The effect of experimental conditions on the detection of spermine in cell extracts and tissues.

The aim of this work was to investigate the effect of experimental conditions on the visibility of polyamines. In solution the chemical shift of the three groups of peaks (at approximately 1.8, 2.1 and 3.1 ppm) were found to be pH dependent. Relaxation times in aqueous solution at pH 7.0, 298 K and 11.74 T were measured to be: putrescine (T(1) = 2.49 s, T(2) = 2.07 s), spermidine (T(1) = 1.27 s, T(2) = 1.05 s) and spermine (T(1) = 1.02 s, T(2) = 0.82 s). Simple spin-echo sequences could not be used to measure T(2) as the spins also experience phase evolution from homonuclear coupling which imposes a modulation on the T(2) decay curve. This modulation is eliminated by using CPMG sequences with an echo spacing of <500 micros. Relaxation times for spermine in solution in presence of metal ions and protein showed that metal ions had little effect on T(2); however, addition of 15 mg/ml bovine serum albumin reduced T(2) of spermine (0.41 s at 298 K and 0.19 s at 277 K) but was not as short as the T(2) of the polyamine peak in prostatic tissue (0.03 s at 277 K). The MR visibility of polyamines in prostate cell extracts, PC-3 xenograft (intact as well as extracted) and intact human prostatic tissues were investigated. Polyamines were not detected in methanol/chloroform extracts, but were visible in perchloric acid extracts of prostate tumour cells. No polyamines were detected in the HR MAS spectra of three samples of whole PC-3 xenograft tissue studied. In summary, the chemical shift of polyamine species is pH dependent, while protein binding causes peak broadening and reduction in T(2). Perchloric acid extraction improves visibility of intracellular polyamines, but whole tissue polyamines are not seen in xenografts without epithelial/ ductal structure. (c) 2009 John Wiley & Sons, Ltd.