An explanation for the mysterious distribution of melanin in human skin: a rare example of asymmetric (melanin) organelle distribution during mitosis of basal layer progenitor keratinocytes.

BACKGROUND: Melanin is synthesized by melanocytes in the basal layer of the epidermis. When transferred to surrounding keratinocytes melanin is the key ultraviolet radiation-protective biopolymer responsible for skin pigmentation. Most melanin is observable in the proliferative basal layer of the epidermis and only sparsely distributed in the stratifying/differentiating epidermis. The latter has been explained as 'melanin degradation' in suprabasal layers. OBJECTIVES: To re-evaluate the currently accepted basis for melanin distribution in human epidermis and to discover whether this pattern is altered after a regenerative stimulus. METHODS: Normal epidermis of adult human skin, at rest and after tape-stripping, was analysed by a range of (immuno)histochemical and high-resolution microscopy techniques. In vitro models of melanin granule uptake by human keratinocytes were attempted. RESULTS: We propose a different fate for melanin in the human epidermis. Our evidence indicates that the bulk of melanin is inherited only by the nondifferentiating daughter cell postmitosis in progenitor keratinocytes via asymmetric organelle inheritance. Moreover, this preferred pattern of melanin distribution can switch to a symmetric or equal daughter cell inheritance mode under conditions of stress, including regeneration. CONCLUSIONS: In this preliminary report, we provide a plausible and histologically supported explanation for how human skin pigmentation is efficiently organized in the epidermis. Steady-state epidermis pigmentation may involve much less redox-sensitive melanogenesis than previously thought, and at least some premade melanin may be available for reuse. The epidermal melanin unit may be an excellent example with which to study organelle distribution via asymmetric or symmetric inheritance in response to microenvironment and tissue demands.

Quantification of clarithromycin, its 14-hydroxy and decladinose metabolites in rat plasma, gastric juice and gastric tissue using high-performance liquid chromatography with electrochemical detection.

A rapid, selective and sensitive HPLC assay has been developed for the simultaneous analysis of clarithromycin, its 14-hydroxy-clarithromycin metabolite, and its decladinose acid degradation product, in small volumes of rat gastric juice aspirate, plasma and gastric tissue. Sample were extracted with n-hexane/2-butanol (4:1) and the internal standard was roxithromycin. A Kromasil ODS 5 micrometer(75x4.6 mm I.D.) column was used with a mobile phase consisting of acetonitrile/aqueous phosphate buffer (pH 7, 0.086 M) (45:55 v/v). The column temperature was 30 degrees C and coulometric detection was used at 850 mV using a screen voltage of 600 mV. The analysis time was less than 8 min. The limits of quantitation for clarithromycin, 14-OH clarithromycin and decladinose clarithromycin were 0.15 microgram ml(-1) or lower in plasma (0.05 ml); 0.16 microgram ml(-1) or lower in gastric juice (0.2 ml); and 0.51 microgram g(-1) or lower for gastric tissue (0.25 g). The method was linear up to at least 20.3, 15.4 and 12.5 microgram ml(-1) for clarithromycin, 14-OH-clarithromycin and decladinose, respectively, in gastric juice aspirate and plasma and up to 40.6, 30.9 and 25.0 microgram g(-1) in gastric tissue. The assay was applied to the measurement of clarithromycin, 14-OH-clarithromycin and, for the first time, decladinose clarithromycin in pharmacokinetic studies of gastric transfer of clarithromycin in individual rats.

Measurement of amoxicillin in plasma and gastric samples using high-performance liquid chromatography with fluorimetric detection.

A rapid, selective and sensitive HPLC assay has been developed for the routine analysis of amoxicillin in rat plasma, gastric juice aspirate and gastric tissue which is applicable to low concentrations of amoxicillin (<1 microg mL(-1)) or small sample volumes. Amoxicillin was converted, via an internal rearrangement, to form a fluorescent product which was subsequently recovered using liquid-liquid extraction. A Kromasil ODS 3 microm (150 x 3.2 mm I.D.) column was maintained at 40 degrees C and used with a mobile phase consisting of methanol-water (55:45, v/v). Fluorimetric detection was at an lambda(ex) of 365 nm and an lambda(em) of 445 nm. The limits of quantitation for amoxicillin were 0.1 microg mL(-1) for gastric juice aspirate (500 microL), 0.5 microg mL(-1) for plasma (50 microL) and 0.075 microg g(-1) for gastric tissue (250 mg). The method was linear up to at least 15 microg mL(-1) in gastric juice aspirate, up to 200 microg mL(-1) in plasma and up to 100 microg g(-1) in gastric tissue, with inter- and intra-day RSDs being less than 19%. The assay has been applied to the measurement of amoxicillin in rat plasma, gastric juice aspirate and gastric tissue for pharmacokinetic studies in individual rats.

Impact of acid secretion, gastritis, and mucus thickness on gastric transfer of antibiotics in rats.

BACKGROUND AND AIMS: The success of Helicobacter pylori eradication regimens depends on gastric pH, inflammation, and mucus thickness. Our aim was to investigate the effects of acid secretion, inflammation, and mucolysis on gastric antibiotic transfer. SUBJECTS AND METHODS: A total of 134 anaesthetised rats were given metronidazole, amoxicillin, or clarithromycin intravenously and gastric contents were aspirated via an indwelling cannula. Acid secretion was controlled by either omeprazole or pentagastrin while gastritis was induced by infection with H pylori or dosing with iodoacetamide. Mucolysis was achieved by instilling pronase into the gastric lumen. RESULTS: Metronidazole transfer increased with acid secretion and fell with omeprazole, independently of gastric pH. Clarithromycin was also transferred with acid but was then rapidly degraded. Omeprazole prevented this degradation, raising gastric luminal concentrations. Omeprazole did not alter amoxicillin transfer. Gastritis induced by H pylori did not alter gastric transfer of metronidazole and amoxicillin but that of clarithromycin was increased by 23%. However, gastritis induced by iodoacetamide reduced clarithromycin transfer without any effect on metronidazole or amoxicillin transfer. Pronase treatment increased amoxicillin transfer fourfold and metronidazole by 66% but reduced clarithromycin transfer by 35%. CONCLUSIONS: Metronidazole and clarithromycin are predominantly transferred with gastric acid rather than by an acid trapping mechanism. Pronase increases the appearance of amoxicillin and metronidazole in gastric secretions.