Modification of low-density lipoprotein during radiolabeling with 99mTc using three labeling methods.

AIM: The mechanisms of native low-density lipoprotein (LDL) uptake by monocytes and macrophages via the specific cholesterol down-regulated LDL-receptor differs form the mechanism responsible for the unregulated scavenging of the modified, for example, oxidized LDL, by the atherosclerotic plaques and foam cells. For this reason, we investigated if the 99mTc-labeled LDL stands for the native or modified molecule. The influence of the LDL sampling methods, isolation, preparation and radiolabeling of lipoproteins on structure modification and the subsequent imaging behavior has as yet not been addressed in detail. METHODS: Herein we present data on the effects of 99mTc labeling on some oxidation relevant parameters of LDL, such as the lag-time, thiobarbituric acid reactive substances (TBARS), relative electrophoretic mobility (REM), baseline dienes (BD), lipid peroxides (POX), free amino-groups (NH2-groups) and free sulphydryl-groups (SH-groups). Three methods of 99mTc labeling were compared: dithionite method (1), borohydride method (2) and ascorbic acid method (3). Data for oxidation parameters are expressed as a percent of freshly isolated native LDL (% native LDL) or as a percent of LDL treated with the labeling buffers and reagents, but in absence of the radioisotope (% control LDL). RESULTS: The levels of BD were most influenced by methods 2 and 3, and remained almost unchanged when the method 1 was used. The lag-time of 99mTc-LDL produced by method 2 doubled but it was decreased by 23% when the method 3 was employed. No change in the lag-time compared to the native LDL was observed with the method 1. The TBARS levels were 3-5 fold higher than in native LDL when methods 1 and 2 were used, but 33% lower in products made by the method 3. The number of thiol groups increased 3 fold in method 1, was only slightly elevated in method 3, but reduced in method 2 compared to native LDL. NH2-groups were increased with all three labeling procedures, but this increase was not considered significant. REM was altered only in products obtained by methods 1 (1.5x increase) and by method 2 (1.25x increase). No fragmentation of Apo B using sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) electrophoresis was observed by 99mTc-LDL produced in any of the METHODS: The increase of lipid peroxide generation was observed only when the method 2 was used. CONCLUSIONS: Of the three tested methods, we found all of them to render LDL oxidatively modified to some extent. Therefore, it appears that the native-LDL imaging with 99mTc-labeled LDL is impossible. Only the ascorbic acid method 3 appears to offer some protection and exerts antioxidant effects.

Modification of low-density lipoprotein by different radioiodination methods.

Scintigraphic imaging of radiolabeled low-density lipoproteins (LDL) is an interesting tool for the understanding of its role in pathomechanism of atherosclerosis. Metabolism of native LDL shows quite different pattern and kinetics as compared to that of modified LDL which is not mediated by classical LDL-receptor and accumulates in atherosclerotic lesions to form lipid-laden foam cells. Therefore we were interested whether radiolabelling of LDL induces structural modifications. We performed the iodine labeling of LDL for scintigraphic imaging of atherosclerosis by three different methods: chloramine-T (A), iodine monochloride (B) and iodogen (C). The highest radiolabelling yield of (125)I was obtained by the iodogen method (75.44+/-13.52%) and the lowest (49.01+/-12.74%) by iodine monochloride. Chloramine T showed a labeling yield of 62.82+/-6.17%. The stability of the tracer was very high with all the methods, persisting up to 6 h (98.83+/-1.2% - 91.38+/-4.7%, 15 min vs 6 h after labeling). For the first time we not only investigated the influence of radiolabelling on relative electrophoretic mobility (REM), but also various oxidation parameters such as baseline dienes (BD), thiobarbituric acid reactive substances (TBARS), endogenous peroxides (POX) and oxidation resistance in the copper-mediated oxidation system (expressed as lag-time) were measured. Furthermore, oxidation- derived fragmentation of the lipoproteins was examined with SDS-PAGE electrophoresis. Data are expressed as % change compared to native LDL before radiolabeling. BD were reduced by 32% using the method (A), but increased by 33% and 47% with the monochloride (B) and iodogen method (C), respectively. The effect on lag-time was comparable for all the three methods, ranging from 25 to 36% reduction in lag-time. TBARS were strongly increased 5-7 fold by all the methods. REM was changed by all three methods. While by methods A and C we have found a moderate increase in REM by 1.75 and 2.0 fold, respectively, and no fragmentation of Apo B was observed, in contrast by method B a dramatic 4.5 fold increase in REM was found. SDS-PAGE-electrophoresis showed strong fragmentation of the apoB only for method B. We conclude, that iodine labeling of LDL induces significant modification of the molecule. Once modified, LDL no longer reflects the native molecule, exhibiting altered functional properties. Using radiolabeled LDL this fact should be considered.

