Biomedical subjects

E J Menzel

Publications and source records attributed to E J Menzel.

At least 19 recordsLinked to original sources

PubMed2005

Troglitazone inhibits long-term glycation and oxidation of low-density lipoprotein.

Troglitazone (T) is a member of a new class of antidiabetic drugs termed thiazolidinediones (TZDs), which has previously been used as an anti-diabetic agent. In this study we investigated the influence of T, a ligand for PPAR-gamma receptor, on copper-catalyzed or cell-mediated oxidation of native, glycated, and glycoxidated low-density lipoprotein (LDL). A dose-dependent inhibition of copper-mediated low-density lipoprotein-oxidation, as monitored by the formation of oxidation-specific fluorescence, was observed for both native and glycated low-density lipoprotein. At the concentration of 20 microg/mL the inhibition amounted from 14.7% to 64.7% by all low-density lipoprotein forms. For glycated low-density lipoprotein we obtained the highest oxidation rate, but the most pronounced inhibition by T was found for glycoxidated low-density lipoprotein (goLDL). Inhibitory effects of T were also investigated by measurement of relative electrophoretic mobility (REM) in the concentration range of 0 to 20 microg/mL. The inhibition of 4h oxidation of native low-density lipoprotein was found in the entire concentration range, but significance was seen at 10 microg/mL. The long-term glycation and glycoxidation of low-density lipoprotein as measured by 5-hydroxymethyl-2-furaldehyde (5-HMF) formation and binding of fructosamine was found to be inhibited by T. In endothelial cell-mediated oxidation of low-density lipoprotein cytotoxicity of T in the concentration range of 0 to 160 microg/mL during 2 to 24 h oxidation was investigated. In the non-cytotoxic concentration range of 5 to 20 microg/mL, a significantly reduced liberation of isoprostane 8-epi-PGF2alpha during 24 h cell-mediated oxidation of low-density lipoprotein and its modifications was found. This inhibitory action of T was most significant in the case of goLDL and amounted to approximately 20% to 60% inhibition at 5 to 20 microg/mL T, respectively. In the concentration range of 40 to 160 microg/mL, however, T showed an increasing cytotoxic action, as evidenced by loss of cell adhesion, loss of cellular protein, morphological changes, and cell disintegration as well as by strongly enhanced troglitazone-mediated isoprostane 8-IP liberation (fivefold to sixfold). T may be used as a model to explore the thiazolidinediones' mechanism on oxidation in a more general aspect for treatment for T2DM, because T is not clinically available.

Adult↗

PubMed2001

Calcium antagonists as inhibitors of in vitro low density lipoprotein oxidation and glycation.

The peroxidation step in lipid transformation is considered to be essential in the pathogenesis of atherosclerosis. Calcium antagonists (CA) appear to have antioxidant effects in addition to their potent vasorelaxant properties. In the present study, we compared the antioxidative efficacy of CA (amlodipine, lacidipine, nifedipine, isradipine, diltiazem, and semotiadil) in the copper-catalysed oxidation of low-density lipoprotein (LDL) with that of glycated(g)/glycoxidated(go) LDL. This issue is of great importance when considering the potential therapeutic use of antioxidant drugs in diabetes-associated vasculopathy. Oxidation of native LDL was inhibited most efficiently (>90%) by lacidipine and semotiadil in the concentration range 10(-4)-10(-3) M. We found, however, a dramatic decrease in antioxidant activity towards g/goLDL as compared to native LDL in all the CA tested. Only lacidipine significantly inhibited copper-mediated oxidation of g/goLDL in the whole concentration range tested (10(-5) M-10(-3) M). This probably resulted from the increased auto-oxidative potential introduced by early and advanced glycation end products (AGE) into the g/goLDL. We noted that coincubation of LDL with 10(-3) M CA and 0.5 M glucose under oxidative/non-oxidative conditions partially or fully restored the antioxidant capacity of the different CA to inhibit the subsequent copper-catalysed oxidation of the modified LDL. This is a clear indication that CA inhibit glycative or glycoxidative LDL changes during the preceding long-term glycation period. The notion that both oxidative changes and long-term glycation effects were reduced by CA was corroborated by fluorescence analysis, AGE-ELISA, quantitation of lipid peroxidation, and thiobarbituric acid reactive substance (TBARS) measurement of long-term g/goLDL. The strongest antioxidative effects during long-term glycation of LDL were seen with isradipine, lacidipine, nifedipine, and semotiadil. Diltiazem was the only CA that could not prevent TBARS formation in LDL during the long-term glycation period. In contrast, Amadori product formation, as measured by the generation of fructosamines, was not significantly reduced by any CA tested. Thus CA, like other antioxidants, significantly retard AGE formation, while initial glycation reactions, such as Amadori product formation, are only weakly inhibited.

