Polyamine metabolism in Menkes kinky hair disease

Clinical investigations of the urinary excretion of putrescine and the polyamines spermidine and spermine in a patient with Menkes kinky hair disease are reported. This disorder, characterized by intra- and extracellular copper deficiency, is associated with significant depression of diamine oxidase and monoamine oxidase activity. Urinary excretion of diamine and polyamines, monitored over a 2-month interval in a 4-month old patient with Menkes kinky hair disease, documented a 3- to 10-fold increase in the excretion of free putrescine, spermidine and spermine as well as the conjugated derivatives of putrescine and spermidine. These observations suggest that abnormalities in diamine and polyamine concentration occur in disease states in which the metabolic transformation of these compounds is impaired.     

Inhibition of intestinal epithelial DNA synthesis and adaptive hyperplasia after jejunectomy in the rat by suppression of polyamine biosynthesis

Transient increases in the activity of ornithine decarboxylase (ODC), the first and rate-limiting enzyme in polyamine biosynthesis, may be critical to initiation of cell growth. We previously reported such increases in ODC activity, and the polyamines, putrescine, and spermidine in rat ileal mucosa between days 1 and 4 after intestinal resection. During this time, there is initiation of mucosal cell hyperplasia, as measured morphologically and biochemically. Intestinal weight and mucosal thickness increase, as do mucosal DNA content and DNA synthesis. In the present study, we gave rats the specific irreversible ODC inhibitor, alpha-difluoromethyl ornithine (DFMO), beginning 3 d before jejunectomy. DFMO completely suppressed the increases in ODC activity and polyamine content in the intestinal mucosa. The suppression in ODC activity was associated with an 87% suppression of DNA synthesis, and resulted in a complete abolition of intestinal adaptation, as manifested by the absence of intestinal weight gain, increase in mucosal thickness, or increase in crypt cell production. Our results indicate that the increases in ODC activity and polyamine biosynthesis are critical for adaptive postresectional crypt cell proliferation in vivo, and that the critical step mediated by polyamines in this adaptive process is the onset of new DNA synthesis.     

Determination of polyamines in urine

A method is described for the determination of putrescine, cadaverine, spermidine and spermine in urine. The procedure involves hydrolysis of 1 ml of urine in 6 N HCl at 120° overnight, adjustment to pH 13, and extraction into isoamyl alcohol. The evaporated solution is treated with dansyl chloride, and the dansyl polyamines are chromatographed on a thin-layer plate of alumina G (Woelm). A solvent of dioxane-chloroform (1:200) allows good separation of putrescine, cadaverine and spermidine, but spermine is not always distinguishable. Dioxane-acetic acid-chloroform (2:1:97) separates cadaverine, spermidine and spermine, but putrescine is not well separated from ammonia. Preliminary treatment of urine with urease should be effected if it is required to eliminate ammonia. Dansyl polyamines are measured fluorimetrically after elution.Normal values are given for each polyamine. Elevated amounts of spermidine, and sometimes of spermine as well, are frequently found in cancer patients. Raised excretions of putrescine and cadaverine are occasionally encountered, the latter possibly due to bacterial action.     

Polyamine distribution in cellular compartments of blood and in aging erythrocytes.

Human blood was separated into pure preparations of erythrocytes, mononuclear leukocytes, polymorphonuclear leukocytes, platelets, and platelet free plasma. The mean concentrations of putrescine, spermidine, and spermine per 10(9) cells were found to be several orders of magnitude higher for leukocytes than erythrocytes. There was no significant difference between leukocyte types. Platelets and plasma had relatively low levels in proportion to the amounts contributed by erythrocytes and leukocytes to whole blood. Human erythrocytes were age-separated by density and the changes in polyamine concentrations in maturing erythrocytes were documented. There were highly significant statistical differences between young and old red blood cells for putrescine, spermidine and spermine. The clinical use of red blood cell polyamines as an indicator of the activity of the bone marrow in anemic states is suggested.     

Plasma polyamines determined by negative-ion chemical ionization/mass spectrometry.

We have developed an accurate and highly sensitive gas-chromatographic/mass-spectrometric procedure for determining di- and polyamines (putrescine, spermidine, and spermine) in plasma and erythrocytes. Deuterium-labeled analogs of putrescine, spermidine, and spermine were synthesized for use as internal standards. Trifluoroacetyl derivatives of the polyamines were formed during the procedure and then detected with negative-ion chemical ionization/mass spectrometry in combination with multiple ion monitoring. Limits of sensitivity ranged from 0.25 to 1.0 pmol of analyte injected into the instrument.     

