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

Abstract

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.

Quantification of metabolite concentrations in benign and malignant prostate tissues using 3D proton MR spectroscopic imaging

“…Note that a surface coil was the only acceptable alternative for the recruitment of healthy volunteers. Our Spm T 1 result lies between published values of ∼200 and 1025 msec measured at 11.7 T . Spm dissolved in deuterium oxide and healthy‐appearing prostate tissues were used in these experiments.…”

“…Concentration 40 188 mM of water in the prostate served as the concentration reference . It was assumed that 12 protons contribute to the Spm intensity at 3.1 ppm …”

1H MR spectroscopic imaging of the prostate at 7T using spectral‐spatial pulses

“…Without additional strong couplings present, this would lead to an in‐phase signal of spermine at 3.1 ppm, independent of TE. However, the spectral shape of the 1.8‐ppm spermine resonance in high resolution spectra is indicative for the presence of nonequivalent spins , and the existence of a strong coupling between these protons has also been shown in theoretical calculations . The strong J‐coupling slightly affects the refocusing effect, but the signal of spermine at 3.1 ppm was mostly absorptive at different TEs.…”

“…The strong J‐coupling slightly affects the refocusing effect, but the signal of spermine at 3.1 ppm was mostly absorptive at different TEs. The other polyamines present in prostate tissue, spermidine, and putrescine show structurally and spectrally close resemblance to spermine . Therefore, it is expected that J‐coupling refocusing also occurs in these metabolites.…”

The reduced mobility of the biogenic amines: trimethylamine, putrescine, cadaverine, spermidine and spermine

“…Several analytical methods gave been used to measure the concentration of biogenic amines in food products and body fluids (vaginal discharge, urine and saliva) [1,5,[17][18][19][20]24]. Most of these techniques are labor intensive, slow, costly or lack sensitivity or specificity.…”