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Departments of
* Medicine, and
Bioengineering, University of California San Diego, La Jolla, California, USA; and The Burnham Institute for Medical Research,
La Jolla, California, USA
1Correspondence: 9500 Gilman Dr., University of California, San Diego, La Jolla, CA 92093-0652, USA. E-mail: gboss{at}ucsd.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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200,000 people dying from sepsis annually in the U.S. The high mortality results in part from severe hypotension secondary to high serum NO concentrations. Reducing NO levels should be beneficial in sepsis, but NOS inhibitors have had a checkered history in animal models, and one such agent increased mortality in a clinical trial. An alternative approach to reduce NO levels in sepsis is to use an NO scavenger, which should leave sufficient free NO for normal physiological functions. Using a well-established model of bacterial sepsis in Drosophila melanogaster, we found that cobinamide, a B12 analog and an effective NO scavenger in vitro, dramatically improved fly survival. Cobinamide augmented the effect of an antibiotic and was beneficial even in immune-deficient flies. Cobinamide’s mechanism of action appeared to be from reducing NO levels and improving cardiac function.—Broderick, K. E., Feala, J., McCulloch, A., Paternostro, G., Sharma, V. S., Pilz, R. B., Boss, G. R. The nitric oxide scavenger cobinamide profoundly improves survival in a Drosophila melanogaster model of bacterial sepsis.
Key Words: septic shock • cobalamin • cardiac function
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Much of the pathophysiology of sepsis results from a systemic inflammatory response to the offending microbial agent (i.e., activation of the host’s innate immune response with production of cytokines and vasoactive substances). Hence, tumor necrosis factor-alpha (TNF-
), interleukin (IL)-1beta (interleukin-1ß), and platelet-activating factor all contribute to development of the septic state (1)
.
Neuronal NOS (nNOS) and endothelial NOS (eNOS) (NOS I and III, respectively), are constitutively expressed in many tissues producing relatively low pico- to nanomolar concentrations of NO in response to increased intracellular calcium (2)
. Inducible NOS (iNOS, NOS II) is expressed in many cell types including granulocytes, monocytes/macrophages, myocytes, vascular endothelial cells, and smooth muscle cells (3)
. Transcription of iNOS is induced many-fold by TNF-, IL-1ß, and bacterial endotoxin [LPS], and the resulting iNOS protein produces almost 1000-fold higher NO concentrations (i.e., nano- to micromolar) than the constitutive enzymes (4)
. A pathological increase in iNOS underlies the extreme elevation of NO in sepsis, with high NO levels clearly contributing to the profound hypotension, impaired cardiac function, and decreased systemic vascular resistance (SVR) that characterize this disorder (5)
.
Nonselective NOS inhibitors that inhibit all three NOS isozymes increase blood pressure and SVR in animal models of sepsis but have had a mixed effect on sepsis-associated survival in animals (6)
. This may relate to their inhibition of eNOS leading to microvascular vasoconstriction and decreased tissue and organ perfusion (7)
. In a large Phase III Clinical Trial, NG-methyl-L-arginine hydrochloride, a nonselective NOS inhibitor, depressed cardiac function and increased mortality, causing the trial to be terminated early (8)
. Selective iNOS inhibitors should avoid some of the problems encountered with nonspecific inhibitors and have yielded favorable results in animal studies (9)
. However, cardiac myocyte iNOS is required for ß-adrenergic responsiveness of the cells (10)
. Thus, even selective iNOS inhibition may be detrimental, and several groups of workers have urged caution in using any type of NOS inhibitor in sepsis (11)
.
