Formation of Polyamines in the Rumen of Goats During Growth

Ornithine decarboxylase (E.G. 4.1.1.17) and S-adenosylmethionine decarboxylase (E.G. 4.1.1.50) and their products putrescine, spermidine and spermine were estimated in the rumen liquid from 3 groups of growing kids and 23 adult goats. Polyamines were also estimated in the feedstuff used. Marked differences in polyamine synthesis in rumen liquid were observed between the different groups of kids. Two groups of kids growing up together with adult goats had at an age of 2–4 months a peak of a few days duration in enzyme activity as well as in polyamine concentration. In these groups ornithine decarboxylase activity reached maximal values of 158±79 s (n = 4) and 100 (66–117) (n = 3) nmol[14CO2]/ml rumen liquid/h at an age of 120 and 77 days, respectively. The corresponding activity in rumen liquid from kids who were isolated from other animals was only about 1/10 of this value. By comparison ornithine decarboxylase activity in adult goats was 30.7±20 (n = 43) nmol[14CO]/ml/h. In rumen liquid from kids grown up together with adults, concentrations of the polyamines reached maximum at about the same time as ornithine decarboxylase activity. The mean maximal concentration of putrescine in the 2 groups was about 350 and 500 nmol/ml, while the corresponding value for spermidine was about 200 nmol/ml in both groups. Relatively constant and high concentration of polyamines were present in the feedstuff used. However, in growing kids the ruminai putrescine and spermidine concentration at times far exceeded those that could be accounted for by the estimated intake of polyamines by the food. The results therefore strongly indicate that polyamines are formed in considerable amounts in rumen content of kids during the phase of rapid growth. Results from a few experiments with calves also indicate that this may be true for cattle. polyamines; putrescine; spermidine; spermine; ornithine-decarboxylase; rumen liquid.     

A simple and sensitive method of analysis for histamine,putrescine, and polyamines without the use of an amino acid analyzer

Histamine, putrescine, spermidine, and spermine in rat tissues and human urine were separated on a CM-cellulose column (0.6 × 10 cm). These amines in the chromatographic eluate were determined by the reactions with o-phthalaldehyde (for histamine), fluorescamine, o-phthalaldehyde-mercaptoethanol, or 2,4,6-trinitrobenzene sulfonate (for putrescine and polyamines). The procedures are rapid and simple when popular instruments are used. The limits of determination by the present method were of the order of 0.1 to 0.2 nmol for histamine and 2 to 4 nmol for putrescine and polyamines.     

Determination of polyamines in human prostate by high-performance liquid chromatography with fluorescence detection

A high-performance liquid chromatographic method for the determination of polyamines in human prostate has been developed. This method is based on pre-column derivatization with dansyl chloride (Dns-Cl). The derivatives were separated on a μBondapak C₁₈ column (250×4.6 mm I.D.; 10 μm), and eluted with methanol and distilled water using a one-step linear gradient. The column eluate was monitored by fluorescence detection (excitation, 370 nm; emission, 506 nm). The within-assay precision of the study (C.V.) was as follows: putrescine (PUT) 2.88%, spermidine (SPD) 2.94% and spermine (SP) 1.17%. The between-assay precision (C.V.) was: PUT 2.66%, SPD 3.06%, SP 2.79%. The recovery was greater than 97%. The detection limit for PUT, SPD and SP were 0.05, 0.08 and 0.06 nmol/ml, respectively. In contrast to other studies, sample or polyamine derivatives did not require extraction with an organic solvent such as ethanol, evaporation under vacuum or other condensation procedures. This is a simple, rapid and sensitive method that can be applied to the determination of polyamines in nearly all biological tissues and body fluids, such as urine and serum.     

Assessment of caspase-4 released free AFC by RP-HPLC and fluorescence detection

A simple RP-HPLC method based on fluorescence detection was developed for the quantitation of 7-amino-4-trifluoro methylcoumarin (AFC) in cell lysates from JEG-3 choriocarcinoma cells for determination of caspase-4 activity. In contrast to the established methods of AFC detection using a fluorescence microplate reader or using a fluorescence photometer, the separation of AFC-signals from interfering fluorescence signals by a reversed phase column affords more precise quantitation of released AFC. This can be important for analyses of cell lysates with low caspase activity or experimental series with marginal differences among samples. By applying this new method, a linear dynamic range of 40pmol/mL to 3nmol/mL with a correlation coefficient of 0.9996 was achieved. Due to the short retention time ( approximately 7min), the determination of AFC by RP-HPLC under isocratic conditions requires small amounts of samples (50microL injection volume), and allows increased sample throughput. This method should be easily applied with little or no modification to other caspase assays by using the same fluorophore.     

