Method for preventing vascular disease

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.

Description

[0001] 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

[0002] 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

[0003] 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.

[0004] 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.

[0005] 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).

[0006] 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.

[0007] 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).

[0008] 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).

[0009] 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).

[0010] 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 &bgr;-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 &bgr;-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.

[0011] 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.

[0012] 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.

[0013] 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.

[0014] 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.

[0015] 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.”

[0016] 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.

[0017] 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

[0018] 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.

[0019] 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.

[0020] 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

[0021] 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).

[0022] 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).

[0023] 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).

[0024] 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).

[0025] 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).

[0026] 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).

[0027] 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).

[0028] 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).

[0029] 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).

[0030] 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).

[0031] 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

[0032] 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.

[0033] 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.

[0034] 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.

[0035] 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.

[0036] 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.

[0037] 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.

[0038] 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.

[0039] 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.

[0040] 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

[0041] Methods for Measurement of Serum Vitamin and Metabolite Levels.

[0042] 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).

[0043] 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.

[0044] 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

[0045] Incidence of Elevated MMA, 2-MCA, HC, and CT Levels in the Geriatric Population.

[0046] 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.

[0047] 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.

[0048] 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 &mgr;mol/L. Creatinine clearance was calculated using the formulation of Cockroft and Gault (1976) Nephron 16:31-41.

[0049] 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: 1 TABLE 1 INCIDENCE OF LOW SERUM VITAMN AND HIGH METABOLITE LEVELS IN GERIATRIC POPULATIONS A-D AND A YOUNGER HEALTHY POPULATION E. Folic Group B12 Acid B6 MMA 2-MCA HC CT A 6% 12% 48% 36% 44% 55% 64% B 6% 19% 53% 47% 39% 59%  6% C 3% 10% 50% 32% 45% 39% 73% D 6%  6% 17% 26% 23% 38% 41% E 2%  1%  1%  3%  6%  2%  4%

[0050] 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.

[0051] 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

[0052] Determination of Serum B12 Folate, MMA, HC, CT and 2-MCA Levels in a Geriatric Outpatient Population.

[0053] 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.

[0054] 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.

[0055] 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%.

[0056] 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.

[0057] 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.

[0058] 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.

[0059] 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

[0060] Determination of Serum B12, Folate, MMA, and HC Levels in a Geriatric Nursing Home Population.

[0061] 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.

[0062] 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.

[0063] 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.

[0064] 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

[0065] Determination of Serum B12 Folate, I4MA, and HC Levels in a Geriatric Population.

[0066] 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).

[0067] 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.

[0068] 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.

