Title:
Method for preventing vascular disease
Kind Code:
A9


Abstract:
A method for orally administering vitamin preparations is described which combine vitamin B12 (B12, cobalamin) and folic acid (folate), with and without pyridoxine (B6), for preventing and treating elevated serum homocysteine (HC), cystathionine (CT), methylmalonic acid (MMA), or 2-methylcitric acid (2-MCA) levels. These metabolites have been shown to be indicative of B12 and/or folic acid deficiencies. Further, it is likely that a B6 deficiency may be present with a B12 or folate deficiency. The method of the invention is also for use in lowering serum HC, CT, MMA, or 2-MCA in patients with or at risk for neuropsychiatric, vascular, renal or hematologic diseases. The method of the present invention eliminates the costly and time consuming steps of distinguishing between vitamin deficiencies once a deficiency is found by measurement of serum metabolite levels. The present invention is of particular benefit to the populations at risk for elevated serum metabolite levels, such as the people over the age of 65, and populations that have or are at risk for neuropsychiatric, vascular, renal and hematologic diseases.



Inventors:
Allen, Robert H. (Denver, CO, US)
Stabler, Sally P. (Denver, CO, US)
Application Number:
10/309761
Publication Date:
03/15/2007
Filing Date:
12/04/2002
Primary Class:
Other Classes:
514/350, 514/251
International Classes:
A61K31/714; A61K31/4415; A61K31/525
View Patent Images:



Primary Examiner:
LUCAS, ZACHARIAH
Attorney, Agent or Firm:
PETER F WEINBERG (DENVER, CO, US)
Claims:
What is claimed is:

1. A method of preventing vascular disease in a human, comprising periodically administering orally a single formulation having between 0.3 and 10 mg vitamin B12 and 0.1 and 0.4 mg folic acid.

2. The method of claim 1 wherein the formulation includes approximately 2 mg vitamin B12 and 0.4 mg folic acid.

3. The method of claim 1 wherein said vascular disease is cardiovascular disease.

4. The method of claim 1 wherein said vascular disease is stroke.

5. The method of claim 1 wherein said vascular disease is peripheral vascular disease.

6. The method of claim 1 wherein said vascular disease is associated with renal disease.

7. A method of preventing vascular disease in a human, comprising periodically administering orally a single formulation having between 0.3 and 10 mg vitamin B12, and 0.1 and 0.4 mg folic acid, and 5 and 75 mg vitamin B6.

8. The method of claim 7 wherein the formulation includes approximately 2 mg vitamin B12 and 0.4 mg folic acid and 25 mg vitamin B6.

9. The method of claim 7 wherein said vascular disease is cardiovascular disease.

10. The method of claim 7 wherein said vascular disease is stroke.

11. The method of claim 7 wherein said vascular disease is peripheral vascular disease.

12. The method of claim 7 wherein said vascular disease is associated with renal disease.

13. A method of preventing vascular disease in a human, comprising periodically administering orally a single formulation having between 0.3 and 10 mg vitamin B12 and 0.4 and 10.0 mg folic acid.

14. The method of claim 13 wherein the formulation includes approximately 2 mg vitamin B12 and 1.0 mg folic acid.

15. The method of claim 13 wherein the formulation includes approximately 2 mg vitamin B12 and 2.0 mg folic acid.

16. The method of claim 13 wherein the formulation includes approximately 2 mg vitamin B12 and 2.5 mg folic acid.

17. The method of claim 13 wherein the formulation includes approximately 1 mg vitamin B12 and 2.0 mg folic acid.

18. The method of claim 13 wherein the formulation includes approximately 1 mg vitamin B12 and 2.5 mg folic acid.

19. The method of claim 13 wherein said vascular disease is cardiovascular disease.

20. The method of claim 13 wherein said vascular disease is stroke.

21. The method of claim 13 wherein said vascular disease is peripheral vascular disease.

22. The method of claim 13 wherein said vascular disease is associated with renal disease.

23. A method of preventing vascular disease in a human, comprising periodically administering orally a single formulation having between 0.3 and 10 mg vitamin B12, and 0.4 and 10 mg folic acid, and 5 and 75 mg vitamin B6.

24. The method of claim 23 wherein the formulation includes approximately 2 mg vitamin B12 and 1.0 mg folic acid and 25 mg vitamin B6.

25. The method of claim 23 wherein the formulation includes approximately 2 mg vitamin B12 and 2.0 mg folic acid and 25 mg vitamin B6.

26. The method of claim 23 wherein the formulation includes approximately 2 mg vitamin B12 and 2.5 mg folic acid and 25 mg vitamin B6.

27. The method of claim 23 wherein the formulation includes approximately 1 mg B12 and 2.0 mg folic acid and 25 mg vitamin B6.

28. The method of claim 23 wherein the formulation includes approximately 1 mg B12 and 2.5 mg folic acid and 25 mg vitamin B6.

29. The method of claim 23 wherein said vascular disease is cardiovascular disease.

30. The method of claim 23 wherein said vascular disease is stroke.

31. The method of claim 23 wherein said vascular disease is peripheral vascular disease.

32. The method of claim 23 wherein said vascular disease is associated with renal disease.

Description:

This application is a continuation of Ser. No. 09/793,214, filed on Feb. 26, 2001, which is a continuation of Ser. No. 09/273,754 filed Mar. 22, 1999, now issued as U.S. Pat. No. 6,297,224, which is a continuation of application no. Ser. No. 09/012,955 filed Jan. 26, 1998 now issued as U.S. Pat. No. 5,795,873, which is a divisional of application no. Ser. No. 07/999,499, which was filed Dec. 29, 1992, now issued as U.S. Pat. No. 5,563,126.

FIELD OF THE INVENTION

This invention relates to the field of nutrition. Specifically, the invention is comprised of new oral vitamin preparations combining vitamin B12 (B12, cobalamin) and folic acid (folate), and vitamin B12, folate, and pyridoxine (B6) for use in patients with elevated serum metabolite levels of homocysteine (HC), cystathionine (CT), methylmalonic acid (MMA), or 2-methylcitric acid (2-MCA). The elevation of these metabolites has been shown to be indicative of tissue deficiencies of B12 and/or folate and/or B6, and related to increased risk of neuropsychiatric, vascular, renal and hematologic diseases. One embodiment of the present invention uses a non-prescription formulation comprising between 0.3-10.0 mg B12 and 0.1-0.4 mg folate, with the preferred embodiment using 2.0 mg B12 and 0.4 mg folate. Another embodiment of the non-prescription formulation uses 0.3-10 mg B12, 0.1-0.4 mg folate, and 5-75 mg B6, with the preferred embodiment using 2.0 mg B12, 0.4 mg folate, and 25 mg B6. Another embodiment of the present invention uses a prescription strength formulation comprising between 0.3-10.0 mg B12 and 0.4-1.0 mg folate, with the preferred embodiment using 2 mg B12 and 1.0 mg folate. In a further embodiment of the present invention, a prescription strength formulation is used comprising 0.3-10 mg B12, 0.4-1.0 mg folate, and 5-75 mg B6, with the preferred embodiment using 2 mg B12, 1.0 mg folate, and 25 mg B6. The formulations of the present invention eliminate the costly and time-consuming steps of distinguishing between vitamin deficiencies once a deficiency is found by measurement of serum metabolite levels. The present invention is of particular benefit to the populations at risk for tissue deficiencies of B12, folate, and B6, such as people over the age of 65, and populations that have or are at risk for neuropsychiatric, vascular, renal and hematologic diseases.

BACKGROUND

Vitamins B12, folate, and B6 are required cofactors in metabolic pathways involving methionine, homocysteine, cystathionine, and cysteine. B12 in the form of 5′-deoxyadenosylcobalamin is an essential cofactor in the enzymatic conversion of methylmalonylCoA to succinylCoA. The remethylation of homocysteine (HC) to methionine catalyzed by methionine synthase requires folate (methyltetrahydrofolate) and B12 in the form of methylcobalamin. HC is condensed with serine to form cystathionine (CT) in a reaction catalyzed by cystathionine □-synthase which requires B6 (pyridoxal phosphate). CT is hydrolyzed in another B6-dependent reaction to cysteine and □-ketobutyrate.

It is important to diagnose and treat B12, folate, and B6 deficiencies because these deficiencies can lead to life-threatening hematologic abnormalities which are completely reversible by proper treatment. B12 deficiency is a multisystem disorder with extremely varied clinical presentation which has been thought to occur in 0.4% of the population, e.g., about 1 million people in the United States. Symptoms of B12 deficiency include significant anemia, displayed for example in decreased hematocrit (e.g., <25%) or hemoglobin (e.g., <8 g %), with macrocytic red blood cells (i.e., mean cell volume generally greater than 100 fl), or neurologic symptoms of peripheral neuropathy and/or ataxia. See, for example, Babior and Bunn (1983) in Harrison's Principles of Internal Medicine, (Petersdorf et al., eds.), McGraw-Hill Book Co., New York; Lee and Gardner (1984) in Textbook of Family Practice, 3rd Ed. (Rakel, ed.), Saunders & Co., Philadelphia). The hematological abnormalities seen are due to intracellular folate deficiency since folate is required for a number of essential enzymatic reactions involved in DNA and RNA synthesis and since the form of folate in serum (5-methyltetrahydrofolate) must be metabolized to tetrahydrofolate by the B12-dependent enzyme methionine synthase before it can be utilized by the RNA- and DNA-related enzymes. While it has been well recognized that individuals with B12 deficiency could display neurologic disorders in the absence of anemia, such situations were believed to be exceptional and rare. See, Beck (1985) in Cecil Textbook of Medicine, 17th Ed., (Wyngaarden and Smith, eds.), W. B. Saunders, Philadelphia, pp. 893-900; Babior and Bunn (1987) in Harrison's Principles of Internal Medicine, 11th Ed., (Braunwald et al., eds.) McGraw-Hill, New York, pp. 1498-1504; Walton (1985) in Brain's Diseases of the Nervous System, 9th Ed., Oxford University Press, Oxford, UK. The neurologic symptoms of B12 deficiency were considered to be late manifestations of the disease most typically occurring after the onset of anemia or, if they occurred first, were soon to be followed by the onset of anemia. See, Woltmann (1919) Am. J. Med. Sci. 157:400-409 Victor and Lear (1956) Am. J. Med. 20:896-911.

