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HORMONES AND SIGNALING

Regulatory mechanisms underlying agmatine homeostasis in humans

¹Department of Pharmacology and Toxicology and ³Institute for Medical Microbiology, Immunology and Parasitology, University of Bonn; ²Department of Internal Medicine I and ⁵Institute of Human Genetics, University Hospital of Bonn; ⁴Department of Surgery, Evangelische Kliniken Bonn, Betriebsstätte Waldkrankenhaus, Bonn, Germany

Submitted 12 June 2008 ; accepted in final form 25 September 2008

	ABSTRACT

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Regulation of agmatine homeostasis has so far only been poorly defined. In the present study, three mechanisms regulating human agmatine homeostasis were investigated. 1) Enzymatic regulation: expression of arginine decarboxylase, diamine oxidase, and ornithine decarboxylase in human colon neoplastic tissue was, at the mRNA level, about 75% and 50% lower and 150% higher, respectively, than in the adjacent normal tissue; expression of agmatinase was unchanged. 2) Bacteria-derived agmatine: ten representative bacteria strains of the human intestinal microbiota considerably differed in agmatine production and its efflux into their surrounding fluid, suggesting that the composition of the intestinal microbiota influences the agmatine availability in the gut lumen for absorption. 3) Regulation of blood plasma agmatine concentration by the human liver: at low concentrations in portal venous blood plasma, agmatine either slightly increased or further decreased in blood plasma through liver passage. Above a threshold of 14 ng/ml agmatine in the portal venous blood plasma, substantial hepatic agmatine removal from blood occurred. Taken together, a perturbation of agmatine homeostasis has been proven to be involved in the regulation of malignant cell proliferation. The amount of agmatine available for absorption, which is an important physiological source of agmatine in the human organism, should differ considerably depending on the composition of the bacterial flora in the chyme since the various species of intestinal bacteria largely differ in their ability to form agmatine. Finally, evidence has been presented that the liver plays a crucial physiological role in the maintenance of agmatine homeostasis in the human organism.

intestinal bacterial microflora; colon cancer; arginine decarboxylase; liver cirrhosis; hepatic agmatine uptake

View larger version (19K): [in this window] [in a new window]   Fig. 1. Substrates and enzymes involved in agmatine homeostasis. Enzymes in boldface were investigated in the present study.

In more detail, human colon cancer tissue was used as an experimental model for tissues with impaired agmatine homeostasis (18). As the first aim, we examined the role of agmatine-synthesizing and -metabolizing enzymes as regulatory factors by determining whether neoplastic colon cancer tissue and the adjacent macroscopically normal tissue differ in the expression of the enzymes arginine decarboxylase (catalyzing the reaction from L-arginine to agmatine), diamine oxidase (catalyzing the degradation of agmatine to 4-guanidinobutyrate), agmatinase (catalyzing the reaction from agmatine to putrescine), and ornithine decarboxylase (catalyzing the reaction from ornithine to putrescine; Fig. 1).

Absorption of bacterial agmatine from the gut lumen is another factor that essentially influences agmatine homeostasis in the mammalian organism (17). Therefore, the second aim of the present study was to compare ten representative intestinal bacterial species with respect to agmatine production and its efflux into the culture medium.

In rat about 60% of agmatine taken up from the stomach and intestine is accumulated in the liver (16) and undergoes enterohepatic circulation. In rat hepatocyte cultures, 50% of agmatine taken up by the cells are transformed to 4-guanidinobutyraldehyde and 10% to polyamines; only 30% are recovered as nonmetabolized agmatine (1). On the other hand, hepatocytes themselves are also capable of synthesizing agmatine (7). Thus the liver appears to play a crucial role in the regulation of the agmatine concentration in the systemic circulation. Data concerning the predominance of biosynthesis or removal of agmatine, i.e., its gain or loss during passage of the blood through the human liver, are not available. In the third part of the study, we therefore analyzed the concentration of agmatine in blood samples drawn simultaneously from the portal and hepatic vein of patients with liver cirrhosis during the therapeutic implantation of an intrahepatic portosystemic stent shunt. To our knowledge, this is the first report on blood agmatine concentrations in samples taken simultaneously from the portal and hepatic vein in humans.