[0069] 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. 2 TABLE 2 SERUM METABOLITE & VITAMIN LEVELS IN A GERIATRIC OUTPATIENT POPULATION Total Patient B12 Folate Homocysteine MMA CT MC 116 66** 9.8 41.8** 1508** 507** 759** 118 79** 9.3 29.6** 2200** 343** 428** 016 155** 7.6 15.3 1316** 208 196 067 163** 6.6 9.9 93 164 69 091 178** 12.0 29.2** 3108** 438** 318** 042 181** 11.3 13.0 452** 300 262** 030 185** 6.6 26.0** 282** 310 223 037 187** 9.4 12.3 160 218 334** 100 187** 9.5 13.6 208 453** 141 036 188* 9.9 16.3** 298** 385** 322** 109 189** 7.6 12.3 127 188 161 007 191** 11.7 67.1** 6349** 619** 1005** 018 193** 5.8 16.7** 412** 272 235** 050 210 4.0 25.3** 464** 727** 121 108 214 6.0 31.1** 264 523** 315** 041 216 7.2 19.1** 418** 360** 288* 126 224 6.5 8.8 103 361** 121 005 231 12.5 17.1** 269 825** 276** 024 235 13.0 18.5** 2946** 232 289** 111 237 6.3 14.6 135 380** 203 023 239 4.1 21.9** 385** 775** 279** 010 256 12.9 11.5 652** 119 144 055 258 6.8 7.5 189 342 185 102 259 10.9 23.9** 1894** 423** 400** 026 260 18.5 20.4** 1949** 295 248** 107 262 13.1 10.1 231 628** 153 038 269 7.6 15.7 222 152 152 140 277 4.0 29.1** 744** 602** 254** 074 278 5.2 24.1** 699** 296 187 002 278 14.6 14.8 554** 259 277** 019 282 8.5 12.4 329** 262 161 035 287 5.8 9.8 230 390** 218 049 290 3.9 33.0** 140 275 138 078 290 10.9 12.5 197 240 209 045 291 8.7 9.5 162 613** 132 092 294 14.9 19.3** 500** 246 167 137 297 6.8 10.1 631** 340 184 072 298 6.7 19.7** 375** 302 246** 149 310 8.3 16.1 314** 199 149 047 312 4.9 15.9 277** 271 173 060 312 9.4 8.0 100 228 203 046 314 6.5 16.2 142 336 125 093 318 6.4 16.5** 304** 361** 130 014 321 14.5 10.7 275** 233 170 088 327 7.1 17.8** 263 507** 258** 032 340 6.6 8.6 150 133 133 147 347 7.6 18.2** 305** 219 265** 001 351 4.7 20.8** 199 402** 223 090 353 4.9 20.7** 144 419** 178 008 358 5.4 11.6 372** 529** 177 104 360 12.7 12.1 260 89 77 110 370 3.0** 17.1** 456** 297 150 103 371 18.7 14.5 257 219 180 056 373 6.5 12.4 236 415** 189 048 374 3.6** 9.7 167 237 230** 131 377 10.9 13.6 256 220 85 122 378 76 21.9** 906** 227 196 004 385 8.6 10.3 109 288 92 120 390 9.8 22.9** 499** 529** 260** 138 405 6.9 14.7 334** 238 188 141 407 8.1 14.3 168 259 263** 101 408 5.9 9.2 160 134 40 145 410 3.7** 25.4** 567** 550 349** 027 415 11.1 10.6 169 278 164 028 418 5.6 34.6** 608** 589** 351** 011 420 10.6 18.8** 683** 1014** 282** 081 421 6.6 16.5** 861** 641** 531** 033 423 4.2 16.3** 156 194 170 057 425 18.3 13.5 209 381** 321** 021 427 18.9 12.1 223 524** 168 135 430 8.8 13.5 284** 412** 180 097 435 15.4 10.9 353** 465** 119 052 438 6.8 15.2 281** 372** 238** 132 448 12.6 16.8** 1931** 394** 250** 086 451 12.1 6.6 139 208 107 148 458 13.9 11.4 187 322 238** 012 466 15.3 8.3 560** 250 144 083 466 12.0 13.7 366** 214 193 133 470 13.8 10.8 290** 275 55 017 475 4.0 39.6** 196 467** 220 053 476 13.4 12.3 226 206 125 009 482 6.5 25.3** 240 470** 214 066 498 9.6 12.9 374** 233 92 031 507 11.0 14.8 173 278 220 099 507 10.4 9.6 124 233 63 128 507 4.6 9.4 294** 324 176 013 514 11.3 15.9 163 151 522 7.8 14.3 370** 324 215 077 523 6.8 17.7** 184 210 214 079 523 15.6 13.0 316** 223 251** 054 524 4.9 10.0 148 230 123 020 524 9.9 14.2 235 366** 190 069 528 7.0 9.7 257 281 83 085 536 4.0 22.5** 97 191 114 084 551 14.2 12.5 166 179 131 082 559 12.3 14.6 208 371** 182 117 560 3.4** 18.8** 102 176 88 061 561 12.7 9.8 170 404** 152 006 567 4.6 16.8** 138 688** 165 129 567 4.9 16.2 363** 495** 331** 003 570 11.4 12.9 189 330 230** 115 576 6.3 17.8** 128 231 95 089 578 10.3 12.0 147 258 236** 143 581 2.6** 42.7** 165 555** 208 114 583 5.1 16.6** 599** 660** 177 080 593 9.5 18.0** 208 289 142 015 598 7.0 12.4 167 381** 95 039 598 9.6 18.1** 691** 719** 354** 070 612 5.6 13.7 197 296 82 051 622 12.9 8.3 119 246 150 139 628 8.5 7.8 145 166 83 150 628 8.6 14.5 295** 315 183 043 635 5.9 13.7 239 272 189 096 651 17.4 9.7 326** 073 657 7.0 9.5 186 283 78 127 665 5.8 8.1 166 344** 147 121 677 10.2 9.5 226 346** 173 034 694 15.9 12.1 406** 592** 584** 124 697 9.7 11.0 63 179 60 123 702 10.4 10.6 186 148 96 113 705 7.6 8.4 107 534** 92 071 709 10.6 11.3 207 584** 141 076 722 8.1 10.5 271 489** 138 044 724 7.3 12.1 212 683** 217 040 731 15.1 7.4 205 149 136 062 741 4.4 18.7** 153 856** 416** 025 741 10.0 12.2 224 344** 121 119 755 5.9 10.1 187 377** 61 075 757 10.0 24.7** 246 345** 276** 098 759 13.8 13.9 380** 239 156 134 769 7.5 10.4 125 131 81 087 773 25.0 10.1 181 285 135 142 788 4.6 12.1 166 273 129 064 792 15.4 8.6 218 299 139 094 793 16.6 10.0 186 179 173 022 808 8.8 14.4 184 271 161 112 812 12.0 9.2 181 184 108 125 817 14.4 11.0 158 242 72 106 862 5.3 9.2 94 300 95 146 890 13.9 11.9 135 058 897 5.3 18.5** 154 460** 80 063 943 17.8 19.7** 277** 642** 306** 095 960 25.3 10.7 135 181 111 152 963 9.4 8.8 198 130 971 15.9 13.5 106 307 84 059 1063 9.4 9.7 129 378** 54 105 1109 11.0 6.1 87 155 64 136 1163 6.0 13.1 250 565** 122 065 1251 14.5 10.7 88 147 88 029 1490 22.2 9.7 129 111 105 144 1536 7.0 17.7** 216 694** 418** 068 1809 12.7 10.4 59 128 39

