Clinica Chimica Acta 344 (2004) 23 – 35 www.elsevier.com/locate/clinchim
Review
Polyamines as clinical laboratory tools A. Gugliucci * Laboratory of Biochemistry, Division of Basic Medical Sciences, Touro University College of Osteopathic Medicine, 1310 Johnson Lane, Mare Island, Vallejo, CA 94592, USA Received 9 February 2004; received in revised form 25 February 2004; accepted 25 February 2004
Abstract Since their discovery by Antoni van Leeuwenhoek in 1678 until the recent development of transgenic mice expressing proteins altering polyamine levels in a tissue-specific manner, polyamines have been the object of intense research efforts which have shed light on several biological and pathological processes. From the discovery of a particular form of proteasome regulation of the catabolism of the key regulatory enzyme in their synthetic pathway, to the experimental cancer treatment or prevention with polyamine antagonists or inhibitors of the latter enzyme, a whole spectrum of interests can be revealed. Still, many aspects of their functions remain elusive and difficulties inherent in their analysis, which relies on sophisticated highperformance liquid chromatographic (HPLC) methods, and the lack of standardization; have hampered the transit from the research realm to the standard clinical laboratory domain. Their assay in biological fluids has been used for cancer diagnosis and for monitoring anticancer treatment. In this article, we attempt to provide an overview of polyamine structure, nutritional value, metabolism, and physiological roles. Next, we will summarize the main analytical methods on which we count, and finally we willaddress their role in diagnosis of cancer as well their proposed role as antioxidant and antiglycation agents. D 2004 Elsevier B.V. All rights reserved. Keywords: Spermine; Spermidine; Glycation; Cancer; Ornithine decarboxylase; Free radicals
1. Introduction The polyamines putrescine, spermidine, and spermine are a group of naturally occurring compounds exerting a large number of biological effects, yet despite several decades of intensive research work, the mode of action of the polyamines at the molecular level is largely unknown [1 – 11]. Polyamines are highly regulated polycations that are essentially in-
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volved in cell growth and differentiation [2– 8]. They bear unique structural features of regularly spaced positive charges interrupted by hydrophobic methylene bridges, as shown in Fig. 1 and Table 1. They influence the transcriptional and translational stages of protein synthesis, stabilize membranes, modulate neurophysiological functions and may act as intracellular messengers. They are synthesized in all eukaryotic cells from their immediate precursor, ornithine. The importance of the polyamines in cell function is reflected in a strict regulatory control of their intracellular levels. Their high concentration in sperm fluid and in circulating blood (especially in erythrocytes), their putative role in cancer develop-
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Fig. 1. Overview of the structure of the di- and polyamines as well as their main known physiological roles.
ment and treatment, their role in nutrition as well as their proposed antiglycation activity, all make these compounds interesting from the clinical biochemist’s standpoint [8 – 14]. Since their discovery by Antoni van Leeuwenhoek in 1678 until the recent development of transgenic mice expressing proteins altering polyamine levels in a tissue-specific manner, polyamines have been the object of intense research efforts, which have shed light on several biological and pathological processes. From the discovery of a particular form of proteasome regulation of the catabolism of the key regulatory enzyme in their synthetic pathway, to the experimental cancer treatment or prevention with polyamine antagonists or inhibitors of the latter enzyme, a whole spectrum of interests can be revealed. Still, many aspects of their functions remain elusive and difficulties inherent in their analysis,
Table 1 pKa of primary and secondary amino groups in polyamines Putrescine Spermidine Spermine
pK1
pK2
pK3
pK4
9.35 9.52 8.90
10.8 10.8 9.79
– 11.56 10.95
– – 11.50
which relies on sophisticated high-performance liquid chromatographic (HPLC) methods, and the lack of standardization; have hampered the transit from the research realm to the standard clinical laboratory domain. Their assay in biological fluids has been used for cancer diagnosis and for monitoring anticancer treatment. In this article, we attempt to provide a succinct overview of polyamine structure, nutritional value, metabolism, and physiological roles. Next, we will summarize the main analytical methods on which we count and finally we will address their role in diagnosis of cancer as well their proposed role as antioxidant and antiglycation agents.