	MATERIALS AND METHODS

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RT-PCR. All patients gave their informed consent to the investigation of their resectates, which were obtained during colon cancer surgery. The study was approved by the Ethics Committee at the Faculty of Medicine of the University of Bonn. Total RNA was isolated from the human colon specimens using the RNeasy Mini Kit (Qiagen, Hilden, Germany) with DNase treatment according to the manufacturer's instructions. Total RNA of each sample was reverse transcribed according to the manufacturer's instructions (RevertAid cDNA Synthesis Kit; Fermentas, St. Leon-Rot, Germany; using random hexamer primers). For quantitative RT-PCR, 35 µl of amplification mixture (Quantitect SyBrGreen Kit, Qiagen) was used, containing 20 ng of reverse-transcribed RNA and 300 nM of the respective primers (arginine decarboxylase, accession no. NM_052998, forward primer: 5'-CTGCCGCAACTACACGTAG-3', reverse primer: 5'-GGACATGGCATAGGTGATGTG-3'; ornithine decarboxylase, accession no. NM_002539, forward primer: 5'-GTGGGTGATTGGATGCTCTTTG-3', reverse primer: 5'-AGGCCCTGACATCACATAGTAG-3'; agmatinase, accession no. NM_024758, forward primer: 5'-CAGCTGGCTGTATTCCTCTG-3', reverse primer: 5'-TATCTGTAGGGATCCAAGGTCG-3'; diamine oxidase, accession no. NM_001091, forward primer: 5'-GTCCTGAACGTGCACTTCG-3', reverse primer: 5'-GCTAACTCATGAGTGACGCTG-3') according to the manufacturer's instructions. Reactions (triplicates, 10 µl) were run on a Mx3000 real-time cycler (Stratagene, La Jolla, CA). The cycling conditions were 15 s polymerase activation at 95°C and 45 cycles at 95°C for 15 s, at 58°C for 30 s and at 72°C for 30 s. Each assay included a standard curve (5 points from 100 to 0.01 ng/35 µl) and no-template controls. The results were analyzed using the Stratagene software (version Mx3000 Pro). The relative mRNA expression (R) was calculated from the ratio of "neoplastic tissue" over "normal tissue", R = E^{Ct control - cancer} (target)/E^{Ct control - cancer} (housekeeper) with the efficiency E = 10^–1/slope measured with a standard curve in all experiments (21). The results for the housekeeping gene β-actin were determined by the same method (β-actin forward primer: 5'-TCCATCATGAAGTGTGACGT-3'; reverse primer: 5'-GAGCAATGATCTTGATCTTCAT-3'). The housekeeping gene stably expressed in all samples was used as an internal standard to normalize mRNA expression, which compensates for differences in sample concentrations and reverse transcription efficiencies. However, in the present experiments, no significant differences were detected comparing normalization to β-actin and to total RNA amount showing negligible variations in reverse transcription and PCR efficiencies. The identity of the PCR products was initially confirmed by agarose gel electrophoresis followed by dideoxy chain termination sequencing and then, after each real-time reaction, by melt-point analysis. Although the enzymes would be detected directly as proteins by Western blotting, this approach was not used because of the lack of commercially available antibodies against arginine decarboxylase or diamine oxidase. The expression of mRNA of the respective enzymes was compared with that found in the adjacent macroscopically normal tissue of the same patient.

To investigate the total sequence of the cDNA encoding arginine decarboxylase in neoplastic and adjacent normal tissue, specific PCR primers were designed to amplify the complete coding sequence of arginine decarboxylase. The primer sequences (forward: 5'-CGGAGTTTGTGTGTTGCATAC-3'; reverse: 5'-ACAGGGCAGAAACACTGCAG-3') were adopted from the human arginine decarboxylase reference sequence (accession number NM_052998). RT-PCR was carried out using cDNA and Pfu-Polymerase (Stratagene). The PCR conditions were 30 s polymerase activation at 95°C and 40 cycles of 30 s denaturation at 94°C, 30 s at the annealing temperature of the primers (58°C), and 2-min extension at 72°C, followed by a final extension period at 72°C for 10 min. PCR products were separated by gel electrophoresis on a 0.8% agarose gel. The bands of interest were cut off the gel, and the cDNA was extracted from the gel using the MinElute Gel Extraction Kit (Qiagen). PCR products were then directly sequenced at MWG Biotech (Ebersberg, Germany).