Antioxidants↗

PubMed2000

Differential scanning calorimetry, biochemical, and biomechanical analysis of human skin from individuals with diabetes mellitus.

The aim of this work was to compare biochemical, two-dimensional biomechanical and calorimetric parameters of diabetic skin vs. control skin. Skin specimens taken from the palms and backs of the hands of aged persons with non-insulin-dependent diabetes mellitus (NIDDM) and of controls (CO) were compared (age range 68-85 years). Only skin specimens from individuals with diabetes mellitus (DM) showed an increased fluorescence specific for the formation of advanced glycation end-products (AGEs) and the presence of tissue AGEs, such as N(e)-(Carboxymethyl)lysine (CML). Differential scanning calorimetry (DSC) revealed an elevation of the heat flow per unit mass during collagen denaturation in diabetic skin samples. However, the temperatures of the heat flow maximum and the onset of the phase transformation were not uniformly altered. Young's moduli were found to be increased in diabetic skin and correlated with AGE-fluorescence and tissue AGEs. The ratio between the Young's moduli, which defines a measure for the degree of anisotropy, was higher for dorsal skins from hands. In dorsal skin specimens from diabetic subjects the degree of anisotropy was more pronounced than in healthy controls. In general, neither of the measured parameters showed any correlation with age. However, E(1) moduli were clearly associated with the duration of diabetes.

Aged↗

PubMed2000

Troglitazone-binding to LDL and its glycated modifications: its role in cell-catalysed and Cu-mediated LDL-oxidation.

Troglitazone (T), an anti-diabetic drug improving insulin resistance, was studied as to its inhibition of copper ion-catalysed oxidation of native, glycated and glycoxidated low-density lipoprotein (LDL). A dose-dependent inhibition was noted in the concentration range 40-160 microg/ml. An almost complete inhibition of oxidation (2-8 h), as monitored by the formation of thiobarbituric acid-reactive substances, was observed for both native and glycated LDL at a concentration of 160 microg/ml T, while the maximal inhibition for glycoxidated LDL amounted only to 60% at this concentration of the drug. This is reflected by differences in the affinity of the drug for the different types of LDL modification: While the binding of T both to native or glycated LDL increased linearly with increasing T concentration and was not saturable in the concentration range tested (0-160 microg/ml), binding of the drug to glycoxidated LDL was already nearly saturated at 10 microg/ml. The nearly complete inhibitory action of T towards oxidation of native and glycated LDL was lost, however, upon increasing the total oxidation time to 24 h. In human umbilical vein endothelial cell-mediated oxidation of LDL, T at a concentration of 20 microg/ml significantly reduced formation of oxidation-dependent fluorescent chromophores and liberation of 8-epi-PGF2alpha. In contrast, generation of thiobarbituric acid-reactive substances was not significantly inhibited. As opposed to copper-mediated LDL-oxidation, different binding of T to LDL-modifications does not govern inhibition of human umbilical vein endothelial cell-mediated LDL-oxidation.

Adult↗

PubMed2000

Alterations of biochemical and two-dimensional biomechanical properties of human skin in diabetes mellitus as compared to effects of in vitro non-enzymatic glycation.

