Here we report the widespread natural occurrence of a known antibiotic and antineoplastic compound, hydroxyurea in animals from many taxonomic groups.
Hydroxyurea occurs in all the organisms we have examined including invertebrates (molluscs and crustaceans), fishes from several major groups, amphibians and mammals. The species with highest concentrations was an elasmobranch (sharks, skates and rays), the little skate Leucoraja erinacea with levels up to 250 μM, high enough to have antiviral, antimicrobial and antineoplastic effects based on in vitro studies. Embryos of L. erinacea showed increasing levels of hydroxyurea with development, indicating the capacity for hydroxyurea synthesis. Certain tissues of other organisms (e.g. skin of the frog (64 μM), intestine of lobster (138 μM) gills of the surf clam (100 μM)) had levels high enough to have antiviral effects based on in vitro studies. Hydroxyurea is widely used clinically in the treatment of certain human cancers, sickle cell anemia, psoriasis, myeloproliferative diseases, and has been investigated as a potential treatment of HIV infection and its presence at high levels in tissues of elasmobranchs and other organisms suggests a novel mechanism for fighting disease that may explain the disease resistance of some groups. In light of the known production of nitric oxide from exogenously applied hydroxyurea, endogenous hydoxyurea may play a hitherto unknown role in nitric oxide dynamics.
Citation: Fraser DI, Liu KT, Reid BJ, Hawkins E, Sevier A, Pyle M, et al. (2015) Widespread Natural Occurrence of Hydroxyurea in Animals. PLoS ONE 10(11): e0142890. https://doi.org/10.1371/journal.pone.0142890
Editor: Markus Michael Bachschmid, Boston University, UNITED STATES
Received: July 14, 2015; Accepted: October 28, 2015; Published: November 24, 2015
Copyright: © 2015 Fraser et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Data Availability: All data are within the paper.
Funding: Funding was provided by Natural Science and Engineering Research Council of Canada www.nserc-crsng.gc.ca.
Competing interests: The authors have declared no competing interests exist.
Hydroxyurea is a remarkable compound that has been known to science since1869 when it was first synthesized . Various studies show it has antiviral, antibacterial, and antineoplastic properties . Its mechanism of action involves inhibition of ribonucleotide reductase (EC 18.104.22.168) which inhibits DNA synthesis  in a variety of organisms. It is, or has been used in the treatment of a variety of neoplastic diseases, sickle cell anemia, psoriasis, myeloproliferative diseases and infectious diseases such as HIV . It is listed as an “essential medicine” by the World Health Organization . Hydroxyurea, however, is virtually unknown in nature with a record of its presence in the bacterium Streptomyces garyphalus as an intermediate in cycloserine synthesis  and a report in human plasma at levels close to the limits of detection (2.6 μM) . We examined the levels of hydroxyurea in tissues of representative of invertebrate and vertebrate groups.
Materials and Methods
Animals collected in Passamaquoddy Bay, New Brunswick, Canada were collected under Department of Fisheries and Oceans Canada permit number 323401. Euthanasia procedures for this specific study were approved by University of Guelph Animal Care Committee Protocol number 11R014. Surf clams, Spisula solidissima, were collected at low tide at Bar Road, St. Andrews, New Brunswick Canada. Lobsters, Homarus americanus were purchased from a local (Guelph, Ontario, Canada) seafood retailer. Hagfish, Eptatretus stouti tissues were donated by D. Fudge, Department of Integrative Biology, University of Guelph. Little skates (L. erinacea Mitchill 1825) of either sex were collected by otter trawl in Passamaquoddy Bay (New Brunswick, Canada), before transport to holding facilities in the Hagen Aqualab, at the University of Guelph (Guelph, Ontario) where they were maintained for several months to several years. Skate eggs were obtained from this colony. African lungfish (Protopterus dolloi) were held and sampled as previously described . Adult rainbow trout (Oncorhynchus mykiss Walbaum 1792) of either sex were purchased from a local fish farm (Belleville, Ontario) and transported to holding facilities at the University of Guelph. Trout were held as previously described [8,9]. Frogs, Lithobates pipiens tissues were donated by P. Smith, Department of Integrative Biology, University of Guelph. Sheep, Ovis aries, tissues were obtained from a local slaughterhouse (Guelph, Ontario, Canada). Animals collected in Passamaquoddy Bay, New Brunswick, Canada were collected with permission of the Department of Fisheries and Oceans Canada permit number 323401.
