Normal-phase high-performance liquid chromatography of tocopherols and tocotrienols. Comparison of different chromatographic columns.

Natural vitamin E is composed of eight different vitamers (alpha-, beta-, gamma- and delta-tocopherols and alpha-, beta-, gamma- and delta-tocotrienols). As these eight vitamers have different antioxidant and biological activities, it is necessary to have quantitative data on each substance separately. The aim of this study was to find universal HPLC columns for the separation of all eight components and to test if a few columns of the same material (different batches) will give reproducible results. Normal-phase HPLC separations of vitamin E compounds in a prepared mixture (containing oat extracts, palm oil and tocopherol standards) were tried on six silica, three amino and one diol columns. As shown by calculations of retention factors (k), separation factors (alpha), numbers of theoretical plates (N) and resolutions (Rs), the best separations were obtained on three silica columns and two amino columns using 4 or 5% dioxane in hexane as the mobile phase as well as on a diol column using 4% tert.-butyl methyl ether in hexane as the mobile phase.

Characterization of C3a receptor-proteins on guinea pig platelets and human polymorphonuclear leukocytes.

The expression of specific membrane receptors for C3a was determined on guinea pig C3a-sensitive (gp R+) platelets and human polymorphonuclear leukocytes (hu PMNL). Binding studies with 125I-labeled C3a from gp or hu sources and Scatchard analysis applied to the binding data revealed the existence of two receptor classes on gp R+ platelets; a high-affinity class with about 200 binding sites/cell and Kd = 1.7 x 10(-9) M, and a relatively low-affinity class with Kd = 10(-8) M and about 500 sites/cell. Hu PMNL express a homogeneous receptor class with Kd = 3 x 10(-8) M and 40,000 sites/cell. Molecular characterization of the C3a receptor on gp R+ platelets was achieved by (a) cross-linking photoaffinity-labeled receptors to bound 125I-labeled C3a; (b) photoaffinity labeling receptors with a 13-amino acid residue C3a analogue 125I-Nap-Ahx-13; and (c) use of chemical cross-linkers like disuccinimidylsuberate to cross-link receptors with 125I-C3a. All three techniques gave rise to very similar labeling patterns. With the photoaffinity labeling methods, a diffuse band pattern was observed with an apparent molecular mass of 95-123 kDa with 125I-C3a as label, and 85-105 kDa with 125I-Nap-Ahx-13 as label. Chemical cross-linking of 125I-C3a revealed three distinct bands with molecular masses of approximately 123, 108 and 95 kDa. Subtracting the contribution of the cross-linked ligands, the C3a receptor on gp R+ platelets appears to be a protein complex, consisting of one to three components with estimated molecular masses between 83-114 kDa.

Lectin histochemical aspects of vascular development in fetal mammalian skin.

The study describes lectin reactivity of the developing vascular network in fetal mammalian skin. Skin samples were taken from fetuses of different gestational age of the pig, the goat and the cat. Clearly positive, endothelial lectin staining of capillaries and smaller vessels could only be obtained during mid gestation. The sequence of lectin reactivity, demonstrating: 1st alpha-D-N-acetylglucosamine and N-acetyl-alpha-D-galactosamine, 2nd alpha-D-galactose and sialic acid, and 3rd alpha-L-fucose, sialic acid and probably alpha-D-mannose/alpha-D-glucose residues, emphasizes different stages of cell surface coat as well as of cell membrane development. Thus, the typical outgrowth period of developing integumental blood vessels is reflected histochemically.

Structural and histochemical aspects of epidermis development of fetal porcine skin.

Epidermal development of fetal porcine skin was studied in fetuses from 41 days of gestation until birth with scanning and electron microscopy techniques as well as histochemical methods, including immunohistochemistry. The porcine fetus develops a relatively thick and solid multilayered cover of epidermal cells, which is not lost before birth. It consists of tightly packed cells of the periderm and the stratum intermedium. The periderm cells are totally filled with filamentous proteins; in the intermediate cells, the filamentous proteins are concentrated in the cell periphery, forming a thick marginal zone. Immunohistochemically, the cytofilaments could be identified as cytokeratins of lower and higher molecular weights. The first thin stratum corneum lamellae are formed below the stratum intermedium at about 80-85 days of gestation.

Development of hair coat and skin glands in fetal porcine integument.

The prenatal development of the hair coat and skin glands was studied in skin samples of ten body regions of porcine fetuses (German Landrace) ranging between 40 and 114 days (birth) of gestation (15-1000 g body weight), using light microscopy and scanning electron microscopy. During the development of hair follicles, central and lateral primary hair follicles as well as secondary hair follicles can be distinguished. Initiation of primary hair follicles begins at 40-41 days of gestational age (15 g BW) and has finished at 73 days (300 g BW). The structural development of the primary hair follicle is described in detail, and is divided into twelve stages, in general agreement with those of other mammals. The secondary hair follicles formed later remain rudimentary and disappear during the birth period. The length, depth, density and angle of slope of the primary hair follicles, and the depth of the apocrine skin glands were measured. The development of hair follicle length is closely correlated with the increase in body weight. There is a similar but changing correlation for the development of hair follicle density, especially in connection with hair follicle initiation. In the development of the apocrine skin glands, a branching of the secretory tubule is conspicuous. Secretion does not begin before birth. The development of the sebaceous glands is closely related to hair follicle maturation, i.e. the formation of the first hair canal. The results are discussed in relation to findings from corresponding studies in small laboratory animals, other domestic mammals and man.

Some observations on dermis development in fetal porcine skin.

The study describes dermis development in fetal porcine skin between 40 d GA and birth. The first fibroblast arrangement occurs about 47 d GA, while between 60-65 d GA the first fibres are visible. They can be identified as collagen about 67 d GA, somewhat later the dermis shows 2 strata. At the end of median gestional life, the first birefringent lattice pattern of collagen fibre bundles is formed. The first elastic fibres cannot be demonstrated before 100 d GA.A clear increase in fibrocyte numbers occurs about 70-80 d GA, the next one at birth. The thickness development of the fetal porcine dermis is closely correlated with the increase in body weight. Additionally RNA and glycogen content developments have been studied in the fetal dermis.