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Assay for vitamin b12 absorption and method of making labeled vitamin b12

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Assay for vitamin b12 absorption and method of making labeled vitamin b12


The invention provides methods for labeling vitamin B12 with 14C, 13C, tritium, and deuterium. When radioisotopes are used, the invention provides for methods of labeling B12 with high specific activity. The invention also provides labeled vitamin B12 compositions made in accordance with the invention.
Related Terms: Vitamin B12

Browse recent The Regents Of The University Of California patents - Oakland, CA, US
Inventors: Peter J. Anderson, Stephen Dueker, Joshua Miller, Ralph Green, John Roth, Colleen Carkeet, Bruce A. Buchholz
USPTO Applicaton #: #20120264174 - Class: 435 86 (USPTO) - 10/18/12 - Class 435 
Chemistry: Molecular Biology And Microbiology > Micro-organism, Tissue Cell Culture Or Enzyme Using Process To Synthesize A Desired Chemical Compound Or Composition >Preparing Compound Containing Saccharide Radical >N-glycoside >Cobalamin (i.e., Vitamin B12, Lld Factor)

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The Patent Description & Claims data below is from USPTO Patent Application 20120264174, Assay for vitamin b12 absorption and method of making labeled vitamin b12.

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CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser. No. 11/234,079, filed Sep. 23, 2005, which claims benefit of U.S. provisional application 60/613,172, filed Sep. 23, 2004. Each application is incorporated by reference herein for all purposes.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

This invention was made with Government support under Grant No. GM34804 awarded by the National Institutes of Health. The Government has certain rights in this invention. The United States Government also has rights in this invention pursuant to Contract No. W-7405-ENG-48 between the United States Department of Energy and the University of California for the operation of Lawrence Livermore National Laboratory.

BACKGROUND OF THE INVENTION

Vitamin B12 (cobalamin, or “Cbl”) deficiency is a significant public health problem, particularly among the elderly. In the United States, there are 37 million people over age 65 and conservative estimates indicate that 2-3% of this population has or will develop pernicious anemia caused by failure of gastric intrinsic factor production and consequent B12 malabsorption (Chanarin, I. The megaloblastic anemias, 2nd edition. “Blackwell Scientific Publications” Oxford (1979); Cannel, R. Archives of Internal Medicine 156:1097-1100 (1996)). Other estimates suggest that the prevalence of B12 deficiency may be as high as 30-40% among the elderly due to food B12 malabsorption caused by chronic gastritis, gastric atrophy, and perhaps other unknown causes (Baik, H. W. et al. Annual Review of Nutrition 19:357 77 (1999)). In addition, surgical procedures such as gastrectomy and ileal resection, inflammatory bowel disease (Crohn\'s disease), radiation therapy for cancers of the abdominal or pelvic region, treatment of gastric reflux with H2 blockers such as omeprazole, and bacterial overgrowth in the small intestine, cause B12 malabsorption syndromes. Recently, we and others have observed an apparently high prevalence of B12 deficiency in both children and young adults in diverse locations, such as Guatemala, Mexico, Kenya, and Israel (Rogers, L. M. et al., FASEB J 13:A251 (1999); Gielchinsky, Y. et al., British Journal of Haematology 115:707-9 (2001); Lindsay, H. Allen, PhD. Personal Communication). The causes of B12 deficiency in these populations are unclear, but may be related to a combination of low intake and unrecognized malabsorption.

The classical pathophysiological manifestations of B12 deficiency include megaloblastic anemia and neurological degeneration related to neuronal demyelination (Green, R. et al., Neurology 45:1435-40 (1995)). Neurological deficits run the gamut from peripheral neuropathy to depression, cognitive disturbances, and dementia (Savage, D. G., Baillier\'s Clinical Haematology 8:657-78 (1995); Van Goor, L. P. et al., Age Ageing 24:536-42 (1995)). Moreover, recent evidence suggests that B12 deficiency may contribute to the risk of vascular disease (related to elevated plasma levels of the vascular risk factor homocysteine) (Refsum, H. et al., Annu Rev Med 49:31-62 (1998)), cancer (particularly breast cancer) (Choi, S-W. Nutrition Reviews 57:250 60 (1999)), and neural tube defects (spina bifida, anencephaly) (Refsum, H. British Journal of Nutrition 85 (supp12):5109-13 (2001)). B12 deficiency may also play a role in the rate of onset of clinical AIDS resulting from HIV infection (Tang, A. M. et al., Journal of Nutrition 127:345-51 (1997); Baum, M. K. et al., AIDS 9:1051-6 (1995))