Test bacteria and culture conditions. The human gastrointestinal tract harbors about 400–600 bacterial species (10). For any given individual, however, 30 to 40 bacterial species contribute for 99% of the fecal microflora (for review, see Ref. 4). The intestinal microbiota is dominated by obligate anaerobes (99%). The predominant species of obligate anaerobes belong to the genera Bacteroides (65%), Bifidobacterium (32%), Eubacterium (3%), and Veillonella (0.3%) species. The predominant aerobe and facultative anaerobe microbiota (1%) comprises Escherichia coli (E. coli) (up to 45%), other Enterobacteriaceae (1%), and Enterococcus (up to 45%), or Lactobacillus (9%) species. As typical representatives of the physiological intestinal microflora (19), the following reference strains were investigated: E. coli, strain Nissle (Mutaflor), Enterococcus faecalis DSM 2981, Bacteroides vulgatus DSM 1447, Bacteroides fragilis DSM 2151, Bacteroides thetaiotaomicron DSM 2255, Eggerthella lenta (= Eubacterium lentum) DSM 2243, Lactobacillus reuteri DSM 20016, Lactobacillus acidophilus DSM 20079, Bifidobacterium bifidum DSM 20082, and Veillonella dispar DSM 20735. After three passages of the stock cultures of E. coli and E. faecalis from their lyophilisate on Columbia agar with 5% sheep blood (Becton Dickinson, Heidelberg, Germany) and incubation in normal air of 36°C, test tubes containing 10 ml of brain heart infusion broth (BHI; Oxoid, Wesel, Germany) were inoculated with colony material of three single colonies. The other test strains were propagated in parallel on Columbia agar with 5% sheep blood and on Schaedler agar and incubated under anaerobic conditions (36°C, 72 h) using the GasPak (R) system (Becton Dickinson). Depending on the strength of growth, up to 20 colonies of each strain were transferred into test tubes containing 10 ml of BHI. After anaerobic incubation for 72 h at 36°C, broth cultures were centrifuged for 10 min at 4°C and 6,000 g. Each bacterial strain was harvested during the late log/beginning stationary phase of growth with a yield of bacterial cells in the range of 1,000,000,000 colony forming units/ml before the centrifugation step. Pellets and supernatants and another tube with the original broth cultures were immediately stored in an ice water bath. Due to differences in the cellular bacterial protein content between the various strains, the absolute amount of bacterial protein in the culture dishes differed between the various bacterial strains (Table 1, right column). Therefore, agmatine content in the pellets and culture media was normalized to the amount of bacterial protein (mg protein, Table 1). The absolute amount of bacterial protein in the culture dishes did not correlate with agmatine content in the pellet or culture medium (Table 1, right column).

Agmatine determination. Protein was precipitated in the blood plasma samples as well as in the supernatants and pellet homogenisates of the bacterial cultures by addition of 0.6 M perchloric acid and 0.1 M hydrochloric acid in methanol (1:1:0.5, vol/vol/vol). After incubation for 15 min at 4°C, each sample was centrifuged for 5 min at 6,000 g, and agmatine in the supernatant was determined by HPLC as described previously (18). The detection limit was about 400 pM. The recovery was about 70%. Day-to-day variation of the peak height of the agmatine standard amounted to 5–17%; the reproducibility, i.e., the deviation of the peak height of the same sample measured twice, amounted to less than 2%.

Statistics. Unless stated otherwise, means ± SE are given. Log2 values were calculated for the relative expression of the enzymes (Ct) determined in the quantitative RT-PCR. Since these log values were distributed normally, the t-test for unpaired data was used to analyze whether expression of the enzymes in the tumor tissue significantly differed from that in the adjacent normal tissue.

Since the agmatine values did not follow a Gaussian distribution, nonparametric Spearman correlation analysis of agmatine levels in portal and hepatic venous blood plasma of the individual patients was performed. In six portal venous blood samples plasma agmatine concentrations were above 14 ng/ml, i.e., they were outside of the mean plus four times standard deviation of agmatine concentration of the 24 other patients, which identified them as belonging to a subgroup. Comparison of these six values with agmatine concentration in the portal venous blood samples of the residual 24 patients (all below 10 ng/ml) by the Mann-Whitney-test significantly (P = 0.0024) indicated that the two groups of samples do not belong to the same main unit.