Kinetic studies of human liver ferrochelatase. Role of endogenous metals

Ferrochelatase activity in human liver has been extensively characterized in the mitochondrial fraction by kinetic study of the enzyme in initial velocity conditions. We found that human liver mitochondrial membranes contain large amounts of endogenous metals that are substrates for the enzyme, leading to a lack of linearity of the activity as function of protein concentration. This lack of linearity is mainly due to a high zinc-chelatase activity with endogenous zinc. Under optimal experimental conditions, the maximum velocity for iron incorporation was 8.7 nmol of protoheme/h/mg of protein, and the maximum velocity for zinc incorporation was 4.3 nmol of zinc-protoporphyrin/h/mg of protein. The Michaelis constant for protoporphyrin IX was (i) dependent on the amount of protein when the overall chelatase reactions were measured but (ii) independent of the amount of protein when only zinc-chelatase activity was measured (Km = 0.5 microM). The Michaelis constants for iron and zinc were 0.35 and 0.08 microM, respectively, and the inhibitory constants for competitive incorporation of iron and zinc were KIFe/Zn = 0.12 microM and KIZn/Fe = 0.58 microM. The affinity of the enzyme for zinc lowers the actual determination of ferrochelatase activity with iron as substrate. Furthermore, when measuring ferrochelatase (e.g. in liver biopsy), endogenous zinc content in the biological sample must be taken into account.     

Androgenic control of polyamine concentrations in rat epididymis.

Unilateral orchidectomy resulted in a significant decrease in tissue content of putrescine and polyamines. However, no differences were detected when the results were expressed in terms of ng g-1 tissue. At 48 h after bilateral orchidectomy, a significant decrease in putrescine content was observed, but spermidine and spermine content were unaffected. The observed decrease in putrescine was prevented by treatment with testosterone propionate, but neither spermidine nor spermine were affected. Bilateral orchidectomy resulted in a significant decrease in the tissue content of putrescine, spermidine and spermine after 7 days. Treatment with testosterone propionate increased the content of putrescine, spermidine and spermine in the epididymis by about 200%, 92% and 34%, respectively. When results were expressed as nmol g-1, a significant decrease after castration in putrescine and spermidine, but not in spermine, was observed. Treatment with testosterone propionate restored putrescine concentration, but had no effect on spermidine and spermine concentrations. In castrated rats treated with testosterone propionate, the anti-androgen flutamide abolished the effect of the androgen on putrescine and spermidine content, but there was no effect on spermine. Acetylputrescine was not detected in the epididymis, while acetylpolyamines were detected at much lower concentrations than polyamines. After bilateral orchidectomy there was a decrease in the tissue content of all acetylpolyamines and an increase in their tissue concentration. The effect of castration on acetylpolyamine content was reversed by testosterone propionate treatment. We conclude that an active synthesis of polyamines occurs in the rat epididymis, and that this process depends upon the androgen environment. Regulation of ornithine decarboxylase activity appears to be the main step that is controlled by androgens.     

Polyamine transport systems in the LLC-PK1 renal epithelial established cell line