OBJECTIVE: The aim of this study was to evaluate whether multiaxial analysis of diabetes-specific biomechanical changes generated in vitro by non-enzymatic glycation of human skin samples from healthy subjects reflect the changes seen in skin from subjects with diabetes mellitus. DESIGN: Descriptive study. BACKGROUND: Non-enzymatic glycation of skin in vitro causes an increased stiffness comparable to in vivo changes seen in diabetic patients. These changes are probably due to increased cross-linking of collagen molecules. METHODS: Skin specimens from 7 subjects with diabetes mellitus and 7 controls (age range: 74-90) were analyzed for biomechanical changes using a multiaxial tensile testing device. Control skins from healthy individuals in the age range of 50-65 yr were artificially glycated. One part of these samples was coincubated with the glycation inhibitor aminoguanidine. Glycation of tissues was determined by measuring fluorescence of solubilized samples. Multiaxial biomechanical analysis allows the determination of maximum (a(I)) and minimum elastic modulus (a(II)). These parameters describe the amplitude of the elastic stress response, which is exponentially related to strain. RESULTS: Principal stresses, both maximum and minimum, were increased in skins from diabetic subjects as compared to controls. The increases of the principal stresses were comparable to those obtained by in vitro glycation of normal skins. CONCLUSION: These results, which can be detected unequivocally with the multiaxial test mode, show that our in vitro model closely reflects changes in skin samples from individuals with diabetes mellitus. Aminoguanidine partially inhibited these as well as biochemical changes. RELEVANCE: Multiaxial testing of in vitro glycated skin samples can be used as a model for in vivo changes caused by diabetes mellitus. In addition, therapeutical effects of aminoguanidine, an inhibitor of non-enzymatic glycation, can be monitored in this model.

Aged↗

PubMed2000

The effects of glycation/glycoxidation on the liberation of 8-epi-PGF2alpha from low density lipoprotein during its in vitro oxidation.

In individuals suffering from diabetes mellitus, low-density lipoprotein (LDL) can undergo glycoxidation, both the surface protein and the unsaturated fatty acids in the particle core experiencing oxidative damage. In these patients, plasma levels of glycated(g)/glycoxidated(go) low-density lipoprotein and of 8-epi-PGF2alpha (8-IP), a relatively stable peroxidation product of arachidonic acid, are increased. This study reports on the modified oxidation of gLDL and goLDL by human umbilical vein endothelial cells or by copper cations in the absence of cells. In both systems, glycated LDL was found to be more easily oxidized than either nLDL or goLDL. In addition, liberation of 8-IP from glycoxidated LDL is significantly reduced, because a large amount of 8-IP is already formed during long-term glycation of LDL in the absence of metal chelators, i.e. during glycoxidation. From these in vitro results we conclude, that 1) gLDL is more prone to cell-mediated oxidation than native or goLDL and 2) the increased in vivo plasma levels of 8-IP observed in diabetes mellitus could be due to prolonged LDL-glycoxidation liberating continuously significant amounts of 8-IP.

Adult↗

PubMed2000

Changes of biochemical and biomechanical properties in Dupuytren disease.

BACKGROUND: The major biochemical characteristic of Dupuytren disease is the progressive and irreversible deposition of excess fibrous collagen characterized by an enhanced type III collagen proportion. OBJECTIVE: To investigate the influence of changes of the collagen spectrum on the biophysical properties of the palmar aponeurosis. DESIGN: Variably affected palmar regions from 30 individuals with Dupuytren disease were classified according to histologic test results and clinical stage. Biochemical, biomechanical, and thermal contracture studies were performed. RESULTS: The relative type III collagen content increased with increasing tissue involvement and was found to correlate with calorimetric and biomechanical properties with the exception of the Young modulus. In experiments on the thermal isometric contracture, the collagen denaturation temperature decreased with increasing type III collagen content, ie, increasing involvement. To study the dependence of biophysical properties from the collagen type distribution independent of structural changes, as seen in Dupuytren disease, we investigated rat skins from animals of an age range characterized by dramatic changes in type III collagen content (0-18 months). Biomechanical data also correlated significantly with type III collagen content in rat skin with the exception of the time constant of stress relaxation. CONCLUSION: In light of these results, we suggest that structural changes, such as reduced collagen fibril diameters, associated with alterations in the type III collagen proportion may influence biophysical properties of connective tissues in the involved palmar aponeurosis in addition to alterations of the cross-linking pattern.

