Method for preventing vascular disease

Description

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

FIELD OF THE INVENTION

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

BACKGROUND

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

SUMMARY OF THE INVENTION

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

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

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

BRIEF DESCRIPTION OF THE DRAWINGS

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

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

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

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

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

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

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

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

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

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

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

DETAILED DESCRIPTION OF THE INVENTION

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

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

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

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

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

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

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

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

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

EXAMPLE 1

Methods for Measurement of Serum Vitamin and Metabolite Levels.

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

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

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

EXAMPLE 2

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

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

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

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

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

TABLE 1
INCIDENCE OF LOW SERUM
VITAMN AND HIGH METABOLITE LEVELS
IN GERIATRIC POPULATIONS A-D AND
A YOUNGER HEALTHY POPULATION E.

		Folic
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%

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

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

EXAMPLE 3

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

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

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

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

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

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

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

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

EXAMPLE 4

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

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

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

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

It requires less time and expense to treat patients with elevated metabolite levels with a combination of vitamin B₁₂ and folate, or a combination of vitamin B_12, folate and vitamin B₆. The utility of the approach of the present invention is appreciated only after it is taught, for the first time in the present disclosure, that a deficiency of one or more of the three vitamins occurs commonly in the elderly population as evidenced by elevation of one or more metabolites, i.e., MMA, 2-MCA, HC and CT.

EXAMPLE 5

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

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

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

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

It requires less time and expense to treat patients with elevated metabolite levels with a combination of vitamin B₁₂ and folate, or a combination of vitamin B_12, folate and vitamin B₆. The utility of the approach of the present invention is appreciated only after it is taught, for the first time in the present disclosure, that a deficiency of one or more of the three vitamins occurs commonly in the elderly population as evidenced by elevation of one or more metabolites, i.e., MMA, 2-MCA, HC and CT.

TABLE 2
SERUM METABOLITE & VITAMIN LEVELS IN A GERIATRIC
OUTPATIENT POPULATION

						Total
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

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**

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