Isolation and quantitation of cadmium-, zinc- and copper-metallothioneins by high-performance liquid chromatography-atomic absorption spectrometry.

The research involves the use of high-performance liquid chromatography and atomic absorption spectrometry to isolate and measure the amounts of cadmium, zinc, and copper-metallothioneins. The study assesses the efficacy of this method by analyzing its results against established values, revealing discrepancies in the certified metal contents of some commercially available MTs.

Research Method

• In this study, Metallothioneins (MTs) were separated and quantified by a process called reversed-phase high-performance liquid chromatography (RP-HPLC).
• This process was combined directly with atomic absorption spectrometry (AAS) to further quantify the metal contents present in the MTs.
• The MTs were then eluted (the process of extracting one material from another by washing with a solvent) from an RP-8 column with a gradient of Tris buffer pH 7.0 and methanol. The existence of these MTs were detected by their absorption of UV light at a wavelength of 220 nm.

Results

• The researchers analyzed commercially available purified MTs from the kidneys of horses and the livers of rabbits for their purity and metal composition.
• They found that one batch of the horse kidney MTs yielded only 50% of the estimated value. This means that the estimated metal content was higher than what was actually present.
• In some cases, the certified metal content provided by the provider of these MTs differed significantly from the values found by the researchers. Hence, calling into question the accuracy of the provided values.
• The researchers also tested their method with MTs from rat liver and fetal bovine liver.
• The method was found to be accurate as the metal contents found in MTs via graphite-furnace atomic absorption spectrometry corresponded well with the values provided by RP-HPLC-AAS.
• Finally, the molar ratios of cadmium, zinc and copper were calculated in MT-1, MT-2, and total MT. This ratio will provide further details on the composition of the MTs.