The content of unbound polyamines in blood plasma and leukocytes of patients with polycythemia vera.

In the blood plasma and isolated leukocytes of 11 patients with polycythemia vera and 3 healthy subjects, the polyamines putrescine, spermidine and spermine were determined. The average values in the leukocytes of the healthy volunteers were found to be 1.8 +/- 1.4 nmol putrescine/10(8) cells, 3.0 +/- 0.9 nmol spermidine/10(8) cells and 12.9 +/- 3.8 nmol spermine/10(8) cells. In the plasma of healthy persons the amounts of the polyamines were below the sensitivity level of the method employed. In 4 patients with polycythemia vera no polyamines were detected. In contrast, in 7 cases 0.1 to 3 nmol polyamines/ml were found. The level of polyamines in the leukocytes of 6 of these patients was decreased and in one patient corresponded to the values found in the normal range (17.7 +/- 6.0 nmol polyamines/10(8) cells). Continued blood-letting therapy on 3 patients led to values approaching the concentrations found in normal subjects in both blood plasma and leukocytes. A decreased amount of these diamines in the leukocytes of the patients was seen to correlate with an elevated concentration in the plasma.     

Polyamines and polyamine-metabolizing enzyme activities in human semen

The content of polyamines putrescine, spermidine and spermine as well as the activities of some polyamine-metabolizing enzymes have been analyzed from normal and pathological human semen samples. Seminal fluid from semen samples showing no apparent abnormalities in semen analyses contained about 0.2 mM of putrescine, 0.1 mM of spermidine and 3 mM of spermine. Seminal plasma was found to be an exceptionally rich source of diamine oxidase activity (EC 1.4.3.6). In addition, polyamine-synthesizing enzyme activities, S-adenosylmethionine decarboxylase and spermidine synthase, were invariably found in seminal plasma. Spermatozoa contained very low or undetectable activities of 5-adenosylmethionine decarboxylase and spermidine synthase; however, a definitive diamine oxidase activity was found also in the cellular component of the semen.The activity of diamine oxidase, and especially that of spermidine synthase, was relatively high in semen samples having low sperm density (less than 20 × 10⁶ spermatozoa per ml). With increasing number of spermatozoa there was a slight decrease in both activities, but as the sperm count exceeded 100 × 10⁶ per ml the activity of diamine oxidase and spermidine synthase sharply increased. The concentration of spermine in seminal plasma showed only minor changes in relation to the number of spermatozoa in semen, the changes being roughly opposite as compared to the changes of the enzyme activities. A significant negative correlation was found between the concentration of putrescine and the activity of diamine oxidase in seminal plasma.Several properties of diamine oxidase from human seminal plasma were found to be comparable to those of other mammalian diamine oxidases. Using radioactive putrescine as the substrate the enzyme activity was inhibited by addition of unlabelled histamine, cadaverine, spermidine and spermine. The enzyme was also powerfully inhibited by various carbonyl reagents and methylglyoxal bis-(guanylhydrazone).     

Pituitary hormones and the small bowel: effect of hypophysectomy on intestinal adaptation to small bowel resection in the rat*

Abstract. The influence of pituitary hormones on intestinal adaptation to small bowel resection was studied by examining jejunal and ileal structure and function in control and in sham-operated rats, and in animals with 50% proximal or distal resection which were divided into three main groups: normally-fed, hypophysectomized, and pair-fed. The pituitary was removed 2 weeks before intestinal surgery and gut structure and function were studied 4 weeks later. The effectiveness of hypophysectomy was confirmed by histological examination of the aspirated pituitary, and by showing a significant subsequent reduction in weight of the testes and adrenals. Food intake and body weight fell significantly after removing the pituitary; intestinal surgery caused a transient further decrease in food intake. Measurements of intestinal villus height and crypt depth, indices of mucosal mass (mucosal wet weight, protein and DNA content/cm intestine), measurements of mucosal a-glucosidase activity, and in vivo galactose absorption/unit length of intestine all showed comparable results. In rats with an intact intestine, resection resulted in mucosal hyperplasia and increased segmental absorption. Following hypophysectomy, there was marked mucosal hypoplasia and hypofunction which seemed to be due largely to associated hypophagia since comparable changes were found in the pair-fed, sham-operated rats. However following pituitary removal, both distal jejunum and proximal ileum retained their capacity to regenerate though the magnitude of this adaptive change was much greater in the resected, pair-fed rats suggesting that hypophagia alone cannot explain the diminished adaptation to resection after hypophysectomy. By inference, pituitary hormones do influence the adaptive response to resection.     

