«y United States Patent Bailey (54) FOOD AND VITAMIN PREPARATION CONTAINING THE NATURAL ISOMER OF REDUCED FOLATES, Steven W. Bail (75) Inventor: 1), Mobile, AL (US) (73) Assignee: South Alabama Medical Science Foundation, Mobile, AL (US) (*) Notice: Subject to any disclaimer, the term of this patent is extended or adjusted under 35 USC 154(b) by 0 days, This patent is subject to a terminal dis claimer. 21) Appl. Nos 9/418,649 22) Filed: Oct. 15, 1999 Related US. ADI (63) Continuation of application No, 09/117 586, fled 3s appi- (60) Provisional application No. 609010.898, fled on Jan. 34, 1996, G1) In. cl? A231. 1/302 (2) US. cL 426/72; 426/549; 426/801; 4263807; 514/258; 514/261 426/72, 549, 801, 4261807; 514258, 261 (58) Field of Search ed US. PATENT DOCUMENTS 66) References 1431,525 10/1922 Hoffman 24082,219, Dickens 3833730 Pedersen et 44755926 6/1988 Lucas al 4834987 5/1989 Lembke eta 5.006055 41991 Miler eta. S)118505 61992 Koltringsr 5'563,126 10/1995 Allen eta. 51524686 41997 Shimoda eta SONTDIS * 12/1999 Bailey et al FOREIGN PATENT DOCUMENTS 0382 019.41 81199 (EP) US06254904B1 6,254,904 BL “Jul. 3, 2001 (0) Patent No: US (4s) Date of Patent: 0.388 927 AL 0382 493.02 10505 ws AL 0 646 322. AL 971990 (eR) 41992 (ED) 5/1994 EP) 41995 (ED) MONAILA 6/1998 UP) SO72918648 1/1995 UP) SOSUTOTSSA 3/1996. CP) OTHER PUBLICATIONS Browa, JP. et a, Gastroenterology 64(2):223-232 (1973). Colman, N. et al, Am J Clin Nutr 28:459-464 (1975). Gregory, JF. et al, Am J Clin Nutr 55:1147-1153 (1992), O'Broin, LD. et all, Am J Clin Nutr 28:438-444 (1975). Perry, J. and Chanarin, 1, Br J Haemo 18:329-339 (1970). Tamura, T, and Stokstad, E.LR,, Br J Haemo 25:513-532 973), IMS World database DRUGLAUNCH (1994) Poncz ot al., Journal of Pediatrics, 98:75-69 (1981). Poncz ot al., The Journal of Pediatrics, 120:948 (1996). accession No, 94:61435 * cited by examiner Primary Examiner—Hiclen Pratt (7A) Attorney, Agent, or Firm —Braman & Rogalskyj, LLP. 67) ABSTRACT A composition for human or animal consumption for sup= plying folate which includes a natural isomer of reduced folate, such as (68)-tetahytrofolic acid, S-metbyl(6S)> letrabydrofolic acid, $-formyl-(6S)-etrahydrofolic acid, l0-formyl-(6R)-tetrshydrofolic acid, 5,10-methylene-(OR)- letrahydrofolie acid, 5,10-methenyl-(6R)-tetrahydrofolie acid, 5-formimino-(68)-tetrahydrofolic acid, and their poly lutamyl derivatives is disclosed. Such compositions include muliivitamin preparations (with or without minerals and other nutrients); breakfast foods such as. prepared real, toaster pastries and breakfast bars; infant formulas; dietary’ supplements and complete diet and weight-loss formulas and bars; animal feed (for example pet foods) and animal feed supplements (such as for poullry feed). The amount of the natural isomer of a reduced folate in a ‘composition for human consumption an range between about 5¢% and about 200% of the daily requirement for folic acid per serving o doseUS 6,254,904 BI 1 FOOD AND VITAMIN PREPARATION CONTAINING THE NATURAL ISOMER OF REDUCED FOLATES ‘The present application is a continuation of US. patent application Ser. No. 09/117,586, filed Jul. 31, 1998, aow US. Pat, No, 5,997,915, which is a 371 of PCT/US97/ (01870, filed Jan. 31, 1997, which claims the benefit of US. Provisional Patent Application Serial No. 600103898, filed Jan. 31, 1996, FIELD OF THE INVENTION ‘The present invention relates generally to the field of nutrition, and more specifically to food and vitamin prepa- rations containing the atural isomer of reduced Folate BACKGROUND OF THE INVENTION ‘The folates are ubiquitous to nearly all forms of life ‘Humans and many other animals lack the capacity to make their own folate which thus is an essential vitamin, one type of essential nurient. Anemia especially during pregnancy land in the geriarie population was an early indication of dietary requirement for folate. A major function of folate is to remove one-carboa units from molecules being metabo- lized and thea deliver them to molecules being syuthesized ‘As an example, folate participates in the formation of the iucleie acids. Further, the activity of DNA is controlled, in pat, by methylation, and the primary methylating agent of | the body (Seidenosylmethionine) is made in a metabolic cycle involving a folate. Many. studies have, therefore, ocused on the relationship of folate status’ to cancer susceptibility, especially colorectal adenoma, ‘The importance of folate to proper growth is clearly evident in the occurrence of neural tube defects in newborn infants. Reports {rom several countries have shown that a _majorty of such cases are associated with low folate levels in the mother. The incidence of these defects a8 well as of | cleft lip’palate is considerably reduced! when women are given folic acid (I) starting early in pregnancy. Recently, a Significant correlation has been discovered between vitamin deficiency, especially of folate, and peripheral vascular disease, @ major cause of death. A high percentage of individuals with this afliction have sbaormal blood levels of homocysteine, a precursor to methionine inthe folate depen= dent siep of the S-adenosylmethonine cycle. Folate defi- ciency has also been linked to defective maturation of & ‘number of different cell types, to nervous system disorders, and 10 decreased immune response. ‘The clear relation of folate intake to health has caused ‘many governmental agencies around the world (such as the USS. National Reasearch CounciD to specify a recommended dietary allowance ("RDA") for folate, Ta the US. these values are used by the Food and Drug Administration to establish the Reference Daily Intake ("RDI") that is listed on {ood labels, currently 0.4 mg for adults. The highest daily amount of folie acid recommenced by & country is 2.0 mg for healthy adults. Many products are available that contain RDI ot near RDI levels of folic acid (I) including most daily ‘multiple vitamins, These can be purchased in solid (ea, tablet, capsule, or powder) or in liguid formulations, both over-the-counter and by prescription. In the US. folic acid (jis also available by itself typically at a dosage of 0.4 ma, but also up to 0.8 mg. in bealih food! stores. Many complete dicts, infant diets, dictary supplements and weight loss products sls0 contain folic acid (I). In some countries folic acid is added to specific food types as determined by health olfcias to provide adequate folate to the general population ‘without risking excess consumption, Many breakfast foods, such as cereals, cereal bars, breakfast drink mixes, breakfast 0 6 s 2 bars and toaster pastries have folic acid (1) added at a modest fraction of the RDI, typically 10-50% of the adult value per serving. In many of these uses the folic acid (I is accom- panied by other vitamins, sometimes at RDI dosages, but uso at lower or much higher levels. Frequently, though not always, essential mineral nurients are also present. Further, many products also include compounds hypothesized 10 have health related value, but which either bave not been officially recognized as effective, or