The risks of B12 deficiency may be accentuated by folic acid fortification. The importance of recognizing B12 deficiency and conditions that cause B12 malabsorption is magnified by the government-mandated addition of folic acid to the United States food supply since Jan. 1, 1998 intended to reduce the risk of neural tube birth defects. A recent national advisory committee on B vitamin intake has cautioned on the need for increased vigilance to detect B12 deficiency and its causes, particularly among the elderly in the wake of the folic acid fortification initiative (Food and Nutrition Board—Institute of Medicine. Dietary reference intakes for thiamin, riboflavin, niacin, vitamin B6, folate, vitamin B12, pantothenic acid, biotin, and choline. National Academy Press, Washington, D.C. (1998)). This is based on the well-recognized observation that folic acid can reverse megaloblastic anemia caused by B12 deficiency while neurological degeneration progresses unabated. This is important because B12 deficiency in the elderly is often not suspected until anemia develops. It is unknown whether the supplemented level of folic acid in the food supply will increase the incidence of neurological damage due to undiagnosed B12 deficiency. Several authoritative sources have cautioned that there is no known safe dose of supplemental folic acid in patients with untreated B12 deficiency (Chanarin, I. The megaloblastic anemias, 2nd edition. “Blackwell Scientific Publications” Oxford (1979); Food and Nutrition Board—Institute of Medicine. Dietary reference intakes for thiamin, riboflavin, niacin, vitamin B6, folate, vitamin B12, pantothenic acid, biotin, and choline. National Academy Press, Washington, D.C. (1998)). Therefore, the institution of folic acid fortification necessitates increased consideration of B12 status, particularly in older adults.

B12 absorbed across the small intestine enters the circulation bound to a transcobalamin. Ingested B12 is released from food sources by the activity of gastric enzymes aided by the low pH of the stomach maintained by gastric acid (Stabler, S. P. In: Present Knowledge in Nutrition, 8th ed. Bowman B A, Russell R M, eds. Washington, D.C.: ILSI (2001)). After its release from food, B12 binds with haptocorrin (also known as “R binder”) secreted by the salivary glands. The B12-R binder complex then travels to the small intestine where, in an alkaline milieu, trypsin and other pancreatic proteases degrade the R binder and release the B12. The B12 subsequently binds with the gastric glycoprotein, IF. The IF-B12 complex binds to specific receptors located on the brush border of the ileal mucosa. Following internalization, the B 12 molecule is released from IF within ileal enterocytes and enters the portal venous blood bound to transcobalamin II (TC II), one of three B12 carrier proteins in the plasma. TC II is produced in endothelial cells and is responsible for receptor-mediated uptake of B 12 by all tissues. Structurally distinct from TC II are the haptocorrins (also known as transcobalamins I and III). These are glycoproteins produced in granulocytes, and appear to play no role in receptor-mediated cellular B12 delivery.

The Schilling Urinary Excretion test has been the standard method of testing for a patient\'s ability to absorb B12 since its introduction in 1953. The test has four stages. In Stage I, the patient takes an oral dose of B12 labeled with a radioactive isotope of cobalt, such as 60Co or 57Co. A period of time, usually 2 to 6 hours, is permitted to pass to allow sufficient time for the labeled B12 to be absorbed into the patient\'s small intestine. At the end of the period, the patient is given an intramuscular injection of an excess of non-labeled B12 to flush labeled B12 that the patient has absorbed into the intestine out of the patient into the urine. The patient collects all of his or her urine for a 24 hour period (a so-called “complete collection”), and the radioactivity in the urine is then measured. In patients with normal absorption, 8 to 40% of the labeled B12 will appear in the urine, while patients with absorption problems will have less than 8%, or frequently none.