Fish were euthanized by cervical section. Tissues were rapidly excised, frozen in liquid nitrogen and stored at –80°C until used. Blood was drawn by cardiac (skates) or caudal (other fish) puncture using heparinized syringes. Erythrocytes were separated from plasma by centrifuging blood at 2,430 g for 10 minutes at 4°C. Sheep tissues were collected from a federally regulated abattoir at the University of Guelph.
Preparation of tissues for use in hydroxyurea and urea assays
Tissues were homogenized in a small volume of ddH2O using a Polytron PT1200 homogenizer set at high speeds (25, 000 rpm) for three 10 second bursts, with a cooling period of 30 seconds between each burst. Homogenized samples were then spun at 9,700 g with a Sorval SA-600 rotor and 4°C for 10 minutes to remove cellular debris. The resulting supernatants and diluted plasma samples were collected and deproteinized with 60% perchloric acid (PCA) to a final concentration of 0.5M PCA. Acidified samples were then spun at 22,000 g for 20 minutes with a Sorval SA-600 rotor and 4°C. The supernatants were collected for use in hydroxyurea and urea assays and the pellets discarded.
Measurement of hydroxyurea and urea in biological samples
Determination of hydroxyurea content in deproteinized plasma and tissue samples followed the colorimetric assay of Fabricius and Rajewsky . Absorbance of hydroxyurea samples was measured at 540 nm using a Cary 300 UV/Vis spectrophotometer (Agilent Technologies). Urea was measured according to the protocol originally described by Rahmatullah and Boyde  at 525 nm. In our study, hydroxyurea was measured chemically by the method of Fabricius and Rajewsky  with analate addition. Its identity was confirmed by gas-chromatography mass spectrometry using the method of Scott et al.  in plasma and liver samples from L erinacea (Fig 1).
A: Fragmentation pattern for derivatized hydroxuyrea (hydroxyurea triTMS). B: Overlaid GC-MS chromatograms of a 10 μg/ml standard of hyroxyurea (maroon), deproteinized liver sample from L. erinacea (green), and deproteinized plasma sample from L. erinacea (red). Peaks at elution time ~5.4 min represents hydroxyurea while peaks at ~ 7.8 min represents tropic acid (10 μg/ml) an internal standard in all three samples. Chromatograms were selective for ions 277 (representative of hydroxyurea triTMS) and 280 (representative of tropic acid-diTMS). X-axis = minutes, Y-axis = kCps.
Gas Chromatography-Mass Spectrometry (GC/MS)
Samples were derivatized as detailed in Scott et al. . Once derivatized, tubes were cooled to room temperature and the contents transferred to autosampler vials containing tapered inserts before being placed in the autoinjector of the GCMS and run. Injections of 1 ml were used for GCMS analysis. GC/MS operating conditions were adapted from those detailed in Table 26.3 of Scott et al. . The GC/MS was operated in selected ion mode after electron impact fragmentation, selective for ion 277 (hydroxyurea tri-TMS).
Results and Discussion
We initially found tissue specific accumulation of hydroxyurea in the elasmobranch L. erinacea with values up to 250 μM in the spiral valve (intestine) (Fig 2a). The extensive literature on the antibiotic and antineoplastic effects of hydroxyurea lead to the obvious conclusion that its biological role in animals is as part of the innate immune system to combat viral and other infections. The nominal concentrations we report for the little skate are in the range of concentrations causing 50% inhibition (ED50) of a variety of processes including DNA synthesis, ribonucleotide reductase activity in viruses and growth of some cell types (Table 1). The use of the values presented in Table 1 in comparison to the values we report here has several caveats that must be considered. Firstly, the times used for determination of the ED50 reported in Table 1 range from 10 minutes to several days in vitro. However, according to Haber’s law, the severity of a toxic effect depends on the total exposure (i.e. exposure concentration multiplied by the duration of exposure) . Maintenance of chronic high levels of hydroxyurea in vivo would thus reduce the concentration needed for a given effect. Thus, the hydroxyurea levels we report would be even more effective in vivo than the values in Table 1 would predict. Secondly, our hydroxyurea tissue concentrations are likely to be underestimates in the vertebrates we examined since hydroxyurea reacts with hemoglobin and some would be destroyed during preparation in tissues with substantial blood supplies . Concentrations in the spleen especially may be higher than measured since, as a storage site for erythrocytes the spleen has the highest concentrations of hemoglobin of any tissue.