LLC-PK1 cells were brought to a quiescent state by treatment with DL-2-difluoromethylornithine (DFMO), a specific inhibitor of L-ornithine decarboxylase (ODC). The inhibition of ODC, which is the key enzyme for polyamine synthesis, strongly reduced the cellular content of putrescine and spermidine. The cells resumed DNA-synthesis followed by mitosis when exogenous putrescine was added. DFMO treatment strongly stimulated the putrescine uptake capability. A kinetic analysis of the initial uptake rates revealed a saturable Na+-dependent and a saturable Na+-independent pathway on top of non-saturable diffusion. The stimulation by DFMO was exclusively due to an effect on the Vmax values of the saturable pathways. The Na+-dependent transporter had a higher affinity for putrescine (apparent Km = 4.7 +/- 0.7 microM) than the Na+-independent transporter (apparent Km = 29.8 +/- 3.5 microM). As a consequence, although the latter transporter had a higher Vmax, the Na+-dependent transport was more important at a physiological putrescine concentration. Putrescine uptake by both transporters was inhibited with similar relative affinities by spermidine, spermine as well as by the antileukemic agent, methylglyoxal bis(guanylhydrazone), but not by amino acids. The activity of the Na+-dependent transporter was very much dependent on SH-group reagents, whereas the Na+-independent transporter was not affected. Both transporters were inhibited by metabolic inhibitors and by ionophores but the Na+-dependent transporter was affected to a greater extent. For both transporters there was a down-regulation in response to exogenous putrescine. This suggests that the polyamine transporters in LLC-PK1 are adaptively regulated and may contribute to the regulation of the cellular polyamine level and cellular proliferation.     

Determination of polyamines in human saliva by high-performance liquid chromatography with fluorescence detection

A high-performance liquid chromatographic method for the determination of polyamines (spermine, spermidine and putrescine) in human saliva was developed. This method is based on pre-column derivatization with o-phthaldialdehyde (OPA). The derivatives were separated on a Nucleosil ODS column (250×4.6 mm I.D.; 5 μm). The gradient elution was performed with two mobile phases A (water) and B (methanol) at a flow rate of 0.8 ml/min. The column eluate was monitored by fluorescence detection (excitation, 360 nm; emission, 510 nm). The within- and between-assay coefficients of variation for all the compounds were below 5%. The detection limits for spermine, spermidine and putrescine were 0.04, 0.05 and 0.06 nmol/ml, respectively. The recovery was greater than 90%. Our analytical technique requires neither preliminary extraction with an organic solvent, nor long multi-step procedures. For saliva samples, this is a simple, rapid and highly reproducible method that can be easily applied to the routine determination of salivary polyamines, whose levels increase early in several pathological conditions.     

Determination of pseudo-alpha- and pseudo-beta-DL-glucose by gas-liquid chromatography, high-performance liquid chromatography, and enzymatic colorimetry with glucose 2-oxidase

Three methods have been developed for measuring pseudo-alpha- and pseudo-beta-DL-glucose (pseudo-beta-D-glucose), synthetic compounds in which the ring oxygens of alpha- and beta-DL-glucose (beta-D-glucose) have been replaced by a methylene group. Moderate sensitivity in the determination of these pseudo-glucoses dissolved in human serum was obtained by GLC (0.1 nmol) and HPLC (0.5 nmol). The colorimetric determination with glucose 2-oxidase, peroxidase, and 2,2'-azino-di-(3-ethylbenzothiazoline-6-sulfonic acid) was satisfactory for the assay of pseudo-alpha- and pseudo-beta-DL-glucose (respective sensitivities: 25 and 5 nmol). The addition of hexokinase to the colorimetric assay system made it possible to eliminate glucose present in the sample, such as serum, and the remaining pseudo-alpha- or pseudo-beta-DL-glucose in the sample solution could then be measured by a colorimetric method using glucose 2-oxidase. The methods described can be used for biochemical studies involving pseudo-alpha- and pseudo-beta-DL-glucose.     

Determination of thiopurine S-methyltransferase activity in erythrocytes using 6-thioguanine as substrate and a non-extraction liquid chromatographic technique

A non-extraction high-performance liquid chromatographic (HPLC) method has been developed for the determination of 6-methylthioguanine (6-MTG), as part of the determination of thiopurine S-methyltransferase activity (TPMT) in erythrocytes. Erythrocyte lysate is added to a glass vial containing substrates and incubation buffer, which is then sealed for the rest of the analysis. Enzyme incubation, sample preparation, and analysis are then undertaken without further sample-handling steps. The need for a solvent extraction step has been overcome by heating the incubate to 85 degrees C to stop the enzyme reaction. The heat inactivation step precipitates protein which upon centrifugation forms a thin film in the bottom of the glass vial enabling the supernatant to be injected directly onto the HPLC system. The assay shows excellent precision and recovery with a within-batch imprecision giving a co-efficient of variation of 2.9% (mean=41.5 nmol 6-MTG/gHb/h, n=10) and 5.1% (mean=12.6 nmol 6-MTG/g Hb/h, n=10). The between-batch imprecision gives a co-efficient of variation of 8.2% (mean=11.1 nmol 6-MTG/gHb/h, n=11) and 7.3% (mean=41.0 nmol 6-MTG/gHb/h, n=16). Determination of the TPMT activity in 120 people shows a range of enzyme activity of 11.3-63.8 nmol 6-MTG/gHb/h with a mean and median activity of 34.8 and 34.2 nmol 6-MTG/gHb/h, respectively. TPMT is increasingly used in clinical practice to ensure optimisation of treatment with thioguanine drugs. This direct HPLC method minimises sample-handling, reduces inherent imprecision, the possibility of laboratory error and with the potential for further automation, makes it ideal for use in a regional referral laboratory.     

