Method for treatment and prevention of deficiencies of vitamins B.sub.12, folic acid, and B.sub.6

FIELD OF THE INVENTION
This invention relates to the field of nutrition. Specifically, the invention is comprised of new oral vitamin preparations combining vitamin B.sub.12 (B.sub.12, cobalamin) and folic acid (folate), and vitamin B.sub.12, folate, and pyridoxine (B.sub.6) 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 B.sub.12 and/or folate and/or B.sub.6, 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 B.sub.12 and 0.1-0.4 mg folate, with the preferred embodiment using 2.0 mg B.sub.12 and 0.4 mg folate. Another embodiment of the non-prescription formulation uses 0.3-10 mg B.sub.12, 0.1-0.4 mg folate, and 5-75 mg B.sub.6, with the preferred embodiment using 2.0 mg B.sub.12, 0.4 mg folate, and 25 mg B.sub.6. Another embodiment of the present invention uses a prescription strength formulation comprising between 0.3-10.0 mg B.sub.12 and 0.4-1.0 mg folate, with the preferred embodiment using 2 mg B.sub.12 and 1.0 mg folate. In a further embodiment of the present invention, a prescription strength formulation is used comprising 0.3-10 mg B.sub.12, 0.4-1.0 mg folate, and 5-75 mg B.sub.6, with the preferred embodiment using 2 mg B.sub.12, 1.0 mg folate, and 25 mg B.sub.6. 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 B.sub.12, folate, and B.sub.6, 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 B.sub.12, folate, and B.sub.6 are required cofactors in metabolic pathways involving methionine, homocysteine, cystathionine, and cysteine. B.sub.12 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 B.sub.12 in the form of methylcobalamin. HC is condensed with serine to form cystathionine (CT) in a reaction catalyzed by cystathionine .beta.-synthase which requires B.sub.6 (pyridoxal phosphate). CT is hydrolyzed in another B.sub.6 -dependent reaction to cysteine and .alpha.-ketobutyrate.
It is important to diagnose and treat B.sub.12, folate, and B.sub.6 deficiencies because these deficiencies can lead to life-threatening hematologic abnormalities which are completely reversible by proper treatment. B.sub.12 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 B.sub.12 deficiency include significant anemia, displayed for example in decreased hematocrit (e.g., <25%) or hemoglobin (e.g., .ltoreq.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 B.sub.12 -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 B.sub.12 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 B.sub.12 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 B.sub.12 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 B.sub.12 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 B.sub.12 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 B.sub.12 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.
B.sub.6 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 .gamma.-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 confusions, 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 B.sub.12 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 B.sub.12 levels are a relatively insensitive determinant of B.sub.12 deficiency in that only 50% of patients with clinically confirmed B.sub.12 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 B.sub.12 levels (Allen (1991) supra), with no evidence of B.sub.12 deficiency and are unlikely to benefit from B.sub.12 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 B.sub.12 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., supra).
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 B.sub.12 and folate, with elevated HC levels seen with both B.sub.12 and folate deficiencies, and elevated MMA levels seen with a B.sub.12 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 (1989() 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 B.sub.12 deficiency (Allen et al. (1991) in Proceedings of the 1st 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 B.sub.6, 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 .beta.-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 hyperhomocysteinaemia 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 .beta.-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 .beta.-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 .beta.-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 B.sub.12 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; Garry et al. (1984) J. Am. Geriatr. Soc. 32:71926; Hanger et al. (1991) J. Am. Geriatr. Soc. 39:1155-1159), and B.sub.6 (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 B.sub.12, folate, and B.sub.6. The hematologic abnormalities seen with B.sub.12 deficiency are indistinguishable from those seen with folate deficiency. Similarly to a B.sub.12 deficiency, B.sub.6 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 B.sub.12 deficiency will respond to treatment with folate alone. However, the neuropsychiatric abnormalities caused by the B.sub.12 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 B.sub.12, folate, and B.sub.6, 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 B.sub.12, folate and B.sub.6, 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 B.sub.12, folate, and B.sub.6.
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 B.sub.12. However, contrary to the present invention, Hathcock and Troendle teach away from combining B.sub.12 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 B.sub.12 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 B.sub.12 deficient patient, in that the 0.5 mg folate may correct the hematologic abnormalities present, but the 0.5 mg B.sub.12 dose may be insufficient to correct a B.sub.12 deficiency due to inadequate intrinsic factor. By contrast, the formulation of the present invention teaches the use of the combination of B.sub.12 and folate, and of B.sub.12, folate and B.sub.6, sufficient to treat either single or multiple deficiencies of B.sub.12, folate, and B.sub.6. 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.sub.12 and/or folate and/or to B.sub.6 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 B.sub.12 and folate, and vitamin B.sub.12, folate and B.sub.6, 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 B.sub.12, folate, and B.sub.6 or for neuropsychiatric, vascular, renal, or hematologic diseases.
One embodiment of the present invention is uses an over-the-counter formulation comprised of between 0.3-10 mg CN-cobalamin (B.sub.12) and 0.1-0.4 mg folate. Another embodiment of the non-prescription formulation uses 0.3-10 mg B.sub.12, 0.1-0.4 folate, and 5-75 mg B.sub.6. Preferred embodiments of the over-the-counter formulation are comprised of about 2.0 mg B.sub.12 and 0.4 mg folate, and 2.0 mg B.sub.12, 0.4 mg folate, and 25 mg B.sub.6, respectively.
Another embodiment of the present invention uses a prescription formulation comprised of between 0.3-10 mg CN-cobalamin (B.sub.12) and 0.4-10.0 mg folate. Another embodiment of the prescription formulation of the present invention uses 0.3-10 mg B.sub.12, 0.4-1.0 mg folate, and 5-75 mg B.sub.6. Preferred embodiments of the prescription formulation use about 2.0 mg B.sub.12 and 1.0 mg folate, and 2.0 mg B.sub.12, 1.0 mg folate, and 25 mg B.sub.6, respectively.

BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows the distribution of serum B.sub.12 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 B.sub.12 values <300 pg/ml (ages 65-99, n=38/152) and a normal population with serum B.sub.12 values <300 pg/ml (ages 17-65, n=10/100).
FIG. 3 shows serum HC levels for a population of elderly outpatients with serum B.sub.12 values <300 pg/ml (ages 65-99, n=38/152) and a normal population with serum B.sub.12 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 B.sub.12 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 B.sub.12 values of <300 pg/ml (ages 65-99, n=10/38).
FIG. 6 shows the distribution of serum B.sub.12 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 B.sub.12 values <300 pg/ml (ages 55-107, n=29/212) and a normal population with serum B.sub.12 values (ages 17-65, n=10/100).
FIG. 8 shows serum HC levels for a population of elderly nursing home patients with serum B.sub.12 values <300 pg/ml (ages 55-107, n=29/212) and a normal population with serum B.sub.12 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 B.sub.12 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 B.sub.12 values <300 pg/ml (ages 55-107, n=14/29).
FIG. 11 shows the distribution of serum B.sub.12 levels for a population of elderly patients (ages 65-99, n=548) and a normal population (ages 22-63, n=117) (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 B.sub.12 (B.sub.12, cobalamin) and folic acid (folate), and vitamin B.sub.12, folate and pyridoxine (B.sub.6). The formulations of the present invention are for use in the treatment of elevated serum levels of one or more 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 metabolites 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 (B.sub.12) 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 B.sub.12, 0.1-0.4 mg folate, and 5-75 mg B.sub.6. Preferred embodiments of the non-prescription formulation are comprised of about 2.0 mg B.sub.12 and 0.4 mg folate, and 2.0 mg B.sub.12, 0.4 mg folate, and 25 mg B.sub.6, respectively.
Another embodiment of the present invention is comprised of a prescription formulation comprised of between about 0.3-10 mg B.sub.12 and 0.4-10.0 mg folate, with the preferred embodiment comprised of about 2.0 mg B.sub.12 and 1.0 mg folate. Another embodiment of the prescription strength formulation is comprised of about 0.3-10 mg B.sub.12, 0.4-10.0 mg folate, and 5-75 mg B.sub.6, with a preferred embodiment comprised of about 2.0 mg B.sub.12, 1.0 mg folate, and 25 mg B.sub.6.
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 B.sub.12, folate, and B.sub.6 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 B.sub.12 and folate, with or without B.sub.6, is preferred over intramuscular injections for patient convenience and ease of administration.
For example, the inclusion of B.sub.12 will be useful as a safeguard for patients misdiagnosed as folate deficient, even though they are actually B.sub.12 deficient, since treatment with folate alone in such patients is extremely dangerous. The danger arises from the fact that treating a B.sub.12 deficient patient with folate alone may reverse or prevent the hematologic abnormalities seen in B.sub.12 deficiency, but will not correct the neuropsychiatric abnormalities of a B.sub.12 deficiency and may actually precipitate them. Even in the absence of intrinsic factor, approximately 1% of a 2.0 mg oral dose of B.sub.12 is absorbed by diffusion. Thus, approximately 20 ug of B.sub.12 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 B.sub.12. The inclusion of folate will be of benefit since B.sub.12 deficiency causes a secondary intracellular deficiency of folate. The inclusion of folate and B.sub.6 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 B.sub.12 deficiency and response of serum MMA and HC to B.sub.12 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 B.sub.12 and folate were measured by a quantitative radioassay method using purified intrinsic factor and purified folate binding protein. Vitamin B.sub.6 was measured by a radioenzymatic assay method wherein serum is incubated with apoenzyme tyrosine-decarboxylase, C.sub.14 labelled tyrosine is added to start the enzymatic reaction which is stopped with HCl. Subsequently the free C.sub.14 -labelled CO.sub.2 is adsorbed by a KOH impregnated filtering paper. The measured C.sub.14 activity is directly proportional to the B.sub.6 (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 .alpha.) and the Kruskall Wallis test. From the result 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 B.sub.12, folate, and B.sub.6 concentrations was calculated as (p.