Relationships among vasculature, mitotic activity, and endothelial nitric oxide synthase (eNOS) in bovine antral follicles of the first follicular wave.

To determine the relationships among vasculature, mitotic activity, and expression of endothelial nitric oxide synthase (eNOS) of antral follicles in Bos indicus, bovine ovaries were obtained on day 6 of the estrous cycle from 10 crossbred (Brahman to Thai native cows) after a synchronized estrus with prostaglandin F2α analogue. Ovaries were fixed, paraffin-embedded, and used for immunofluorescence detection of factor VIII (a marker of endothelial cells). Immunostaining of eNOS and proliferating cell nuclear antigen (PCNA) were performed with specific monoclonal antibodies. Vasculature and positive staining of eNOS and PCNA were quantitatively evaluated with the image analysis. Follicles were classified by size (small, medium, and large) and by structure as healthy and atretic follicles (n = 82). The expression of factor VIII and eNOS were detected greater in the blood vessels of the theca layers of the healthy follicles than those in atretic follicles. The labeling indices (LIs) in granulosa and theca cells were greater (P < 0.05) in the healthy small and medium follicles than in the healthy large follicles. Vasculature, capillary area density, and capillary number density were positively correlated with eNOS expression and the LIs of granulosa and theca cells but were negatively correlated with the healthy follicle size. During the growing phase of antral follicle in Bos indicus, relationships among vasculature, mitotic activity, and eNOS were observed predominantly in healthy antral follicles. Thus, these data highlight the importance of vasculature, cell proliferation, and eNOS expression of growing and atretic follicles in the first follicular wave.

Influence of enzyme distribution and diffusion on permeation profile of prodrug through viable skin: theoretical aspects for several steady-state fluxes in two transport directions.

A physical modeling and theoretical simulation aspect for the simultaneous transport and metabolism of prodrug in viable skin were described to understand the influence of enzyme distribution and diffusion. The physical model was formulated assuming that the viable epidermis and dermis have distinct diffusional and metabolic characteristics and that the metabolic reaction in each layer follows a first-order kinetics. The differential equations were analytically solved, and the steady-state flux of prodrug into receiver and that of metabolite into receiver and donor and the total flux in forward (epidermis to dermis) and backward (dermis to epidermis) directions were derived. The flux of prodrug in the forward direction always equals that in the backward direction. The metabolite flux into receiver became transport direction-dependent when the diffusional characteristic of epidermis was different from that of dermis regardless of enzyme distribution. The metabolite flux into donor in the backward direction relative to that in the forward direction increased with increase of dermis/epidermis ratio of any parameters among metabolic rate constant, partition coefficient and diffusion coefficient of prodrug and metabolite. The difference of total flux between the 2 transport directions was caused by the difference in metabolic rate constant, partition coefficient and diffusion coefficient of prodrug between epidermis and dermis. The higher any parameters were for dermis, the higher was total flux in the backward direction.

Simultaneous transport and metabolism of nicotinic acid derivatives in hairless mouse skin.

In vitro simultaneous transport and metabolism of three ester prodrugs of nicotinic acid (NA), methyl nicotinate (MN), ethyl nicotinate (EN) and butyl nicotinate (BN) were studied using excised skin from hairless mouse. Hydrolysis studies of these esters with and without skin homogenate were also done at 37 degrees C. Both the ester and NA were detected in all receiver solutions in permeation studies, and no chemical hydrolysis of the esters was found, indicating that the esters were hydrolyzed during the skin permeation process. The total (ester+NA) flux from a saturated solution of ester prodrugs was higher than that of NA and was highest for MN, followed by EN and BN, whereas the total permeability coefficient of ester prodrugs increased from MN to BN. A difference in the NA/total flux ratio was found among these prodrugs; thus, esterase activity was also dependent on the alkyl chain length of the esters. The total flux from each ester solution increased linearly with the donor concentration. NA flux from MN and EN solutions increased with an increase in the donor concentration and reached a plateau at the high concentration range, suggesting that metabolic saturation occurred. NA fluxes at the plateau were similar among ester prodrugs and corresponded to the Vmax estimated from the hydrolysis experiment. The order of donor concentration at which NA reached a plateau also corresponded to the order of Km. It was confirmed that a difference in alkyl chain length of the ester prodrugs affected not only permeability but also metabolism in the skin permeation process.

Species difference in simultaneous transport and metabolism of ethyl nicotinate in skin.

The objective of this research was to compare the characteristics of skin permeation and metabolism of ethyl nicotinate (EN) among humans and several animal models. In vitro simultaneous skin permeation and metabolism experiment of EN was done in side by side diffusion cells at 37 degrees C. An EN hydrolysis experiment was carried out using skin homogenate and kinetic parameters (Vmax and K(m)) were estimated by computer data-fitting to Michaelis-Menten equation. Both EN and a metabolite, nicotinic acid (NA), were detected in all receiver solutions in permeation studies and no significant chemical hydrolysis was found, indicating that enzymatic hydrolysis of EN occurred during the skin permeation process. Difference in total (EN + NA) flux, from EN-saturated solution, was less than double among various species. The ratio of NA flux to total flux was highest for rat (0.94) followed by hairless rat, mouse, human and hairless mouse (0.76, 0.23, 0.19 and 0.13), and thus a great species difference was found in skin esterase activity. Total flux increased linearly with increase in donor concentration for all species. For hairless rat, mouse and hairless mouse, NA fluxes increased with increase in EN donor concentration and reached a plateau, suggesting that metabolic saturation occurred in skin. Species difference in NA fluxes and EN donor concentration in which the NA flux reached a plateau were also found. In rats, kinetic parameters for EN hydrolysis using skin homogenate were significantly higher than those in mice. These results suggest that species difference in permeation profiles of EN might primarily reflect the difference in esterase activity. To predict skin permeability in human using an animal model, the species difference in skin metabolism should be taken into consideration.