James V. Hunt

GLUCOSE MODIFICATION OF HUMAN SERUM ALBUMIN : A STRUCTURAL STUDY

Structural changes associated with the exposure of human serum albumin (HSA) to glucose with or without the presence of Cu (II) have been characterized using a bank of methods for structural analysis including circular dichroism (CD), amino acid analysis (AAA), fluorescence measurements, SDS-PAGE, and boronate binding (which is a measure of Amadori product formation). We show that in the short-term (10 d) incubation mixtures, HSA is resistant to Cu (II)-mediated oxidative damage and that the early products of glycation of HSA had minimal effects on the folded structure. Amino acid analysis showed that there was no formation of advanced glycation endproducts (AGE), which can be measured by loss of lysine. This remained the case in longer term incubation of HSA (56 d) in the hyperglycemic concentration range (5-25 mM glucose) despite increased levels of Amadori product (60% boronate binding) and the formation of glycophore (Excitation 350, Emission 425). At high, nonphysiological concentrations (100 mM and 500 mM) of glucose, glycophore formation increased and 3 and 11 mol Lysine-glucose adduct/mol HSA were converted to AGE, respectively. This was accompanied by increased damage to tryptophan and protein-protein crosslinking but only minor tertiary structural change. In the presence of Cu (II), however, AGE formation was accompanied by extensive damage to histidine and tryptophan side chains, main chain fragmentation, and loss of both secondary and tertiary structure. Thus, changes in structure appear to be the result of oxidation as opposed to glycation, per se.

Ascorbic acid oxidation: A potential cause of the elevated severity of atherosclerosis in diabetes mellitus?

The exposure of mouse peritoneal macrophages to cholesterol linoleate‐containing, artificial lipoproteins can lead to intracellular ceroid accumulation. This can be used as a model to study the role of oxidation in macrophage uptake of lipoproteins containing unsaturated fatty acids, considered by many as a primary event in atherosclerotic plaque formation. Our studies show that ascorbic acid can both inhibit and promote the formation of ceroid in such a model system. The transition metal copper (Cu(II)) further elevates ceroid accumulation and EDTA, a metal chelator, inhibits it. When trace levels of transition metals are present, low concentrations of ascorbic acid can elevate ceroid formation. This pro‐ and antioxidant characteristic of ascorbic acid was confirmed by monitoring the generation of oxidants by various concentrations of ascorbic acid, assessed by benzoic acid hydroxylation or the fragmentation of BSA. We discuss these observations in the context of an apparent increase in ascorbic acid oxidation and elevated severity of atherosclerosis in diabetes mellitus.

Aminoguanidine and its pro-oxidant effects on an experimental model of protein glycation

Recent reports show a pro-oxidant activity of aminoguanidine. Aminoguanidine is able to generate hydrogen peroxide in the presence of Cu (II). These observations have been confirmed by the present studies in that aminoguanidine is, indeed, able to generate oxidants similar in reactivity to the hydroxyl radical and is also able to fragment BSA in a Cu (II)-dependent manner. Studies on glycated bovine serum albumin show that aminoguanidine can affect a number of parameters associated with the nonenzymatic glycation of protein. This includes an ability to decrease glucose attachment and levels of protein fluorescence termed glycophore, resulting from protein glycation. Aminoguanidine also increases the generation of dicarbonyl compounds by glycated protein. All of these effects on parameters of glycation appear to be Cu (II) dependent. Further studies show that one effect of protein glycation is to decrease its susceptibility to proteolysis. The reverse is true of protein oxidation, which has previously been shown to increase the susceptibility of proteins to proteolytic digestion. Evidence is presented suggesting that aminoguanidine is able to enhance the proteolytic digestion of glycated BSA, a protein shown to be protease resistant. Our observations are discussed within the context of current concepts of protein glycation in the development of diabetic complications and aminoguanidines potential use as a prophylactic agent in diabetes mellitus.

