequential deformations responded faster than other cell lines by increasing their ALP activity only after 1 day of stretching. Like MG-63 cells, proliferation of the ROS 17/2.8 rat osteosarcoma cell line was not affected by sequential deformations. This study suggests that short, repeated deformations defined to mimic rehabilitation exercises recommended after prostheses implantation are more likely to exert beneficial effects on implanted bone than continuous strains.

BACKGROUND:Loss of collagen and elastin is observed in the elderly. In geriatric inpatients, chronic protein malnutrition could induce susceptibility to additional morbidity such as pressure sores.OBJECTIVE:The purpose of this study was to explore the relationship between nutritional and inflammatory status and the production of tissue inhibitor of matrix metalloproteinase 1 (TIMP-1).METHODS:Chronically ill elderly inpatients, without or with pressure sores, were enrolled. Nutritional protein markers, acute phase reactants, and TIMP-1 were determined, and changes in these biological parameters were compared.RESULTS:Chronic inflammatory process and protein malnutrition were observed in all enrolled patients. The severity of these two pathophysiological processes was independent of the occurrence of pressure sores. The serum prealbumin and albumin levels were lower in patients with pressure sores than in those without. In addition, the general increase in the TIMP-1 level was independent of the occurrence of pressure sores. The TIMP-1 level was mainly related to the prognostic inflammatory and nutritional index.CONCLUSIONS:The general increase in acute-phase reactants observed in the elderly could be related to the intercurrent diseases. The generally low serum albumin level, lowest in patients with pressure sores, may be considered evidence of protein malnutrition and hypercatabolism. Regarding the morbidity, the increase in TIMP-1 levels could be explained as an adaptive process to prevent intrinsic protein expenditure of extracellular matrices.

La cholécystokinine fait l'objet depuis plusieurs années de nombreuses publications scientifiques car il s'agit d'un ensemble de peptides régulateurs dont l'action se situe à plusieurs niveaux de l'organisme, avec une fonction importante dans le domaine nutritionnel et un rôle dans la régulation de la prise alimentaire par le déclenchement de la sensation de satiété. Cet article passe en revue de façon détaillée la biosynthèse et la structure de cette famille de peptides, les récepteurs de la cholécystokinine ainsi que les aspects digestifs, nutritionnels, métaboliques et de contrôle de la satiété de ce peptide régulateur. Les aspects psychocomportementaux de la CCK ne seront pas ou peu abordés ici.

Pentosidine, an advanced glycation end product (AGE), was assayed by HPLC in serum proteins from patients with Alzheimer type dementia (AD), patients with diabetes mellitus (D), and healthy (C) age-matched old subjects (mean age from each group = 84 years). Serum pentosidine was significantly different between the three groups despite similar renal function (serum creatinine < 160 micromol/L). In all groups of patients, pentosidine was independent of glycated hemoglobin (HbA1C) and the early glycation marker fructosamine and appeared to be an independent marker, mainly bound to serum albumin. Pentosidine could be an important factor useful for the diagnosis of Alzheimer's disease.

The reaction of fibroblasts to mechanical forces generated by fibroblasts themselves in anchored collagen lattices was studied. Fibroblasts were cast in collagen gels. Free retracted gels (RG) were compared with stressed gels (SG) in 3-day and 14-day experiments. As previously described, SG showed an increase in protein, mainly collagen, biosynthesis. Matrix metalloproteinases (pro-MMP-2 and MMP-2) were studied by zymography. Certain cell membrane components, integrin alpha(2) and phosphatidylserine, were studied by flow cytometry with antibodies against the integrin alpha(2) subunit and with annexin V binding. Mechanical stress stimulated production of pro-MMP-2 both in the short-term (3-day) and the longer term (14-day) cultures. However, the pro-enzyme was not more activated and there was no difference in the amount of MMP-2 between RG and SG. There was only an increase with time under both conditions. The stressed fibroblasts reacted early with an increase in the integrin alpha(2) subunit, but the stimulated cells disappeared from the 14-day cultures. The number of cells measured in terms of the amount of DNA decreased between day 3 and day 14, mainly in the SG due to cytolysis. This cell stress was related to an alteration in the plasma membrane detected by the annexin V marker.

Ascorbic acid (vitamin C) is a primary antioxidant for cells. But, ascorbic acid added to culture medium is not readily available to cells in culture, because it is unstable in aqueous media. We determined the conditions required to obtain and maintain a constant concentration of ascorbate in the culture medium using ascorbate and ascorbate-phosphate. The study was carried out with human fibroblasts and the amounts of ascorbate in the culture medium were determined by high performance liquid chromatography. A mixture of 0.25 mmol/L ascorbate and 0.45 mmol/L ascorbate-phosphate provided a constant concentration of ascorbate in the culture medium. This constant ascorbate concentration proved to be nontoxic for cells and stimulated cell growth in the short term and long term.