A: The distribution of hydroxyurea and urea in the blood plasma and tissues of adult little skates (L. erinacea, n = 5). Hydroxyurea was non-detectable in erythrocytes. Values are means ± standard error (SE) of the mean. Values with the same letter above the bar are not significantly different from each other. B: Whole body hydroxyurea concentrations (mean μM ± SE) measured in little skate (L. erinacea) embryos at stages 2 and 3 of development according to criteria described by Hoff . Values are from whole embryos with yolk sacs removed from both stage 2 and 3 embryos. Values are means ± standard error (SE) of the mean. Values with the same letter above the bar are not significantly different from each other.
Values are reported in either micromolar (μM) or millimolar (mM).
The values we report for L. erinacea are in the range that would affect some viral and bacterial processes (Fig 2a and Table 1). Generally, viral processes are more susceptible to inhibition by hydroxyurea than bacterial or mammalian cell lines (Table 1) . Interestingly, our plasma concentrations for L. erinacea (87 μM) correspond to the range maintained to treat human HIV Type 1 patients (10–130 μM): the range that inhibits HIV in vitro .
Elasmobranchs are an ancient vertebrate group with unusual physiological and biochemical characteristics . They are the earliest known vertebrate group to have an adaptive immune system using antibodies. Among the features of their biology that has attracted public interest is their anecdotal resistance to disease, especially cancer. There is little hard science to validate such claims but the available literature records few viral and bacterial diseases from this group in spite of a considerable interest . Among vertebrates, the incidence of neoplasia is lowest in elasmobranchs [35,36]. Reports of the unusual occurrence of bacteria in plasma  and tissues  of apparently healthy elasmobranchs may also be due to the effects of hydroxyurea in preventing bacterial growth.
Although the mechanism of synthesis of hydroxyurea in vivo is not currently known, the presence of hydroxyurea in embryos from eggs of L. erinacea and the increase in concentration as the embryo grows (Fig 1b) provides evidence of the capacity for hydroxyurea synthesis in L. erinacea.
Levels of hydroxyurea in tissues of other species including invertebrates and vertebrates are for the most part lower than those of the little skate (Fig 3a–3g). Similar to the situation in the skate, the distribution is tissue specific. In the surf clam, S. solidissima levels were high in mantle (100 μM) and in the lobster, H. americanus high levels were found in the intestine (138 μM). In the non-elasmobranch vertebrates, levels were generally low with the highest levels being found in skin (64 μM) of the frog L. pipiens. In the lungfish, P. annectens, the highest levels were found in the gills (38 μM). In the trout, O. mykiss, the highest levels were found in the pyloric caecae (32 μM). In the sheep, O. aries, the highest levels were in kidney (25 μM). If one assumes that endogenous hydroxyurea confers some defense against viral or other infection, significantly higher levels in some tissues may mean these are sites that need to be defended most.
Values with the same letter above the bar are not significantly different from each other. Tissues of the clam were not included in the statistical analysis due to the low n values. There were no differences between tissues of the Pacific hagfish or trout. Due to the low n value for plasma, gill and eggs of the lungfish is these tissues were not included in the analysis and have no letter above the bar.
The tissue specific distribution of hydroxyurea indicates either it can be synthesized locally or transported and concentrated. Its structural similarity to urea (both are polar, with a low molecular weight differing only in the presence of a hydroxyl group) suggests it could be transported by the same carriers as urea but the tissue distribution of these 2 compounds in L. erinacea is very different (Fig 2a). The main organic osmolyte of marine elasmobranchs is urea that is accumulated to levels more than one thousand times that of hydroxyurea  (Fig 2a). In general, tissue specific differences in urea content are small (~20%). Hydroxyurea concentrations, on the other hand can vary by as much as 25 fold between tissues. Thus there must be transporters that can distinguish urea and hydroxyurea and these are known in mammals . In vitro studies show hydroxyurea is far less permeant than urea in mouse erythrocytes due to the capacity of the urea transporter B (UT-B) to distinguish between them . Several carriers for hydroxyurea have been identified including the organic anion transporting polypeptides (OATP) OATP1A2, OATP1B1 and OATP1B3, organic cation transorters (OCT), OCTN1 and OCTN2 and urea transporters A and B . Active transport of hydroxyurea by OCT1B3 has also been suggested .