Rapid method for the determination of total 5-methyltetrahydrofolate in blood by liquid chromatography with fluorescence detection

A liquid chromatographic method is described for the determination of total 5-methyltetrahydrofolate (5-MTHF) in whole-blood samples. The method was applied to a survey of whole-blood total 5-MTHF levels of women at child-bearing age. To determine whole-blood total 5-MTHF content, a whole-blood sample was frozen and thawed to break red blood cells and the 5-MTHF polyglutamates were released and hydrolyzed into 5-MTHF monoglutamate by endogenous polyglutamates hydrolase in the plasma. In brief, an aliquot of 0.1 ml whole-blood sample was mixed with 0.3 ml 57 mmol/l ascorbic acid and incubated at 37 degrees C for 60 min, then diluted with 0.6 ml buffer solution (0.2 mol/l potassium phosphate dibasic and 30 mmol/l mercaptoethanol, pH 8.5). After the sample was heated at 100 degrees C for 10 min and centrifuged, the supernatant was analyzed by reversed-phase liquid chromatography with fluorescence detection. The recoveries from spiked samples were from 95 to 105% with within-day and day-to-day relative standard deviations less than 6.5%. The detection limit was estimated to be 30 nmol/l based on three times the noise level (peak to peak). Application of the method to a survey of whole-blood total 5-MTHF levels of women at child-bearing age showed that the method was reliable and suitable for the determination of blood total 5-MTHF.     

Determination of polyamines in digestive and reproductive tissues of adult Asterias vulgaris (Echinodermata: Asteroidea)

1. The polyamines spermine, spermidine and putrescine were extracted from tissues of Asterias vulgaris and quantitated using thin layer chromatography. 2. In the pyloric caeca, mean (+/- SE) levels of spermine, spermidine and putrescine were 138(15), 86(24) and 415(77) nmol/g wet wt tissue, respectively. In the testes, levels were 95(12), 13(6) and less than 6 nmol/g wet wt. In the ovaries, levels were 105(9), 11(0.8) and 15(8) nmol/g wet wt. 3. High levels of polyamines in the pyloric caeca may be related to secretion or excretion in this tissue. 4. We hypothesize that polyamines will be important in the regulation of cellular activity in these tissues during the annual reproductive cycle.     

Subcellular localization, inhibiting specificity and catalytic properties of several aminooxidases from the human placenta

Human placenta was shown to contain practically all known types of aminooxidase, i.e., Membrane-bound and soluble monoamine oxidases A that predominantly oxidize serotonin (Km approximately 0.05 and 0.2 mM) and tyramine (Km approximately 0.03 and 0.085 mM), partly oxidize phenylethylamine (Km approximately 0.013 and 0.1 mM) and slightly oxidize benzylamine; Monoamine oxidase B and its intermediate form, B', with equal sensitivity towards the inhibitors, Lilly 51641 and deprenyl. The main substrates for these enzymes are phenylethylamine (Km = 0.011 mM for the membrane-bound and 0.019 mM for the soluble enzymes); Membrane-bound and soluble benzylamine oxidases that are stable to MAO inhibitors but are highly labile towards semicarbazide and aminoguanidine and that predominantly oxidize benzylamine. The Km value for the soluble enzyme is 0.19 mM, its specific activity is 0.058 nmol aldehyde/min/mg protein, which markedly exceeds that for serum benzylamine oxidase (i.e., 0.014 nmol/min/mg) and thus excludes its serum origin; Diamine oxidase that oxidizes putrescine (Km = 0.025 mM), histamine and cadaverine and only slightly oxidizes benzylamine. One characteristic feature of the placenta is the presence of soluble MAO as well as MAO incorporated into the endoplasmic reticulum membrane (microsomes). In all probability, these enzymes are precursors of the mitochondrial enzyme. The concentration of MAO A in the mitochondria is approximately 1.3%, that in microsomes--approximately 1%, kcat = 270 and 320 min-1, respectively.     