2. Polyamines: structure (Fig. 1) The naturally occurring polyamines (diamine putrescine, triamine spermidine, and tetramine spermine) are ubiquitous polycations [1– 8]. They are present in all prokaryotic and eukaryotic cells thus far studied. They stabilize nucleic acids and stimulate their replication. Spermidine and spermine can bridge the major and minor grooves of DNA, acting as a clamp holding together either two different
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molecules or two distant parts of the same molecule [15 – 18]. Polyamines are essential for growth processes, and they have also been associated with carcinogenesis [19 – 21]. In 1678, van Leuwenhoek [22] reported crystals in sperm samples when left to dry, which we now know were spermine-phosphate crystals. Polyamines were re-discovered more than once over the next two centuries. Charcot reported crystals in blood samples with very high leucocyte counts, later named Charcot– Leyden crystals [23]. In 1878, Schreiner [24] identified those crystals as phosphates of a new organic base, although he proposed the wrong formula. Landenburg and Abel were the first to use the name spermine in 1888 [25]. In 1926, Dudley achieved its his synthesis, he also synthesized spermidine, and next then determined that the amine also existed in tissues [26]. No stretch of imagination is required to understand that putrescine and cadaverine had been isolated from tissues after bacterial decomposition [27]. As shown in Fig. 1, putrescine is actually a diamine; however, due to its role as obligatory precursor of the others, it is also considered a polyamine. Spermine, bearing four amino groups, is found in superior eukaryotes, while the others are ubiquitous.
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3. Cellular levels: de novo synthesis Adequate cellular polyamine levels are achieved by a careful balance between biosynthesis, degradation, and uptake of the amines [3,5,7,28 –34]. Polyamines are bound to macromolecules (mainly nucleic acids) and this pool is in equilibrium with a free polyamine pool, which accounts for up to 7– 10% of the total cell content (illustrated in Fig. 2 below). Some of the regulatory mechanisms involved in maintaining a balance in the cellular polyamine pools are truly unique. The polyamine biosynthetic pathway consists of two highly regulated enzymes, (a) ornithine decarboxylase (ODC) (1 in Fig. 2); (b) S-adenosylmethionine decarboxylase (2 in Fig. 2); (c) two constitutively expressed enzymes, spermidine synthase (3 in Fig. 2), and spermine synthase (4 in Fig. 2). Out of these four enzymes, the two decarboxylases represent unique mammalian enzymes with an extremely short half-life and dramatic inducibility in response to growth promoting stimuli. Of note, as we depict in Fig. 2, this pathway depends on adequate supply of ornithine, usually the
Fig. 2. Overview of polyamine metabolism. Note that polyamine pools are tightly controlled by synthesis, catabolism and uptake. Antizyme is a protein regulator of the activity of the main enzyme in polyamine synthesis but also a targeting system for proteasome catabolism of this enzyme. Note the link of polyamine pathway and the urea cycle and the key participation of SAM as an aminopropyl donor.