[0070] 3 TABLE 3 SERUM METABOLITE & VITAMIN LEVELS IN A GERIATRIC NURSING HOME POPULATION Patient B12 Folate Homocysteine Methylmalonic Acid NH170   8** 14.0 34.8**  3365** NH129  40** 7.4 40.9**  6245** NH156  44** 22.4 17.4**  1130** NH139  56** 9.7 20.9**  1180** NH132  67** 7.6 92.4** 12641** NH176  129** 9.2 20.3**  1156** NH196  136** 6.2 41.0**  1077** NH109  139** 9.8 20.9**  1294** NH203  146** 4.3 12.2  437** NH141  161** 13.4 12.2  223 NH178  172** 8.2 5.9  141 NH103  189** 5.5 13.1  362** NH181  196** 6.3 14.7  296** NH160  206 11.9 12.5  640** NH197  221 24.0 10.5  654** NH073  222 3.6** 19.8**  490** NH110  227 5.5 13.7  1297** NH010  228 4.0 21.1**  413** NH012  234 8.7 16.0  596** NH037  236 11.5 22.5** 11299** NH114  238 12.8 13.2  442** NH211  240 6.0 14.1  166 NH075  250 9.3 12.1  170 NH172  255 7.2 14.4  552** NH148  259 5.7 19.2**  317** NH138  264 9.2 16.7**  340** NH150  264 4.0 13.7   98 NH099  272 5.5 12.5  125 NH124  275 6.9 11.5   87 NH179  301 7.6 7.1  143 NH135  302 6.5 23.4**  397** NH087  304 7.8 10.8  327** NH180  304 5.8 10.5  237 NH209  306 7.6 11.9  105 NH107  310 3.3** 8.6  148 NH081  320 4.3 23.6**  470** NH068  324 7.9 13.4  243 NH183  325 7.7 11.1  144 NH033  330 13.8 7.7  149 NH161  333 8.5 11.3  385** NH192  337 10.7 9.5  209 NH136  340 6.7 18.2**  409** NH191  342 20.2 13.4  271 NH137  343 4.0 15.6  183 NH182  346 8.2 14.4  448** NH020  347 8.4 10.4  149 NH165  351 18.5 11.8  425** NH095  352 8.5 14.5  366** NH194  361 4.3 20.3**  305** NH106  362 4.8 12.9  298** NH060  367 4.7 16.4**   71 NH009  368 5.1 15.9  325** NH071  382 4.9 12.9  330** NH080  390 6.1 15.0  171 NH013  407 6.7 12.4  310** NH126  409 9.2 17.4**  137 NH030  411 11.2 10.4  844** NH210  413 8.6 11.9  210 NH158  414 5.7 16.2  508** NH027  416 10.2 15.5  769** NH003  424 16.5 9.5  167 NH187  429 4.7 8.8  439** NH022  430 10.5 14.0  214 NH082  436 10.6 17.7**  340** NH162  438 6.1 19.2**  180 NH021  439 5.3 15.1  191 NH056  447 11.7 10.9  184 NH119  448 3.2** 14.1  241 NH120  448 5.6 12.0  138 NH186  450 4.7 23.1**  213 NH064  451 6.9 10.6  237 NH057  453 14.6 10.4  282** NH131  454 8.1 16.2  258 NH059  462 6.0 9.1  147 NH202  465 3.3** 17.0**  393** NH134  475 15.3 11.6  321** NH083  475 7.4 10.6  178 NH199  479 15.1 10.4  141 NH042  482 6.0 15.0  141 NH200  491 13.6 9.8  154 NH213  497 8.1 10.0   92 NH143  500 5.2 22.1**  175 NH031  502 6.4 16.1  151 NH188  504 12.5 15.1  1461** NH171  504 10.7 12.9  344** NH008  505 4.6 9.9  185 NH102  506 16.6 9.1  236 NH145  512 7.7 22.2**  161 NH093  514 5.1 17.7**  185 NH118  524 25.0 10.1  314** NH185  524 8.7 12.1   84 NH111  527 5.1 18.4**  250 NH149  530 12.6 18.2**  531** NH011  534 8.1 12.5  654** NH128  540 4.3 11.6  120 NH035  547 7.5 9.8  193 NH005  551 17.7 5.0  365** NH212  552 11.9 12.1  202 NH007  554 6.4 26.1**  646** NH086  554 9.5 5.1  127 NH069  555 22.7 6.8  