If the amount of radiolabeled B12 in the urine reveals that the B12 has not been absorbed well from the oral dose, the cause for the malabsorption must be determined. Thus, after a sufficient time has passed to permit most of the excess B12 administered in Stage I to be eliminated from the intestinal tract, Stage II of the test is conducted. In this Stage, radiolabeled B12 is administered again, this time with an oral dose of intrinsic factor. Proper absorption indicates that the problem is a lack of intrinsic factor. If the B12 is again not absorbed, the problem could be, for example, bacterial overgrowth with consumption of B12, sprue, celiac disease, or liver disease. Thus, if the Stage II results are abnormal, Stage III is conducted. In this Stage, the patient is put on antibiotics for two weeks and the B12 absorption measured again to see if excess bacterial growth is interfering with B12 absorption. Finally, if absorption is still abnormal, the Stage IV test involves administering pancreatic enzymes in conjunction with radioactive B12 to determine if the malabsorption is due to a lack of these enzymes, another rare cause of B12 malabsorption.

While the Schilling test has been the standard test for decades, its use has become rare. The test is cumbersome, relatively unreliable and expensive. The test requires the use of B12 labeled with radioactive cobalt, which is difficult to procure and to dispose of Radioactive cobalt emits gamma radiation, a penetrating form of radiation which is only attenuated using very dense protective shielding (often lead); this exposes the patients and medical personnel to a small but quantifiable level of gamma radiation from the labeled B12. Moreover, the urine samples provided by the patients following dosage must be treated as radioactive waste. The increasingly stringent regulation of radioactive waste, the undesirability of subjecting patients to gamma radiation, and the extra handling care required for a gamma emitter all increase the expense of the test and reduce the desirability of administering it.

Additionally, because the first stage of the test involves administering an injection of B12 to the patient, it is necessary to wait a significant time for the B12 from that shot to be eliminated before the patient can be administered a second test to determine whether lack of intrinsic factor is the reason for the deficiency. This waiting period causes problems with patient compliance and, of course, undesirably delays treatment of the underlying condition. Further, since the test is based on urine collection, it is problematic for use with people who have renal problems, since lack of labeled B12 in the urine may reflect renal insufficiency rather than absorption problems. Unfortunately, the prevalence of renal problems tend to be higher in older people, who are also the population who most need to be tested for B12 absorption problems.

In some instances, the problem is that the patient cannot absorb B12 well from food, but can absorb so-called crystalline B12 well. In these instances, the patient can get adequate B12 simply by taking it in pill form, avoiding the need for monthly injections.

B12 labeled with radioactive carbon or hydrogen would be a useful alternative to B12 labeled with radioactive cobalt. 14C, for example, emits beta particles, rather than gamma radiation, and is thus safer for lab personnel to handle, as well as safer for patients. B12 labeled with carbon or hydrogen is not commercially available. 14C-labeled B12 was made by Boxer et al. as early as 1951 (Arch Biochem. 30(2):470-1). The B12 was labeled by labeling the cyano group and making cyanocobalamin. Unfortunately, since the cyano-group is cleaved off in the body, B12 labeled in this manner is not useful for metabolic studies or for tracking B12 absorption. B12 can also be labeled with 14C by growing bacteria on media labeled with 14C and then separating out the B12; unfortunately, in many cases, relatively little B12 is made in comparison to other bacterial metabolic products, resulting in a small amount of radiolabeled B12 and a large amount of radiolabeled waste material. Moreover, even in cases where the organism makes a relatively high percentage of B12 compared to other products, only a small proportion of the B12 is labeled in comparison to the total amount of B12. Since there is a limit to how much B12 can be absorbed even by persons with normal absorption of B12, to be useful, a high percentage of the B12 molecules should be labeled for the results of any studies to be accurate. (The degree to which a substance is labeled is referred to as its “specific activity.”) The recommended daily intake of vitamin B12 is 2.4 μg. Larger doses have the possibility of altering the B12 status before a diagnosis can be made, and a dose with a vitamin B12 mass less than 10 μg is therefore desirable, with doses of 5 μg or smaller being desirable. Thus, it is desirable to use B12 with a high specific activity.

It would be desirable to have a radiolabeled B12 that is not labeled with a gamma emitter. It would further be desirable to be able to produce a radiolabeled B12 in a manner that does not create large amounts of radioactive waste in its production in comparison to the amount of labeled B12 and that has high specific activity. It would be further useful to be able to have a test for B12 deficiency that would permit determination of the cause of B12 deficiency. The present invention fulfills these and other needs.