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product of the urea cycle arginase, which is then, in a way another key element for adequate polyamine synthesis. Another important element is the universal aminopropyl donor S-adenosyl methionine, a key coenzyme in this pathway. As we depict in Fig. 3, the catalysis of ornithine decarboxylation is mediated by the dimeric form of the enzyme ODC using pyridoxal phosphate as a coenzyme, lysine residues play an important role in dimer stabilization and in the classic binding to the coenzyme [29 –31]. 3.1. Ornithine decarboxylase is the key enzyme in polyamine synthesis: sui generis regulation 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 regulation of ODC is not mediated by posttranslational or allosteric mechanisms. Instead, its steady steady-state concentration depends mainly on transcription, mRNA translation and catabolism [28 – 31]. The polyamine biosynthetic enzyme ODC is degraded by the 26S proteasome via an ubiquitinindependent pathway in mammalian cells [32 –36]. Its degradation is greatly accelerated by association with the polyamine-induced regulatory protein antizyme 1
(AZ1), which binds to ODC, inhibits its activity, and targets it for proteasome degradation (Fig. 1). This unusual pathway remains an exception only found so far in polyamine metabolism. 3.2. The protein antizyme 1 controls ODC proteasome degradation Antizyme 1, a 26-kDa protein is regulated by polyamine concentration at the level of translation by programmed ribosomal frameshifting [37,38]. Recently, a further level of complexity was found: there is a protein inhibitor of antizyme 1 (AZ1), as depicted in Fig. 2. The biological half-life of the two regulatory enzymes ornithine decarboxylase and S-adenosylmethionine decarboxylase (5 – 60 min) are among the shortest known for mammalian enzymes, allowing the cell to rapidly change the cellular polyamine levels and underlying the critical importance of this regulation [5– 7,30– 36]. Reduction of the cellular polyamine pool rapidly induces growth arrest. In addition, cells may undergo apoptosis when the polyamine pools are essentially depleted [38]. An excessively high polyamine concentration, on the other hand, is toxic to the cell and may in some situations induce apoptosis [39 – 42]. Thus, it is imperative for cells to maintain their polyamine pools within rather narrow limits. The development and introduction of specific inhibitors
Fig. 3. Hypothetical diagram of ornithine decarboxylase (ODC) in its dimeric catalytic form. Note the role of pyridoxal phosphate as a coenzyme and lysine residues as key participants in stabilization and catalytic efficiency.
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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. Cellular polyamine homeostasis is achieved through a careful balance between biosynthesis, degradation, and uptake.
4. Polyamine catabolism is less well characterized (Fig. 4) Catabolism of polyamines is less well characterized, especially with regard to relative physiological hierarchy of the pathways in humans [5,9 –11,43,44].
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Many enzymes can oxidize polyamines to aldehydes, some of them are extracellular, and therefore it is not clear what role they may play in intracellular polyamine catabolism. Human urine contains unmodified polyamines together with oxidation and acetylation products as well as cyclic derivatives [5]. Two main pathways for polyamine catabolism have been described: an interconversion pathway and a terminal polyamine catabolism [5,9 – 11]. The interconversion pathway is a cyclic process, which controls polyamine turnover and is depicted in Fig. 4. In conjunction with polyamine transport, it regulates intracellular polyamine homeostasis. Putrescine, the precursor of spermidine and spermine, is exclusively
Fig. 4. Overview of polyamine catabolism. Note the role of acetyl transferase and polyamine oxidases.
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formed by decarboxylation of ornithine—as far as de novo synthesis is concerned. As pointed out above, spermidine and spermine synthase form spermidine from putrescine, and spermine from spermidine, by transfer of aminopropyl residues from decarboxylated S-adenosylmethionine. In the catabolic branch of the interconversion cycle depicted in Fig. 4, spermine is degraded to spermidine, and spermidine to putrescine [5,11]. The first step in this sequence is acetylation in the N1 position. This is followed by oxidative splitting of the acetylated polyamines, whereby the aminopropyl residues, which originated from decarboxylated S-adenosylmethionine are removed. The enzyme catalyzing this step is a polyamine oxidase (FADdependent). Terminal polyamine catabolism is catalyzed by Cu2(+)-dependent amine oxidases, of which only diamine oxidase has been well defined [43 – 46]. By oxidative deamination of a primary amino group, each intermediate of the interconversion cycle can be transformed into an aldehyde, which is further oxidized to an amino acid or a gamma-lactam [45,46]. The products of the terminal catabolism as well as the acetylated polyamines are subject to renal excretion. In addition to intracellularly synthesized polyamines, polyamines from various tissues and from exogenous sources (such as the gastrointestinal tract) may be utilized by those tissues, which have a high demand.
5. Cellular levels: uptake and dietary sources 5.1. Polyamine uptake is a saturable, carriermediated, and energy-dependent process As we mentioned above, intracellular polyamine pools are actively regulated by de novo synthesis, degradation, excretion, and import from extracellular sources [5,10,47,48]. Polyamine-specific carriers (Figs. 1 and 4) are widely distributed in prokaryotes and eukaryotes. Mammalian polyamine transport activity is also acutely controlled by cell cycle events and hormonal stimulation. Polyamine transport is a saturable, carrier-mediated, and energy-dependent process [49]. Although its physiological properties have been extensively studied, the molecular characteristics of the diamine and polyamine carrier proteins have only been elucidated in prokaryotes.
