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.
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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.
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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
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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.