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A new assay for the screening of hypochlorite/hypochlorous acid (XOCl) scavengers, based on the reversed-phase high performance liquid chromatographic analysis of human serum albumin (HSA, 0.2% in 100 mM sodium phosphate, pH 7), before and after oxidation by XOCl (1.6 mM), was developed. XOCl induced a significant decrease of the area under the chromatographic peak of HSA at 280 nm due to the oxidation of the aromatic amino acids tryptophan and tyrosine, as suggested by the literature and by the chromatographic analyses and the electrochemical study performed here. The assay was validated by testing known XOCl scavengers such as ascorbic acid, cysteine, glutathione, S-methylglutathione and alpha-lipoic acid and other antioxidants such as carnosine and chlorogenic acid, which inhibited the oxidation of HSA. Quantitative activities were calculated using an original formula based on the changes of the area of the albumin peak. Electrochemical data collected here in a homogeneous medium showed that the anodic potentials of the antioxidants tested are less positive (ascorbic acid, chlorogenic acid and cysteine) or similar (alpha-lipoic acid) compared with those of the aromatic residues (tryptophan and tyrosine) of HSA oxidized by XOCl. However, as expected, carnosine, glutathione and S-methylglutathione were inactive at a glassy-carbon, gold or platinum electrode., (Copyright 2002 John Wiley & Sons, Ltd.)

Lonidamine (LND) or [1-(2,4-dichlorobenzyl)-1H-indazole-3-carboxylic acid] is an anticancer and an antispermatogenic drug whose mechanism of action is still incompletely understood. LND is effective against a number of tumors, including head, neck and breast cancers, probably because of the inhibition of mitochondrial electron transport and the enzyme hexokinase and to the induction of apoptosis. Instead, the antispermatogenic activity of LND appeared to be related not only to its energolytic activity but also to other effects activities such as the inhibition of specific chloride channels in the epididymis and the disruption of the inter-Sertoli-germ cell junctions, leading to premature release of germ cells. In addition, we recently reported that, in the rat, LND at the dose of 100 mg/Kg b.w. p.o., a fully active but well tolerated dose, caused specific changes of the testicular and epididymal macroglobulins (alpha(2)-macroglobulin, alpha(1) inhibitor-3 and alpha(1)-macroglobulin). Further studies are needed to elucidate the mechanism of action of LND, the lead compound of an interesting class of antispermatogenic drugs based on the core structure of 1-(2,4-dichlorobenzyl)-indazole-3-carboxylic acid.

Neither quercetin (Q), nor 3-O-acylquercetines, up to 100 microg/mL, had any significant activity on selected gram-positive strains (Staphylococcus aureus, Bacillus subtilis, Listeria ivanovi, Listeria monocytogenes, Listeria serligeri), gram-negative strains (Escherichia coli, Shigella flexneri, Shigella sonnei, Salmonella enteritidis, Salmonella tiphymurium) and yeasts (Candida albicans and Candida glabrata). In addition, we confirmed the known anti-HIV activity of Q (80% inhibition at 40 microM), which might depend on the free hydroxyl in the C-3 position, as suggested by the lack of activity of the 3-O-acylquercetines. Finally, we described an interesting inhibitory activity on Candida rugosa lipase by Q (IC(16)=10(-4) M) and its esters (3-O-acylquercetines) which, in vivo, could play an important role against lipase producing microorganisms. In particular, 3-O-acyl-quercetines, being more active (IC(16)=10(-4)-10(-6) M) and more lipophilic, could be more effective than Q when applied to the skin or mucosae, and deserve to be studied further.

The activity of nonsteroidal antiinflammatory drugs (NSAIDs) in rheumatoid arthritis is not only due to the inhibition of the production of prostaglandins, which can even have beneficial immunosuppressive effects in chronic inflammatory processes. Since we speculated that these drugs could also act by protecting endogenous proteins against denaturation, we evaluated their effect on heat-induced denaturation human serum albumin (HSA) in comparison with several fatty acids which are known to be potent stabilizers of this protein. By the Mizushimas assay and a recently developed HPLC assay, we observed that NSAIDs were slightly less active [EC50 to approximately 10(-5)-10(-4) M] than FA and that the HPLC method was less sensitive but more selective than the turbidimetric assay, i.e. it was capable of distinguishing true antiaggregant agents like FA and NSAIDs from substances capable of inhibiting the precipitation of denatured protein aggregates. In conclusion, this survey could be useful for the development of more effective agents in protein condensation diseases like rheumatic disorders, cataract and Alzheimers disease.