5.2. Our exogenous supply of polyamines is obtained through the diet or by synthesis in the intestinal flora The polyamines are found in fruits and vegetables, foods of animal origin (milk, eggs, fish, and meat), and fermented food products (cheese, beer, and sauerkraut) [5,50 –53]. Polyamines play an essential role in the development of the intestine [50]. It is well documented that dietary polyamines induce many of the biochemical and morphological changes involved in postnatal intestine maturation [50 – 53]. The systemic requirements for these substances are elevated during phases of intense growth or increased demand; thus, the nutritional supply can be crucial during the evolution of processes that involve a high degree of loss combined with deficits in endogenous biosynthesis [51 – 53]. On the other hand, increased tissue and organ polyamine concentrations correlate with diseases of neoplastic origin [54 –66]. It has been hypothesized that polyamine inhibition could be a therapeutic mechanism for such conditions. Polyamine levels in serum and urine of healthy human beings decline progressively with increasing age. Some major enzymes involved in the polyamine biosynthetic pathway show similar trends [67]. Hormonal induction of ornithine decarboxylase activity is strongly reduced in organs of aged animals. There is also some evidence for an age-related decrease in the level of ornithine decarboxylase and its inducibility in mammalian cells cultured in vitro [67].
6. Polyamines in the circulation: why do erythrocytes take up polyamines? Although leukocytes have the highest concentration per cell, the main circulating pool of polyamines resides in erythrocytes due to their highest relative concentration in blood [68 –72]. Serum polyamines associate with proteins and lipoproteins by virtue of their unique chemical properties. Binding to lipoproteins implies interaction of these cations with anionic charges on phospholipids on the surface of lipoproteins, namely HDL and LDL secondarily [14]. Polyamines can also form covalent adducts with proteins
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via the transglutaminase reaction, a process described for apoB and fibronectin [73]. With regard to erythrocytes, polyamines are actively taken up by specific membrane transporters [68,69,74]. Polyamines are associated with RBC membranes and cytosol [74]. Given the fact that mature red blood cells lack nuclei and do not have any biosynthetic activity, the existence of this uptake pathway has been explained on the assumption that polyamines play a protein stabilizing and/or antioxidant role [74]. We have recently put forward a hypothesis that complements and broadens this explanation, which we will summarize later [8].
7. Polyamines and cancer As mentioned above, rapidly growing tissues and chiefly tumor cells display high activities of ODC and S-adenosylmethionine decarboxylase. These facts recommended PA biosynthesis as a viable target for antineoplastic therapy. Growth-associated genes, such as c-fos and c-myc proto-oncogenes, are activated during cellular proliferative processes [77]. This activation is due to signals transmitted from the cellular membrane to its nucleus. Following mitogenic stimulation, a simultaneous activation of polyamine biosynthesis and transcription of the c-fos proto-oncogene has been described. Likewise, malignant transformation leads to an increase in polyamine biosynthesis, deregulation of ODC, and the amplification of proto-oncogenes; but the special role of each of the polyamines on proto-oncogene expression has not been explored [75 – 79]. Selective inhibitors have been developed for literally all enzymes of PA metabolism. Difluromethyl ornithine (DFMO, an ODC competitive inhibitor) and other compounds, for instance, became key tools in the elucidation of the PA metabolic system, but only few of them were efficient as inhibitors of tumor growth [75 – 79]. An interesting approach put forward is the chemoprevention of cancer with polyamine antimetabolites, a process that appears to work in many experimental animal models. Nevertheless, a real breakthrough in the treatment of human cancer has not yet occurred [57 – 66,75 – 79]. It should be noted that many radioprotectants have structures closely related to the polyamines. These structures are so