for which optimal ‘amouais have not been se. Products such as those deseribed above are meant fo fill an important and wide spread need for folate, especially mong those whose dictary habits ‘would otherwise preclude intake of a sufficient amount of this vitamin, Folie acid (1) is a component of many animal and pet foods. It is also included in powders or liquids used as animal feed supplements, often in combination with other nutrients. For example, the National Research Council NRO) recommends diets containing 0.2 mg and 1.0 mg of| folie acid (1) per ke of dry diet (assuming 5 keal metabo lizable energy per gram) for dogs and cats, respectively. For chicks the NRC has recommended 0.55 ma folie acid per kg ‘of diet, although recent literature suggests that the optimal ‘value is about thrve times higher than this, The form of folate currently added to all commercial vitamin preparations or whieh is added to Foods, folie acid () (also keown as pleroyl-L-glutamic acid), is aot one of the major forms found in natural Fresh foods. The structure of folie acid (D differs from the most abundant natural folate in several aspects. Fist, the side-chain of natural folates in ilmost all fresh foods contains more than one L-ghatamic acid moiety. Frequently, five to seven (but covering a ‘considerable span of more or fewer) ofthis amino acid are Tinked together into a polyglutamate chain. It is well known, however, that the primary form by which folates arc absorbed has only a single glutamate residue. Cleavage of the extra glutamates of dietary folates is usually accom plished by an enzyme in the digestive tract. In this aspect folic acid (I) is pot at a disadvantage in comparison 10 naturally occurring folates. The second ference between folic acd (and natural folates is that whereas the pteridine ring af the former (Dis fully oxidized, natural flates in fresh uncooked foods are mostly presen a the elaydro forms. Almost all of the {known piysio-logical funetions of folate are performed by teirshydrofolic acid, (68)-FH, (I), or by a one carbon derivative of it ilustated as Tollows: S-methy1(65)41L, {h, "S-formyL(6S)-FH, (IV), LO-formyl(6R)-FH, (V), 5,10-methylene-(6R)-FH, (VI), 5,10-methenyl(6R)-FH, Sii-metnlen(6R)-FH, (UN, 5 10-metheny (OR) FH, mula or each of these compourtls is provided below. wh SOHOIE &US 6,254,904 BI AOL ‘These i no known direet cofactor function for Flic acid (1) itself in humans. Some (6S)-etrahydrofolic acid. poly- slutamate is found in plants or animals, but the majority of 0 6 s 28. §-Methy!-terabytlrofoli acid in high doses (for 4 folate is polyglutamate forms of either Ssmethyl-, S-formyl- (65)-etrahydrofolic acid, and in some cases. 10-formyl- letrahydrofolic acid, Presumably, most of the folic acid (I) found in biological food sources results from oxidation, especially on storage. Whea Folie acid (I) is absorbed by the digestive tract it is eventually reduced to active (68)- ‘etrahyclofolic acid (I) by the enzyme dihydrofolate reduc tase. The oral bioavailability of folic acid (1) as been shown to be widely variable, The literature contains reports of individuals having poor intestinal upiake of folic acid (I) ‘who respond normally to intramuscular injection of folic ‘cid (D, or bad normal serum folate status prior to any folic acid challenge. Several small seale investigations in which the values have been averaged have concluded thatthe oral uptake of several of the reduced folates is similar to folic ‘acid (1). However, there is reason to believe that a segment (of the population possesses adequate oral response to reduced folates, but not to oral folic acid (I). '5-Formyl-tetrahydrofolic acid (also known as leucovorin or Tolinic acid) bas long been used in therapeutic doses for several diseases. Examples include rescue from the toxicity ‘of methotrexate chemotherapy, aod the synergistic combi- nation with fluorouracil for reatment of Various eancers. I is also given t teat acute anemia not due to B,, mg/day) has been patented for treatment of depression (and ‘other neurological disorders) (EP382019 and EP388827 to Le Grazie 1990, and EP482493 to Le Gree 1992) That reduced folates have been overlooked as an improved source for providing the RDA level is in part due to the stereochemistry ofthese compounds, Ia addition to the Single chiral center of the L-glutamte chain in folic acid (I), the tetrahydrofolates contain a second stereochemical center stearbon-6. Chemical reduction of folie acc (D) produces a nearly racemic mixture of the 1Wo isomers at this postion, ‘This is ia contrast to the reduced folates found in suture which all consist of a single diastereoisomer, all having the same L-configuration at earbon-6. (Compounds II-VI are shown as the natural isomer). For many years only the racemic 6(R,S) mixture of $-formyltetraiydrofolic. acid (leucovorin) has been used for therapy of diseases. Recently, however, concern over the possible effects of the unnatural Somer component as resulted in the commercial introdue= tion of the pure natural isomer for these high dose disease treatments by Lederle, although at very high cost. Most therapeutic regimes utilizing leucovorin asta few weeks or perhaps moaths. The elfect of a long term exposure 10 the Unnatural isomer of reduced folates is uaknown, For example, although litle 5-formyl-(OR)-tetrahydrofolic acid is absorbed, there is considerable uplake of the unnatural Somer of S-melhylteteahydrofolie acid by the intestinal trect and other cells of the body which with continuous intake may lead to adverse consequences. ‘Uniil recently, processes for making the natural isomer of reduced folates have been limited in scale, or costly, or both, ‘These include chromatographic separation, enzymatic reduction, and fractional crystallization. The use of reduced folates as’ daily source of vitamin requires a method that is, applicable to large seale production of the natural isomer having high purity at a cost that will not place a burden on the average consumer, SUMMARY OF THE INVENTION “The present invention relates to a composition which includes one oF more natural isomers of reduced folate and {nutritional substance. The one or more natural isomers of reduced folate is selected from the group consisting of (6S)-tetrahydrofolie acid, 5-methyl-(68)-tetrabydeofolic acid, S-formyl-(68)tetrahydrofolic acid, 10-formyl-(6R)-US 6,254,904 BI 5 tctrahydrofolie acid, 5,10-methylene-(6R)-tetrahydrofolie acid, 5,10-methenyl-(6R)-tetrahydrofolic acid, S-formimino.