Another approach to reduce the high NO levels in septic shock is to use an NO scavenger (6
, 12)
. When used at an appropriate concentration, an NO scavenger should neutralize only the pathologically elevated amounts of NO, and thus physiologically relevant amounts of NO will remain. Most of the work with NO scavenging has been with hemoglobin (Hb), but free extracellular Hb is nephrotoxic (6)
. Thus, other NO scavengers have been considered for sepsis, including cobalamin, which improves survival in rodents treated with LPS (13)
. We showed previously that cobinamide, the penultimate precursor in cobalamin biosynthesis lacking cobalamin’s dimethylbenzimidazole ribonucleotide tail (see Supplemental Fig. 1), has a 100-fold greater affinity for NO than cobalamin, and that each cobinamide molecule neutralizes two molecules of NO (14)
. Moreover, we showed that cobinamide is a highly effective NO scavenger in cultured cells and an isolated organ system (15)
. We now show that cobinamide markedly enhances survival in a D. melanogaster model of sepsis.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Escherichia coli strain DH 5- was from Invitrogen (Carlsbad, CA, USA) and S. aureus strain 29213 was from the American Type Culture Collection (ATCC, Manassas, VA, USA). Diethylenetriamine NONOate (Deta-NONOate) was from Cayman Scientific (Ann Arbor, MI, USA) and gentamicin, LPS, and N-monomethyl-L-arginine monoacetate (L-NMMA) were from Sigma Chemical Co. (St. Louis, MO, USA). NO gas > 99.9% pure was from Matheson (Montgomeryville, PA, USA), and was passed through 5 M NaOH prior to use to remove nitrogen oxide species (14)
. The membrane-permeable cGMP analogs 8-bromo-cGMP (8-Br-cGMP) and 8-chlorophenylthio-cGMP (8-CPT-cGMP) were from Biolog, Inc. (Hayward, CA, USA), the guanylate cyclase inhibitor [1H-(1,2,4)oxadiazolo(4,3–1)quinoxalin-1-one] (ODQ) was from Calbiochem (San Diego, CA, USA), and FlyNap was from Carolina Biological Supply Co. (Burlington, NC, USA). Dihydroxo-cobinamide (cobinamide) was produced and purified from hydroxo-cobalamin (Sigma Chemical Co.) as described previously (15)
; its purity was >95% as determined by spectral analysis and HPLC (15)
. Beveled 33 gauge needles and 2.5 µl glass syringes were from Hamilton Company (Reno, NV, USA).
Growth of D. melanogaster on cobinamide-supplemented food
Cobinamide-containing fly food was prepared as described by heating standard fly food paste to 40°C and adding cobinamide to a final concentration of 100 µM (15)
. Flies were grown on the supplemented food from the first instar larval stage prior to use in experiments. We observed no toxicity from the cobinamide even when flies were grown for more than 10 generations on the supplemented food.
Injection of D. melanogaster
Drosophila melanogaster were anesthetized on ice and injected into the dorsal thorax with 1 µl of the test agent using a 33 gauge needle mounted on a 2.5 µl syringe. For injecting E. coli and S. aureus, bacteria were grown overnight in LB broth to stationary phase (optical density 1.2 AU at 660 nm) and 1 µl of the bacterial suspension was injected. The number of bacteria in the suspension was determined by colony count after limiting dilution. Cobinamide, Deta-NONOate, LPS, L-NMMA, 8-Br-cGMP, 8-CPT-cGMP, and ODQ were prepared in water and 1 µl of the indicated concentration of the agent was injected. NO gas was dissolved in fully deoxygenated water to a saturating concentration of 1.95 mM (14)
. In coinjection experiments, cobinamide was premixed with the indicated agent immediately prior to injection. Once injected, flies were returned to standard culture bottles and observed for 72 h. Flies injected with only water or LB broth were fully awake and mobile within 30 min of injection. Starting 2 h postinjection, flies were assessed for viability and scored as dead if they were motionless for at least 5 min and did not respond to shaking of their vial.
Effect of cobinamide on bacterial growth in vitro and in vivo
The effect of cobinamide on bacterial growth in vitro was assessed by adding cobinamide at concentrations of 1, 10, and 100 µM to logarithmically growing suspensions of E. coli or S. aureus or by growing bacteria on agar containing cobinamide at the same concentrations (the agar was prepared in a similar manner as preparing cobinamide-containing fly food). The effect of cobinamide on bacterial growth in vivo was assessed by extracting flies 6 h postbacterial injection and determining the number of bacteria in the extracts by colony count after limiting dilution.