However, it has recently been shown that the textbook description of severe megaloblastic anemia and combined systems disease of the nervous system is the rarest presentation of B12 deficiency at the present time (Stabler et al. (1990) Blood 76:871-881; Carmel (1988) Arch. Int. Med. 148:1712-1714 Allen (1991) in Cecil Textbook of Medicine, 19th Ed., (Wyngaarden and Smith, et al. eds.), W. B. Saunders, Philadelphia, pp. 846-854.). Therefore, contrary to previous teachings, patients that may benefit from B12 therapy may have minimal to no hematologic changes while manifesting a wide variety of neurologic and psychiatric abnormalities (Lindenbaum et al. (1988) N. Engl. J. Med. 318:1720-1728; Greenfield and O'Flynn (1933) Lancet 2:62-63). This is particularly true for populations at risk for B12 deficiency, such as the elderly population (Pennypacker et al. (1992) J. Am. Geriatric Soc. 40: (in press).

The incidence of folate deficiency in the population is unknown, but has been thought to occur commonly in individuals with various degrees of alcoholism. The hematologic abnormalities seen with folate deficiency, such as macrocytic anemia, are indistinguishable from those seen with B12 deficiency. Folate is required for a number of essential enzymatic reactions involved in DNA and RNA synthesis, and is particularly important in rapidly dividing cells like those in the bone marrow.

B6 is required for the first step in heme synthesis and serves a major role in transamination reactions of amino acid metabolism, in decarboxylations, and in the synthesis of the neuroactive amines histamine, tyramine, serotonin, and □-aminobutyric acid (GABA). Clinical manifestations include microcytic hypochromic anemia, characteristic skin changes of dermatitis and acrodynia, muscular weakness, and a variety of neuropsychiatric abnormalities including hyperirritability, epileptiform convulsions, depression and confusion (Newberne and Conner (1989) in Clinical Biochemistry of Domestic Animals, Academic Press, San Diego, pp. 796-834).

Vitamin deficiencies are generally determined by measurement of serum levels. Normal serum B12 levels are 200-900 pg/ml, with levels of less than 100 pg/ml being said to indicate clinically significant deficiency (Beck (1985) supra) However, serum B12 levels are a relatively insensitive determinant of B12 deficiency in that only 50% of patients with clinically confirmed B12 deficiency have levels less than 100 pg/ml, 40% are 100-200 pg/ml, and at least 5-10% have values in the 200-300 pg/ml range. Diagnosis is further complicated by the fact that 2.5% of normal subjects (6,250,000 people in the U.S.) have low serum B12 levels (Allen (1991) supra), with no evidence of B12 deficiency and are unlikely to benefit from B12 therapy (Schilling et al. (1983) Clin. Chem. 29:582; Stabler (1990) supra).

Normal serum folate levels are 2.5-20 ng/ml, with levels less than 2.5 ng/ml indicating the possibility of clinically significant deficiency. Like B12 serum levels, however, serum folate levels are a relatively insensitive measure in that only 50-75% of patients with folate deficiency have levels less than 2.5% ng/ml, with most of the remaining 25-50% being in the 2.5-5.0 ng/ml range (Allen (1991) in Cecil Textbook of Medicine, 19th Ed., su ra) The development of sensitive serum metabolite assays for HC, CT, MMA, and 2-MCA has allowed the relationship between metabolite levels and vitamin deficiencies to be investigated (Stabler et al. (1987) Anal. Biochem. 162:185-196; Stabler et al. (1986) J. Clin. Invest. 77:1606-1612; Stabler et al. (1988) J. Clin. Invest. 81:466-474). It has been found that elevated serum levels of HC and MMA are clinically useful tests of functional intracellular deficiencies of B12 and folate, with elevated HC levels seen with both B12 and folate deficiencies, and elevated MMA levels seen with a B12 deficiency (Allen et al. (1990) Am. J. Hematol. 34:90-98 Lindenbaum et al. (1990) Am. J. Hematol. 34:99-107; Lindenbaum et al. (1988) N. Engl. J. Med. 318:1720-1728; Beck (1991) in Neuropsychiatric Consequences of Cobalamin Deficiency, Mosby Year Book 36:33-56 Moelby et al. (1990) 228:373-378; Ueland and Refsum (19890 J. Lab. Clin. Med. 114:473-501; Pennypacker et al. (1992) supra). Increased serum levels of CT are seen in both deficiencies and 2-MCA is elevated in B12 deficiency (Allen et al. (1991) in Proceedings of the 1 st International Congress on Vitamins and Biofactors in Life Science, Kobe (Japan) ; Allen et al. (1993) Metabolism (in press)). HC and CT may be elevated in patients with intracellular deficiency of B6, but this has not been as well documented (Park and Linkswiler (1970) J. Nutr. 100:110-116; Smolin and Benvange (1982) J. Nutr. 112:1264-1272).

Elevated serum metabolite levels are observed in disease states other than classic vitamin deficiencies. For example, elevated HC levels have been observed in the presence of vascular disease. The homocysteine theory of atherosclerosis, formulated by McCully and Wilson (1975) Atherosclerosis 22:215-227, suggests that high levels of HC are responsible for the vascular lesions seen in homocystinuria, a genetic defect caused by a deficiency in the enzyme cystathionine □-synthase. The theory also implies that moderate elevations of HC might be associated with increased risk for vascular disease (Ueland et al. (1992) in Atherosclerotic Cardiovascular Disease, Hemostasis, and Endothelial Function (Francis, Jr., ed.), Marcel Dekker, Inc., New York, pp. 183-236). Moderate hyperhomocysteinemia has been shown to be frequently present in cases of stroke and to be independent of other stroke risk factors (Brattstrom et al. (1992) Eur. J. Clin. Invest. 22:214-221). Clinical and experimental evidence demonstrates that patients who are homozygotes for cystathionine □-synthase deficiency have a markedly increased incidence of vascular disease and thrombosis. A number of studies (see, Clarke et al. (1991) N. Engl. J. Med. 324:1149-1155) strongly suggest that heterozygotes for a deficiency of cystathionine β-synthase also have an increased incidence of vascular disease and thrombosis and that such heterozygotes may constitute as many as one-third of all patients who develop strokes, heart attacks, or peripheral vascular disease under age 50. It is also likely that such heterozygotes are also at increased risk for vascular disease and thrombosis after age 50. Since the incidence of heterozygosity for cystathionine β-synthase deficiency is estimated to be 1 in 60-70, this means that there are approximately 4 million heterozygotes in the U.S. It is also possible that patients with vascular disease due to other causes, such as hypercholesterolemia, would also benefit from a decrease in their serum HC levels even if their existing levels are only slightly elevated or actually within the normal range.

Renal disease is another condition that gives rise to elevated levels of serum metabolites. Approximately 75% of patients with renal disease have elevated serum concentrations of HC, CT, MMA, and 2-MCA. Since patients with renal disease have a significant incidence and marked acceleration of vascular disease, it might be beneficial to lower their serum metabolite levels, especially that of HC.

An increasing prevalence of low serum B12 concentrations with advancing age has been found by many but not all investigators (Bailey et al. (1980) J. Am. Geriatr. Soc. 28:276-278 Eisborg et al. (1976) Acta Med. Scand. 200:309-314; Niisson-Ehle et al. (1989) Dig. Dis. Sci. 34:716-723; Norman (1985) 33:374; Hitzhusen et al. (1986) Am. J. Clin. Pathol. 85:3236), folate (Magnus et al. (1982) Scan. J. Haematol. 28:360-366; Blundell et al. (1985) J. Clin. Pathol. 38:1179-1184 Elwood et al. (1971) Br. J. Haematol. 21:557-563; Garryet al. (1984) J. Am. Geriatr. Soc. 32:71926; Hanger et al. (1991) J. Am. Geriatr. Soc. 39:1155-1159), and B6 (Ranke et al. (1960) J. Gerontol. 15:41-44; Rose et al. (1976) Am. J. Clin. Nutr. 29:847-853; Baker et al. (1979) J. Am. Geriatr. Soc. 27:444-450). Moreover, prevalence estimates for these vitamin deficiencies vary widely depending on the population groups studied. It has been unclear whether this increased prevalence is a normal age related phenomena or a true reflection of tissue vitamin deficiency and whether the low serum vitamin concentrations are a reliable indicator of functional intracellular deficiency.