(6S)-ctrahydrofolic acid, and polyglutamyl ‘erivatives thereof. ‘The nutritional substance is. a food preparation, an essential nutrient preparation, or a combi- ration thereof. Whea the cutrtional substance is & food preparation, the food preparation includes wo or more food ‘components. Each gram of the food preparation has a natural ‘molar amount, N, of tbe one or more natural isomers of reduced folate, N being greater than or equal to 7210, and teach gram of the composition has «total molar amount, T, of the one or more natural isomers of reduced folate greater than’ N, When the nutritional substance isan essential fautrient preparation, the essential nutrient preparation includes a vitamin other than ascorbie acid “The present invention also relates to method for increas- ing the folate content of a nutritional substance. The method includes providing a nutritional substance selected from the group consisting of a food preparation, an essential nutrient preparation, and combinations thereof. The method further Includes incorporating into the nutritional substance a molar amount of one oF more natural isomers of reduced folate selected from the group consisting of (68)-etrahydrofalic acid, S-methyl-(6S}tettahydrofolie acid, 5-formyl-(68)- tetahydrofolic acid, 10-formyl-(6R)-teirabydrofolic acid, 5,10-methylene-(6R)-tetrahydrofol (GR)-tetrahydrofolie aid, 5-formimino-(6S)-etrahydrofolic acid, and polyglutamy! derivatives thereof. When the nutri- tional substance is a food preparation, the food preparation comprises two or more Food components, When the nutri= tional substance is an essential nutrient preparation, the essential nutrient preparation comprises vitamin other than ascorbic ac ‘Assignificant number of people are folate deficient espe~ cially vulnerable are those Whose life style does not include Sufficient fresh food sources of folates. An object of this invention is nutritional compositions in which the natural isomer of telrabyrofolic acid, or a derivative thereof, is substituted forthe usual folie acid (I) for the satisfaction ot partial satisfaction of the dietary requirement for this vita- ‘min. While some may not be greatly aflected by the inclu- sion of reduced folates in mullivitamia preparations and breakfast foods, still a substantial number of people, and thus the average health of the population, will be improved by addressing the needs of those foe whom folie acid (1) bioavailability is poor. Consumer confidence with regard to consumption of a food or other nutritional product will be increased with the knowledge that the folate content is chemically ential to the most abwaclant natural forms of this vitamin, except for the advantageous absence of mul- tiple glutamate residues. A further advantage is that health agencies will be aided in recommending optimal levels ‘when a more uniformly absorbed form of folate is widely used. DETAILED DESCRIPTION OF THE INVENTION ‘The present invention relates to a composition which includes one or more natural isomers of reduced folate and 44 nueitional substance. Natural isomers of reduced folate Suitable for use in the present invention include, for example, (6S)-tetrahydrofolic acid, 5-meihyl-(6S)- tetrahydrofolic acid, S-formyl-(6S)-etrahydrofolic acid, 10-formyl-(6R)-tetrahydeofolie acid, 5,10-methylene-(6R)- tetrahydrofolic acid, 5,10-methenyl-(6R)-tetrahydrofolie acid, and. 5-formimino-(6S)-tetrahydrofolic acid. Other natural isomers of reduced folate suitable for use in the present invention inchide the polyglutamyl, such as the iglutamyl, triglutamyl, tetraglutamyl, pentaglutamyl, and hhexaglutamyl, derivatives of (6S)-tetrahydrofolic acid, c acid, 5,10-methenyl- 35 0 6 6 S-methyl-(68)-teteahydrofolic acid, leirahydrofolie acid," 10-formyl(6R)teibydeololic seid, 5,l0-methylene-(6R)-etraydrofolic acid, 5,10smethenyl- GR)-tetrahydrofolic acid, and 5-foemimino-(6S)- tewahydeofolic seid. Any of all of the natural omers of reduced folate can be present in its ehirally pure form, or iteratively, the composition ean optionally contain a molae ‘moat of one or more unnatural isomers of edced folate, Such as (OR)-telrahydrofolic acid, 5-methyl-(OR)- lctrahydrofolic acid, S-forinyl(6S)-terabydrofolie ack, TO-fomyl-(68)-tctrhydeofolic acid, 5,10-methylene-(68)- teirydrotolie acid, 3,10-meibenyl-(68 teabydeofolc cid, S-formimino-(6R)-tetahydrofolic ack, and. poly= slulsmyl derivatives thereof The molar amount ofthe at fal isomer of reduced folate ean be equal to the molar mount of its corresponding unnatural isomer (as where the Unnatural and natural isomer are present ss a racemic mixture), oF, preferably, the natural isomer of reduced folate fan he present in a molar amount greater than the molar amount of the corresponding unnatural, isomer, The total molar aniouat of the one oe more natural isomers of reduced Folate present in the composition can be between 5° and 200% Of « human daly requirement for folate per a cus- ‘omarly consumed quantity of the composition AS used herein, the total molar amouat of the one oF more natural Romers of reduced folate includes ‘natural isomers of reduoed flats whieh are naturally resent inthe nutritional Substance as well as natural isomers of reduced foltes Which might have been added to the nutritional substance ‘The customarily consumer! quantity of various compositions depends, of course on the nature ofthe composition, Where {he composition includes a food preparation the customarily consumed quantity is the amowat of the food preparation eustomarily consumed per eating oecasion, for example, as Set forth by the U.S. Food and Deug Adlmiistation forthe purpene of establishing realistic and consistent serving sizes forse in food labeling. Examples of customarily consemed quantities for various food groups can be found in 21 CER $101.12, whieh is hereby incorporated by reference "The human daily requirement for folate varies from person o person, depending on factors such as body weigh, ge, heal, sex, and he like, Suitable values for the human dily requzemeat for folate include RDI and RDA values, promulgated, respectively, by the FDA. and the: Natcnal Research Council (*NRC"), Presently, RDI values, expressed in tems of micrograms of folic acid (oanomoles of folic acid), are 400 gag (907 moles) for adults and 800 ag (1814 amoles) for pregnant women, In view of this, the ‘compositions ofthe present invention ean have between 45 fad 1814 nomoles of between 91 and 3625 moles of folate per customarily consumed quantity of the nueitional sub- Stance. Current RDA values are’ published in National Research Council: Reconmended Daily Allowances, 10h 4, Washington, D.C. (1989), which is hereby incorporated by reference. They are, again expressed in lerms of micto= rams of lolic ack! (nanomoles of Tlic sid) 25 yx (37 moles) for infants 0-6 months of age, 35 ug (79 amos) for infants 6 mons to one year of age, 30g (113 amoles) foe infants 1-3 years of aze, 100 jg (227 nmoles) for infants 7-10 years of age, 20 (454 mmoles) for mae als, 180 ‘ng (408 novos) for female adults other thn pregnant adult females, and 400 ag (907 anoles) for pregnant females. In view of this typical compositions of the present invention an have heiween 2.8 and 113 nmotes, between 4 and 159 moles, between 28 and 227 nmoles, between L1 and 454 nimoles, between 22 and 9G7amoles, between 20 and 816 moles, or between 45 and 1812 moles of folate per customarily consumed quantity of the nutritional substance Suitable valies for the human daly requirement for foltes sce ako established by the World Health Organization a8 7103 nmolesikg of body weight. For pregnant women theUS 6,254,904 BI 7 value calculated based upon