+-.2 p (1-p)/n.times.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 B.sub.12, folate, and B.sub.6 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 B.sub.12 and folate were measured as described in Example 1 with a vitamin B.sub.12 /folate dual RIA kit (CT301/CT302 Amersham Buchier, UK). Vitamin B.sub.6 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 .mu.mol/L. Creatine 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 VITAMIN AND HIGHMETABOLITE LEVELS IN GERIATRIC POPU-LATIONS A-D AND A YOUNGER HEALTHYPOPULATION E. FolicGroup B.sub.12 Acid B.sub.6 MMA 2-MCA HC CT______________________________________A 6% 12% 48% 36% 44% 55% 64%B 6% 19% 53% 47% 39% 59% 66%C 3% 10% 50% 32% 45% 39% 73%D 6% 6% 17% 26% 23% 38% 41%E 2% 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 B.sub.12, 1.1 mg folate, and 5 mg B.sub.6, 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 B.sub.12, folate, and B.sub.6 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 B.sub.12, folate and B.sub.6. 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 B.sub.12, folate, and B.sub.6, 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 B.sub.12, 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 B.sub.12 deficiency in geriatric outpatients as determined by both low serum B.sub.12 levels and elevations of MMA and HC, and to determine the response to B.sub.12 treatment. Blood samples were obtained on 152 consecutive geriatric outpatients, ages 65-99. Control values were determined from 100 subjects, ages 17-65. Serum B.sub.12, 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: B.sub.12 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 B.sub.12 and folate were measured as described in Example 1 using a Corning Immophase kit (CIBA-Corning, Medfield, Mass.) with the normal range defined as 200-800 pg/ml for B.sub.12 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 B.sub.12 level .ltoreq.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 B.sub.12 levels. More than half of the subjects with low or low-normal serum B.sub.12 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 B.sub.12 levels were treated with weekly injections of 1.0 mg B.sub.12. Parenteral B.sub.12 administration caused elevated metabolite levels to fall to or towards normal (FIGS. 4 and 5) in every subject treated with B.sub.12. It appears that the true prevalence of previously unrecognized B.sub.12 deficiency in this elderly populations was at least 14.5%.
It can be seen from the data presented in Table 2 that serum B.sub.12 levels are insensitive for screening B.sub.12 deficiencies since similar numbers of patients with low normal serum B.sub.12 levels of 201-300 pg/ml compared with patients with low B.sub.12 levels (.ltoreq.200 pg/ml) had markedly elevated metabolites which fell with B.sub.12 treatment. Further, this study shows that elderly patients have a high incidence (at least 14.5%) of unrecognized B.sub.12 deficiency, detectable by measurement of serum HC and MMA levels in patients with serum B.sub.12 levels <300 pg/ml.
A further finding in this study emphasizes the need to treat elevated metabolite levels with a combination of vitamin B.sub.12 and folate with or without B.sub.6. Some of the patients exhibiting elevated metabolite levels did not fully respond to B.sub.12 treatment. This may indicate a concomitant deficiency of folate and/or B.sub.6. The lack of response to B.sub.12 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 B.sub.12 .ltoreq.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 B.sub.12 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 B.sub.12 and folate levels are normal. B.sub.6 levels were not performed in this study, but B.sub.6 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 B.sub.12 and folate, or a combination of vitamin B.sub.12, folate and vitamin B.sub.6. 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 B.sub.12, Folate, MMA, and HC Levels in a Geriatric Nursing Home Population