It is difficult, expensive and time-consuming to distinguish between deficiencies of vitamins B12, folate, and B6. The hematologic abnormalities seen with B12 deficiency are indistinguishable from those seen with folate deficiency. Similarly to a B12 deficiency, B6 deficiencies also result in hematologic as well as neuropsychiatric abnormalities. The traditional methods of determining deficiencies by measurement of serum vitamin levels are often insensitive. As a result, in order to determine if and which vitamin deficiency is present, a patient will be treated with one vitamin at a time and the response to that vitamin determined by normalization of serum vitamin levels and the correction of hematologic abnormalities. These steps are then repeated with each vitamin. This method of treatment is both expensive and time-consuming. In the presence of multiple deficiencies, the diagnosis of vitamin deficiencies is further confused and give rise to the dangerous possibility that only one deficiency will be treated. For example, the hematologic abnormalities seen with a B12 deficiency will respond to treatment with folate alone. However, the neuropsychiatric abnormalities caused by the B12 deficiency will not be corrected and may indeed by worsened.

It has now been discovered for the first time that the prevalence of intracellular deficiencies of vitamins B12, folate, and B6, alone or in combination, is substantially higher than that previously estimated by measurement of serum vitamin concentrations. The present disclosure establishes that tissue deficiencies of one or more of the vitamins B12, folate and B6, as demonstrated by the elevated metabolite concentrations, occurs commonly in the elderly population even when serum vitamin levels are normal. Based on this new discovery, the present invention addresses the problem of distinguishing between vitamin deficiencies when low, low-normal, or normal serum vitamin concentrations are found by providing formulations for the treatment of high serum metabolites and at-risk populations for combinations of one or more tissue deficiencies of vitamins B12, folate, and B6.

Hathcock and Troendle (1991) JAMA 265:96-97, have suggested the treatment of pernicious anemia with an oral pill containing 300 to 1000 ug or more per day of B12. However, contrary to the present invention, Hathcock and Troendle teach away from combining B12 therapy with folate, since “if the oral cobalamin therapy should fail to maintain adequate levels, folate might provide protection against development of anemia while permitting nerve damage from cobalamin deficiency.”

U.S. Pat. No. 4,945,083, issued Jul. 31, 1990 to Jansen, entitled: Safe Oral Folic-Acid-Containing Vitamin Preparation, describes a oral vitamin preparation comprising 0.1-1.0 mg B12 and 0.1-1.0 mg folate for the treatment or prevention of megaloblastic anemia. This formulation presents a problem in the case of a B12 deficient patient, in that the 0.5 mg folate may correct the hematologic abnormalities present, but the 0.5 mg B12 dose may be insufficient to correct a B12 deficiency due to inadequate intrinsic factor. By contrast, the formulation of the present invention teaches the use of the combination of B12 and folate, and of B12, folate and B6, sufficient to treat either single or multiple deficiencies of B12, folate, and B6. The present invention does not rely on the determination of vitamin deficiencies by the measurement of serum vitamin levels, but uses the more sensitive measurement of elevated serum metabolites of HC, CT, MMA, and 2-MCA, shown to be related to the presence of B 12 and/or folate and/or to B6 deficiencies or to the presence of the increased risk of neuropsychiatric, vascular, renal, and hematologic diseases.

It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention as claimed.

SUMMARY OF THE INVENTION

This invention includes a method for orally administering two new vitamin preparations containing vitamin B12 and folate, and vitamin B12, folate and B6, for the treatment of patients with elevated serum metabolites, such as homocysteine, cystathionine, methylmalonic acid, and 2-methylcitric acid, as well as populations at risk for tissue deficiencies in one or more of the vitamins B12, folate, and B6 or for neuropsychiatric, vascular, renal, or hematologic diseases.

One embodiment of the present invention uses an over-the-counter formulation comprised of between 0.3-10 mg CN-cobalamin (B12) and 0.1-0.4 mg folate. Another embodiment of the non-prescription formulation uses 0.3-10 mg B12, 0.1-0.4 mg folate, and 5-75 mg B6. Preferred embodiments of the over-the-counter formulation are comprised of about 2.0 mg B12 and 0.4 mg folate, and 2.0 mgB12, 0.4 mg folate, and 25 mg B6, respectively.

Another embodiment of the present invention uses a prescription formulation comprised of between 0.3-10 mg CN-cobalamin (B12) and 0.4-10.0 mg folate. Another embodiment of the prescription formulation of the present invention uses 0.3-10 mg B12, 0.4-10.0 mg folate, and 5-75 mg B6. Preferred embodiments of the prescription formulation use about 2.0 mg B12 and 1.0 mg folate, and 2.0 mg B12, 1.0 mg folate, and 25 mg B6, respectively.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the distribution of serum B12 levels for a population of elderly outpatients (ages 65-99, n=152) and a normal population (ages 17-65, n=100).

FIG. 2 shows serum MMA levels for a population of elderly outpatients with serum B12 values <300 pg/ml (ages 65-99, n=38/152) and a normal population with serum B12 values <300 pg/ml (ages 17-65, n=10/100).

FIG. 3 shows serum HC levels for a population of elderly outpatients with serum B12 values <300 pg/ml (ages 65-99, n=38/152) and a normal population with serum B,2 values <300 pg/ml (ages 17-65, n=10/100).

FIG. 4 shows serum MMA levels before and after treatment with parenteral cobalamin for a population of elderly outpatients with elevated MMA values and serum B12 values <300 pg/ml (ages 65-99, n=15/38).

FIG. 5 shows serum HC levels before and after treatment with parenteral cobalamin for a population of elderly outpatients with elevated HC values and serum B12 values of <300 pg/ml (ages 65-99, n=10/38).

FIG. 6 shows the distribution of serum B12 levels for a population of elderly nursing home patients (ages 55-107, n=212) and a normal population (ages 17-65, n=100).

FIG. 7 shows serum MMA levels for a population of elderly nursing home patients with serum B12 values <300 pg/ml (ages 55-107, n=29/212) and a normal population with serum B12 values (ages 17-65, n=10/100).

FIG. 8 shows serum HC levels for a population of elderly nursing home patients with serum B12 values <300 pg/ml (ages 55-107, n=29/212) and a normal population with serum B12 values <300 pg/ml (ages 17-65, n=10/100).

FIG. 9 shows serum MMA levels before and after treatment with parenteral cobalamin for a population of elderly nursing home patients with serum B12 values <300 pg/ml (ages 55-107, n=14/29).

FIG. 10 shows serum HC levels before and after treatment with parenteral cobalamin for a population of elderly nursing home patients with serum B12 values <300 pg/ml (ages 55-107, n=14/29).

FIG. 11 shows the distribution of serum B12 levels for a population of elderly patients (ages 65-99, n=548) and a normal population (ages 22-63, n=1 17) (Framingham study).

DETAILED DESCRIPTION OF THE INVENTION

Reference will now be made in detail to the presently preferred embodiments of the invention, which, together with the following examples, serve to explain the principles of the invention.

This invention uses new oral vitamin formulations combining vitamin B12 (B12, cobalamin) and folic acid (folate), and vitamin B12, folate and pyridoxine (B6). The formulations of the present invention are for use in the treatment of elevated serum levels of one or more of the metabolites homocysteine (HC), cystathionine (CT), methylmalonic acid (MMA), or 2-methylcitric acid (2-MCA). The use of the formulations of the present invention further include as a method of lowering serum metabolite levels of one or more of HC, CT, MMA, or 2-MCA, where these metabolite levels are not elevated but the patients are at risk for or have neuropsychiatric, vascular, renal, or hematologic diseases.

One embodiment of the present invention uses a non-prescription formulation comprised of between about 0.3-10 mg CN-cobalamin (B12) and 0.1-0.4 mg folate. Another embodiment of the present invention uses a non-prescription formulation comprised of between about 0.3-10 mg B12, 0.1-0.4 mg folate, and 5-75 mg B6. Preferred embodiments of the non-prescription formulation are comprised of about 2.0 mg B12 and 0.4 mg folate, and 2.0 mg B12, 0.4 mg folate, and 25 mg B6, respectively.

Another embodiment of the present invention is comprised of a prescription formulation comprised of between about 0.3-10 mg B12 and 0.4-10.0 mg folate, with the preferred embodiment comprised of about 2.0 mg B12 and 1.0 mg folate. Another embodiment of the prescription strength formulation is comprised of about 0.3-10 mg B12, 0.4-10.0 mg folate, and 5-75 mg B6, with a preferred embodiment comprised of about 2.0 mg B12, 1.0 mg folate, and 25 mg B6.

The formulations of the present invention are for the treatment and prevention of elevated metabolite levels in at risk populations, such as the elderly, and people that have or are at risk for neuropsychiatric, vascular, renal and hematologic diseases. The present invention eliminates the costly and time consuming need to differentiate between B12, folate, and B6 deficiencies.

The administration of a daily dose of the vitamin formulations of the present invention provides better long-term normalization of serum HC and other metabolites than prior art formulations, and eliminates the difficulty in differentiating between deficiencies of two or three of the vitamins, the difficulty in diagnosing multiple deficiencies of two or three of the vitamins, and the expense of doing so. Further, the administration of an oral preparation of B12 and folate, with or without B6, is preferred over intramuscular injections for patient convenience and ease of administration.