Ascorbic Acid and Diabetes Mellitus

Despite the introduction of insulin and hypoglycemic agents the future of a diabetic individual can be an uncertain one with the probability of a diminished quality and length of life (Entmacher et al., 1964; Pell and D’Alonzo, 1971; Entmacher, 1975). The diabetic patient is prone to a number of complications affecting the vascular system, kidney, retina, peripheral nerves, lens, and skin. The diabetic individual has a 25-fold increased risk of blindness, a 20-fold increase in the risk of renal failure, and a two- to sixfold increase in the risk of coronary heart disease (Klein et al., 1985). There is also a 20-fold increase in the risk of amputation as a result of gangrene. The incidence of peripheral neuropathy can range between 6 and 86% depending on diagnostic criteria and also upon duration of disease. The cause of diabetic complications was initially an open question with the assumption that the cause was the same as that which led to the initial loss of blood sugar control (Pirart, 1978; DCCT, 1993) and abnormalities in lipid metabolism (Randle et al., 1963; Bevilacqua et al., 1990), both of which characterize the syndrome. Complications subsequently became associated with hyperglycemia since elevated plasma glucose levels became increasingly viewed as the single outstanding feature which distinguished a diabetic from a nondiabetic individual.

Apolipoprotein oxidation in the absence of lipid peroxidation enhances LDL uptake by macrophages

A characteristic of the antioxidant, probucol, is its inability to inhibit apolipoprotein B fragmentation in low density lipoprotein (LDL), despite a pronounced ability to inhibit lipid oxidation on relatively lengthy exposure to Cu(II). Here we show that a short exposure of LDL to hydrogen peroxide and Cu(II) leads to 125I‐labelled apolipoprotein B fragmentation, the production of malondialdehyde and hydroperoxides and leads to increased uptake by macrophages on subsequent culture. However, pre‐loading LDL with probucol protects LDL from lipid oxidation but not protein fragmentation or macrophage uptake. The use of probucol to conduct studies on apolipoprotein B oxidation without extensive lipid oxidation may prove useful when studying LDL apolipoprotein damage on exposure to an aqueous free radical insult.

Differing effects of probucol and vitamin E on the oxidation of lipoproteins, ceroid accumulation and protein uptake by macrophages.

Studies using 125I-low density lipoprotein (125I-LDL) show that probucol (10 microM) and alpha-tocopherol (100 microM) inhibit protein degradation in LDL exposed to Cu (II) in vitro. The inhibitory effect of alpha-tocopherol on protein fragmentation exceeded that of probucol. On the other hand, probucol was more able to inhibit lipid peroxidation. The subsequent uptake of Cu (II)-oxidised 125I-LDL by murine peritoneal macrophages (MPM) was virtually unaffected by the presence of probucol during LDL oxidation. The same was not true for alpha-tocopherol which led to lower levels of 125I-LDL uptake by MPM. Thus, it appears that although the antioxidant activity of probucol exceeds that of alpha-tocopherol for lipid oxidation, the reverse is true for protein degradation and, perhaps more significantly, for subsequent macrophage uptake. Further studies used artificial lipoproteins composed of cholesteryl linoleate or cholesteryl arachidonate complexed with bovine serum albumin. Culture of these artificial lipoproteins with MPM resulted in protein uptake, protein degradation, cholesterol oxidation to cholest-5-en-3 beta,7 beta-diol and the intracellular accumulation of ceroid in MPM. The presence of alpha-tocopherol (0-100 microM) inhibited all of these processes. Probucol (0-10 microM) inhibited ceroid accumulation and cholesterol oxidation to the same degree as alpha-tocopherol (0-100 microM) but had no effect upon protein degradation and protein uptake. Control studies of lipoproteins incubated without cells showed that protein degradation by cell-independent processes was also inhibited by alpha-tocopherol, but not by probucol. These observations are discussed in the context of the role of lipoprotein oxidation in atherogenesis.

Flow cytometric measurement of ceroid accumulation in macrophages

Flow cytometry has been examined as a method for quantitative measurement of the accumulation in macrophages of ceroid, an autofluorescent polymer composed of oxidised protein and lipid. Murine peritoneal macrophages were cultured in the presence of cholesteryl linoleate- or arachidonate-bovine serum albumin (CL/BSA or CA/BSA) complexes. Ceroid accumulation was greater from CA/BSA than from CL/BSA and was dependent upon both time and cell plating density. Inclusion of vitamin E with the complexes diminished the accumulation of ceroid fluorescence after exposure to either CL/BSA or CA/BSA. Controls included exposure of macrophages to BSA, alone and with vitamin E, both of which led to some fluorescence at a similar wavelength to that used to monitor ceroid accumulation (Ex: 351.1-363.8 nm/Em: 490 nm and upwards). Ceroid accumulation can be monitored semi-quantitatively by staining techniques. However, such methods are relatively crude and give little information about the amount of ceroid within cells. Flow cytometry, on the other hand, can give a quantitative assessment of cellular ceroid accumulation, provided experiments are conducted with appropriate controls. The findings are discussed in the context of human atherosclerosis and of future investigation of cell-mediated lipid oxidation and its potential antagonists.