A rapid gas chromatographic method for the determination of plasma polyamines and its application to the prediction of tumour response to chemotherapy

Isobutyloxycarbonyl derivatives of the polyamines, spermidine, spermine and putrescine in 1 ml plasma were analysed on a mixed phase, 1.5% SE-30, 0.15% FFAP, column by temperature-programmed gas chromatography using a nitrogen-sensitive glass bead detector. Levels in 60 normal subjects (mean ±2 S.D.) were 0.13 ± 0.08 μmol/l for spermidine, 0.02 ± 0.04 for spermine and 0.18 for putrescine. Elevated spermidine levels were found pre-treatment in only 20% of patients with advanced metastatic cancer, reflecting the lack of sensitivity of polyamines as markers for malignant disease. However, serial studies in 26 patients undergoing remission induction chemotherapy showed that a significant rise in plasma spermidine within 48 h correlated with subsequent clinical response (p < 0.001). Tumour response to chemotherapy can be predicted by the assay of plasma polyamines.     

Ornithine decarboxylase and polyamines in cholecystokinin-induced pancreatic growth in rats: effects of α-difluoromethylornithine and the CCK receptor antagonist L-364,718

Acute and long-term changes of ornithine decarboxylase and polyamines during pancreatic adaptation in response to cholecystokinin administration (1 microgram kg-1 body wt every 8 h) were studied in rats. alpha-difluoromethylornithine, an irreversible and specific inhibitor of ornithine decarboxylase, was applied simultaneously to elucidate the essential role of polyamines in pancreatic growth. In the cholecystokinin-treated animals ornithine decarboxylase activity was increased after 2 h, reached a maximum after 8 h (444.6 pmol 14CO2 h-1 mg-1 DNA, about 65-fold greater than controls, P less than 0.001) followed by a significant increase of putrescine after 6 h and spermidine after 24 h while spermine remained unchanged. The trophic parameters increased in the following time sequence: thymidine kinase (12 h), DNA polymerase (24 h), pancreatic weight (2 days), protein (2 days) and DNA (5 days). alpha-difluoromethylornithine significantly delayed the increase in ornithine decarboxylase, putrescine and spermidine as well as all trophic parameters. Increases in ornithine decarboxylase, polyamines and all trophic parameters were completely inhibited by simultaneous application of the CCK receptor antagonist L-364,718. These data indicate an important role for ornithine decarboxylase and polyamines in cholecystokinin-induced pancreatic growth in rats.     

Enhanced Yield of Food Colourant Annatto from Seeds of Bixa orellana L.: The Efficacy of Polyamines Floral Spray

Annatto is one of the important natural food colourant widely used in dairy industry. The efficiency of polyamines on augmentation of annatto pigment production in field cultivated Bixa orellana L. (Achiote) crop has been investigated. Analysis of annatto pigment profile in seeds of harvested fruits indicates an increase in annatto pigment content upon foliar treatment with the three polyamines viz, putrescine, spermidine and spermine over the untreated control plants. The augmentation of total pigment level was in the range of 16?C72, 27.9?C83.7 and 34.5?C70.79?% for floral spray of spermidine, spermine and putrescine respectively over that of water sprayed control flowers of B. orellana plant. Similarly, bixin content was 20.9?C69, 36.2?C56.15, 45.72?C65.32?% for spermidine, spermine and putrescine respectively over control flowers of Bixa plant. The best response for enhanced annatto pigment content (4.17?%) at 60?days after flowering and bixin (3.54?%) was evident with 2.5???M of spermine treatment, next best being 1???M of putrescine. This ensures polyamines as an agriculturally important phytochemical, which is an easily adoptable methodology for enhancing annatto pigment yield in the standing crop, for commercial purposes.     

Determination of erythrocytic polyamines by reversed-phase liquid chromatography.