An important consideration in understanding the impact of retaining high levels of hydroxyurea in tissues is its inhibitory effect of ribonucleotide reductase (RR), an enzyme needed by all cells for DNA synthesis. Mammalian RR is less susceptible to inhibition by hydroxyurea than viral or bacterial RR (Table 1). This would be important for inhibition of viral and bacterial replication without affecting mammalian cell growth. Hydroxyurea thus could provide a level of protection against viral and other challenges as part of the innate immune defense mechanism.
Our findings of hydroxyurea in a mammal, although at levels 5–10 fold lower than in the elasmobranch, may be particularly important for understanding mammalian disease resistance. Exogenously applied hydroxyurea has been shown to stimulate nitric oxide production in mammalian systems [41,42]. Nitric oxide synthase plays a key role in the killing of pathogenic organisms by phagocytes  although the mechanism is not known . We suggest a role for naturally occurring hydroxyurea in phagocyte function via the following mechanism.
Nitric oxide is produced from arginine by nitric oxide synthase (NOS) via the intermediate hydroxyarginine. Arginase is known to react with hydroxyarginine in vitro to produce hydroxyurea instead of urea . We propose that in vivo some hydroxyarginine is diverted to hydroxyurea synthesis by arginase and the hydroxyurea converted to NO as depicted in Fig 4. This mechanism helps explain the paradoxical colocalization of arginase and NOS in cells such as human endothelial cells . In endothelial cells arginine metabolism is highly compartmentalized  and arginase is known to compete with NOS for arginine .
Our finding that hydroxyurea occurs in many animal groups at levels that could act as a defense against viral and other challenges implies: a) a new component to the innate immune system of animals that may explain superior disease resistance of some groups and b) a new intermediate in the pathway for NO production in animals.
We thank Dr. Armen Charchoglyan of the Mass Spectrometry Facility at the University of Guelph for technical assistance. We also thank Peter Smith for donation of frogs from the undergraduate physiology teaching lab at the University of Guelph. We also thank Brian McDougall, meat lab manager and his team, Department of Animal and Poultry Science, University of Guelph for the sheep tissue samples.
Conceived and designed the experiments: JSB DIF KTL. Performed the experiments: BJR EH AS MP PHRO JWR. Wrote the paper: DIF JSB.
- 1. Dresler WFC, Stein R (1869) Ueber den hydroxylharnstoff. Justus Liebigs Amm Chemie 150: 1317–1322.
- 2. Parker JN, Parker PM (2004) Hydroxyurea. A medical dictionary, bibliography, and annotated research guide to internet references. San Diego California: ICON Health Publications.
- 3. Elford HL (1968) Effect of hydroxyurea on ribonucleotide reductase. Biochemical and Biophysical Research Communications 33: 129–135. pmid:4301391
- 4. World Health Organization (2013) WHO model list of essential medicines 18th list. World Health Organization.
- 5. Svensson ML, Valerie K, Gatenbeck S (1981) Hydroxyurea, a natural metabolite and an intermediate in D-cycloserine biosynthesis in Streptomyces garyphalus. Archives of Microbiology 129:210–212.
- 6. Kettani T, Cotton F, Gulbis B, Ferster A, Kumps A 2009) Plasma hydroxyurea determined by gas chromatography-mass spectrometry. Journal of Chromatography 877B:446–450.
- 7. Frick NT, Bystriansky JS, Ip YK, Chew SF, Ballantyne JS (2008) Carbohydrates and amino acid metabolism following 60 days of fasting and aestivation in the African lungfish, Protopterus dolloi. Comparative Biochemistry and Physiology 151A:85–92.
- 8. Robinson JW, Yanke D, Mizra J, Ballantyne JS (2011) Plasma free amino acid kinetics in rainbow trout (Oncorhynchus mykiss) using a bolus injection of 15N-labelled amino acids. Amino Acids 40:689–696. pmid:20676903
- 9. Morgan RL, Ballantyne JS, Wright PA (2003) Regulation of a renal urea transport with salinity in a marine elasmobranch Raja erinacea. Journal of Experimental Biology 206:3285–3292. pmid:12909709
- 10. Fabricius E, Rajewsky MF (1971) Determination of hydroxyurea in mammalian tissues and blood. Revue Europeene D’Etudes Cliniques et Biologiques 16:679–683.
- 11. Rahmatullah M, Boyde TRC (1980) Improvements in the determination of urea using diacetylmonoxime; methods with and without deproteinisation. Clinica Chimica Acta 107:3–9.