Effect of glucose-mediated LDL oxidation on the P388D1 macrophage-like cell line

Oxidised human low density lipoprotein (LDL) is thought to play a role in the development of atherosclerosis. Recent reports suggest that glucose-derived oxidants are capable of oxidising LDL. In this report, the effect of glucose-mediated oxidation of LDL upon the macrophage like cell line, P388D(1), was examined. Glucose-mediated oxidation of LDL was assessed by changes in the electrophoretic mobility of LDL and by analysis of lipid content using gas chromatography. The presence of Cu(II) (0.5 microM) was essential for the oxidation of LDL. The oxidation was potentiated by glucose in a dose- and time-dependent manner. At the concentration of LDL used (1 mg/ml), high concentrations of glucose (up to 500 mM) were required to oxidise LDL. The electrophoretic mobility of LDL correlated with the degree of lipid oxidation; both correlated with an inhibitory effect of oxidised LDL upon P388D(1) DNA synthesis. Diethylenetriaminepentaacetic acid (DETAPAC), a transition metal chelator, and aminoguanidine (AMG), an anti-glycation agent, inhibited the oxidation of LDL and attenuated the effects on DNA synthesis. Thus, glucose can mediate transition metal-dependent oxidation of LDL to a level that can affect P388D(1) cells, a mechanism which might have relevance to accelerated atherosclerosis in diabetic patients.

The Inhibition of Foam Cell Formation by Sodium Diethyldithiocarbamate

A prominent feature of human atherosclerosis is the lipid-laden foamy macrophage, which often also contains the insoluble pigment, ceroid. The culture of macrophage-like cells, P388D1s, with artificial lipoproteins composed of cholesteryl linoleate (CL) and bovine serum albumin (BSA) results in foam cell formation with lipoprotein uptake and the intracellular accumulation of ceroid. Ceroid accumulation is accompanied by the oxidation of the cholesterol ester as monitored by gas chromatography. The sodium salt of diethyldithiocarbamic acid (DDC) at 1-5 microM effectively inhibited lipoprotein uptake, cholesteryl linoleate oxidation and ceroid accumulation in cultures of P388D1. Further studies showed that intracellular ceroid accumulation appeared to require the presence of cystine in the medium. Lipoprotein oxidation by this macrophage-like cell therefore appears to involve a mechanism dependent on cystine metabolism which is consistent with previous reports of macrophage-mediated lipoprotein oxidation. Studies on CL/BSA-induced ceroid accumulation in human monocytes also showed that DDC behaved in much the same manner. This inhibitory effect of DDC on foam cell formation, often considered a primary event of atherosclerosis, at concentrations as low as 1 microM, suggests the need for further, more comprehensive, studies on this compounds activities.

LIPID OXIDATION, LIPOPROTEIN CELL-ASSOCIATION AND CEROID ACCUMULATION IN P388D1 MACROPHAGE-LIKE CELLS

Flow cytometry can be used to quantify the accumulation of ceroid in macrophages, the result of cellular handling of certain lipoproteins. Using P388D1 cells, a murine-derived macrophage-like cell line, the effect of the lipophilic antioxidant, DL-alpha-tocopherol, upon the uptake and accumulation of ceroid by the cells was monitored on culture with artificial lipoproteins containing a single lipid species. Ceroid accumulation was greater for artificial lipoprotein composed of BSA complexed with cholesteryl arachidonate, than with cholesteryl linoleate. alpha-Tocopherol inhibited the ceroid accumulation, which was also dependent upon cell density. Thus, since these findings are similar to recent observations in primary cultures of murine peritoneal macrophages, it would appear that macrophage-like cell lines such as P388D1 cells are appropriate for the study of potential agonists and antagonists of lipid oxidation. Culture of P388D1 cells with oxidised human low-density lipoprotein (LDL) also resulted in ceroid formation, shown to be dependent upon the level of LDL oxidation as assessed by thiobarbituric acid-reactivity, the xylenol orange assay of peroxides and gas chromatographic analysis of cholesterol and fatty acid content. Ceroid accumulation reflected changes in the level of LDL oxidation better than did the cell association of oxidised radiolabelled LDL, monitored as that bound and retained by the cell.