134 NH121  555 8.2 10.0  112 NH117  571 6.6 9.7  351** NH055  581 14.8 9.1  265 NH025  581 5.2 15.3  181 NH104  583 3.9 14.6  1699** NH173  583 11.2 10.6  160 NH177  584 6.2 5.7  111 NH207  586 8.5 16.4**  243 NH070  591 5.4 12.0  168 NH038  592 8.0 8.8  230 NH049  599 10.7 21.7**  238 NH062  606 4.5 7.7   96 NH153  608 7.7 13.6  221 NH206  611 6.6 16.4**  400** NH018  614 6.3 10.9  123 NH163  616 5.0 9.6  132 NH189  619 7.6 12.0  158 NH045  620 21.0 12.4  265 NH074  621 10.2 9.2  172 NH054  623 8.0 9.8  121 NH152  625 8.2 7.8  206 NH140  637 21.7 13.6  300** NH050  642 16.3 13.5  275** NH089  644 7.7 16.7**  444** NH036  649 7.9 10.7   68 NH097  651 6.6 13.4  426** NH016  656 4.1 61.0**  356** NH053  657 14.2 10.6  320** NH066  658 7.7 11.4  228 NH051  659 4.0 10.7  216 NH108  671 5.8 24.0**  823** NH058  673 6.0 11.2  392** NH028  675 22.3 9.1  105 NH204  678 4.7 10.2  148 NH169  679 6.9 19.2**  267 NH032  681 12.7 5.9   99 NH065  682 11.0 13.5  176 NH061  683 13.4 9.6  190 NH116  685 9.0 7.5  244 NH015  699 6.8 16.8**  236 NH157  711 10.0 12.8  198 NH155  715 10.0 17.6**  308** NH034  715 7.9 11.4  179 NH040  717 10.5 15.7  256 NH105  718 6.0 13.2  308** NH048  719 8.0 10.8  207 NH084  720 6.8 9.4  169 NH115  724 16.3 9.4  161 NH205  734 8.5 13.3  232 NH113  738 11.7 10.3  171 NH154  738 13.7 9.6  123 NH167  741 17.0 6.6  129 NH190  752 5.2 14.1  254 NH067  760 22.5 9.5  232 NH014  767 8.9 7.3  100 NH072  768 8.3 6.9  131 NH133  772 8.8 20.4**  219 NH122  778 6.0 10.4  108 NH076  781 12.1 14.9  282** NH147  785 7.5 24.5**  411** NH026  786 9.7 8.3  146 NH151  789 24.4 11.1  182 NH198  797 10.9 10.7  158 NH088  801 6.4 18.3**  184 NH004  806 11.3 8.8   96 NH024  818 5.1 14.1  219 NH100  826 16.4 10.5  103 NH078  831 7.2 10.3  266 NH052  844 19.6 8.0  193 NH142  848 18.6 12.1  398** NH002  862 9.4 11.3  212 NH091  891 4.9 12.6  169 NH127  897 22.0 8.4  132 NH096  901 9.3 5.2  104 NH201  910 25.0 15.7  424** NH184  941 21.5 10.8  170 NH208  945 20.2 9.8  111 NH130  968 22.4 10.4  339** NH164  989 8.0 16.8**  102 NH077 1006 15.1 9.2  188 NH017 1015 11.9 9.5  175 NH029 1053 18.6 11.4  161 NH023 1055 9.3 9.7  193 NH047 1079 6.4 11.4  106 NH043 1082 14.5 13.9  144 NH195 1088 36.9 12.2  150 NH193 1092 8.2 15.7  225 NH046 1093 9.2 18.8**  186 NH101 1108 3.9 8.1  139 NH098 1117 11.3 12.5   88 NH168 1124 25.2 15.0  203 NH006 1126 6.9 8.1  159 NH144 1135 8.0 21.9**  262 NH044 1159 26.8 10.2  109 NH175 1162 7.8 12.0  210 NH146 1179 9.8 10.1  129 NH112 1238 10.3 15.0  347** NH001 1304 13.1 6.9  142 NH166 1337 13.4 8.3   67 NH079 1346 18.0 12.0  248 NH041 1528 20.7 8.2  155 NH063 1559 15.0 7.0   66 NH159 1566 6.6 15.5  451** NH125 1703 8.2 20.6**  153 NH094 1768 15.9 8.4  182 NH123 2028 10.2 16.8**  206 NH174 2106 13.3 12.8  280** NH039 2227 23.8 8.9  119 NH019 2297 11.1 15.5  177 NH092 2360 5.7 9.8  131 NH085 3141 22.0 26.9**  1947**