BRIEF

SUMMARY

OF THE INVENTION

The invention provides methods for labeling vitamin B12, comprising providing a facultative anaerobe bacterium (i) which has a eut operon, (ii) which requires B12 to grow on a medium in which ethanolamine or propandiol is the only carbon source, and (iii) cannot synthesize B12 under aerobic conditions, (b) providing a medium having ethanolamine or propandiol as the only carbon source, (c) providing labeled cobinamide (“Cbi”), or a labeled dimethylbenzimidazole (“DMB”), or both, and (d) culturing the bacterium on said media in the presence of said Cbi and said DMB, under aerobic conditions, thereby resulting in the formation of labeled B12. The bacterium can be a Salmonella species. In some embodiments, the Salmonella species is S. enterica. The label can be, for example, 13C, deuterium, and tritium. The label can be on the DMB.

In other embodiments, the invention provides methods of determining absorption of vitamin B12 in a subject, comprising: (a) administering B12 labeled with 14C, 13C, deuterium, or tritium to the subject; (b) taking a blood sample from said subject, (c) subjecting the blood sample to mass spectroscopy and measuring elevation of 14C, 13C, deuterium or tritium associated with the labeled B12 above background 14C, 13C, deuterium, or tritium concentrations, respectively and (d) comparing said concentration of labeled B12 to the concentration of labeled B12 found in subjects with normal B12 absorption. In some embodiments, the mass spectroscopy is accelerator mass spectroscopy. In some embodiments, the label is 14C or tritium.

In yet other embodiments, the invention provides methods of studying vitamin B12 metabolism in a subject, comprising: (a) administering B12 labeled with 14C, 13C, deuterium, or tritium to said subject; (b) taking a blood sample from said subject, (c) subjecting said blood sample to mass spectroscopy and measuring elevation of 14C, 13C, deuterium or tritium associated with the labeled B12 above background 14C, 13C, deuterium, or tritium concentrations, respectively and (d) comparing said concentration of labeled B12 to the concentration of labeled B12 found in subjects with normal B12 absorption. The vitamin B12 metabolism can be plasma clearance, turnover rate, enzyme kinetic measurement, or uptake and distribution of the vitamin.

In another aspect, the invention provides a radioactively labeled vitamin B12 composition having a specific activity of at least about 100 mCi/mol wherein the label is 14C, and physiologically acceptable compositions comprising such labeled vitamin B12 compositions. In some embodiments, the radioactively labeled vitamin B12 composition has a specific activity of at least 1 Ci/mol. In other embodiments, the radioactively labeled vitamin B12 composition has a specific activity of at least 10 Ci/mol, often at least 25 Ci/mol or at least 40 Ci/mol. In exemplary embodiments, 14C-labeled B12 has a specific activity of 50-62Ci/mol. In other embodiments, the 14C-labeled B12 has a specific activity of 37 Ci/mol.

B12 compositions of the invention also include tritium-labeled vitamin B12 compositions, and physiologically suitable compositions comprising such tritium-labeled B12 compositions. The specific activity is often at least 25 Ci/mol. Typically, the tritium-labeled vitamin B12 of the invention has a specific activity that is in the range from about 3000 to about 5000 Ci/mol. In some embodiments the specific activity is about 55,000 Ci/mol, e.g., 57, 400, Ci/mol.

The invention also provides a labeled vitamin B12 composition that has a nonradioactive label incorporated, e.g., 13C or deuterium. Such labeled vitamin B12 compositions typically have at least over 10% of the label incorporated into them. Typically, nonradioactively-labeled B12 molecules of the invention have over 50%, often over 60-70%, and typically 90%-100% incorporated label.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram of a B12 molecule. The corrin ring is the structure comprising the cobalt at the core of the molecule, while 5,6-dimethylbenzimidazole is the nucleotide ligand at the lower middle of the figure. The R group is most often methyl, deoxyadenosyl, cyano, or hydroxo.

FIG. 2 shows a radiochromatograom of 14C-vitamin B12 from S. typhimurium following growth with 14C-DMB.

FIG. 3 shows the UV/vis Spectrum of the B12 peak from the bacterial extract (upper line) and a vitamin B12 standard (lower line).

