Putrescine detection

The polyamines putrescine, cadaverine, spermidine, and spermine, which are seen at elevated levels in some victims of cancer, were separated on a Technicon (The Technicon Company Chauncey, NY) TSM Amino Acid Analyzer packed with an 8% divinylbenzene-co-polystyrene sulfonated resin with post-column ninhydrin detection.111 Amines such as ethanolamine, noradrenaline, hexamethylene diamine, methoxytryptamine, spermine, and spermidine were separated from amino acids on a DC-4A cation exchange resin.112 A similar approach, using a Beckman Model 121M amino acid analyzer equipped with an AA-20 column, was also successful.113 A Polyamin-pak strong cation exchange column (JASCO) was eluted with a citrate buffer for the detection of putrescene, spermine, cadaverine, and 1,5-diaminohex-ane from rat thymus.114 A post-column o-phthaldehyde detection system was used. 

Kamei et al. [45] separated spermine, spermidine, putrescine, and cadav-erine in an ion-pair reversed-phase LC system and detected the hydrogen peroxide formed in the reaction catalyzed by the enzymes putrescine oxidase and polyamine oxidase with POCL. The same analytes were determined in a later study [46], together with the acetyl derivatives. The sensitive determination of uric acid, selectively converted to hydrogen peroxide by uricase, has been investigated by several authors [37, 47],... 

IEC was applied to determine biogenic polyamines such as putrescine (4a), cadaverine (4b), tyramine (5), histamine (6), spermidine (38), agmatine (39) and tryptamine (40), contained in aqueous trichloroacetic extracts of leafy vegetables, such as cabbage and lettuce. A cation exchange column loaded with potassium ions and a special buffer were used. Spermidine (38) was the major amine detected in this group (7-15 Xg/g fresh weight)144. 

Okuzumi et al. (1990) investigated the relationship between microflora on horse mackerel (Trachurus japonicus) and the dominant spoilage bacteria. The results of their study showed that Pseudomonas I/II, Pseudomonas III/IV-NH, Vibrio, and Photobacterium were dominant when high levels of putrescine, cadaverine, and histamine were detected. 

Dimethylamine (109), putrescine (111), and spermidine (110), isolated from various insects (Table VIII), were obtained as p,p -nitrophenylazobenzoyl, p-phenylazobenzenesulfonyl, and I-dimethylaminonaphthalene-5-sulfonyl (dan-syl) derivatives and picrates or were detected by high-performance liquid chromatography (HPLC) using the ion-exchange resin (106,343). V,V-Dimethyl-3-phenylethylamine (131) from spiders of the genus Sclerobunus (Table VIII) has been identified by mass spectral comparison with a synthetic sample (117). 

Figure 8.15 Electrophoretic separation of biogenic amines using a counterionic Indirect Fluorescence Detection (IFD) system, (a) putrescine, (b) histamine, and (c) tryptamine [127],...

The zig-zag mixer was then used as a pre-column reactor to derivatize biogenic amines such as histamine and tyramine with o-phthaldialdehyde and to detect the corresponding products with micellar electro kinetic chromatography [150]. A separation of four amines, histamine, tyramine, putrescine and tryptamine, was demonstrated in that way. 

A number of putrescine derivatives have been detected in nature for which one or two cinnamic acid analogs with amide linkages are known 4-coumaroylputrescine (2), di-4-coumaroylputrescine (3), feruloylpu-trescine (4) (subaphylline), diferuloylputrescine (5), caffeoylputrescine (6) (paucine), dicaffeoylputrescine (7), sinapoylputrescine (8), and disinapoyl-putrescine (9). Paucine, one of the first diaminoalkane alkaloids known since 1894 as a component of the seed of Pentaclethra macrophylla, was hydrolyzed (40% KOH - H20) to give putrescine and caffeic acid. Structure 6 was deduced (37) from this data together with spectroscopic data, especially mass spectra. Another derivative of putrescine, subaphylline (4), first isolated from Salsola subaphylla, was shown to be the monoferuloyl derivative of putrescine by hydrolysis (30% K0H-H20) (38). The structure elucidation of the other derivatives of putrescine (2, 3, 5, 7-9) mentioned before was undertaken in a manner similar to that of 4 and 6. Several compounds were synthesized and compared to the natural products and are summarized in Table II of Section V. 