body weight should be increased by about 454 to about 680 nmoles, “The {otal molar amount ofthe one or more natural isomers of reduced folate present in the composition can, alternatively, be between 54% and 3000% of an animal daily ‘requirement for folate per a customarily consumed quantity ff the nutritional substance. The animal, whose dai requirement for folate is referred w above, can bey for example, a dog, a cat, a chicken, a cattle, a domestic animal, 4 goal, a horse, a mink, a fox, a sheep, ot a swine. Suitable values for the an animal's daily requirement for folate are promulgated, for example, by the NRC in Nusrient Require- nents of Domestic Animats (Washingon:National Academy Press) particularly in those publications having the follow: ing subtitles: “Nutrient Requirements of Becf Cattle," Sev- «nth Revised Edition (1996, ISBN 0-309-05426-5); *Nutri- cent Requirements of Cats,” Revised Edition (1986, ISBN (0-309-03682-8); "Nutrieal Requirements of Dairy Cattle,” ixth Revised Eultion, Update (1989, ISBN 0-309.03826- ); “Nutrient Requirements of Dogs, Revised (1985, ISBN. 0-309-03496-5); “Nutsient Requirements of Fish,” (ISBN (0-309-04891-5); “Nutrient Requirements of Goals: Angora, Dairy, and Meat Goats in Temperate and Tropical Countries, “(ISBN 0-309-03185-0); “Nutcient Requirements of Horses,” Filth Revised Edition, (1989, ISBN 0300-03989. 4); "Nutrient Requirements of Laboratory Animals,” Fourth Revised Edition (1995, ISBN 0-309-05126-6) Requirements of Mink and Foxes,” Second Revised Elton, (1982, ISBN’ 0-309-03325-X), "Nutrient Requirements of | Poultry.” Ninth Revised Edition, (1994, ISBN 0-309-04892- 3); “Nutrient Requirements of Sheep,” Sixth Revised Edition, (1985, ISBN. 0-309.03596-1); and “Nutrient Requirements of Swine,” Ninth Revised Edition, (1988, ISBN 0-309-03770-8) (collectively referred 10 as "NRC Animal Nuteient Requirements”), which are hereby incor- porated by reference. As indicated above the nutritional substance ean be a food preparation or an essential quttieat preparation. Essential fulrieat preparations are materials which contain one or ‘more essential nutrients. Where only one essential nutrient is present in the essential nutrient preparation, that essential futrieat can be a vitamin other than ascorbic acid, ‘The cesential nutrient preparation can, alternatively, include a vitamin other than ascorbic acid and, in addition, ascorbic acid, As used herein, essential nutrients are those nutieats which are required to sustain bealth but which cannot be effectively produced by one or more animals of by humans, Examples of essential nutrients are compiled in a number of| published sources, including Modern Nutrition in Health ‘and Disease, Sth ed, Shils etal, eds., Philadelpbia:Lea and Fehiger (1994), which is hereby incorporated by reference, [Essential nutients are meant to include essential vitamins and provitamins thereof, essential fats, essential minerals, Such as those minerals Tor which daily values have beet recommended, and essential amino acids. One example of fan essential urient preparation is a formulation which ‘contains a vitamin and a caloric content of les than 2.5 cal per dry gram, preferably less than 2 cal per dry gram, most preferably less than 1.8 eal per dry gram. Essential nutrient preparations also include those materials which contain at Teast one vitamia in an amount greater than 15%, preferably greater than 20%, more preferably greater than 40% of the US. adult RDA’ for that essential nutrient per gram of sential nutrient preparation. Still other suitable essential ‘ulriet preparations contain atleast «wo vitamins, each in fan amount greater than 10%, preferably greater than 15%, ‘more preferably greater than 20% of the U.S. adult RDA for 3 per gram of essential qurient prepa ial qutrieat- preparations are_com= monly referred to as dietary supplements, vitamin supplements, and mineral supplements, multipie vitamin “Nuteient 25 6 s 8 supplements, andl the like, and ar typically avilable come menial io the form of pills, tablets, capes, powders, symips, and suspensions. Preferably, the essential inal hmpostion contains atleast one csental mutica in an Amount greater than 25%, more preferably greater ban 50%, od most preterably grater than of eqial to 1004 ofthe daily requirement for that essential aurent per customarily onsumet! quant ofthe exental nutrient preparation 'As indicated above, the nutritional substance can also be 1 food preparation, Food preparations are mera which Contain one or more mind seid eaebobyerat, oe fat, which fre suitable for human or animal constmption, and hich fre no essential muri! preparations Ts prefered that the food preparation be a two or more component food prep ‘ation For example, wo or more component food prepa- oe Eafe] tase (esl eens omer foods. One component foods are foods which ae derived Substantially fom & single natal source. A small percent teat the ove-compooent food canbe derived from a scobed futur soutecs ut that peeentoge. by weighs preerably Iess than 5%, more preferably fess than than 1%, more referable than 0.12, One component foods ince, for Example, sugar, unswectened juicer nec or puree from a single species of plant, such as unswectened apple juice (including a blend of different varieties of apple juice), frapefrat juice, orange julce, apple sauce, apnct nectar tonto jit tomatosavce, tomato pure, andthe like. Grain plants of a single species and materials produced from grain plants of a single species, sich a8 corn syrup, tye our, ‘heat flour, oa banyan the like are also considered to be ‘ne component foods. Alternatively, the two oF more com= ponent food preparition cin he a mite of one o mors one Component foods and ooe or more essential nutiens Prefers, she amount of at least one ofthe one oF more sential nutrients present in the two component food is frente than te amon’ ofthe a least one essential mation {hati naturally present collectively inthe one or more one ‘component foods. or example, where the csontal autres sre vitamin X and vitamin ¥ sh where the one component food is orange juice and where the orange ice naturally contains vitamin X and vitamin ¥ in amounts “Nand SRY", respectively, is prefered th the, composizion contain vitamin X ind vtemin Yin amas “Tx and" Ty, respectively, so that Tx is greater than Nx, Ty is greater than Ns or both rood preparations particularly well suited tothe practice of the present invention include breakfas foods, such as prepared creas, toaster pastries, and breakfast dink mixes tnfant omolasy dietary supplement; complet diet formic lis; nd weightloss prepartions, sich as weightloss drinks ana weightloss bar The food preparation can be one which naturally consins no natural bomer of reduced folate, Altematively, it can ontain'a-patual golae amount of a natural omer of reduced folate. For purposes ofthis application the molar mount of natural Somer of reduced folate 1. collectively the numberof moles of (68)ettahydrofolc ack, S-metc (Syictryceafolie asd, S-formyl-(68)-tettabydrofaic acid, 10-formy!