Abstract
A simple and highly sensitive procedure is described enabling the simultaneous determination of biogenic polyamines (PAs) and their related monoacetyl derivatives in stomach tissue. The method is based on HPLC using octane sulfonate as an ion-pairing agent employed in acetate buffers at pH 4.5. The application is accompanied with fluorescence detection followed by post-column derivatization with o-phthaldialdehyde at room temperature (20±0.5°C). N1- and N8-acetylspermidines (ASPD) can be determined with this method in the same run without performing any special procedures or pre-purification in concentrations exceeding 8.5 pmoles. The variability in reproducibility of the day-today precision and duplicate determination, and simultaneous determination of standard mixture and biological samples were found < 2%. The mean (± s.e.mean) retention times (n=12) for putrescine (Put), N1-ASPD, N8-ASPD, spermidine (Spd) and spermine (Spm) are 8.97±0.025; 17.64±0.063; 18.99±0.133; 28.20±0.070 and 39.81±0.098 min, respectively. The method was applied to determine PAs and specifically N1- and N8-ASPD in glandular part of stomach tissue of fasting rats (STFR) without any interference with endogenous aminoacids, histamine, and other reactive moieties. PAs and both mono-ASPD have been successfully determined in the STFR and the values are as follows: Put 37.2±10.1; N1-ASPD 5.88±0.48; N8-ASPD 4.43±0.94; Spd 750.7±22.7 and Spm 618.2±37.4 nmole/g of wet tissue. Information on gastric tissue polyamines and their acetylated derivatives may be useful in understanding the mechanism of drugs or agents that play some part in gastric ulcer production or its repair mechanisms.
Highlights
The polyamines spermidine, spermine, their precursor diamine putrescine and their acetylated derivatives are considered to be universally distributed in the living cells and have been postulated to play an important role in the control of cellular growth
We report a specific modified HPLC-fluorescence assay method of Seiler and Knödgen [25] with modifications by Marchant et al [30] for diamines, polyamines and their acetylated derivatives in pmole range in gastric tissue in a single run
The results show that the fluorescence quantum yield of the derivatives is different with different polyamines
Summary
The polyamines spermidine, spermine, their precursor diamine putrescine and their acetylated derivatives are considered to be universally distributed in the living cells and have been postulated to play an important role in the control of cellular growth. Polyamines are implicated to play an important role in cell proliferation, ulcer healing [1, 2], acid secretion inhibition [3] and gastroprotection by epidermal growth factor [4] that has been demonstrated to stimulate gastric mucus synthesis [4,5,6,7]. TLC technique usually require extensive sample pretreatment and labor and variability in tissue sample contents and their excretory products can cause problem with the analysis
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