Preliminary studies were made on the incorporation of C14-labeled glycine and proline into tissue cultures of rabbit stromal cells, excised rabbit corneas, and rabbit corneas in vivo. Some evidence has been presented that this incorporation reflects synthesis, chiefly into proteins. With rabbit corneas in vitro, no evidence was found that the presence of epithelium increased the incorporation of amino acid into constituents of the stroma. During in vivo incorporation it was found that the proteins of the endothelium were about twice and the proteins of the epithelium about four times as active as those of the stroma.
The normal rabbit cornea was found to contain an average of 22.7% solids (77.3% moisture), nitrogen comprising 14.7% of the dry weight. On a dry weight basis the cornea was composed of 10% as epithelium, 87% as stroma, and 3% as endothelium plus Descemet's membrane. The solid and nitrogen content of the stroma was nearly identical with that of the whole cornea. Endothelium plus Descemet's membrane and epithelium layers were relatively low in nitrogen content (10 and 12% of the dry weight, respectively) while the epithelium was relatively high in solids (30%). The partition of nitrogen, as protein and nonprotein, soluble or insoluble in physiological saline, was estimated for each corneal layer.
The normal rabbit cornea was found to contain an average of 22.7% solids (77.3% moisture), nitrogen comprising 14.7% of the dry weight. On a dry weight basis the cornea was composed of 10% as epithelium, 87% as stroma, and 3% as endothelium plus Descemet's membrane. The solid and nitrogen content of the stroma was nearly identical with that of the whole cornea. Endothelium plus Descemet's membrane and epithelium layers were relatively low in nitrogen content (10 and 12% of the dry weight, respectively) while the epithelium was relatively high in solids (30%). The partition of nitrogen, as protein and nonprotein, soluble or insoluble in physiological saline, was estimated for each corneal layer.
Background: Asthma is one of the common conditions clinicians encounter in children. Various studies report the association of poor vitamin D (Vit D) status of mothers during pregnancy, with an increased risk of asthma in their children, especially in HIV-positive mothers. Simultaneous measurement of Vit D metabolites, rather than measuring total Vit D, would improve assessment of Vit D metabolism in these children. This study aimed to validate a high-performance liquid chromatographic (HPLC) method using ultraviolet detection for the measurement of five Vit D isoforms (25 OHD2 and D3; 1,25 [OH]2D2 and D3 and 24,25 [OH]2D) simultaneously.
Methodology: Serum samples from 80 paediatric patients with asthma were subjected to solid phase extraction and 4-phenyl-1,2,4-triazole-3,5-dione-derivatisation. Chromatographic separation was achieved with a pentafluoro-phenylpropyl (PFPP) analytical column. Total 25 OH Vit D was measured on an automated immunochemistry analyser for comparison purposes.
Results and discussion: Good linearity was achieved for 25 OHD2, 25 OHD3 and 24,25 (OH)2D with r2 ≥ 0.99, with limits of detection ≤ 5.0 ng/ml and lower limits of quantitation ≤ 10.0 ng/m. The 1,25 (OH)2D metabolites, present in the blood at pg/ml levels, could not be quantified within the required ranges. Comparing total 25 OH Vit D immunoassay and HPLC results showed a poor correlation (r2 = 0.073), possibly due to cross-reactivity of the antibodies in the immunoassay with other Vit D isoforms.
Conclusion: This method provides a precise and accurate, with a potential for high throughput, determination of Vit D isoforms in the routine laboratory.
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