2, University of Tehran, Tehran, Iran
Cell labeling with superparamagnetic iron oxides (SPIO) is becoming a routine procedure in cellular magnetic resonance imaging (MRI). Quantifying the intracellular iron in labeled cells is a prerequisite for determining the number of accumulated cells by quantitative MRI studies. To establish the most sensitive and reproducible method for measuring iron concentration in magnetically labeled cells, we investigated and compared four different methods using an ultraviolet-visible (UV/VIS) spectrophotometer. Background spectra were obtained for 5 and 10 M hydrochloric acids, a mixture of 100 mM citric acid plus ascorbic acid and bathophenanthroline sulphonate (BPS), and a mixture of 5 M hydrochloric acid plus 5% ferrocyanide. Spectra of the same solutions containing either 10 or 5 µg/mL iron oxides were also created to determine the peak absorbance wavelengths for the dissolved iron. In addition, different known iron concentrations were used to obtain calibration lines for each method. Based on the calibration factors, iron was measured in samples with a known amount of iron and in labeled cells. Methods based on the use of 10 M hydrochloric acid underestimated iron concentration in all experiments; for this method to give an accurate measurement, iron concentration in sample needs to be at least 3 µg/mL.
Cellular magnetic resonance imaging (MRI) techniques enable in vivo imaging and tracking of in vivo-administered magnetically labeled cells. Labeling of cells with ferumoxides or other superparamagnetic iron oxides (SPIO) or ultra-small SPIO (USPIO) is therefore an important step and is becoming a routine procedure in cellular MRI (1,2,3,4,5,6,7). Accurate MRI quantification of in vivo-accumulated labeled cells requires a reliable and sensitive method for determining the concentration of intracellular iron in labeled cells before their in vivo administration. Quantitative analysis of intracellular iron concentration can be accomplished using a variety of analytical methods. Still, one of the most important factors that needs to be considered when quantifying intracellular iron concentration is the sensitivity of the method and its ability to determine the low concentrations of iron.
Available commercial kits for determining iron concentration in various types of solutions (e.g., QuantiChrom Iron Assay kit; BioAssay Systems, Hayward, CA, USA) are not sensitive enough for measuring low iron concentrations that are usually used for labeling cells with the USPIO nanoparticles. Nuclear magnetic resonance (NMR) relaxometric methods can also be employed to measure the concentration of iron, and these are based on the linear relationship between iron content and NMR relaxation rates 1/T1 or 1/T2 (8). By using either custom designed equipment or commercially available MRI scanners, T1 and T2 relaxation rates obtained from samples are compared with the known iron concentration in serial dilutions of iron solution that is used for generating standard calibration curve. However, the MRI method is rather cumbersome, and MRI equipment is not readily available for routine use in many cell biology laboratories. Inductively coupled plasma mass spectrometry (ICP-MS) and optical emission spectroscopy (OES) are rapid, sensitive analytical tools that have also been used to determine iron concentration in cells (9,10). Most of the described techniques can be performed in standard laboratory settings; however ICP-MS is not available in all laboratories that are involved in magnetic labeling of cells, and the samples for ICP-MS are usually sent out to core facility or analytical laboratory for analysis. Various groups have utilized different methods to determine iron concentration in biological samples, and while ICP-MS may be considered as a more accurate method, so far none of the methods has been identified as gold standard for measuring intracellular iron concentration. Moreover, sensitivity of method in determining nanogram levels of iron is another important factor to be considered when choosing the appropriate method.
Among various methods that are available for determining the concentration of iron in labeled cells (11,12,13,14), the most commonly used are spectrophotometric methods that rely on acid digestion of cells followed by the Ferrozine-based assay for iron. Ferrozine is an iron-chelating agent that forms a complex with ferrous iron (Fe2+) and exhibits characteristic UV/VIS absorption at 562 nm. However, many investigators who have used spectrophotometric methods differ in regard to the chemical composition as well as the wavelength to be used in the assay (6,15). In addition, methods using different concentrations of hydrochloric acid alone or in combination with potassium ferrocyanide have been reported (15). Moreover, there has been no consensus among the investigators on the absorbance wavelength even when only hydrochloric acid was used (15,16). Previously, our group has also reported various methods for determining iron concentration in labeled cells such as MRI imaging relaxometry-based method and Ferrozine-based spectrophotometric assay (6,7,8,14).
To establish the most sensitive and reproducible method for determining dissolved iron concentration, we investigated and compared four distinct UV/VIS spectrophotometric methods. This was achieved by determining the following three parameters: (i) peak absorbance values for solutions alone that are used in each method; (ii) peak absorbance values for iron dissolved in the solution specific for each method; and (iii) separate calibration line calculation for each method, based on the peak absorbance values of iron dissolved in specific solution for the given method. Finally, to confirm the accuracy and validity of methods, we measured the concentration of iron in the samples of known iron concentration and separately tested the recovery of iron by each method.