Simultaneous determination of histamine and polyamines by capillary zone electrophoresis with 4-fluor-7-nitro-2,1,3-benzoxadiazole derivatization and fluorescence detection

Capillary zone electrophoresis (CZE) with fluorescence detection was applied to the simultaneous determination of histamine and polyamines including spermine, spermidine, diaminopropane, putrescine, cadaverine, diaminohexane with 4-fluor-7-nitro-2,1,3-benzoxadiazole (NBD-F) as the fluorescent derivatization reagent. The seven NBD-F labeled amines was separated within 200 s using 85 mM phosphate running buffer at pH 3.0. The concentration limits of these amines ranged from 5.1 x 10(-8) M for spermine to 2.1 x 10(-8) M for histamine. The relative standard deviations for migration time and peak height were less than 1.5% and 6.0%, respectively. The method was successfully applied to the analysis of biogenic amines in the lysate of tobacco mesophyll protoplasts, and spermidine and putrescine were detected in the lysate with satisfying recovery.     

A sequential injection analysis/chemiluminescent plant tissue-based biosensor system for the determination of diamine

In this paper, a new chemiluminescent plant tissue-based biosensor for diamine detection was presented by employing sequential injection analysis (SIA), which facilitates precise fluidic handling and lower consumption of sample and reagents. Pea-seedling tissue acted as the molecular recognition element and was packed in a mini-PTFE column and further incorporated in the SIA system. The analysis of diamines, such as putrescine and cadaverine, is based on an enzymatic conversion which takes place in the plant tissue column to produce hydrogen peroxide. The formed hydrogen peroxide was detected by a chemiluminescence reaction involving luminol and Co(2+). Under the optimal conditions, the linear calibration graphs were obtained within 0.2-80 microM (putrescine) and 0.5-100 microM (cadaverine). The detection limits of 0.03 and 0.06 microM were achieved for putrescine and cadaverine, respectively, along with the relative standard deviations of 2.14% and 3.08% (n=11) and a sampling frequency of 40 h(-1). The present biosensor has been used for the analysis of diamine in fish samples with an acceptable accuracy.     

Effect of nitric oxide and putrescine on antioxidative responses under NaCl stress in chickpea plants

Chickpea plants were subjected to salt stress for 48 h with 100 mM NaCl, after 50 days of growth. Other batches of plants were simultaneously treated with 0.2 mM sodium nitroprusside (NO donor) or 0.5 mM putrescine (polyamine) to examine their antioxidant effects. Sodium chloride stress adversely affected the relative water content (RWC), electrolyte leakage and lipid peroxidation in leaves. Sodium nitroprusside and putrescine could completely ameliorate the toxic effects of salt stress on electrolyte leakage and lipid peroxidation and partially on RWC. No significant decline in chlorophyll content under salt stress as well as with other treatments was observed. Sodium chloride stress activated the antioxidant defense system by increasing the activities of peroxidase (POX), catalase (CAT) superoxide dismutase (SOD) and ascorbate peroxidase (APX). However no significant effect was observed on glutathione reductase (GR) and dehydro ascorbate reductase (DHAR) activities. Both putrescine and NO had a positive effect on antioxidant enzymes under salt stress. Putrescine was more effective in scavenging superoxide radical as it increased the SOD activity under salt stress whereas nitric oxide was effective in hydrolyzing H₂O₂ by increasing the activities of CAT, POX and APX under salt stress.     

Effects of putrescine and ethephon on some oxidative stress enzyme activities and proline content in salt stressed spinach leaves

The effects of putrescine and ethephon on peroxidase (POD; EC 1.11.1.7), polyphenol oxidase (PPO; EC 1.14.18.1), catalase (CAT; EC 1.11.1.6) activities and proline content in spinach leaves under saline stress were investigated. In control conditions, putrescine increased PPO and CAT activities and proline content, but decreased POD activity. Ethephon increased these three enzyme activities but did not affect proline content. In saline conditions, putrescine increased POD and CAT activities and proline content, while it decreased PPO activity. Ethephon increased both PPO and CAT activities and proline content, but decreased POD activity. Putrescine and ethephon have opposite effects on the enzyme activities and proline accumulation because they acts as antagonists.     