For example, the inclusion of B12 will be useful as a safeguard for patients misdiagnosed as folate deficient, even though they are actually B12 deficient, since treatment with folate alone in such patients is extremely dangerous. The danger arises from the fact that treating a B12 deficient patient with folate alone may reverse or prevent the hematologic abnormalities seen in B12 deficiency, but will not correct the neuropsychiatric abnormalities of a B12 deficiency and may actually precipitate them. Even in the absence of intrinsic factor, approximately 1% of a 2.0 mg oral dose of B12 is absorbed by diffusion. Thus, approximately 20 ug of B12 would be absorbed from the formulations of the present invention which would be more than adequate even in patients with pernicious anemia who have lost their intrinsic factor-facilitated absorption mechanism for B12. The inclusion of folate will be of benefit since B12 deficiency causes a secondary intracellular deficiency of folate. The inclusion of folate and B6 will also be of benefit in patients with mixed vitamin deficiencies.

The formulations of the present invention may be administered as a non-injectable implant or orally. Non-injectable use may be as a patch. Formulations for oral administration are preferably encapsulated. Preferably, the capsule is designed so that the formulation is released gastrically where bioavailability is maximized. Additional excipients may be included to facilitate absorption of the vitamin formulations. Diluents, flavorings, low melting point waxes, vegetable oils, lubricants, suspending agents, tablet disintegrating agents, and binders may also be employed.

Example 1 describes the methods used to measure serum vitamin and metabolite levels. Example 2 describes a new study conducted with 412 subjects over the age of 65 with a variety of medical conditions correlating the incidence of low serum vitamin levels with elevated serum metabolite levels. A study determining the incidence of undetected B12 deficiency and response of serum MMA and HC to B12 treatment in a geriatric outpatient population is described in Example 3. Example 4 describes a similar study conducted with a geriatric nursing home population, and Example 5 describes a similar study conducted with another geriatric population.

EXAMPLE 1

Methods for Measurement of Serum Vitamin and Metabolite Levels.

Serum vitamin assays. Serum vitamins B12 and folate were measured by a quantitative radioassay method using purified intrinsic factor and purified folate binding protein. Vitamin B6 was measured by a radioenzymatic assay method wherein serum is incubated with apoenzyme tyrosine-decarboxylase, C14 labelled tyrosine is added to start the enzymatic reaction which is stopped with HCl. Subsequently the free C14-labelled CO2 is adsorbed by a KOH impregnated filtering paper. The measured C14 activity is directly proportional to the B6 (pyridoxal phosphate) concentration (Laboratory Bioscientia, Germany).

Serum metabolite assays. Serum metabolite assays for homocysteine and methylmalonic acid were conducted by the capillary gas chromatography and mass spectrometry methods of Marcell et al. (1985) Anal. Biochem. 150:58; Stabler et al. (1987) supra, and Allen et al. (1990) Am. J. Hematol. 34:90-98. Serum cystathionine levels were assayed by the method of Stabler et al. (1992) Blood (submitted). Serum 2-methylcitric acid was assayed by the method of Allen et al. (1993) Metabolism supra.

Statistical methods. Statistical analysis was done with the SAS statistical package (version 6.06). Nonparametric data for two or more groups were tested with the two sample Wilcoxon rank sum test (with Bonferroni's correction for the significance level ax) and the Kruskall Wallis test. From the results of the healthy young subjects reference intervals were calculated. Since the frequency distribution of the values of each parameter were markedly abnormal they were transformed to normal distributions using log transformation. The sample prevalence p with 95% confidence intervals of low serum vitamins B12, folate, and B6 concentrations was calculated as (p±2p (1−p)/n×100 wherein n is the total sample size, p is the number of low serum vitamin concentrations/n; low serum concentrations are defined as <mean −2 S.D.

EXAMPLE 2

Incidence of Elevated MMA, 2-MCA, HC, and CT Levels in the Geriatric Population.

The serum concentrations of B12, folate, and B6 were measured in 412 subjects over the age of 65 (subgroups A-D), and in 99 healthy control subjects aged 20-55 years (subgroup E). The geriatric subgroups were defined as follows: A, 110 patients with atherosclerosis; B, 98 patients with neuropsychiatric disorders; C, 102 patients with atherosclerosis and multiple diseases including rheumatoid arthritis and diabetes; D, 102 subjects who were healthy.

Venous blood was obtained from all subjects in the morning after an overnight fast. The blood was spun within one hour after collection and the serum was transported in dry ice to the central laboratory. Serum vitamins B12 and folate were measured as described in Example 1 with a vitamin B12/folate dual RIA kit (CT301/CT302 Amersham Buchier, UK). Vitamin B6 and serum metabolites were measured as described in Example 1.

Since renal function can influence serum metabolite concentrations (Ueland and Refsum (1989) supra Moelby et al. (1992) Scand. J. Clin. Lab. Invest. 52:351-354), serum creatinine concentrations were measured in all subjects by the Jaffe photometric method (Laboratory Bioscientia, Germany). Normal range was 62-124 μmol/L. Creatinine clearance was calculated using the formulation of Cockroft and Gault (1976) Nephron 16:31-41.

Normal ranges for serum vitamin and metabolite levels were determined by the mean ±2 standard deviations after log normalization using the values from subgroup E. Results are shown in Table 1:

TABLE 1
INCIDENCE OF LOW SERUM
VITAMN AND HIGH METABOLITE LEVELS
IN GERIATRIC POPULATIONS A-D AND
A YOUNGER HEALTHY POPULATION E.
Folic
GroupB12AcidB6MMA2-MCAHCCT
A6%12%48%36%44%55%64%
B6%19%53%47%39%59% 6%
C3%10%50%32%45%39%73%
D6% 6%17%26%23%38%41%
E2% 1% 1% 3% 6% 2% 4%

There was a rough correlation with low vitamin levels and elevated metabolites, but many of the patients with elevated metabolites had low normal or normal vitamin levels. Correlations between clinical abnormalities within groups A, B, and C were not present. Patients were treated with weekly injections of a multi-vitamin preparation containing 1.0 mg B12, 1.1 mg folate, and 5 mg B6, resulting in a marked lowering or normalization of elevated metabolite levels in virtually every elderly patient.

These data support the conclusions that there is an increased incidence of low levels of serum B12, folate, and B6 in the geriatric population, and that serum MMA, 2-MCA, HC and CT are elevated in an even higher percentage of geriatric patients. The presence of elevated levels of one or more of the metabolites HC, CT, MMA, or 2-MCA indicate a tissue or intracellular deficiency of one or more of the vitamins B12, folate and B6. It not possible to tell without expensive, time-consuming, and extensive testing which one vitamin or pair of vitamins, or whether all three vitamins are deficient. These observations, together with the fact that elevated metabolite levels are corrected by parenteral therapy with a combination of vitamins B12, folate, and B6, indicate that a tissue deficiency of one or more of these vitamins occurs commonly in the geriatric population and that measurement of serum vitamin levels alone is an inadequate method for identifying such deficiencies.

EXAMPLE 3

Determination of Serum B12 Folate, MMA, HC, CT and 2-MCA Levels in a Geriatric Outpatient Population.

A study was conducted with 152 elderly outpatient subjects to measure the prevalence of B12 deficiency in geriatric outpatients as determined by both low serum B12 levels and elevations of MMA and HC, and to determine the response to B12 treatment. Blood samples were obtained on 152 consecutive geriatric outpatients, ages 65-99. Control values were determined from 100 subjects, ages 17-65. Serum B12 folate, MMA, HC, CT, and 2-MCA levels were obtained for each patient, shown in Table 2. The significance of the results marked as “**” in Table 2 are as follows: B12 levels of <200 pg/ml; folate <3.8 ng/ml; homocysteine >16.2 uM; MMA >271 nM; CT >342 nM; and 2-MCA >228 nM. Serum MMA, HC, CT, and 2-MCA levels were measured as described in Example 1. Serum B12 and folate were measured as described in Example 1 using a Coming humophase kit (CIBA-Corning, Medfield, Mass.) with the normal range defined as 200-800 pg/ml for B12 and 3.8 ng/ml for folate. After evaluation, patients received weekly parenteral cyanocobalamin injections (1,000 ug IM) for 8 weeks, followed by monthly injections. Repeat laboratory and clinical assessments were administered at 8 weeks and at 6 months.

Results show that 25% of the subjects had a serum B12 level <300 pg/ml and 8.5% had a low level of <200 pg/ml. FIG. 1 shows the shift seen in elderly subject towards lower serum B12 levels. More than half of the subjects with low or low-normal serum B12 levels had elevations of MMA (FIG. 2) and/or HC (FIG. 3) greater than 3 S.D. above the means in normals and representing 14.5% of the total screened population.

Patients with low and low/normal serum B12 levels were treated with weekly injections of 1.0 mg B12. Parenteral B12 administration caused elevated metabolite levels to fall to or towards normal (FIGS. 4 and 5) in every subject treated with B12. It appears that the true prevalence of previously unrecognized B12 deficiency in this elderly population was at least 14.5%.