A study was conducted with 212 elderly nursing home patients to determine serum B.sub.12, 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 B.sub.12 levels (FIG. 6), elevated serum MMA (FIG. 7) and HC (FIG. 8) levels. Parenteral administration of B.sub.12 1 mg per week for 8 weeks to those with serum B.sub.12 <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 B.sub.12 and folate, with or without B.sub.6. Some of the patients exhibiting elevated metabolite levels did not fully respond to B.sub.12 treatment. This may indicate a concomitant deficiency of folate and/or B.sub.6. The lack of response to B.sub.12 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 B.sub.12 >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 B.sub.12 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 B.sub.12 and folate levels are normal. B.sub.6 levels were not performed in this study, but B.sub.6 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 B.sub.12 and folate, or a combination of vitamin B.sub.12, folate and vitamin B.sub.6. 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 B.sub.12, Folate, MMA, 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 B.sub.12, 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 B.sub.12 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 B.sub.12 >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 B.sub.12 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 B.sub.12 and folate levels are normal. B.sub.6 levels were not performed in this study, but B.sub.6 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 B.sub.12 and folate, or a combination of vitamin B.sub.12, folate and vitamin B.sub.6. 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 AGERIATRIC OUTPATIENT POPULATION. Homo- TotalPatient B12 Folate cysteine 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 196067 163** 6.6 9.9 93 164 69091 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 223037 187** 9.4 12.3 160 218 334**100 187** 9.5 13.6 208 453** 141036 188** 9.9 16.3** 298** 385** 322**109 189** 7.6 12.3 127 188 161007 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** 121108 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** 121005 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** 203023 239 4.1 21.9** 385** 775** 279**010 256 12.9 11.5 652** 119 144055 258 6.8 7.5 189 342 185102 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** 153038 269 7.6 15.7 222 152 152140 277 4.0 29.1** 744** 602** 254**074 278 5.2 24.1** 699** 296 187002 278 14.6 14.8 554** 259 277**019 282 8.5 12.4 329** 262 161035 287 5.8 9.8 230 390** 218049 290 3.9 33.0** 140 275 138078 290 10.9 12.5 197 240 209045 291 8.7 9.5 162 613** 132092 294 14.9 19.3** 500** 246 167137 297 6.8 10.1 631** 340 184072 298 6.7 19.7** 375** 302 246**149 310 8.3 16.1 314** 199 149047 312 4.9 15.9 277** 271 173060 312 9.4 8.0 100 228 203046 314 6.5 16.2 142 336 125093 318 6.4 16.5** 304** 361** 130014 321 14.5 10.7 275** 233 170088 327 7.1 17.8** 263 507** 258**032 340 6.6 8.6 150 133 133147 347 7.6 18.2** 305** 219 265**001 351 4.7 20.8** 199 402** 223090 353 4.9 20.7** 144 419** 178008 358 5.4 11.6 372** 529** 177104 360 12.7 12.1 260 89 77110 370 3.0** 17.1** 456** 297 150103 371 18.7 14.5 257 219 180056 373 6.5 12.4 236 415** 189048 374 3.6** 9.7 167 237 230**131 377 10.9 13.6 256 220 85122 378 7.6 21.9** 906** 227 196004 385 8.6 10.3 109 288 92120 390 9.8 22.9** 499** 529** 260**138 405 6.9 14.7 334** 238 188141 407 8.1 14.3 168 259 263**101 408 5.9 9.2 160 134 40145 410 3.7** 25.4** 567** 550** 349**027 415 11.1 10.6 169 278 164028 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 170057 425 18.3 13.5 209 381** 321**021 427 18.9 12.1 223 524** 168135 430 8.8 13.5 284** 412** 180097 435 15.4 10.9 353** 465** 119052 438 6.8 5.2 281** 372** 238**132 448 12.6 16.8** 1931** 394** 250**086 451 12.1 6.6 139 208 107148 458 13.9 11.4 187 322 238**012 466 15.3 8.3 560** 250 144083 466 12.0 13.7 366** 214 193133 470 13.8 10.8 290** 275 55017 475 4.0 39.6** 196 467** 220053 476 13.4 12.3 226 206 125009 482 6.5 25.3** 240 470** 214066 498 9.6 12.9 374** 233 92031 507 11.0 14.8 173 278 220099 507 10.4 9.6 124 233 63128 507 4.6 9.4 294** 324 176013 514 11.3 15.9 163151 522 7.8 14.3 370** 324 215077 523 6.8 17.7** 184 210 214079 