Reflection contrast microscopy for high resolution detection of (3)H-estradiol in ultrathin sections of human stratum corneum.

A single autoradiographical method for light and electron microscopy (LM and EM) is presented. Human skin, containing (3)H-estradiol ((3)H-E2) after an in vitro permeation experiment, was processed via a non-extractive tissue preparation protocol, comprising cryo-fixation, freeze-drying, osmium tetroxide vapor fixation, and Spurr resin embedding. Semithin sections were processed for LM autoradiography, while ultrathin sections were processed both for high-resolution LM and EM autoradiography. The autoradiographs were visualized by bright-field microscopy (BFM), reflection contrast microscopy (RCM), and transmission electron microscopy to evaluate the potentials of RCM visualization in high-resolution LM autoradiography. RCM visualization of ultrathin vs. semithin resin sections showed an improved stratum corneum morphology. Histological staining was superfluous. The localization of (3)H-E2 in human stratum corneum using high-resolution LM autoradiography and RCM was as accurate as with high-resolution EM autoradiography.

The effect of rheological behaviour of a topical anaesthetic formulation on the release and permeation rates of the active compound.

The objective of this study was to investigate the possibility of developing a topical cream that allows maximum release rate of the active compound while having suitable consistency, i.e., sufficient apparent plasticity. A submicron (o/w) emulsion containing a model compound was investigated in the presence and absence of different polymers: sodium carboxymethylcellulose (CMC), Carbopol 934P (C934), polyethylene glycol 400 (PEG400) and polyethylene glycol 4000 (PEG4000). Various concentrations of the polymers were used in order to produce different rheological behaviours. The amount of drug passing through the membrane was measured as a function of time, using static diffusion cells with either Silastic sheeting 500-1 or guinea pig skin as membrane. The emulsion without polymer was used as reference. Rheological measurements were performed, giving the viscosity and the apparent yield stress of the formulations. Furthermore, theoretical values for diffusion coefficients and diffusion pathways were estimated and compared with the experimental data to discuss different diffusion models. Gelling polymers have been shown to produce an increase in the macroviscosity, thus inhibiting the diffusion of the oil droplets in the formulation without affecting the molecular diffusion. However, we suggest that when a compound of limited solubility is emulsified, the intact oil droplets contribute to the transport of the compound through the formulation. Thus, both release and permeation rates are decreased as the apparent yield stress, i.e., the macroviscosity of the formulation, is increased sufficiently by addition of gelling polymers.

Visualization of percutaneous 3H-estradiol and 3H-norethindrone acetate transport across human epidermis as a function of time.

Developing transdermal therapeutic systems for estradiol and norethindrone acetate raised questions about the steroids penetration pathway across and retention in the skin. This paper describes the distribution of 3H-estradiol and 3H-norethindrone acetate in human stratum corneum after topical application to dermatomed skin in vitro. The study involved (a) permeation experiments to determine the steroid flux, (b) autoradiographical visualization of the steroid distribution in the same skin samples, and (c) a correlation between flux and skin distribution in time. On correlating the steroid flux with intraepidermal steroid distribution, it was concluded that both permeants were bound in the skin tissue. The steroids were preferentially located in or close to the intercellular lipids of the stratum corneum, indicating that both transport and binding occurred via this domain of the stratum corneum. This study demonstrated the importance of correlating drug flux with intraepidermal drug distribution as a function of time.

Validation of freeze-drying to visualize percutaneous 3H-estradiol transport: the influence of skin hydration on the efficacy of the method.

A study was made of the validity of freeze-drying to visualize the distribution of 3H-estradiol in human stratum corneum after topical application of a dry dose, a patch or a buffer solution. Each of these donor formulations was applied to human dermatomed skin for 24 h using Franz permeation cells. Subsequently, small pieces of skin were subjected to cryofixation, freeze-drying, osmium tetroxide vapor fixation, Spurr resin embedding and electron microscopic autoradiography. Stratum corneum from dry dose and patch application experiments was well preserved by freeze-drying, allowing an accurate localization of 3H-estradiol. In contrast, stratum corneum from buffer solution experiments suffered from cryofixation artifacts due to excessive hydration of the skin. The corresponding autoradiographs showed strong redistribution of 3H-estradiol. Thus, the visualization method under investigation has its limitations regarding the hydration level of the skin.

Different zonal distribution of the asialoglycoprotein receptor, the alpha 2-macroglobulin receptor/low-density-lipoprotein receptor-related protein and the lipoprotein-remnant receptor of rat liver parenchymal cells.

Periportal and perivenous parenchymal cells were isolated by the digitonin-pulse perfusion method. The digitonin-pulse perfusion was shown to lead to selective lysis of the correct zone with a straight and sharp border of two to three cells. The mean ratios of alanine aminotransferase activity (a marker for periportal parenchymal cells) and glutamine synthetase activity (a perivenous marker) of periportal to perivenous parenchymal cells were 1.76 and 0.025 respectively. Cells were incubated in vitro with 125I-asialo-orosomucoid (ASOR), 125I-trypsin-activated alpha 2-macroglobulin (alpha 2M-T) or 125I-beta-migrating very-low-density lipoprotein (beta-VLDL), in order to determine the zonal distribution of the asialoglycoprotein receptor (ASGPr), the alpha 2-macroglobulin receptor/low-density-lipoprotein receptor-related protein (alpha 2Mr/LRP) and the lipoprotein-remnant receptor, respectively. Maximum binding capacity for 125I-ASOR on parenchymal cells showed a periportal/perivenous ratio of 0.70. The periportal/perivenous ratio of Bmax. values of binding of 125I-alpha 2M-T to parenchymal cells was 1.51. The Bmax. values of binding of 125I-beta-VLDL, however, were about equal for both cell populations. It is concluded that the maximum binding capacity of the ASGPr on isolated periportal parenchymal cells is 0.70 times that of perivenous parenchymal cells. The 1.51-fold higher expression of the alpha 2Mr/LRP on periportal cells, compared with perivenous parenchymal cells, indicates a zonal specialization for the uptake of the suggested multiple ligands. In contrast, the observed homogeneous distribution of the lipoprotein-remnant receptor is in accordance with the suggestion that lipoprotein remnants bind to a specific receptor, which is different from the alpha 2Mr/LRP. The zonal heterogeneity in the expression of receptors suggests that receptor-dependent uptake pathways are under zonal control, leading to intrahepatic heterogeneity in the removal of ligands from the blood circulation.

Rainbow trout corpuscles of Stannius: stanniocalcin synthesis in vitro.

In fresh-water rainbow trout, Oncorhynchus mykiss (formerly called Salmo gairdneri), experimentally induced mild hypercalcemia results in release of immunoreactive stanniocalcin from the corpuscles of Stannius (CS) and stimulated synthetic and releasing activities of the glands as measured in vitro. Pulse-chase experiments showed that stanniocalcin (STC) is a 56-kDa glycoprotein, processed from a 64-kDa precursor, prostanniocalcin (PSTC). PSTC and STC are homodimeric molecules that are readily split into monomers in the presence of reducing agents such as 2-mercaptoethanol. The monomeric form of PSTC and STC contains an approximately 5- to 6-kDa glycomoiety. Neither this sugar residue nor the NH2-terminal amino acid sequences of PSTC or STC proved to contain antigenic sites for the antiserum used in this study. Two-dimensional gel electrophoresis indicated the presence of several isoforms of PSTC and STC molecules that may reflect different stages of maturation of the (pro)hormone.