(6R)-tetrahydrofolic acid, 5,10-methylene- (GR)-tetranydrofolic. acid, 5,10-methenyl-(OR)- {eirabydrotolie ack, 5foeeimino.(68)ttrabydeotols acid, {nt polyglsiamy! derivatives thereat) contsined per gram of food is designated “Nor some foods, the molar smount of natural isomer of reduced folate vaturaly present fy Keown, For ethers the molar amount of natural isomer of reduced folate can be determined by a qumber of sensitive dna specifi methods, such as microbial growth dependence, folate bindivg protein based assay, highopectormance Tig chromatography CHPLC) and. gas" chromatography (“GC”), Suitable methods are described, for example, inUS 6,254,904 BI Cossins, B.A, “Polates in and Prerins, Vol. 1, Blakley et al eds, New York:fohn Wey Sons, pp. I-60 (98H, which netey eo: rated by relerenee ‘The moar amount of nawal isomer of reed folate wesent in he compenition ofthe present invention i greater {hat the molar amount of natural somer of reduced Tote present inthe food. preparation. For purposes of thi pplication, the molar amount of natural omer of reduced folate (Ce, collectively, the number of moles of (68)- teirahydrofolic acid, S-methy1-(5S)-etrabvdrofolic cid, rayl-(6S)-ietabydrofolic. acid, 10-formyl-(GR)- {cirahydrololic. seid, 3,10-melbylene-(GR)-ttrahydro Seid, 5,10-methenyl-GR)-tetrahydrofolic. acid, $formimino68).tetrshydrovolic acid, and. polygitamyt dlerivatives thereof) contained per gram of compesiton is designated “T~,Thus, Tnevessarily must be greater than N, Preferably, TIN is greater than 103%; more preferably, iis stealer that 110%) and, most preferably, its greater than 120%. {As indicated above, the composition can, optionally, include one or more Uanatural Iomers of reduced Folate ‘When present in the composition, the one or more unnatural isomers of reduced folate is prefeeably present in a molar amount which i les than T eins N. The present invention also relates to a method for inceas- ing the folate content of 4 nuvtional substance. The aut tional substance ean be food preparation, an essential nutrient preparation, or «combination of food preparation and an essential nutrient preparation. The folate content of {he nutetional substance is increased by incorporating one oF ‘more natural isomers of reduced folate into or with the nutritional substance. This can be achieved by methods well, Know ia the ar of food and essential nutrient preparation, sich 3s by homegenizing, coating, spraying, coarsely mixing, tossing, kneading, pilling, and extruding one or more uonatural isomer of reduced folate, singly or in Combination, onto or with the nutetional substance ‘One of more of the one or more natural isomers of reduced folate that are added to the nutritional substance in ‘tecordance with the prescat invention ean be substantially hirally pure or eich'of the one or more natura isomers of reduoed folate ean be chiral pure. Altematively, one or ‘mote of the one or more natural isomers of reduced folate can be present ina mixlure wih one of more unnatural isomers of reduced folate. The molar amount of the one of ‘mote natural somers of reduced folate and the one o¢ more Unnatueal isomers of reduced folate proseat inthe mixture ludded (o the nutritional substance can be the same, a8 i the tase where a racemic misture is added, or they can be siffecent. Preferably the molar amount ofthe natural isomer exceeds the molar amount of the unoatural omer. Addi= tionally or alternatively, wanaturl isomer of reduced Toate can be incorporated ina separate step subsequent to or prior to incorporating the one or more natural isomers of reduced folate into the nutetional substance. I is prefered thatthe collective molar amount of unnatural isomer of reduced Tolate added before, during, andlor after the addition of natural isomer of reduced folate be less than the collective ‘molar amount of matral isomer added Natural isomers of reduced folate that are substantially chirlly pure can be prepared by aay suitable method, Including, or example, by the method described in US. Pat No. 5,350,851 to Bailey et a, which is hereby incorporated by reference. ‘When the nutritional substance is food preparation, ia addition to incoeporating a natural isomer of reduced folate, One oF more essential nutrients, optionally, can be incorpo- {ated into the food preparation. "The essential nutrients ean, Torexample, be alded tothe food preparation before, during, or after addition of the natural isomer of reduced folate 0 6 10 ‘The compositions of the present invention can be used to increase a subject’s dietary Intake of folate by administering the composition tothe subject. The subject caa be an animal, such as.a dog or a eat; alleratively, the subject can be human. Certain classes of individuals are viewed 10 be especially benefited by increasing dietary intake of folate. ‘These include pregnant females; females who have had a miscarriage; females who have carried a fetus having a neural tube defect, a cleft lip defect, or a eleft palate defect; ‘and humans who sulfer vascular disesse "The compositions can also be used 10 reat a subject aMlicted with intestinal malabsorption, especially folate mal- absorption, When treating a subject aflicted with intestinal malabsorption, the amouat of composition administere preferably effective to cause an increase in the subject's bloed folate level. More preferably, the amount aclministered is effective to produce blood folate levels in an normal range, as determined by conventional blood-folate analysis methods, such as with the Quanta Phase TI assay from BioRad Laboratories, Hercules, Calif. ‘The compositions can be administered enterally, such as orally, intragastriely, or transpyloricly. Many faciors that may modify the action of the composition can be taken into account by those skilled in the af; e., body weight, sex, dict, time of administration, coute of administeaton, rate of excretion, condition ofthe subject, and reaction sensitivities and severities. Administration cad be earried out co ‘ously or periodically, such as once daily, or once with every meal. ‘Compositions containing the natural isomer of reduced folates ate preferably for enteral consumption (enteral including oral, intragastric, or tanspylorie), and are of any, food preparation, food supplement, essential nutrient preparation, or vitamin preparation, Typical examples of Such Toexl or vitamin preparations ave those 1 which folic acid (I) is curently adkled for use by either humans or other animals. Natural isomer means a tettahydrofolate having the natural configuration at both the glitamate a- andthe pileridine 6-carhons, More specifically, these compositions are, but are nol limited to, multivitamin (with or without minerals and other nutrients) preparations (such prepara: tions can be in solid, liquid or suspension forms); breakfast foods such as prepared cereals, breakfast drink mixes, toaster pastries and breakfast ars; infant formulas; dietary supplements and! complete diet and weight-loss formulas ‘and bars; animal feed or animal feed supplements. (for ‘example, for poultry), and pet foods, The natural isomer of