Binding of [99mTc]chondroitin sulfate to scavenger receptors on human chondrocytes as compared to binding of oxidized [125I]LDL on human macrophages.

Chondroitin sulfate (CS) used for treatment of osteoarthritis exerts distinct effects on human articular chondrocytes in vitro. We performed a binding analysis with 99mTc-labeled CS (Condrosulf, a commercial CS preparation containing calcium stearate) and cultured human chondrocytes in order to evaluate the presence of specific receptors. Saturation binding at 37 degrees C for 2 h revealed the presence of high-affinity binding sites for CS with a Kd of 2.3 x 10(-9) mol/L and a Bmax of 5.0 x 10(8). Extensive dialysis of Chondrosulf led to a decrease of the binding affinity by 52.5 +/- 19.5% and of the number of CS binding sites/cell by 62.0 +/- 14.0%, demonstrating that the additive present in the Condrosulf preparation enhances CS binding. The nature of the binding site is not yet known but evidence exists in the literature that the scavenger receptor CD36, thoroughly investigated on macrophages, is also found on chondrocytes and might be involved in CS binding. Therefore, we undertook a comparative binding study with human monocytes and labelled LDL and oxidized LDL, the latter being a postulated atherogenic agent in atherosclerosis. For [125I]-LDL binding we found a Kd of 0.45 x 10(-8) mol/L and a Bmax of 0.14 x 10(6) on quiescent monocytes and for [125I]-(ox)LDL binding a Kd of 1.8 x 10(-8) mol/L and a Bmax of 1.3 x 10(6) using LPS-activated monocytes. These data are comparable to the binding affinity found for lipoprotein-proteoglycan-complexes and hence are an indication but not a proof that CD36 is involved in CS binding to human chondrocytes.

Prostaglandins and lipid modification.

Postaglandins(PG) and low-density lipoproteins (LDL) both are playing a key role in atherogenesis. Their interaction at the local vascular level is of central relevance in plaque formation and progression. Details of these complex actions however, still need to be elucidated. Lipoproteins are influencing the PG-production of arterial wall cells and platelets, while PGs in turn have been shown to regulate lipoprotein receptor binding and entry into the arterial wall. Modification of LDL severely influences arterial wall trapping and foam cell formation. During LDL-modification, isoprostanes, a new family of compounds generated by free radical catalysed action, independent of cyclooxygenase, are formed. 8-epi PGF(2alpha) the most well known member exerts a great variety of proatherogenic actions, among them vasoconstriction and platelet activation; it also serves as a mitogen and stimulator of endothelin release. The influence of various eicosanoids on lipoprotein modification, however, has not been assessed yet.

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.

Methylene blue-enhanced stability of (99mTc)HMPAO and simplified quality control--a comparative investigation.

(99mTc)HMPAO is a radiopharmaceutical used for SPECT imaging of regional cerebral perfusion and for labeling cellular blood elements. The addition of methylene blue enhanced the stability of lipophilic (99mTc)HMPAO complex up to 3 h after reconstitution, 86.9+/-4.2% compared to 49.8+/-8.9% for the non-stabilised complex and persists over time (86.2+/-3.5% after 15 min compared to 78.2+/-4.0% after 3 h). The method widely used for estimation of radiochemical purity is a standard chromatographic procedure which is quite time-consuming taking about 30 min. Comparison of the more rapid and simple solvent extraction method with octanol, which needs only about 10 min to complete. showed a good correlation with the chromatographic method (84.4+/-4.4% compared to 89.1+/-4.3%). Using ethyl acetate as solvent instead of octanol gave a slightly higher extraction rate of the lipophilic complex (91.5+/-5.5% compared to 89.1+/-4.3%). Further, the ethyl acetate extraction method results in an overestimation, extracting partly secondary complex. It is confirmed, that the stability of the lipophilic (99mTc)HMPAO complex can be increased with methylene blue. The octanol extraction method, using higher extraction volume (up to 3 ml), is recommended as a fast and efficient approach for the quality control in daily clinical routine.

Why is glycated LDL more sensitive to oxidation than native LDL? A comparative study.