Aging↗

PubMed2000

[Polymeric chondroitin sulfate vs. monomeric glucosamine for the treatment of osteoarthritis].

Two potentially chondro-protective substances are compared with each other: polymeric chondroitin sulfate and monomeric glucosamine. Chondroitin-sulfate is characterized by tropism for cartilage, i.e. it is preferentially incorporated into cartilagenous tissue. This quality could be of interest in the SPECT-analysis of osteoarthritic knee joints. In contrast to the classic agents Ibuprofen and Indomethacin, the antiinflammatory action of chondroitin-sulfate is less prominent, but the drug does not show any toxic side effects. Glucosamine on the other hand is a multifunctional precursor of the glycosaminoglycan synthesis in general. It lacks the cartilage tropism of chondroitin-sulfate. Into what sort of a glycosaminoglycan glucosamine is eventually integrated, depends therefore on the nature of the cells participating in the biosynthesis.

Aggrecans↗

PubMed1999

Comparison of palmar aponeuroses from individuals with diabetes mellitus and Dupuytren's contracture.

It is well known that Dupuytren's contracture is often associated with diabetes mellitus. Palmar fascia from individuals with diabetes mellitus and/or Dupuytren's contracture as well as controls were subjected to differential scanning calorimetry, biomechanical and biochemical analysis. The collagen denaturation temperature of the palmar aponeurosis from individuals with diabetes mellitus in the presence (71.0 degrees C) or absence of Dupuytren's contracture (70. 6 degrees C) was increased as compared with controls (68.5 degrees C), while this parameter was significantly reduced (about 3.5 degrees C) in contracture bands of Dupuytren's contracture. Stress relaxation experiments revealed that the viscous fraction was slightly reduced in diabetes mellitus (6.5%) vs. controls (8.3%), whereas in Dupuytren's contracture, irrespective of additional diabetes mellitus, a pronounced increase of this parameter was seen (36.5% vs. 24.5%) in the presence of diabetes mellitus. The time constants were significantly elevated by both disorders, this increase being more pronounced in Dupuytren's contracture. Taken together, these changes can be explained by increased cross-linking in diabetes mellitus, while in Dupuytren's contracture other structural changes, such as increased collagen type III content and loss of fascicular organization, play an additional role besides the finding of reduced cross linking.

Aged↗

PubMed1999

Inhibition of cytokine production and adhesion molecule expression by ibuprofen is without effect on transendothelial migration of monocytes.

The present study focusses on the effects of ibuprofen and its enantiomers on cytokine production by peripheral blood monocytes and endothelial cells as well as on the potential modulation of ADM-expression by human umbilical vein endothelial cells and the concomitant effects on monocyte transendothelial migration as measured by a cell migration assay system. This consists of an endothelial cell monolayer on a solid collagen substrate, i.e. an artificial vessel wall construct. We observed a significant inhibition by 100 microg/ml ibuprofen of VCAM-1 expression by endothelial cells while ELAM-1 and ICAM-1 expression was not influenced. However, we could not see any concomitant inhibitory effects on the spontaneous migration of monocytes after preincubating the endothelial cell monolayer with ibuprofen up to concentrations of 100 microg/ml and activating with suboptimal and optimal concentrations of TNF-alpha. Our monocyte transendothelial migration system reflects very sensitively endothelial cell-activation even by very low TNF-alpha concentrations. (S)- and (R)-ibuprofen were equal in their inhibitory/activating effects on cytokine production, with the exception of stronger IL-8 induction in endothelial cells by (R)-ibuprofen as compared to its chiral analogue.

Cell Adhesion Molecules↗

PubMed1999

Novel C5-dependent mechanism of neutrophil stimulation by bioincompatible dialyzer membranes.