reduced folates can also be used in products which health officials might in the future deem an appropriate vehicles for satisfaction ofthe daly folate requirement. The composition ‘of the present invention can include 4 natural isomer of tetrahydrofolate, such as I-VIIL, preferably the mono- tlutamate form, in a vitamin formulation preferably con= lining. at least one other vitamin (Cor example another vitamin such as a B vitamin) other than ascorbic acid (vitamin ©), although ascorbic acid can be an additional ‘component, Such nueienis or vitamins can be these tended for either buman or animal use. Other ingredients may also be present, such as fillers, binding agents, stabilizers, sweeteners, including nutitive sweeteners (e, sucrose, sor. bitol and ether polyols) and on-nutetive sweeteners (e.8. Saccharia, aspartame, and acesulfame K), colorants, flavors, Dulles, salts, coatings, andthe like that are known to those skilled ia the art of vitimin formulation. For many applications of the described reduced folates (IT through VIII) 5-methyl-(6S)-etrahydrofolic acid (ID), 5-formyl(6S)-etrahydrofolic acid (IV), and 5,10-methenyl- (6R)-tetrahydrofolic acid (VID) are. prefersed. All of the reduced folates are to dillering extent subject to oxidation by air. Several factors influence this susceptibility, foremostUS 6,254,904 BI WL being derivatization of the S-nitrogen which considerably slows oxidation, The S-methyl- and 5-formyl derivatives are the most abundant forms of folate found in most tissues of | the body. The predominate folate in human blood i Semethyl(68)-ttrahytrofolic acid (IL). In using tetrahydrofolates for increasing the folate in a ulritional composition, several factors should be consid= cred. Many nutrients are unstable to processing, including several of the vitamins, For example, vitamins A, By (Chiamin), and ascorbic acid are especially labile under some conditions, Many procedures are known for enbancing the Stability of the various nutrients such as pH and moisture Contcol of the composition. Components: which are 10 a Alegre incompatible with each other, for example ascorbic acid with the more oxidized forms of iron or copper, ean be ‘made to be present heterogeneously in the composition, [Nutrients which are unstable to heat ate often added after baking steps; for example, vitamins are often sprayed onto breakfast cereals subsequent to toasting. Nutrients which sre unstable to air ean be packaged in a reduced oxygen condition, and/or in containers that have low or no perme: ability 10 oxygen, These and other procedures known t0 those skilled in the art are wseful for maintaining folates in their natural reduced form. ‘The rate of oxidation of a reduced folate in the presence of air is increased in water solutions, The shelf life of liquid. 25 preparations will be geeally extended if they are preferably kept in airtight containers, Replacing ait with an inert gas such as nitrogen or argon also retards loss. Reduced folaies can also be protected from oxidation by a aumber of reducing agenis and antioxidans, the most relevant of these being other vitamins that are often included in multivitamin ‘mixtures or nutritional compositions. Ascorbic acid has been used for protection of reduced folates in biochemical experi= ‘ments and procedures for the laboratory analysis of biologi- cal samples. Such protection need not be limited 10 use of | ascorbic acid of other vitamins; other agents suitable for human or animal consumption are useful, for example jso-ascorbie acid and certain thiols, such as glutathione, Further, known packaging and formulation technologies which increase the stability of compounds such as ascorbic acid or other air labile materials (for example, coated forms, blister packaging, and use of reduced metals or metal complexes) are useful for the maintenance of reduced Tolates. The salt form of reduced folate also somewhat affects stability and solubility, and this can be optimized for the needs of each prociuct. The pH of the final composition can also be optimized according tothe stability properties of| the particular reduced folate derivative used aad of the other components present, as is well understood in the arts of processing nutrients and of folate compounds. For example, in the presence of moisture 5,t0-methenyl(6R)-FH, (VID) ccan be transformed into 10-formyl(6R)-FI, and S-formyl- (6S)-FH (IV) (the latter also a preferred compound) in a pH dependent manner. Compostions containing 5,10-metbenyl- (GR)-FH, (VI1) ase most stable to oxidation when either substantially dry and/or have an acidity less than about pH 4. With proper attention to the above factors, the lability of | reduced folates need not limit the life of a produ cially with IT and TV which are more resistant than ascorbic faci to many oxidation reactions, ‘The substitution of a reduced folate for folie acid (I) should take into account the differences. in molecular ‘weights ofthe various forms, For example, the current US, Reference Daily Intake of O.4 mg of Folic acid (I) corre: sponds 19 0.91 micromole using an anhyérous molecular ‘weight of 441.4, The effective molecular weight of reduced Tolates depends upon the derivative employed! (ic. I-VI), the salt form, and Water content. For example, 0.91 micro- mole of S-formyl(65)-tetrahyirofolic acid (IV) calcium 6 s 2 sultenabydate woul! weigh 0.545 mg, and 091 mieto- mole of Ssmethy1(68)ctabydeoolc aid (ID dsadium Sit would weigh 0.56 mg. Several salt forms of the redsced Totes are deserted in the Irate, soch a5 Hydrochonde, sodium, potassium, magnesium, csllum, and others and having various water conten. For cach of ies forme similar Callin can be made, The moun required to achieve th mole equivalent oa desired faction Sf the RDI would then be ta fraction of this new weight ‘Asan example 25% RDI of Smethy- (6) etrabydofoic SEG dain st would be 0.230.486.0114 mg ths mmole equvalen of 040 mg of folie ahd (I). Previows ivesigitions of groups of individuals haying 2. aocmal "ptske of folie sce) have shown tat the bios of ihe reduced folaes ip similar on a nok bass "As mentioned above los of nlints during procesting, specially of foods well Known to those sled fn hk a ‘Ab ollen practical procedure iy the addition of an inal fxeoss, an “overage, of particular mitint or nutrient, Sth hat the inl poeproessing amount is tte desired ievel Many highly scmitve and specie methods. ar known (sich as microbial rowth dependence, folate binds ing protein based says, HPLC and GC) forthe analysis of tole, in ot the ede and onze ors ta well a for thelr various derivatives, These asays permit ajsiment of the added amount of tbs aural homer of edhced folate Sas to ye the desired foal amount subsequent 10 oe essing ad packaging. The range ofthe natural some of 9 reduced late inte corponion of this invention i pref “rably tat fing between abou 5% and about 2007¢ of the RDI of humus for Tout, and should be ‘compass both hesitation were allowance is [processing loss, and also where no such allowance i made Ripe RDA dosages ae specified for diferent groupe of people, for