- 12. Scott DK, Neville K, Garg U (2010) Determination of hydroxyurea in serum or plasma using gas chromatography-mass spectrometry (GC-MS). In: Garg U, Hammett-Stabler CA, editors. Methods in Molecular Biology: Volume 603. Clinical Applications of Mass Spectrometry. Humana Press p. 279–287.
- 13. Gaylor DW (2000) The use of Haber`s law in standard setting and risk assessment. Toxicology 149:17–19. pmid:10963857
- 14. Roa D, Kopsombut P, Aguinaga MdP, Turner EA (1997) Hydroxyurea-induced denaturation of normal and sickle cell hemoglobins in vitro. Journal of Clinical Laboratory Analysis 11:208–213. pmid:9219062
- 15. Pfeiffer SE, Tolmach LJ (1967) Inhibition of DNA synthesis in HeLa cells by hydroxyurea. Cancer Research 27:124–129. pmid:6020353
- 16. Young CW, Hodas S (1964) Hydroxyurea: inhibitory effect on DNA metabolism. Science 146:1172–1174. pmid:14203679
- 17. Yarbro JW, Kennedy BJ, Barnum CP (1965) Hydroxyurea inhibition of DNA synthesis in ascites tumor. Proceedings of the National Academy of Sciences of the United States of America 53:1033–1035. pmid:5222544
- 18. Rosenkranz HS, Gutter B, Becker Y (1973) Studies on the developmental cycle of Chlamydia trachomatis: selective inhibition by hydroxyurea. Journal of Bacteriology 115:682–690. pmid:4725618
- 19. Fontecave M, Lepoivre M, Elleingand E, Gerez C, Guittet O (1998) Resveratrol, a remarkable inhibitor of ribonucleotide reductase. FEBS Letters 421:277–279. pmid:9468322
- 20. Berglund O, Sjoberg BM (1979) Effect of hydroxyurea on T4 ribonucleotide reductase. Journal of Biological Chemistry 254:253–254. pmid:368052
- 21. Mohler WC (1964) Cytotoxicity of hydroxyurea (NSC-32065) reversible by pyrimidine deoxyribosides in a mammalian cell line grown in vitro. Cancer Chemotherapy Reports 34:1–6.
- 22. Veale D, Cantwell BMJ, Kerr N, Upfold A, Harris AL (1988) Phase 1 study of high-dose hydroxyurea in lung cancer. Cancer Chemotherapy and Pharmacology 21:53–56. pmid:3342464
- 23. Gale GR, Kendall SM, McLain HH, DuBois S (1964) Effect of hydroxyurea on Pseudomonas aeruginosa. Cancer Research 24:1012–1019. pmid:14195338
- 24. Nozaki A, Morimoto M, Kondo M, Oshima T, Numata K, Fujisawa S, et al. (2010) Hydroxyurea as an inhibitor of hepatitis C virus RNA replication. Archives of Virology 155:601–605. pmid:20204428
- 25. Lori F, Malykh A, Cara A, Sun D, Weinstein JN, Lisziewicz J, et al. (1994) Hydroxyurea as an inhibitor of human immmunodeficiency virus type 1 replication. Science 266:801–805. pmid:7973634
- 26. Schlapfer E, Fischer M, Ott P, Speck RF (2003) Anti-HIV-1 activity of leflunomide: a comparison with mycophenolic acid and hydroxyurea. AIDS 17:1613–1620. pmid:12853743
- 27. Rosenkranz HS, Rose HM, Morgan D, Hsu KC (1966) The effect of hydroxyurea on virus development. II. Vaccinia virus. Virology 28:510–519. pmid:5328227
- 28. Martinez-Rojano H, Macilla-Ramirez J, Quinonez-Diaz L, Galindo-Sevilla N (2008) Activity of hydroxyurea against Leishmania mexicana. Antimicrobial Agents and Chemotherapy 52:3642–3647. pmid:18694950
- 29. Sinclair WK (1965) Hydroxyurea: differential lethal effects on cultured mammalian cells during the cell cycle. Science 150:1729–1731. pmid:5858026
- 30. Hoff GR (2009) Embryo developmental events and the egg case of the Aleutian skate Bathyraja aleutica (Gilbert) and the Alaska skate Bathyraja parmifera (Bean). Journal of Fish Biology 74:483–501. pmid:20735574
- 31. Larsen IK, Sjoberg BM, Thelander L (1982) Characterization of the active site of ribonucleotide reductase of Escherichia coli, bacteriophage T4 and mammalian cells by inhibition studies with hydroxyurea analogues. European Journal of Biochemistry 125:75–81. pmid:7049700
- 32. Villani P, Maserati R, Regazzi MB, Giacchino R, Lori F (1996) Pharmacodynamics of hydroxyurea in patients infected with human immunodeficieny virus type 1. Journal of Clinical Pharmacology 36:117–121. pmid:8852387
- 33. Ballantyne JS, Robinson JW (2011) Physiology of sharks, skates and rays. In: Farrell AP, editor. Encyclopedia of fish physiology: from genome to environment. Volume 3. San Diego: Academic Press p. 1807–1818.