Measurement of nitrite and nitrate in D. melanogaster
NO has an extremely short half-life and is rapidly oxidized to nitrite and nitrate in the presence of oxygen. Hence, one of the most common methods for assessing NO production in vivo is to measure nitrite and nitrate concentrations using the Griess reagent (17)
. Using a kit from Active Motif based on an enhanced Griess reagent, we measured nitrite and nitrate as described (15)
. Briefly, 4 h after injection with the indicated agent, flies were anesthetized on ice, decapitated, and torsos were extracted in the kit lysis buffer by homogenization and sonication; the extracts were centrifuged and nitrite and nitrate were measured in the supernatant. To retard oxidation of NO present in the flies at the time of extraction, we deoxygenated the lysis buffer and added 1 mM sodium dithionite to the buffer; we showed in control experiments that neither procedure had any effect on subsequent measurement of nitrite and nitrate.
In experiments where flies were coinjected with cobinamide, we reduced the cobinamide from its standard +3 valency state [Co(III)] to the +2 valency state [Co(II)] by adding a 10-fold excess of ascorbic acid (which had no effect on the flies or on the subsequent measurement of nitrite and nitrate). It was necessary to reduce the cobalt because, as mentioned earlier, 1 mol of cobinamide[Co(III)] neutralizes 2 mol of NO: during neutralization of the first NO molecule, the Co(III) is reduced to Co(II) oxidizing NO to nitrite (14)
; the latter would raise the background in the nitrite and nitrate measurement. The resulting cobinamide[Co(II)] binds one NO, and thus can still be used as an NO scavenger.
Assessment of D. Melanogaster heart rate
We measured the heart rate of Drosophila using light microscopy as described previously (18)
. Briefly, flies were injected as described above with the indicated agent in the absence or presence of cobinamide. Four hours later, the flies were anesthetized with 50% triethylamine (FlyNap) and placed on a microscope slide, dorsal side down, with wings spread. An inverted microscope was used to record a 20 s moving image of each fly heart while subjected to heat stress at 35°C to induce a maximal rate. Customized software was used to extract a 2-dimensional time-space, or M mode, representation of cardiac contraction from image intensities measured along a line perpendicular to the heart tube. The frequency of contraction was derived from the average intensity of pixels on the line. Minor modifications to the described M mode analysis (18)
were necessary to account for the slower rates observed under some conditions. In addition to using Fourier analysis of the images, which can miss slow contraction frequencies among low frequency noise, a beat detection algorithm was used that detects and counts the number of systolic contractions within the M mode image (19)
.
Statistical analysis
Differences between sets of data were compared using a nonpaired t test.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 21)
. Using this model, we infected flies with 1 x 108 colony forming units of either a Gram-positive (Staphylococcus aureus) or Gram-negative (Escherichia coli) organism (Fig. 1
). Consistent with previous work (20
, 21)
, we found that flies succumbed to both bacteria, with S. aureus being more toxic than E. coli (Fig. 1
, compare open circles in panels A, B, noting different time scale). Injecting flies with water or bacterial culture broth was without effect, indicating that the bacteria, and not the injection process, killed the flies (Fig. 1A
, inverted triangles). Flies that had been grown on food supplemented with cobinamide, both before and after bacterial injection, showed a striking improvement in survival compared to flies not provided cobinamide (Fig. 1A, B
, compare hexagons, flies fed cobinamide, to open circles). Flies fed cobinamide before injection only or after injection only were less resistant to the bacteria but still had a marked increase in survival (Fig. 1B
, diamonds and upright triangles; similar results were found in S. aureus). Cobinamide was considerably more effective than cobalamin in these studies, increasing the percentage of flies that survived by at least 4-fold over that of cobalamin.
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In the treatment of sepsis, it is unlikely that oral administration of a drug would result in a sufficiently rapid effect, and the drug would most likely need to be administered parenterally. We therefore altered the experimental protocol and coinjected cobinamide with the bacteria; we could not test the effect of administering cobinamide at various times after bacterial injection because the flies could not withstand two successive injections. We found that coinjecting cobinamide at a final concentration of 500 µM in the bacterial broth induced the same striking improvement in survival as observed in flies provided oral cobinamide before and after bacterial injection (Fig. 1B
, squares; similar results were found for S. aureus). Lower concentrations of cobinamide were less effective, while 1 mM cobinamide had a slightly greater effect. The fluid volume of a fly is 10 µl (22)
, suggesting that the initial drug concentration was 50 µM in flies injected with 500 µM cobinamide; however, we had found earlier that cobinamide is excreted rapidly by the Malpighian tubules of Drosophila, and thus the cobinamide concentration in the flies was likely less during the course of the experiment (15)
. We found no evidence of cobinamide toxicity either in flies acutely injected with cobinamide or in ones fed cobinamide for multiple generations.