[0071] 4 TABLE 4 SERUM METABOLITE & VITAMIN LEVELS IN A GERIATRIC POPULATION Patient B12 Folate Homocysteine MMA 495  77** 10.0 65.4** 3145** 484  84** 10.0 77.5** 6820** 522  100** 3.6** 15.5  967** 455  115** 1.9** 21.8**  170 493  135** 4.4 16.9**  421** 528  145** 3.9 38.3**  729** 510  155** 4.6 14.1  804** 502  155** 2.1** 16.9**  347** 412  160** 18.5** 33.8** 1301** 409  160** 4.8 16.8**  164 470  165** 9.2 19.9** 1468** 460  165** 6.8 11.5  142 437  170** 4.9 16.5**  813 439  170** 1.2** 21.3**  502** 525  175** 11.5 15.3 1058** 442  175** 4.2 17.5**  328** 456  180** 7.3 11.1  206 450  180** 5.0 11.8  196 477  185** 3.4** 31.4**  369** 508  190** 4.1 19.5**  335** 423  190** 2.5** 19.0**  329** 462  190** 3.8 11.6  276** 523  190** 5.6 16.8**  207 482  190** 2.9** 25.1**  179 459  190** 5.3 19.6**  167 543  195** 4.3 13.5  470** 520  195** 1.7** 22.2**  309** 431  195** 7.2 13.5  251 513  200 5.0 25.0** 1184** 534  200 4.9 32.6** 1080** 515  200 4.9 17.3**  478** 531  200 5.1 26.8**  466** 516  200 3.6** 17.8**  279** 526  200 1.6** 23.5**  171 471  205 5.7 22.0**  542** 413  205 2.6** 20.4**  304** 497  205 3.3** 19.4**  258 539  205 4.1 15.4  247 544  205 12.5 11.7  233 540  205 4.0 17.1**  185 517  205 2.2** 15.0  151 496  210 3.7** 15.2 1103** 488  210 16.5 21.8**  600** 416  215 12.5 10.0  197 434  220 7.1 24.8**  439** 545  220 11.5 14.4  407** 547  220 5.3 17.5**  396** 408  220 3.2** 16.4**  357** 449  220 3.7** 13.7  272** 507  220 8.5 10.0  179 458  225 10.5 21.1**  964** 491  225 7.2 16.0  472** 529  230 2.0** 61.1 1172** 415  230 3.2** 28.9**  377** 453  230 3.6** 19.8**  336** 448  230 5.2 13.1  319** 498  230 5.9 20.1**  255 533  230 5.7 11.7  151 466  235 35.0 12.1  617** 537  235 5.7 10.7  394** 483  235 8.6 16.6**  344** 512  235 3.9 12.5  190 452  240 4.7 26.5** 1068** 454  240 5.2 11.9  201 535  240 4.4 15.3  195 421  245 10.5 12.5  464** 469  245 6.2 20.0**  448** 474  245 7.3 10.3  327** 486  245 9.2 12.6  156 536  250 22.5 20.3** 1068** 475  250 5.6 23.0  456** 511  250 2.7** 23.1**  398** 465  250 4.1 23.1**  323** 506  250 5.2 11.5  252 417  250 5.5 25.2**  241 524 1250 2.5** 14.4  212 411  250 9.9 11.5  200 492  250 5.2 10.7  182 548  250 2.9** 12.4  179 441  250 4.5 8.5  147 480  255 4.8 16.9**  558** 532  255 7.0 14.8  419** 464  255 11.5 12.9  400** 494  255 6.2 12.1  293** 106  255 4.5 11.7  203 546  260 5.5 14.7  662** 541  260 5.4 30.8**  426** 420  260 5.3 13.6  347** 500  260 6.7 14.0  330** 538  260 9.3 17.3**  298** 457  260 2.9** 12.6  286** 472  260 8.3 13.8  278** 424  260 8.3 10.1  242 433  260 6.8 10.5  197 425  265 7.3 14.7  724** 468  265 3.8 16.7**  289** 435  265 7.4 14.0  150 499  265 2.2** 12.4  131 432  270 4.3 28.3**  432** 521  270 3.7** 15.3  349** 549  270 4.21 12.4  343** 518  270 10.0 10.1  276** 418  270 26.0 9.4  213 419  270 6.5 12.5  212 428  270 4.2 18.7**  189 443  270 8.8 12.0  187 446  270 11.0 8.1  157 461  275 7.6 15.1  663** 440  275 4.9 12.9  248 436  275 6.3 30.1**  233 530  275 7.4 13.6  231 438  275 4.6 8.5  221 527  275 7.5 10.5  219 444  275 4.0 12.2  180 429  280 5.3 15.3  463** 503  280 4.4 25.7**  421** 485  280 3.5** 15.6  381** 410  280 14.5 10.0  201 487  280 3.9 10.5  166 430  280 9.2 8.8  161 519  285 3.9 22.2**  919** 476  285 10.5 12.8  339** 509  285 5.4 13.0  331** 501  285 5.5 12.4  252 542  285 6.9 15.5  242 445  285 7.2 14.9  