	RESULTS

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Human colon tissue. Resectates from 10 patients (5 women, 5 men; median age 71 yr, range 60–92 yr) with colon carcinoma who underwent surgery were investigated. Expression of mRNA encoding arginine decarboxylase and diamine oxidase in neoplastic tissue specimens was decreased to about one-fourth and one-half, respectively, of that found in the adjacent macroscopically normal tissue (Fig. 2). In contrast, expression of mRNA for ornithine decarboxylase was increased about 2.5-fold (Fig. 2). No significant difference was observed in the expression of agmatinase between neoplastic tissue and adjacent normal tissue (Fig. 2).

View larger version (9K): [in this window] [in a new window]   Fig. 2. Expression of arginine decarboxylase (ADC), ornithine decarboxylase (ODC), diamine oxidase (DAO), and agmatinase (AGMAT) at the mRNA level in neoplastic colon tissue compared with the adjacent normal colon tissue; mRNA levels were determined by quantitative RT-PCR. *P < 0.05, **P < 0.01, ***P < 0.001 (unpaired t-test; n = 10 for each enzyme).

Production of agmatine by selected bacteria and efflux into the culture medium. Most of the bacteria investigated (exceptions: Eggerthella lenta and Enterococcus faecalis, see below) are capable of producing considerable amounts of agmatine. This can be derived from the finding that it is present both in the bacteria themselves and in the culture medium; agmatine in the medium mainly reflects efflux of the compound from the bacteria into the medium but may, to a limited extent, also be due to efflux of agmatine from dead bacterial cells. The culture media of the various strains of bacteria largely differed in their agmatine content. A very high content was determined in the media of Bacteroides vulgatus, Lactobacillus reuteri, Lactobacillus acidophilus, and Veillonella dispar (Table 1). In the culture media of Bacteroides fragilis, Bacteroides thetaiotaomicron, Bifidobacterium bifidum, and E. coli Nissle, lower but still reasonably high levels of agmatine content were detected. Eggerthella lenta and Enterococcus faecalis are exceptions in that only almost negligible amounts of agmatine were present in the bacteria, and that agmatine in the culture medium of these bacteria was also much lower (with large variations between the individual values) than in the case of the other bacteria (Table 1).

For each of the latter strains of bacteria, the apparent rate of agmatine efflux was determined as a measure of the ability of agmatine to flow out of the bacteria into the surrounding medium. The apparent rate of efflux was calculated as the percentage of agmatine in the culture medium related to the total agmatine, i.e., agmatine in the culture medium plus the residual agmatine in the bacteria. In the case of Bacteroides fragilis and Bacteroides thetaiotaomicron, the apparent rate of efflux amounted to 60–70%, but it was more than 95% in the case of all other strains of bacteria. These apparent rates represent the percentage of the agmatine produced by the bacteria that would be available for absorption from the gut.

Agmatine synthesis and metabolism in patients with liver cirrhosis. Agmatine was detected in the portal and hepatic venous blood of the patients at median concentrations of 2.4 ng/ml plasma and 2.9 ng/ml plasma, respectively (ranges: 0–221.5 ng/ml plasma and 0–12.5 ng/ml plasma, respectively; n = 30 each; Fig. 3A). There was no significant negative or positive correlation between agmatine concentrations in portal and hepatic venous blood plasma in the individual patients when all patients were analyzed (Spearman correlation coefficient r = –0.16, P = 0.40). An increase in agmatine concentration in the blood occurred approximately as often and to a similar degree as a decrease in agmatine concentration (Fig. 3). Subgroup analysis according to the severity of liver cirrhosis as indicated by the Child-Pugh score also failed to reveal significant correlations between agmatine concentrations in portal and hepatic venous blood plasma (Fig. 3B).

View larger version (13K): [in this window] [in a new window]   Fig. 3. Agmatine concentrations in specimens of portal venous (closed symbols) and hepatic venous (open symbols) blood plasma. Blood samples were drawn from both veins of each patient simultaneously. Agmatine concentrations in corresponding blood samples from the same patient are connected by solid lines. A: agmatine in corresponding blood samples from all patients (=30) irrespective of the severity of the impairment of their liver function. B: agmatine in corresponding blood samples from patients grouped according to the severity of their liver cirrhosis as indicated by the Child-Pugh score (Child A: n = 4; Child B: n = 18; Child C: n = 8).