Clinically, an NO scavenger such as cobinamide would be combined with other treatment modalities in a septic patient. We therefore assessed the combination of cobinamide with the antibiotic gentamicin, and found that cobinamide significantly augmented the beneficial effect of gentamicin on fly survival in S. aureus-infected flies (Fig. 2
A). Thus, cobinamide could enhance the effect of antibiotics in the treatment of sepsis.
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To determine whether cobinamide would be beneficial in an immunocompromised host, we took advantage of the power of Drosophila genetics and the availability of multiple deficient lines. We studied flies with a mutation in Imd, which is part of innate fly immunity and regulates production of antimicrobial peptides (23
, 24)
. As found earlier (16
, 23
, 24)
, Imd– flies succumbed more rapidly to bacterial infection than WT flies (Fig. 2B
). Coinjecting cobinamide with bacteria into Imd– flies significantly prolonged fly survival (Fig. 2B
), indicating that cobinamide was effective even under conditions of an impaired immune response.
Since NOS inhibitors have been used in experimental sepsis, we assessed the effect of L-NMMA, a nonselective NOS inhibitor, on survival of flies infected with E. coli. When injected at concentrations 
1 µM, L-NMMA was toxic to flies and decreased the survival of flies injected with E. coli (Fig. 3
), whereas when injected at concentrations between 0.01 and 0.1 µM, L-NMMA caused a small, statistically nonsignificant increase in survival of E. coli-infected flies (Fig. 3)
.
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Mechanism of cobinamide rescue of D. melanogaster from bacterial sepsis
To study the mechanism of cobinamide’s beneficial effect on bacterially infected flies, we first assessed whether cobinamide could be directly toxic to bacteria. At concentrations as high as 100 µM, cobinamide had no effect on the logarithmic growth of E. coli or S. aureus in vitro. To determine whether cobinamide might have an effect in vivo, we quantitatively cultured bacteria from flies 6 h postbacterial injection and found the same number of bacteria in cobinamide-treated flies as in untreated flies. Thus, cobinamide did not appear to have a bactericidal or bacteriostatic effect.
To test the hypothesis that cobinamide was functioning as an NO scavenger, we first assessed whether cobinamide is effective in other states of excess NO, such as in flies treated with an NO donor or in LPS-treated flies. LPS is toxic to flies and activates the Drosophila NOS gene, thereby increasing the NO concentration (25)
. We found that injecting 1 µl of 1 mM Deta-NONOate or 5 mg/ml LPS killed flies with kinetics similar to that when injecting flies with E. coli and that cobinamide markedly increased survival in the Deta-NONOate- and LPS-injected flies both when the flies were fed cobinamide or when coinjected with the drugs (Figs. 4
A, B). We found similar results when flies were injected with pure NO gas dissolved in buffer, making it unlikely that cobinamide was reversing a potential toxic effect of the diethylene-triamine backbone of Deta-NONOate. In a second set of experiments, we assessed the effect of cobinamide on the concentrations of nitrite and nitrate in injected flies, since the latter are stable catabolic products of NO and can be used to assess NO concentrations over time (17)
. Injecting flies with E. coli, S. aureus, or LPS increased nitrite and nitrate concentrations by 4- or 5-fold over those found in flies injected with vehicle alone, consistent with induction of the Drosophila NOS gene (Fig. 5
A). In flies coinjected with cobinamide, the increase in nitrite and nitrate concentrations was <2-fold (Fig. 5A
). Injecting flies with Deta-NONOate increased nitrite/nitrate levels >8-fold, which was reduced to 3-fold when flies were treated with cobinamide (Fig. 5A
). Thus, cobinamide appeared to scavenge NO in the flies. In a final set of experiments we assessed the effect of cobinamide in flies treated with the cGMP analogs 8-Br-cGMP and 8-CPT-cGMP. Cyclic GMP is downstream from NO in the NO/guanylate cyclase signal transduction pathway, and cobinamide would not be expected to affect cGMP-induced changes. Consistent with this postulate, we found no effect of cobinamide on 8-Br-cGMP and 8-CPT-cGMP toxicity in flies (Fig. 5B
). The increased toxicity of 8-CPT-cGMP compared to 8-Br-cGMP may be because the former compound is less susceptible to phosphodiesterases than the latter compound. Since bacteria, an NO donor, and cGMP analogs killed flies with similar kinetics, this suggests that their lethal effects may involve a similar mechanism (or mechanisms).
