Separation of delta6- and delta9,11-estradiol: analytical method development, validation and practical application.

For estradiol (E2) the separation of the degradation products delta6- and delta9,11-E2 is especially challenging due to their structural similarity. There is no method described in the literature yet which adequately addresses this problem. The present study describes a HPLC method for the separation and quantitation of E2 and its degradation products 6alpha-hydroxy-E2, 6beta-hydroxy-E2, 6-keto-E2, delta9,11-E2, beta-equilenol and delta6-E2. The method employs a Kinetex PFP analytical column, using methanol and deionized water as mobile phases. Different UV- and fluorescence detection modes were used for maximal sensitivity and specificity. The applicability and capability of the method was demonstrated for Vagifem tablets. Finally, the method was validated with respect to selectivity, sensitivity, linearity, precision and accuracy.

A 40 kDa inhibitor of protein kinase C purified from bovine brain.

An inhibitor of protein kinase C has been purified to homogeneity from bovine brain cytosol by a four-step method. It is heat stable, has an apparent molecular mass of 40 kDa and is composed of two polypeptide chains of 19 kDa.

Virus-induced amyloidosis in scrapie involves a change in covalent linkages in the preamyloid.

Preparations of the preamyloid and the amyloid protein from normal and scrapie hamster brains show different solubilization behaviours towards Triton X-114 extraction. The normal isoform is completely extractable from microsomal membranes by the detergent, whereas the pathological one is not. Both forms can be isolated using preparative SDS electrophoresis as the final step in order to remove all non-covalently associated materials. After removal of the SDS these purified proteins retain their solubility differences against Triton X-114. This demonstrates that at least one distinct modification of the preamyloid protein--which has to be covalent in nature--must have occurred to account for the strong aggregation tendency of the pathological isoform.

Effects of multiple ejaculations after extended periods of sexual abstinence on total, motile and normal sperm numbers, as well as accessory gland secretions, from healthy normal and oligozoospermic men.

Four healthy donors provided three ejaculates on one day to deplete their sperm reserves and also after abstention periods of 1, 2, 4, 7, 10 and 14 days. The percentage of spermatozoa that were normally formed, progressively motile and which swelled in hypotonic medium remained unchanged throughout this period and the increase in total numbers of viable spermatozoa for 10 days prompted a comparison of seven donors and six oligozoospermic men. Multiple ejaculations after 10 days of abstinence for the patients with low normal, motile sperm numbers increased the total number, number of normal and number of motile spermatozoa by a mean of 230, 370 and 520%, respectively.

Characterisation of antisera raised against species-specific peptide sequences from scrapie-associated fibril protein and their application for post-mortem immunodiagnosis of spongiform encephalopathies.

Transmissible spongiform encephalopathies (TSE), such as scrapie or Creutzfeldt-Jakob disease (CJD), are fatal neurodegenerative diseases of the central nervous system caused by a yet unidentified virus. They are accompanied by a brain specific amyloidosis, during which a host coded protein irreversibly aggregates to form the scrapie-associated fibrils. The diagnosis of TSE relies on histopathological detection of spongiform lesions, on electron microscopical detection of fibrils, or on the immunological detection of SAF protein, which is the most specific diagnostic marker. In order to improve the diagnosis of TSE, we developed a protocol for rapid tissue fractionation and enrichment of SAF protein which subsequently allows the specific detection of SAF protein by western blotting and immunodetection. Using some new antisera raised against synthetic peptides with sequences specific for the hamster, sheep, cattle and human SAF protein, several samples can be diagnosed for TSE within 24 hours, starting with only 10-100 mg of brain tissue from different species.

Evaluation of methaemalbumin in acute pancreatitis.

Methaemalbumin (MHA) was measured in 62 patients with acute pancreatitis. In 26 MHA-positive patients the occurrence of renal and pulmonary complications was 65% and 58%, respectively, compared with 3% and 6% in 36 MHA-negative patients. Fatality rate was 54% in MHA-positive and 6% in MHA-negative patients. In 17 MHA-positive cases haemorrhagic pancreatitis was proven at laparotomy or postmortem examination. Thus MHA determination proved to be a valuable diagnostic and prognostic parameter in acute pancreatitis.

Specificity of increased amylase to creatinine clearance ratio in acute pancreatitis.

The amylase to creatinine clearance ratio was found to be normal in 11 of 33 patients with acute pancreatitis. The ratio was elevated in 10 of 19 patients with renal insufficiency. Thus, it does not seem to be a specific index in the diagnosis of acute pancreatitis.