Ascorbate and tocopherol are important antioxidants that protect cells against oxidative stress. The interaction of ascorbate and α-tocopherol in cells is difficult to detect as both ascorbate and α-tocopherol are unstable in vitro in a biological medium. We examined the interactions between human dermal fibroblasts, ascorbate and α-tocopherol to determine the effects of the vitamins on growth and cell viability. The interaction of ascorbate and α-tocopherol was studied in a fibroblast culture medium during 48h. Ascorbate and α-tocopherol were detected by fluorimetry after high-performance liquid chromatography (HPLC). Cell growth and cell viability were studied by cell numeration after trypan blue staining. The ascorbate concentration fell in presence of α-tocopherol in cell culture medium under all experimental conditions, with or without cells. Ascorbate partly protected α-tocopherol but only in presence of cells. Cell viability was preserved by α-tocopherol whereas ascorbate enhanced fibroblast growth. The synergy between ascorbate and α-tocopherol corresponds to a consumption of ascorbate which spares α-tocopherol but only in presence of cells.

A new experimental method has been used to study the behaviour of human osteoblasts cultured on bioceramics subjected to mechanical strains. The ceramics were alumina, hydroxyapatite (HA) and a duplex system composed of hydroxyapatite-covered alumina. The system applied 400 microdeformations for a 6-h period with a cycle frequency of 0.5 Hz to osteoblasts growing on ceramic-covered disks. The effects of strains on short-term cell viability, cell growth, alkaline phosphatase (ALP) activity, and collagen biosynthesis were assessed. When possible, the parameters (lactate dehydrogenase) were studied along the experiment in samples of the culture medium, in the other cases by comparison of stretched and unstretched cultures on the same ceramics with the same cell line. In relationship with the coating, mechanical strains resulted in a decrease in DNA corresponding to cell number, an LDH release during straining, an unchanged (alumina) or decreased (HA and duplex) ALP activity, a decrease (HA and duplex) of collagen and total protein synthesis or an increase of it (alumina). The stress-producing device and its associated protocol are shown to be suitable for investigating the behaviour of cells, cultured on biomaterials subjected to mechanical strain.

Adult human osteoblastic cells were grown in a native type I collagen gel. Proliferation and viability analyses showed that cells rapidly stopped dividing and became blocked in the G0G1 phase (91% on day 13). Carboxyfluorescein diacetate cell staining and flow cytometry showed that osteoblasts were viable for the first 16 days and then viability decreased (58% viable cells on day 22). Osteoblasts were able to retract the matrix. Betaglycerophosphate (βGP) stimulated the deposition of mineral particles in the collagen network, and electron probe microanalysis showed that they were principally calcium and phosphorus, with a Ca/P ratio of about 1.7. Various times of βGP supply were tested. We compared 10 mM βGP added only once at day 0, or continuously from day 0, day 8, or day 21. Mineralization was observed in conditions where βGP was added at day 0. Furthermore, 10 mM βGP added once during gel preparation was sufficient to induce mineralization with mineral accumulation up to day 15 whereas the speed of the gel contraction decreased. In every condition, cultures expressed high alkaline phosphatase (ALP) levels as early as day 3, which decreased afterwards. These kinetics might explain why the other conditions did not prove favorable to the mineralization process. The model was used to study the influence of blocking gel retraction. Blocking retraction delayed the ALP activity decrease, but had no effect on mineralization. In conclusion, human adult osteoblasts cultured in native collagen gel stopped proliferation and underwent mineralization very early. This model should be used to investigate the influence of effectors on the early stages of culture.

Glycyl-L-proline (gly-pro) is an end product of collagen metabolism that is further cleaved by prolidase (EC 3.4.13.9); the resulting proline molecules are recycled into collagen or other proteins. We postulated a relationship between defective gly-pro hydrolysis, increased collagen degradation and skin destruction. This relationship was tested using HPLC to measure the gly-pro in urine. 24 hour urine samples were collected from 27 old people (86 ± 6 years old), of whom 15 were suffering from skin pressure sores of the sacrum or calcaneus. The urine from patients with pressure sores contained significantly more gly-pro than the urine from the control. A cut-off at 7μmol/ mmol creatinine gave the test a positive predictive value of 70%. Collagen breakdown was also increased as indicated by the increase of hydroxyproline (hyp) in the urine. But this breakdown seemed to stop at the gly-pro step.