example. pregnant and non-pregnant women, Further, the RDI level although eying oa RDA values, cab tedlferent fom RDA valves. The sve range of*betweet shout 5% and sbost 2008 of the RDI for folate” should be ken to operate independently on each of these separate or their foreign equivalents, as presently sated or as modo nthe ft. For the purpose [tthis invention these sever! speciieations shall be refered to asthe daily requirement for folate. Unless the rosom- ineaded etary allowance fo folate nina is increase, the maxiount Hin aunt of the natuea omer of & reluced folate in composition for human sae in satisiying the daly reqorement for folate preferaly shoul pot exceed Shout 45 micromele per dose or castomarly conumed Scving. However for Uavidual aliced with inestial malatsomplion, such as esac disease or topical suc, Compositions Containing higher amounts of the natural So" tier ofa reduced folate wil be Uctl. For the purpose of this invention an ssenil trent composition ean be a ditary supplement or the like, the Siena fate component of which derived fom Sebstantaly pore tirabpdrofolic ack or derivative threo, Such as compours It through VIL. Essential trent com: pesitons encompassed by this avenion comprise the at tal Somer of a reduced folate preferably within the above described range along with other vitamins andlor other nuleents which are preferably each present in an amount thats coosideredo be sao Io formulating compentions fae Shima consumption manufacturers often, comideray Exceed the dosage recommended by the NRC Tor folate (by IWhfotd, 20-14, oF more in some cases), ot onto coveteome loses ding processing, but alk io cover occ. Sons of posible increased ned Tor folate such 28 ding ‘ntiioti retment Other vitamin nd ntrent components am be present in amit that wary considerably fom NRC recommendations, The following examples are even 0 further illusrate the ievention, snd ae no intended to inst is scope in any wy.US 6,254,904 BI 13 EXAMPLES 1) A typical ready 10 at breakfast cereal: com (andior cather gesins), sugar, salt, malt favoring, such that 30g Serving provides about 2 g of proicin, 26. total Sutbohyafte and 330 mg of sodiua, ako consining pet Serving sie vitamin A palmitate (154 of RDI, ascorbic acid (G59 Of RDD, redueed iron (43% of RDD, vitamin D (10% SE RDP), thiamin hydrochloride (25% of RD, riboflavin 5% of RDD, niscinamide 25% of RDD, pyridoxine hydrochloride (250 of RDN, and 0.114 mg of 5-methyl6 (S)etehydeofoic acid (ID disodium salt (the mole equiva Jeot of 0.1 mg folie acid, 25% of RDI) 2) A typical daily multivitamin tablet: caleium carbonate, ascorbic acid (60 mg, 100% RDP, gelatin, vitamin E acetate GOLU, 100% RD), starch, niaeinamide 20 mg, 100% RDN, hjdroxypropyl-methylcellulose, calcium pastothen- ate (10mg, 100% RDI), calcium silicate, hydroxypropylellulose, pyridoxine hydrochloride (2 ma, 100F% RDN, cioflavin (1.7 ma, 100% RDI, thiamin mono- niate (15' mg, 100% RDP, beta carotene & vitamin A acetate (5000 1.U,, 10076 RD}, sodium hexametaphosphate, ‘magnesium stearate, vitamin D400 LU., 100% RDI), Vitamin By (6 ug, 100% RDN), lecithin, and 0.437 mg of $Sumnethyl6(S)-etahydrofolic aid (IM) magnesium salt (ihe ‘mole equivalent of 04 mg folie acs, 100% of RDI). 3) Atypical daily multivitamin and migerals table: es cium phosphate (130 mg of elemental calcium), magnesium hnydroxide & stearate (100 mg, 25% RDI), cellos, potas- sium chloride, ascorbic acid (60 mg, 10% RDI), gelatin, ferrous fuimarate (18 mg elemental ron, 100% RDN, zine sulfate (15 mg, 100% RDI, modified cellulose gum, vitamin E acetate (30 .U, 100% RDM), cic acid, niacinamide (20 mg, 100% RDI), magnesium stearate, hydroxypropyl- ‘methylcellulose, caeium pantethenate (10.mg, 100% RDD), Sclenium yeast, polyvinylpyrrolidone, hydroxypropyleellulose, manganese sulfate, silica, copper oxide (2 mis, 100% RD}, chromium yeasi, molybdenum Yyeist, pyridoxine hydrachioride (2 mg, 100% RDI), ribo Tavig (L7 mg, 100% RDP, thiamin movoniuate (1'5 ms, 1007% RDP, beta carotene & vitamin A acetate (5000 LU, 100F% RDN, potassium ide (150 yas, 10% RDI, sium hhexametaphosphate, biotin (30 ag, 10% RDN), vitamin D 400 TU: 100% RD, viamia'B,. (6 ns, 100% RD, lecithin, and 0.545 mg 5-lonmyl46s)-tettahydroflic acd (i) eaicium ssitpentahyarate (the mole equivalent of 0.4 mg of folie acid, 100% RDP “4) A typical daily multivitamin and minerals tablet for colder sults caleium carbonate, calcium phosphate (200 mg, Ca, 20% RDI, 48 mg phosphorous, 5% RD), magnesium oxide, magnesium Stearate (100 mg, 25% RDI, potassium chloride (80 mg, 2% RDI), microrystlline cellulose, ascor- bie acid (60 mg, 100% RDD, gelatin, d'talla-toeopberyl acetate (45 LU, 150% RB), modified food starch, ‘maltodextrin, crospovidone, reduced iron (4 mg, 22 RDI), hydroxypropy! methyleellose, niacinamide (20 mg, 100% RDI), zine oxide (15 mg, 100% RDN), calcium pantothenate, ‘manganese sulfate G5 mg), vitamin D 400 LU. 100% RDI), ttanivm dioxide, vitamin Aand f-carotene (5000 LU, 100% RDD, stearic acid, pyridoxine hyrochloride G ma, 150% RDD, shoflavin (1.7 mg, 100% RDD, silicon dioxide, copper oxide @ mg, 100% RDD, dextrose, thiamin mono nitrate (15 mg, 100% RDI, tethy citrate, polysorbate 80, ctthromium ehioride (130 jg), artificial eolor, potassium iodide ((150 ug, 100% RDD, Sodium measilcate (2 mg). sium molybdate (160 ug), borates, sodium selenate 20 ea), biotin 30g, 1%% RDD, sodium metavanadate (10 13), yamocobalamin (25 sg, 417% RDI, nickelows sulfate (3 ig), and phytonadione, and_5,10-methenyl-(GR)- {ctrahydrofolie acid hydrochloride (VIIYO.44 mg, the mole equivalent of 0 mg of Folie acid, 100% RDI}. 6 14 5) A tical complete diet drink: water, sugar, calcium and sodium caseinates, maltodextrin, high‘oleie safflower oil, soy protein, soy oil, canola ol, cocoa, sodium and potassium Ciirates,calciuin carbonate and phosphate (250 mg Ca, 25% RDD, magnesium chloride and phosphate (100 mg Mg, 25% RDP), sodium chloride, soy lecithin, choline chloride lavor, ascorbic acid (30 mg, 50% RDI), carrageenan, zine sulfate 6 mg, 37% RDI), ferrous sulfate (4.5 mg Fe, 25% RDN), alla-tocopberyl acetate (11.3 LU., 37.7% RDI), niacinamide (mg, 25% RDN, calcium pantothenate (2.5 mg, 25% RD), manganese sulfate (1.3 mg), copper silt 25% RDI), vitamin ‘A palimtate (1250 1.U., 25% RDI), thiamin hydrochloride (0375 mg, 25% RDN, pyridoxine hydrochloride (0.5 mg, 25% RDI), riboflavin (0.425 mg, 25% RD), biotin (75 43, 2566 RDI), sodium molybdate (38 jg), chromium chloride @5 12), potassium iodide (37.5 jag, 25% RDI, sodium seleaate (18 18), phylloquinone (vitamin K,), eyanocobal- amin (1.5 ug, 25% RDD, vitamin D, (100 TU., 25% RDN, apd 0.136 mg 5-Formy!-(6S)-etrabydrofolic acid (IV) cal cium salt-pentahydrate (Ihe mole equivalent of 0.1 mg of folic acid, 25% RDM), packaged in an air-tight container, ad supplying about 225 calories, 6) A typical enhanced B-vitamin/etralydrofolate tablet dlibasie calcium phosphate, pyridoxine hydrochloride (50 mg, 2.500% RDI, cellulose, stearic acid, magnesium 25 stearate, and 0.912 mg of S-methyl-6(8)-tetrahydrofolie acid All sodium sat (he mole equivalent of 0.8 mg folic acid, 200% of RDI for adults, 100% RDA for pregnant women). 