FIG. 4 shows accelerator mass spectrometry (ASM) detection of 14C in human plasma. Units are expressed as femtomolar 14C-B12. Measurements were performed on 30 μl of plasma, the entire sample set consumed less than 1 mL of whole blood.

FIG. 5 shows recovery of 14C in urine and fecal specimens, and cumulative recovery of 14C. After seven days, 15.9% of the dose was recovered in the urine and stool, consistent with the slow body turnover of vitamin B12.

DETAILED DESCRIPTION

OF THE INVENTION I. Introduction

Surprisingly, the present invention provides methods for producing 13C- or 14C, or tritium-radiolabeled vitamin B12 without creating large amounts of radiolabeled waste compared to the amount of B12. The invention provides for labeled B12 with high specific activity, that is, that a high percentage of the B12 molecules are labeled relative to those that are not. Further, the invention provides methods of assaying B12 absorption that overcome some of the procedural problems with the Schilling test. Moreover, the invention also provides methods for determining whether a person has malabsorption of B12 that uses such limited amounts of radiolabeled B12 that in some embodiments, the body samples taken for measurement do not qualify as radioactive waste under U.S. regulations, markedly easing the handling and paperwork requirements for conducting the test. The methods of the invention therefore overcome some of the problems that have reduced recent use of the Schilling test, and are expected to result in improved monitoring of the B12 absorption of the at-risk population, thereby improving the public health.

A further advantage of the invention is that 14C has a half life of thousands of years, while 60Co has a half life of 5.26 years and 57Co, which is currently the most commonly used isotope, has a half life of 270 days. Thus, cobalt-labeled B12 has a relatively short shelf life and cannot be maintained for substantial periods, while the half life of 14C-labeled B12 is so long that it can be considered as stable for the uses contemplated herein.

A. Controlled C and H Labeling of B12

“Vitamin B12” or “B12” refers to a “family of substances composed of tetrapyrrole rings surrounding a central cobalt atom with nucleotide side chains attached to the cobalt. The overall group name is cobalamin, with each of the different cobalt-linked upper axial ligands conferring a different name: methyl (methylcobalamin), hydroxyl (hydrocobalamin) H2O (aquacobalamin), cyanide (cyanocobalamin) and 5-deoxyadenosine (deoxyadenosylcobalamin).” Klee, G., Clin. Chem. 46(8B):1277-1283 (2000) at page 1277. The current recommended international nomenclature is that the term “B12” refers to cyanocobalamin. Cyanocobalamin is not a natural version of the vitamin; rather, it is a synthetic form made by reacting other forms of cobalamin with cyanide. The reaction changes them to cyanocobalamin, and stabilizes them. It is the form of the vitamin commonly found in dietary supplements, such as multivitamins.

In a first embodiment, the invention provides methods for producing B12 (cobalamin, or “Cbl”) labeled with 14C or tritium in a manner useful for determining B12 absorption, without creating amounts of radiolabeled waste that are large in proportion to the amount of labeled B12. The methods can also be used to label the B12 with 13C-, which can be used, for example, for analysis of B12 metabolism by nuclear magnetic resonance (NMR).

As noted in the Background section, 14C labeled cyanocobalamin (a form of B12 stabilized with cyanide) was made in 1951. See Boxer et al., supra. The labeling, however, was on the cyano group, which is labile and is cleaved off in the body. Cobalamin labeled using this method therefore dissociates from the cyanide group before the B12 is eliminated in the urine, and cannot be used in absorption studies based on urine analysis. For example, it cannot be used in place of 60cobalt- or 57cobalt-labeled cobalamin in the Schilling test.

Cobalamin can be labeled with 14C- or tritium by growing B12-producing bacteria on media radiolabeled with a variety of radiolabeled precursors and then separating out the B12 by standard techniques. But this method has several disadvantages. The label is promiscuously distributed into a multitude of molecules. Since B12 constitutes only a small proportion of the metabolic product of most microorganisms, this method yields a very small amount of labeled B12 and a much larger amount of radiolabeled waste. To compensate for this inefficiency, large fermentation or growth vessels would need to be used to realize a useful quantity of labeled B12. For example, Brown and Shemin (J. Biol Chem, 248(23):8015-8021 (1973)) synthesized 14C B12 in Propionibacterium shermanii grown in a medium containing L-[methyl-14C]-methionine. The total volume of the culture medium was 9 liters with the specific activity of 10 μCi/mmole.