The enzyme spermidine synthetase catalyzes the conversion of putrescine to spermidine. The separation of the substrate from the product was accomplished on a reversed phase column (Ci8) using a mobile phase of 60% methanol (v/v). The compounds in the eluent were detected by scintillation counting or by UV at 254 nm as shown in Figure 9.101 (labeled Blank). 

Following derivatization with o-phthalaldehyde, TV1 -acetylspermidine, and putrescine are separated on a Beckman Ultrasphere ion-pair column (4.6 mm x 250 mm, 5 /urn). Solvent A was composed of 0.1 M sodium acetate and 10 mM octanesulfonic acid adjusted to pH 4.5 with acetic acid. Solvent B contained 0.2 M sodium acetate (pH 4.5)-acetonitrile (10 3, v/v) with 10 mM octanesulfonic acid. A linear gradient from 35% A and 65% B to 100% B was achieved in 10 minutes, followed by continued flow of solvent B for 15 minutes. The flow rate was 1 mL/min. Postminutes, column derivatization of polyamines with o-phthalaldehyde was accomplished with a pump. Fluorescence detection was used, with excitation and emission wavelengths of 340 and 455 nm. 

Recently, multiple PCR systems have been published that use different pairs of primers to simultaneously detect lactic acid bacteria producers of histamine, tyra-mine and putrescine, the majority wine biogenic amines (Marcobal et al. 2005b Constantini et al. 2006). 

Marcobal, A., de las Rivas, B., Moreno-Arribas, M.V. Munoz, R. (2005b). Multiplex-PCR method for the Simultaneous Detection of Acid Lactic Bacteria Produdng Histamine, Tyramine and Putrescine, Three Major Biogenic Amines. J. Eood Prot., 68, 874-878. 

Another type of electronic nose was developed at Lund University in Sweden specifically to detect the odors associated with spoiled fish. In this case the sensor uses two enzymes linked to a polymer to detect the presence of amines such as histamine, putrescine, and cadaverine. Such amines are produced by microbes as fish spoil. 

Putrescine A-methyltransferase (PMT, EC 2.1.1.53) catalyses the first specific step in the biosynthesis of tropane alkaloids, cocaine and nicotine [123]. Putrescine is methylated by PMT via SAM (S-adenosylmethionine) transferring the methyl group from SAM to an amino group of putrescine. Fig. (1). This enzyme has been isolated from roots of both Nicotiana tabacum and D. stramonium [124], and the activity of this enzyme is restricted to the roots of Solanaceous species corroborated by results describing a root pericycle-specific activity in A. belladonna [125]. Nevertheless, more recently, a low mRNA pmt transcript level in leaves of N. tabacum, with a rise in transcript level after mechanical wounding has been detected [126]. 

Hair may also be an important tool for the diagnosis and monitoring of various disease states. For example, the concentrations of polyamines (e.g., putrescine, spermidine, and spermine) in the hair may be helpful in diagnosing and assessing disease activity in women with cervical or ovarian cancer. Assessing the level of polyamines in the hair shaft is preferred to measuring them in plasma and urine because the polyamine levels can vary during the day in plasma and urine. Increased levels of porphyrins in hair have been detected in patients with porphyria... 

Fig. 3-164. Separation of various polyamines. — Separator column IonPac CS1 eluent see Table 3-25 flow rate 0.6 mL/min detection fluorescence after reaction with o-phthaldialdehyde injection volume 20 pL solute concentrations 4.4 ppm putrescine, 5.1 ppm cadaverine, 7.3 ppm spermidine, and 10.1 ppm spermine.

Another source of post-mortem GHB production is putrescine (1,4-butanediamine), a biogenic polyamine initially detected in decaying animal tissues, but now known to be present in all cells, both eukaryotic and prokaryotic, where it is important for cell... 

Amorim et al. (1976, 1977) using thin-layer chromatography isolated and identified polyamines from green coffee beans putrescine and spermine. These products are degraded during roasting, except putrescine which could be detected in the light roasted coffee. 

Identified in green and roasted beans by Amorim et al. (1977) by fluorometry and densitometry of the dansyl (5-dimethylamino-l-naphthalenesulfonyl) derivative by TLC. Unlike to the other polyamines, putrescine could be detected in the light-roasted coffee (1-3 ppm), but the polyamine content does not... 

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