Background: Both urinary and biliary stones can contain calcium. Bile salts (BA), which are known to bind Ca2+, are commonly used to dissolve the latter but not the former., Methods: The effect of physiologic BA on calcium oxalate (CaOx) precipitation was evaluated by a recently developed method., Results: The Ca2+ binding properties of BA were confirmed by small but significant decreases in pH observed following addition of CaCl2 to bile acids solutions. More importantly, BA inhibited CaOx precipitation with effective concentrations of approximately 10-3 mol/L. The clinical relevance of the latter observation is presently unknown but it is of note that in the same in vitro assay, the activity of BA appeared comparable to that of citric acid, the most common drug for urolithiasis. Although BA do not reach mmol/L levels in urine, they are known to change the physicochemical properties of this fluid, possibly slowing down the crystal growth process. However, the hypothetical therapeutic use of BA in former stone patients would present at least two major problems: (i) hepatotoxicity and (ii) lithogenic activity, due to hyperoxaluria subsequent to increased intestinal absorption of oxalate., Conclusion: The ability of BA in effectively binding calcium ions and in inhibiting the precipitation of CaOx appears of interest from both a physiopathologic and a pharmacologic point of view, even if it does not currently seem exploitable for prophylactic or therapeutic purposes.

A HPLC method for the determination of lonidamine in serum and testis, suitable for pharmacological studies in the rat and other mammals, has been developed. Briefly, 0.5 mL of serum or about 0.2 g of testicular tissue were extracted with ethyl acetate and evaporated to dryness under nitrogen. The residue was redissolved in methanol and an aliquot was injected onto a C18 column eluted with a mobile phase consisting of acetonitrile:water (51:49, v/v), containing 0.1% trifluoroacetic acid. The eluate was monitored at 230 nm with a sensitivity of 0.05 AUFS. By this method, the pharmacokinetics and the serum and testicular levels of the drug up to 120 h after the administration of one single dose (100 mg/kg body weight) of lonidamine to Sprague-Dawley rats have been studied. Results were highly variable, as previously reported, but a very good linear correlation was found between the serum and the testicular levels, suggesting that, in the rat, and possibly in the human, testicular levels could be estimated based on the serum concentrations.

In a recent study (Leone et al., 2000) we reported that lonidamine (LND), an antispermatogenic drug, affected the concentration of selected testicular and epididymal proteins in the rat. Thus, the effect of LND on alpha2-macroglobulin (alpha2-M) and on other two acute phase proteins (APP), hemopexin (HPX) and alpha1-antitrypsin (alpha1-AT) was examined here. LND was administered orally at the dose of 100 mg/kg, the animals were killed after 24 and 48 hr and the samples were analyzed by immunoblotting. The drug did not induce any significant change of alpha2-M in the serum or testis and of HPX and alpha1-AT in the serum, testis or epididymis. Thus, the antispermatogenic action of LND was not accompanied by a significant change of these inflammatory markers, even if it did cause a decrease of alpha1-inhibitor-3, a negative APP, as previously reported (Leone et al., 2000).

Two methods for the analysis of antioxidants, based on polyacrylamide gel electrophoresis (PAGE) and gel permeation high performance liquid chromatography (HPLC) were developed. Both of them exploit the variations of the signal (band or peak) given by human serum albumin (0.2% w/v in 100 mM sodium phosphate pH 7) upon oxidation with hypochlorite (1% of a solution containing 4% active Cl), quantitatively determined by densitometric analysis or peak integration. Based on such changes, two formulas were defined which allowed the determination of the antioxidant activity of ascorbic acid (EC(50,PAGE)=4.8x10(-4) M, EC(50,HPLC)=3.6x10(-4) M), glutathione (EC(50,PAGE)=1.5x10(-4) M, EC(50,HPLC)=2.0x10(-4) M) and melatonin (EC(50,PAGE)=5.2x10(-4) M, EC(50,HPLC)=3.2x10(-4) M), chosen as reference compounds. A good correlation was found between the activities of these substances in the two assays, which are also in good agreement with literature data, indicating that the two methods are essentially equivalent. These assays could be useful for the screening of new antioxidant drugs for pathological conditions such as cataract, rheumatic diseases, atherosclerosis and Alzheimer's disease.