It can be seen from the data presented in Table 2 that serum B12 levels are insensitive for screening B12 deficiencies since similar numbers of patients with low normal serum B12 levels of 201-300 pg/ml compared with patients with low B12 levels (<200 pg/ml) had markedly elevated metabolites which fell with B12 treatment. Further, this study shows that elderly patients have a high incidence (at least 14.5%) of unrecognized B12 deficiency, detectable by measurement of serum HC and MMA levels in patients with serum B12 levels <300 pg/ml.

A further finding in this study emphasizes the need to treat elevated metabolite levels with a combination of vitamin B12 and folate with or without B6. Some of the patients exhibiting elevated metabolite levels did not fully respond to B12 treatment. This may indicate a concomitant deficiency of folate and/or B6. The lack of response to B12 treatment could result from a deficiency of one, a pair, or all three vitamins. However, it would be expensive and time-consuming to attempt to distinguish between the vitamin deficiencies.

Another, and perhaps the most important, finding in this study is the large number of patients with serum B12>300 pg/ml who have elevated values for one or more metabolites as indicated by a “**” next to the individual values. As can readily be seen in Table 2, there are many examples of elevated value for MMA and/or 2-MCA at all levels of serum B12 including the mid-normal (300-600 pg/ml), the high-normal (600-800 pg/ml), and even the elevated (>800 pg/ml) ranges. The same is true for elevations of HC and CT. In some patients the serum folate is low, indicating that folate deficiency may be present, but in many cases both B12 and folate levels are normal. B6 levels were not performed in this study, but B6 deficiency would not be expected to cause elevations of MMA or 2-MCA. Thus in many patients it is not clear which vitamin, or pair of vitamins, or whether all three vitamins is or are deficient. One could pick a single vitamin, often at random, with which to treat a patient for several weeks or months, and then repeat measurement of metabolite levels to determine if a partial or full correction had occurred. If there was no response, one could try another vitamin, or if there was a partial response one could add a second vitamin, and then repeat metabolite measurement after several weeks or months. If there was still no response, one could try the third vitamin, or if there was a partial response, one could try a different pair of vitamins. Eventually one could determine whether an individual vitamin, a particular pair of vitamins, or all three vitamins were required to normalize or maximally reduce the metabolite levels, but it would often require months or even a year to make this determination. Such a determination would be expensive. In addition, a patient who was optimally treated with a single vitamin or pair of vitamins might subsequently develop a deficiency of one or even two of the other vitamins as evidenced by a re-elevation or increase in the levels of one or more metabolites. Therapeutic testing could be reinitiated and continued as described above, although this would also be time-consuming and expensive.

It requires less time and expense to treat patients with elevated metabolite levels with a combination of vitamin B12 and folate, or a combination of vitamin B12, folate and vitamin B6. The utility of the approach of the present invention is appreciated only after it is taught, for the first time in the present disclosure, that a deficiency of one or more of the three vitamins occurs commonly in the elderly population as evidenced by elevation of one or more metabolites, i.e., MMA, 2-MCA, HC and CT.

EXAMPLE 4

Determination of Serum B12, Folate, MMA, and HC Levels in a Geriatric Nursing Home Population.

A study was conducted with 212 elderly nursing home patients to determine serum B12, folate, MMA, and HC levels (Table 3). The significance of the results shown in Table 3 marked with “**” are as described for Table 2 (Example 3). The control group consisted of 100 subjects between the ages of 17-65 years. As in the study described in Example 3, the elderly population exhibited a shift to lower serum B12 levels (FIG. 6), elevated serum MMA (FIG. 7) and HC (FIG. 8) levels. Parenteral administration of B12 1 mg per week for 8 weeks to those with serum B12<300 pg/ml caused elevated MMA (FIG. 9) and HC (FIG. 10) levels to fall to or towards normal.

As in the study reported in Example 3, a further finding in this study emphasizes the need to treat elevated metabolite levels with a combination of vitamins B12 and folate, with or without B6. Some of the patients exhibiting elevated metabolite levels did not fully respond to B12 treatment. This may indicate a concomitant deficiency of folate and/or B6. The lack of response to B12 treatment could result from a deficiency of one, a pair, or all three vitamins. However, it would be expensive and time-consuming to attempt to distinguish between the vitamin deficiencies.

Again, an important finding in this study is the large number of patients with serum B12>300 pg/ml who have elevated values for one or more metabolites as indicated by a “**” next to the individual values. As is seen in Table 3, there are many examples of elevated values for MMA at all levels of serum B12 including the mid-normal (300-600 pg/ml), the high-normal (600-800 pg/ml), and even the elevated (>800 pg/ml) ranges. The same is true for elevations of HC. In some patients the serum folate is low, indicating that folate deficiency may be present, but in many cases both B12 and folate levels are normal. B6 levels were not performed in this study, but B6 deficiency would not be expected to cause elevations of MMA. Thus, again it is not clear which vitamin, or pair of vitamins, or whether all three vitamins is or are deficient. One could pick a single vitamin with which to treat a patient for several weeks or months, and then repeat measurement of metabolite levels to determine if a partial or full correction had occurred. If there was no response, one could try another vitamin, or if there was a partial response one could add a second vitamin, and then repeat metabolite measurement after several weeks or months. If there was still no response, one could try the third vitamin, or if there was a partial response, one could try a different pair of vitamins. Eventually one could determine whether an individual vitamin, a particular pair of vitamins, or all three vitamins were required to normalize or maximally reduce the metabolite levels, but it would often require months or even a year to make this determination. Such a determination would be expensive. In addition, a patient who was optimally treated with a single vitamin or pair of vitamins might subsequently develop a deficiency of one or even two of the other vitamins as evidenced by a re-elevation or increase in the levels of one or more metabolites. Therapeutic testing could be reinitiated and continued as described above, although this would also be time-consuming and expensive.

It requires less time and expense to treat patients with elevated metabolite levels with a combination of vitamin B12 and folate, or a combination of vitamin B12, folate and vitamin B6. The utility of the approach of the present invention is appreciated only after it is taught, for the first time in the present disclosure, that a deficiency of one or more of the three vitamins occurs commonly in the elderly population as evidenced by elevation of one or more metabolites, i.e., MMA, 2-MCA, HC and CT.

EXAMPLE 5

Determination of Serum B12 Folate, I4MA, and HC Levels in a Geriatric Population.

A study was conducted with 548 elderly subjects from the Framingham study between the ages of 65-99 to determine serum B12, folate, MMA, and HC levels (Table 4). The significance of the results shown in Table 4 (marked with “**”) are as described for Table 2 (Example 2).

As in the study described in Examples 3 and 4, the elderly population exhibited a shift to lower serum B12 levels (FIG. 11), and elevated serum MMA and HC levels. The elderly population also exhibited a high incidence (9.5%) of low serum folate levels (Table 4). As in the studies reported in Examples 2, 3 and 4, the incidence of tissue or intracellular vitamin deficiencies based on elevated metabolite levels was higher than that predicted from measurement of serum vitamin levels.

As in Examples 3 and 4 above, these results confirm the importance of the finding that there are a large number of patients with serum B12>300 pg/ml who have elevated values for one or more metabolites as indicated by a “**” next to the individual values. As is seen in Table 4, there are many examples of elevated MMA values at all levels of serum B12 including the mid-normal (300-600 pg/ml), the high-normal (600-800 pg/ml), and even the elevated (>800 pg/ml) ranges. The same is true for elevations of HC. In some patients the serum folate is low, indicating that folate deficiency may be present, but in many cases both B12 and folate levels are normal. B6 levels were not performed in this study, but B6 deficiency would not be expected to cause elevations of MMA. Thus, again it is not clear which vitamin, or pair of vitamins, or whether all three vitamins is or are deficient. One could pick a single vitamin with which to treat a patient for several weeks or months, and then repeat measurement of metabolite levels to determine if a partial or full correction had occurred. If there was no response, one could try another vitamin, or if there was a partial response one could add a second vitamin, and then repeat metabolite measurement after several weeks or months. If there was still no response, one could try the third vitamin, or if there was a partial response, one could try a different pair of vitamins. Eventually one could determine whether an individual vitamin, a particular pair of vitamins, or all three vitamins were required to normalize or maximally reduce the metabolite levels, but it would often require months or even a year to make this determination. Such a determination would be expensive. In addition, a patient who was optimally treated with a single vitamin or pair of vitamins might subsequently develop a deficiency of one or even two of the other vitamins as evidenced by a re-elevation or increase in the levels of one or more metabolites. Therapeutic testing could be reinitiated and continued as described above, although this would also be time-consuming and expensive.

It requires less time and expense to treat patients with elevated metabolite levels with a combination of vitamin B12 and folate, or a combination of vitamin B12, folate and vitamin B6. The utility of the approach of the present invention is appreciated only after it is taught, for the first time in the present disclosure, that a deficiency of one or more of the three vitamins occurs commonly in the elderly population as evidenced by elevation of one or more metabolites, i.e., MMA, 2-MCA, HC and CT.