- 34. Smith M, Warmolts D, Thoney D, Hueter R (2004) Elasmobranch husbandry manual. Captive care of sharks, rays and their relatives. Columbus Ohio: Ohio Biological Survey, Inc.
- 35. Harshbarger JC, Charles AM, Spero PM (1981) Collection and analysis of neoplasms in sub-homeothermic animals from a phyletic point of view. In: Dawe CJ, Harshbarger JC, Kondo S, Sugimura T, Takayama S, editors. Phyletic approaches to cancer. Tokyo: Japanese Society of Science Press; p. 357–384.
- 36. Harshbarger JC (1977) Role of the registry of tumors in lower animals in the study of environmental carcinogenesis in aquatic animals. Annals of the New York Academy of Sciences 298:280–289.
- 37. Mylniczenko ND, Harris B, Wilborn RE (2007) Blood culture results from healthy captive and free-ranging elasmobranchs. Journal of Aquatic Animal Health 19:159–167. pmid:18201057
- 38. Grimes DJ, Grimes DJ (1990) Review of human pathogenic bacteria in marine animals with emphasis on sharks. Elasmobranchs as living resources: advances in the biology, ecology, systematics, and the status of the fisheries.Washington: NOAA; p. 63–69.
- 39. Zhao D, Sonawane ND, Levin MH, Yang B (2007) Comparative transport efficiencies of urea analogues through urea transporter UT-B. Biochimica et Biophysica Acta 1768:1815–1821. pmid:17506977
- 40. Walker AL, Franke RM, Sparreboom A, Ware RE (2011) Transcellular movement of hydroxyurea is mediated by specific solute carrier transporters. Experimental Hematology 39:446–456. pmid:21256917
- 41. King SB (2003) The nitric oxide producing reactions of hydroxyurea. Current Medicinal Chemistry 10:437–452. pmid:12570692
- 42. King SB (2004) Nitric oxide production from hydroxyurea. Free Radical Biology and Medicine 37:737–744. pmid:15304249
- 43. Babior BM (2000) Phagocytes and oxidative stress. American Journal of Medicine 109:33–44. pmid:10936476
- 44. Slauch JM (2011) How does the oxidative burst of macrophages kill bacteria? Still an open question. Molecular Microbiology 80:580–583. pmid:21375590
- 45. Boucher JL, Custot J, Vadon S, Delaforge M, Lepoivre M, Tenu JP, et al. (1994) Nω-hydroxy-L-arginine, an intermediate in the L-arginine to nitric oxide pathway is a strong inhibitor of liver and macrophage arginase. Biochemical and Biophysical Research Communications 203:1614–1621. pmid:7945311
- 46. Topal G, Brunet A, Walch L, Boucher JL, David-Dufilho M (2006) Mitochondrial arginase II modulates nitric-oxide synthesis through nonfreely exchangeable L-arginine pools in human endothelial cells. Journal of Pharmacology and Experimental Therapeutics 318:1368–1374. pmid:16801455
- 47. Mariotti F, Petzke KJ, Bonnet D, Szezepanski I, Bos C, Huneau JF, et al. (2013) Kinetics of the utilization of dietary arginine for nitric oxide and urea synthesis: in sight into the arginine-nitric oxide metabolic system in humans. American Journal of Clinical Nutrition 97:972–979. pmid:23535108
- 48. Mori M (2007) Regulation of nitric oxide synthesis and apoptosis by arginase and arginine recycling. Journal of Nutrition 137:1616S–1620S. pmid:17513437
- 49. Burkitt MJ, Raafat A (2006) Nitric oxide generation from hydroxyurea: significance and implications for leukemogenesis in the management of myeloproliferative disorders. Blood 107:2219–2222. pmid:16282342