The methods of the present invention solve these problems. They permit providing B12 labeled in a manner useful for testing for malabsorption of B12, without creating relatively large amounts of labeled waste products. As noted, the method of Brown and Shemin resulted in specific activity of 10 μCi/mmole. By contrast, the methods described herein provide a specific activity equivalent to 40,000 to 60,000 μCi/mmole. As a result, even a microscale incubation in 0.1-0.5 L flasks can provide greater total radioactive yields of 14C- or tritiated-B12 than less efficient labeling techniques that are orders of magnitude greater in volume. The method herein also permit efficient synthesis of 13C-labeled B12.

The methods of the invention can achieve at least 10% incorporation of the label, e.g., 14C, 13C, tritium, or deuterium into a vitamin B12 composition. Often, at least 20-40% incorporation is achieved and frequently the method achieves at least 50% or 60% incorporation of the label. Typical levels of incorporation of the label into vitamin B12 is at least 70% or greater, often 90% incorporation or greater. Accordingly, the invention also provides labeled B12 compositions that have a high specific activity, for example, with respect to 14C-labeled compounds, a specific activity of at least about 5 to about 10 Ci/mol, typically greater than about 20 Ci/mol or about 30 Ci/mol, and often greater than about 40 to about 60 Ci/mol. The specific activity of tritium-labeled B12 of the invention is typically between about 1480 to about 5000 Ci/mol.

Vitamin B12 compositions in accordance with the invention comprise multiple vitamin B12 molecules. As used herein, the percent incorporation of a label refers to the average number of labeled B12 molecules in a B12 composition. For example, 100% incorporation refers to a B12 compositions that is labeled, on average, with at least one heavy atom, e.g., 14C, tritium, deuterium, or 13C, per molecule at a particular site. As appreciated by one of skill in the art, multiple sites can also be substituted with a heavy atom. In some embodiments, incorporation is at least 10%, typically at least 30%, 40%, or 50-70% incorporation. Preferably, incorporation is greater than 70%, e.g., 80%, 90%, 95% or about 100%. The percent incorporation can be determined by known methods, e.g., mass spectrometry or NMR. Percent incorporation can alternatively be expressed as the average number of molecules labeled at a particular site. For example, 80% incorporation can be expressed as an average of 0.8 atoms of label per B12 molecule in a B12 composition in which there is one heavy atom labeling site.

The methods involve exploiting the metabolic pathways of organisms that have the eut operon and which use dimethylbenzimidazole (“DMB”) as the lower ligand of B12. The eut operon is well known in the art and is conserved in many bacteria. In Salmonella enterica serovar Typhimurium LT2 and other Salmonella spp. it includes 17 genes that are expressed only in the presence of ethanolamine plus B12. See, e.g., Kofoid, et al., J Bacteriol 181:5317-29 (1999). The operon has been carefully studied, particularly in S. enterica serovar Typhimurium. See, e.g., Roof and Roth, J Bacteriol 174:6634-6643 (1992), Roof and Roth, J Bacteriol 170:3855-3863 (1988), Roof and Roth, J Bacteriol 171:3316-3323 (1989), Sheppard and Roth, J Bacteriol 176:1287-96 (1994). The present inventors have previously elucidated and published the functions of various proteins encoded by genes in the eut operon. The discussion below will note portions of this information that are useful in understanding the present invention.

Preferably, the bacteria are facultative anaerobes, such as members of the family Enterobacteriaceae. In preferred embodiments, the facultative anaerobe is a Salmonella species. In some preferred embodiments, the facultative anaerobe is Salmonella enterica.

Adenosyl-cobalamin (Ado-B12, molecular weight 1570 Daltons) is a cofactor for several enzymes in Salmonella species, such as S. enterica. In Salmonella, the cofactor is synthesized only in the absence of oxygen, and functions in methionine synthesis and in two degradative pathways, the degradation of propanediol and the degradation of ethanolamine. Ethanolamine is degraded to acetaldehyde by a B12-dependent enzyme, and the acetaldehyde is then added to CoA to form acetyl-CoA. This pathway utilizes the products of the eut operon.