523 15.6 13.0 316** 223 251**054 524 4.9 10.0 148 230 123020 524 9.9 14.2 235 366** 190069 528 7.0 9.7 257 281 83085 536 4.0 22.5** 97 191 114084 551 14.2 12.5 166 179 131082 559 12.3 14.6 208 371** 182117 560 3.4** 18.8** 102 176 88061 561 12.7 9.8 170 404** 152006 567 4.6 16.8** 138 688** 165129 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 95089 578 10.3 12.0 147 258 236**143 581 2.6** 42.7** 165 555** 208114 583 5.1 16.6** 599** 660** 177080 593 9.5 18.0** 208 289 142015 598 7.0 12.4 167 381** 95039 598 9.6 18.1** 691** 719** 354**070 612 5.6 13.7 197 296 82051 622 12.9 8.3 119 246 150139 628 8.5 7.8 145 166 83150 628 8.6 14.5 295** 315 183043 635 5.9 13.7 239 272 189096 651 17.4 9.7 326**073 657 7.0 9.5 186 283 78127 665 5.8 8.1 166 344** 147121 677 10.2 9.5 226 346** 173034 694 15.9 12.1 406** 592** 584**124 697 9.7 11.0 63 179 60123 702 10.4 10.6 186 148 96113 705 7.6 8.4 107 534** 92071 709 10.6 11.3 207 584** 141076 722 8.1 10.5 271 489** 138044 724 7.3 12.1 212 683** 217040 731 15.1 7.4 205 149 136062 741 4.4 18.7** 153 856** 416**025 741 10.0 12.2 224 344** 121119 755 5.9 10.1 187 377** 61075 757 10.0 24.7** 246 345** 276**098 759 13.8 13.9 380** 239 156134 769 7.5 10.4 125 131 81087 773 25.0 10.1 181 285 135142 788 4.6 12.1 166 273 129064 792 15.4 8.6 218 299 139094 793 16.6 10.0 186 179 173022 808 8.8 14.4 184 271 161112 812 12.0 9.2 181 184 108125 817 14.4 11.0 158 242 72106 862 5.3 9.2 94 300 95146 890 13.9 11.9 135058 897 5.3 18.5** 154 460** 80063 943 17.8 19.7** 277** 642** 306**095 960 25.3 10.7 135 181 111152 963 9.4 8.8 198130 971 15.9 13.5 106 307 84059 1063 9.4 9.7 129 378** 54105 1109 11.0 6.1 87 155 64136 1163 6.0 13.1 250 565** 122065 1251 14.5 10.7 88 147 88029 1490 22.2 9.7 129 111 105144 1536 7.0 17.7** 216 694** 418**068 1809 12.7 10.4 59 128 39______________________________________
TABLE 3______________________________________SERUM METABOLITE & VITAMIN LEVELS IN AGERIATRIC 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 223NH178 172** 8.2 5.9 141NH103 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 166NH075 250 9.3 12.1 170NH172 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 98NH099 272 5.5 12.5 125NH124 275 6.9 11.5 87NH179 301 7.6 7.1 143NH135 302 6.5 23.4** 397**NH087 304 7.8 10.8 327**NH180 304 5.8 10.5 237NH209 306 7.6 11.9 105NH107 310 3.3** 8.6 148NH081 320 4.3 23.6** 470**NH068 324 7.9 13.4 243NH183 325 7.7 11.1 144NH033 330 13.8 7.7 149NH161 333 8.5 11.3 385**NH192 337 10.7 9.5 209NH136 340 6.7 18.2** 409**NH191 342 20.2 13.4 271NH137 343 4.0 15.6 183NH182 346 8.2 14.4 448**NH020 347 8.4 10.4 149NH165 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** 71NH009 368 5.1 15.9 325**NH071 382 4.9 12.9 330**NH080 390 6.1 15.0 171NH013 407 6.7 12.4 310**NH126 409 9.2 17.4** 137NH030 411 11.2 10.4 844**NH210 413 8.6 11.9 210NH158 414 5.7 16.2 508**NH027 416 10.2 15.5 769**NH003 424 16.5 9.5 167NH187 429 4.7 8.8 439**NH022 430 10.5 14.0 214NH082 436 10.6 17.7** 340**NH162 438 6.1 19.2** 180NH021 439 5.3 15.1 191NH056 447 11.7 10.9 184NH119 448 3.2** 14.1 241NH120 448 5.6 12.0 138NH186 450 4.7 23.1** 213NH064 451 6.9 10.6 237NH057 453 14.6 10.4 282**NH131 454 8.1 16.2 258NH059 462 6.0 9.1 147NH202 465 3.3** 17.0** 393**NH134 475 15.3 11.6 321**NH083 475 7.4 10.6 178NH199 479 15.1 10.4 141NH042 482 6.0 15.0 141NH200 491 13.6 9.8 154NH213 497 8.1 10.0 92NH143 500 5.2 22.1** 175NH031 502 6.4 16.1 151NH188 504 12.5 15.1 1461**NH171 504 10.7 12.9 344**NH008 505 4.6 9.9 185NH102 506 16.6 9.1 236NH145 512 7.7 22.2** 161NH093 514 5.1 17.7** 185NH118 524 25.0 10.1 314**NH185 524 8.7 12.1 84NH111 527 5.1 18.4** 250NH149 530 12.6 18.2** 531**NH011 534 8.1 12.5 654**NH128 540 4.3 11.6 120NH035 547 7.5 9.8 193NH005 551 17.7 5.0 365**NH212 552 11.9 12.1 202NH007 554 6.4 26.1** 646**NH086 554 9.5 5.1 127NH069 555 22.7 6.8 134NH121 555 8.2 10.0 112NH117 571 6.6 9.7 351**NH055 581 14.8 9.1 265NH025 581 5.2 15.3 181NH104 583 3.9 14.6 1699**NH173 583 11.2 10.6 160NH177 584 6.2 5.7 111NH207 586 8.5 16.4** 243NH070 591 5.4 12.0 168NH038 592 8.0 8.8 230NH049 599 10.7 21.7** 238NH062 606 4.5 7.7 96NH153 608 7.7 13.6 221NH206 611 6.6 16.4** 400**NH018 614 6.3 10.9 123NH163 616 5.0 9.6 132NH189 619 7.6 12.0 158NH045 620 21.0 12.4 265NH074 621 10.2 9.2 172NH054 623 8.0 9.8 121NH152 625 8.2 7.8 206NH140 