TABLE 2
SERUM METABOLITE & VITAMIN LEVELS IN A GERIATRIC
OUTPATIENT POPULATION
Total
PatientB12FolateHomocysteineMMACTMC
11666**9.841.8**1508**507**759**
11879**9.329.6**2200**343**428**
016155**7.615.31316**208196
067163**6.69.99316469
091178**12.029.2**3108**438**318**
042181**11.313.0452**300262**
030185**6.626.0**282**310223
037187**9.412.3160218334**
100187**9.513.6208453**141
036188*9.916.3**298**385**322**
109189**7.612.3127188161
007191**11.767.1**6349**619**1005**
018193**5.816.7**412**272235**
0502104.025.3**464**727**121
1082146.031.1**264523**315**
0412167.219.1**418**360**288*
1262246.58.8103361**121
00523112.517.1**269825**276**
02423513.018.5**2946**232289**
1112376.314.6135380**203
0232394.121.9**385**775**279**
01025612.911.5652**119144
0552586.87.5189342185
10225910.923.9**1894**423**400**
02626018.520.4**1949**295248**
10726213.110.1231628**153
0382697.615.7222152152
1402774.029.1**744**602**254**
0742785.224.1**699**296187
00227814.614.8554**259277**
0192828.512.4329**262161
0352875.89.8230390**218
0492903.933.0**140275138
07829010.912.5197240209
0452918.79.5162613**132
09229414.919.3**500**246167
1372976.810.1631**340184
0722986.719.7**375**302246**
1493108.316.1314**199149
0473124.915.9277**271173
0603129.48.0100228203
0463146.516.2142336125
0933186.416.5**304**361**130
01432114.510.7275**233170
0883277.117.8**263507**258**
0323406.68.6150133133
1473477.618.2**305**219265**
0013514.720.8**199402**223
0903534.920.7**144419**178
0083585.411.6372**529**177
10436012.712.12608977
1103703.0**17.1**456**297150
10337118.714.5257219180
0563736.512.4236415**189
0483743.6**9.7167237230**
13137710.913.625622085
1223787621.9**906**227196
0043858.610.310928892
1203909.822.9**499**529**260**
1384056.914.7334**238188
1414078.114.3168259263**
1014085.99.216013440
1454103.7**25.4**567**550349**
02741511.110.6169278164
0284185.634.6**608**589**351**
01142010.618.8**683**1014**282**
0814216.616.5**861**641**531**
0334234.216.3**156194170
05742518.313.5209381**321**
02142718.912.1223524**168
1354308.813.5284**412**180
09743515.410.9353**465**119
0524386.815.2281**372**238**
13244812.616.8**1931**394**250**
08645112.16.6139208107
14845813.911.4187322238**
01246615.38.3560**250144
08346612.013.7366**214193
13347013.810.8290**27555
0174754.039.6**196467**220
05347613.412.3226206125
0094826.525.3**240470**214
0664989.612.9374**23392
03150711.014.8173278220
09950710.49.612423363
1285074.69.4294**324176
01351411.315.9163
1515227.814.3370**324215
0775236.817.7**184210214
07952315.613.0316**223251**
0545244.910.0148230123
0205249.914.2235366**190
0695287.09.725728183
0855364.022.5**97191114
08455114.212.5166179131
08255912.314.6208371**182
1175603.4**18.8**10217688
06156112.79.8170404**152
0065674.616.8**138688**165
1295674.916.2363**495**331**
00357011.412.9189330230**
1155766.317.8**12823195
08957810.312.0147258236**
1435812.6**42.7**165555**208
1145835.116.6**599**660**177
0805939.518.0**208289142
0155987.012.4167381**95
0395989.618.1**691**719**354**
0706125.613.719729682
05162212.98.3119246150
1396288.57.814516683
1506288.614.5295**315183
0436355.913.7239272189
09665117.49.7326**
0736577.09.518628378
1276655.88.1166344**147
12167710.29.5226346**173
03469415.912.1406**592**584**
1246979.711.06317960
12370210.410.618614896
1137057.68.4107534**92
07170910.611.3207584**141
0767228.110.5271489**138
0447247.312.1212683**217
04073115.17.4205149136
0627414.418.7**153856**416**
02574110.012.2224344**121
1197555.910.1187377**61
07575710.024.7**246345**276**
09875913.813.9380**239156
1347697.510.412513181
08777325.010.1181285135
1427884.612.1166273129
06479215.48.6218299139
09479316.610.0186179173
0228088.814.4184271161
11281212.09.2181184108
12581714.411.015824272
1068625.39.29430095
14689013.911.9135
0588975.318.5**154460**80
06394317.819.7**277**642**306**
09596025.310.7135181111
1529639.48.8198
13097115.913.510630784
05910639.49.7129378**54
105110911.06.18715564
13611636.013.1250565**122
065125114.510.78814788
029149022.29.7129111105
14415367.017.7**216694**418**
068180912.710.45912839

TABLE 3
SERUM METABOLITE & VITAMIN LEVELS IN A GERIATRIC
NURSING HOME POPULATION
PatientB12FolateHomocysteineMethylmalonic Acid
NH170  8**14.034.8** 3365**
NH129 40**7.440.9** 6245**
NH156 44**22.417.4** 1130**
NH139 56**9.720.9** 1180**
NH132 67**7.692.4**12641**
NH176 129**9.220.3** 1156**
NH196 136**6.241.0** 1077**
NH109 139**9.820.9** 1294**
NH203 146**4.312.2 437**
NH141 161**13.412.2 223
NH178 172**8.25.9 141
NH103 189**5.513.1 362**
NH181 196**6.314.7 296**
NH160 20611.912.5 640**
NH197 22124.010.5 654**
NH073 2223.6**19.8** 490**
NH110 2275.513.7 1297**
NH010 2284.021.1** 413**
NH012 2348.716.0 596**
NH037 23611.522.5**11299**
NH114 23812.813.2 442**
NH211 2406.014.1 166
NH075 2509.312.1 170
NH172 2557.214.4 552**
NH148 2595.719.2** 317**
NH138 2649.216.7** 340**
NH150 2644.013.7  98
NH099 2725.512.5 125
NH124 2756.911.5  87
NH179 3017.67.1 143
NH135 3026.523.4** 397**
NH087 3047.810.8 327**
NH180 3045.810.5 237
NH209 3067.611.9 105
NH107 3103.3**8.6 148
NH081 3204.323.6** 470**
NH068 3247.913.4 243
NH183 3257.711.1 144
NH033 33013.87.7 149
NH161 3338.511.3 385**
NH192 33710.79.5 209
NH136 3406.718.2** 409**
NH191 34220.213.4 271
NH137 3434.015.6 183
NH182 3468.214.4 448**
NH020 3478.410.4 149
NH165 35118.511.8 425**
NH095 3528.514.5 366**
NH194 3614.320.3** 305**
NH106 3624.812.9 298**
NH060 3674.716.4**  71
NH009 3685.115.9 325**
NH071 3824.912.9 330**
NH080 3906.115.0 171
NH013 4076.712.4 310**
NH126 4099.217.4** 137
NH030 41111.210.4 844**
NH210 4138.611.9 210
NH158 4145.716.2 508**
NH027 41610.215.5 769**
NH003 42416.59.5 167
NH187 4294.78.8 439**
NH022 43010.514.0 214
NH082 43610.617.7** 340**
NH162 4386.119.2** 180
NH021 4395.315.1 191
NH056 44711.710.9 184
NH119 4483.2**14.1 241
NH120 4485.612.0 138
NH186 4504.723.1** 213
NH064 4516.910.6 237
NH057 45314.610.4 282**
NH131 4548.116.2 258
NH059 4626.09.1 147
NH202 4653.3**17.0** 393**
NH134 47515.311.6 321**
NH083 4757.410.6 178
NH199 47915.110.4 141
NH042 4826.015.0 141
NH200 49113.69.8 154
NH213 4978.110.0  92
NH143 5005.222.1** 175
NH031 5026.416.1 151
NH188 50412.515.1 1461**
NH171 50410.712.9 344**
NH008 5054.69.9 185
NH102 50616.69.1 236
NH145 5127.722.2** 161
NH093 5145.117.7** 185
NH118 52425.010.1 314**
NH185 5248.712.1  84
NH111 5275.118.4** 250
NH149 53012.618.2** 531**
NH011 5348.112.5 654**
NH128 5404.311.6 120
NH035 5477.59.8 193
NH005 55117.75.0 365**
NH212 55211.912.1 202
NH007 5546.426.1** 646**
NH086 5549.55.1 127
NH069 55522.76.8 134
NH121 5558.210.0 112
NH117 5716.69.7 351**
NH055 58114.89.1 265
NH025 5815.215.3 181
NH104 5833.914.6 1699**
NH173 58311.210.6 160
NH177 5846.25.7 111
NH207 5868.516.4** 243
NH070 5915.412.0 168
NH038 5928.08.8 230
NH049 59910.721.7** 238
NH062 6064.57.7  96
NH153 6087.713.6 221
NH206 6116.616.4** 400**
NH018 6146.310.9 123
NH163 6165.09.6 132
NH189 6197.612.0 158
NH045 62021.012.4 265
NH074 62110.29.2 172
NH054 6238.09.8 121
NH152 6258.27.8 206
NH140 63721.713.6 300**
NH050 64216.313.5 275**
NH089 6447.716.7** 444**
NH036 6497.910.7  68
NH097 6516.613.4 426**
NH016 6564.161.0** 356**
NH053 65714.210.6 320**
NH066 6587.711.4 228
NH051 6594.010.7 216
NH108 6715.824.0** 823**
NH058 6736.011.2 392**
NH028 67522.39.1 105
NH204 6784.710.2 148
NH169 6796.919.2** 267
NH032 68112.75.9  99
NH065 68211.013.5 176
NH061 68313.49.6 190
NH116 6859.07.5 244
NH015 6996.816.8** 236
NH157 71110.012.8 198
NH155 71510.017.6** 308**
NH034 7157.911.4 179
NH040 71710.515.7 256
NH105 7186.013.2 308**
NH048 7198.010.8 207
NH084 7206.89.4 169
NH115 72416.39.4 161
NH205 7348.513.3 232
NH113 73811.710.3 171
NH154 73813.79.6 123
NH167 74117.06.6 129
NH190 7525.214.1 254
NH067 76022.59.5 232
NH014 7678.97.3 100
NH072 7688.36.9 131
NH133 7728.820.4** 219
NH122 7786.010.4 108
NH076 78112.114.9 282**
NH147 7857.524.5** 411**
NH026 7869.78.3 146
NH151 78924.411.1 182
NH198 79710.910.7 158
NH088 8016.418.3** 184
NH004 80611.38.8  96
NH024 8185.114.1 219
NH100 82616.410.5 103
NH078 8317.210.3 266
NH052 84419.68.0 193
NH142 84818.612.1 398**
NH002 8629.411.3 212
NH091 8914.912.6 169
NH127 89722.08.4 132
NH096 9019.35.2 104
NH201 91025.015.7 424**
NH184 94121.510.8 170
NH208 94520.29.8 111
NH130 96822.410.4 339**
NH164 9898.016.8** 102
NH077100615.19.2 188
NH017101511.99.5 175
NH029105318.611.4 161
NH02310559.39.7 193
NH04710796.411.4 106
NH043108214.513.9 144
NH195108836.912.2 150
NH19310928.215.7 225
NH04610939.218.8** 186
NH10111083.98.1 139
NH098111711.312.5  88
NH168112425.215.0 203
NH00611266.98.1 159
NH14411358.021.9** 262
NH044115926.810.2 109
NH17511627.812.0 210
NH14611799.810.1 129
NH112123810.315.0 347**
NH001130413.16.9 142
NH166133713.48.3  67
NH079134618.012.0 248
NH041152820.78.2 155
NH063155915.07.0  66
NH15915666.615.5 451**
NH12517038.220.6** 153
NH094176815.98.4 182
NH123202810.216.8** 206
NH174210613.312.8 280**
NH039222723.88.9 119
NH019229711.115.5 177
NH09223605.79.8 131
NH085314122.026.9** 1947**