A simple and rapid semiautomated procedure for determining polyamines in erythrocytes by high-performance liquid chromatography is described. Putrescine, spermidine, and spermine are converted to fluorescent dansyl derivatives, extracted with cyclohexane, and separated in less than 10 min on a reversed-phase C18 ODS column, with an acetonitrile-water gradient as the mobile phase. The method showed a coefficient of variation of 2.73% for spermidine and 3.27% for spermine. The respective reference values, evaluated in 10 healthy patients, were 7.88 (SD 2.09) and 5.42 (SD 1.55) mumol/L of packed erythrocytes. Only negligible amounts of putrescine were found.     

Inhibition of in vitro pyrraline formation by L-arginine and polyamines

Glycation of biomolecules such as proteins, nucleic acids and lipids leading to the formation of advanced glycation end products (AGEs) may be a major contributor to the pathological manifestations of diabetes mellitus. Several studies have shown that the chemical inhibition of AGEs formation results in attenuation of diabetic complications. We tested the in vitro inhibition of pyrraline formation on bovine serum albumin and L-lysine by L-arginine and the polyamines putrescine, cadaverine, spermidine and spermine. Among the inhibitors, L-arginine and spermine potently inhibited pyrraline formation. This effect could be related to the presence of the guanidino group in L-arginine and four amino groups in spermine, but this inhibitory effect was also shown by putrescine, cadaverine and spermidine, suggesting that these natural compounds may have a novel therapeutic potential in preventing diabetic complications. A significant unexpected observation emerged when experiments were carried out with aminoguanidine. It showed increased absorbance produced by a non-identified compound whose peak appears at 285 nm, but this aspect remains to be investigated.     

Simple, sensitive assay of polyamines by high-performance liquid chromatography with electrochemical detection after post-column reaction with immobilized polyamine oxidase

This simple, rapid liquid-chromatographic assay of urinary polyamines (putrescine, spermidine, spermine, and cadaverine) involves electrochemical detection with a post-column immobilized enzyme, polyamine oxidase (EC 1.4.3.6) from soybean seedlings. Polyamines are separated by isocratic ion-pairing reversed-phase chromatography, then enzymatically converted, with release of hydrogen peroxide, via the post-column reactor with immobilized polyamine oxidase; the hydrogen peroxide is detected by electrochemical oxidation on a platinum electrode. The detection limits for injected putrescine, spermidine, and spermine were 0.3, 0.5, 0.6, and 4 pmol, respectively, with linear ranges of two to three orders of magnitude. Reproducibility was also good, with CV values less than 7%. The efficiency of the immobilized enzyme column was not decreased after analysis of 300 urine samples. Putrescine and spermidine excretion in urine from patients with blood cancers and solid cancers was significantly increased.     

Serum and Urine Polyamines in Normal and in Short Children

The serum and urine polyamines putrescine, spermidine, and spermine were measured in 112 normal subjects from 0 to 70 yr of age, and in three groups of short children from 7 to 20 yr: 21 growth hormone (GH) deficient patients, 20 normal variant short stature children, and 9 girls with 45, X Turner's syndrome. Urine polyamines were expressed as micromoles per gram of creatinine or per kilogram body weight, and serum polyamines were expressed as nanomoles per milliliter.In normals, the three polyamines were highest in urine and serum at birth. The mean levels declined progressively with age, the rate of change decreasing with age. The mean for the normal subjects, and its 95% confidence and prediction intervals, were estimated from birth to age 70 for each serum and urine polyamine.In GH-deficient children, serum and urine values were significantly lower (P < 0.05) than the age-specific normal values (with the exception of serum spermidine and spermine), averaging 25-55% below normal. This abnormality was corrected during 1 wk of treatment with human GH.In Turner's syndrome, serum and urine values were significantly reduced (P < 0.05), averaging 35-80% below age-specific normals. GH treatment had no corrective effect.In 6 of 20 normal variant short stature children, polyamine levels were significantly (P < 0.01) subnormal, averaging 50-80% below age-specific normals in both serum and urine. Treatment with GH had no corrective effect.These data show that levels of polyamines in serum and urine are correlated with linear growth primarily during the first decade of life. Subnormal polyamine levels are generally associated with growth retardation.     