It is well established that oxidative modification of low-density lipoprotein (LDL) plays a causal role in human atherogenesis and the risk of atherosclerosis is increased in patients with diabetes mellitus. To examine the influence of different agents which may influence LDL-glycation and oxidation, experiments including glycation with glucose, glucose 6-phosphate, metal chelators (EDTA) and antioxidants (BHT) were performed. The influence of time dependence on the glycation process and the alteration of the electrophoretic mobility of LDL under diverse glycation and/or oxidation conditions was also investigated. The formation of conjugated dienes and levels of lipid peroxides in these different LDL-modifications were estimated. The copper-induced oxidation of LDL in vitro was determined by measurement of thiobarbituric acid reactive substances (TBARS) and expressed as nmol MDA/mg of LDL protein. We found that glycated LDL is more prone to oxidation than native LDL. Using native LDL, the maximal oxidation effect was found to reach a value of 49.72 nmol MDA/mg protein after 8 h. The maximum oxidation of the 31 days, glycated LDL with glucose was 71.76 nmol MDA/mg protein amounting to 144.33% of the value found for native LDL. In the case of glucose 6-phosphate glycation, the maximum oxidation under the same conditions amounted to 173.77% of the value found for native LDL. To measure the extent of glycation, fluorescence of advanced glycation end products (AGEs) was determined (370 nm excitation and 440 nm emission). The most potent glycation agent was glucose 6-phosphate leading to the formation of very high amounts of AGEs. This process was promoted in the absence of EDTA, which prevents the oxidative cleavage of modified Amadori products (ketoamines) to AGEs. We therefore conclude that both processes, glycation and oxidation, result in the modification of LDL. The lower the glycation-rate (+/- EDTA) as measured by relative fluorescence units RFU (generation of AGEs), the lower the additional oxidation rate after glycation as measured by TBARS (generation of MDA equivalents). Glycation and/or oxidation change the electrophoretic mobility of LDL.

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.

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.

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.

Influence of acetylsalicylic acid on oxidation of native and glycated low-density lipoprotein.

It is generally accepted that oxidation of low-density lipoproteins (LDL) is a causal factor in the development of atherosclerosis. Non-enzymatic glycosylation of LDL, i.e."glycation", plays a central role in late complications of diabetes mellitus and may initiate and/or accelerate the oxidation process. Therefore, the inhibition of this processes is of major therapeutic relevance. The influence of acetylsalicylic acid (ASA) on the oxidation of native and glycated LDL was studied in vitro. LDL (0.25 mg protein/ml ) was oxidatively modified with 5.0 microM CuSO4. Only at "supratherapeutical" ASA concentrations in the range 0.06-2.0 mg /ml we found a significant concentration-dependent inhibition of LDL oxidation both for native and glycated LDL, which was from 0.2 mg/ml upwards significantly more marked for native LDL than for glycated LDL. The maximal inhibitory effect occurred at 2.0 mg/ml with 89.6% inhibition of LDL-oxidation for native LDL and 64.4% for glycated LDL. At 0.2 mg/ml ASA the respective inhibitory values were 38.5% and 31.0%. For glycated LDL the ASA doses of maximal- and approximately 50%-inhibition, as found for native LDL, were chosen to investigate the inhibitory effect on 2,4,8 and 24 hours oxidation of glycated LDL to monitor the time-dependency of inhibition by ASA. This revealed that ASA only delayed, not permanently inhibited LDL oxidation.

The role of antioxidants in the long-term glycation of low density lipoprotein and its Cu2+-catalyzed oxidation.

In the present study we investigated the influence of antioxidants such as EDTA, alpha-tocopherol, troglitazone and acetylsalicylic acid on the long-term-glycation of LDL and its copper ion-catalyzed oxidation. We observed that (a) all antioxidants inhibited AGE-formation, while Amadori product formation was only diminished by extreme concentrations of acetylsalicylic acid, (b) glycated LDL was more susceptible to copper-catalyzed oxidation than unglycated LDL, and (c) the oxidation of native LDL was more dramatically inhibited by the antioxidants than that of glycated LDL. The observed differences may be a consequence of the significantly higher endogenous content in hydroperoxides of glycated LDL as compared to native LDL. Therapeutic implications of these findings regarding vitamin E, which is supposed to slow atherogenesis and the development of microvascular complications in diabetes, are obvious: Vitamin E-monotherapy, while blocking oxidative and AGE-modification of LDL, is unable to inhibit its AP-formation. As a consequence, tocopherol is susceptible to increased consumption by AP-associated radical production in hyperglycemic patients, which could be checked in part by the tocopherol-protecting agent troglitazone and/or by acetylsalicylic acid.

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.

Effects of PGI2 and analogues (taprostene, iloprost) on oxidation of native and glycated LDL.

Oxidation and glycation of low-density lipoprotein (LDL) has been claimed to play a central role in the pathogenesis of atherosclerosis. Therefore, the inhibition of this processes is of major therapeutic importance. In the present paper the influence of prostaglandin (PG)I2, and its stable analogues taprostene and iloprost on copper-induced oxidation of native, glycated and glycoxidated LDL was investigated. The results show, that the most pronounced effect on inhibition of native LDL-oxidation was obtained by taprostene in the whole concentration range tested (0.2 microg-10 microg/ml) reaching a maximal inhibition of 95% at 10 microg/ml. Examining glycoxidated LDL the inhibitory effect on oxidation was less pronounced reaching only about 10%. In case of glycated LDL, however, no significant inhibitory effect on oxidation was seen. Iloprost was effective as inhibitory agent against oxidation of native LDL at concentrations of 10 and 20 microg/ml, showing a maximal inhibition of 86% at a concentration of 20 microg/ml. Iloprost was ineffective on oxidation of glycated and glycoxidated LDL. Examining the extremely short-lived PGI2 itself, no significant inhibitory effect on oxidation of native, glycated or glycoxidated LDL, however, was seen. This finding might be of relevance for patients with diabetes mellitus, showing a decreased endogenous PGI2-production in particular those with bad metabolic control and high concentrations of circulating advanced glycosylation end products (AGEs).