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similar, in fact, that some of these agents use the polyamine transporter to enter the cell [80]. Polyamines play a clear role in DNA stabilization and appear to protect it directly from free radical damage [80,81]. Undoubtedly, there is a dual role in polyamine physiology and they probably play a surrogate or permissive role in cancer development. Selective enzyme inhibitors as anticancer drugs have essentially proven to be inefficacious mainly due to the sophistication of the system which regulates intracellular polyamine pools. The selective impairment of tumor growth is also prevented by the ubiquitous occurrence of the PAs, and our ability to utilize exogenous polyamines. DFMO has had disappointing results in most therapeutic attempts to use it as single drug, but based on its low toxicity, it may have potential in cancer chemoprevention. It has been claimed that DFMO may improve the efficacy of some of the existing cytotoxic drugs. So as to inhibit tumor growth, several reactions or regulatory functions of polyamine metabolism have to be impaired at the same time. In this regard, one more recent target is polyamine uptake. Cells take up polyamines from plasma when the de novo pathways are blocked. During the nineties 1990s, polyamine derivatives and structural analogues have been prepared, which inhibit cell growth at low micromolar concentrations. Bis(ethyl)norspermine is a prototype of polyamine-mimetics [82]. However, no therapeutically useful drug has been identified so far. Extracellular PAs, and particularly extracellular spermine can be cytotoxic and neurotoxic at high concentrations [83 – 85]. A major problem in the improvement of polyamine analogues to therapeutically useful drugs is finding structures, which do not share neurotoxic properties with spermine. Several tetramines are present in the early phases of clinical trials.
8. Circulating polyamines in clinical chemistry: methods (Fig. 5) Polyamine measurement in biological fluids and tissues may have clinical relevance, especially in cancer patients [55 – 59]. In clinical chemistry, the fluid most frequently employed for polyamine analysis is, of course, blood. As described above, over 90%
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of circulating polyamines are in RBCs (5– 50 Amol/l), while serum polyamines concentration amount to less than 0.1 Amol/l [56 – 58]. Free polyamine concentrations, together with their acetyl conjugates, increase significantly in the biological fluids and the affected tissues of cancer patients. Their concentrations decrease with the improvement in the patient’s condition on multiple therapy [53,55 – 59]. As briefly outlined in Fig. 5, several chromatographic techniques are used in monitoring concentrations of polyamines in cancer. Among the wide panel of analytical methods developed for the quantification of polyamines, HPLC separation of polyamines after derivatization with dansyl chloride remains the most commonly used method [57 – 59,86]. Besides, high-resolution capillary column gas chromatography (GC) is increasingly used over packed column GC, and in recent years, capillary zone electrophoresis has also gained some importance in polyamine determinations [86]. Very recently, a new method has been evaluated which avoids the cumbersome chromatographic steps (Fig. 5). Atmospheric pressure chemical ionization mass spectrometry (MS) can be used to detect and quantify biologically relevant polyamines after dansylation, without chromatographic separation. Positive-ion mass spectra for each dansylated polyamine were generated after optimization by flow injection analysis
(FIA). FIA coupled with MS detection by selected ion monitoring greatly increased the sensitivity of the polyamine detection [87]. In these opinion of these authors hands, the method is linear over a wide range of polyamine concentrations and allows detection of quantities as low as 5 fmol, which represents 50-fold more sensitivity than the usual HPLC/fluorimetry procedure. A good correlation between these two methods was observed. It remains to be determined if the process has enough practicality to be adopted by a critical mass or researchers.
9. Polyamines in oncology and oncohematology (Fig. 6) As described earlier, ODC is expressed early in the cell cycle and has a short half-life [29,33 – 37]. For these reasons, it has been proposed as a marker of proliferation, ; some of the main uses are depicted in Fig. 6 [60 –66]. In a recent study aiming aimed at determining the chemosensitivity of hematological neoplasias, ODC was detected in hematological cancer cells by quantitative immunohistochemical analyses using an ODC antibody and an FITC-linked second antibody [88]. The authors detected drug resistance in patients who subsequently died. Leukemia and lymphoma patients showed different sensitivities to certain drugs, while lymphocytes from
Fig. 5. Overview of the main methods employed in clinical and research environments for the measurement of polyamines in erythrocytes or body fluids.
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Fig. 6. Polyamines as clinical laboratory tools. Main fields that have been explored for polyamines as diagnostic or disease course-monitoring tools.
normal individuals were sensitive to all drugs tested. The method also permitted testing of the effect of new drugs on the proliferation of lymphocytes from hematological cancer patients.