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Cbi improves cardiac function in bacterially injected flies
Increased NO in sepsis suppresses mammalian cardiac function through the guanylate cyclase/cGMP/cGMP-dependent protein kinase signal transduction pathway (8
, 10
, 26)
. We found that injecting Drosophila with E. coli or 8-CPT-cGMP significantly reduced the flies’ heart rate (Fig. 6
A). These measurements were performed 4 h postinjection because this amount of time was required for the heart rate to return to baseline in control flies injected with water; flies already dead at this time were excluded from the analysis, and so the observed effect could have been even greater. We could not perform measurements on S. aureus-injected flies because by 4 h postinjection most S. aureus-injected flies were already dead (as shown in Fig. 1A
). Cobinamide alone had no affect on the heart rate of Drosophila, and returned the rate fully to normal in E. coli-injected flies (Fig. 6A
). As would be expected for an NO scavenger, cobinamide had no affect on the 8-CPT-cGMP-induced reduction in cardiac rate (Fig. 6A
). These data suggest that cobinamide was rescuing bacterially injected flies at least in part by improving cardiac function. As further evidence that the decrease in heart rate in bacterially injected flies was from activating the NO/guanylate cyclase/cGMP signal transduction pathway, we found that the guanylate cyclase inhibitor ODQ also returned the heart rate to normal in E. coli-injected flies (Fig. 6B
). Shown in Fig. 6C
is a representative M mode analysis of the beating heart of Drosophila.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. The only new drug specifically approved for treating sepsis is recombinant activated protein C (drotrecogin alfa), and because it is associated with an increased risk of serious bleeding, it is approved only for patients with severe sepsis who are predicted to have a high mortality rate (27)
. Novel approaches to treating sepsis are needed urgently.
Since high NO levels clearly are a major cause of the profound hypotension and poor organ perfusion in sepsis, it was logical that NOS inhibitors were tried in this disorder. However, since these agents inhibit intracellular NO production, they could have a detrimental effect by impairing normal NO functions, such as maintenance of microvascular flow. This may be why nonselective NOS inhibitors increased mortality in some animal models of sepsis and had a detrimental effect in human sepsis (6
, 8
, 28
29
30)
. Similar to mammalian systems, we found that a NOS inhibitor, at low micromolar concentrations, was toxic to D. melanogaster whereas nontoxic concentrations of the NOS inhibitor did not significantly improve survival of bacterially infected flies. Impairment of normal NO functions should not occur with an NO scavenger when used at an appropriate concentration because it should leave sufficient free NO available. We estimated free NO concentrations at various NO and cobinamide concentrations using the COMICS program, which calculates free ligand concentrations knowing the KA of the binding partner (31)
. Because NO concentrations are in the high nano- to low micromolar range in sepsis, we chose 0.1, 1, and 5 µM as the total NO concentration and calculated the free NO concentration at 1 and 10 µM cobinamide (see Supplemental Table 1). Even at a cobinamide concentration 10-fold greater than that of NO (e.g., 1 µM cobinamide and 0.1 µM NO or 10 µM cobinamide and 1 µM NO), the free NO concentration was 9 pM, which is in the physiological range of NO produced by eNOS and nNOS.
Drosophila is increasingly being recognized as an excellent model for human disease and drug discovery (32
, 33)
. Compared to mammalian models, flies are inexpensive, reproduce rapidly, and are easily mutated. A Drosophila model for bacterial sepsis has been used for >30 years to study host defense mechanisms, including innate immunity (16
, 23
, 24
, 34)
. We took advantage of the power of fly genetics and studied the effects of cobinamide in bacterially infected immune-deficient flies. We would have liked to have studied NOS-deficient flies, which should exhibit some resistance to bacterial infection, but unfortunately these flies die in late embryonic and early larval stages (35)
.