Binding of long-term glycated low density lipoprotein and AGE-albumin by peripheral monocytes and endothelial cells.

Modification of low density lipoprotein (LDL) and plasma or tissue proteins by non-enzymatic glycation culminating in the formation of advanced glycation endproducts (AGEs) is one of the essential pathomechanisms leading to diabetes-associated long-term complications. We compared binding of glycated, glycoxidated and oxidated LDL by peripheral monocytes in activated and quiescent form. Interaction via specific receptors was different for glycated as compared to (glyc)oxidated LDL-modifications. In addition, binding of glycated LDL to quiescent and activated human umbilical vein endothelial cells was studied. In patients with insulin-dependent diabetes mellitus (IDDM), AGE-binding was significantly increased as compared to healthy individuals. Specific and non-specific monocyte binding mechanisms were detected, and both were significantly increased in IDDM patients. Specific and non-specific binding strategies possibly act in concert to eliminate circulating AGEs, which are instrumental in the development and progress of microangiopathic and macroangiopathic complications of diabetes mellitus.

Do E-series prostaglandins and their metabolites influence oxidation of native and glycated low-density lipoproteins?

Oxidation of lipoproteins, and, in particular, low-density lipoproteins (LDL), has been shown to play a significant role in the pathogenesis of atherosclerosis. Oxidized LDL are endocytosed via scavenger receptors to form lipid-laden foam cells. The non-enzymatic reaction of glucose with proteins and lipoproteins results in a modified LDL involved in the pathogenesis of late complications in diabetes mellitus. In the present paper, the influence of various E-series prostaglandins (PGE1; 13,14-dihydro PGE1; 13,14-dihydro 15-keto PGE1; and PGE2) on oxidation of native and glycated LDL was investigated. The effect of these agents in the concentration range from 1 pg/mL to 1.6 micrograms/mL on copper-induced oxidation of native and glycated LDL was tested. The concentration of each agent causing the maximal effect on oxidation of native LDL, as measured by the formation of thiobarbituric acid-reacting substances, was chosen to estimate the effect on 2, 4, 8, and 24 h oxidation of glycated LDL. The study was performed with LDL isolated by sequential ultracentrifugation from normolipidemic individuals. LDL (0.25 mg protein/mL) was oxidatively modified with 5 microM CuSO4. The glycosylation of LDL was performed by incubation of LDL with 500 mM glucose for varying periods of time ranging from 10 to 31 days. Our results show that only 13,14-dihydro PGE1 significantly inhibits copper-induced oxidation of native LDL, while the other examined E-series prostaglandins in vitro are ineffective as reducing agents in LDL-oxidation.

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.

Cholinergic deficit induced by ethylcholine aziridinium (AF64A) transiently affects somatostatin and neuropeptide Y levels in rat brain.

The question whether during the process of cholinergic degeneration somatostatin- and/or neuropeptide Y-containing neurons in rat hippocampus and cortex react to the withdrawal of cholinergic function was addressed. After bilateral intracerebroventricular injection of the cholinotoxin ethylcholine aziridinium (AF64A; 1 or 2 nmol/ventricle) in rats, the activity of choline acetyltransferase (ChAT) started to decline in the hippocampus within 24 h. The reduction of ChAT activity reached its maximum within 4 days (34 and 55% after 1 and 2 nmol of AF64A/ventricle, respectively) and persisted during the observation period of 14 days. In the parietal cortex, ChAT activity decreased by 23% 4 days after 2 nmol of AF64A/ventricle. The loss in ChAT activity was accompanied by a transient decline in the levels of somatostatin and a transient increase in the levels of neuropeptide Y in both brain areas. In the hippocampus, the reduction in somatostatin content was most pronounced after 2 days (by 22 and 33% after 1 and 2 nmol of AF64A/ventricle, respectively). Within 14 days, somatostatin levels returned to control values. Neuropeptide Y levels increased slightly by approximately 25% of control values in the hippocampus. The changes described were present in both the dorsal and ventral subfields of the hippocampus. Similar but less pronounced changes in levels of both neuropeptides were observed in the parietal cortex. The present data provide further evidence for a close neuronal interrelationship between cholinergic and somatostatin- and/or neuropeptide Y-containing neurons in rat hippocampus and parietal cortex.