Maria Grazia Leone

Simultaneous determination of hydrocortisone, dexamethasone, indomethacin, phenylbutazone and oxyphenbutazone in equine serum by high-performance liquid chromatography

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.

Quantification of prostaglandin D synthetase in cerebrospinal fluid: a potential marker for brain tumor.

Prostaglandin D synthetase (PGD‐S; prostaglandin‐H2 D‐isomerase, EC 5,3,99,2), a 30 kDa glycoprotein also known as β‐trace protein that catalyzes the formation of prostaglandin D2 (PGD2) from PGH2, was purified to apparent homogeneity from human cerebrospinal fluid (CSF) using a two‐step procedure involving HPLC on a Vydac C8 reversed‐phase column and high performance electrophoresis chromatography (HPEC) using a 10% T SDS‐polyacrylamide gel. The purity of PGD‐S isolated from CSF was confirmed by silver stained SDS‐polyacrylamide gel and direct protein microsequencing (NH2‐APEAQVSVQPNFQ). A highly specific polyclonal antibody was prepared against this protein for immunoassay development. Using an ELISA, it was found that the concentration of PGD‐S in CSF did not alter significantly in different pathological conditions of the central nervous system (CNS). These include dementia (n=9), hydrocephalus (n=4), neuropathy (n=11), optic neuritis (n=4), multiple sclerosis (n=11), and demyelinating syndrome (n=11), when compared to normal individuals (n=12); however, the level of PGD‐S in the CSF obtained from patients with brain tumor (n=11), was reduced by as much as 2‐fold when compared to control samples (n=12) illustrating PGD‐S is a potentially useful marker for brain tumor.

Lipocalin type prostaglandin D-synthase: which role in male fertility?

Lipocalin type prostaglandin-D-synthase (L-PGDS), also called beta-trace, is an extracellular protein very abundant in compartments beyond blood-tissue barriers, such as the cerebrospinal fluid, the aqueous humor, the amniotic fluid and the seminal fluid. In the latter fluid the major function of L-PGDS does not seem to be the synthesis of prostaglandin D(2) (PGD(2)) from its precursor PGH(2), which is very unstable in aqueous solution. Instead, seminal L-PGDS, an important carrier of bile pigments, retinoids, thyroid hormones and essential fatty acids, would contribute to providing, beyond the blood-testis barrier, thyroid hormones and retinoids to the developing germ cells in the seminiferous tubules and the maturing spermatozoa in the epididymis.

Inhibition of heat-induced denaturation of albumin by nonsteroidal antiinflammatory drugs (NSAIDs): Pharmacological implications

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∼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.

Effects of the major alkaloid of Hydrastis canadensis L., berberine, on rabbit prostate strips

Previous studies (Palmery et al., 1996) have shown that Hydrastis canadensis L. displays adrenolytic activity possibly ascribable to berberine (Ber), its principal alkaloid. The aim of this study was to compare the activity of H. canadensis extract (HCE) with that of Ber on rabbit prostate strips. Both HCE and Ber inhibited prostate contraction mediated by norepinephrine (NE) and phenilephrine (PHE). The effect of HCE is less pronounced than that of Ber at the same dose on NE‐induced prostate contraction. These results suggest that other factors with adrenergic activity may modulate NE response. Moreover, HCE seems to be, at least at some doses, more effective than Ber on PHE‐mediated prostate contraction. Studies on the effect of other alkaloids present in the extract will contribute to the understanding of this phenomenon. This study also points to a possible therapeutic use of Ber in prostatic hypertrophy. Copyright

Corneal Toxicity of Xylazine and Clonidine, in Combination with Ketamine, in the Rat