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 68NH097 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 228NH051 659 4.0 10.7 216NH108 671 5.8 24.0** 823**NH058 673 6.0 11.2 392**NH028 675 22.3 9.1 105NH204 678 4.7 10.2 148NH169 679 6.9 19.2** 267NH032 681 12.7 5.9 99NH065 682 11.0 13.5 176NH061 683 13.4 9.6 190NH116 685 9.0 7.5 244NH015 699 6.8 16.8** 236NH157 711 10.0 12.8 198NH155 715 10.0 17.6** 308**NH034 715 7.9 11.4 179NH040 717 10.5 15.7 256NH105 718 6.0 13.2 308**NH048 719 8.0 10.8 207NH084 720 6.8 9.4 169NH115 724 16.3 9.4 161NH205 734 8.5 13.3 232NH113 738 11.7 10.3 171NH154 738 13.7 9.6 123NH167 741 17.0 6.6 129NH190 752 5.2 14.1 254NH067 760 22.5 9.5 232NH014 767 8.9 7.3 100NH072 768 8.3 6.9 131NH133 772 8.8 20.4** 219NH122 778 6.0 10.4 108NH076 781 12.1 14.9 282**NH147 785 7.5 24.5** 411**NH026 786 9.7 8.3 146NH151 789 24.4 11.1 182NH198 797 10.9 10.7 158NH088 801 6.4 18.3** 184NH004 806 11.3 8.8 96NH024 818 5.1 14.1 219NH100 826 16.4 10.5 103NH078 831 7.2 10.3 266NH052 844 19.6 8.0 193NH142 848 18.6 12.1 398**NH002 862 9.4 11.3 212NH091 891 4.9 12.6 169NH127 897 22.0 8.4 132NH096 901 9.3 5.2 104NH201 910 25.0 15.7 424**NH184 941 21.5 10.8 170NH208 945 20.2 9.8 111NH130 968 22.4 10.4 339**NH164 989 8.0 16.8** 102NH077 1006 15.1 9.2 188NH017 1015 11.9 9.5 175NH029 1053 18.6 11.4 161NH023 1055 9.3 9.7 193NH047 1079 6.4 11.4 106NH043 1082 14.5 13.9 144NH195 1088 36.9 12.2 150NH193 1092 8.2 15.7 225NH046 1093 9.2 18.8** 186NH101 1108 3.9 8.1 139NH098 1117 11.3 12.5 88NH168 1124 25.2 15.0 203NH006 1126 6.9 8.1 159NH144 1135 8.0 21.9** 262NH044 1159 26.8 10.2 109NH175 1162 7.8 12.0 210NH146 1179 9.8 10.1 129NH112 1238 10.3 15.0 347**NH001 1304 13.1 6.9 142NH166 1337 13.4 8.3 67NH079 1346 18.0 12.0 248NH041 1528 20.7 8.2 155NH063 1559 15.0 7.0 66NH159 1566 6.6 15.5 451**NH125 1703 8.2 20.6** 153NH094 1768 15.9 8.4 182NH123 2028 10.2 16.8** 206NH174 2106 13.3 12.8 280**NH039 2227 23.8 8.9 119NH019 2297 11.1 15.5 177NH092 2360 5.7 9.8 131NH085 3141 22.0 26.9** 1947**______________________________________
TABLE 4______________________________________SERUM METABOLITE AND VITAMIN LEVELS IN AGERIATRIC POPULATION.Patient B.sub.12 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** 170493 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** 164470 165** 9.2 19.9** 1468**460 165** 6.8 11.5 142437 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 206450 180** 5.0 11.8 196477 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** 207482 190** 2.9** 25.1** 179459 190** 5.3 19.6** 167543 195** 4.3 13.5 470**520 195** 1.7** 22.2** 309**431 195** 7.2 13.5 251513 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** 171471 205 5.7 22.0** 542**413 205 2.6** 20.4** 304**497 205 3.3** 19.4** 258539 205 4.1 15.4 247544 205 12.5 11.7 233540 205 4.0 17.1** 185517 205 2.2** 15.0 151496 210 3.7** 15.2 1103**488 210 16.5 21.8** 600**416 215 12.5 10.0 197434 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 179458 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** 255533 230 5.7 11.7 151466 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 190452 240 4.7 26.5** 1068**454 240 5.2 11.9 201535 240 4.4 15.3 195421 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 156536 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 252417 250 5.5 25.2** 241524 250 2.5** 14.4 212411 250 9.9 11.5 200492 250 5.2 10.7 182548 250 2.9** 12.4 179441 250 4.5 8.5 147480 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 203546 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 242433 260 6.8 10.5 197425 265 7.3 14.7 724**468 265 3.8 16.7** 289**435 265 7.4 14.0 150499 265 2.2** 12.4 131432 270 4.3 28.3** 432**521 270 3.7** 15.3 349**549 270 4.1 12.4 343**518 270 10.0 10.1 276**418 270 26.0 9.4 213419 270 6.5 12.5 212428 270 4.2 18.7** 189443 270 8.8 12.0 187446 270 11.0 8.1 157461 275 7.6 15.1 663**440 275 4.9 12.9 248436 275 6.3 30.1** 233530 275 7.4 13.6 231438 275 4.6 8.5 221527 275 7.5 10.5 219444 275 4.0 12.2 180429 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 201487 280 3.9 10.5 166430 280 9.2 8.8 161519 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 252542 285 6.9 15.5 242445 285 7.2 14.9 237427 285 4.0 17.1** 233490 290 4.7 13.9 230451 290 2.1** 20.0** 226414 290 7.0 9.7 117467 290 4.1 6.5 68463 295 5.8 12.3 