	DISCUSSION

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The present study aimed at analyzing regulatory mechanisms underlying agmatine homeostasis in pathophysiology and physiology. The first aspect investigated was based on data from in vitro and in vivo studies, which suggested an association of a distortion of intracellular agmatine homeostasis with neoplastic transformation and tumor growth (2, 18, 25). Since a modification in the quantitative pattern of agmatine-synthesizing and -metabolizing enzymes (Fig. 1) may play a crucial role in the pathophysiology of such a disorder, we searched for changes in the expression of these enzymes in neoplastic colon cancer tissue compared with adjacent macroscopically normal tissue. The present study is the first one to show that the expression of mRNA encoding the key agmatine forming enzyme arginine decarboxylase was 75% lower in the neoplastic specimens of the colon than in the adjacent tissue. This observation is compatible with the suggestion that the reduced expression of this enzyme is the most important reason for the reduced agmatine content, which was previously determined in neoplastic colon cancer tissue (18). At the mRNA level, the agmatine-forming enzyme arginine decarboxylase was not genetically altered in the cancer tissue, ruling out the possibility that mutations of arginine decarboxylase are causally involved in the lowered agmatine content and neoplastic transformation. Although it cannot be completely excluded that a reduced function of the agmatine transporter might have contributed to the reduced agmatine content in the neoplastic tissue, this possibility seems to be unlikely because, in previous investigations, we observed no major differences in uptake rates between tumor cell lines and nontumor cell lines (6).

Expression of the mRNA encoding the agmatine-degrading enzyme diamine oxidase (Fig. 1) was also studied. It was found to be reduced by 50% (Fig. 2). A possible interpretation is that this reduction reflects the attempt of the cell to counterbalance the reduced formation of agmatine. If this interpretation also held true for other agmatine-degrading enzymes, their expression may also be decreased, as previously shown for agmatinase in a cell line of renal carcinoma cells (3). However, in the colon tumor tissues analyzed in the present study, agmatinase expression was not changed (Fig. 2), suggesting that this enzyme may be of minor importance for agmatine homeostasis in colon. In agreement with such an assumption, the expression of agmatinase is high in the kidney and liver but very low in the colon (9, 13).

The expression of the key enzyme in the polyamine synthesis pathway, ornithine decarboxylase (Fig. 1; for review, see Ref. 20) was markedly increased in the colon carcinoma tissue investigated in the present study. This observation is in agreement with the results of previous studies demonstrating that ornithine decarboxylase mRNA expression (8) and activity (11, 24) are increased in adenocarcinoma tissues compared with normal tissues. The increases in expression and activity of ornithine decarboxylase lead to increased polyamine levels in the cells followed by tumor cell proliferation.

Taken together, the present results in conjunction with previous ones reported in the literature (see also Introduction) support the view that agmatine plays a crucial role in the pathophysiology of tumor cell proliferation in humans. In the present paper, the expression of the key agmatine-forming enzyme, arginine decarboxylase, and of the key polyamine-synthesizing enzyme, ornithine decarboxylase, in colon cancer tissue was compared with the expression of the corresponding enzymes in the normal tissue adjacent to the malignant tumor. In view of these changes in enzyme expression, it may be concluded that intracellular agmatine homeostasis is impaired: agmatine content is reduced, leading to enhanced tumor cell proliferation mediated by the elevated intracellular polyamine content resulting from the increased availability of ornithine decarboxylase. Thus agmatine, by acting as a functional antagonist of polyamines, is involved in the regulation of tumor cell proliferation and growth. It is conceivable that agmatine, in addition to this pathophysiological regulatory mechanism, may also play a role in the control of "normal cell" proliferation.