237 427  285 4.0 17.1**  233 490  290 4.7 13.9  203 451  290 2.1** 20.0**  226 414  290 7.0 9.7  117 467  290 4.1 6.5  68 463  295 5.8 12.3  296** 473  295 7.5 14.4  290** 505  295 4.1 12.4  257 198  300 11.5 10.9  323** 195  300 9.8 12.2  216 207  305 7.7 13.2  330**  67  305 8.6 15.4  312**  50  305 9.0 11.6  235  70  305 12.5 12.7  228 113  305 5.6 13.5  201  39  305 6.9 19.7**  170  3  305 4.2 11.5  135 325  305 14.5 9.4  94 368  310 4.7 15.9  371** 322  310 7.8 15.3  362** 295  310 7.2 13.8  305** 347  310 5.8 16.5**  266 313  310 6.1 16.5**  219 355  310 5.5 15.4  138 291  310 4.5 15.2  125 478  315 23.0 17.7**  857**  53  315 5.8 12.1  505** 240  315 6.7 12.3  394**  14  315 9.6 14.2  331** 137  315 7.8 24.3**  306** 254  315 8.7 17.0**  285** 109  315 3.7** 16.5**  263 252  315 5.2 10.1  241 186  315 4.1 15.4  238 183  315 5.5 10.7  195 390  315 6.9 10.0  188 267  315 2.2** 12.0  124 310  320 12.0 13.8  395**  31  320 17.0 12.9  334**  88  320 4.8 13.8  217 403  320 9.6 11.3  162  60  320 6.2 11.4  155 315  320 6.4 9.9  136 175  325 6.3 17.8**  486** 317  325 22.0 14.0  294**  18  325 6.3 11.1  241 247  325 13.5 13.2  231 223  325 9.2 12.6  203 132  325 3.7** 15.4  184 168  325 4.3 10.2  174 238  325 5.5 9.9  166 117  325 5.2 15.0  154 404  330 2.5** 33.1** 1085** 138  330 4.8 11.3  360** 316  330 3.6** 10.2  272**  61  330 5.1 12.5  242 333  330 34.0 9.2  235  16  330 4.6 13.3  211 276  330 5.7 11.9  200 391  330 4.1 8.4  184 362  330 9.2 11.7  178  1  330 9.9 8.9  170 379  335 16.0 12.1  471** 147  335 9.0 9.7  427**  89  335 8.0 15.3  385** 211  335 5.0 12.2  374**  45  335 5.9 16.3**  250  47  335 5.0 13.6  249 402  335 4.7 13.5  230 314  335 7.6 9.7  203 150  335 4.8 11.2  119 120  340 1.9** 21.0**  775** 284  340 7.2 25.6**  439** 230  340 14.0 11.4  419** 149  340 8.8 18.9**  337** 269  340 3.9 16.2  302** 197  340 10.5 12.8  233  19  340 9.6 11.0  232 422  340 3.1** 14.4  188 196  340 11.5 8.9  169  40  345 8.7 14.6  610** 244  345 8.6 15.8  461** 287  345 5.7 18.1**  427** 100  345 8.3 14.8  403** 383  345 4.3 27.2**  284**  62  345 19.5 9.6  250 350  345 8.0 10.0  249  65  345 8.0 10.2  247 307  345 16.5 11.6  208  69  345 17.0 9.9  197 328  345 7.5 8.9  192  43  345 6.0 13.2  191 222  345 6.1 9.2  175 306  345 4.3 17.2**  160 154  345 7.1 10.2  148  94  350 4.8 16.1  302** 201  350 6.1 9.9  200  13  350 5.1 10.9  193 236  355 7.2 14.8  309** 191  355 5.8 15.3  257 481  355 5.2 17.1**  134  92  360 4.2 25.2**  321** 324  360 3.8 16.6**  264  87  360 3.3** 13.3  200  46  360 5.4 11.1  179 289  360 9.5 7.9  129 392  360 5.1 10.3  125 320  365 6.4 17.3**  240 134  365 13.5 11.8  238 239  365 7.7 13.2  236 326  365 6.0 10.9  180 364  365 4.1 13.9  154 218  365 7.5 11.2  126 216  365 6.2 12.2  119 248  365 5.7 13.3  117 375  370 4.1 20.7**  532** 288  370 6.4 18.8**  436** 161  370 6.3 11.2  340** 244  370 19.5 9.8  286** 330  370 18.0 12.2  228 334  370 12.5 8.7  172 275  370 6.9 12.7  162  54  375 7.3 10.1  583** 185  375 9.3 10.5  386**  52  375 8.1 15.5  291** 366  375 5.0 12.5  280**  93  375 3.3** 16.2  248 151  375 2.9** 12.3  235  85  375 6.7 14.8  217 294  375 7.0 12.2  184 361  375 7.9 10.7  179 318  375 5.5 13.7  160 386  375 7.6 10.4  153 304  375 9.1 9.4  132 228  380 7.7 17.1**  320** 110  380 4.0 7.2  135 204  380 5.7 10.6  91 348  385 2.3** 17.4**  368** 146  385 11.5 12.5  253 260  385 5.5 13.7  