296**473 295 7.5 14.4 290**505 295 4.1 12.4 257198 300 11.5 10.9 323**195 300 9.8 12.2 216207 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 228113 305 5.6 13.5 201 39 305 6.9 19.7** 170 3 305 4.2 11.5 135325 305 14.5 9.4 94368 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** 266313 310 6.1 16.5** 219355 310 5.5 15.4 138291 310 4.5 15.2 125478 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** 263252 315 5.2 10.1 241186 315 4.1 15.4 238183 315 5.5 10.7 195390 315 6.9 10.0 188267 315 2.2** 12.0 124310 320 12.0 13.8 395** 31 320 17.0 12.9 334** 88 320 4.8 13.8 217403 320 9.6 11.3 162 60 320 6.2 11.4 155315 320 6.4 9.9 136175 325 6.3 17.8** 486**317 325 22.0 14.0 294** 18 325 6.3 11.1 241247 325 13.5 13.2 231223 325 9.2 12.6 203132 325 3.7** 15.4 184168 325 4.3 10.2 174238 325 5.5 9.9 166117 325 5.2 15.0 154404 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 242333 330 34.0 9.2 235 16 330 4.6 13.3 211276 330 5.7 11.9 200391 330 4.1 8.4 184362 330 9.2 11.7 178 1 330 9.9 8.9 170379 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 249402 335 4.7 13.5 230314 335 7.6 9.7 203150 335 4.8 11.2 119120 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 232422 340 3.1** 14.4 188196 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 250350 345 8.0 10.0 249 65 345 8.0 10.2 247307 345 16.5 11.6 208 69 345 17.0 9.9 197328 345 7.5 8.9 192 43 345 6.0 13.2 191222 345 6.1 9.2 175306 345 4.3 17.2** 160154 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 193236 355 7.2 14.8 309**191 355 5.8 15.3 257481 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 179289 360 9.5 7.9 129392 360 5.1 10.3 125320 365 6.4 17.3** 240134 365 13.5 11.8 238239 365 7.7 13.2 236326 365 6.0 10.9 180364 365 4.1 13.9 154218 365 7.5 11.2 126216 365 6.2 12.2 119248 365 5.7 13.3 117375 370 4.1 20.7** 532**288 370 6.4 18.8** 436**161 370 6.3 11.2 340**224 370 19.5 9.8 286**330 370 18.0 12.2 228334 370 12.5 8.7 172275 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 248151 375 2.9** 12.3 235 85 375 6.7 14.8 217294 375 7.0 12.2 184361 375 7.9 10.7 179318 375 5.5 13.7 160386 375 7.6 10.4 153304 375 9.1 9.4 132228 380 7.7 17.1** 320**110 380 4.0 7.2 135204 380 5.7 10.6 91348 385 2.3** 17.4** 368**146 385 11.5 12.5 253260 385 5.5 13.7 211136 385 3.6** 19.8** 205338 385 5.0 16.2 180376 385 3.6** 13.7 154194 385 12.5 7.9 153504 385 38.0 9.5 138160 390 8.1 24.7** 475**354 390 11.5 12.8 212 25 390 5.1 11.3 205387 390 8.7 8.4 162 86 390 21.0 12.6 133133 390 3.9 11.3 113331 395 12.0 20.1** 638**130 395 10.5 10.8 256 82 395 2.8** 9.8 236119 395 12.5 16.3** 209380 395 10.5 14.3 159373 395 5.5 11.6 152256 395 10.5 9.9 149384 395 7.3 14.7 116105 400 19.0 10.5 322**251 400 4.8 14.9 289**352 400 11.5 9.6 181279 400 4.5 11.7 170339 400 7.4 13.6 168381 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 207268 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 218396 410 5.6 16.1 190356 410 1.9** 27.6** 149237 410 3.6** 16.6** 122112 410 5.5 8.9 107259 410 4.7 11.6 99176 415 5.2 21.9** 453**193 415 10.5 11.3 163323 415 6.1 9.6 163202 415 11.5 9.4 150398 415 8.0 12.6 134321 420 5.2 10.7 383**142 420 29.0 8.3 234327 420 3.2** 14.6 203342 420 7.3 9.4 156170 420 20.5 10.3 142345 420 29.5 13.2 136302 420 8.6 8.8 128115 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 241365 425 6.7 16.7** 237139 425 12.5 10.4 224143 425 8.1 13.5 216426 425 19.5 14.5 201303 425 3.0** 14.5 154388 425 6.2 12.3 135127 425 6.7 8.4 100262 430 10.0 12.1 323**270 430 4.8 12.9 293**514 430 4.3 12.9 197341 430 3.5** 19.9** 190278 430 5.2 10.8 182370 430 11.0 15.3 174 55 430 7.6 11.0 162274 430 5.0 8.2 131367 430 17.5 8.0 126 98 430 13.5 12.8 125337 435 13.5 14.1 395**309 435 8.7 12.9 349**305 435 17.5 15.4 187144 435 25.0 8.9 167 34 435 8.6 7.6 157234 435 9.7 9.2 116123 440 9.6 12.2 622**200 440 4.8 12.4 257250 440 7.5 12.9 248107 440 6.3 14.7 183300 440 6.5 7.9 123374 445 5.4 14.0 247372 445 11.0 11.0 181 36 445 4.0 10.0 181271 445 7.2 10.4 124242 445 15.5 9.6 112264 445 6.0 10.7 100172 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 182357 455 6.3 14.4 296**319 455 17.0 10.2 147308 455 15.0 9.8 131235 455 23.0 9.0 114349 455 9.2 8.3 82178 460 5.6 20.6** 473**312 460 4.7 14.4 197 79 460 5.0 10.4 173131 460 18.0 10.2 162243 460 2.6** 11.6 160261 465 7.7 10.6 252378 465 5.4 13.2 