Substitution of 0.462 µM-1 cm-1 and cell count numbers into Equation 1 yielded MCH values. The 7-day K562 induction time course (Figure 4B) produced a mean maximum MCH value of 7.31 pg/cell in induced samples compared to only 0.983 pg/ cell in the uninduced controls (Day 7). The induced value is significantly lower than the normal red blood cell level of 27–33 pg/cell (31), which has only been approached previously in K562 cells with glutamine starvation combined with hemin treatment (32). The 5-day MEL induction time course (Figure 4C) resulted in a mean maximum MCH of 4.47 pg/cell in induced cultures and 0.583 pg/cell in uninduced cultures (Day 5). These values indicate that cellular hemoglobin levels are much lower in MEL cells than K562 cells; however, this is not unexpected as the average volume of a K562 cell is more than three times that of an MEL cell.
The results of additional studies that validate the ability of the CLARiTY system to quantitate hemoglobin in MEL cells under various conditions can be found in the Supplementary Material. Specifically, the ef fects of hypoxia (Supplementary Figure S2), glutamine starvation (Supplementary Figure S3), iron and ALA supplementation (Supplementary Figure S4A), and SA treatment (Supplementary Figure S4B) on heme production and cell proliferation are described.
The CLARiTY, pyridine hemochromogen, and fluorescence assays each have unique strengths and weaknesses for the measurement of hemoproteins. The pyridine hemochromogen assay has the largest linear range of operation and the highest analytical sensitivity, but it is also the most labor-intensive of the three approaches. The ability of the fluorescence assay to detect extremely low concentrations of heme is well-known, yet the processes of derivatization and associated dilution considerably of fset this at tribute. Additionally, fluorescence requires calibration on a day-to-day basis due to variables such as temperature and lamp intensity. In contrast, the consistency of the CLARiTY between sample types was clearly established here in defining equivalent εHb values for whole-cell resuspensions of mouse blood and differentiating MEL and K562 cells. The CLARiTY also maintained normalized limits of detection and quantitation that were comparable, if not superior, to the other assays. Although the linear range of the CLARiTY is smaller than that of the other methods, this parameter appears to be sample-dependent and is significantly larger for whole-cell samples than for standard solutions of hemoglobin. Further, preliminary data collected on the CLARiTY shortly before publication of this work indicate that an empirically based absorbance correction method from Javorfi et al. (33) can produce a greater LOL and linear dynamic range than the Fry Equation. Perhaps most importantly, the CLARiTY system is able to quantitate protein-associated heme in situ quickly and without sample derivatization or destruction, permitting downstream applications and/or analyses. Author contributions
J.R.M., H.A.D., and R.B.P. designed the study. J.R.M. per formed the experiments, processed the data, conducted the statistical analysis, and wrote the manuscript. H.A.D. edited the manuscript, provided laboratory resources, and obtained funding. J.S.B. provided technical support and assisted with data processing.
Funding for this project was provided by NIH grant R01-DK096051. We would like to thank A. Medlock, K. Mohler, and T. Dailey for their assistance with the CLARiTY system and cell culture studies. We acknowledge the comments and suggestions provided by J. DeSa Lorenz, R. Desa, P. Boxrud, and K. Solntsev from Olis, Inc. during the presubmission process. This paper is subject to the NIH Public Access Policy.
The authors declare no competing interests.
Address correspondence to Harry A. Dailey, Biomedical and Health Sciences Institute, Paul D. Coverdell Center, 500 DW Brooks Drive, Athens, GA 30602-7394. E-mail: email@example.com
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