Purpose: To compare the corneal toxicity of xylazine (XYL)/ketamine (KET) with that of clonidine (CLO)/KET in the rat, in the presence or not of the α2-adrenergic antagonist yohimbine (YOH). Methods: XYL (10 mg/kg) and CLO (0.15 mg/kg) were administered subcutaneously in the rat in combination with KET (50 mg/kg), in the presence or not of YOH (2 mg/kg). Results: The corneas immediately lost transparency and luster, but recovered within 120 min. By both light and electron microscopy, a marked stromal edema and alterations of all layers were observed. In addition, XYL/KET altered the permeability of the cornea as indicated by the augmented levels of 14C-indomethacin, topically administered 30 min after the anesthetic combination. Conclusions: The mechanism of the corneal toxicity of XYL and CLO in the rat is unclear but we speculate that: (a) proptosis and inhibition of normal blinking did not play a major role because topical application of hyaluronic acid did not protect against it; corneal decompensation, edema and opacification could be due to (b) osmotic or (c) mechanical endothelial stress: the first resulting from the sudden increase of the glucose concentration in the aqueous humor due to the well-known inhibition of insulin release by α2-adrenergic agonists, and the second from the acute elevation of intraocular pressure caused by these α2-adrenergic mydriatics in the rat; (d) addition, XYL and CLO could act by directly interacting with local α2- or, possibly, α1-adrenergic receptors, whose function is still not clear but probably essential for corneal homeostasis.

CHANGES IN CONCANAVALIN A-REACTIVE PROTEINS IN NEUROLOGICAL DISORDERS

Changes of glycosylation of cerebrospinal fluid proteins such as α2‐macroglobulin, and prostaglandin D synthase were studied by lectin blotting, using concanavalin A, in multiple sclerosis (n = 42) and neuropathies (n = 20) in comparison to neurological controls (n = 22). The concanavalin A‐reactivity of α2‐macroglobulin, which was increased in the neuropathies but not in multiple sclerosis compared to controls, correlated with the total concanavalin A‐reactivity in controls and neuropathies but not in multiple sclerosis, indicating that the protein could be abnormally glycosylated in the latter disease. Although the concentration and the concanavalin A‐reactivity of prostaglandin D synthase were not significantly different in the three groups, the two parameters correlated only in neuropathies but not in controls or multiple sclerosis, probably due to the high heterogeneity of the protein. These changes deserve to be studied in further detail in view of their potential clinical applications. J. Clin. Lab. Anal. 13:158–165, 1999.

Development of an enzyme-linked immunosorbent assay, using a monoclonal antibody against α2-macroglobulin, for the diagnosis of systemic lupus erythematosus

Objectives: To develop an enzyme-linked immunosorbent assay (ELISA) using a monoclonal antibody (mab) directed against abnormally glycosylated serum α2-macroglobulin (α2-M) from patients with systemic lupus erythematosus (SLE). Design and methods: Serum α2-M purified by HPLC from patients with SLE was injected in a Balb/c, CB6 F1 female mouse and hybrid cell lines were screened using α2-M Glu-C fragments derived from SLE and normal donors (NHS). A mab was selected and used to develop an ELISA by which sera from NHS (n = 14), SLE (n = 34), rheumatoid arthritis (n = 15), Sjogren’s syndrome (n = 11), mixed connective tissue diseases (n = 12), and liver diseases (n = 11) were analyzed. Results: The affinity of the mab for α2-M from SLE, but not from the other diseases, was higher compared to NHS, as demonstrated by immunoblotting and ELISA. Conclusions: The ELISA was capable of recognizing changes of glycosylation of α2-M in SLE and may be useful for its differential diagnosis.

Effects of lonidamine on testicular and epididymal proteins in the rat.

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.

Micropurification of β- and γ-crystallins from rabbit aqueous humor

Abstract Soluble crystallins are normally present in the aqueous humor, originating from the lens, and their concentration may increase in certain conditions such as cataract, possibly contributing to aqueous outflow pathway obstruction, leading to glaucoma. Whether the stability and the tendency of aqueous crystallins to aggregate are different in patients with certain forms of open-angle glaucoma has not so far been established, mainly due to the lack of a suitable purification procedure from this fluid in which crystallins are present at very low concentration together with dozens of other proteins. About 4 μg each of β- and γ-crystallins were obtained from 20 ml of rabbit aqueous humor by C 8 reversed-phase high-performance liquid chromatography (HPLC) and high-performance electrophoresis chromatography (HPEC). The identity of the proteins was confirmed by amino acid analysis following sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and electrophoretic blotting onto polyvinylidene fluoride membranes, with or without previous digestion with Staphylococcus aureus protease V 8 .