221 49 465 47.0 10.8 179226 465 7.7 10.2 173377 465 5.6 8.5 143253 465 10.0 7.0 138 76 470 12.5 14.8 304**203 470 15.0 7.6 233296 470 23.5 11.0 161382 470 5.3 11.1 109 6 475 10.5 12.5 232 75 475 4.5 8.1 150332 475 9.4 10.0 144290 475 14.0 9.1 143128 475 5.9 9.3 133124 475 6.0 13.5 111177 475 8.8 9.1 106126 480 11.0 11.0 212283 480 5.2 10.6 175209 480 10.5 10.5 175293 480 6.8 15.5 135121 485 4.7 20.0** 345**282 485 12.0 10.9 236 71 485 13.5 8.1 168385 485 9.0 14.1 128190 495 9.9 10.4 410**210 495 8.6 12.0 243155 495 5.9 10.4 219336 495 13.5 9.9 135280 500 8.7 14.5 334** 96 500 4.7 10.8 237145 500 5.9 17.5** 233199 500 4.2 13.8 199489 500 11.5 9.7 198217 500 6.4 9.6 166 90 500 7.5 8.5 106164 510 5.2 23.8** 408**343 510 4.5 13.7 284** 42 510 4.9 7.4 233351 510 8.5 11.0 207299 510 12.0 8.0 104 99 520 10.5 25.8** 322**114 520 30.0 10.9 220369 520 29.0 16.7** 206 37 520 10.5 8.6 191215 520 6.7 16.8** 151401 520 7.5 12.6 148229 520 7.9 11.0 116135 520 3.2** 8.3 88 81 530 6.8 14.8 372** 91 530 14.5 10.6 228167 530 23.5 9.2 176181 530 5.5 9.3 171 56 530 20.0 8.3 163 5 530 13.5 8.1 159180 540 12.0 9.0 216311 540 4.1 13.3 214389 540 3.9 13.9 169125 540 5.5 13.0 159 35 540 22.5 11.0 123104 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 263163 550 6.7 14.3 219 66 550 10.5 11.6 206 29 550 17.5 9.6 191227 550 7.9 11.7 154 38 550 7.5 11.9 152241 550 10.5 9.8 100102 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 108255 560 8.8 9.9 93122 570 66.0 13.8 304**208 570 34.0 10.2 255 23 570 21.5 8.3 241447 570 25.0 10.0 164225 570 5.7 12.2 154174 570 7.1 11.0 127 11 570 19.0 8.9 113165 580 10.5 14.8 226182 580 8.9 8.2 189245 590 15.5 10.0 262 83 590 17.5 8.3 199166 590 11.5 9.4 188158 590 7.3 10.7 166187 590 4.5 11.0 146156 590 23.5 11.3 112231 600 9.5 9.0 192 78 600 11.5 9.4 151329 610 15.0 7.3 312** 57 610 16.0 11.9 286** 7 610 12.0 10.4 195277 610 9.5 7.8 153108 620 13.5 8.4 191205 620 18.0 7.5 145263 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 143399 630 14.0 11.0 90266 640 5.1 15.7 364** 12 640 24.5 9.0 233152 640 8.1 10.0 209405 640 7.0 12.8 186 27 640 22.5 8.4 136258 640 8.3 11.2 120249 640 8.7 9.1 81297 650 16.0 10.0 279**192 650 4.9 14.9 213257 650 3.3** 16.3** 208184 650 12.5 9.9 193 58 650 18.5 10.7 172301 650 16.0 15.5 162397 650 12.5 8.4 146272 650 11.0 7.4 120153 650 7.1 13.1 116406 650 6.6 5.8 81 10 660 9.0 7.6 154 26 660 22.0 8.3 132265 670 3.9 19.3** 509**359 670 21.0 8.3 269 48 670 32.0 9.9 262335 670 11.5 8.1 121189 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 266353 710 11.5 11.4 206281 710 10.5 9.6 185 2 710 8.0 8.5 109212 740 20.0 11.1 250 8 740 12.0 11.5 216206 750 12.5 8.3 116101 770 14.5 12.7 372**344 770 32.0 11.7 297** 20 770 35.0 10.1 245407 770 10.5 12.0 110360 780 2.7** 20.9** 157232 790 15.5 10.1 151141 790 12.5 9.5 74129 800 8.7 11.7 211188 800 15.0 12.3 174400 800 12.5 10.3 156 24 810 23.0 7.5 194173 830 35.0 11.4 243214 830 21.5 12.0 187 63 830 13.8 8.8 185148 830 45.0 7.1 146 84 830 23.5 7.0 136179 830 16.5 6.6 96171 840 23.5 11.2 195 28 870 5.8 15.9 197233 870 7.9 12.7 169221 870 40.0 7.0 126371 880 20.0 8.5 152213 890 10.5 18.0** 231358 900 21.0 8.3 149298 910 15.5 10.2 221118 910 100.0 9.7 170479 950 11.5 12.1 188 30 950 6.2 10.5 170159 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 180157 1250 12.0 14.0 206363 1350 28.0 10.4 190 22 1400 13.5 10.4 233 64 1400 31.0 9.7 149169 1450 15.0 9.5 150______________________________________

Number	Name	Date
4940658	Allen et al.	Jul 1990
4945083	Jansen, Jr.	Jul 1990
5374560	Allen et al.	Dec 1994

	Number	Date
Parent	727628	Jul 1991
Parent	333124	Apr 1989
Parent	933553	Nov 1986

Method for treatment and prevention of deficiencies of vitamins B.sub.12, folic acid, and B.sub.6

Information

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Date Filed

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Agents

CPC

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Abstract

Description

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Parent Case Info

US Referenced Citations (3)

Non-Patent Literature Citations (2)

Related Publications (1)

Continuation in Parts (3)

Entry
Gilman et al. "The Pharmacological Basis of Therapeutics", Published 1980 By MacMillan (pp. 1333-1340).
Barness, American Journ. of Clin. Nutr. (20) 1967 pp. 573-577.