10. Polyamines and the short bowel syndrome After intestinal resection, adaptation, a progressive recovery from the short bowel syndrome, a malabsorptive disorder, may be seen. Research has focused on optimizing remnant intestinal function through dietary or pharmacologic interventions. Polyamines, as previously mentioned, play a key role in intestinal development and recovery [50,65].
11. Polyamines as glycation inhibitors? Our hypothesis Diabetes mellitus has a high incidence of microand macrovascular complications. These complications are related to hyperglycemia and its corollary, glycation, the derivatization of amino groups in proteins by reactive carbonyl groups [89 –92]. Despite the effort throughout the last decade to generalize intense treatment aiming to achieve euglycemia, the last data prove that goal has not been reached.
Therapeutic agents that might counter glycation become then desirable adjuvants for diabetes treatment. Actually, not only in diabetes, but in uremia and atherosclerosis as well, that increased levels of reactive carbonyl compounds with protein modifications (‘‘carbonyl stress’’) are part of the pathogenic process. Research for safe drugs that are capable of inhibiting glycation is being actively pursued by several groups [93 – 98]. The toxicity or the side effects of many of them has become a concern. To our knowledge, no research so far had addressed the putative antiglycation effect of polyamines. Due to their chemical structure, polyamines emerge, a priori, as very good candidates to quench carbonyls. Based on their ubiquity, polycationic nature, essential role in growth, relatively high concentrations in some tissues (up to 2 mmol/l in skin), and high concentrations in sperm (up to 10 mmol/l in a fluid containing also 10 mmol/l fructose, a very aggressive glycating agent), we hypothesized that polyamines inhibit glycation and that might be another of their molecular functions [8]. Our most recent findings show that spermine and spermidine inhibit in vitro structural modifications induced by glycation in selected appropriate target systems [8]. This may have implications in biology and clinical medicine, since as modulation of polyamine uptake, dietary allowances or metabolism could be envisaged as avenues to explore in diabetes
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long-term complications management. Spermine is present in millimolar concentrations in nuclei, closely associated with chromosomes, and may then act protecting DNA and histones from glycation. It has been demonstrated that spermine, can act directly as a free radical scavenger and the role of those in advanced glycation is well established [80,81,92]. Red blood cells lack DNA and are almost completely devoid of RNA, yet most of circulating polyamines are actually carried by RBC, which possess polyamine transporters and actively concentrate polyamines [68,69]. If these polyamines are not bound to nucleic acids, what is their role? One proposed role that has been tested is that they reinforce the antioxidant systems of the RBC [74]. RBCs live 120 days with no renewal of their proteins and are subjected to several oxidative stresses, but also to the variations in glycemia since as their glucose transporter GLUT I, is insulin-independent. These facts would support our hypothesis for an alternate role of polyamines in metabolism, namely RBC might accumulate polyamines to quench glycating agents. Our laboratory is presently addressing these hypotheses [8]. If proven right, dietary recommendations mainly ensue that favor food containing high amounts of polyamines in diabetes, spermine, and spermidine are readily bioavailable in foods such as meats, poultry, cheeses, and green peas, to name a few. Polyamines could alternatively be used as supplements to lower glycation. As discussed above, many polyamine analogues are already in trial for other uses that could be engineered to scavenge carbonyls while having lesser unwanted side effects on cell proliferation. To shed more light on polyamine physiology, its modulation and putative diagnostic and therapeutic uses, studies on transgenic mice expressing protein altering polyamine levels in a tissue-specific manner are granted. These models have become available and show considerable promise for evaluation of the roles of polyamines in biology. Mice with large increases in ODC, S-adenosylmethionine decarboxylase or antizyme, a protein regulating polyamine synthesis by reducing polyamine transport and ODC, have been produced [99]. This decade will surely bring further insight into the role of polyamine modulation in human pathology.
Acknowledgements This work was supported by an intramural grant from Touro University, California, to A.G. The author is grateful to Ms. A. Garramela for her assistance in the preparation of the manuscript.
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