In the context of absorption of agmatine from the gut, a second important aspect contributing to agmatine homeostasis in humans, namely the availability of bacteria-derived agmatine in the gut lumen, has been subject of the present study. In fact, agmatine formed by the resident intestinal microflora is a major physiological source of the compound in the mammalian organism including humans. To get an idea about the availability of agmatine from this source, the production of agmatine by ten typical representatives of the human intestinal microflora and the efflux of this agmatine into the surrounding culture medium (in analogy to the chyme in the case of the gut) were determined. As a result, it was demonstrated here for the first time that intestinal bacteria species largely differ in their ability to form agmatine (Table 1). Actinobacterium Eggerthella lenta even failed to reproducibly form any agmatine. Accordingly, a predominance of bacteria species that produce only low amounts of agmatine may cause a reduced availability of agmatine in the chyme for its absorption into the organism and its cells. Such a low agmatine concentration in the chyme may aggravate the above-mentioned tumor-promoting effect of the low cellular agmatine content because of the low arginine decarboxylase expression in tumor cells. As a note of caution, it has to be reminded that the growth conditions in vitro are very different from the environment within the human intestine. Thus the ability of the strains investigated to produce agmatine in the gut lumen could differ significantly from their production in vitro.

The third regulatory mechanism influencing agmatine homeostasis in humans investigated here refers to hepatic elimination of the agmatine absorbed from the gut lumen and to hepatic formation. For ethical reasons, such an investigation could not be carried out in healthy individuals, but simultaneous withdrawal of blood from the portal and hepatic veins as a basis for such a study became possible in patients with liver cirrhosis who underwent therapeutic implantation of an intrahepatic portosystemic stent shunt. In these patients with liver cirrhosis, it was an open question whether the remaining hepatic function is sufficient to modify the level of agmatine in the blood plasma during its passage through the liver. Since, in fact, blood samples were always taken from the portal and hepatic veins at the same time, possible periodic changes in absorption cannot account for differences of agmatine levels between the corresponding blood samples. In the blood of all patients, agmatine could be detected at concentrations similar to those previously reported for humans (5, 28). The agmatine levels found in the patients were also comparable to those in rat blood determined in our laboratory with the same methods (means ± SE: 0.97 ± 0.23 ng/ml plasma; n = 38).

Due to the high amount of agmatine synthesized by bacteria in, and absorbed from, the gut lumen, loss of agmatine in blood was expected to occur as a result and indicator of its predominant metabolism during passage through the liver. However, the agmatine levels in portal and hepatic venous blood plasma were, when considering all cases, not negatively correlated. This held true irrespective of the degree of impairment of liver function by the cirrhosis as indicated by the Child-Pugh scores (Fig. 3, A and B). To provide an explanation for this phenomenon, it must be taken into account that the hepatocyte not only metabolizes agmatine but is also capable of synthesizing this compound (7, 29). Obviously, at low agmatine concentrations in the portal venous blood plasma, agmatine synthesis can dominate over its metabolism, reflected by the increase in agmatine concentration during passage through the liver. In the patients investigated here, such increases of agmatine level in hepatic venous blood plasma over the value in portal venous blood plasma occurred approximately as often as the reverse process, i.e., metabolization.

However, it should be noted that a significant negative correlation of agmatine levels in portal and hepatic venous blood plasma was observed for the subgroup of six patients who had markedly higher agmatine levels in their portal venous blood plasma (>14 ng/ml) than the other 24 patients (< 10 ng/ml; Fig. 3). In those six patients, agmatine metabolism clearly predominated over synthesis, leading to a loss of agmatine in blood during passage through the liver. Thus the agmatine level in hepatic venous blood plasma was adjusted by the liver to the same range as in the corresponding blood samples from the other patients.

Concluding remarks. Important physiological and pathophysiological regulatory mechanisms underlying agmatine homeostasis and its disorders, respectively, have been disclosed in the present study in humans. Thus an impairment of agmatine homeostasis has been proven to be involved in the regulation of malignant cell proliferation. Our data demonstrate for the first time that the expression of arginine decarboxylase, the key enzyme for the biosynthesis of agmatine, is reduced in neoplastic tissues in humans. It appears probable that agmatine, in addition to this pathophysiological relevance, may also be involved in the physiological control of normal cell proliferation and growth. The amount of agmatine formed by the resident intestinal microflora is an important physiological source of agmatine in the human organism. The various species of intestinal bacteria largely differ in their ability to form agmatine. Accordingly, the amount of agmatine available for absorption should differ considerably depending on the composition of the bacterial flora in the chyme. Predominance of bacteria producing only low amounts of agmatine available for absorption from the gut lumen may lead to an increase in the tumor cell proliferation-promoting effect associated with the decreased expression of arginine decarboxylase (see above). Finally, evidence has been presented that the liver plays a crucial physiological role in the maintenance of agmatine homeostasis in the human organism by its ability to take up and metabolize agmatine on the one hand and synthesize it on the other. Even the impaired liver function in patients with liver cirrhosis is sufficient to adjust agmatine concentrations in the hepatic venous blood plasma. This occurs irrespective of the degree of functional impairment of the cirrhotic liver, as defined by the different Child-Pugh score. Accordingly, a rather constant supply of all tissues of the organism with agmatine for cellular uptake is made possible.