As shown in FIG. 1, cofactor B12 includes a corrinoid ring with a central cobalt atom; the molecule includes an upper ligand (“R” in FIG. 1 where “R” is an adenosyl group) covalently attached to the central cobalt by the 5′ carbon of ribose. The lower B12 ligand is coordinated with cobalt and covalently attached to the ring by a nucleotide loop. A variety of lower ligands are used in different bacteria: purine, pyrimidines, and dimethylbenzimidazole (“DMB”). When Salmonella grows aerobically and is provided with the B12 precursor cobinamide, it adds DMB as the lower ligand. The Salmonella genome includes at least three genes that can add an adenosyl group to cobalamin to form Ado-B12, the cofactor needed for ethanolamine use. The eutT gene encodes an ATP:cobalamin adenyl transferase. The gene was identified and its protein shown to produce Ado-B12 for both metabolism of ethanolamine and continued induction of the eut operon.

In particular, the methods of the invention exploit the discovery that a strain of S. enterica serovar Typhimurium LT2, TT24,733 (genotype: cbiD24::MudJ), cannot grow in aerobic conditions when ethanolamine as the only carbon source without the presence of both cobinamide (“Cbi,” a B12 precursor lacking the lower ligand. Cbi is usually given as dicyanocobinamide) and DMB, yet the strain retains the other relevant enzymes, allowing the strain to use exogenously supplied cobinamide and DMB to make B12. The Salmonella bacteria were grown aerobically on ethanolamine is the sole carbon source. Thus, they must form cofactor Ado-B12 from the DMB and cobinamide so that they may metabolize the ethanolamine to grow. As noted above, this requires the presence of the cofactor Ado-B12. Salmonella cannot synthesize B12 de novo under aerobic conditions. Further, they cannot grow aerobically on ethanolamine when solely cobinamide is provided. Growth of these organisms can be restored, however, by adding DMB. Thus, bacteria on an ethanolamine media in aerobic conditions and provided with 13C, 14C-, deuterium or tritium labeled DMB and cobinamide will take up the labeled DMB and cobinamide so that they can metabolize the ethanolamine. Persons of skill will recognize that, conversely, bacteria on an ethanolamine media in aerobic conditions and provided with 13C, 14C-, deuterium, or tritium labeled cobinamide and DMB will take up the labeled cobinamide and DMB so that they can metabolize the ethanolamine. Alternatively, both the cobinamide and the DMB can be labeled.

Use of the mutant strain is not necessary for the practice of the invention, but is preferred since it removes the concern that localized anaerobic conditions might form in the medium that might permit the organism to synthesize unlabeled B12 through the normal pathway. This increases the specific activity of the resulting B12. Use of non-mutant strains is also expected to result in B12 with sufficient specific activity to be used in absorption assays. As noted, however, non-mutant strains may also produce some unlabeled B12 in pockets of anaerobic conditions in the media, which may modestly dilute the labeled B12 with unlabeled B12. With S. enterica Typhimurium, this possibility can be reduced by not providing an alternate electron acceptor, such as tetrathionate, in the grown medium.

In the studies underlying the present invention, the DMB was labeled with 14C specifically in the carbon of the small benzimidazole ring, C-2. Conveniently, this can be done by acquiring a precursor of DMB, dimethylphenylenediamine (Sigma, St. Louis, Mo.) and inserting a 13C or 14C carbon from formic acid. Formic acid, sodium salt [14C] is commercially available, for example, from Moravek Biochemicals, Brea, Calif. Any carbons or hydrogens in DMB can be labeled instead, however, so long as the DMB as a molecular entity is labeled. Alternatively, the Cbi can be labeled, instead of the DMB, or both can be labeled. The labeled molecule can then be recovered by standard techniques, such as those described in Florent J. and Ninet, L. Vitamin B12. In: Microbial Technology, 2nd ed. Peppler J H, Perlman D, eds. New York: Academic Press, 1979.



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Application #
US 20120264174 A1
Publish Date
10/18/2012
Document #
13523662
File Date
06/14/2012
USPTO Class
435 86
Other USPTO Classes
International Class
12P19/42
Drawings
6


Vitamin B12


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