The objective of the study was to evaluate the contribution of reactive oxygen intermediate formation for receptor modulation on neutrophils by the cellulosic dialyzer membrane cuprophan (CU). In patients dialyzed with CU, CD11b and CD66b upregulation on neutrophils (by 104.3 +/- 37.9% and 85.7 +/- 31.1%, respectively), and a downregulation of L-selectin (by 44.9 +/- 26.9%) was seen, whereas expression of CD11a remained unaltered. Hemodialysis with polysulfone did not bring about major changes in surface receptor expression. In vitro incubation of isolated neutrophils in the presence of serum with hollow fibers of CU or polysulfone showed similar results: Only CU resulted in upregulation of CD11b and CD66b expression (by 65.5 +/- 18.7% and 60.1 +/- 24%) and a decrease in CD62L expression (by 60.6 +/- 18.2%). In contrast to receptor alterations, generation of reactive oxygen intermediate by CU occurred in the absence of serum. Inhibition experiments with soluble complement receptor 1, which produced only partial inhibition of receptor up-/down-regulation, indicated the existence of also other than alternate complement-dependent mechanisms for neutrophil activation. By using C5-depleted serum instead of normal human serum, up-/down-regulation of CD11b, CD62L, and CD66b by CU was dramatically reduced, whereas C3-depleted serum did not produce that effect. C5-deficient serum repleted with purified C5, as well as purified C5 alone, was able to induce receptor modulation by CU comparable to normal human serum. L-Methionine, a specific inhibitor for the oxidative activation of C5, blocked the modulatory effect of CU in assays with purified C5 as well as with serum. As a result, in addition to the alternative pathway of complement, a C5-dependent mechanism probably activated by neutrophil-derived reactive oxygen intermediate leads to receptor modulation and subsequent generation of the well known side effects of bioincompatible dialyzer membranes.

Adult↗

PubMed1998

Hyaluronidase and its substrate hyaluronan: biochemistry, biological activities and therapeutic uses.

This is an overview of the biochemistry, biological function and therapeutic uses of hyaluronidase and its substrate, hyaluronate. We focus on the role of hyaluronate and its receptor CD44 in cell-cell and cell-matrix adhesion and cell activation as well as on the putative role of hyaluronate and hyaluronidase in morphogenesis. Variants of CD44 and their putative role in tumor metastasis are also included. Other topics that are discussed are the chemical and enzymatic nature of hyaluronidase, i.e. the mode of substrate degradation, pharmacodynamical and pharmacokinetic aspects of this enzyme and its role as spreading factor. Purification methods, possible contaminations and techniques of activity determinations are mentioned as well as the physiological role of hyaluronidase and tumor-associated alterations in serum and tissue enzyme levels. As far as therapeutic applications are concerned, we discuss uses of hyaluronidase in ophthalmology and regional anesthesia as well as pain management in osteoarthritis using hyaluronate.

Cell Adhesion↗

PubMed1998

Autoantibodies to oxidised low density lipoproteins in IDDM are inversely related to metabolic control and microvascular complications.

Diabetes mellitus is associated with an increased risk of atherosclerosis. The oxidation of low-density lipoproteins (LDL) is considered a key event in the initiation of atherosclerosis. To investigate LDL oxidation in vivo we measured autoantibodies to oxidised LDL (oxLDL) in 94 patients with insulin-dependent diabetes mellitus (IDDM), compared to 27 age-matched, healthy control subjects. Patients and control subjects were screened for autoantibodies using a solid phase ELISA, comparing the binding to oxLDL with that to native LDL (nLDL). In patients with IDDM the oxLDL/nLDL antibody ratio was significantly higher than in control subjects (means+/-SEM: 2.24+/-0.26 vs 1.17+/-0.17, p < 0.03). Antibody-negative patients had a longer diabetes duration (13.5+/-1.3 vs 9.1+/-1.1 years, p < 0.01) and higher actual and mean HbA1c levels compared to antibody-positive patients (8.8+/-0.2 vs 7.9+/-0.2%, p < 0.005 and 8.3+/-0.2 vs 7.7+/-0.2%, p < 0.03; respectively). In patients with a high microangiopathy score, the antibody ratio was lower than in patients without complications (1.04+/-0.10 vs 2.40+/-0.29, p < 0.01). OxLDL specific immune complexes were found exclusively in antibody-negative as compared to antibody-positive patients (18.3 vs 0 %; p < 0.01). Our data demonstrate an inverse relationship between free oxLDL antibodies and the severity of the disease. This apparent paradox can be explained in part by our demonstration of oxLDL immune complexes, masking free antibodies.