	GRANTS

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This work was supported by grants of the Deutsche Krebshilfe and the Doktor-Robert-Pfleger-Stiftung.

	ACKNOWLEDGMENTS

 
We thank Petra Spitzlei for skilful technical assistance. Present affiliation of Michael Schepke: HELIOS Klinikum Siegburg GmbH, Siegburg, Germany.

	FOOTNOTES

 

Address for reprint requests and other correspondence: G. Molderings, Inst. of Human Genetics, Univ. Hospital of Bonn, Wilhelmstrasse 31, D-53111 Bonn, Germany (e-mail: molderings{at}uni-bonn.de)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

	REFERENCES

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1. Cabella C, Gardini G, Corpillo D, Testore G, Bedino S, Solinas SP, Cravanzola C, Vargiu C, Grillo MA, Colombatto S. Transport and metabolism of agmatine in rat hepatocyte cultures. Eur J Biochem 268: 940–947, 2001.[Web of Science][Medline]
2. Choi YS, Cho YD. Effects of agmatine on polyamine metabolism and the growth of prostate tumor cells. J Biochem Mol Biol 32: 173–180, 1999.[Web of Science]
3. Dallmann K, Junker H, Balabanov S, Zimmermann U, Giebel J, Walther R. Human agmatinase is diminished in the clear cell type of renal cell carcinoma. Int J Cancer 108: 342–347, 2004.[CrossRef][Web of Science][Medline]
4. Guarner F. Enteric flora in health and disease. Digestion 73, Suppl 1: 5–12, 2006.[CrossRef][Web of Science][Medline]
5. Halaris A, Zhu H, Feng Y, Piletz JE. Plasma agmatine and platelet imidazoline receptors in depression. Ann NY Acad Sci 881: 445–451, 1999.[CrossRef][Web of Science][Medline]
6. Heinen A, Brüss M, Bönisch H, Göthert M, Molderings GJ. Pharmacological characteristics of the specific transporter for the endogenous cell growth inhibitor agmatine in six tumor cell lines. Int J Colorectal Dis 18: 314–319, 2003.[Web of Science][Medline]
7. Horyn O, Luhovyy B, Lazarow A, Daikhin Y, Nissim I, Yudkoff M, Nissim I. Biosynthesis of agmatine in isolated mitochondria and perfused rat liver: studies with 15N-labelled arginine. Biochem J 388: 419–425, 2005.[CrossRef][Web of Science][Medline]
8. Hu HY, Liu XX, Jiang CY, Lu Y, Liu SL, Bian JF, Wang XM, Geng Z, Zhang Y, Zhang B. Ornithine decarboxylase gene is overexpressed in colorectal carcinoma. World J Gastroenterol 11: 2244–2248, 2005.[Medline]
9. Iyer RK, Kim HK, Tsoa RW, Grody WW, Cederbaum SD. Cloning and characterization of human agmatinase. Mol Genet Metab 75: 209–218, 2002.[CrossRef][Web of Science][Medline]
10. Janda JM, Abbott SL. The Enterobacteria. Washington DC: ASM, 2006.
11. Linsalata M, Caruso MG, Leo S, Guerra V, D'Attoma B, Di Leo A. Prognostic value of tissue polyamine levels in human colorectal carcinoma. Anticancer Res 22: 2465–2469, 2002.[Web of Science][Medline]
12. Matés JM, Sánchez-Jiménez F, López-Herrera J, de Castro IN. Regulation by 1,4-diamines of the ornithine decarboxylase activity induced by ornithine in perifused tumor cells. Biochem Pharmacol 42: 1045–1052, 1991.[CrossRef][Web of Science][Medline]
13. Mistry SK, Burwell TJ, Chambers RM, Rudolph-Owen L, Spaltmann F, Cook WJ, Morris SM. Cloning of human agmatinase. An alternate path for polyamine synthesis induced in liver by hepatitis B virus. Am J Physiol Gastrointest Liver Physiol 282: G375–G381, 2002.[Abstract/Free Full Text]