The mechanism responsible for the antispermatogenic activity of lonidamine (LND) [1-(2,4-dichlorobenzyl)-1H-indazole-3-carboxylic acid], a drug with low systemic toxicity and lack of significant hormonal effects, is still unclear but may be related to alterations of Sertoli cell proteins. Here, we confirmed that a single oral dose of LND (100 mg/kg b.w.) to sexually mature Sprague-Dawley rats causes shrinkage and weight reduction of the testes after 48 h. These macroscopic changes correlated with histologic alterations revealed by light microscopy, consistent with partially reversible inhibition of spermatogenesis. When the testes and the epididymides of animals treated with or without LND were homogenized and analyzed by the Bradford assay, a significant increase of total protein content was observed after 24 and 48 h. When these homogenates were analyzed by concanavalin blotting, specific changes of the major rat macroglobulins, i.e. alpha(1)-inhibitor-3, alpha(2)-macroglobulin, and alpha(1)-macroglobulin, were noted. In particular, LND caused a decrease of testicular alpha(1)-inhibitor-3, but not an increase of testicular alpha(2)-macroglobulin, indicating a mild local inflammatory response to the drug.

The capability of alpha-crystallin (alpha-C), a known molecular chaperon, of protecting beta-C and gamma-C against heat-induced aggregation was studied by gel permeation high performance liquid chromatography. The activity was calculated using a formula based on the changes in the areas under the chromatographic peaks of these proteins, which appeared well separated. When heat-induced aggregation was studied in the range 22-90 degrees C, beta-C appeared more stable than gamma-C. The activity of alpha-C in stabilizing gamma-C but not beta-C was already relevant at 60 degrees C, but the maximum activity was higher (about 35%) for beta-C than for gamma-C. This method could be useful for studying the effect of drugs with potential anti-cataract activity on heat-induced aggregation of individual lens proteins.

Ethyl acetate extracts of equine serum, containing 0-5 microg/ml of hydrocortisone (HYD), dexamethasone (DEX), oxyphenbutazone (OPB), indomethacin (IND), phenylbutazone (PB) and probenecid as internal standard, were evaporated with nitrogen, resuspended in methanol and analyzed by HPLC, using a C-18 column equilibrated with 51:49 acetonitrile-water, 0.1% trifluoroacetic acid, at 1 ml/min. The eluate was monitored at 254 nm. The selectivity (inter-assay C.V.<4%), sensitivity (limits of quantitation of 0.25 microg/ml for HYD, DEX and IND, 0.5 microg/ml for PB and 1 microg/ml for OPB, despite the occurrence of significant degradation of OPB and PB during the analysis) and precision (intra-assay and inter-assay C.V.'s of about 3-6 and 9-15%, respectively) of the method appeared appropriate for anti-doping control of racehorses.

Quantitative and qualitative changes of serum proteins, apart from glycation, have not been sufficiently studied in streptozotocin-induced diabetic rats (D), the most common experimental model for diabetes. Thus, we decided to analyze the serum of diabetic rats by concanavalin A-blotting in comparison with rats with acute inflammation induced by fermented yeast (Y), in which characteristic alterations of serum proteins have been described. Two months after the streptozotocin treatment, the blood glucose levels were highly elevated (456+/-24 vs. 124+/-10 mg/dl, p<0.001, n=12), the body weight was significantly lower than normal (279+/-10 vs. 392+/-6 g, p<0.001, n=12), and serum proteins appeared to be highly glycated (p<0.001) when analyzed by the fructosamine assay, without any significant change in the total serum protein concentration. Analysis by concanavalin A-blotting, revealed a significant decrease of alpha1-inhibitor-3 (alpha1-I3, p<0.05) and an increase of the beta chain of haptoglobin (beta-Hp, p<0.05) in both D and Y rats (n=3) compared with control animals. However, acute inflammation caused a marked rise of two prominent acute phase proteins, alpha2-macroglobulin and hemopexin, which did not change appreciably in diabetic rats. Further work will be necessary to evaluate the physiopathological significance of these phenomena which could result from changes of both concentration and glycosylation of the aforementioned proteins.

Hyaluronic acid (HA) is known to increase the ocular bioavailability of ophthalmic drugs not only for its viscous properties but also for its specific affinity for ocular mucins. This phenomenon, called bio- or mucoadhesion, can be evaluated in vitro by mechanical tests which, however, require considerable amounts of mucin (M) that are difficult to obtain from ocular surfaces. Thus, we developed an alternative method, based on gel permeation liquid chromatography, to examine the interaction of HA with microgram quantities of mucin. HA (from human umbilical cord or rooster comb) were fractionated using a Sepharose CL-4B column, before and after incubation with porcine gastric mucin (PGM), and the fractions were analyzed by a specific assay based on the histological dye Stains-all. PGM interacted with high molecular weight (M.W). HA, causing the displacement of low M.W., non-covalently bound, HA fragments, which were eluted under a distinct chromatographic peak. By quantitating the relative area of this peak, an evaluation of the mucoadhesion of HA could be obtained. This method could be useful to study the interaction between HA and microgram quantities of ocular M (mucin), obtained from individual patients or normal subjects.