TABLE 4
SERUM METABOLITE & VITAMIN LEVELS IN A GERIATRIC
POPULATION
PatientB12FolateHomocysteineMMA
495 77**10.065.4**3145**
484 84**10.077.5**6820**
522 100**3.6**15.5 967**
455 115**1.9**21.8** 170
493 135**4.416.9** 421**
528 145**3.938.3** 729**
510 155**4.614.1 804**
502 155**2.1**16.9** 347**
412 160**18.5**33.8**1301**
409 160**4.816.8** 164
470 165**9.219.9**1468**
460 165**6.811.5 142
437 170**4.916.5** 813
439 170**1.2**21.3** 502**
525 175**11.515.31058**
442 175**4.217.5** 328**
456 180**7.311.1 206
450 180**5.011.8 196
477 185**3.4**31.4** 369**
508 190**4.119.5** 335**
423 190**2.5**19.0** 329**
462 190**3.811.6 276**
523 190**5.616.8** 207
482 190**2.9**25.1** 179
459 190**5.319.6** 167
543 195**4.313.5 470**
520 195**1.7**22.2** 309**
431 195**7.213.5 251
513 2005.025.0**1184**
534 2004.932.6**1080**
515 2004.917.3** 478**
531 2005.126.8** 466**
516 2003.6**17.8** 279**
526 2001.6**23.5** 171
471 2055.722.0** 542**
413 2052.6**20.4** 304**
497 2053.3**19.4** 258
539 2054.115.4 247
544 20512.511.7 233
540 2054.017.1** 185
517 2052.2**15.0 151
496 2103.7**15.21103**
488 21016.521.8** 600**
416 21512.510.0 197
434 2207.124.8** 439**
545 22011.514.4 407**
547 2205.317.5** 396**
408 2203.2**16.4** 357**
449 2203.7**13.7 272**
507 2208.510.0 179
458 22510.521.1** 964**
491 2257.216.0 472**
529 2302.0**61.11172**
415 2303.2**28.9** 377**
453 2303.6**19.8** 336**
448 2305.213.1 319**
498 2305.920.1** 255
533 2305.711.7 151
466 23535.012.1 617**
537 2355.710.7 394**
483 2358.616.6** 344**
512 2353.912.5 190
452 2404.726.5**1068**
454 2405.211.9 201
535 2404.415.3 195
421 24510.512.5 464**
469 2456.220.0** 448**
474 2457.310.3 327**
486 2459.212.6 156
536 25022.520.3**1068**
475 2505.623.0 456**
511 2502.7**23.1** 398**
465 2504.123.1** 323**
506 2505.211.5 252
417 2505.525.2** 241
52412502.5**14.4 212
411 2509.911.5 200
492 2505.210.7 182
548 2502.9**12.4 179
441 2504.58.5 147
480 2554.816.9** 558**
532 2557.014.8 419**
464 25511.512.9 400**
494 2556.212.1 293**
106 2554.511.7 203
546 2605.514.7 662**
541 2605.430.8** 426**
420 2605.313.6 347**
500 2606.714.0 330**
538 2609.317.3** 298**
457 2602.9**12.6 286**
472 2608.313.8 278**
424 2608.310.1 242
433 2606.810.5 197
425 2657.314.7 724**
468 2653.816.7** 289**
435 2657.414.0 150
499 2652.2**12.4 131
432 2704.328.3** 432**
521 2703.7**15.3 349**
549 2704.2112.4 343**
518 27010.010.1 276**
418 27026.09.4 213
419 2706.512.5 212
428 2704.218.7** 189
443 2708.812.0 187
446 27011.08.1 157
461 2757.615.1 663**
440 2754.912.9 248
436 2756.330.1** 233
530 2757.413.6 231
438 2754.68.5 221
527 2757.510.5 219
444 2754.012.2 180
429 2805.315.3 463**
503 2804.425.7** 421**
485 2803.5**15.6 381**
410 28014.510.0 201
487 2803.910.5 166
430 2809.28.8 161
519 2853.922.2** 919**
476 28510.512.8 339**
509 2855.413.0 331**
501 2855.512.4 252
542 2856.915.5 242
445 2857.214.9 237
427 2854.017.1** 233
490 2904.713.9 203
451 2902.1**20.0** 226
414 2907.09.7 117
467 2904.16.5 68
463 2955.812.3 296**
473 2957.514.4 290**
505 2954.112.4 257
198 30011.510.9 323**
195 3009.812.2 216
207 3057.713.2 330**
 67 3058.615.4 312**
 50 3059.011.6 235
 70 30512.512.7 228
113 3055.613.5 201
 39 3056.919.7** 170
3 3054.211.5 135
325 30514.59.4 94
368 3104.715.9 371**
322 3107.815.3 362**
295 3107.213.8 305**
347 3105.816.5** 266
313 3106.116.5** 219
355 3105.515.4 138
291 3104.515.2 125
478 31523.017.7** 857**
 53 3155.812.1 505**
240 3156.712.3 394**
 14 3159.614.2 331**
137 3157.824.3** 306**
254 3158.717.0** 285**
109 3153.7**16.5** 263
252 3155.210.1 241
186 3154.115.4 238
183 3155.510.7 195
390 3156.910.0 188
267 3152.2**12.0 124
310 32012.013.8 395**
 31 32017.012.9 334**
 88 3204.813.8 217
403 3209.611.3 162
 60 3206.211.4 155
315 3206.49.9 136
175 3256.317.8** 486**
317 32522.014.0 294**
 18 3256.311.1 241
247 32513.513.2 231
223 3259.212.6 203
132 3253.7**15.4 184
168 3254.310.2 174
238 3255.59.9 166
117 3255.215.0 154
404 3302.5**33.1**1085**
138 3304.811.3 360**
316 3303.6**10.2 272**
 61 3305.112.5 242
333 33034.09.2 235
 16 3304.613.3 211
276 3305.711.9 200
391 3304.18.4 184
362 3309.211.7 178
1 3309.98.9 170
379 33516.012.1 471**
147 3359.09.7 427**
 89 3358.015.3 385**
211 3355.012.2 374**
 45 3355.916.3** 250
 47 3355.013.6 249
402 3354.713.5 230
314 3357.69.7 203
150 3354.811.2 119
120 3401.9**21.0** 775**
284 3407.225.6** 439**
230 34014.011.4 419**
149 3408.818.9** 337**
269 3403.916.2 302**
197 34010.512.8 233
 19 3409.611.0 232
422 3403.1**14.4 188
196 34011.58.9 169
 40 3458.714.6 610**
244 3458.615.8 461**
287 3455.718.1** 427**
100 3458.314.8 403**
383 3454.327.2** 284**
 62 34519.59.6 250
350 3458.010.0 249
 65 3458.010.2 247
307 34516.511.6 208
 69 34517.09.9 197
328 3457.58.9 192
 43 3456.013.2 191
222 3456.19.2 175
306 3454.317.2** 160
154 3457.110.2 148
 94 3504.816.1 302**
201 3506.19.9 200
 13 3505.110.9 193
236 3557.214.8 309**
191 3555.815.3 257
481 3555.217.1** 134
 92 3604.225.2** 321**
324 3603.816.6** 264
 87 3603.3**13.3 200
 46 3605.411.1 179
289 3609.57.9 129
392 3605.110.3 125
320 3656.417.3** 240
134 36513.511.8 238
239 3657.713.2 236
326 3656.010.9 180
364 3654.113.9 154
218 3657.511.2 126
216 3656.212.2 119
248 3655.713.3 117
375 3704.120.7** 532**
288 3706.418.8** 436**
161 3706.311.2 340**
244 37019.59.8 286**
330 37018.012.2 228
334 37012.58.7 172
275 3706.912.7 162
 54 3757.310.1 583**
185 3759.310.5 386**
 52 3758.115.5 291**
366 3755.012.5 280**
 93 3753.3**16.2 248
151 3752.9**12.3 235
 85 3756.714.8 217
294 3757.012.2 184
361 3757.910.7 179
318 3755.513.7 160
386 3757.610.4 153
304 3759.19.4 132
228 3807.717.1** 320**
110 3804.07.2 135
204 3805.710.6 91