Adult↗

PubMed1998

Differential expression of receptors for advanced glycation end products on monocytes in patients with IDDM.

Accelerated modification of proteins by glucose terminating in the formation of advanced glycation endproducts (AGEs) is one of the main pathogenetic mechanisms of diabetes-associated complications. One pathway by which AGEs may exert their effects is by interaction with specific receptors initially identified on macrophages, monocytes and endothelial cells. As AGE-induced autocrine upregulation of AGE receptors has been observed in vitro, we hypothesized that AGE-binding might be enhanced in diabetic patients to compensate for the elevated levels of circulating AGEs. We therefore examined the expression of AGE-binding sites on peripheral monocytes, serum levels of AGEs and AGE-induced cytokine production in patients with insulin-dependent diabetes mellitus (IDDM) compared to age-matched, healthy control subjects. In patients, AGE-binding capacity was significantly increased and there was only one class of binding sites, as revealed by Scatchard analysis (1.8 x 10(5) vs 1.4 x 10(5) binding sites per cell). Affinity of binding was, however, similar (Ka 1.5 x 10(6) vs 1.4 x 10(6) mol(-1)). Saturation of binding was reached at 2.0-3.0 micromol/l with AGE-bovine serum albumin (BSA) as ligand. In contrast, cytometry using fluorescein isothiocyanate-labelled AGE-proteins showed no saturability and reversibility of AGE-binding up to 80 micromol/l, indicating non-specific binding in this concentration range. Again, this non-specific binding was significantly higher in IDDM patients. In addition, we found much higher levels of circulating AGEs in patients as compared to controls and studied possible functional consequences of increased AGE binding in vitro, monocyte stimulation by AGEs triggering cytokine release to a similar extent in patients and controls, i.e. independently of the AGE-binding capacity. Our finding of an enhanced overall AGE-binding capacity of peripheral monocytes in IDDM could be instrumental in limiting the plasma concentration of AGEs, the non-specific binding coming into play after saturation of specific binding sites by higher plasma AGE-levels. Both binding strategies may act in concert as "damage limitation mechanisms" in the development of AGE-dependent diabetic complications.

Adult↗

PubMed1998

Two-dimensional stress-relaxation behavior of human skin as influenced by non-enzymatic glycation and the inhibitory agent aminoguanidine.

In order to simulate the in vivo stress alterations of diabetic skin in an in vitro model, we examined the viscoelastic properties of long-term glycated human skin samples. Since skin is subjected to biaxial tension, we used two-dimensional multiaxial testing which better reflects the in vivo situation than the uniaxial testing mode. For native skin samples from the abdominal region we found a direction-dependent elastic stress strain behavior. The viscous stress component was separated from the elastic stress component by relaxation tests at consecutive incremental steps of radial strains. We hypothesize that glycation-induced changes in the tissue stiffness are generated in a direction-dependent mode. A marked increase of the direction-dependent stiffness was found upon long-term incubation with glucose-6-phosphate. This increase was statistically significant for the maximum principal elastic stress component which was highly correlated with the degree of non-enzymatic collagen modification. The viscous fractions obtained from two-dimensional relaxation tests at consecutive radial strains were inversely correlated with non-enzymatic modification. Only at 30% radial strain a significant decrease of the viscous fraction engendered by glucose-6-phosphate was observed together with a direction-dependent significant increase of the expectation value of the time constant. The biomechanical and biochemical effects of long-term glycation could be partially reversed by aminoguanidine, a potential therapeutic agent for patients with diabetes mellitus. Our findings suggest that additional cross-links generated by long-term glycation cause two-dimensional biomechanical alterations in human skin, which can be unequivocally detected by multiaxial testing.

Aged↗

PubMed1998

Comparison of normal and in vitro aging by non-enzymatic glycation as verified by differential scanning calorimetry.