14. Molderings GJ. The many faces of agmatine. Pharmacol Rep 58: 266–267, 2006.[Web of Science]
15. Molderings GJ, Bönisch H, Göthert M, Brüss M. Agmatine and putrescine uptake in the human glioma cell line SK-MG-1. Naunyn-Schmiedebergs Arch Pharmacol 363: 671–679 2001.[CrossRef][Web of Science][Medline]
16. Molderings GJ, Heinen A, Menzel S, Göthert M. Exposure of rat isolated stomach and rats in vivo to [¹⁴C]agmatine: accumulation in the stomach wall and distribution in various tissues. Fundam Clin Pharmacol 16: 219–225, 2002.[CrossRef][Web of Science][Medline]
17. Molderings GJ, Heinen A, Menzel S, Lübbecke F, Homann J, Göthert M. Gastrointestinal uptake of agmatine: distribution in tissues and organs and pathophysiologic relevance. Ann NY Acad Sci 1009: 44–51, 2003.[CrossRef][Web of Science][Medline]
18. Molderings GJ, Kribben B, Heinen A, Schröder D, Brüss M, Göthert M. Intestinal tumor and agmatine (decarboxylated arginine): low content in colon carcinoma tissue specimens and inhibitory effect on tumor cell proliferation in vitro. Cancer 101: 858–868, 2004.[CrossRef][Medline]
19. Murray PR, Baron EJ, Jorgensen JH, Landry ML, Pfaller MA. Manual of Clinical Microbiology. Washington DC: ASM, 2007.
20. Pegg AE. Regulation of ornithine decarboxylase. J Biol Chem 281: 14529–14532, 2006.[Abstract/Free Full Text]
21. Pfaffl MW. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 29: e45, 2001.[Abstract/Free Full Text]
22. Philip R, Campbell E, Wheatley DN. Arginine deprivation, growth inhibition and tumour cell death: 2. Enzymatic degradation of arginine in normal and malignant cell cultures. Br J Cancer 88: 613–623, 2003.[CrossRef][Web of Science][Medline]
23. Pugh RN, Murray-Lyon IM, Dawson JL, Pietroni MC, Williams R. Transection of the oesophagus for bleeding oesophageal varices. Br J Surg 60: 646–649, 1973.[Web of Science][Medline]
24. Rozhin J, Wilson P, Bull A, Nigro N. Ornithine decarboxylase activity in rat and human colon. Cancer Res 44: 3226–3230, 1984.[Abstract/Free Full Text]
25. Satriano J, Matsufuji S, Murakami Y, Lortie MJ, Schwartz D, Kelly CJ, Hayashi S, Blantz RC. Agmatine suppresses proliferation by frameshift induction of antizyme and attenuation of cellular polyamine levels. J Biol Chem 273: 15313–15316, 1998.[Abstract/Free Full Text]
26. Satriano J, Isome M, Casero RA Jr, Thomson SC, Blantz RC. Polyamine transport system mediates agmatine transport in mammalian cells. Am J Physiol Cell Physiol 281: C329–C334, 2001.[Abstract/Free Full Text]
27. Wolf C, Brüss M, Hänisch B, Göthert M, von Kügelgen I, Molderings GJ. Molecular basis for the antiproliferative effect of agmatine in tumor cells of colonic, hepatic, and neuronal origin. Mol Pharmacol 71: 276–283, 2007.[Abstract/Free Full Text]
28. Zhang WZ, Kaye DM. Simultaneous determination of arginine and seven metabolites in plasma by reversed-phase liquid chromatography with a time-controlled ortho-phthaldialdehyde precolumn derivatization. Anal Biochem 326: 87–92, 2004.[CrossRef][Web of Science][Medline]
29. Zhu MY, Iyo A, Piletz JE, Regunathan S. Expression of human arginine decarboxylase, the biosynthetic enzyme for agmatine. Biochim Biophys Acta 1670: 156–164, 2004.[Medline]

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