211 136  385 3.6** 19.8**  205 338  385 5.0 16.2  180 376  385 3.6** 13.7  154 194  385 12.5 7.9  153 504  385 38.0 9.5  138 160  390 8.1 24.7**  475** 354  390 11.5 12.8  212  25  390 5.1 11.3  205 387  390 8.7 8.4  162  86  390 21.0 12.6  133 133  390 3.9 11.3  113 331  395 12.0 20.1**  638** 130  395 10.5 10.8  256  82  395 2.8** 9.8  236 119  395 12.5 16.3**  209 380  395 10.5 14.3  159 373  395 5.5 11.6  152 256  395 10.5 9.9  149 384  395 7.3 14.7  116 105  400 19.0 10.5  322** 251  400 4.8 14.9  289** 352  400 11.5 9.6  181 279  400 4.5 11.7  170 339  400 7.4 13.6  168 381  405 6.7 12.4  294** 285  405 7.0 14.2  281** 340  405 3.6** 19.6**  275**  51  405 6.5 14.3  233  33  405 6.5 9.6  207 268  405 3.3** 14.9  205  73  405 5.2 13.1  172  17  410 7.5 16.2  473** 286  410 4.7 18.8**  415** 140  410 5.9 21.7**  302** 116  410 6.8 14.5  218 396  410 5.6 16.1  190 356  410 1.9** 27.6**  149 237  410 3.6** 16.6**  122 112  410 5.5 8.9  107 259  410 4.7 11.6  99 176  415 5.2 21.9**  453** 193  415 10.5 11.3  163 323  415 6.1 9.6  163 202  415 11.5 9.4  150 398  415 8.0 12.6  134 321  420 5.2 10.7  383** 142  420 29.0 8.3  234 327  420 3.2** 14.6  203 342  420 7.3 9.4  156 170  420 20.5 10.3  142 345  420 29.5 13.2  136 302  420 8.6 8.8  128 115  425 6.3 22.2**  628**  97  425 12.5 19.8**  313** 246  425 8.7 15.1  241  72  425 10.5 13.5  241 365  425 6.7 16.7**  237 139  425 12.5 10.4  224 143  425 8.1 13.5  216 426  425 19.5 14.5  201 303  425 3.0** 14.5  154 388  425 6.2 12.3  135 127  425 6.7 8.4  100 262  430 10.0 12.1  323** 270  430 4.8 12.9  293** 514  430 4.3 12.9  197 341  430 3.5** 19.9**  190 278  430 5.2 10.8  182 370  430 11.0 15.3  174  55  430 7.6 11.0  162 274  430 5.0 8.2  131 367  430 17.5 8.0  126  98  430 13.5 12.8  125 337  435 13.5 14.1  395** 309  435 8.7 12.9  349** 305  435 17.5 15.4  187 144  435 25.0 8.9  167  34  435 8.6 7.6  157 234  435 9.7 9.2  116 123  440 9.6 12.2  622** 200  440 4.8 12.4  257 250  440 7.5 12.9  248 107  440 6.3 14.7  183 300  440 6.5 7.9  123 374  445 5.4 14.0  247 372  445 11.0 11.0  181  36  445 4.0 10.0  181 271  445 7.2 10.4  124 242  445 15.5 9.6  112 264  445 6.0 10.7  100 172  450 11.5 14.9  607**  32  450 11.5 13.6  362** 346  450 13.5 15.8  330**  41  450 8.5 11.4  194  95  450 5.1 12.5  182 357  455 6.3 14.4  296** 319  455 17.0 10.2  147 308  455 15.0 9.8  131 235  455 23.0 9.0  114 349  455 9.2 8.3  82 178  460 5.6 20.6**  473** 312  460 4.7 14.4  197  79  460 5.0 10.4  173 131  460 18.0 10.2  162 243  460 2.6** 11.6  160 261  465 7.7 10.6  252 378  465 5.4 13.2  221  49  465 47.0 10.8  179 226  465 7.7 10.2  173 377  465 5.6 8.5  143 253  465 10.0 7.0  138  76  470 12.5 14.8  304** 203  470 15.0 7.6  233 296  470 23.5 11.0  161 382  470 5.3 11.1  109  6  475 10.5 12.5  232  75  475 4.5 8.1  150 332  475 9.4 10.0  144 290  475 14.0 9.1  143 128  475 5.9 9.3  133 124  475 6.0 13.5  111 177  475 8.8 9.1  106 126  480 11.0 11.0  212 283  480 5.2 10.6  175 209  480 10.5 10.5  175 293  480 6.8 15.5  135 121  485 4.7 20.0**  345** 282  485 12.0 10.9  236  71  485 13.5 8.1  168 385  485 9.0 14.1  128 190  495 9.9 10.4  410** 210  495 8.6 12.0  243 155  495 5.9 10.4  219 336  495 13.5 9.9  135 280  500 8.7 14.5  334**  96  500 4.7 10.8  237 145  500 5.9 17.5**  233 199  500 4.2 13.8  199 489  500 11.5 9.7  198 217  500 6.4 9.6  166  90  500 7.5 8.5  106 164  510 5.2 