7) A typical poulcy feed vitamin supplement: (amounts per ky of diet) vitamin A (trans retnyl acetate, 5500 1), Vitamin E (11 LU), menadione sod bisule (IL mg), Vitamin D, (1100 £1}, ribolavin (4.4 mg), vitamin By, 10 ig), viamin B, (3.0 tip), thiamin mononitrate (2.2'm), biotin (0.3 ma), ethoxyguin (125 mg), and 2.0 mg S-formsl- (6S)-tcitahydiofolic acid (V) calcium salt-pentahydrate (ihe mole equivaleat of LS mg of folie ack) 8) Atypical dry cat food: ground yellow cor, com gluten meal, soybean meal, poulry by-product meal, animal fat, fish neal, meat and bone meal, ground wheat, phosphoric acd calcium carbonate, died animal digest, salt, brewers dried yeast, potassium chloride, dried whey solubles, cho- line chloride, dried skimmed mil, taurine, L-lysine, zine oxide, ferrous sulfate nisin, vitamin A, vtamnia D,, vitamin By, caleium pantothenate, citric aid, manganese sulfate, ribaflavin supplement, biotin, copper salt, thiamine mosonitate, pyridoxine hydrochiorie, menadione sedium bisulfate complex, suel thatthe crude protein is aot ess than 31%, crude fat isnot less than 8%, etude fiber i not more than’4.5%, moisture is not more than 12%, ealeium is nol less than 1.2%, phosphorous i not less than 1.0%, sodium chloride isnot more than 1.5%, the metabolizable enesyy i about 3,600 keal kg, taurine, ion, vitamins A Dy Ba ae E areal least 100% of levels recommended by the Assoeia- tion of American Feed Coateol Officials, and containing not less than 0.97 mgyky dict S-methsl-0(5)Xlraydrofolic acid (UP calcium salt dihydrate (he mote equivalent of 08 mgikg dit of folic acid) 9) A typical soy based infant formula: 75.5% water; 13% sucrose; 6.6% oleo oil: coconut, high oleie (saflower oF Sunflower), and soybean oils; 38% soy protein isolates (protein 27 g fat 5.3 g, carbohydrate 102, linoleie acid 500 mg); potassium citrate and bicarbonate (potassium 105 ma); monobasic potassium and dibasic calcium phosphates (phosphorous 63 img); soy lecithin: taurine; calcium cartae {eenan; calcium hydroxide, eloride and citrate (calcium 90 mg), sodium chloride (sodium 30 mg); L-methionine; zine (Z08mg),ferows (Fe 8 mg), and manganese (Ma 30g) sulfates; copper salt (Cu 70 3 taurine; L-carnitine; potas Sium iodide (19 lg); ascorbic acid (S3 mg) choline chloride; alpha-tocopheryl acetate (14 1.0.) niaeinamideUS 6,254,904 BI 15 (750 2); vitamin A palmitate and beta-carotene (300 1.0); calcium pantothenate (450 4); thiamin hydrochloride (100 14g); rbollavin (150 aa); pyridoxine hydrochloride (62.5 ua); vitamin K, (15 1g); biotin (5.5 ig); vitamin D (60 LU); eyanocobalamin (0.3 uz); and 9:1 ug of S-methyl-6(S)- fetralydrofolie acid (III) calcium salt dihydrate (the mole ‘equivalent of 7.5 ug of folic acid), packaged in an air-tight container (amounts are per 150 mi of 1:1 diluted Formals). One skilled in the act will readily appreciate that the present invention is well adapted to carry out the objects and Obtain the ends and advantages mentioned. While the above description contains many specificities, these should not be construed as limitations on the scope of the invention, but rather as an exemplification of preferred embodiments thereof. Changes therein and other uses will occur 10 those skilled in the at which are encompassed within the spirit of| the invention as defined by the seape of the claims and theit legal equivalents, ‘What is claimed: 1, A composition comprising: fone or more natural isomers of reduced folate selected from the group consisting of (68)-tetrahydrofolie acid, Semethyl(6S)-tetrahydrofolic acid, 5-formyl-(6S)- tetrabycrofolic acid, 10-formyl-(6R)-teteahydnofolic acid, 5,10-methylene-(6R)-tetrahydrofolic acid, 5,10- smther tetrahydrofolic acid, and polyglutamyl derivatives thereof 4 nutritional substance selected from the group consisting, ‘of a food preparation, an essential nutrient preparation, and combinations thereof, wherein, when the nutritional substance is a food preparation, the food preparation comprises two or ‘more food components and each gram of said food preparation has a natural molar amount, N, of said one ‘or more natural isomers of reduced folate, wherein N is ‘greater or equal to zero and wherein each gram of said ‘composition has total molar amount, T,of said one oF ‘more natural isomers of reduced folate geeater than Ni and wherein, when the nutritional substance is an essential ‘nutrient preparation, the essential nulrien! preparation comprises « vitamin other than ascorbie acid. 2./A composition according to claim 1, wherein ssid nutritional substance is a food preparation. 1-(6R)-Cerabydrofolic acid, S-formimino(6S)- °S 0 16 3. A composition according to claim 2, wherein said natural isomer of reduced folate is (6S)-etrahydrofolic acid ‘or a polygiutamyl derivative thereof 4. A composition according to claim 2, wherein said natural isomer of reduced folate is S-methyl-(6S)- letrahydrofolic acid or a polyglutamyl derivative thereo. '5. A composition according © claim 2, wherein said natural isomer of reduced folate is S-formyl-(6S)= {etrahydrofolic acid or a polyslutamyl derivative thereof. 6. A composition according to claim 2, wherein said natural isomer of reduced folate is 10-formyl-(OR)- ‘etrahydrofolic acid or a polygiutamyl derivative thereof. 7. A composition according 10 claim 2, wherein said natural isomer of reduced folate is 5,10-methylene-(6R)- lctrahydrofolic acid or a polyglutamyl derivative thereof. 8. A composition according to claim 2, whervin said natural isomer of reduced folate is 5,10-methenyl-(6R)- Ietrahydrofolie acid or a polyglutamsl derivative thereo. 9. A composition according to claim 2, wherein said natural isomer of reduced folate is S-formimino-(65)- tetrahydrofolic acid or a polyglutamyl derivative thereof. 10. A composition according to claim 1, wherein said nutritional substance is an essential nutrient preparation. IL. A composition according to claim 10, wherein said natural isomer of reduced folate is (6S)-etrahydrofolic acid ‘or a polyglutamyl derivative thereo. 12. A composition according to claim 10, wherein said natural isomer of reduced Tolate is S-methyl-(6S)- tetrahydrofolic acid or a polyglutamyl derivative thereof. 13. A composition according to claim 10, wherein said natural isomer of reduced folate is 5-formyl-(6S)- Ielrahydrofolie acid or a polyglutamsl derivative thereof. 14. A composition according to claim 10, wherein said natural isomer of reduced folate is 10-formyl-(OR)- Ielrahydrofolie acid or a polyglutamyl derivative thereo. 15. A composition according to claim 10, wherein said natural isomer of reduced folate is 3,10-merhylene-(6R)> Ietrahydrofolic acid or a polyglutamyl derivative thereo. 16. A composition according to claim 10, wherein said natural isomer of reduced Folate is 5,10-methenyl-(6R)- {etrahydrofolie acid or a polyglutamyl derivative therco. 17. A composition according to claim 10, wherein said natural isomer of reduced folate is S-formimino-(6S)- letrahydrofolic acid or a polyghutamyl derivative thereof.