The biomechanical parameters of rat tail tendons (RTTs) from 35-, 64-, 180- and 900-day-old animals, corresponding to the early maturation phase, the mature and the senescent state were determined. The increase of maximum stiffness, ultimate stress and the elastic fraction of stress was most pronounced in the maturation phase. Differential scanning calorimetry (DSC) experiments were performed showing an almost linear increase of the collagen denaturation temperature in the age range 35-139 days. After 14 days incubation in glucose, we observed a marked increase of the biomechanical parameters in the young, an increase of maximum stiffness in mature, and only slight alterations of the biomechanical behavior in senescent RTTs. Both glucose incorporation and formation of advanced glycation end products were most prominent in 35-day-old RTTs. These biochemical findings were in excellent agreement with the enhancement of the collagen denaturation temperature after the incubation phase. Results suggest that the validity of the term 'accelerated aging' depends on the experimental approach, i.e. biomechanical tests, thermal isometric contraction or DSC.

Aging↗

PubMed1997

The role of oxidative stress in the long-term glycation of LDL.

Advanced glycation is a major pathway for the posttranslational modification of plasma and tissue proteins. The initiating reaction is the nonenzymatic addition of sugars such as glucose to the primary amino groups of proteins, i.e., mainly to lysine residues. These "early" Schiff base and Amadori products then undergo a series of inter- and intramolecular rearrangements to produce the "late" products termed advanced glycation end products (AGEs). Incubation of LDL with glucose or glucose-6-phosphate produces AGE moieties on both the lipid and apolipoprotein B components. In addition, we tried to generate AGE-LDL by reaction with AGE-peptides (< 10 kD) obtained by enzymatic digestion of long-term glycated fibronectin as a model for connective tissue AGE-peptides. AGE-formation can be assessed by monitoring of fluorescence (370/440 nm) which is easily differentiated from the much lower autofluorescence of oxidized low density lipoproteins (oxLDL). Alternatively, AGE formation was detected by an AGE-specific ELISA using antibodies elicited in rabbits against bovine AGE-RNAse. In the present study we investigated the influence of oxidative stress on the long-term glycation of LDL and the modulation of LDL-oxidation by AGE-modification. We observed (a) that the rate of AGE formation is reduced by BHT/EDTA both on LDL and serum albumin (glycation vs. glycoxidation), (b) long-term glycated LDL is more readily oxidized than unglycated LDL, (c) oxLDL is more prone to AGE-modification, (d) AGE-modification of LDL strongly alters its epitope spectrum and (e) that aminoguanidine at higher concentrations (1-10 mM) inhibits copper-catalyzed LDL oxidation in the way of a classical antioxidant.

Animals↗

PubMed1997

Characteristics of CD14 shedding from human monocytes. Evidence for the competition of soluble CD14 (sCD14) with CD14 receptors for lipopolysaccharide (LPS) binding.

The accumulation of sCD14 shed from human monocytes in vivo might correlate with other inflammatory parameters and could be of importance in overcoming a sepsis situation. The development of the sCD14 titer in the supernatant of monocyte-enriched MNC cultures isolated from healthy volunteers was studied utilizing a commercially available sCD14 ELISA. These culture experiments revealed the prolonged liberation of sCD14 into the supernatant during a period of several days. A medium-exchange schedule of 2-3 days was found to be superior to a longer incubation period with respect to the sCD14 yield. PMA initially enhanced the CD14 shedding slightly, but after a few hours it strongly repressed the process. Such a reduction was also achieved by protein synthesis inhibitors (cycloheximide, actinomycin D). Additionally, we monitored the concentration of sCD14, CRP, IL-6 and IL-8 in human sera from healthy persons or patients suffering from severe burn injuries with or without sepsis. Our results indicate that sCD14 is strongly correlated with IL-6, but not with IL-8. sCD14 titers were higher in the group of patients with both burn injuries and sepsis. From experiments with monocyte-enriched MNC cultures isolated from healthy volunteers and medium supplemented with sera containing sCD14 as well as radiolabeled LPS, we conclude that the enhanced shedding of CD14 in vivo during sepsis is probably not able to reduce the binding of LPS to monocytes.

Binding, Competitive↗