23.8**  408** 343  510 4.5 13.7  284**  42  510 4.9 7.4  233 351  510 8.5 11.0  207 299  510 12.0 8.0  104  99  520 10.5 25.8**  322** 114  520 30.0 10.9  220 369  520 29.0 16.7**  206  37  520 10.5 8.6  191 215  520 6.7 16.8**  151 401  520 7.5 12.6  148 229  520 7.9 11.0  116 135  520 3.2** 8.3  88  81  530 6.8 14.8  372**  91  530 14.5 10.6  228 167  530 23.5 9.2  176 181  530 5.5 9.3  171  56  530 20.0 8.3  163  5  530 13.5 8.1  159 180  540 12.0 9.0  216 311  540 4.1 13.3  214 389  540 3.9 13.9  169 125  540 5.5 13.0  159  35  540 22.5 11.0  123 104  550 10.5 16.5**  544** 393  550 4.9 11.9  339** 394  550 23.0 14.0  278** 292  550 6.9 16.2  263 163  550 6.7 14.3  219  66  550 10.5 11.6  206  29  550 17.5 9.6  191 227  550 7.9 11.7  154  38  550 7.5 11.9  152 241  550 10.5 9.8  100 102  550 9.7 8.6  91  77  560 24.0 14.8  554** 162  560 10.5 11.8  275** 273  560 8.7 9.4  180  80  560 6.3 11.2  108 255  560 8.8 9.9  93 122  570 66.0 13.8  304** 208  570 34.0 10.2  255  23  570 21.5 8.3  241 447  570 25.0 10.0  164 225  570 5.7 12.2  154 174  570 7.1 11.0  127  11  570 19.0 8.9  113 165  580 10.5 14.8  226 182  580 8.9 8.2  189 245  590 15.5 10.0  262  83  590 17.5 8.3  199 166  590 11.5 9.4  188 158  590 7.3 10.7  166 187  590 4.5 11.0  146 156  590 23.5 11.3  112 231  600 9.5 9.0  192  78  600 11.5 9.4  151 329  610 15.0 7.3  312**  57  610 16.0 11.9  286**  7  610 12.0 10.4  195 277  610 9.5 7.8  153 108  620 13.5 8.4  191 205  620 18.0 7.5  145 263  620 9.8 10.2  101  9  630 4.9 11.4  300** 111  630 8.3 11.1  276**  68  630 11.5 8.9  143 399  630 14.0 11.0  90 266  640 5.1 15.7  364**  12  640 24.5 9.0  233 152  640 8.1 10.0  209 405  640 7.0 12.8  186  27  640 22.5 8.4  136 258  640 8.3 11.2  120 249  640 8.7 9.1  81 297  650 16.0 10.0  279** 192  650 4.9 14.9  213 257  650 3.3** 16.3**  208 184  650 12.5 9.9  193  58  650 18.5 10.7  172 301  650 16.0 15.5  162 397  650 12.5 8.4  146 272  650 11.0 7.4  120 153  650 7.1 13.1  116 406  650 6.6 5.8  81  10  660 9.0 7.6  154  26  660 22.0 8.3  132 265  670 3.9 19.3**  509** 359  670 21.0 8.3  269  48  670 32.0 9.9  262 335  670 11.5 8.1  121 189  680 6.6 17.9**  358** 220  680 15.5 10.9  115  15  690 13.5 13.4  159  44  700 20.0 12.7  244  21  700 13.5 10.2  129  74  700 15.0 7.1  65  4  710 29.0 8.5  266 353  710 11.5 11.4  206 281  710 10.5 9.6  185  2  710 8.0 8.5  109 212  740 20.0 11.1  250  8  740 12.0 11.5  216 206  750 12.5 8.3  116 101  770 14.5 12.7  372** 344  770 32.0 11.7  297**  20  770 35.0 10.1  245 407  770 10.5 12.0  110 360  780 2.7** 20.9**  157 232  790 15.5 10.1  151 141  790 12.5 9.5  74 129  800 8.7 11.7  211 188  800 15.0 12.3  174 400  800 12.5 10.3  156  24  810 23.0 7.5  194 173  830 35.0 11.4  243 214  830 21.5 12.0  187  63  830 13.8 8.8  185 148  830 45.0 7.1  146  84  830 23.5 7.0  136 179  830 16.5 6.6  96 171  840 23.5 11.2  195  28  870 5.8 15.9  197 233  870 7.9 12.7  169 221  870 40.0 7.0  126 371  880 20.0 8.5  152 213  890 10.5 18.0**  231 358  900 21.0 8.3  149 298  910 15.5 10.2  221 118  910 100.0 9.7  170 479  950 11.5 12.1  188  30  950 6.2 10.5  170 159 1000 9.5 8.7  281** 219 1050 37.0 14.3  313** 103 1050 12.5 10.3  154  59 1150 17.5 7.3  180 157 1250 12.0 14.0  206 363 1350 28.0 10.4  190  22 1400 13.5 10.4  233  64 1400 31.0 9.7  149 169 1450 15.0 9.5  150

Claims

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.