Effect of α-difluoromethylornithine on polyamine and DNA synthesis in regenerating rat liver: Reversal of inhibition of DNA synthesis by putrescine

The possibility that α-difluoromethylornithine, a specific, irreversible inhibitor of ornithine decarboxylase could be used to prevent the rise in hepatic putrescine and spermidine content following partial hepatectomy was tested. Administration of α-difluoromethylornithine at a dose of 400 mg/kg every 4 h reduced hepatic putrescine to <2 nmol/g, but had only a small effect on the rise in spermidine seen at 28 h after partial hepatectomy. Such treatment also reduced the rise in DNA synthesis produced by partial hepatectomy by up to 70%. The inhibitory effect towards DNA synthesis could be reversed by administration of putrescine which increased the hepatic putrescine content to about 30–40% of that in the regenerating control livers. These results suggest that accumulation of putrescine rather than spermidine is needed for DNA synthesis after partial hepatectomy. They also suggest that part, but not all of the rise in putrescine normally seen in the liver after partial hepatectomy is needed for the enhanced DNA synthesis associated with liver regeneration. Experiments with lower doses of α-difluoromethylornithine showed that a substantial part of the rise in hepatic ornithine decarboxylase activity could be abolished without affecting either the rise in spermidine content or the increase in DNA synthesis after partial hepatectomy.     

Determination of polyamines in human tissues by precolumn derivatization with 9-fluorenylmethyl chloroformate and high-performance liquid chromatography

A high-performance liquid chromatography (HPLC) assay with fluorescence detection was developed for the determination of the polyamines putrescine, spermidine, spermine in samples of human spinal cord, cerebellum, cerebrospinal fluid (CSF), skeletal muscle, and muscle microdialysates without an extensive sample preparation. The precolumn derivatization was performed with 9-fluorenylmethyl chloroformate (FMOC), and the derivatizated polyamines were stable for at least 14 h at 4 degrees C. All polyamines were separated within 35 min. The method was checked for linearity, and mean correlation coefficient values of 0.995, 0.999, and 0.991 were achieved for putrescine, spermidine, and spermine, respectively. The within- and between-assay coefficient of variation percentages evaluated in standard solutions varied between 1.0 and 4.9% and between 1.3 and 6.9%, respectively. The corresponding values obtained in samples of human spinal cord were between 1.0 and 5.0% and between 0.6 and 5.8%. The values of the recovery, evaluated in spinal cord tissue, varied between 83.7 and 93.5%.     

Polyamines in Helianthus annuus L. during Germination under Salt Stress

The level of the three main polyamines putrescine, spermidine, and spermine and the biosynthetic enzyme arginine decarboxylase (ADC) decreased in Helianthus annuus L. seedlings subjected to increasing (50, 100, and 150 mm) NaCl concentrations. The pattern of polyamines in control plants increased during the initial 72 h and then reached a plateau. The putrescine level showed an increase of 370% after 72 h of development. The lower salt treatment slightly diminished the overall polyamine content. The highest NaCl concentration (150 mm) induced a strong putrescine diminution (from 381 to 78.9 nmol g⁻¹ FW) at 72 h whereas a small decrease in ADC activity was detected. ODC was detected in neither control nor treated plantlets during the experimental period. The level of spermidine also decreased, but the magnitude of the decay was less pronounced than putrescine. The fact that ODC was not detected and ADC activity followed a pattern similar to that of putrescine led us to suppose that the variation in putrescine content could be attributed entirely to the decrease in ADC activity. α-Difluoromethylarginine and α-difluoromethylornithine (ADC and ODC inhibitor, respectively) did not inhibit but delayed the onset of germination of sunflower seeds, and α-difluoromethylornithine increased the content of spermidine and spermine. The present data suggest that polyamines could be involved in the germination process of H. annuus seeds and in response to salt stress. Received April 14, 1997; accepted July 10, 1997