Polyamines: from molecular biology to clinical applications

The polyamines putrescine, spermidine and spermine represent a group of naturally occurring compounds exerting a bewildering number of biological effects, yet despite several decades of intensive research work, their exact physiological function remains obscure. Chemically these compounds are organic aliphatic cations with two (putrescine), three (spermidine) or four (spermine) amino or amino groups that are fully protonated at physiological pH values. Early studies showed that the polyamines are closely connected to the proliferation of animal cells. Their biosynthesis is accomplished by a concerted action of four different enzymes: ornithine decarboxylase, adenosylmethionine decarboxylase, spermidine synthase and spermine synthase. Out of these four enzyme, the two decarboxylases represent unique mammalian enzymes with an extremely short half life and dramatic inducibility in response to growth promoting stimuli. The regulation of ornithine decarboxylase, and to some extent also that of adenosylmethionine decarboxylase, is complex, showing features that do not always fit into the generally accepted rules of molecular biology. The development and introduction of specific inhibitors to the biosynthetic enzymes of the polyamines have revealed that an undisturbed synthesis of the polyamines is a prerequisite for animal cell proliferation to occur. The biosynthesis of the polyamines thus offers a meaningful target for the treatment of certain hyperproliferative diseases, most notably cancer. Although most experimental cancer models responds strikingly to treatment with polyamine antimetabolites--namely, inhibitors of various polyamine synthesizing enzymes--a real breakthrough in the treatment of human cancer has not yet occurred. It is, however, highly likely that the concept is viable. An especially interesting approach is the chemoprevention of cancer with polyamine antimetabolites, a process that appears to work in many experimental animal models. Meanwhile, the inhibition of polyamine accumulation has shown great promise in the treatment of human parasitic diseases, such as African trypanosomiasis.     

Analysis of erythrocytic polyamines by automated reverse-phase liquid chromatography

A sensitive and reproducible method for quantitation of polyamines in erythrocytes by high-performance liquid chromatography (HPLC) is described. Spermidine and spermine are first converted to fluorescent dansyl derivatives and are then separated in less than 15 minutes on a C18 reverse-phase cartridge using methanol in water mobile phase. Sensitivity of the method is 20 pmol with recoveries that average 96% and 95% for spermidine and spermine, respectively. Precision study revealed an average mean coefficient of variation of 5.42 ± 2.95 (mean ± S.D.) from four levels of the polyamines (8.2 to 354.9 nmol/g hemoglobin [Hgb]). The normal ranges for 30 apparently healthy men and women, aged 20–60 years, were 2.9–33.7 nmol/g Hgb (± 2 S.D. from mean) for spermidine and 0-20.9 nmol/g Hgb for spermine. The quantity of putrescine was negligible. Feasibility of this method was evaluated in serial specimens from an advanced-stage patient with prostate cancer who was receiving multiple-modality therapy. Results revealed that this HPLC method can be used in quantifying circulating polyamines in clinical specimens.     

Clinical relevance of polyamines as biochemical markers of tumor kinetics.

The polyamines, spermidine and spermine, and their diamine precursor, putrescine, constitute a unidirectional biosynthetic pathway whose biosynthetic enzymes and accumulation patterns appear to play important roles in the regulation of growth processes. Concentrations of these compounds in physiological fluids are low or undetectable under normal conditions, are elevated in patients with metastatic cancer, and are thought to reflect growth (putrescine concentrations) and cell turnover (spermidine concentrations) of the organism. Cancers are a broad spectrum of diseases in which there are altered growth fractions and cell-turnover fractions, and therefore cancer chemotherapeutic agents have been developed to take tumor kinetics into account. Because changes in polyamines in physiological fluids reflect cell kinetics, this review compiles evidence of their efficacy as biochemical markers of cancer and suggests their possible usefulness to clinicians in rapidly assessing tumor response to chemotherapy or to multimodality therapy.     

Polyamine compartmentalization in various human disease states

Whole blood was separated into preparations of erythrocytes, mononuclear leukocytes, polymorphonuclear leukocytes, platelets and plasma. Each preparation was analyzed for the concentration of the polyamines putrescine, spermidine and spermine. This was done in 17 controls, 14 patients with psoriasis, four patients with hereditary elliptocytosis, two patients with chronic lymphocytic leukemia and one patient each with lung cancer, non-Hodgkin's lymphoma, sickle cell anemia with mild psoriasis, and progeria. In patients with elevated blood polyamine levels, absorption onto erythrocytes was relatively common, and the spermine/spermidine ratio was useful in localizing abnormalities and characterizing the nature of the polyamine alteration. Proliferative states were associated with elevated spermine/spermidine ratios relative to controls while this relationship was reversed in erythropathies such as hereditary elliptocytosis and sickle cell anemia.