Iron and 8-Isoprostane Levels in Acute and Chronic Wounds Sim Yeoh-Ellerton, Michael C. Stacey  Journal of Investigative Dermatology  Volume 121, Issue 4, Pages 918-925 (October 2003) DOI: 10.1046/j.1523-1747.2003.12471.x Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Ferritin levels in acute and chronic wound fluid. The results for the ferritin assay on 22 pairs of chronic wound fluids showed a significant decrease in the level of ferritin during the healing of chronic leg ulcers (Wilcoxon; p<0.05). There were significantly higher levels of ferritin in chronic wound fluid at both healing phases compared to acute wound fluid (Mann–Whitney U test; p<0.001). The immunoturbidimetric assay, Tina-quant-Ferritin, was conducted using 10 μL of wound fluid. Chronic wound fluid was diluted 1 in 2 with 0.9% sodium chloride solution and acute wound fluid was assayed undiluted. The turbidimetric measurement of absorbance 0–700 nm is directly proportional to the concentration of ferritin in the wound fluid. Journal of Investigative Dermatology 2003 121, 918-925DOI: (10.1046/j.1523-1747.2003.12471.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Transferrin levels in acute and chronic wound fluid. The transferrin levels for the 22 pairs of chronic wound fluid (nonhealing and healing phases) and 12 samples of acute wound fluid showed significantly higher transferrin levels in acute wound fluid compared to nonhealing chronic wound fluid (Mann–Whitney U test; p<0.01) as denoted by an asterisk. A wound fluid volume of 2 μL was sufficient for the assay, and both acute and chronic wound fluids were assayed neat using the Tina-quant assay with a Hitachi 911 automated analyzer. Journal of Investigative Dermatology 2003 121, 918-925DOI: (10.1046/j.1523-1747.2003.12471.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Iron levels in different wound fluid types using the Ferrozine assay and ICP-AES. The Ferrozine assay was performed on eight pairs of chronic nonhealing and healing wound fluid samples and eight day 3 samples of acute wound fluid. ICP-AES was used to investigate the iron levels in 14 pairs of chronic wound fluid and 13 day 3 samples of acute wound fluid. The two independent iron measurement methods showed that total iron levels were not significantly different between the wound fluid types. Iron determination by Ferrozine assay was conducted using 100 μL of wound fluid diluted 1 in 2 with Milli-Q water. Acidification and protein precipitation was carried out using 40 μL of 10% HCl and 3.5% HCl/5% TCA, respectively. Iron in the supernatant (10 μL) was reduced using 3.5% HCl/5% TCA and 30 μL of reductant solution, and color development was achieved with 30 μL of Ferrozine reagent and 30 μL of ammonium acetate solution. ICP-AES was conducted using 100 μL of wound fluid, which was acidified and protein was precipitated as described for the Ferrozine assay. The supernatant collected was made up to 2 mL with Milli-Q water and analyzed at a wavelength of 261.187 nm. Journal of Investigative Dermatology 2003 121, 918-925DOI: (10.1046/j.1523-1747.2003.12471.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Distribution of ferritin in leg ulcer in normal skin. (A) Ferritin staining in nonhealing leg ulcer tissue. Immunohistochemistry staining for ferritin in a nonhealing leg ulcer tissue biopsy showed extensive distribution of ferritin in both the dermis and the epidermis. Ferritin staining (brown) appeared to be located intracellularly and extracellularly in the matrix as depicted at 20× magnification. (B) Ferritin staining in normal skin tissue. Immunohistochemistry staining for ferritin in normal skin tissue was considerably less or not evident in the tissue as depicted at 20× magnification. Journal of Investigative Dermatology 2003 121, 918-925DOI: (10.1046/j.1523-1747.2003.12471.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Distribution of ferric ion in leg ulcer and normal skin tissue. (A) Ferric ion staining in nonhealing leg ulcer tissue. Using Perls' Prussian blue staining, there appears to be extensive ferric ion in the dermal region of the leg ulcer tissue and very little or none evident in the epidermis. Staining (blue) was predominately distributed extracellularly in the matrix. (B) Ferric ion staining in normal skin tissue. For comparison, this is a Perls' Prussian blue staining on a normal skin tissue showing very little or no positive staining for ferric ion in the dermis or epidermis. Journal of Investigative Dermatology 2003 121, 918-925DOI: (10.1046/j.1523-1747.2003.12471.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 8-Isoprostane levels in acute and chronic wound fluids measured by EIA and GC-ECNI-MS (median and 95% CI). A total of 13 pairs of chronic wound fluid and 15 samples of acute wound fluid were analyzed by EIA. There was no significant change in the level of free 8-isoprostane observed during the healing of chronic leg ulcers. Comparison with acute wounds, however, showed that 8-isoprostane levels were significantly higher by 3–4-fold (p<0.001) in both healing stages of chronic wounds compared to acute wounds. The same trends were also observed using GC-ECNI-MS based on four pairs of chronic wound fluid and five samples of acute wound fluid. Chronic wound fluid at both healing stages exhibited higher levels of 8-isoprostane by comparison to acute wound fluid and no changes in levels during the healing of the ulcer. Determination of 8-isoprostane was carried using 150 μL of acute and chronic nonhealing and healing wound fluid. The same sample preparation was carried out prior to assay for both methods as described in Materials and Methods and the extracted sample was read at 405 nm for the EIA and detected by monitoring m/z 569 and 573 for 8-iso-PGF2α-d4. Journal of Investigative Dermatology 2003 121, 918-925DOI: (10.1046/j.1523-1747.2003.12471.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 7 Total antioxidant status in acute and chronic wound fluid (median and 95% CI). The antioxidant activity in wound fluid was assessed by measuring the total antioxidant status of 15 pairs of chronic wound fluid and 20 samples of acute wound fluid. A higher level of total antioxidant was observed in chronic nonhealing wound fluid compared to healing wound fluid and between both chronic wound fluids and acute wound fluid. For accuracy and reproducibility, Randox total antioxidant control was used during the assay. The wound fluid used in the assay was analyzed in neat concentration. Journal of Investigative Dermatology 2003 121, 918-925DOI: (10.1046/j.1523-1747.2003.12471.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions