Spermidine Values Research Articles

Modulation of nutrient acquisition and polyamine pool in salt-stressed wheat (Triticum aestivum L.) plants inoculated with arbuscular mycorrhizal fungi

Two wheat (Triticumaestivum L.) cultivars, Sids 1 and Giza 168, were grown under non-saline or saline conditions (4.7 and 9.4 dS m−1) with and without arbuscular mycorrhizal fungi (AMF) inoculation. Salt stress considerably decreased root colonization, plant productivity and N, P, K+, Fe, Zn and Cu concentrations, while it increased Na+ level, particularly in Giza 168. Mycorrhizal colonization significantly enhanced plant productivity and N, P, K+, Fe, Zn and Cu acquisition, while it diminished Na+ uptake, especially in Sids 1. Salinity increased putrescine level in Giza 168, however, values of spermidine and spermine increased in Sids 1 and decreased in Giza 168. Mycorrhization changed the polyamine balance under saline conditions, an increase in putrescine level associated with low contents of spermidine and spermine in Giza 168 was observed, while Sids 1 showed a decrease in putrescine and high increase in spermidine and spermine. Moreover, mycorrhizal inoculation significantly reduced the activities of diamine oxidase and polyamine oxidase in salt-stressed wheat plants. Modulation of nutrient acquisition and polyamine pool can be one of the mechanisms used by AMF to improve wheat adaptation to saline soils. This is the first report dealing with mycorrhization effect on diamine oxidase and polyamine oxidase activities under salt stress.

Role of dietary polyamines in a phase III clinical trial of difluoromethylornithine (DFMO) and sulindac for prevention of sporadic colorectal adenomas

Background:The polyamine-inhibitory regimen difluoromethylornithine (DFMO)+sulindac has marked efficacy in preventing metachronous colorectal adenomas. Polyamines are synthesised endogenously and obtained from dietary sources. Here we investigate dietary polyamine intake and outcomes in the DFMO+sulindac colorectal adenoma prevention trial.Methods:Dietary polyamine data were available for 188 of 267 patients completing the study. Total dietary polyamine content was derived by the sum of dietary putrescine, spermine and spermidine values and categorised into two groups: highest (>75–100%) vs the lower three quartiles (0–25, 25–50 and 50–75%). Baseline tissue polyamine concentration and ODC1 genotype were determined. Logistic regression models were used for risk estimation.Results:A significant interaction was detected between dietary polyamine group and treatment with regard to adenoma recurrence (P=0.012). Significant metachronous adenoma risk reduction was observed after DFMO+sulindac treatment in dietary polyamine quartiles 1–3 (risk ratio (RR) 0.19; 95% confidence interval (CI) 0.08–0.42; P<0.0001) but not in quartile 4 (RR 1.51; 95% CI 0.53–4.29; P=0.44). However, a lower number of events in the placebo group within dietary quartile 4 confound the aforementioned risk estimates.Conclusion:These preliminary findings reveal complex relationships between diet and therapeutic prevention, and they support further clinical trial-based investigations where the dietary intervention itself is controlled.

Contents of biologically active polyamines in duck meat and giblets after slaughter and their changes during meat storage and cooking

Dietary polyamines (PA), putrescine (PUT), spermidine (SPD) and spermine (SPM), participate in numerous physiological roles, particularly in protein and DNA biosynthesis, but also in tumour growth. Reliable information on their content in foods is thus needed. We therefore determined PA content in duck meat and giblets (n=15) after extraction with perchloric acid, as dansyl derivatives, using a UPLC method. Mean PUT contents ranged between 3.0 and 5.2mgkg−1 in meat and liver. Mean SPD values were 10.5, 5.8, 30.9, 4.1 and 4.1mgkg−1 and SPM values were 49.4, 24.1, 77.0, 17.5 and 12.1mgkg−1 in breast, thigh, liver, heart and gizzard, respectively, 24h after slaughter. SPD and SPM contents decreased by about 25% during 6-month freeze-storage and by about 30–40% during 9-day cold-storage under aerobic or a modified atmosphere packagings. PA losses were higher in roasted than in stewed breast meat. Consumption of duck meat and particularly of liver can participate considerably in PA daily intake.

Role of dietary polyamines in a phase III clinical trial of DFMO and sulindac for prevention of metachronous colorectal adenomas.

1523 Background: Our group has previously demonstrated that polyamine-inhibitory regimen difluoromethylornithine (DFMO) plus sulindac vs. placebo has marked efficacy in preventing metachronous colorectal adenomas in a phase-III trial. Polyamines are synthesized endogenously but also obtained from diet. Here we investigate the role of dietary polyamines in the phase III placebo controlled colorectal adenoma prevention trial with DMFO + sulindac. Methods: Dietary polyamine data were available for 222 of 375 baseline study patients, and for 188 of 267 patients completing the study. A dietary questionnaire completed by each patient was collected at the initiation of the study. Total dietary polyamine content was derived by the sum of dietary putrescine, spermine and spermidine values and further categorized into highest (75%-100%) quartile group vs. a group with lower three quartiles (0-25, 25-50 and 50%-75%). Baseline tissue polyamine concentration was determined using HPLC methodology. Linear and logistic regression analyses were used for risk estimation. Results: A significant direct association was observed between dietary polyamine intake and tissue spermidine (p = 0.02) and spermine concentrations (p = 0.04) at baseline. Higher dietary polyamine group at baseline was associated with adenomas > 1 cm (43.6 % vs. 26.4 %; p = 0.01),high grade adenomas (32.7% vs. 20.4 %; p = 0.06) and advanced adenomas (52.7% vs. 35.9 %; p = 0.02). Among all patients, a significant interaction was observed between treatment, dietary polyamines and the risk of metachronous adenomas (p = 0.01). Therefore we stratified the final analyses (n = 188) by polyamine intake groups. We observed a significant risk reduction of metachronous adenomas from treatment with DFMO+sulindac in the lower dietary polyamine group (RR = 0.19 [0.09-0.40] p < 0.0001) and no benefit in the higher dietary polyamine group (RR = 1.04 [0.32-3.36] p = 0.94). Conclusions: Controlling dietary polyamines may be an effective strategy for preventing the occurrence of colorectal adenomas and colorectal cancer. Author Disclosure Employment or Leadership Position Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Cancer Prevention Pharmaceuticals Cancer Prevention Pharmaceuticals

Contents of biologically active polyamines in chicken meat, liver, heart and skin after slaughter and their changes during meat storage and cooking

Dietary polyamines, putrescine, spermidine (SPD) and spermine (SPM), participate in an array of important physiological roles, including tumour growth. Thus, reliable information on polyamine content in foods has been needed. We therefore determined polyamine contents in chilled chicken meat and giblets (n = 20) and skin (n = 10) 24 h after slaughter. The polyamines were determined, after extraction with perchloric acid, as dansyl derivatives, using an HPLC method. Mean SPD values were 4.8, 10.2, 11.4, 48.7 and 12.1 mg kg−1 and SPM values were 36.8, 38.0, 24.3, 133 and 82.7 mg kg−1 in breast, thigh, skin, liver and heart, respectively. Significant statistical correlations between SPD and SPM contents were observed in breast, thigh, skin and liver, whereas correlations were insignificant in heart. An increase of SPD and SPM was apparent in breasts and thighs stored at −18 °C for 6 months; however, it was significant only for SPM in thighs. The losses of both SPD and SPM were statistically insignificant during storage of aerobically packaged breasts up to 9 days at +2 °C. A significant decrease of SPM to about 60% of the initial contents was observed in both vacuum-packaged and in modified atmosphere (20% CO2 and 80% O2)-stored breasts on day 21 at +2 °C. For both SPD and SPM, roasting, grilling and frying of fresh breasts caused losses of about 40–60% of the initial contents (higher than boiling and stewing). Similarly, losses of SPM, due to roasting of breasts frozen for 3 or 6 months, were higher than those caused by stewing. Putrescine was detected only sporadically and at levels close to the detection limit of 1.0 mg kg−1 (fresh matter).

Urinary polyamine levels in patients with gastrointestinal malignancy.

Polyamine levels (putrescine, spermidine, and spermine) were determined in 24-hour urine samples by a high voltage electrophoresis technique. Eleven of 14 patients with widespread metastatic gastrointestinal cancer had two or more elevated polyamine values. Two patients with cancer confined to the colon and two with cancer spread within the pelvis had two or more elevated polyamine values. Three patients who had all known disease removed by surgery, two patients with gastric lymphoma, and one patient with a vilous adenoma of the colon had normal values. Individual values of spermidine and spermine appeared to be elevated more frequently than putrescine values in these patients.

Metabolic Stability of α-Methylated Polyamine Derivatives and Their Use as Substitutes for the Natural Polyamines

Metabolically stable polyamine derivatives may serve as useful surrogates for the natural polyamines in studies aimed to elucidate the functions of individual polyamines. Here we studied the metabolic stability of alpha-methylspermidine, alpha-methylspermine, and bis-alpha-methylspermine, which all have been reported to fulfill many of the putative physiological functions of the natural polyamines. In vivo studies were performed with the transgenic rats overexpressing spermidine/spermine N(1)-acetyltransferase. alpha-Methylspermidine effectively accumulated in the liver and did not appear to undergo any further metabolism. On the other hand, alpha-methylspermine was readily converted to alpha-methylspermidine and spermidine; similarly, bis-alpha-methylspermine was converted to alpha-methylspermidine to some extent, both conversions being inhibited by the polyamine oxidase inhibitor N(1), N(2)-bis(2,3-butadienyl)-1,4-butanediamine. Furthermore, we used recombinant polyamine oxidase, spermidine/spermine N(1)-acetyltransferase, and the recently discovered spermine oxidase in the kinetic studies. In vitro studies confirmed that methylation did not protect spermine analogs from degradation, whereas the spermidine analog was stable. Both alpha-methylspermidine and bis-alpha-methylspermine overcame the proliferative block of early liver regeneration in transgenic rats and reversed the cytostasis induced by an inhibition of ornithine decarboxylase in cultured fetal fibroblasts.

Trypanothione biosynthesis in Leishmania major

Trypanothione plays a crucial role in regulation of intracellular thiol redox balance and in defence against chemical and oxidant stress. Crithidia fasciculata requires two enzymes for the formation of trypanothione, namely glutathionylspermidine synthetase (GspS; EC 6.3.1.8) and a glutathionylspermidine-dependent trypanothione synthetase (TryS; EC 6.3.1.9), whereas Trypanosoma cruzi and Trypanosoma brucei use a broad-specificity trypanothione synthetase to make trypanothione from glutathione (GSH) and spermidine. Here, we report the identification of two genes in Leishmania major with similarity to previously identified GSPS and TRYS. GSPS is an apparent pseudogene containing two frame shift mutations and two stop codons, whereas TRYS is in a single open-reading frame. The enzyme encoded by TRYS was expressed and found to catalyse formation of trypanothione with GSH and either spermidine or glutathionylspermidine. When GSH is varied as substrate the enzyme displays substrate inhibition (apparent K m = 89 μM, K i s = 1 mM, k cat = 2 s −1). At a fixed GSH concentration, the enzyme obeys simple hyperbolic kinetics with the other substrates with apparent K m values for spermidine, glutathionylspermidine and MgATP of 940, 40 and 63 μM, respectively. Immunofluorescence and sub-cellular fractionation studies indicate that TryS localises to the cytosol of L. major promastigotes. Phylogenetic analysis of the GspS and TryS amino acid sequences suggest that in the trypanosomatids, TryS has evolved to replace the GspS/TryS complex in C. fasciculata. It also appears that the L. major still harbours a redundant GSPS pseudogene that may be currently in the process of being lost from its genome.

Properties of trypanothione synthetase from Trypanosoma brucei.

Trypanothione [N(1),N(8)-bis(glutathionyl)spermidine] plays a central role in defence against oxidant damage, ribonucleotide metabolism and in resistance to certain drugs in trypanosomatids. In Crithidia fasciculata, synthesis of trypanothione involves sequential conjugation of two molecules of glutathione (GSH) to spermidine by two enzymes: glutathionylspermidine synthetase (GspS; EC 6.3.1.8) and trypanothione synthetase (TryS; EC 6.3.1.9), whereas in Trypanosoma cruzi both steps are catalysed by an unusual TryS with broad substrate specificity. To determine which route operates in T. brucei, we have cloned and expressed a single copy gene with similarity to C. fasciculata and T. cruzi TRYS. The purified recombinant protein catalyses formation of trypanothione from either spermidine and GSH, or glutathionylspermidine and GSH. The enzyme displays high substrate inhibition with GSH as variable substrate (apparent K(m)=56 microM, K(i)(s)=37 microM, k(cat)=2.9s(-1)). At a fixed subsaturating GSH concentration (100 microM), the enzyme obeys simple hyperbolic kinetics yielding apparent K(m) values for spermidine, glutathionylspermidine and MgATP of 38, 2.4, and 7.1 microM, respectively. Recombinant TryS can also catalyse conversion of spermine to glutathionylspermine and bis(glutathionyl)spermine, as recently reported for T. cruzi. The enzyme has amidase activity that can be inhibited by iodoacetamide. Studies using GSH and polyamine analogues identified GSH as the critical determinant for recognition by the amidase domain. Thus, the biosynthesis and degradation of trypanothione are similar in African and American trypanosomes, and different from the insect trypanosomatid, C. fasciculata.

Voltage-Dependent Blockade of Connexin40 Gap Junctions by Spermine

The effects of spermine and spermidine, endogenous polyamines that block many forms of ion channels, were investigated in homotypic connexin (Cx)-40 gap junctions expressed in N2A cells. Spermine blocked up to 95% of I j through homotypic Cx40 gap junctions in a concentration- and transjunctional voltage ( V j)-dependent manner. V j was varied from 5 to 50 mV in 5-mV steps and the dissociation constants ( K m) were determined from spermine concentrations ranging from 10 μM to 2 mM. The K m values ranged from 4.9 mM to 107 μM for 8.6 ≤ V j ≤ 37.7 mV, within the physiological range of intracellular spermine for V j ≥ 20 mV. The K m values for spermidine were ≥ 5 mM. Estimates of the electrical distance ( δ) for spermine ( z = +4) and spermidine ( z = +3) were 0.96 and 0.76 respectively. Cx40 single channel conductance was 129 pS in the presence of 2-mM spermine and channel open probability was significantly reduced in a V j-dependent manner. Similar concentrations of spermine did not block I j through homotypic Cx43 gap junctions, indicating that spermine selectively blocks Cx40 gap junctions. This is contrary to our previous findings that large tetraalkylammonium ions, also known to block several forms of ion channels, block junctional currents ( I j) through homotypic connexin Cx40 and Cx43 gap junctions.

Motor depressant effects of systemically administered polyamines in mice: involvement of central NMDA receptors

The systemic administration of polyamines (s.c.) produced a dose-dependent motor depression. With high doses the depressant effect was long-lasting and the animals showed signs of toxicity. ED 50 values for spermidine and putrescine were 38, 90 and 251 mg/kg respectively. The motor depression induced by the systemic administration of N-methyl- d-aspartate (NMDA; 25 mg/kg i.p.) was used as a model for studying the interactions between polyamines and the NMDA receptor. Results indicate that (1) the motor effects elicited by NMDA are very similar to those induced by polyamines at ED 50 doses; (2) polyamines, even at non-active doses, potentiate the motor depressant effect induced by NMDA; (3) the NMDA receptor antagonist, (5 R,10 S)-(+)-5-methyl-10,11-dihydro-5 H-dibenzo[ a,d]cyclohepten-5,10-imine (MK-801; 0.5 mg/kg i.p.), abolishes the depressant effect elicited by NMDA and by polyamines, even at toxic doses; (4) amphetamine (1.5 mg/kg i.p.) does not counteract the motor depressant effects of NMDA or polyamines. On the other hand, the adenosine receptor antagonist, theophylline (30 mg/kg i.p.), counteracts NMDA- but not polyamine-induced motor depression. The concentration of polyamines in the brain is modified after their systemic administration at high doses and at the ED 50 dose of putrescine. In conclusion, the data suggest that the NMDA receptor could be a target mediating the motor effect elicited by polyamines. They also show that the quantitative analysis of the motor effects elicited by non-convulsant doses of NMDA might be a powerful tool for studying in vivo the interaction between neurotransmission systems involved in the regulation of motor activity.

Seasonal Fluctuations in Free Polyamines in Scots Pine Needles

Seasonal fluctuations in the free polyamines putrescine, spermidine and spermine were monitored in the needles of six specimens of Scots pine (Pinus sylvestris L.) in Central Finland from March, 1991 to December, 1992. Putrescine was the major polyamine in the needles and spermine occurred in the lowest concentrations. Current year needles showed higher polyamine levels than current + 1 year needles. The highest putrescine values were measured in winter and lowest in summer, but considerable variations were observed between the trees in winter. Spermidine concentrations were highest in spring. Low spermidine values were observed in most trees usually in December, but also in April and August. Spermidine did not show as regular a fluctuation as did putrescine. Low spermine values coincided with low temperatures in winter, and spermine increased during April and May in both years. The ratio putrescine/spermidine in the needles showed less variation between the trees than the absolute polyamine levels. Increase in put/spd ratio was observed in December, 1991, between March and May, 1992 and October, 1992.

Expression of spermidine/spermine N1-acetyltransferase in growing Yoshida AH-130 hepatoma cells

Activity and messenger RNA levels of spermidine/spermine N1-acetyltransferase, the rate-limiting enzyme of the polyamine interconversion pathway, were investigated in host liver and in Yoshida AH-130 ascites hepatoma cells as a function of tumor growth phases. Enzyme activity reached maximal values at day 10 in host liver (2.0-fold increase) and at days 10 and 14 in hepatoma cells (4.2- and 5.4-fold increases)--that is, when the cellular growth was nearly arrested. At day 10 the messenger RNA levels of spermidine/spermine N1-acetyltransferase were augmented concomitantly; they were about two and four times higher, respectively, in host liver and tumor cells than in control liver. The in vitro transcription rate seemed to be constant during hepatoma cell growth. Treatment of the animals with N1,N2-bis-(2,3-butadienyl)-1,4-butanediamine (MDL 72527), a specific inhibitor of polyamine oxidase, caused large accumulation of N1-acetylspermidine in hepatoma cells and in the ascitic fluid; the maximal values were reached at day 14. The levels of putrescine in inhibitor-treated rats decreased in hepatoma cells (day 5) and in ascitic fluid (days 5 and 14), whereas values of spermidine and spermine remained unchanged. The proposed role for spermidine/spermine N1-acetyltransferase-enhanced expression is to regulate the cellular polyamine pool by causing their excretion as acetylderivatives from tumor cells into the ascitic fluid, even if putrescine seems also to be excreted. Eventual repeat uptake of putrescine by hepatoma cells could contribute to the control of cellular polyamine levels.

Regulation of polyamine transport by polyamines and polyamine analogs.

Regulation of polyamine transport in murine L1210 leukemia cells was characterized in order to better understand its relationship to specific intracellular polyamines and their analogs and to quantitate the sensitivity by which it is controlled. Up-regulation of polyamine uptake was evaluated following a 48-hr treatment with a combination of biosynthetic enzyme inhibitors to deplete intracellular polyamine pools. The latter declined gradually over 48 hr and was accompanied by a steady increase in spermidine (SPD) and spermine (SPM) transport as indicated by rises in Vmax to levels approximately 4.5 times higher than control values. Restoration of individual polyamine pools during a 6-hr period following inhibitor treatment revealed that SPD and SPM uptake could not be selectively affected by specific pool changes. The effectiveness of individual polyamines in reversing inhibitor-induced stimulation of uptake was as follows: putrescine < SPD < SPM = the SPM analog, N1, N12-bis(ethyl)spermine (BESPM). In contrast to stimulation of transport, down-regulation by exogenous polyamines or analogs occurred rapidly and in response to subtle increases in intracellular pools. Following a 1-hr exposure to 10 microM BESPM, Vmax values for SPD and SPM fell by 70%, whereas the analog pool increased to only 400-500 pmol/10(6) cells--about 15-20% of the total polyamine pool (approximately 2.8 nmol/10(6) cells). SPM produced nearly identical regulatory effects on transport kinetics. Both BESPM and SPM were even more effective at down-regulating transport that had been previously stimulated four to fivefold by polyamine depletion achieved with enzyme inhibitors. A dose response with BESPM at 48 hr revealed a biphasic effect on uptake whereby concentrations of analog < 3 microM produced an increase in SPD and SPM Vmax values, whereas concentrations 3 microM and higher produced a marked suppression of these values. Cells treated with 3 microM BESPM for 2 hr and placed in analog-free medium recovered transport capability in only 3 hr. Thus, whereas stimulation of polyamine transport is a relatively insensitive and slowly responsive process that tends to parallel polyamine depletion, down-regulation of polyamine transport by exogenous polyamines and analogs and its reversal are rapidly responsive events that correlate with relatively small (i.e., 15-20%) changes in intracellular polyamine pools.

Polyamine N-acetyltransferase from Fasciola hepatica

A cytosolic polyamine N-acetyltransferase which catalyses polyamine and diamine acetylation has been partially purified from the liver fluke Fasciola hepatica. The enzyme has an apparent M r of 50 000 and unlike the corresponding mammalian liver counterpart is capable of putrescine acetylation. Among the substrates tested, spermidine had the highest reaction rate but putrescine had a lower K m value. The K m values for spermidine, spermine, norspermidine, putrescine, cadavarine and 1,3-diaminopropane were 20 μM, 1.30 mM, 20 μM, 7 μM, 10 μM and 50 μM, respectively. Acetylated polyamines were also substrates for the trematode acetylase, but histones were inactive. The partially purified enzyme had no deacetylase activity. The K m for acetyl-CoA was 4.4 μM. Coenzyme A was strongly inhibitory with a K i value of 5.3 μM. Bis(benzyl)polyamine analogue MDL 27695 was a potent competitive inhibitor of the enzyme with a K i of 22 μM. Inhibition by 1,4-dimethylputrescine was non-competitive and had a K i value of 15 μM. The trematode acetylase is highly dependent on sulfhydryl groups for its activity. As had been reported in nematodes, polyamine acetylation could represent a process by which trematodes convert excess polyamines to forms suitable for transport and excretion. On the other hand, this could be the regulatory step of a functional interconversion pathway in these parasites.

Increased synthesis of N1-acetylspermidine in hepatic preneoplastic nodules and hepatomas

Hepatic preneoplastic nodules and hepatomas obtained in a multistep protocol of rat hepatocarcinogenesis (diethylnitrosamine, 2-acetylaminofluorene, and partial hepatectomy) showed high values of spermidine N 1-acetyltransferase activity, the rate-limiting enzyme in polyamine interconversion. Such an increase was associated with the appearance of N 1-acetylspermidine, an enhancement in putrescine, and a decline in spermine. Such changes suggest an activation of the interconversion pathway of high into lower polyamines in hepatic preneoplastic nodules and hepatomas.

Spermidine radioimmunoassay in monitor of precancerous lesions of esophagus

Serum spermidine was assayed by radioimmunoassay in different stages of esophageal carcinogenesis in the population from high risk area of esophageal cancer, Linxian County. The serum spermidine values were 76.94 +/- 74.38 ng/ml in 36 normal individuals; 115.71 +/- 113.45 ng/ml in 35 patients with marked epithelial hyperplasia (MEH) and 294.48 +/- 135.36 ng/ml in 31 patients with early esophageal cancer. Patients with MEH were given intervention treatment by Aminoretinoic Ester or Anticancer B or placebo (Starch) as controls. One year later, samples from the population were collected again for serum spermidine measurement. The values were 95.8 +/- 68.2 ng/ml in 27 normals; 125.1 +/- 72.9 ng/ml in 62 patients with MEH treated by Anticancer B; 125.6 +/- 117.2 ng/ml in 64 patients with MEH by Aminoretinoic Ester; 162.4 +/- 76.6 ng/ml in 62 controls and 210.5 +/- 182.9 ng/ml in 44 patients with early esophageal cancer. The results showed that spermidine radioimmunoassay could reflect the tendency of esophageal precancerous changes toward cancer or back to normal. They can be taken as a mid-way monitor indicator for tumor-blocking drugs. Also, it could be of value in the early diagnosis of esophageal cancer.

Decreased Urinary Polyamines in Patients with Psoriasis Treated with Etretinate

Oral administration of the aromatic retinoid etretinate is effective therapy for psoriasis and other epidermal hyperproliferative disorders. Since polyamine metabolism is known to be important in cell growth and differentiation, we measured urinary levels of the polyamines putrescine, spermidine, and spermine as a reflection of cutaneous polyamine metabolism in 19 psoriatic patients treated with etretinate for 16 weeks. Using thin-layer chromatography, polyamine determinations were performed on urine collected pretherapy, during therapy, and 8 weeks after therapy was concluded. Good to excellent clearing of psoriasis occurred in 18 of 19 patients. All urinary polyamines showed a downward trend in the first week of therapy, prior to significant clinical improvement. At week 16 of therapy, the greatest reduction in mean urinary polyamine content occurred. Mean putrescine levels decreased from pretherapy to week 16 by 27% (p less than 0.001), mean spermidine values fell by 34% (p less than 0.001), and mean spermine levels declined by 37% (p = 0.005). These data are consistent with the hypothesis that etretinate inhibits polyamine biosynthesis.

Determination of polyamines in human blood by electron-capture gas—liquid chromatography

The present work was undertaken to develop a sensitive and selective method for the estimation of putrescine, spermidine and spermine in human blood employing electroncapture gas—liquid chromatography. Polyamines were derivatized with heptafluorobutyric anhydride. The heptafluorobutyric derivatives of polyamines could be well resolved within 15 min under a temperature programme. The detection limit was 0.1 pmol for putrescine and cadaverine, and 0.02 pmol for spermidine and spermine. The method was applied to polyamine determinations in erythrocytes from human blood. For pre-separation of the polyamines from other compounds, a simple clean-up method utilizing an activated Permutit has been devised. Major interfering substances could be removed by the batchwise Permutit treatment. The mean values of spermidine and spermine concentrations, and the spermidine/ spermine ratio in erythrocytes obtained from normal subjects ( n = 11) were similar to reported values. The analytical procedure is thought to be applicable to various biological materials.

Purification and characterization of extracellular polyamine oxidase produced by Penicillium sp. No. PO-1

An extracellular polyamine oxidase produced by Penicillium sp. No. PO-1 was completely purified using the chromatofocusing method with a very high yield (93%) of the activity. The enzyme was composed of two identical subunits ( M r 64000) and contained FAD. The optimal pH for activity was approx. 4.0. The enzyme oxidized spermidine and spermine. K m and V max values for spermidine were respectively 8.2 μM and 16.4 μmol H 2O 2/mg protein per min. Corresponding values for spermine were 5.3 μ M and 13.3 μmol H 2O 2/mg protein per min. The enzyme attacked the secondary amino group of spermidine and spermine, and produced putrescine, 3-aminopropionaldehyde and H 2O 2. The enzyme activity was completely inhibited by phenylhy- drazine. However, sulfhydryl reagents showed no effect on the activity. It is expected that the enzyme will be useful in determining the amount of polyamine in body fluids.