Of nonvertebrate animals, Drosophila is particularly appropriate to use in sepsis studies because, as in mammals, NO and cGMP contribute to regulation of the heart and circulatory system (36
, 37)
. A difference between flies and mammals, however, is that flies cannot generate a reflex tachycardia in response to decreased flow of hemolymph as mammals do in response to hypotension. E. coli and 8-CPT-cGMP suppressed cardiac rate in the flies to approximately the same extent, pointing to cardiac impairment as a potential toxic mechanism. This is analogous to mammalian systems where high NO levels during bacterial sepsis lead to decreased cardiac contractility and circulatory collapse; in addition, bradycardia can occur in human sepsis and is especially common in neonatal sepsis (38)
.
Cobinamide is a contaminant of vitamin B12 preparations and can be absorbed across hog ileum independent of intrinsic factor (39)
. Once absorbed, cobinamide binds tightly to haptocorrin but poorly to transcobalamin II (40)
. Since the serum levels of haptocorrin and transcobalamin II are similar, a significant amount of cobinamide can be present in serum, and cobalamin analogs, including cobinamide, have been detected in the serum, bile, and tissues of animals and humans, accounting for 5–50% of total corrinoids (41
, 42)
. Studies in rabbits show that, like cobalamin, the vast majority of injected cobinamide is taken up by the liver within minutes; after hepatic release, the tissue distribution of cobinamide and cobalamin was similar, and both were excreted in the urine and feces (43)
. Hence, the pharmacokinetics of cobinamide are similar to that of cobalamin.
At concentrations of up to 200 µM, cobinamide did not inhibit the growth of mouse leukemic cells or human monocytes and lymphocytes (44
, 45)
. In whole animals, cobinamide had no effect on the growth of baby chicks when administered parenterally at 40-fold the dose of cobalamin; it did inhibit chick growth when given orally, suggesting it interfered with cobalamin absorption (46)
. Cobinamide administered subcutaneously to rats at a continual rate of 2 µg/h for 14 days had no apparent toxic effects, and the drug did not inhibit the two mammalian cobalamin-dependent enzymes methionine synthase or methylmalonyl-coenzyme A mutase (47)
. In cultured human and rodent fibroblasts, we found no evidence of cobinamide toxicity up to concentrations of 50 µM, and toxicity could be reversed fully by coadministering cobalamin (15)
. Thus, cobinamide appears to be relatively nontoxic in mammalian systems, and any toxicity can be overcome by cobalamin.
In conclusion, we found that cobinamide is highly effective in rescuing D. melanogaster from bacterial sepsis. Cobinamide appeared to function by scavenging NO and reversing inhibitory effects of NO/cGMP on cardiac function. Since NO/cGMP suppress cardiac function in mammals, the D. melanogaster bacterial sepsis model appears remarkably similar to mammalian systems, and cobinamide may be an excellent drug candidate for the treatment of sepsis.
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ACKNOWLEDGMENTS |
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Received for publication January 12, 2006. Accepted for publication April 17, 2006.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-amino-L-arginine, an inhibitor of nitric oxide synthase, raises vascular resistance but increases mortality rates in awake canines challenged with endotoxin. J. Exp. Med. 176,1175-1182This article has been cited by other articles:
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  K. E. Broderick, L. Alvarez, M. Balasubramanian, D. D. Belke, A. Makino, A. Chan, V. L. Woods Jr., W. H. Dillmann, V. S. Sharma, R. B. Pilz, et al. Nitrosyl-Cobinamide, a New and Direct Nitric Oxide Releasing Drug Effective In Vivo Experimental Biology and Medicine, December 1, 2007; 232(11): 1432 - 1440. [Abstract] [Full Text] [PDF]
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K. E. Broderick, M. Balasubramanian, A. Chan, P. Potluri, J. Feala, D. D. Belke, A. McCulloch, V. S. Sharma, R. B. Pilz, T. D. Bigby, et al. The Cobalamin Precursor Cobinamide Detoxifies Nitroprusside-Generated Cyanide Experimental Biology and Medicine, June 1, 2007; 232(6): 789 - 798. [Abstract] [Full Text] [PDF]
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