348 3852.3**17.4** 368**
146 38511.512.5 253
260 3855.513.7 211
136 3853.6**19.8** 205
338 3855.016.2 180
376 3853.6**13.7 154
194 38512.57.9 153
504 38538.09.5 138
160 3908.124.7** 475**
354 39011.512.8 212
 25 3905.111.3 205
387 3908.78.4 162
 86 39021.012.6 133
133 3903.911.3 113
331 39512.020.1** 638**
130 39510.510.8 256
 82 3952.8**9.8 236
119 39512.516.3** 209
380 39510.514.3 159
373 3955.511.6 152
256 39510.59.9 149
384 3957.314.7 116
105 40019.010.5 322**
251 4004.814.9 289**
352 40011.59.6 181
279 4004.511.7 170
339 4007.413.6 168
381 4056.712.4 294**
285 4057.014.2 281**
340 4053.6**19.6** 275**
 51 4056.514.3 233
 33 4056.59.6 207
268 4053.3**14.9 205
 73 4055.213.1 172
 17 4107.516.2 473**
286 4104.718.8** 415**
140 4105.921.7** 302**
116 4106.814.5 218
396 4105.616.1 190
356 4101.9**27.6** 149
237 4103.6**16.6** 122
112 4105.58.9 107
259 4104.711.6 99
176 4155.221.9** 453**
193 41510.511.3 163
323 4156.19.6 163
202 41511.59.4 150
398 4158.012.6 134
321 4205.210.7 383**
142 42029.08.3 234
327 4203.2**14.6 203
342 4207.39.4 156
170 42020.510.3 142
345 42029.513.2 136
302 4208.68.8 128
115 4256.322.2** 628**
 97 42512.519.8** 313**
246 4258.715.1 241
 72 42510.513.5 241
365 4256.716.7** 237
139 42512.510.4 224
143 4258.113.5 216
426 42519.514.5 201
303 4253.0**14.5 154
388 4256.212.3 135
127 4256.78.4 100
262 43010.012.1 323**
270 4304.812.9 293**
514 4304.312.9 197
341 4303.5**19.9** 190
278 4305.210.8 182
370 43011.015.3 174
 55 4307.611.0 162
274 4305.08.2 131
367 43017.58.0 126
 98 43013.512.8 125
337 43513.514.1 395**
309 4358.712.9 349**
305 43517.515.4 187
144 43525.08.9 167
 34 4358.67.6 157
234 4359.79.2 116
123 4409.612.2 622**
200 4404.812.4 257
250 4407.512.9 248
107 4406.314.7 183
300 4406.57.9 123
374 4455.414.0 247
372 44511.011.0 181
 36 4454.010.0 181
271 4457.210.4 124
242 44515.59.6 112
264 4456.010.7 100
172 45011.514.9 607**
 32 45011.513.6 362**
346 45013.515.8 330**
 41 4508.511.4 194
 95 4505.112.5 182
357 4556.314.4 296**
319 45517.010.2 147
308 45515.09.8 131
235 45523.09.0 114
349 4559.28.3 82
178 4605.620.6** 473**
312 4604.714.4 197
 79 4605.010.4 173
131 46018.010.2 162
243 4602.6**11.6 160
261 4657.710.6 252
378 4655.413.2 221
 49 46547.010.8 179
226 4657.710.2 173
377 4655.68.5 143
253 46510.07.0 138
 76 47012.514.8 304**
203 47015.07.6 233
296 47023.511.0 161
382 4705.311.1 109
6 47510.512.5 232
 75 4754.58.1 150
332 4759.410.0 144
290 47514.09.1 143
128 4755.99.3 133
124 4756.013.5 111
177 4758.89.1 106
126 48011.011.0 212
283 4805.210.6 175
209 48010.510.5 175
293 4806.815.5 135
121 4854.720.0** 345**
282 48512.010.9 236
 71 48513.58.1 168
385 4859.014.1 128
190 4959.910.4 410**
210 4958.612.0 243
155 4955.910.4 219
336 49513.59.9 135
280 5008.714.5 334**
 96 5004.710.8 237
145 5005.917.5** 233
199 5004.213.8 199
489 50011.59.7 198
217 5006.49.6 166
 90 5007.58.5 106
164 5105.223.8** 408**
343 5104.513.7 284**
 42 5104.97.4 233
351 5108.511.0 207
299 51012.08.0 104
 99 52010.525.8** 322**
114 52030.010.9 220
369 52029.016.7** 206
 37 52010.58.6 191
215 5206.716.8** 151
401 5207.512.6 148
229 5207.911.0 116
135 5203.2**8.3 88
 81 5306.814.8 372**
 91 53014.510.6 228
167 53023.59.2 176
181 5305.59.3 171
 56 53020.08.3 163
5 53013.58.1 159
180 54012.09.0 216
311 5404.113.3 214
389 5403.913.9 169
125 5405.513.0 159
 35 54022.511.0 123
104 55010.516.5** 544**
393 5504.911.9 339**
394 55023.014.0 278**
292 5506.916.2 263
163 5506.714.3 219
 66 55010.511.6 206
 29 55017.59.6 191
227 5507.911.7 154
 38 5507.511.9 152
241 55010.59.8 100
102 5509.78.6 91
 77 56024.014.8 554**
162 56010.511.8 275**
273 5608.79.4 180
 80 5606.311.2 108
255 5608.89.9 93
122 57066.013.8 304**
208 57034.010.2 255
 23 57021.58.3 241
447 57025.010.0 164
225 5705.712.2 154
174 5707.111.0 127
 11 57019.08.9 113
165 58010.514.8 226
182 5808.98.2 189
245 59015.510.0 262
 83 59017.58.3 199
166 59011.59.4 188
158 5907.310.7 166
187 5904.511.0 146
156 59023.511.3 112
231 6009.59.0 192
 78 60011.59.4 151
329 61015.07.3 312**
 57 61016.011.9 286**
7 61012.010.4 195
277 6109.57.8 153
108 62013.58.4 191
205 62018.07.5 145
263 6209.810.2 101
9 6304.911.4 300**
111 6308.311.1 276**
 68 63011.58.9 143
399 63014.011.0 90
266 6405.115.7 364**
 12 64024.59.0 233
152 6408.110.0 209
405 6407.012.8 186
 27 64022.58.4 136
258 6408.311.2 120
249 6408.79.1 81
297 65016.010.0 279**
192 6504.914.9 213
257 6503.3**16.3** 208
184 65012.59.9 193
 58 65018.510.7 172
301 65016.015.5 162
397 65012.58.4 146
272 65011.07.4 120
153 6507.113.1 116
406 6506.65.8 81
 10 6609.07.6 154
 26 66022.08.3 132
265 6703.919.3** 509**
359 67021.08.3 269
 48 67032.09.9 262
335 67011.58.1 121
189 6806.617.9** 358**
220 68015.510.9 115
 15 69013.513.4 159
 44 70020.012.7 244
 21 70013.510.2 129
 74 70015.07.1 65
4 71029.08.5 266
353 71011.511.4 206
281 71010.59.6 185
2 7108.08.5 109
212 74020.011.1 250
8 74012.011.5 216
206 75012.58.3 116
101 77014.512.7 372**
344 77032.011.7 297**
 20 77035.010.1 245
407 77010.512.0 110
360 7802.7**20.9** 157
232 79015.510.1 151
141 79012.59.5 74
129 8008.711.7 211
188 80015.012.3 174
400 80012.510.3 156
 24 81023.07.5 194
173 83035.011.4 243
214 83021.512.0 187
 63 83013.88.8 185
148 83045.07.1 146
 84 83023.57.0 136
179 83016.56.6 96
171 84023.511.2 195
 28 8705.815.9 197
233 8707.912.7 169
221 87040.07.0 126
371 88020.08.5 152
213 89010.518.0** 231
358 90021.08.3 149
298 91015.510.2 221
118 910100.09.7 170
479 95011.512.1 188
 30 9506.210.5 170
15910009.58.7 281**
219105037.014.3 313**
103105012.510.3 154
 59115017.57.3 180
157125012.014.0 206
363135028.010.4 190
 22140013.510.4 233
 64140031.09.7 149
169145015.09.5 150